U.S. patent application number 17/659274 was filed with the patent office on 2022-08-11 for treating neural disease with tyrosine kinase inhibitors.
This patent application is currently assigned to GEORGETOWN UNIVERSITY. The applicant listed for this patent is GEORGETOWN UNIVERSITY. Invention is credited to Charbel Moussa.
Application Number | 20220249481 17/659274 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220249481 |
Kind Code |
A1 |
Moussa; Charbel |
August 11, 2022 |
TREATING NEURAL DISEASE WITH TYROSINE KINASE INHIBITORS
Abstract
Provided herein are methods of treating or preventing a
neurodegenerative disease, a myodegenerative disease or a prion
disease in a subject comprising administering a tyrosine kinase
inhibitor.
Inventors: |
Moussa; Charbel;
(Germantown, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GEORGETOWN UNIVERSITY |
Washington |
DC |
US |
|
|
Assignee: |
GEORGETOWN UNIVERSITY
Washington
DC
|
Appl. No.: |
17/659274 |
Filed: |
April 14, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16909075 |
Jun 23, 2020 |
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17659274 |
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15148760 |
May 6, 2016 |
10709704 |
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16909075 |
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14398379 |
Oct 31, 2014 |
9474753 |
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PCT/US2013/039283 |
May 2, 2013 |
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15148760 |
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61641441 |
May 2, 2012 |
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61771515 |
Mar 1, 2013 |
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International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 45/06 20060101 A61K045/06; A61K 31/496 20060101
A61K031/496 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under grant
number AG30378 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of treating Lewy Body disease in a subject in need
thereof, comprising: selecting a subject with Lewy Body disease or
at risk for Lewy Body disease; and administering to the subject an
effective amount of a tyrosine kinase inhibitor or a
pharmaceutically acceptable salt thereof, wherein the tyrosine
kinase inhibitor is not Gleevec, and wherein the tyrosine kinase
inhibitor or a pharmaceutically acceptable salt thereof crosses the
blood brain barrier.
2. The method of claim 1, wherein the tyrosine kinase inhibitor is
selected from the group consisting of nilotinib or a
pharmaceutically acceptable salt thereof, bosutinib or a
pharmaceutically acceptable salt thereof, and a combination
thereof.
3. The method of claim 1, wherein the effective amount of the
tyrosine kinase inhibitor or a pharmaceutically acceptable salt
thereof is less than about 10 mg/kg.
4. The method of claim 1, wherein the tyrosine kinase inhibitor or
a pharmaceutically acceptable salt thereof is administered
daily.
5. The method of claim 1, further comprising administering a second
therapeutic agent to the subject.
6. A method of inhibiting or preventing toxic protein aggregation
in a neuron of a subject with Lewy Body disease, comprising
contacting the neuron in the subject with an effective amount of a
tyrosine kinase inhibitor or a pharmaceutically acceptable salt
thereof, wherein the tyrosine kinase inhibitor is not Gleevec, and
wherein the tyrosine kinase inhibitor or a pharmaceutically
acceptable salt thereof crosses the blood brain barrier.
7. The method of claim 6, wherein the tyrosine kinase inhibitor is
selected from the group consisting of nilotinib or a
pharmaceutically acceptable salt thereof, bosutinib or a
pharmaceutically acceptable salt thereof, and a combination
thereof.
8. The method of claim 6, wherein the protein is selected from the
group consisting of alpha-synuclein and insoluble Parkin.
9. The method of claim 6, wherein the effective amount of the
tyrosine kinase inhibitor or a pharmaceutically acceptable salt
thereof is less than about 10 mg/kg.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/909,075, filed Jun. 23, 2020, which is a continuation
of U.S. patent application Ser. No. 15/148,760, filed May 6, 2016,
now U.S. Pat. No. 10,709,704, issued Jul. 14, 2020, which is a
continuation application of U.S. patent application Ser. No.
14/398,379, filed Oct. 31, 2014, now U.S. Pat. No. 9,474,753,
issued Oct. 25, 2016, which is a U.S. national stage application
under 35 U.S.C. .sctn. 371 of PCT/US2013/039283, filed May 2, 2013,
which claims the benefit of U.S. Provisional Application No.
61/641,441, filed May 2, 2012, and U.S. Provisional Application No.
61/771,515, filed Mar. 1, 2013. The above-listed applications are
hereby incorporated herein by this reference in their
entireties.
BACKGROUND
[0003] Neurodegenerative diseases include genetic and sporadic
disorders associated with progressive nervous system dysfunction.
It has been estimated that one of four Americans will develop a
neurodegenerative condition in their lifetimes. Generally, however,
the underlying mechanisms causing the conditions are not well
understood and few effective treatment options are available for
preventing or treating neurodegenerative diseases. Similarly,
treatment options for myodegenerative disease and prion disease are
also limited.
SUMMARY
[0004] Provided herein is a method of treating or preventing a
neurodegenerative disease, a myodegenerative disease or a prion
disease in a subject, comprising selecting a subject with a
neurodegenerative disease of the central nervous system, a
myodegenerative disease or a prion disease or at risk for a
neurodegenerative disease of the central nervous system, a
myodegenerative disease or a prion disease and administering to the
subject an effective amount of a tyrosine kinase inhibitor, wherein
the tyrosine kinase inhibitor is not Gleevec, and wherein the
tyrosine kinase inhibitor crosses the blood brain barrier.
[0005] Further provided is a method of inhibiting or preventing
toxic protein aggregation in a neuron, a muscle cell or a glial
cell comprising contacting the neuron, the muscle cell or the glial
cell with an effective amount of a tyrosine kinase inhibitor,
wherein the tyrosine kinase inhibitor is not Gleevec and wherein
the tyrosine kinase inhibitor crosses the blood brain barrier.
[0006] Also provided is a method of rescuing a neuron from
neurodegeneration, a muscle from myodegeneration or a glial cell
from degeneration comprising contacting the neuron, the muscle cell
or the glial cell with an effective amount of a tyrosine kinase
inhibitor, wherein the tyrosine kinase inhibitor is not Gleevec and
wherein the tyrosine kinase inhibitor crosses the blood brain
barrier.
[0007] Further provided herein is a method of treating amyotrophic
lateral sclerosis or frontotemporal dementia in a subject,
comprising selecting a subject with amyotrophic lateral sclerosis
or frontotemporal dementia, wherein the subject has a TDP-43
pathology, and administering to the subject an effective amount of
a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor
is not Gleevec and wherein the tyrosine kinase inhibitor crosses
the blood brain barrier.
[0008] Also provided is a method of promoting parkin activity in a
subject, comprising selecting a subject with a disorder associated
with decreased Parkin activity and administering to the subject an
effective amount of a small molecule that increase parkin activity,
wherein the small molecule is not Gleevec.
[0009] Further provided is a method of treating or preventing a
neurodegenerative disease in a subject, comprising selecting a
subject with a neurodegenerative disease or at risk for a
neurodegenerative disease, determining that the subject has a
decreased level of parkin activity relative to a control, and
administering to the subject an effective amount of a small
molecule that increases parkin activity, wherein the small molecule
is not Gleevec.
[0010] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0011] FIG. 1 is a diagram showing the cellular mechanisms
associated with parkin activity in neurodegenerative conditions
(left) and upon intervention with tyrosine kinase inhibitors
(right). Intervention activates parkin activity to promote
clearance of autophagic vacuoles.
[0012] FIG. 2 is a diagram showing that amyloid accumulation leads
to autophagic induction and sequestration in phagophores. In
transgenic or amyloid expressing animals parkin interaction with
beclin-1 is reduced, leading to decreased maturation of phagophore
into autophagosomes and autophagic defects. Kinase inhibition
activates parkin and increases its interaction with beclin-1,
resulting in maturation of phagophores into phagosomes and
clearance. Subcellular fractionation via metrazimide gradients to
isolate the phagophore (AV-10), autophagosomes (AV-20) and the
lysosomes was used to show how the cell handles amyloid
accumulation and clearance.
[0013] FIG. 3 shows that parkin interacts with beclin-1 in wild
type but not parkin-/- mice: Proximity Ligation Assay (PLA) in situ
on 20 mm thick brain sections showed parkin and beclin-1
interaction in A) C57BL/6 mice but not B) parkin-/- mice (control),
indicating that parkin interacts with beclin-1. PLA in situ on 20
mm thick brain sections showed parkin and beclin-1 interaction in
C) Tg-A53T and D) Tg-APP mice treated with DMSO, E) Tg-A53T and F)
Tg-APP treated with 10 mg/kg nilotinib for 3 weeks, G) Tg-A53T and
H) Tg-APP treated with 5 mg/kg bosutinib for 3 weeks.
[0014] FIG. 4 is a graph representing ELISA levels of human
A.beta..sub.1-42 in brain lysates of triple mutant APP-AD mice
(Tg-APP) treated with either 1 mg/kg or 5 mg/kg Nilotinib once
every two days for 6 weeks. N=10 animals. P<0.05. ANOVA, with
Neuman Keuls multiple comparison. An asterisk indicates a
significant difference compared to DMSO. Bars are mean.+-.SD.
[0015] FIG. 5 is a graph representing ELISA levels of human
A.beta..sub.1-42 in brain lysates of triple mutant APP-AD mice
(Tg-APP) treated with either 1 mg/kg or 5 mg/kg bosutinib once
every two days for 6 weeks. N=10 animals. P<0.05. ANOVA With
Neuman Keuls multiple comparison. An asterisk indicates a
significant difference as compared to DMSO. Bars are
mean.+-.SD.
[0016] FIG. 6 is a graph representing ELISA levels of human
.alpha.-synuclein in brain lysates of A53T mice (A53T-Tg) treated
with 5 mg/kg Bosutinib once a day for 3 weeks. N=10 animals.
P<0.05. ANOVA, with Neuman Keuls multiple comparison. An
asterisk indicates a significant difference as compared to DMSO.
Bars are mean.+-.SD.
[0017] FIG. 7 is a graph representing ELISA levels of human
.alpha.-synuclein in brain lysates of A53T mice (A53T-Tg) treated
with either 1 mg/kg or 5 mg/kg Bosutinib once every 2 days for 6
weeks. N=10 animals. P<0.05. ANOVA, with Neuman Keuls multiple
comparison. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0018] FIG. 8 is a graph representing ELISA levels of human
.alpha.-synuclein in blood of A53T mice (A53T-Tg) treated with
either 1 mg/kg or 5 mg/kg Bosutinib once every 2 days for 6 weeks.
N=10 animals. P<0.05. ANOVA, with Neuman Keuls multiple
comparison. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0019] FIG. 9 is a graph representing ELISA levels of human
.alpha.-synuclein in brain lysates of A53T mice (A53T-Tg) treated
with either 1 mg/kg or 5 mg/kg Nilotinib once every second day for
6 weeks. N=10 animals. P<0.05. ANOVA, with Neuman Keuls multiple
comparison. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0020] FIG. 10 is a graph representing ELISA levels of human
.alpha.-synuclein in blood of A53T mice (A53T-Tg) treated with
either 1 mg/kg or 5 mg/kg Nilotinib once every second day for 6
weeks. N=10 animals. P<0.05. ANOVA, with Neuman Keuls multiple
comparison. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0021] FIG. 11 shows A) a graph representing ELISA levels of human
A.beta..sub.1-42, B) a graph representing human A.beta.1-40 in
brain lysates of triple mutant APP-AD mice (Tg-APP) treated with 5
mg/kg Bosutinib every day for 3 weeks, C) a graph representing
ELISA levels of mouse parkin and D) a graph representing mouse
phosphorylated Tau (Ser 396) in brain lysates of triple mutant
APP-AD mice (Tg-APP) treated with 5 mg/kg Bosutinib every day for 3
weeks. N=10 animals. P<0.05. ANOVA With Neuman Keuls multiple
comparison. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0022] FIG. 12 is a graph representing ELISA levels of human
A.beta..sub.1-42 in brain lysates of lentiviral A.beta..sub.1-42
injected mice (wild type and parkin-/- for 3 weeks and treated with
5 mg/kg Bosutinib every day for 3 additional weeks. N=10 animals.
P<0.05. ANOVA with Neuman Keuls multiple comparison. An asterisk
indicates a significant difference as compared to DMSO. Bars are
mean.+-.SD.
[0023] FIG. 13 shows that .alpha.-synuclein expression in the brain
increases its blood level and tyrosine kinase inhibition reverses
these effects in a parkin-dependent manner. Mice were injected
stereotaxically (bilaterally) with lentiviral .alpha.-synuclein
into the substantia nigra for 3 weeks. Then, half of the animals
were injected with 10 mg/Kg nilotinib and the other half with DMSO.
The effects of .alpha.-synuclein expression and tyrosine kinase
inhibition on A) brain and B) blood levels of .alpha.-synuclein
were compared. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0024] FIG. 14 shows that .alpha.-synuclein expression in the brain
increases its blood level and tyrosine kinase inhibition reverses
these effects in a parkin-dependent manner. Mice were injected
stereotaxically (bilaterally) with lentiviral .alpha.-synuclein
into the substantia nigra for 3 weeks. Then, half of the animals
were injected with 5 mg/Kg bosutinib and the other half with DMSO.
The effects of .alpha.-synuclein expression and tyrosine kinase
inhibition on A) brain and B) blood levels of .alpha.-synuclein
were compared. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0025] FIG. 15 shows that .alpha.-synuclein induced loss of
dopamine and homovanillic acid (HVA) levels. Tyrosine kinase
inhibition reversed these effects and improved motor performance.
Mice were injected stereotaxically (bilaterally) with lentiviral
.alpha.-synuclein into the substantia nigra for 3 weeks. Then, half
the animals were injected with 10 mg/kg Nilotinib or 5 mg/Kg
Bosutinib and the other half with DMSO. The effects of
.alpha.-synuclein expression and tyrosine kinase inhibition on A)
dopamine and homovanillic acid (HVA) levels (ELISA) were compared.
The effects of treatment on B) motor performance were evaluated
using rotarod. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0026] FIG. 16 shows that A.beta..sub.1-42 accumulates in AV-10 in
Tg-APP animals but drug treatment enhances autophagic clearance via
deposition of A.beta..sub.1-42 in AV-20 and lysosome. Histograms
show A.beta..sub.1-42 in subcellular fractions, including
autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20
(autophagosomes) and lysosomes. Transgenic 3.times.APP mice were
injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO
once a day for 3 consecutive weeks. Brain tissues were fractionated
to isolate AVs and human specific ELISA was performed to determine
protein contents. N=5 animals per treatment.
[0027] FIG. 17 shows that A.beta..sub.1-40 accumulates in AV-20 in
Tg-APP animals but drug treatment enhances autophagic clearance via
deposition of A.beta..sub.1-40 in AV-20 and lysosome. Histograms
show A.beta..sub.1-40 in subcellular fractions, including
autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20
(autophagosomes) and lysosomes. Transgenic 3.times.APP mice were
injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO
once a day for 3 consecutive weeks. Brain tissues were fractionated
to isolate AVs and specific ELISA was performed to determine
protein contents. N=5 animals per treatment.
[0028] FIG. 18 shows that P-Tau accumulates in AV-10 in Tg-APP
animals but drug treatment enhances autophagic clearance via
deposition of p-Tau in AV-20 and lysosome, which contains
degradative enzymes. Histograms show Tau hyper-phosphorylation
(p-Tau) at serine 396 in subcellular fractions, including
autophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20
(autophagosomes) and lysosomes. Transgenic 3.times.APP mice were
injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO
once a day for 3 consecutive weeks. Brain tissues were fractionated
to isolate AVs and mouse-specific ELISA was performed to determine
protein contents. N=5 animals per treatment.
[0029] FIG. 19 shows that drug treatment increases parkin activity
leading to protein clearance including parkin itself. Histograms
show parkin in subcellular fractions, including autophagic
vacuole-10 (AV-10; phagophores+autophagosomes), AV-20
(autophagosomes) and lysosomes. Transgenic 3.times.APP mice were
injected IP with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib or DMSO
once a day for 3 consecutive weeks. Brain tissues were fractionated
to isolate AVs and mouse specific ELISA was performed to determine
protein contents. Parkin accumulates in AV-10 in Tg-APP animals but
drug treatment enhances autophagic clearance via deposition of
parkin in AV-20 and lysosome, which contains degradative enzymes.
N=5 animals per treatment.
[0030] FIG. 20 shows that autophagic clearance is parkin-dependent.
Histograms show A.beta..sub.1-42 in subcellular fractions,
including autophagic vacuole-10 (AV-10;
phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes.
Wild type or parkin-/- mice were injected with lentiviral
A.beta..sub.1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib
or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional)
consecutive weeks. Brain tissues were fractionated to isolate AVs
and human specific ELISA was performed to determine protein
contents. A.beta..sub.1-42 accumulates in AV-10 in lentivirus
injected brains but drug treatment enhances autophagic clearance
via deposition of A.beta..sub.1-42 in AV-20 and lysosome. N=5
animals per treatment.
[0031] FIG. 21 shows that P-Tau at serine 396 accumulates in AV-10
in lentivirus injected brains but drug treatment enhances
autophagic clearance via deposition of p-Tau in AV-20 and lysosome,
where it is degraded. Histograms show p-Tau in subcellular
fractions, including autophagic vacuole-10 (AV-10;
phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes.
Wild type or parkin-/- mice were injected with lentiviral
A.beta..sub.1-42 for 3 weeks and treated IP with 10 mg/kg Nilotinib
or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional)
consecutive weeks. Brain tissues were fractionated to isolate AVs
and mouse specific. ELISA was performed to determine protein
contents. Autophagic clearance is parkin-dependent. N=5 animals per
treatment.
[0032] FIG. 22 shows that .alpha.-synuclein accumulates in AV-10 in
lentivirus injected brains but drug treatment enhances autophagic
clearance via deposition of .alpha.-synuclein in AV-20 and
lysosome, which contains degradative enzymes. Histograms show
.alpha.-synuclein in subcellular fractions, including autophagic
vacuole-10 (AV-10; phagophores+autophagosomes), AV-20
(autophagosomes) and lysosomes. Wild type or parkin-/- mice were
injected SN with lentiviral .alpha.-synuclein for 3 weeks and
treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO
once a day for 3 (additional) consecutive weeks. SN tissues were
fractionated to isolate AVs and human specific ELISA was performed
to determine protein contents. Autophagic clearance is
parkin-dependent. N=5 animals per treatment.
[0033] FIG. 23 shows that P-Tau accumulates in AV-10 in lentivirus
injected brains but drug treatment enhances autophagic clearance
via p-Tau deposition in AV-20 and lysosome, which contains
degradative enzymes. Histograms show p-Tau at serine 396 in
subcellular fractions, including autophagic vacuole-10 (AV-10;
phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes.
Wild type or parkin-/- mice were injected SN with lentiviral
.alpha.-synuclein for 3 weeks and treated IP with 10 mg/kg
Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3
(additional) consecutive weeks. SN tissues were fractionated to
isolate AVs and mouse specific ELISA was performed to determine
protein contents. Autophagic clearance is parkin-dependent. N=5
animals per treatment.
[0034] FIG. 24 shows that .alpha.-synuclein accumulates in AV-10 in
A53T brains but drug treatment enhances autophagic clearance via
.alpha.-synuclein deposition in AV-20 and lysosome. Histograms show
.alpha.-synuclein in subcellular fractions, including autophagic
vacuole-10 (AV-10; phagophores+autophagosomes), AV-20
(autophagosomes) and lysosomes, containing digestive enzymes.
Transgenic A53T mice were injected IP with 10 mg/kg Nilotinib or 5
mg/Kg Bosutinib or DMSO once a day for 3 consecutive weeks. Brain
tissues were fractionated to isolate AVs and human specific ELISA
was performed to determine protein contents. N=5 animals per
treatment.
[0035] FIG. 25 shows that P-Tau accumulates in AV-10 in A53T brains
but drug treatment enhances autophagic clearance via p-Tau
deposition in AV-20 and lysosome. Histograms show p-Tau at Serine
396 in subcellular fractions, including autophagic vacuole-10
(AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) and
lysosomes, containing digestive enzymes. Transgenic A53T mice were
injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO
once a day for 3 consecutive weeks. Brain tissues were fractionated
to isolate AVs and mouse specific ELISA was performed to determine
protein contents. N=5 animals per treatment.
[0036] FIG. 26 shows that parkin accumulates in AV-10 in A53T
brains but drug treatment enhances autophagic clearance via parkin
deposition in AV-20 and lysosome. Histograms show parkin in
subcellular fractions, including autophagic vacuole-10 (AV-10;
phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes,
containing digestive enzymes. Transgenic A53T mice were injected IP
with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for
3 consecutive weeks. Brain tissues were fractionated to isolate AVs
and mouse specific ELISA was performed to determine protein
contents. N=5 animals per treatment.
[0037] FIG. 27 is a diagram illustrating howTyrosine kinase
inhibition increases parkin activity and facilitates autophagic
clearance of p-Tau. This process requires Tau stabilization of
intact microtubules. Tyrosine kinase activation, p-Tau accumulation
and impaired autophagy are recognized in neurodegeneration.
Decreased parkin solubility and accumulation with intracellular
A.beta. and p-Tau in autophagic vacuoles in AD brains occurs, while
exogenous parkin facilitates autophagic clearance in animal
models.
[0038] FIG. 28 shows A) phosphorylated c-Abl at tyrosine 412 (T412)
and B) endogenous parkin expression merged in C) hippocampus of 6
month old C57BL/6 mice treated IP with DMSO daily for 3 weeks. FIG.
28 also shows D) decreased phosphorylated c-Abl at tyrosine 412
(T412) and E) increased endogenous parkin expression merged in F)
hippocampus of 6 month old C57BL/6 mice treated IP with 5 mg/kg
Bosutinib daily for 3 weeks.
[0039] FIG. 29 shows A) parkin and B) A.beta. expression merged in
C) cortex of 6 month old Tg-APP mice treated with DMSO or 5 mg/kg
Bosutinib (D-F) once a day for 3 weeks. Using a different
combination of antibodies (see figure G-I showing expression of
parkin (G) and A.beta. (H) in the hippocampus of Tg-APP mice
treated DMSO. J-H show the increase in parkin level in animals
treated for 3 weeks once a day with Bosutinib (J) along with
decreased plaque levels (K and L) in the hippocampus.
[0040] FIG. 30 shows plaque A.beta. stained with 6E10 antibody and
counterstained with DAB in the brain of Tg-APP animals treated IP
with DMSO once a day for 3 weeks.
[0041] FIG. 31 shows plaque A.beta. stained with 6E10 antibody and
counterstained with DAB in the brain of Tg-APP animals treated IP
with 5 mg/kg Bosutinib once a day for 3 weeks.
[0042] FIG. 32 shows that Bosutinib decreases .alpha.-synuclein
levels in transgenic mice expressing A53T throughout the brain. A-D
show human .alpha.-synuclein expression in lentiviral LacZ injected
(for 3 weeks) substantia nigra with A) DMSO and B) 5 mg/kg
Bosutinib once a day for 3 weeks. C and D show human
.alpha.-synuclein expression in lentiviral .alpha.-synuclein
injected (for 3 weeks) substantia nigra with C) DMSO and D) or
Bosutinib once a day for 3 weeks. E-H show Tyrosine Hydroxylase
(TH) expression in lentiviral LacZ injected (for 3 weeks)
substantia nigra with E) DMSO and F) 5 mg/kg Bosutinib once a day
for 3 weeks. G and H show TH expression in lentiviral
.alpha.-synuclein injected (for 3 weeks) substantia nigra with G)
DMSO and H) or Bosutinib once a day for 3 weeks. .alpha.-synuclein
decreases TH neurons and Bosutinib rescues these cells. I-L show
human .alpha.-synuclein expression in A53T mice in I) Cortex, J)
Striatum, K) Brainstem and L) Hippocampus treated with DMSO for 3
weeks. M-P show human .alpha.-synuclein expression in A53T mice in
M) cortex, N) striatum, 0) brainstem and P) hippocampus treated
with 5 mg/kg Bosutinib for 3 weeks.
[0043] FIG. 33 provides graphs representing performance on a Morris
water maze test (in seconds) showing that IP treatment with 5 mg/kg
Bosutinib once daily for 3 weeks improved cognitive behavior in
mice injected bilaterally with lentiviral A.beta..sub.1-42 for 3
weeks prior to drug treatment. Bosutinib treated mice found the
platform (A) but DMSO treated mice spent more time in NW area,
where they were initially placed or the NE or SW without
effectively finding the platform area. Bosutninb improved cognitive
performance in a parkin-dependent manner as the parkin-/- mice did
seemed not to learn much. B) shows that Bosutinib treated mice
traveled less distance with less speed, but entered the platform
area more than DMSO treated mice.
[0044] FIG. 34 shows that tyrosine kinase inhibitors increase
parkin activity levels. A) shows ELISA levels of parkin activity in
human M17 neuroblastoma cells treated with either 10 mg/kg
Nilotinib or 5 mg/kg Bosutinib for 24 hrs. N=12. P<0.05.ANOVA,
with Neuman Keuls multiple comparison. An asterisk indicates a
significant difference as compared to DMSO. Bars are mean.+-.SD. B)
shows parkin levels (ELISA) in brain lysates of wild type mice
injected with lentiviral .alpha.-synuclein for 3 weeks and then
treated with 10 mg/kg Nilotinib once every two days for 3 weeks.
N=10 animals. P<0.05.ANOVA, with Neuman Keuls multiple
comparison. An asterisk indicates a significant difference as
compared to DMSO. Bars are mean.+-.SD.
[0045] FIG. 35 is a Western blot analysis of brain lysates from
Tg-APP mice treated with 5 mg/kg Bosutinib for 3 additional weeks.
These blots show decreased levels of c-Abl, increased parkin and
alteration of different molecular markers of autophagy, indicating
that A.beta. alters normal autophagy and Bosutinib boosts autophagy
to clear A.beta..sub.1-42.
[0046] FIG. 36 is a Western blot analysis of brain lysates from
Tg-APP mice treated with 5 mg/kg Bosutinib for 3 weeks. These blots
show alterations in the levels of molecular markers of
autophagy.
[0047] FIG. 37 is a Western blot analysis of brain lysates from
Tg-APP mice treated with 5 mg/kg Bosutinib for 3 additional weeks.
These blots show decreased levels of C-terminal fragments (CTFs)
and phospho-tyrosine.
[0048] FIG. 38 is a Western blot analysis of brain lysates from
Tg-APP mice treated with 5 mg/kg Bosutinib once a day for
additional weeks. These blots show decreased levels of different
Tau isotopes.
[0049] FIG. 39 is a Western blot analysis of brain lysates from
wild type mice expressing lentiviral A.beta..sub.1-42 (3 weeks)
with and without Bosutinib (5 mg/kg) treatment for 3 additional
weeks. These blots show levels of different molecular markers of
autophagy, indicating that A.beta..sub.1-42 alters normal autophagy
and Bosutinib boosts autophagy to clear A.beta..sub.1-42.
[0050] FIG. 40 is a Western blot analysis of brain lysates from
wild type mice expressing lentiviral A.beta..sub.1-42 (3 weeks)
with and without Bosutinib treatment for 3 additional weeks. These
blots show decreased levels of ubiquitin (top blot) and pan
phospho-tyrosine (second blot) and SIAH2, suggesting that Bosutinib
is a broad tyrosine kinase inhibitor.
[0051] FIG. 41 is a Western blot analysis of brain lysates from
wild type mice expressing lentiviral A.beta..sub.1-42 (3 weeks)
with and without Bosutinib treatment for 3 additional weeks. These
blots show decreased levels of different Tau isotopes.
[0052] FIG. 42 is a Western blot analysis of brain lysates from
wild type mice expressing lentiviral .alpha.-synuclein (3 weeks)
with and without Bosutinib treatment for 3 additional weeks. Blots
show in order increased .alpha.-synuclein in lentiviral synuclein
injected animals, along with decreased c-Abl levels and
phosphorylation, increased parkin levels and markers of autophagy,
including P62, HDAC6, LC3 and ATG12 compared to loading controls
tubulin and MAP2.
[0053] FIG. 43 shows that parkin is insoluble in post-mortem
striatum of human PD patients. A) Histograms represent ELISA
measurement of human parkin in the caudate of PD patients and
control subjects. B) is a WB analysis on 4-12% SDS-NuPAGE gel of
soluble human post-mortem striatal lysates in PD patients and
control subjects, showing parkin (1st blot) and ubiquitinated
proteins (2nd blot) compared to actin loading control. C)
Histograms represent quantification of blots. D) is a WB analysis
on 4-12% SDS NuPAGE gel showing the levels of insoluble parkin (1st
blot), phospho-parkin (2nd blot), ubiquitinated proteins (3rd
blot), and actin (4th blot). E) Histograms represent quantification
of blots. Asterisks indicate a significant difference. F) Box plot
represents individual samples of human PD patients and age-matched
controls. Histograms are mean.+-.SD expressed as % to control.
ANOVA, Neumann Keuls with multiple comparison, or non-parametric
t-Test. P<0.05. N=12 PD patients and 7 control subjects.
[0054] FIG. 44 shows immunostaining of human tissues with human and
GFAP antibodies. Immunostaining of 20 .mu.m thick paraffin embedded
serially sectioned brains with A) human anti-parkin (PRK8) staining
and counterstaining with nuclear marker DAPI showing cytosolic
protein, B) co-staining with parkin and glial marker GFAP showing
parkin expression in astrocytes, C) TH staining in the caudate of a
control subject, D) parkin staining and counterstaining with
nuclear marker DAPI showing cytosolic protein, E). co-staining with
parkin and glial marker GFAP showing parkin expression in
astrocytes, F). TH staining in the caudate of a PD/AD patient, G)
parkin staining and counterstaining with DAPI showing cytosolic
protein, H) co-staining with parkin and glial marker GFAP showing
parkin expression in astrocytes, I) TH staining in the midbrain/SN
of a control subject, J) parkin staining and counterstaining with
DAPI showing cytosolic protein, K) co-staining with parkin and
glial marker GFAP showing parkin expression in astrocytes, L) TH
staining in the midbrain/SN of a PD patient. M). human anti-parkin
(AB5112) staining and counterstaining with nuclear marker DAPI
showing cytosolic protein, N) co-staining with parkin and glial
marker GFAP showing parkin expression in astrocytes, 0) TH staining
in the caudate of a control subject.
[0055] FIG. 45 shows subcellular fractionation in frozen human PD
brain tissues. A) shows human anti-parkin (AB5112) staining and
counterstaining with nuclear marker DAPI showing cytosolic protein.
B) shows neuronal marker MAP-2 staining and DAPI and C) shows
merged parkin and MAP-2 in stained serial sections. D) shows TH in
the midbrain/SN of a control subject. E) shows human anti-parkin
(AB5112) staining and counterstaining with nuclear marker DAPI
showing cytosolic protein. F) shows neuronal marker MAP-2 staining
and DAPI and G) shows merged parkin and MAP-2 in serial sections
stained with H) TH in the midbrain/SN of a PD with Dementia
patient. I) shows a WB analysis on 4-12% SDS NuPAGE gel of human
striatal lysates showing expression of LC3-I and LC3-II (first
panel), LC3-B (second panel) compared to actin loading control
(bottom panel) J) shows histograms representing densitometry
analysis of blots. K) shows a Western blot in subcellular extracts
showing LC3-B in AV-10 and AV-20 and LAMP-3 in lysosomal fraction,
as well as mitochondrial marker COX-IV and nuclear marker PARP-1.
Graphs represent subcellular fractionation and ELISA measurement of
L) human .alpha.-synuclein, M) human parkin and N) human p-Tau
(AT8). Asterisks indicate significantly different to control.
ANOVA, Neumann Keuls with multiple comparison, P<0.05. N=12 PD
patients and 7 control subjects.
[0056] FIG. 46 shows lentiviral expression of .alpha.-synuclein
leads to p-Tau and parkin activity reverses these effects. A) is
aWB analysis on 4-12% SDS-NuPAGE gel of rat striatal extracts
showing levels of parkin (top blot) and .alpha.-synuclein (middle
blot) expression and actin levels (lower blot). B) shows histograms
representing quantification of human .alpha.-synuclein levels by
ELISA. C) shows histograms representing quantification of human
parkin activity. D) is an ELISA measurement of rat p-Tau.
Thioflavin-S staining of 20 .mu.m striatal sections in lentiviral
E) parkin, F) .alpha.-synuclein and G) parkin+.alpha.-synuclein
injected brains. Human .alpha.-synuclein staining of 20 .mu.m
sections cut serially with the thioflavin-S sections is shown in
for lentiviral K) parkin, L) .alpha.-synuclein and M)
parkin+.alpha.-synuclein injected brains. Asterisks indicate
significantly different. Histograms are mean.+-.SD expressed as %
control. ANOVA, Neumann Keuls with multiple comparison, P<0.05.
N=8 animals per treatment for WB and ELISA, 8 for IHC.
[0057] FIG. 47 shows that wild type, but not T240R, parkin reverses
.alpha.-synuclein-induced accumulation of autophagosomes. Electron
micrographs of striatal sections in rat brains injected with A)
Lentiviral LacZ (Lv-LacZ) as control, B) Lentiviral
.alpha.-synuclein (Lv-Syn), C) Lentiviral
parkin+lentiviral-.alpha.-synuclein (Lv-Syn+Lv-Par), vacuoles
contain debris and D) Lentiviral .alpha.-synuclein+lentiviral T240R
(Lv-Syn+Lv-T240R). Asterisk indicates autophagic vacuoles. N=8.
Graphs represent subcellular fractionation and ELISA measurement of
E) .alpha.-synuclein and F) p-Tau in gene transfer animal models.
ANOVA, Neumann Keuls with multiple comparison, P<0.05. N=5
animals per treatment for subcellular fractionation.
[0058] FIG. 48 shows that functional parkin, not mutant T240R
reverses .alpha.-synuclein alteration of normal autophagy. A) shows
a WB analysis on 4-12% SDS NuPAGE gel of rat striatal lysates
showing expression of beclin (first panel), Atg7 (second panel) and
Atg12 (third panel) compared to actin loading control (bottom
panel) in animals injected with Lv-LacZ, Lv-Par, Lv-Syn and
Lv-Par+Lv-Syn. B) shows aWB analysis of rat striatal brain lysates
showing expression of LC3-B (first panel), and HDAC6 (second panel)
compared to actin loading control (bottom panel) in animals
injected with Lv-LacZ, Lv-Par, Lv-Syn and Lv-Par+Lv-Syn. Staining
of 20 .mu.m thick cortical brain sections injected with C)
Lentiviral parkin (Lv-Par), D) Lentiviral .alpha.-synuclein
(Lv-Syn) E) Lentiviral parkin+lentiviral .alpha.-synuclein
(Lv-Par+Lv-Syn) and F) Lentiviral T240R+lentiviral
.alpha.-synuclein (Lv-T240R+Lv-Syn) is shown. G) shows histograms
representing stereological counting of LC3-B positive cells in the
striatum. H) is a Western blot analysis on 4-12% SDS NuPAGE gel
with P62 antibody. Asterisks indicate a significant difference.
Histograms are mean.+-.SD converted to % control. ANOVA, Neumann
Keuls with multiple comparison, P<0.05. N=8 animals per
treatment for WB and ELISA, 8 for IHC.
[0059] FIG. 49 shows that parkin is increased in AD brains. A)
shows aWB analysis on 4-12% SDS-NuPAGE gel of human post-mortem
cortical lysates in AD. B) shows histograms representing human
parkin levels measured by ELISA. C) is aWB analysis on 4-12%
SDS-NuPAGE gel showing expression level of parkin's possible
targets for degradation, including ubiquitinated proteins (top
blot), tubulin (2nd blot) and Cyclin E (3rd blot) and actin (4th
blot). D) shows histograms representing blot quantification by
densitometry. E) is aWB analysis on 4-12% SDS-NuPAGE gel showing
insoluble proteins extracted in 4M urea, including total parkin
(top blot) and phosphorylated parkin at Serine 378 (2nd blot) and
actin (3rd blot). F) shows Histograms representing blot
quantification by densitometry. Asterisks indicate a significant
difference. Histograms are mean.+-.SD expressed as % control. All
bands were quantified relative to actin levels. ANOVA, Neumann
Keuls with multiple comparison, P<0.05.
[0060] FIG. 50 shows increased intraneuronal A.beta..sub.1-42 and
parkin co-localization in the hippocampus of AD brains. IHC of
paraffin embedded 30 .mu.m thick sections of human hippocampus from
control subject (case #1252) stained with A) Human anti-41-42
antibody+DAPI and B) Anti-parkin antibody+DAPI are shown. C) is a
merged figure showing co-staining of A.beta..sub.1-42 and parkin.
IHC of sections of hippocampus from AD patient (case #1774) stained
with D) Human anti-A.beta..sub.1-42 antibody+DAPI and E) Human
anti-parkin antibody+DAPI are shown. F) is a merged figure showing
co-staining of A.beta..sub.1-42 and parkin. IHC of sections of
hippocampus from AD patient (case #1861) stained with G) 4G8
anti-A.beta..sub.1-42 antibody+DAPI and H) human anti-parkin
antibody+DAPI are shown. and I) is a merged figure showing
co-staining of (4G8) A.beta..sub.1-42 and parkin.
[0061] FIG. 51 shows that parkin co-localizes with intraneuronal
A.beta..sub.1-42 in the cortex of AD brains. IHC of paraffin
embedded 30 .mu.m thick sections of human entorhinal cortex from AD
patient (case #1833) stained with A) human anti-A.beta..sub.1-42
antibody+DAPI and B) anti-parkin antibody+DAPI are shown. C) is a
merged figure showing co-staining of A.beta..sub.1-42 and parkin.
IHC of sections of human neocortex from AD patient (case #1851)
stained with D) human anti-A.beta..sub.1-42 antibody+DAPI and E)
anti-parkin antibody+DAPI are shown. F) is a merged figure showing
co-staining of A.beta..sub.1-42 and parkin. IHC of sections of
necortex from AD patient (case #1861) stained with G) 4G8
anti-A.beta..sub.1-42 antibody+DAPI and H) human anti-parkin
antibody+DAPI are shown. I) is a merged figure showing co-staining
of (4G8) A.beta..sub.1-42 and parkin.
[0062] FIG. 52 shows that parkin, A.beta..sub.1-42 and p-Tau
accumulate in autophagic vacuoles of AD brains. A) is aWB analysis
on 4-12% SDS-NuPAGE gel of human post-mortem cortical lysates in AD
probed with anti-LC3 antibody showing LC3-I and LC3-II (1st blot)
and LC3-B (2nd blot) and actin (3rd blot). B) shows histograms
representing blot quantification by densitometry. C) is aWB
analysis of Metrazimide-isolated fractions from frozen brain tissue
showing lysosomal marker LAMP-3 in the floating fraction and
detection of LC3-B in AV-10 and AV-20. Graphs represent ELISA
measurement in autophagic vacuoles of human D) A.beta..sub.1-42, E)
A.beta..sub.1-40, F) p-Tau (AT8) and G) parkin. Asterisks indicate
a significant difference. Histograms are mean.+-.SD expressed as %
control. All bands were quantified relative to actin levels. ANOVA,
Neumann Keuls with multiple comparison, P<0.05.
[0063] FIG. 53 shows that parkin decreases the level of lentiviral
A.beta..sub.1-42 and p-Tau in gene transfer animal models. A) is
aWB analysis on 4-12% SDS NuPAGE gel showing the expression levels
of parkin and A.beta..sub.1-42, analyzed with a synthetic peptide
as a molecular weight and antibody control. B) shows histograms
represent quantification of human parkin by ELISA. C) shows a human
A.beta..sub.1-42 ELISA 2 weeks after lentiviral injection. D) shows
ELISA measurement of rat p-Tau 2 and 4 weeks post-injection.
Thioflavin-S staining of 20 .mu.m cortical sections in lentiviral
E) LacZ, F) A.beta..sub.1-42 and G). parkin+A.beta..sub.1-42
injected brains is also shown. Asterisks indicate a significant
difference. Histograms are mean.+-.SD expressed as % to control.
All bands were quantified relative to actin levels. ANOVA, Neumann
Keuls with multiple comparison, P<0.05. N=8 animals per
treatment for WB and ELISA, 8 for IHC.
[0064] FIG. 54 shows that parkin clears A.beta..sub.1-42-induced
accumulation of autophagic vacuoles. Electron micrographs of
cortical sections in rat brains injected with A) Lentiviral LacZ
(Lv-LacZ) as control, B) lentiviral parkin (Lv-Par), C) lentiviral
A A.beta..sub.1-42 (Lv-A A.beta..sub.1-42) (arrows indicate
vacuoles) and D) lentiviral parkin+lentiviral A A.beta..sub.1-42
(Lv-A A.beta..sub.1-42+Lv-Par) (vacuole contains debris) are shown.
N=8. Graphs represent subcellular fractionation (Blot) and ELISA
measurement of E) A.beta..sub.1-42 and F) p-Tau in gene transfer
animal models. All bands were quantified relative to actin levels.
ANOVA, Neumann Keuls with multiple comparison, P<0.05. N=5
animals per treatment for subcellular fractionation.
[0065] FIG. 55 shows that intracellular A.beta..sub.1-42 impairs
normal autophagy and parkin facilitates autophagic clearance. A) is
aWB analysis on 4-12% SDS NuPAGE gel of rat cortical lysates
showing expression of beclin (first panel), Atg7 (second panel) and
Atg12 (third panel) and actin loading control (bottom panel) in
animals injected with Lv-LacZ, Lv-Par, Lv-A.beta..sub.1-42 and
Lv-Par+Lv-A.beta..sub.1-42. B) is aWB analysis of rat cortical
brain lysates showing expression of LC3-B (first panel), and HDAC6
(second panel) and actin loading control (bottom panel) in animals
injected with Lv-LacZ, Lv-Par, Lv-A.beta..sub.1-42 and
Lv-Par+Lv-A.beta..sub.1-42. Staining of 20 .mu.m thick cortical
brain sections injected with C) lentiviral LacZ (Lv-LacZ), D)
lentiviral parkin (Lv-Par) E) lentiviral A.beta..sub.1-42
(Lv-A.beta..sub.1-42) and F) lentiviral parkin+lentiviral
A.beta..sub.1-42 (Lv-Par+Lv-A.beta..sub.1-42) are shown. G) shows
histograms representing stereological counting of LC3-B positive
cells in the cortex. H) is a WB analysis of 4-12% SDS NuPAGE gel
showing P62 levels. Asterisks indicate a significant difference.
Histograms are mean.+-.SD expressed as % control. All bands were
quantified relative to actin levels. ANOVA, Neumann Keuls with
multiple comparison, P<0.05. N=8 animals per treatment for WB
and ELISA, 8 for IHC.
[0066] FIG. 56 shows that c-Abl activation is associated with
accumulation of .alpha.-synuclein. A WB on 10% SDS-NuPAGE gel shows
A) lentiviral .alpha.-synuclein expression (1st blot), total c-Abl
(2nd blot) and tyrosine 412 (T412) phosphorylated c-Abl (3rd blot)
and actin (N=9). B) shows total c-Abl (1st blot) T412 c-Abl (2nd
blot) and actin in human post-mortem striatal extracts, N=9 PD and
7 controls, p<0.02, two-tailed t-test. C) shows densitometry of
human WBs. D) is aWB on 4-12% SDS-NuPAGE gel that shows total c-Abl
(1st blot) and tyrosine 412 (T412) phosphorylated c-Abl (2nd blot),
and mouse .alpha.-synuclein expression (3rd blot) and actin (N=9).
E) is a graph representing quantification of Mass Spec analysis of
brain Nilotinib (N=5/time point). Graphs represent caspase-3
activity in F) lentiviral .alpha.-synuclein and LacZ injected mice
(N=14) with and without Nilotinib, and G) 6-8 month old transgenic
A53T mice (N=15) and wild type age-matched controls (N=64) with and
without Nilotinib. *Significantly different, ANOVA, Neumann Keuls
multiple comparison, p<0.05.
[0067] FIG. 57 shows that Nilotinib clears .alpha.-synuclein and
protects SN Tyrosine hydroxylase (TH) neurons. Immunohistochemical
staining of 20 .mu.m thick brain sections show human
.alpha.-synuclein in A) lentiviral injected LacZ+Nilotinib mice, B)
mice injected with lentiviral .alpha.-synuclein into the SN and
treated with DMSO and C) mice injected with lentiviral
.alpha.-synuclein and treated with Nilotinib. Immunohistochemical
staining of 20 .mu.m thick brain sections show Tyrosine Hydroxylase
in D) lentiviral injected LacZ+Nilotinib mice, G is higher
magnification from a different animal and E) mice injected with
lentiviral .alpha.-synuclein and treated with DMSO. H) is higher
magnification from a different animal. F) shows mice injected with
lentiviral .alpha.-synuclein and treated with Nilotinib. I) is
higher magnification from a different animal. J) shows Nissl
counter-stained cells in LacZ+Nilotinib, K). .alpha.-synuclein+DMSO
and L). .alpha.-synuclein+Nilotinib.
[0068] FIG. 58 shows that Nilotinib clears accumulation of
autophagic vacuoles in SN of lentiviral .alpha.-synuclein mice.
Transmission electron microscopy of SN neurons shows accumulation
of cytosolic debris and autophagic vacuoles (AVs) in Lentiviral
.alpha.-synuclein expressing mice with DMSO treatment (see FIGS. 58
A, C and E). FIGS. 58 B, D&F) show appearance of larger AVs in
Nilotinib treated mice.
[0069] FIG. 59 shows that Nilotinib attenuates .alpha.-synuclein
levels in A53T mice. Immunohistochemical staining of 20 .mu.m thick
brain sections shows abundant expression of human .alpha.-synuclein
in 6-8 month old transgenic A53T mice treated with DMSO in the A)
striatum, B) brainstem C) cortex and D) hippocampus of different
animals. Daily IP injection of Nilotinib for 3 weeks shows decrease
of human .alpha.-synuclein in the E) striatum, F) brainstem G)
cortex and H) hippocampus.
[0070] FIG. 60 shows that Nilotinib activates parkin and induces
autophagic clearance. A) is a graph representing MTT-based cell
viability in human M17 neuroblastoma cells (N=12) transfected with
A.beta..sub.1-42 (or LacZ) cDNA for 24 hr, and then treated with 10
.mu.M Nilotinib for an additional 24 hr. B) is a graph representing
proteasome activity via Chymotrypsin-like assays using 20 .mu.M 20S
proteasome inhibitor lactacystin as a specificity control in human
neuroblastoma cells (N=12) with and without Nilotinib. C) is a
Human A.beta..sub.1-42 ELISA before and after Nilotinib treatment
in B35 rat neuroblastoma cells (N=12) in media, soluble (STEN
buffer) and insoluble (30% formic acid) lysates in the presence and
absence of shRNA beclin-1. D) is aWB of soluble cell lysates (from
C) showing beclin-1, parkin and LC3 levels with and without
Nilotinib (N=12). E) is a graph represents parkin E3 ubiquitin
ligase function in B35 neuroblastoma cells treated with DMSO or
Nilotinib for 24 hr. Recombinant E1-E2-E3 (positive) or KO
(negative) were used as specificity controls. F) is a graph
representing caspase-3 activity in 1 year old C57BL/6 (N=64) (wild
type) or parkin-/- mice (N=16-19) injected with lentiviral
A.beta..sub.1-42 and treated (IP) with 10 mg/kg for 3 weeks. *
Significantly different, ANOVA with Neumann Keuls multiple
comparison, p<0.05.
[0071] FIG. 61 shows that Nilotinib clearance of brain amyloid is
associated with parkin activation. A graph represents ELISA levels
of A) soluble and insoluble human A.beta..sub.1-42 and B) ELISA
levels of soluble and insoluble human A.beta.1-40 in the brain of
8-12 months old Tg-APP mice (N=9) injected (IP) with 10 mg/kg once
a day for 3 weeks. C) is a graph that represents ELISA levels of
mouse p-Tau in the brain of 8-12 months old Tg-APP mice (N=9). D)
is a graph tat represents ELISA levels of soluble and insoluble
mouse parkin in the brain of 8-12 months old Tg-APP mice (N=9)
injected (IP) with 10 mg/kg (daily for 3 weeks) and parkin-/- brain
extracts as specificity control. E) is aWB analysis on 4-12% SDS
Nu-PAGE gels of brain extracts from Tg-APP treated with Nilotinib
or DMSO showing APP, c-Abl, p-c-AB1 and CTFs and MAP-2 as control
(N=11). F) is aWB of post-mortem cortical extracts of AD patients
(N=12 AD and 7 control) on 10% SDS Nu-PAGE and G) is a graph that
represents densitometry and ratio of c-Abl and p-c-Abl and parkin.
* Significantly different, non-parametric t-test, P<0.05. Also
shown is a graph representing ELISA levels of H) soluble and
insoluble human A.beta..sub.1-42, and I) ELISA levels of mouse
p-Tau in the brain of mice (N=9) injected (IP) with 10 mg/kg (3
weeks). * Significantly different, ANOVA with Neumann Keuls
multiple comparison, p<0.05.
[0072] FIG. 62 shows that Nilotinib promotes autophagic clearance
of amyloid. WB of brain extracts on 4-12% Nu-Page SDS gels are
shown for A) in lentiviral A.beta..sub.1-42 in wild type
mice.+-.Nilotinib showing, c-Abl, p-c-Abl, LC3-B and LC3 relative
to MAP-2 and B) parkin, beclin-1, Atg-5 and 12 relative to tubulin
(N=9). Western blot analysis of brain extracts on 10% Nu-Page SDS
gels for C) Tg-APP.+-.Nilotinib showing, parkin, LC3B, LC3, Atg-5
and beclin-1 relative to tubulin and D) total Tau, ATB, AT180, Ser
396 and Ser 262 relative to actin (N=12) are also provided. E) is a
Western blot analysis of brain extracts on 4-12% Nu-Page SDS gels
in lentiviral A.beta..sub.1-42 in parkin-/- mice.+-.Nilotinib
showing, parkin, beclin-1, LC3 and LC3A relative to tubulin and F)
is a Western blot analysis of Atg-5 and Atg12 relative to MAP-2
(N=7).).* Significantly different, ANOVA with Neumann Keuls
multiple comparison, p<0.05.
[0073] FIG. 63 shows that Nilotinib increases parkin level and
decreases plaque load. Staining of 20 .mu.m brain sections shows
plaque formation within various brain regions in A-D) Tg-APP+DMSO
and E-H) Nilotinib group after a 3-week treatment. Staining of 20
.mu.m thick brain sections shows I) parkin and J) A.beta..sub.1-42
K) is a merged figure in hippocampus of Tg-APP mice after 3 weeks
of DMSO treatment. L) shows parkin, M) shows A.beta..sub.1-42 and
N) is a merged figure in hippocampus of Tg-APP mice after 3 weeks
of Nilotinib treatment. 0) shows parkin, P) shows A.beta..sub.1-42
and Q) is amerged figure in the cortex of Tg-APP mice after 3 weeks
of DMSO treatment. R) shows parkin, S) shows A.beta..sub.1-42 and
T) is amerged figure in cortex of Tg-APP mice after 3 weeks of
Nilotinib treatment. Staining of 20 .mu.m brain sections shows
intracellular A.beta..sub.1-42 within the U). hippocampus of
lentiviral A.beta..sub.1-42 injected mice, inset higher
magnification, and V) Nilotinib clearance of intracellular
A.beta..sub.1-42 (inset is higher magnification). Staining of 20
.mu.m brain sections shows intracellular A.beta..sub.1-42 within
the W) cortex of lentiviral A.beta..sub.1-42 injected mice, inset
higher magnification, and X) Nilotinib clearance of intracellular
A.beta..sub.1-42 (inset is higher magnification).
[0074] FIG. 64 shows that Nilotinib eliminates plaques in
lentiviral A.beta..sub.1-42 injected wild type but not parkin-/-
mice. Staining of 20 .mu.m brain sections shows plaque formation
within various brain regions in different A-C) lentiviral
A.beta..sub.1-42+DMSO wild type mice and D-F) Nilotinib group after
3-week treatment. G-I) show lentiviral A.beta..sub.1-42+DMSO in
parkin-/- mice and J-L) show the Nilotinib group after 3-week
treatment. Transmission electron microscopy shows autophagic
defects in different lentiviral A.beta..sub.1-42+DMSO wild type
brains within M) hippocampus showing distrophic neurons, N) cortex
showing accumulation of autophagic vacuoles, 0) hippocampus showing
enlarged lysosomes. Lentiviral A.beta..sub.1-42+Nilotinib wild type
brains within P) hippocampus, Q) cortex showing clearance of
autophagic vacuoles, R) hippocampus. Lentiviral
A.beta..sub.1-42.+-.Nilotinib in parkin-/- brains within S&V)
hippocampus showing distrophic neurons, T&W) cortex showing
accumulation of autophagic vacuoles and U&X), hippocampus
showing accumulation of autophagic vacuoles, are also shown.
[0075] FIGS. 65A-E show that Nilotinib ameliorates cognition in a
parkin-dependent manner. A) represents the results of a Morris
water maze test after 4 days of training (trials) in lentiviral
A.beta..sub.1-42-injected.+-.Nilotinib wild type (N=14) and
parkin-/- (N=7) mice. B) shows graphs representing the total number
of entry into platform area and distance travelled. C) represents
the results of a Morris water maze test after 4 days of training
(trials) in Tg-APP.+-.Nilotinib (N=12) mice, including heat maps
for each group showing overall performance. D) shows graphs
representing total number of entry into platform area and distance
travelled. E) represents the results of an object recognition test
in Tg-APP.+-.Nilotinib (N=12) and lentiviral
A.beta..sub.1-42-injected.+-.Nilotinib in parkin-/- (N=7). The
recognition index was calculated as (time exploring one of the
objects/time exploring both objects) .times.100 for acquisition
session, and (time exploring new object/time exploring both
familiar and novel objects) .times.100 for the recognition session
given 1.5 hrs later.
* Significantly different, ANOVA with Neumann Keuls multiple
comparison, P<0.05, Significant effect of Nilotinib on
recognition in Tg-APP group, pairwise T-test p<0.001.
[0076] FIG. 66 shows that Nilotinib increases parkin level and
crosses the blood brain barrier. Parkin levels by ELISA in wild
type mice and lentiviral A.beta..sub.1-42.+-.Nilotinib using
parkin-/- brain extracts as a specificity control (N=12) are
shown.
[0077] FIG. 67 shows that Nilotinib eliminates thioflavin-S
staining. Thioflavin staining of 20 .mu.m brain sections shows
plaque formation within various brain regions in different A-D)
Tg-APP+DMSO and E-H) Nilotinib group after 3-week treatment.
[0078] FIG. 68 shows that parkin ubiquitinates A.beta..sub.1-42 to
mediate its degradation. Staining of 20 .mu.m thick sections shows
formation of 6E10-positive plaques in A.beta..sub.1-42 expressing
group 6 weeks post-injection in A) A.beta..sub.1-42 wild type
mice+DMSO, B) A.beta..sub.1-42 wild type mice+Nilotinib, C)
A.beta..sub.1-42 parkin-/- mice+DMSO and D) A.beta..sub.1-42
parkin-/- mice+Nilotinib. Higher magnification showing 6E10
positive cells are provided in E) A.beta..sub.1-42 wild type
mice+DMSO, F) A.beta..sub.1-42 wild type mice+Nilotinib, G)
A.beta..sub.1-42 parkin-/- mice+DMSO, H) A.beta..sub.1-42 parkin-/-
mice+Nilotinib. I) shows a graph representing quantification of
plaque size using image J to delineate boundaries around individual
plaques using 15-25 plaques (2 plaques per animal) and J) shows
stereological counting of A.beta..sub.1-42 positive cells (N=12
animals). K) is a graph representing parkin activity (N=6). *
Significantly different, ANOVA with Neumann Keuls multiple
comparison, p<0.05.
[0079] FIG. 69 shows that TDP-43 inhibits proteasome activity and
alters parkin levels. Western blot analysis of soluble cortical
brain lysates from different litters of mixed male and female
TDP-43 transgenic mice and non-transgenic control littermates on
4-12% SDS NuPAGE gel are provided showing A) human TDP-43 levels
probed with 2E2-D3 antibody (1st blot), total parkin (2nd blot),
ubiquitin (3rd blot) and actin (4th blot) levels. B) shows that the
pellet was re-suspended in 4M urea to extract the insoluble protein
fraction and Western blot was performed showing insoluble parkin
(1st blot) and insoluble TDP-43 (2nd blot) compared to actin
loading control (3rd blot). C) shows a densitometry analysis of A
and B blots showing soluble and insoluble parkin protein levels
normalized to actin and the ratio of soluble to insoluble parkin.
D) shows an ELISA measurement of parkin level in soluble (STEN
extracts) and insoluble (4M Urea) brain extracts compared to
parkin-/- brain extracts as a specificity control. E) is aWestern
blot analysis of cortical brain lysates on 4-12% SDS NuPAGE gel
showing soluble protein levels of the E3 ubiquitin ligase SIAH2
(1st blot) and its target protein HIF-1.alpha. (2nd blot) compared
to actin loading control. F) shows densitometry analysis of blots
in D normalized to actin control, N=4, ANOVA with Neumann Keuls,
P<0.05. G) shows Western blot analysis of M17 cell lysates on
4-12% SDS NuPAGE gel showing human TDP-43 levels (1st blot), total
parkin (2nd blot), ubiquitin (3rd blot) SIAH2 (4th blot) and actin
levels (5th blot) in cells expressing TDP-43 and wild type parkin.
H) is a Western blot analysis of M17 cell lysates on 4-12% SDS
NuPAGE gel showing human TDP-43 levels (1st blot), total parkin
(2nd blot), ubiquitin (3rd blot) SIAH2 (4th blot) and actin levels
(5th blot) in cells expressing LacZ and wild type parkin. I) shows
Histograms represent the chymotrypsin proteasome activity in M17
neuroblastoma cells. * Significantly different, ANOVA, Neumann
Keuls, P<0.05, N=6 for cells.
[0080] FIG. 70 shows that Lentiviral expression of TDP-43 in rat
motor cortex results in detection of TDP-43 in preganglionic
cervical spinal cord inter-neurons. Staining of 20 .mu.m thick
sections from rat brain injected with lentiviral TDP-43 in the
right hemisphere and lentiviral LacZ in the left hemisphere showing
A) neurons in rat motor cortex stained with anti-TDP-43 antibody
that detects both human and rat TDP-43 and DAPI-stained nuclei in
lentiviral LacZ-injected and B) TDP-43 injected hemisphere are
shown. C) shows that neurons in rat motor cortex stained with
anti-TDP-43 antibody that detects human TDP-43 and DAPI-stained
nuclei in lentiviral LacZ-injected and D) TDP-43 injected
hemisphere. E) is a schematic representation of injected motor
cortex relative to contralateral spinal cord region and
dorso-cortical spinal tract (DCST). Staining of 20 .mu.m thick
sections showing pre-ganglionic cervical spinal cord inter-neurons
stained with F). hTDP-43 mouse monoclonal antibody (Abnova) that
recognizes human TDP-43 and G). Anti-TDP-43 rat polyclonal antibody
(ProteinTech) that recognizes both human and rat, and DAPI-stained
nuclei contralateral to lentiviral TDP-43-injected cortex and H
(TDP-43) and I (hTDP-43) contralateral to LacZ injected hemisphere
are shown. Staining of 20 .mu.m thick sections showing fibers in
DCST stained with J) mouse monoclonal hTDP-43 and DAPI and K)
rabbit polyclonal anti-TDP-43 antibody DAPI contralateral to
lentiviral TDP-43-injected cortex are also shown. L shows TDP-43
and M shows hTDP-43. TDP-43 staining and DAPI in DCST contralateral
to LacZ injected hemisphere was also performed. N) shows toluidine
blue stained DCST contralateral to lentiviral TDP-43-injected
cortex compared to 0) LacZ injected hemisphere. Lv: lentivirus.
[0081] FIG. 71 shows that lentiviral parkin increases cytosolic
co-localization of ubiquitin and TDP-43. Staining of 20 .mu.m thick
sections from rat brain injected with lentiviral TDP-43 in the
right hemisphere and lentiviral LacZ in the left hemisphere shows
A) neurons in rat motor cortex stained with mouse monoclonal
(Millipore) anti-parkin, B) rabbit polyclonal anti-TDP-43
antibodies, C) parkin, TDP-43 and DAPI in lentiviral LacZ-injected
hemisphere. D) shows neurons in rat motor cortex stained with mouse
monoclonal anti-ubiquitin and E) rabbit polyclonal anti-TDP-43
antibodies. F) shows ubiquitin, TDP-43 and DAPI in lentiviral
TDP-43-injected hemisphere. G) shows neurons in rat motor cortex
stained with mouse monoclonal anti-parkin and H) rabbit polyclonal
anti-TDP-43 antibodies. I) shows parkin, TDP-43 and DAPI in animals
co-injected with lentiviral TDP-43 and parkin. J) shows neurons in
rat motor cortex stained with mouse monoclonal anti-ubiquitin and
K) rabbit polyclonal anti-TDP-43 antibodies. L) shows ubiquitin,
TDP-43 and DAPI stained nuclei in animals co-injected with
lentiviral TDP-43 and parkin. Neurons in rat motor cortex stained
with M) mouse monoclonal anti-parkin antibodies, N) rabbit
polyclonal anti-TDP-43 antibodies, and O) parkin, TDP-43 and DAPI
stained nuclei in animals injected with lentiviral parkin alone are
shown. Lv: lentiviral.
[0082] FIG. 72 shows that parkin mediates K48 and K63-linked
ubiquitination of TDP-43. Western blot of input samples from
cortical brain lysates analyzed on 4-12% SDS NuPAGE gel show A)
parkin expression levels (1st blot), ubiquitin bound protein levels
(2nd blot) and TDP-43 levels (3rd blot), compared to actin loading
control in rat cortex injected with lentiviral LacZ, TDP-43,
parkin, TDP-43+parkin and TDP-43+T240R mutant. A total of 100 mg
cortical brain samples were immuno-precipitated using rabbit
polyclonal anti-TDP-43 and probed (1:1000) with anti-ubiquitin
antibody (4th blot) compared to actin loading control (5th blot)
from input samples. B) shows a Western blot of input samples and
immuno-precipitated parkin (top blot) and TDP-43 (bottom blot) from
transgenic mice used to measure parkin E3 ubiquitin ligase
activity. C) shows histograms representing parkin E3 ubiquitin
ligase activity in the presence and absence of human TDP-43
immuno-precipitated from TDP-43 transgenic mice, compared to E3
ubiquitin ligase activity using recombinant parkin (sPar),
poly-ubiquitin chain as control and a synthetic E1-E2-E3 control
combination. N=8, P<0.05, ANOVA Neumann Keuls. D) is aWB
analysis showing ubiquitinated TDP-43 in the presence of K48 and
K63 and E) is aWB analysis showing ubiquitinated parkin at K48 and
K63. F) shows histograms representing the chymotrypsin proteasome
activity in fresh cortical brain lysates from rats injected with
lentiviral LacZ, parkin, TDP-43 and TDP-43+parkin. * indicates a
significant difference, ANOVA, Neumann Keuls, P<0.05, N=8. G).
Western blot analysis of cortical brain lysates on 4-12% SDS NuPAGE
gel showing HDAC6 (1st blot) and P62 levels (2nd blot) and actin
control (3rd blot) are provided. H) is a densitometry analysis of
blots in E from gene transfer animal models. * Indicates
significantly different, ANOVA, Neumann Keuls, P<0.05, N=8.
[0083] FIG. 73 shows that TDP-43 forms a multi-protein complex with
parkin and HDAC6. Western blot of input samples from cortical brain
lysates in transgenic A315T mice and control littermates analyzed
on 4-12% SDS NuPAGE gel showing A) shows human TDP-43 expression
levels (1st blot) and immuno-precipitation of TDP-43 showing TDP-43
(2nd blot), parkin (3rd blot) and HDAC6 (4th blot) forming a
protein complex. B) represents the reverse immune-precipitation
experiment, where Western blot of input samples from cortical brain
lysates in transgenic A315T mice and control littermates analyzed
on 4-12% SDS NuPAGE show parkin expression levels (1st blot) and
immuno-precipitation of parkin showing TDP-43 (2nd blot), parkin
(3rd blot) and HDAC6 (4th blot). GFP fluorescence and nuclear
DAPI-staining in living human M17 neuroblastoma cells C) shows
cells transfected with GFP-TDP-43 alone showing GFP fluorescence
within the nucleus. D &E) show cells transfected with
GFP-TDP-43 and parkin showing GFP fluorescence in cytosol and
cellular processes. Inset in D shows higher magnification. F) shows
cells transfected with GFP-TDP-43 and parkin treated with 5 .mu.M
HDAC6 inhibitor, tubacin for 24 hours showing GFP fluorescence
within DAPI-stained nuclei. G). shows cells transfected with
GFP-TDP-43 for 24 hours and treated with tubacin for an additional
24 hours. H) shows cells transfected with GFP-TDP-43 and T240R,
showing lack of GFP fluorescence with parkin mutant. I) shows
qRT-PCR showing Park2 mRNA in M17 cells transfected with LacZ
TDP-43, parkin and TDP-43+parkin. J) shows quantification of
qRT-PCR showing relative Park2 mRNA levels normalized to GADPH and
expressed as % control. N=4, P<0.05, ANOVA, Neumann Keuls. K)
shows qRT-PCR showing Park2 mRNA in rat cortex injected with LacZ
(un-injected control), TDP-43, parkin and TDP-43+parkin. L) shows
quantification of qRT-PCR showing relative Park2 mRNA levels
normalized to GADPH and expressed as % control. N=4, P<0.05,
ANOVA, Neumann Keuls. M) shows qRT-PCR showing Park2 mRNA in
TDP43-Tg and control cortex. N) shows quantification of qRT-PCR
showing relative Park2 mRNA levels normalized to GADPH and
expressed as % control. N=3, P<0.05, ANOVA, Neumann Keuls.
[0084] FIG. 74 is a schematic showing potential effects of parkin
on TDP-43 localization.
[0085] FIG. 75 shows the distribution of GFP-tagged TDP-43 in M17
cells transfected with 3 mg cDNA for 24 hrs and then treated with
Nilotinib (10 mM) or Bosutinib (5 mM) and HDAC6 inhibitor Tubacin
(5 mM) for additional 24 hrs. Inserts (B&D) represent higher
magnification images showing translocation of GFP-tagged TDP-43
from nucleus (A) into the cytosol (B&D, and inserts), while
tubacin impairs translocation (C&E).
DETAILED DESCRIPTION
[0086] Provided herein are methods of treating or preventing a
neurodegenerative disease, a myodegenerative disease or a prion
disease. Neurodegenerative diseases include amyotrophic lateral
sclerosis, Alzheimer's disease, frontotemporal dementia,
frontotemporal dementia with TDP-43, frontotemporal dementia linked
to chromosome-17, Pick's disease, Parkinson's disease, Huntington's
chorea, mild cognitive impairment, Lewy Body disease, multiple
system atrophy, progressive supranuclear palsy, and cortico-basal
degeneration in a subject. The methods include the use of tyrosine
kinase inhibitors. The methods also include the use of tyrosine
kinase inhibitors wherein the tyrosine kinase inhibitor is not
Gleevec and wherein the tyrosine kinase inhibitor crosses the blood
brain barrier. The methods also include the use of tyrosine kinase
inhibitors, wherein the tyrosine kinase inhibitors are not c-Abl
tyrosine kinase inhibitors or are not specific c-Abl
inhibitors.
[0087] Provided herein is a method of treating or preventing a
neurodegenerative disease in a subject, comprising selecting a
subject with a neurodegenerative disease of the central nervous
system, a myodegenerative disease or a prion disease or at risk for
a neurodegenerative disease of the central nervous system, a
myodegenerative disease or a prion disease and administering to the
subject an effective amount of a tyrosine kinase inhibitor, as
described throughout. Optionally, the tyrosine kinase inhibitor is
not Gleevec and the tyrosine kinase inhibitor crosses the blood
brain barrier. For example, the tyrosine kinase inhibitor is
selected from the group consisting of nilotinib, bosutinib, and a
combination thereof.
[0088] In the methods provided herein, neurodegenerative diseases
of the central nervous system include, but are not limited to,
Amyotrophic Lateral Sclerosis, Alzheimer's Disease, Parkinson's
Disease, frontotemporal dementia, Huntington's Disease, Mild
Cognitive Impairment, an .alpha.-Synucleinopathy, a Tauopathy or a
pathology associated with intracellular accumulation of TDP-43.
[0089] In the methods provided herein, myodegenerative diseases
include, but are not limited to, inclusion body myositis (IBM),
spinal-bulbar muscular atrophy (SBMA), and motor neuron disease
(MND).
[0090] In the methods provided herein, prion diseases or
transmissible spongiform encephalopathies (TSEs) include, but are
not limited to, Creutzfeldt-Jakob Disease (CJD), Variant
Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker
Syndrome, Fatal Familial Insomnia and Kuru in humans. Animal prion
diseases include, but are not limited to, Scrapie, Bovine
Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD),
Transmissible mink encephalopathy, Feline spongiform encephalopathy
and Ungulate spongiform encephalopathy.
[0091] Examples of tyrosine kinase inhibitors include, but are not
limited to, nilotinib, bosutinib, or a combination thereof.
Nilotinib (or AMN-107), which is sold as TASIGNA.RTM. (Novartis,
Basel Switzerland), and Bosutinib (or SKI-606) (Pfizer, New York,
N.Y.) are Bcr-Abl tyrosine kinase inhibitors developed as
alternatives to the Bcr-Abl tyrosine kinase inhibitor and CML
treatment, Imatinib. Nilotinib is an Abelson kinase inhibitor
(c-Abl kinase), whereas Bosutinib is a dual Src and c-Abl kinase
inhibitor. These agents are cancer therapeutics that block cellular
proliferation of cancer cells and are currently used primarily in
the treatment of chronic myelogenous leukemia (CML).
[0092] In neurodegenerative disorders, normal autophagic flux is
altered, resulting in the accumulation of autophagic vacuoles or
autophagosomes. This is shown in the Examples where the
accumulation of vacuoles is seen in human patients with decreased
parkin solubility activity. Normal autophagy is a dynamic
multi-step process that prevents protein accumulation via
sequestration into autophagic vacuoles (autophagosomes). Subsequent
fusion of the autophagosomes with lysosomes results in protein
degradation. Interruption of this process results in accumulation
of protein aggregates and neurodegeneration. Parkin is an E3 ligase
involved in proteasomal and autophagic degradation via protein
ubiquitination and autophagosome maturation.
[0093] Tyrosine kinase inhibition activates parkin-mediated
clearance of aggregated proteins and/or activates ubiquitination.
Activation of parkin by tyrosine kinase inhibitors up-regulates
protein levels of beclin, thus facilitating autophagic clearance.
For example, nilotinib, bosutinib, or a combination thereof
activates parkin-mediated clearance of aggregated proteins and/or
activates ubiquitination. Significantly, both nilotinib and
bosutinib cross the blood brain barrier and promote parkin activity
in the central nervous system. Parkin activity promotes autophagic
clearance of amyloid beta and alpha-synuclein and causes protective
mechanisms for parkin ubiquitination, for example, sequestration of
TDP-43 associated with amyotrophic lateral sclerosis (ALS) and
frontotemporal dementia. Furthermore, the tyrosine kinase
inhibitors rescue brain cells from apoptotic death in
neurodegenerative disease. In the case of ALS, the inhibitors
increase ubiquitination of TDP-43 and translocate it from the
nucleus, where it interacts deleteriously with mRNA and thousands
of genes, to the cytosol where it is sequestered.
[0094] The method optionally includes selecting a subject with a
neurodegenerative disease or at risk for developing a
neurodegenerative disease. One of skill in the art knows how to
diagnose a subject with or at risk of developing a
neurodegenerative disease. For example, one or more of the follow
tests can be used genetic test (e.g., identification of a mutation
in TDP-43 gene) or familial analysis (e.g., family history),
central nervous system imaging (e.g., magnetic resonance imaging
and positron emission tomography), clinical or behavioral tests
(e.g., assessments of muscle weakness, tremor, or memory),
laboratory tests.
[0095] The method optionally further includes administering a
second therapeutic agent to the subject. The second therapeutic
agent is selected from the group consisting of levadopa, a dopamine
agonist, an anticholinergic agent, a monoamine oxidase inhibitor, a
COMT inhibitor, amantadine, rivastigmine, an NMDA antagonist, a
cholinesterase inhibitor, riluzole, an anti-psychotic agent, an
antidepressant, and tetrabenazine.
[0096] By way of example, provided herein is a method of treating
amyotrophic lateral sclerosis or frontotemporal dementia in a
subject. The method includes selecting a subject with amyotrophic
lateral sclerosis or frontotemporal dementia, wherein the subject
has a TDP-43 pathology, and administering to the subject an
effective amount of the tyrosine kinase inhibitor. The TDP-43
pathology can be, for example, a TDP-43 mutation. For example, the
tyrosine kinase inhibitor is a tyrosine kinase inhibitor that is
not Gleevec and crosses the blood brain barrier. In another
example, the tyrosine kinase inhibitor is selected from the group
consisting of nilotinib, bosutinib, and a combination thereof.
TDP-43 pathology occurs in ALS and frontotemporal dementia and an
elevated level of TDP-43 in the cytoplasm has been noted in some
cases of ALS and frontotemporal dementia. Mutations in the gene
that encodes the TDP-43 protein (known as TARDBP) have been
discovered in some individuals with ALS and frontotemporal
dementia. Thus, mutated TDP-43 or mutations in TARDBP can serve as
biomarkers for a subject at risk for ALS or frontotemporal
dementia.
[0097] Also provided herein is a method of promoting parkin
activity in a subject. The method includes selecting a subject with
a disorder associated with decreased parkin activity and
administering to the subject an effective amount of the tyrosine
kinase inhibitor. For example, the tyrosine kinase inhibitor is a
tyrosine kinase inhibitor that is not Gleevec and crosses the blood
brain barrier. In another example, the tyrosine kinase inhibitor is
selected from the group consisting of nilotinib, bosutinib, and a
combination thereof.
[0098] Methods for measuring parkin activity are known in the art.
See, for example, Schlossmacher and Shimura ("Parkinson's disease:
assays for the ubiquitin ligase activity of neural Parkin," Methods
Mol. Biol. 301: 351-69 (2005)); Morrison et al. ("A simple cell
based assay to measure Parkin activity," J. Neurochem. 116(3):
342-9 (2011)) and Burns et al. (Hum. Mol. Genet. 18 3206-3216
(2009)).
[0099] Further provided is a method of treating or preventing a
neurodegenerative disease in a subject, comprising selecting a
subject with a neurodegenerative disease or at risk for a
neurodegenerative disease, determining that the subject has a
decreased level of parkin activity relative to a control, and
administering to the subject an effective amount of a small
molecule that increases parkin activity, wherein the small molecule
is not Gleevec. For example, the small molecule can be a tyrosinse
kinase inhibitor, such as, for example, a tyrosine kinase inhibitor
that crosses the blood brain barrier. The tyrosine kinase inhibitor
can also be selected from the group consisting of nilotinib,
bosutinib, and a combination thereof.
[0100] The term effective amount, as used throughout, is defined as
any amount necessary to produce a desired physiologic response. The
effective amount is generally less than the amount used in
chemotherapeutic methods to treat cancer or leukemia, but is an
amount sufficient to activate parkin. Thus, the dosage of the
tyrosine kinase inhibitor in the present methods is optionally
lower than a chemotherapeutic dosage of the inhibitor. For example,
the dosage is optionally less than about 10 mg/kg and can be 8, 7,
6, 5, 4, 3, 2, or 1 mg/kg. One of skill in the art would adjust the
dosage as described below based on specific characteristics of the
inhibitor and the subject receiving it.
[0101] Furthermore, the duration of treatment can be longer in the
present methods than the duration of chemotherapeutic treatment,
for example cancer treatment. For example, administration to a
subject with or at risk of developing a neurodegenerative disease
could be at least daily (e.g., once, twice, three times per day)
for weeks, months, or years so long as the effect is sustained and
side effects are manageable.
[0102] There are several ways to activate parkin. Parkin
immuno-precipitation and incubation with a series of activating and
ligating enzymes (E and E2) and ATP result in parkin
auto-ubiquitination, and confer activity to ubiquitinate targets
like Abeta and TDP-43. So, in order to increase parkin activity,
parkin expression must be increased. This can be achieved by viral
introduction of parkin which leads to over-expression of the
protein and increased activity. As shown in the Examples, this
method repeatedly increases protein degradation via the proteasome
and/or autophagy. Parkin can also be activated by administration of
a tyrosine kinase, such as, for example, nilotinib or bosutinib,
which leads to increased levels of parkin and increased
activity.
[0103] Effective amounts and schedules for administering the
tyrosine kinase inhibitor can be determined empirically and making
such determinations is within the skill in the art. The dosage
ranges for administration are those large enough to produce the
desired effect in which one or more symptoms of the disease or
disorder are affected (e.g., reduced or delayed). The dosage should
not be so large as to cause substantial adverse side effects, such
as unwanted cross-reactions, cell death, and the like. Generally,
the dosage will vary with the type of inhibitor, the species, age,
body weight, general health, sex and diet of the subject, the mode
and time of administration, rate of excretion, drug combination,
and severity of the particular condition and can be determined by
one of skill in the art. The dosage can be adjusted by the
individual physician in the event of any contraindications. Dosages
can vary, and can be administered in one or more dose
administrations daily.
[0104] The tyrosine kinase inhibitor is administered systemically
and preferably orally.
[0105] Also provided herein is a method of inhibiting or preventing
toxic protein aggregation in a neuron and/or rescuing a neuron from
degeneration. The method includes contacting the neuron with an
effective amount of a tyrosine kinase inhibitor. For example, the
tyrosine kinase inhibitor is a tyrosine kinase inhibitor that is
not Gleevec and crosses the blood brain barrier. In another
example, the tyrosine kinase inhibitor is selected from the group
consisting of nilotinib, bosutinib, and a combination thereof. The
toxic protein aggregate optionally comprises one or more of an
amyloidogenic protein, alpha-synuclein, tau, insoluble Parkin,
TDP-43, a prion protein or toxic fragments thereof. By
amyloidogenic protein is meant a peptide, polypeptide, or protein
that has the ability to aggregate. An example of an amyloidogenic
protein is .beta.-amyloid.
[0106] The contacting is performed in vivo or in vitro. The in vivo
method is useful in treating a subject with or at risk of
developing toxic protein aggregates and comprises administering the
tyrosine kinase inhibitor as described above. The in vitro method
is useful for example in treating neural cells prior to
transplantation. The tyrosine kinase inhibitor is generally added
to a culture medium. Optionally, the target neurons are contacted
with a second therapeutic agent as described above.
[0107] Also provided herein is a method of inhibiting or preventing
toxic protein aggregation in a muscle cell and/or rescuing a muscle
cell from degeneration. Further provided is a method of inhibiting
or preventing toxic protein aggregation in a glial cell and/or
rescuing a glial cell from degeneration. The method includes
contacting the glial cell with an effective amount of a tyrosine
kinase inhibitor. For example, the tyrosine kinase inhibitor is a
tyrosine kinase inhibitor that is not Gleevec and crosses the blood
brain barrier.
[0108] The disclosure also provides a pharmaceutical pack or kit
comprising packaging and/or one or more containers filled with one
or more of the ingredients of the pharmaceutical compositions.
Instructions for use of the composition can also be included.
[0109] Provided herein is a pharmaceutical composition comprising
an effective amount of the tyrosine kinase inhibitor in a
pharmaceutically acceptable carrier. The term carrier means a
compound, composition, substance, or structure that, when in
combination with a compound or composition, aids or facilitates
preparation, storage, administration, delivery, effectiveness,
selectivity, or any other feature of the compound or composition
for its intended use or purpose. For example, a carrier can be
selected to minimize any degradation of the active ingredient and
to minimize any adverse side effects in the subject. Such
pharmaceutically acceptable carriers include sterile biocompatible
pharmaceutical carriers, including, but not limited to, saline,
buffered saline, artificial cerebral spinal fluid, dextrose, and
water.
[0110] Depending on the intended mode of administration, the
pharmaceutical composition can be in the form of solid, semi-solid,
or liquid dosage forms, such as, for example, tablets,
suppositories, pills, capsules, powders, liquids, aerosols, or
suspensions, preferably in unit dosage form suitable for single
administration of a precise dosage. The compositions will include a
therapeutically effective amount of the compound described herein
or derivatives thereof in combination with a pharmaceutically
acceptable carrier and, in addition, can include other medicinal
agents, pharmaceutical agents, carriers, or diluents. By
pharmaceutically acceptable is meant a material that is not
biologically or otherwise undesirable, which can be administered to
an individual along with the selected compound without causing
unacceptable biological effects or interacting in a deleterious
manner with the other components of the pharmaceutical composition
in which it is contained.
[0111] As used herein, the term carrier encompasses any excipient,
diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or
other material well known in the art for use in pharmaceutical
formulations. The choice of a carrier for use in a composition will
depend upon the intended route of administration for the
composition. The preparation of pharmaceutically acceptable
carriers and formulations containing these materials is described
in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed.
University of the Sciences in Philadelphia, Lippincott, Williams
& Wilkins, Philadelphia Pa., 2005. Examples of physiologically
acceptable carriers include buffers such as phosphate buffers,
citrate buffer, and buffers with other organic acids; antioxidants
including ascorbic acid; low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.RTM. (ICI, Inc.; Bridgewater, N.J.),
polyethylene glycol (PEG), and PLURONICS.TM. (BASF; Florham Park,
N.J.).
[0112] Compositions containing the compound described herein or
pharmaceutically acceptable salts or prodrugs thereof suitable for
parenteral injection can comprise physiologically acceptable
sterile aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions, and sterile powders for reconstitution into sterile
injectable solutions or dispersions. Examples of suitable aqueous
and nonaqueous carriers, diluents, solvents or vehicles include
water, ethanol, polyols (propyleneglycol, polyethyleneglycol,
glycerol, and the like), suitable mixtures thereof, vegetable oils
(such as olive oil) and injectable organic esters such as ethyl
oleate. Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersions and by the use of
surfactants.
[0113] These compositions can also contain adjuvants such as
preserving, wetting, emulsifying, and dispensing agents. Prevention
of the action of microorganisms can be promoted by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents,
for example, sugars, sodium chloride, and the like can also be
included. Prolonged absorption of the injectable pharmaceutical
form can be brought about by the use of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0114] Solid dosage forms for oral administration of the compounds
described herein or pharmaceutically acceptable salts or prodrugs
thereof include capsules, tablets, pills, powders, and granules. In
such solid dosage forms, the compounds described herein or
derivatives thereof is admixed with at least one inert customary
excipient (or carrier) such as sodium citrate or dicalcium
phosphate or (a) fillers or extenders, as for example, starches,
lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders,
as for example, carboxymethylcellulose, alignates, gelatin,
polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for
example, glycerol, (d) disintegrating agents, as for example,
agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain complex silicates, and sodium carbonate, (e) solution
retarders, as for example, paraffin, (f) absorption accelerators,
as for example, quaternary ammonium compounds, (g) wetting agents,
as for example, cetyl alcohol, and glycerol monostearate, (h)
adsorbents, as for example, kaolin and bentonite, and (i)
lubricants, as for example, talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, or
mixtures thereof. In the case of capsules, tablets, and pills, the
dosage forms can also comprise buffering agents.
[0115] Solid compositions of a similar type can also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethyleneglycols, and the like.
[0116] Solid dosage forms such as tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells, such
as enteric coatings and others known in the art. They can contain
opacifying agents and can also be of such composition that they
release the active compound or compounds in a certain part of the
intestinal tract in a delayed manner. Examples of embedding
compositions that can be used are polymeric substances and waxes.
The active compounds can also be in micro-encapsulated form, if
appropriate, with one or more of the above-mentioned
excipients.
[0117] Liquid dosage forms for oral administration of the compounds
described herein or pharmaceutically acceptable salts or prodrugs
thereof include pharmaceutically acceptable emulsions, solutions,
suspensions, syrups, and elixirs. In addition to the active
compounds, the liquid dosage forms can contain inert diluents
commonly used in the art, such as water or other solvents,
solubilizing agents, and emulsifiers, as for example, ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,
dimethylformamide, oils, in particular, cottonseed oil, groundnut
oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol,
tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid
esters of sorbitan, or mixtures of these substances, and the
like.
[0118] Besides such inert diluents, the composition can also
include additional agents, such as wetting, emulsifying,
suspending, sweetening, flavoring, or perfuming agents.
[0119] Suspensions, in addition to the active compounds, can
contain additional agents, as for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, or mixtures of these substances, and the
like.
[0120] Compositions of the compounds described herein or
pharmaceutically acceptable salts or prodrugs thereof for rectal
administrations are optionally suppositories, which can be prepared
by mixing the compounds with suitable non-irritating excipients or
carriers such as cocoa butter, polyethyleneglycol or a suppository
wax, which are solid at ordinary temperatures but liquid at body
temperature and therefore, melt in the rectum or vaginal cavity and
release the active component.
[0121] Throughout, treat, treating, and treatment refer to a method
of reducing or delaying one or more effects or symptoms of a
neurodegenerative disease or disorder. The subject can be diagnosed
with disease or disorder. Treatment can also refer to a method of
reducing the underlying pathology rather than just the symptoms.
The effect of the administration to the subject can have the effect
of but is not limited to reducing one or more symptoms of the
neurodegenerative disease or disorder, a reduction in the severity
of the neurological disease or injury, the complete ablation of the
neurological disease or injury, or a delay in the onset or
worsening of one or more symptoms. For example, a disclosed method
is considered to be a treatment if there is about a 10% reduction
in one or more symptoms of the disease in a subject when compared
to the subject prior to treatment or when compared to a control
subject or control value. Thus, the reduction can be about a 10,
20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in
between.
[0122] As utilized herein, by prevent, preventing, or prevention is
meant a method of precluding, delaying, averting, obviating,
forestalling, stopping, or hindering the onset, incidence,
severity, or recurrence of the neurodegenerative disease or
disorder. For example, the disclosed method is considered to be a
prevention if there is a reduction or delay in onset, incidence,
severity, or recurrence of neurodegeneration or one or more
symptoms of neurodegeneration (e.g., tremor, weakness, memory loss,
rigidity, spasticity, atrophy) in a subject susceptible to
neurodegeneration as compared to control subjects susceptible to
neurodegeneration that did not receive an agent that activates
parkin. The disclosed method is also considered to be a prevention
if there is a reduction or delay in onset, incidence, severity, or
recurrence of neurodegeneration or one or more symptoms of
neurodegeneration in a subject susceptible to neurodegeneration
after receiving an agent that promotes parkin activity as compared
to the subject's progression prior to receiving treatment. Thus,
the reduction or delay in onset, incidence, severity, or recurrence
of neurodegeneration can be about a 10, 20, 30, 40, 50, 60, 70, 80,
90, 100%, or any amount of reduction in between.
[0123] As used throughout, by subject is meant an individual.
Preferably, the subject is a mammal such as a primate, and, more
preferably, a human. Non-human primates are subjects as well. The
term subject includes domesticated animals, such as cats, dogs,
etc., livestock (for example, cattle, horses, pigs, sheep, goats,
etc.) and laboratory animals (for example, ferret, chinchilla,
mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary
uses and medical formulations are contemplated herein.
[0124] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed methods and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutations of these compounds may not be explicitly
disclosed, each is specifically contemplated and described herein.
For example, if a method is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
in the method are discussed, each and every combination and
permutation of the method, and the modifications that are possible
are specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. This concept applies to
all aspects of this disclosure including, but not limited to, steps
in methods using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed, it is understood
that each of these additional steps can be performed with any
specific method steps or combination of method steps of the
disclosed methods, and that each such combination or subset of
combinations is specifically contemplated and should be considered
disclosed.
[0125] Publications cited herein and the material for which they
are cited are hereby specifically incorporated by reference in
their entireties. A number of embodiments have been described.
Nevertheless, it will be understood that various modifications may
be made. Accordingly, other embodiments are within the scope of the
following claims.
EXAMPLES
Example 1
[0126] Methods for the animal experiments described herein are
detailed in Examples 2-5. Cell culture experiments are referenced
below and explained in Burns et al. (Human Molecular Genetics.
2009) and Rebeck et al. (J. Biol. Chem. 2010, 285:7440-7446).
Additional details are provided in the brief description of the
figures. Using these methods, cellular mechanisms (FIG. 1)
associated with parkin activity in neurodegenerative conditions and
upon intervention with tyrosine kinase inhibitors were studied.
These studies revealed that tyrosine kinase inhibition activates
parkin and increases its interaction with beclin-1, resulting in
maturation of phagophores into phagosomes and clearance (FIG. 2).
It was also shown that parkin interacts with beclin-1 in wild type,
but not parkin -/-mice (FIG. 3). As shown in FIGS. 4-5, 3X APP mice
treated with either Nilotinib or Bosutinib resulted in reduced
A.beta..sub.1-42 in the brain lysates of these mice as compared to
treatment with DMSO. Also, as shown in FIGS. 6-8, treatment of A53T
mice (A53T-Tg) with Bosutinib at different dosages and dosage
schedules resulted in a decrease in human .alpha.-synuclein in the
brain lystates of these mice, as compared to treatment with DMSO.
Further, as shown in FIGS. 9-10, treatment of A53T mice (A53T-Tg)
with Nilotinib at different dosages and dosage schedules resulted
in a decrease in human .alpha.-synuclein in the brain lystates of
these mice, as compared to treatment with DMSO. Decreases in human
soluble A.beta..sub.1-42 and human soluble A.beta..sub.1-40 in the
brain lysates of triple mutant APP-AD mice were also observed after
treatment with Bosutinib (FIGS. 11A and B). Treatment with
Bosutinib also resulted in increased parkin levels and decreased
levels of phosphorylated Tau (FIGS. 11C and D).
[0127] In other experiments, M17 cells transfected with Tau cDNA
were treated with Nilobinib and Tubacin (an HDAC6 inhibitor).
Treatment with Nilotinib resulted in a decrease in human Tau, a
decrease in human A.beta..sub.1-42 and a decrease in
.alpha.-synuclein as compared to transfected cells.
[0128] Treatment of lentiviral A.beta..sub.1-42-injected mice with
bosutinib also resulted in decreased levels of A.beta..sub.1-42 in
brain lysates (FIG. 12).
[0129] In another experiment, mice were injected stereotaxically
(bilaterally) with lentiviral .alpha.-synuclein into the substantia
nigra for 3 weeks. Then, half of the animals were injected with 10
mg/Kg nilotinib and the other half with DMSO. The effects of
.alpha.-synuclein expression and tyrosine kinase inhibition on
brain (FIG. 13A) and blood (FIG. 13B) levels of .alpha.-synuclein
were compared. As shown in FIG. 13, .alpha.-synuclein expression in
the brain increases its blood level and tyrosine kinase inhibition
reverses these effects in a parkin-dependent manner.
[0130] In another experiment, mice were injected stereotaxically
(bilaterally) with lentiviral .alpha.-synuclein into the substantia
nigra for 3 weeks. Then, half of the animals were injected with 5
mg/Kg Bosutinib and the other half with DMSO. The effects of
.alpha.-synuclein expression and tyrosine kinase inhibition on
brain (FIG. 14A) and blood (FIG. 14B) levels of .alpha.-synuclein
were compared. As shown in FIG. 14, .alpha.-synuclein expression in
the brain increases its blood level and tyrosine kinase inhibition
reverses these effects in a parkin-dependent manner.
[0131] In another study, mice were injected stereotaxically
(bilaterally) with lentiviral .alpha.-synuclein into the substantia
nigra for 3 weeks. Then half the animals were injected with 10
mg/kg Nilotinib or 5 mg/Kg Bosutinib and the other half with DMSO.
As shown in FIG. 185, the effects of .alpha.-synuclein expression
and tyrosine kinase inhibition on dopamine and homovanillic acid
(HVA) levels (ELISA) were compared. The effects of treatment on
motor performance were evaluated using rotarod (FIG. 15B). This
study shows that .alpha.-synuclein induced loss of dopamine and
homovanillic acid (HVA) levels. Tyrosine kinase inhibition reversed
these effects and improved motor performance.
[0132] In another study, transgenic A53T mice that express human
.alpha.-synuclein throughout the brain (excluding substantia nigra)
were injected with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO
once daily for 3 weeks. The effects of .alpha.-synuclein expression
and tyrosine kinase inhibition on dopamine and homovanillic acid
(HVA) levels (ELISA) were compared. The effects of treatment on
motor performance were tested using rotarod. .alpha.-synuclein did
not induce loss of Dopamine and HVA (due to absence of
.alpha.-synuclein expression in dopamine producing neurons in these
mice. Tyrosine kinase inhibition increased dopamine and HVA. Motor
performance also increased.
[0133] Studies were also performed to show that A.beta..sub.1-42
and A.beta..sub.1-40 accumulate in AV-10 in Tg-APP animals, but
drug treatment enhances autophagic clearance via deposition of
A.beta..sub.1-42 or A.beta..sub.1-40, respectively, in AV-20 and
lysosomes (See FIGS. 16 and 17, respectively). Additional studies
shows that p-Tau and parkin also accumulate in AV-10 in Tg-APP
animals, but drug treatment enhances autophagic clearance via
deposition of p-Tau or parkin in AV-20 and lysosomes (See FIGS. 18
and 19, respectively).
[0134] In another study, it was shown that A.beta..sub.1-42 and
p-Tau at serine 396 accumulate in the brains of mice injected with
lentiviral A.beta..sub.1-42, but drug treatment enhances autophagic
clearance via deposition of A.beta..sub.1-42 or p-Tau in AV-20 and
lysosomes (See FIGS. 20 and 21, respectively). Also shown is that
p-Tau and .alpha.-synuclein accumulate in the brains of mice
injected with lentiviral .alpha.-synuclein, but drug treatment
enhances autophagic clearance via deposition of p-Tau or
.alpha.-synuclein in AV-20 and lysosomes (See FIGS. 22 and 23,
respectively). Further shown is that .alpha.-synuclein and p-Tau
accumulate in AV-10 of A53T brains, but drug treatment enhances
autophagic clearance via deposition of p-Tau or .alpha.-synuclein
in AV-20 and lysosomes (See FIGS. 24 and 25, respectively). Parkin
also accumulates in the brains of A53T mice, but as shown in FIG.
26, drug treatment enhances autophagic clearance via deposition of
parkin in AV-20 and lysosomes. As shown in FIG. 27, tyrosine kinase
inhibition increases parkin activity and facilitates autophagic
clearance of p-Tau. This process requires Tau stabilization of
intact microtubules. Tyrosine kinase activation, p-Tau accumulation
and impaired autophagy are recognized in neurodegeneration.
Decreased parkin solubility and accumulation with intracellular
A.beta. and p-Tau in autophagic vacuoles in AD brains occurs, while
exogenous parkin facilitates autophagic clearance in animal
models.
[0135] In another study, wild type or parkin-/- mice were injected
with lentiviral Tau.+-.A.beta..sub.1-42 for 3 weeks and treated IP
with 10 mg/kg Nilotinib or DMSO once a day for 3 (additional)
consecutive weeks. Brain tissues were fractionated to isolate AVs
and human specific ELISA was performed to determine
A.beta..sub.1-42 contents. A.beta..sub.1-42 accumulates in AV-10 in
lentivirus injected brains but drug treatment enhances autophagic
clearance via deposition of A.beta..sub.1-42 in AV-20 and lysosome.
It was also observed that autophagic clearance is parkin-dependent.
Further, this study shows that Tau expression leads to
A.beta..sub.1-42 accumulation in AV10 and AV20, but not in
lysosomes, indicating decreased fusion between autophagosomes and
lysosomes.
[0136] In another study, wild type or Tau-/- mice were injected
with lentiviral A.beta..sub.1-42 for 3 weeks and treated IP with 10
mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3
(additional) consecutive weeks. Brain tissues were fractionated to
isolate AVs and human specific ELISA was performed to determine
protein contents. Results showed that A.beta..sub.1-42 accumulates
in AV-10 in lentivirus injected brains but drug treatment enhances
autophagic clearance via deposition of A.beta..sub.1-42 in AV-20
and lysosomes. Autophagic clearance is less efficient in Tau null
animals with A.beta..sub.1-42 accumulation in AV-10 and AV-20.
[0137] In another study, wild type or parkin-/- mice injected with
lentiviral human Tau.+-.A.beta..sub.1-42 for 3 weeks and treated IP
with 10 mg/kg Nilotinib or 30 .mu.L DMSO once a day for 3
(additional) consecutive weeks. Brain tissues were fractionated to
isolate AVs and mouse specific ELISA was performed to determine
protein contents. Results showed that P-Tau at serine 396
accumulates in AV-10 in lentivirus injected brains but drug
treatment enhances autophagic clearance via deposition of p-Tau in
AV-20 and lysosomes, where it is degraded.
[0138] In another study, wild type or parkin-/- mice were injected
with lentiviral Tau.+-.A.beta..sub.1-42 for 3 weeks and treated IP
with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for
3 (additional) consecutive weeks. Brain tissues were fractionated
to isolate AVs and human specific ELISA was performed to determine
protein contents. Results showed that P-Tau at serine 396
accumulates in AV-10 in lentivirus injected brains, but drug
treatment enhances autophagic clearance via deposition of p-Tau in
AV-20 and lysosomes, where it is degraded.
[0139] FIG. 28 shows A) phosphorylated c-Abl at tyrosine 412 (T412)
and B) endogenous parkin expression merged in C) hippocampus of 6
month old C57BL/6 mice treated IP with DMSO daily for 3 weeks. FIG.
28 also shows D) decreased phosphorylated c-Abl at tyrosine 412
(T412) and E) increased endogenous parkin expression merged in F)
hippocampus of 6 month old C57BL/6 mice treated IP with 5 mg/kg
Bosutinib daily for 3 weeks.
[0140] FIG. 29 shows A) parkin and B) A.beta. expression merged in
C) cortex of 6 months old Tg-APP mice treated with DMSO or 5 mg/kg
Bosutinib (D-F) once a day for 3 weeks. Using a different
combination of antibodies (see figure G-I show expression of parkin
(G) and A.beta. (H) in the hippocampus of Tg-APP mice treated DMSO.
J-H show the increase in parkin level in animals treated for 3
weeks once a day with Bosutinib (J) along with decreased plaque
levels (K and L) in the hippocampus.
[0141] FIG. 30 shows plaque A.beta. stained with 6E10 antibody and
counterstained with DAB in the brain of Tg-APP animals treated IP
with DMSO once a day for 3 weeks.
[0142] FIG. 31 shows plaque A.beta. stained with 6E10 antibody and
counterstained with DAB in the brain of Tg-APP animals treated IP
with 5 mg/kg Bosutinib once a day for 3 weeks. A decrease in plaque
formation in the animals treated with Bosutinib as compared to the
animals treated with DMSO was observed.
[0143] FIG. 32 shows that Bosutinib decreases .alpha.-synuclein
levels in transgenic mice expressing A53T throughout the brain.
FIGS. 32A-D show human .alpha.-synuclein expression in lentiviral
LacZ injected (for 3 weeks) substantia nigra with A) DMSO and B) 5
mg/kg Bosutinib once a day for 3 weeks. C and D show human
.alpha.-synuclein expression in lentiviral .alpha.-synuclein
injected (for 3 weeks) substantia nigra with C) DMSO and D) or
Bosutinib once a day for 3 weeks. FIGS. 32E-H show tyrosine
hydroxylase (TH) expression in lentiviral LacZ injected (for 3
weeks) substantia nigra with E) DMSO and F) 5 mg/kg Bosutinib once
a day for 3 weeks. G and H show TH expression in lentiviral
.alpha.-synuclein injected (for 3 weeks) substantia nigra with G)
DMSO and H) or Bosutinib once a day for 3 weeks. synuclein
decreases TH neurons and Bosutinib rescues these cells. FIGS. 32I-L
show human .alpha.-synuclein expression in A53T mice in I) Cortex,
J) Striatum, K) Brainstem and L) Hippocampus treated with DMSO for
3 weeks. FIGS. 32M-P show human .alpha.-synuclein expression in
A53T mice in M) cortex, N) striatum, 0) brainstem and P)
hippocampus treated with 5 mg/kg Bosutinib for 3 weeks.
[0144] Performance tests were also done. As shown in FIGS. 33A and
B, IP treatment with 5 mg/kg Bosutinib once daily for 3 weeks
improved cognitive behavior in mice injected bilaterally with
lentiviral A.beta..sub.1-42 for 3 weeks prior to drug treatment.
Bosutinib treated mice found the platform (A) but DMSO treated mice
spent more time in NW area, where they were initially placed or the
NE or SW area, without effectively finding platform area. Bosutninb
improved cognitive performance in a parkin-dependent manner as the
parkin-/- mice did not seem to learn much. FIG. 41B shows that
Bosutinib treated mice traveled less distance with less speed but
entered the platform area more than DMSO treated mice.
[0145] Studies also showed that parkin activity was increased in
human M17 neuroblastoma cells after treatment with Nilotinib or
Bosutinib (FIG. 34A). Treatment with Nilotinib also resulted in
increased parkin levels in the brain lysates of wild type mice
injected with lentiviral .alpha.-synuclein prior to treatment (FIG.
34B).
[0146] Western blot analysis of brain lysates from wild type mice
treated with Bosutinib revealed that Bosutinib boosts autophagy and
degrades ubiquitinated proteins. Western blot analysis of brain
lysates from Tg-APP mice treated with 5 mg/kg Bosutinib for 3
additional weeks showed decreased levels of c-Abl, increased parkin
and alteration of different molecular markers of autophagy,
indicating that A.beta. alters normal autophagy and Bosutinib
boosts autophagy to clear A.beta..sub.1-42 (FIG. 35). Western blot
analysis of brain lysates from Tg-APP mice treated with Bosutinib
showed alterations in the levels of molecular markers of autophagy
(FIG. 36). Western blot analysis of brain lysates from Tg-APP mice
treated with Bosutinib also showed decreased levels of C-terminal
fragments (CTFs) and phosphor-tyrosine (FIG. 37).
[0147] Western blot analysis of brain lysates from Tg-APP mice
treated with 5 mg/kg Bosutinib once a day for additional weeks
showed decreased levels of different Tau isotopes (FIG. 38).
Western blot analysis of brain lysates from wild type mice
expressing lentiviral A.beta..sub.1-42 (3 weeks) with and without
Bosutinib (5 mg/kg) treatment for 3 additional weeks, showed
decreased c-Abl and increased parkin levels with Bosutinib
treatment, indicating that A.beta..sub.1-42 activates c-Abl and
Bosutinib activates parkin.
[0148] Western blot analysis of brain lysates from wild type mice
expressing lentiviral A.beta..sub.1-42 (3 weeks) with and without
Bosutinib (5 mg/kg) treatment for 3 additional weeks showed levels
of different molecular markers of autophagy, indicating that
A.beta..sub.1-42 alters normal autophagy and Bosutinib boosts
autophagy to clear A.beta..sub.1-42 (FIG. 39). Western blot
analysis of brain lysates from wild type mice expressing lentiviral
A.beta..sub.1-42 (3 weeks) with and without Bosutinib treatment for
3 additional weeks, showed decreased levels of ubiquitin (top blot)
and pan phospho-tyrosine (second blot) and SIAH2, indicating that
Bosutinib is a broad tyrosine kinase inhibitor (FIG. 40).
[0149] Western blot analysis of brain lysates from wild type mice
expressing lentiviral A.beta..sub.1-42 (3 weeks) with and without
Bosutinib treatment for 3 additional weeks showed decreased levels
of different Tau isotopes (FIG. 41). Western blot analysis of brain
lysates from wild type mice expressing lentiviral .alpha.-synuclein
(3 weeks) with and without Bosutinib treatment for 3 additional
weeks was also performed. This blots show increased
.alpha.-synuclein in lentiviral synuclein injected animals, along
with decreased c-Abl levels and phosphorylation, increased parkin
levels and markers of autophagy, including P62, HDAC6, LC3 and
ATG12 compared to loading controls tubulin and MAP2 (FIG. 42).
Example 2: Parkin Inactivation in Parkinson's Disease
[0150] To determine the role of parkin and its association with
baseline autophagy in sporadic PD, human postmortem nigrostriatal
tissues were analyzed via fractionation to determine protein
solubility and the effects of parkin on autophagic clearance in
lentiviral gene transfer animal models were investigated. Whether
lentiviral expression of .alpha.-Synuclein affects autophagy and if
parkin activity reverses .alpha.-Synuclein effects was
investigated. Animal models expressing lentiviral .alpha.-Synuclein
were studied and it was found that parkin expression decreases
.alpha.-Synuclein levels in the absence of ubiquitination. Whether
parkin expression regulates .alpha.-Synuclein clearance via
autophagic degradation was studied.
[0151] Human postmortem brain tissues. Human postmortem caudate and
midbrain regions from 22 PD patients and 15 age matched control
subjects were obtained from John's Hopkins University brain bank.
The age, sex, stage of disease and postmortem dissection (PMD) are
summarized for each patient in Table 1 and 2. The cause of death is
not known. To extract the soluble fraction of proteins, 0.5 g of
frozen brain tissues were homogenized in 1.times.STEN buffer (50 mM
Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM
PMSF and protease and phosphatase cocktail inhibitor), centrifuged
at 10,000 g for 20 min at 4.degree. C., and the supernatants were
collected. All samples were then analyzed by ELISA (see below) or
Western blot using 30 .mu.g of protein. To extract the insoluble
fraction, the pellet was re-suspended in 4M urea solution and
centrifuged at 10,000 g for 15 min, and the supernatant was
collected and 30 .mu.g of protein was analyzed by Western blot.
Western blots were quantified by densitometry using Quantity One
4.6.3 software (Bio-Rad, Hercules, Calif.). Densitometry was
obtained as arbitrary numbers measuring band intensity. Data were
analyzed as mean.+-.Standard deviation, using Two-tailed t-test
(P<0.02) and ANOVA, Neumann Keuls with multiple comparisons
(P<0.05) to compare PD and control groups.
[0152] Immunohistochemistry on slides from human patients was
performed on 30 .mu.m thick paraffin embedded brain slices
de-paraffinized in Xylenes 2.times.5 minutes and sequential ethanol
concentration, blocked for 1 hour in 10% horse serum and incubated
overnight with primary antibodies at 4.degree. C. After 3.times.10
minute washes in 1.times.PBS, the samples were incubated with the
secondary antibodies for 1 hr at RT, washed 3.times.10 minutes in
1.times.PBS. Parkin was immunoprobed (1:200) with mouse anti-parkin
(PRK8) antibody that recognizes a.a. 399-465 (Signet Labs, Dedham,
Mass.) or rabbit polyclonal (1:200) anti-parkin (AB5112) antibody
that recognizes a.a. 305-323 (Millipore) and counterstained with
DAPI. Map 2 was probed (1:300) with mouse monoclonal antibody
(Pierce). Glial Fibrillary Acid Protein (GFAP) was probed (1:200)
with mouse (GA5) Mouse mAb #3670 (Cell Signaling) or (1:200) rabbit
polyclonal (ab4674) antibody (Abcam). Tyrosine Hydroxylase (TH) was
probed (1:100) with rabbit polyclonal (AB152) antibody (Millipore)
and counterstained with DAB.
[0153] Stereotaxic injection. Lentiviral constructs were used to
generate the animal models as explained in Burns et al. (Hum. Mol.
Genetics 18: 3206-3216 (2009); Khandelwal et al. (Mol.
Neurodegener. 5: 47 (2010) and Herman and Moussa (Autophagy
7:919-921 (2011). Stereotaxic surgery was performed to inject the
lentiviral constructs into the striatum of 2-month old male
Sprague-Dawley rats. N=8 animals were used in each treatment. A
total of 116 animals were used in these studies. All procedures
were approved by the Georgetown University Animal Care and Use
Committee (GUACUC).
[0154] Western blot analysis. To extract the soluble protein
fraction, brain tissues were homogenized in 1.times.STEN buffer,
centrifuged at 10,000.times.g for 20 min at 4.degree. C., and the
supernatants containing the soluble fraction of proteins were
collected. To extract the insoluble fraction the pellet was
re-suspended in 4M urea or 30% formic acid and adjusted to pH 7
with 1N NaOH and centrifuged at 10,000.times.g for 20 min at
4.degree. C., and the supernatant containing the insoluble fraction
was collected and analyzed by Western blot. Total parkin was
immunoprobed (1:1000) with PRK8 antibody as indicated (Burns et
al., 2009) and phospho-parkin was probed (1:1000) with anti-Ser 378
antibodies (Pierce). .alpha.-Synuclein was probed with rabbit
monoclonal (1:1000) antibody (Santa Cruz). Autophagy antibodies,
including beclin-1 (1:1000), autophagy like gene (Atg)-7 (1:1000),
Atg12 (1:1000) and LC3-B (1:1000), were used to probe according to
autophagy antibody sampler kit 4445 (Cell Signaling, Inc). Histone
deacetylase 6 (HDAC6) was probed (1:500) using rabbit polyclonal
anti-HDAC6 (Abcam). Rabbit polyclonal anti-SQSTM1/p62 (Cell
Signaling Technology) was used (1:500). A rabbit polyclonal
(Pierce) anti-LC3 (1:1000) and rabbit polyclonal (Thermo
Scientific) anti-actin (1:1000) were used. LAMP-3 was probed
(1:500) rabbit polyclonal antibody (Aviva Systems). Rabbit
anti-ubiquitin (Santa Cruz Biotechnology) antibody (1:1000) was
used. Mitochondrial protein COX-IV was probed (1:1000) with rabbit
polyclonal (ab16056) antibody (Abcam) and human poly ADP-ribose
polymerase (PARP-1) was probed (1:1500) with monoclonal (MA3-950)
antibody (Pierce).
[0155] Immunohistochemistry--These methods were performed on 20
micron-thick 4% paraformaldehyde (PFA) fixed striatal rat brain
sections and compared between treatments. Parkin was probed (1:200)
with Rabbit polyclonal antibody (Chemicon). Rabbit polyclonal LC3-B
(1:100) was used to probe LC3-B (Cell Signaling, Inc). Thioflavin-S
and nuclear DAPI staining were performed according to
manufacturer's instructions (Sigma). Stereological methods--were
applied by a blinded investigator using unbiased stereology
analysis (Stereologer, Systems Planning and Analysis, Chester, Md.)
to determine the total positive cell counts in 20 cortical fields
on at least 10 brain sections (.about.400 positive cells per
animal) as indicated in (Burns et al., 2009, Khandelwal et al.,
2010, Herman and Moussa, 2011).
[0156] .alpha.-Synuclein, parkin and p-Tau enzyme-linked
immunosorbent assay (ELISA)-Specific ELISA (Invitrogen) were
performed using 50 .mu.l (1 .mu.g/.mu.1) of brain lysates detected
with 50 .mu.l primary antibody (3 h) and 100 .mu.l anti-rabbit
secondary antibody (30 min) at RT. Parkin levels using specific
human ELISA (MYBioSource), and p-Tau and .alpha.-Synuclein levels
were measured using human specific ELISA (Invitrogen) according to
manufacturers' protocols.
[0157] Subcellular fractionation to isolate autophagic
vacuoles--0.5 g of Frozen human or animal brains were homogenized
at low speed (Cole-Palmer homogenizer, LabGen 7, 115 Vac) in
1.times.STEN buffer and centrifuged at 1,000 g for 10 minutes to
isolate the supernatant from the pellet. The pellet was
re-suspended in 1.times.STEN buffer and centrifuged once to
increase the recovery of lysosomes. The pooled supernatants were
then centrifuged at 100,000 rpm for 1 hour at 4.degree. C. to
extract the pellet containing autophagic vacuoles (AVs) and
lysosomes. The pellet was then re-suspended in 10 ml (0.33 g/ml)
50% Metrizamide and 10 ml in cellulose nitrate tubes. A
discontinuous Metrizamide gradient was constructed in layers from
bottom to top as follows: 6 ml of pellet suspension, 10 ml of 26%;
5 ml of 24%; 5 ml of 20%; and 5 ml of 10% Metrizamide (Marzella et
al., 1982). After centrifugation at 10,000 rpm for 1 hour at
4.degree. C., the fraction floating on the 10% layer (Lysosome) and
the fractions banding at the 24%/20% (AV 20) and the 20%/10% (AV10)
Metrizamide inter-phases were collected by a syringe and
examined.
[0158] Transmission Electron Microscopy--Brain tissue were fixed in
(1:4, v:v) 4% paraformaldehyde-picric acid solution and 25%
glutaraldehyde overnight, and then washed 3.times. in 0.1M
cacodylate buffer and osmicated in 1% osmium tetroxide/1.5%
potassium ferrocyanide for 3 h, followed by another 3.times. wash
in distilled water. Samples were treated with 1% uranyl acetate in
maleate buffer for 1 h, washed 3.times. in maleate buffer (pH 5.2),
then exposed to a graded cold ethanol series up to 100% and ending
with a propylene oxide treatment. Samples are embedded in pure
plastic and incubated at 60.degree. C. for 1-2 days. Blocks are
sectioned on a Leica ultracut microtome at 95 nm, picked up onto
100 nm formvar-coated copper grids, and analyzed using a Philips
Technai Spirit transmission EM. All sections were acquired and
analyzed by a blind investigator.
Results
[0159] Decreased parkin solubility in postmortem striatum of
sporadic PD patients. To determine the role of parkin in the brain
of sporadic PD patients, human postmortem striatal (caudate)
tissues from 12 PD patients and 7 age-matched controls as described
in Table 1 were analyzed. ELISA measurement of soluble human parkin
revealed a significant (P<0.05) decrease (36%) in parkin levels
in PD caudate/striatum compared to control (FIG. 43A). Western blot
analysis of soluble striatal extracts confirmed the decrease in
parkin levels in PD patients compared to control (FIGS. 43B and C,
54%). No differences in parkin levels were detected in PD cortex.
Probing with anti-ubiquitin antibody showed a higher smear of
ubiquitinated proteins in PD striatum compared to control (FIG.
43B). However, all samples with PMD greater than 16 h showed
significantly (P<0.02, two-tailed t-test) higher levels of
ubiquitin (48%) in both groups and higher parkin levels (25%) with
PMD greater than 13 h within the PD group. To further investigate
whether the decreased degradation of proteins results in alteration
of solubility, the insoluble proteins were extracted in 4M urea. An
increase in the level of parkin was detected in the insoluble
fraction (FIGS. 43D and E, 82%) in contrast to the soluble extract,
which was hardly detected. Parkin phosphorylation at serine 378,
which was not detected in the soluble fraction, was observed in the
insoluble extract (FIGS. 43D and E, 114%). Additionally, more
ubiquitinated proteins (FIG. 43D, 3rd blot) were also detected in
the insoluble fraction. The variations among the samples are
represented to show variation among individual samples, including
soluble, insoluble and phospho-parkin (FIG. 43F). Taken together
these data show decreased parkin solubility and increased
phosphorylation in PD.
[0160] Altered parkin expression and loss of tyrosine hydroxylase
neurons in the nigrostriatum of sporadic PD patients. To determine
whether parkin expression is altered in sporadic PD, human
postmortem midbrain sections from 10 PD patients and 8 control
subjects as identified in Table 2 were examined. To determine the
difference in parkin staining between PD and control brains, serial
brain sections collected from each case were probed with human
anti-parkin antibody (PRK8) that recognizes a.a. 399-495 and
counterstained with either GFAP or DAPI. Confocal microscopy was
used and diffuse parkin cytosolic staining was observed in the
caudate (FIG. 44A) and within GFAP-stained astrocytes of control
brain sections (FIG. 44B), and TH staining (FIG. 54C) was also
observed in the caudate of a control subject (case 1683). However,
intense cytosolic staining in the caudate (FIG. 44D, arrow), and
within astrocytes (FIG. 44E), with diminished TH staining (FIG.
44F) were observed in a PD/AD patient (case 2215). To ascertain
that parkin or GFAP staining were not due to auto-fluorescence in
human slides, the slides were incubated with and without secondary
and or primary antibodies and the absence of non-specific antibody
binding was determined via confocal microscopy. Parkin expression
was further examined in midbrain/SN brain regions. Diffuse parkin
cytosolic staining (FIG. 44G) and within GFAP-stained astrocytes
(FIG. 44H) with TH staining (FIG. 44I) were observed in serial
sections of midbrain/SN of control brain (case 1855). Intense
cytosolic parkin staining (FIG. 54J, arrow), and within astrocytes
(FIG. 44K), with significantly diminished TH staining (FIG. 44L)
were observed in a PD patient (case 2315). Another combination of
antibodies using the AB5112 clone that detects parkin at a.a.
305-323 and GFAP antibodies was used to verify otherwise results.
Intense cytosolic parkin staining (FIG. 44M, arrow), and within
astrocytes (FIG. 44N), with significantly diminished TH staining
(FIG. 44O) were observed in a PD/dementia patient (case 2243).
MAP-2 was used as a neuronal marker and co-stained with parkin
(DAPI counterstain) and TH. Parkin staining (FIG. 45A) was diffuse
within the cytosol and was largely localized to MAP-2 labeled
neurons (FIGS. 45B & C) in the midbrain/SN of a control subject
(case 1277). TH staining was also detected in serial brain sections
(FIG. 45D). However, more intense and less diffuse parkin staining
was detected in the cytosol of DAPI stained cells (FIG. 45E) and
parkin staining was localized to MAP-2 stained neurons (FIG.
45F&G), with significantly decreased TH staining (FIG. 45H) in
the midbrain/SN of a PD/Dementia patient (case 2267).
[0161] Alteration of baseline autophagy in post-mortem striatum of
PD patients. To determine whether the change in parkin solubility
is associated with changes of baseline autophagy, the level of some
autophagic markers in human PD striatal extracts was examined. The
markers of the autophagic cascade were examined, including
microtubule-associated light chain protein 3 (LC3). Probing with
anti-LC3 antibody suggested an increase in LC3-II levels compared
to LC3-I (FIG. 45I&J, 1.sup.st blot, 78%, N=12 PD and 7
control), indicating possible conversion and lipidation of LC3.
LC3-I is abundant and stable in the brain, the ratio of LC3-II to
LC3-I or the amount of LC3-II can be used to monitor the amount of
autophagosome. LC3 is expressed as three isoforms in mammalian
cells, LC3-A, LC3-B and LC3-C. Because LC3-II itself is degraded by
autophagy the amount of LC3 was measured using an antibody specific
for the LC3-B isoform. An increase in the level of LC3-B was
detected in human striatal extracts from PD patients (N=12)
compared to control (N=7) subjects (FIG. 45I&J, 2.sup.nd blot,
48%. P<0.05, ANOVA, Neumann Keuls). Subcellular fractionation
was performed to isolate autophagic vacuoles and lysosomes the
levels of .alpha.-Synuclein, parkin and p-Tau were measured using
quantitative ELISA. First it was determined whether the subcellular
fractionation assay successfully extracted autophagosomes from
lysosomes in frozen human tissues. Western blot analysis on PD
patients brain lysates showed the lysosome-associated membrane
glycoprotein 3 (LAMP-3) in the floating fraction containing
lysosomes (FIG. 45K, 1.sup.st blot), while both the AV-10 and AV-20
fractions contained LC3-B (FIG. 45K, 2.sup.nd blot), suggesting
that frozen human brains contain autophagic vacuoles and our
fractionation did isolate autophagosomes from lysosomes. Probing
for mitochondrial marker cytochrome c oxidase-IV (COX-IV, FIG. 45K,
3.sup.rd blot) and nuclear marker Poly ADP-ribose polymerase
(PARP-1, FIG. 45K, 4.sup.th blot) was also performed, and markers
were detected in all fractions, suggesting that brain samples
contained intact organelles. A comprehensive assay that clearly
shows mitochondria in autophagosomes or lysosomes must be performed
with both IHC co-labeling with LC3-COX-IV (autophagosome) or
cathepsin-D-COX-IV (Lysosome) coupled with immuno-EM to determine
mitochondrial accumulation in separate autophagic vacuoles. An
ELISA was used to measure protein levels in subcellular extracts.
The level of .alpha.-Synuclein was significantly increased
(P<0.05, N=12 PD and 7 control) in AV-10 (31%) and AV-20 (64%)
compared to control (FIG. 45L, ANOVA, Neumann Keuls), but no
.alpha.-Synuclein was detected in the lysosomal fraction.
Interestingly, ELISA measurement of parkin levels also showed a
significant increase in AV-10 (FIG. 45M, 24%) and AV-20 (FIG. 45M,
23%) and a slight non-significant (9%) increase in the lysosome in
PD (N=12) compared to control (N=7) subjects. The levels of p-Tau
were measured as another protein marker that is occasionally
associated with PD pathology. Similarly, no p-Tau was detected in
the lysosome but the levels of p-Tau were significantly increased
in AV-10 (54%) and AV-20 (64%) compared to control (FIG. 45N, N=12
PD and 7 control). These data show accumulation of un-degraded
proteins in autophagosomes in PD.
[0162] Parkin attenuates .alpha.-Synuclein-induced protein
accumulation in the striatum. Because increased parkin insolubility
and decreased soluble parkin levels were observed in association
with alteration of autophagy in PD striatum, it was sought to
over-express parkin and determine whether functional parkin can
reverse .alpha.-Synuclein effects on autophagic clearance. A gene
transfer animal model targeting .alpha.-Synuclein expression to the
striatum of 2-month old rats was used. Lentiviral parkin led to
significant increases (FIG. 46A, 53% by densitometry, N=8,
P<0.05) in parkin levels and lentiviral .alpha.-Synuclein led to
significant increases (41%) in .alpha.-Synuclein levels.
Co-expression of parkin with .alpha.-Synuclein attenuated the
levels of monomeric .alpha.-Synuclein (FIG. 46A) and reduced the
level of higher molecular weight proteins back to control (LacZ) 4
weeks post injection (Khandelwal et al., 2010). No changes in total
parkin levels were observed in brains injected with lentiviral
.alpha.-Synuclein (FIG. 46A, 1st blot) and no-phosphorylated parkin
was detected in rat brains. Independent studies were performed to
confirm changes in .alpha.-Synuclein levels using quantitative
ELISA specific for human .alpha.-Synuclein. The levels of human
.alpha.-Synuclein were significantly increased (FIG. 46B, 54%, N=8)
in the striatum of animals injected with lentiviral
.alpha.-Synuclein compared to LacZ or parkin. Co-injection with
lentiviral .alpha.-Synuclein and parkin reversed the levels of
human .alpha.-Synuclein back to control. Lentiviral delivery of
parkin into the striatum resulted in a significant increase in
parkin when it was expressed alone (FIG. 46C, 44%, N=8) or in the
presence of .alpha.-Synuclein (53%, N=8).
[0163] Changes in rat p-Tau were determined using ELISA. Expression
of human .alpha.-Synuclein leads to a significant increase (FIG.
46D, 34%, N=8) in p-Tau in the rat striatum, but co-expression of
parkin reverses p-Tau back to control. Lentiviral expression of
.alpha.-Synuclein in the striatum leads to detection of
thioflavin-S positive staining (FIG. 46F), compared to lentiviral
parkin alone (FIG. 46F). However, co-expression of parkin with
.alpha.-Synuclein prevents the appearance of thioflavin-S positive
staining (FIG. 46G); suggesting that parkin attenuation of
.alpha.-Synuclein levels can eliminate thioflavin-S positive
species in this animal model. To ascertain that thioflavin-S
staining is associated with .alpha.-Synuclein expression, striatal
sections were stained with human .alpha.-Synuclein antibody and
showed no .alpha.-Synuclein staining in sections cut serially with
the thioflavin-S sections from lentiviral parkin injected rats
(FIG. 46K), compared to an abundant level of .alpha.-Synuclein in
lentiviral .alpha.-Synuclein injected rats, congruent with
thioflavin-S staining (FIG. 46L), while parkin co-expression led to
disappearance of human .alpha.-Synuclein in the rat striatum (FIG.
46M).
[0164] Wild type functional parkin, not mutant T240R, mediates
clearance of .alpha.-Synuclein-induced autophagic vacuoles. It was
sought to determine whether .alpha.-Synuclein expression can change
normal autophagy, leading to formation of autophagic vacuoles in
vivo. EM images of striatal sections showed no vacuoles in
lentiviral LacZ injected animals (FIG. 47A) 4 weeks post injection.
Lentiviral expression of .alpha.-Synuclein led to cytosolic
accumulation of vacuoles (FIG. 47B, asterisks), suggesting that
.alpha.-Synuclein expression alters autophagy in the rat striatum.
Co-expression of parkin with .alpha.-Synuclein led to formation of
autophagic vacuoles containing debris (FIG. 47C). To ascertain
whether parkin function mediates clearance of autophagic vacuoles,
non-functional T240R parkin was used, which is a mutant form that
loses its E3 ubiquitin ligase activity, leading to ARJPD.
Co-expression of mutant T240R parkin with .alpha.-Synuclein did not
prevent the accumulation of cytosolic vacuoles (FIG. 47D,
asterisks), suggesting that parkin mediates autophagic clearance
via its E3 ubiquitin ligase function.
[0165] Levels of human .alpha.-Synuclein and p-Tau were measured
using quantitative ELISA in subcellular fractions. A significant
increase (62%, P<0.05, N=5) in the level of .alpha.-Synuclein
was detected in AV-10 (FIG. 47E) and AV-20 (19%) compared to LacZ
injected animals. However, co-expression of parkin eliminated
.alpha.-Synuclein from AV-10 and significantly increased its levels
in AV-20 (45%) and lysosomes (24%) compared to LacZ (FIG. 47E).
Co-expression of .alpha.-Synuclein with T240R resulted in
significantly elevated (51%) levels of .alpha.-Synuclein in AV-10,
and unlike wild type parkin, failed to show any deposition in
AV-20, which is enriched in autophagosomes or lysosomes.
Significantly increased levels (P<0.05, N=5) of p-Tau were
detected in AV-10 in animals injected with .alpha.-Synuclein (34%)
or .alpha.-Synuclein+T240R (39%) compared to LacZ. However, wild
type parkin expression led to a significant increase of p-Tau in
AV-10 (19%) and lysosome (21%) compared to LacZ, .alpha.-Synuclein
and .alpha.-Synuclein+T240R (FIG. 47F). No parkin as measured by
ELISA was detected in subcellular fractions in these animal models,
suggesting that parkin accumulation in autophagic vesicles can take
place over a protracted time period in PD.
[0166] Functional parkin, not mutant T240R, regulates autophagic
clearance in the striatum of .alpha.-Synuclein expressing animals.
To determine the mechanisms by which parkin can mediate clearance
of autophagic vacuoles in the rat striatum, molecular markers of
the autophagic pathway were examined. WB analysis showed no
difference in beclin-1 levels in animals injected with lentiviral
LacZ, parkin or .alpha.-Synuclein alone (FIG. 48A). A significant
increase in beclin-1 levels (54% by densitometry, N=8, P<0.05)
was observed when parkin was co-expressed with .alpha.-Synuclein,
suggesting that parkin responds to .alpha.-Synuclein-induced
stress. The levels of Atg7 and Atg12 were also significantly
increased by 41% and 33%, respectively, in parkin+.alpha.-Synuclein
injected animals (FIG. 48A) compared to animals injected with LacZ,
parkin or .alpha.-Synuclein alone. No changes in LC3-B levels were
observed between animals injected with lentiviral LacZ or parkin
alone (FIG. 48B) but .alpha.-Synuclein expression significantly
increased (51%) LC3 levels (FIG. 48B), suggesting increased amount
of autophagosomes. Co-expression of parkin and .alpha.-Synuclein
decreased the levels of LC3-B (29% by densitometry, N=8,
P<0.05), suggesting degradation of LC3-B-containing autophagic
vacuoles. No changes were also observed in HDAC6 levels (FIG. 48B)
between animals injected with LacZ, parkin or .alpha.-Synuclein
alone, but HDAC6 level was significantly increased (37%) levels
(FIG. 48B) when animals were co-injected with parkin and
.alpha.-Synuclein together, suggesting that parkin expression
facilitates fusion between autophagosomes and lysosomes. No
differences in the levels of molecular markers of autophagy were
observed when mutant T240R parkin was injected either alone or with
.alpha.-Synuclein. These data show that parkin E3 ubiquitin ligase
activity may up-regulate protein levels of the beclin-1-dependent
autophagic cascade, facilitating autophagic clearance.
[0167] The EM and WB data was supplemented with
immunohistochemistry to determine the presence of LC3-B. Staining
with anti-LC3-B antibody showed no reactivity in the striatum of
animals injected with lentiviral parkin (FIG. 48C). Lentiviral
expression of .alpha.-Synuclein led to an increase in
immunoreactivity to LC3-B (FIG. 48D). Stereological counting of
LC3-B positive cells revealed a significant increase (FIG. 48G.
43%, P<0.05, N=8) in striata injected with .alpha.-Synuclein.
Co-injection of lentiviral parkin with .alpha.-Synuclein (FIG. 48E)
resulted in disappearance of LC3-B from the striatum. To further
ascertain that functional E3 ubiquitin ligase parkin mediates
autophagic changes, LC3-B antibodies were co-injected with
.alpha.-Synuclein and mutant T240R parkin (FIG. 48F) in striatal
sections and no elimination of LC3-B staining was observed in these
animals. Stereological counting of LC3-B stained cells in the
striatum co-injected with .alpha.-Synuclein and T240R showed a
significant increase (37%) in LC3-B reactivity compared to LacZ
(FIG. 48F&G). To further determine whether wild type parkin
leads to clearance of ubiquitinated proteins via autophagy we
stained with anti-P62 antibody. The levels of P62 were
significantly (P<0.05, N=8) increased when .alpha.-Synuclein
(41% by densitometry relative to actin) was expressed compared to
LacZ (FIG. 48F). However, parkin co-expression led to complete
disappearance of P62 staining, suggesting autophagic degradation of
ubiquitinated proteins.
[0168] These studies show decreased parkin solubility in the
striatum of sporadic PD patients, independent of early onset
disease-causing mutations. In conclusion, decreased parkin
solubility can reflect diminished parkin function, which can lead
to alteration of baseline autophagy, including parkin,
.alpha.-Synuclein and p-Tau clearance. Lentiviral expression of
.alpha.-Synuclein leads to p-Tau and accumulation of autophagic
vacuoles. These data demonstrate an association between
.alpha.-Synuclein and autophagic dysfunction in PD, and indicate a
beneficial role for parkin in autophagic clearance. Thus, parkin's
role in autophagic clearance can be exploited as a therapeutic
strategy in PD.
TABLE-US-00001 TABLE 1 Description and clinical diagnosis of human
PD patients and control subjects`s tissues analyzed by Western blot
and ELISA. BRC # FDX Age Sex Race PMD FR Area 399 Control 79 F W 24
Caudate 417 Control 80 F W 6 Caudate 487 Control 73 M W 22 Caudate
515 Control 62 M W 19 Caudate 705 Control 73 M W 9 Caudate 1277
Control 80 F W 8 Caudate 2052 Control 79 M W 16 Caudate 1690 PD 76
M W 18 Caudate 1731 PD 77 M W 16 Caudate 2140 PD with dementia 84 F
W 11 Caudate 2067 PD with dementia, 76 M W 19 Caudate cerebrovas.
dis (NC) 2019 PD with dementia, 83 M W 16.5 Caudate cerebrovas. dis
1989 PD with dementia, 84 M W 5 Caudate LBD neocortical 2074 PD,
cerebrovascular 85 F W 9 Caudate disease 1758 PD, DLB 81 M W 11
Caudate 1948 PD, DLB 77 M W 5 Caudate 1796 PD, Lewy Body 81 M W
8.75 Caudate CHG Limbic, porencephalic cyst 1877 PD, Lewy Body 80 M
W 19 Caudate CHG neocortical 1955 PD, Lewy Body 84 M B 13 Caudate
CHG neocortical
TABLE-US-00002 TABLE 2 Description and clinical diagnosis of human
PD patients and control subjects stained with immonuhistohemistry.
BRC # FDX CERAD BRAAK Age Sex Race PMD FX 1062 Control 58 M B 14
Hippocampus MB 1252 Control 70 M W Hippocampus MB 1277 Control 0 80
F W 8 Caudate, hippocampus, MB 1352 Control 78 F 14 Caudate,
hippocampus, MB 1615 Control 72 M W 20 Caudate 1683 Control 1 91 F
W 8 Caudate 1855 Control 77 M W Caudate, hippocampus, MB 2201
Control 0 2 85 F W 27 Caudate, hippocampus, MB 2215 PD with
dementia, B 4 88 M W 9 Caudate, MB AD probable 2235 PD, tauopathy
86 F W 26 Caudate, MB non-AD, cerebrovas. dis (NC) 2243 PD with
dementia 0 0 68 M W 50 Caudate, MB 2253 PD, contusion 0 1 64 F W 15
Caudate, MB 2267 PD with dementia, 0 1 75 M W 22 Caudate, MB
neocortex 2290 PD A 2 82 M W 53 Caudate, MB 2292 PD with B 4 77 M W
8 Caudate, MB dementia, AD probable 2312 PD 0 0 56 M W 21 MB 2315
PD 0 2 84 M W 8.5 Caudate, MB 2352 PD with 0 2 83 F W 163 Caudate,
MB dementia, cerebrovas. dis (NC)
Example 3--Parkin Inactivation in Alzheimer's Disease
[0169] The role of parkin in post-mortem brain tissues from 21
Alzheimer's disease patients and 15 control subjects was
investigated. In order to determine the role of parkin in A.beta.
clearance, gene transfer animals expressing lentiviral
A.beta..sub.1-42 with and without parkin were generated and
autophagic mechanisms were examined.
Materials and Methods
[0170] Human postmortem brain tissues. Human postmortem hippocampal
and cortical regions from 21 AD patients and 15 age matched control
subjects were obtained from John's Hopkins University brain bank.
The age, sex, stage of disease and postmortem dissection (PMD) are
summarized for each patient in Table 3 and 4. The cause of death is
not known. To extract the soluble fraction of proteins, 0.5 g of
frozen brain tissues were homogenized in 1.times.STEN buffer (50 mM
Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM
PMSF and protease cocktail inhibitor), centrifuged at 10,000 g for
20 min at 40 C, and the supernatants were collected. All samples
were then analyzed by ELISA (see below) or Western blot using 30
.mu.g of protein. To extract the insoluble fraction, the pellet was
re-suspended in 4M urea solution and centrifuged at 10,000 g for 15
min, and the supernatant was collected and 30 .mu.g of protein was
analyzed by Western blot. Western blots were quantified by
densitometry using Quantity One 4.6.3 software (Bio Rad).
Densitometry was obtained as arbitrary numbers measuring band
intensity. Data were analyzed as mean.+-.Standard deviation, using
Two-tailed t-test (P<0.02) and ANOVA, Neumann Keuls with
multiple comparisons (P<0.05) to compare AD and control groups.
Insoluble parkin co-localizes with intracellular A.beta..
[0171] Immunohistochemistry on slides from human patients was
performed on 30 .mu.m thick paraffin embedded brain slices
de-paraffinized in Xylenes 2.times.5 minutes and sequential ethanol
concentration, blocked for 1 hour in 10% horse serum and incubated
overnight with primary antibodies at 4.degree. C. After 3.times.10
minute washes in 1.times.PBS, the samples were incubated with the
secondary antibodies for 1 hr at RT, washed 3.times.10 minutes in
1.times.PBS. A.beta..sub.1-42 was probed (1:200) with rabbit
polyclonal specific anti-A.beta..sub.1-42 antibody (Zymed) that
recognizes a.a.1-42, and (1:200) mouse monoclonal antibody (4G8)
that recognizes a.a. 17-24 (Covance) and counterstained with DAPI.
Parkin was immunoprobed (1:200) with mouse anti-parkin (PRK8)
antibody that recognizes a.a. 399-465 (Signet Labs, Dedham, Mass.)
and rabbit polyclonal (1:200) anti-parkin (AB5112) antibody that
recognizes a.a. 305-622 (Millipore) and counterstained with DAPI.
Because human tissues may exhibit a high level of
auto-fluorescence, other experiments were performed with the
absence of either primary or secondary antibodies to determine the
specificity of immunostaining.
[0172] Stereotaxic injection. Lentiviral constructs were used to
generate the animal models as explained in Rebeck et al. (J. Biol.
Chem. 285, 7440-7446 (2010)) and the identity of the
A.beta..sub.1-42 species generated was confirmed by mass
spectroscopy. Stereotaxic surgery was performed to inject the
lentiviral constructs encoding LacZ, parkin or A.beta..sub.1-42
into the M1 primary motor cortex of 2-month old male Sprague-Dawley
rats. All animals were anesthetized (50 mg/kg body weight) with a
cocktail of Ketamine and Xylazine (50:8). The stereotaxic
coordinates were according to Paxinos and Watson rat brain atlas.
Lentiviral stocks were injected through a Micro syringe pump
controller (Micro4) using total pump (World Precision Instruments,
Inc.) delivery of 6 .mu.l at a rate of 0.2 .mu.l/min. In one side
of the brain animals were injected with 1) a lentiviral-LacZ vector
at 2.times.10.sup.10 multiplicity of infection (m.o.i); 2) with
1.times.10.sup.10 m.o.i lentiviral-parkin and 1.times.10.sup.10
m.o.i lentiviral-LacZ; 3) 1.times.10.sup.10 m.o.i
lentiviral-A.beta.1-Insoluble parkin co-localizes with
intracellular A.beta..sub.1-42 and 1.times.10.sup.10 m.o.i
lentiviral-LacZ; or 4) and 1.times.10.sup.10 m.o.i
lentiviral-A.beta..sub.1-42 and 1.times.10.sup.10 m.o.i
lentiviral-parkin. All procedures were approved by the Georgetown
University Animal Care and Use Committee (GUACUC). A total of 84
rats were used in these studies.
[0173] Western blot analysis. To extract the soluble protein
fraction, brain tissues were homogenized in 1.times.STEN buffer (50
mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20
mM PMSF and protease cocktail inhibitor), centrifuged at
10,000.times.g for 20 min at 40.degree. C., and the supernatants
containing the soluble fraction of proteins were collected. To
extract the insoluble fraction the pellet was re-suspended in 4M
urea or 30% formic acid and adjusted to pH 7 with 1N NaOH and
centrifuged at 10,000.times.g for 20 min at 40 C, and the
supernatant containing the insoluble fraction was collected and
analyzed by Western blot. The supernatants were analyzed by WB on
SDS NuPAGE 4-12% Bis-Tris gel (Invitrogen, Inc.). Protein
estimation was performed using Bio-Rad protein assay (Bio-Rad
Laboratories Inc., Hercules, Calif.). Total parkin was immunoprobed
(1:1000) with PRK8 antibody as indicated [43] and phospho-parkin
was probed (1:1000) with anti-Ser 378 antibodies (Pierce).
A.beta..sub.1-42 was immunoprobed with (1:600) mouse 6E10 antibody
(Signet Labs, Mass.), analyzed alongside a synthetic peptide
(AnaSpec, Calif., USA). Rabbit polyclonal antibodies anti-beclin
(1:1000), autophagy like gene (Atg)-7 (1:1000), Atg12 (1:1000) and
LC3-B (1:1000) were used to probe autophagy proteins using antibody
sampler kit 4445 (Cell Signaling, Inc). Histone deacetylase 6
(HDAC6) was probed (1:500) using rabbit polyclonal anti-HDAC6
(Abcam). Rabbit polyclonal anti-SQSTM1/p62 (Cell Signaling
Technology) was used (1:500). Lysosomal-associated membrane protein
3 (LAMP-3) was probed (1:500) with rabbit polyclonal antibody
(ProteinTech). A rabbit polyclonal (Pierce) anti-LC3 (1:1000) and
rabbit polyclonal (Thermo Scientific) anti-actin (1:1000) were
used.). Rabbit polyclonal (1:1000) Cyclin E (Thermo Scientific),
rabbit polyclonal (1:1000) tubulin (Thermo Scientific) and mouse
monoclonal (1:500) anti-ubiquitin (Santa Cruz Biotechnology) were
used. Map 2 was probed (1:1000) mouse monoclonal antibody
(Pierce).
[0174] Western blots were quantified by densitometry using Quantity
One 4.6.3 software (Bio Rad). Densitometry was obtained as
arbitrary numbers measuring band intensity. Data were analyzed as
mean.+-.standard deviation, using ANOVA, with Neumann Keuls
multiple comparison between treatment groups. A total number of N=8
was used in each treatment.
[0175] Immunohistochemistry was performed on 20 micron-thick 4%
paraformaldehyde (PFA) fixed cortical brain sections and compared
between treatments. A.beta..sub.1-42 was probed (1:200) with rabbit
polyclonal specific anti-A.beta..sub.1-42 antibody (Zymed). Rabbit
polyclonal LC3-B (1:100) was used to probe LC3-B (Cell Signaling,
Inc). Thioflavin-S and nuclear DAPI staining were performed
according to manufacturer's instructions (Sigma).
[0176] Stereological methods were applied by a blinded investigator
using unbiased stereology analysis (Stereologer, Systems Planning
and Analysis, Chester, Md.) to determine the total positive cell
counts in 20 cortical fields on at least 10 brain sections
(.about.400 positive cells per animal) from each animal. These
areas were selected across different regions on either side from
the point of injection, and all values were averaged to account for
the gradient of staining across 2.5 mm radius from the point of
injection. An optical fractionator sampling method was used to
estimate the total number of positive cells with multi-level
sampling design. Cells were counted within the sampling frame
determined optically by the fractionator and cells that fell within
the counting frame were counted as the nuclei came into view while
focusing through the z-axis.
[0177] A.beta., parkin and p-Tau enzyme-linked immunosorbent assay
(ELISA)-Specific p-Tau, A.beta..sub.1-40 and A.beta..sub.1-42 ELISA
(Invitrogen) were performed using 50 .mu.l (1 .mu.g/.mu.l) of brain
lysates detected with 50 .mu.l human p-Tau (AT8) or A.beta. primary
antibody (3 h) and 100 .mu.l anti-rabbit Insoluble parkin
co-localizes with intracellular A.beta. antibody (30 min) at RT.
Extracts were incubated with stabilized Chromogen for 30 minutes at
RT and solution was stopped and read at 450 nm, according to
manufacturer's protocol. Parkin levels were measured using specific
human ELISA (MYBioSource) was measured using human specific ELISA
(Invitrogen). All ELISA were performed according to manufacturers'
protocols.
[0178] Subcellular fractionation to isolate autophagic
vacuoles--Animal brains were homogenized at low speed (Cole-Palmer
homogenizer, LabGen 7, 115 Vac) in 1.times.STEN buffer and
centrifuged at 1,000 g for 10 minutes to isolate the supernatant
from the pellet. The pellet was re-suspended in 1.times.STEN buffer
and centrifuged once to increase the recovery of lysosomes. The
pooled supernatants were then centrifuged at 100.000 rpm for 1 hour
at 40.degree. C. to extract the pellet containing autophagic
vacuoles (AVs) and lysosomes. The pellet was then re-suspended in
10 ml (0.33 g/ml) 50% Metrizamide and 10 ml in cellulose nitrate
tubes. A discontinuous Metrizamide gradient was constructed in
layers from bottom to top as follows: 6 ml of pellet suspension, 10
ml of 26%; 5 ml of 24%; 5 ml of 20%; and 5 ml of 10% Metrizamide.
After centrifugation at 100,000 rpm for 1 hour at 40 C, the
fraction floating on the 10% layer (Lysosome) and the fractions
banding at the 24%/20% (AV 20) and the 20%/10% (AV10) Metrizamide
inter-phases were collected by a syringe and examined.
[0179] Transmission Electron Microscopy--Brain tissue were fixed in
(1:4, v:v) 4% paraformaldehyde-picric acid solution and 25%
glutaraldehyde overnight, then washed 3.times. in 0.1M cacodylate
buffer and osmicated in 1% osmium tetroxide/1.5% potassium
ferrocyanide for 3 h, followed by another 3.times. wash in
distilled water. Samples were treated with 1% uranyl acetate in
maleate buffer for 1 h, washed 3.times. in maleate buffer (pH 5.2),
then exposed to a graded cold ethanol series up to 100% and ending
with a propylene oxide treatment. Samples are embedded in pure
plastic and incubated at 60.degree. C. for 1-2 days. Blocks are
sectioned on a Leica ultracut microtome at 95 nm, picked up onto
100 nm formvar-coated copper grids, and analyzed using a Philips
Technai Spirit transmission EM. All images were collected by a
blind investigator.
[0180] Soluble parkin is decreased in post-mortem AD brain tissues.
To determine whether parkin levels are changed in other regions of
AD brain, frozen post-mortem cortical tissues from 12 AD patients
and 7 age matched control subjects were examined. The clinical
diagnosis and post-mortem dissection (PMD) are summarized in Table
3. No information was provided about the cause of death. Western
blot (WB) analysis with anti-parkin antibody (PRK8) revealed a
significant decrease (52%, P<0.05) in soluble parkin level in
the cortex of AD brain (FIGS. 49A & D). To ascertain that the
decrease in parkin level is not due to neuronal loss, an anti-MAP-2
antibody was used as a neuronal marker and loading control (FIG.
49A). Quantitative parkin ELISA showed a significant decrease (46%)
in soluble parkin levels (FIG. 49B, P<0.05), suggesting that
parkin levels may be reduced in AD. Further analysis using
two-tailed t-test (P<0.02) showed no differences within AD or
control samples with age, but parkin levels were significantly
(P<0.05) reduced (21%) in all samples with PMD greater than 15
hours.
[0181] Potential parkin function was investigated via examination
of the level of ubiquitinated proteins and possible targets of
parkin E3 ubiquitin ligase activity, including tubulin and Cyclin
E. The levels of ubiquitinated proteins (FIG. 49C, 1st blot) was
increased in WB of soluble AD cortical extracts compared to control
subjects, suggesting lack of degradation of ubiquitinated proteins.
No significant differences (t-test, P<0.02) were observed within
the AD group, but variation was noticeable among control subjects,
with older subjects showing smears of ubiquitinated proteins
similar to AD (FIG. 49C, 1st blot lane 1 (case#399) and lane 3
(case#1277). Significantly increased levels of tubulin (2nd blot,
63%, FIG. 49D, P<0.05) and Cyclin E (3rd blot, 34%, FIG. 49D)
were also observed in AD compared to control subjects. The
insoluble protein fraction of human postmortem cortical tissues was
then extracted in 4M urea and western blot was performed. Little
parkin was detected in the insoluble fraction of control brains,
but total parkin was significantly increased (130%, P<0.05) in
AD brains, suggesting parkin insolubility (FIGS. 49E & F). We
also detected significantly (P<0.05) increased levels (143%) of
phosphorylated parkin at serine-378 relative to actin in AD brains
but not control subjects (FIG. 49E, 2nd blot and FIG. 49F),
suggesting that parkin phosphorylation may be associated with
decreased solubility.
[0182] Parkin co-localizes with intraneuronal A.beta..sub.1-42 in
the hippocampus and cortex in AD. To investigate whether parkin
expression is altered in human AD brains, a mouse monoclonal
anti-parkin (PRK8) antibody that recognizes a.a. 399-465 and rabbit
polyclonal anti-human A.beta..sub.1-42 antibody that recognizes
a.a. 1-42 were used. Hippocampal staining showed intraneuronal
A.beta..sub.1-42 (FIG. 50A) and parkin (FIG. 50B) in nuclear
DAPI-stained neurons in control human subjects, and both parkin and
A.beta..sub.1-42 were detected within the same cells (FIG. 50C).
The levels of intraneuronal expression of A.beta..sub.1-42 were
increased in the hippocampus of AD patients (FIG. 50D), without
noticeable detection of amyloid plaques. Co-staining showed
increased intracellular parkin levels (FIG. 50E) in AD hippocampus
compared to control subjects (FIG. 50B), suggesting accumulation of
parkin in AD brains. Both intracellular parkin and A.beta..sub.1-42
were co-localized in hippocampal neurons (FIG. 50F). To ascertain
the specificity of these results in human sections alternate rabbit
polyclonal anti-parkin (AB5112) antibody that recognizes a.a.
305-622 and mouse monoclonal anti-human A.beta..sub.1-42 (4G8)
antibody that recognizes a.a. 17-42 were used. Hippocampal staining
showed intraneuronal A.beta..sub.1-42 (FIG. 50G) and parkin (FIG.
50H) in nuclear DAPI-stained neurons in AD, and both parkin and
A.beta..sub.1-42 were detected within the same cells (FIG. 50J)
without noticeable detection of amyloid plaques.
[0183] Other brain regions were examined where extracellular
plaques were detected to ascertain whether parkin co-localizes with
intracellular A.beta..sub.1-42. Staining with anti-A.beta..sub.1-42
antibody showed plaque formation in the entorhinal cortex as well
as intracellular A.beta..sub.1-42 accumulation (FIG. 51A),
suggesting presence of both intracellular and extracellular
A.beta..sub.1-42 in AD cortex. Parkin staining was also observed
within nuclear DAPI-stained neurons in the entorhinal cortex (FIG.
51B), but parkin co-localized only with A.beta..sub.1-42 containing
neurons and not with extracellular A.beta..sub.1-42 plaques (FIG.
51C, arrows). Further analysis of the neocortex also resulted in
detection of intracellular accumulation and plaque A.beta..sub.1-42
(FIG. 51D) in AD, as well as intracellular parkin expression (FIG.
51E). Similarly, parkin and A.beta..sub.1-42 were co-localized
(FIG. 51F, arrows) intracellularly in AD cortex. An alternate
combination of antibodies was used (as above) and plaque formation
was detected in the cortex as well as intracellular
A.beta..sub.1-42 accumulation (FIG. 51G), suggesting presence of
both intracellular and extracellular A.beta..sub.1-42 in AD cortex.
Parkin staining within nuclear DAPI-stained neurons in AD cortex
was also observed (FIG. 51H), but parkin co-localized only with
A.beta..sub.1-42 containing neurons and not with extracellular
A.beta..sub.1-42 plaques (FIG. 51I, arrows).
[0184] Accumulation of parkin, A.beta. and p-Tau in autophagosomes
in AD brain. To determine whether parkin co-staining with
intracellular A.beta..sub.1-42 is associated with autophagic
activities in AD, anti-microtubule-associated light chain protein 3
(LC3) antibodies were used as probles and sub-cellular
fractionation was performed to measure the levels of amyloid
proteins in autophagic organelles using quantitative ELISA. Probing
with anti-LC3 antibody suggested a significant increase in LC3-II
compared to LC3-I (28%) levels (FIG. 52A, 1st blot & FIG. 52B),
indicating possible lipidation of LC3. The amount of LC3 compared
to actin was measured using an antibody specific for the LC3-B
isoform. A significant increase (33%, P<0.05) in the level of
LC3-B was detected in human cortical extracts from AD patients
compared to control subjects (FIG. 52A, 2nd blot & FIG.
52B).
[0185] To ascertain that sub-cellular fractionation leads to
isolation of autophagic vacuoles, WB was performed with lysosomal
marker using anti-LAMP-3 antibody that showed lysosomal fraction in
the top layer of Metrazimide gradient (FIG. 52C, top blot), and
anti-LC3B (2nd blot), which detected autophagosomes in both the 10%
(AV-10) and 20% (AV-20) gradient fractions. The levels of A.beta.
and p-Tau were measured using quantitative ELISA in these
fractions. A significant increase (89%, P<0.05) in the level of
A.beta..sub.1-42 was detected in AV-10 (FIG. 52D) and AV-20 (78%),
which are enriched in autophagosomes in AD compared to control.
Similarly, a significant increase (110%, P<0.05) in the level of
A.beta..sub.1-40 was detected in AV-10 (FIG. 52E) and AV-20 (89%)
in AD compared to control. No A.beta. was detected in the lysosomal
fraction. The levels of p-Tau were measured as another protein
marker associated with AD. No p-Tau was detected in the lysosome
but the levels of p-Tau (AT8) were significantly increased in AV-10
(81%) and AV-20 (140%) compared to control (FIG. 52F). Because
AV-20 is enriched in autophagosomes, these data show accumulation
of un-degraded proteins in autophagosomes in AD. Surprisingly, the
level of parkin was significantly increased (P<0.05) in AV-10
(64%) and AV-20 (52%) compared to control (FIG. 52G), but not in
the lysosomal fraction, suggesting that accumulated parkin
co-localizes with A.beta. and p-Tau in autophagic compartments.
[0186] Lentiviral A.beta..sub.1-42 induces p-Tau and amyloidogenic
protein and exogenous parkin reverses these effects. Because parkin
insolubility and co-localization was detected with intraneuronal
A.beta..sub.1-42 in AD brain, lentiviral gene transfer was used to
co-express A.beta..sub.1-42 and parkin in the rat cortex and the
effects of these proteins on autophagy were investigated. It was
observed that lentiviral delivery led to an increase (50% by
densitometry, N=8) of parkin expression (FIG. 53A, 1.sup.st blot)
and A.beta..sub.1-42 clearance 2 weeks post-injection of lentiviral
parkin together with A.beta..sub.1-42 (FIG. 53A, 2.sup.nd blot). No
changes in total parkin levels were observed in brains injected
with lentiviral A.beta..sub.1-42 (FIG. 53A, 1st blot), and no
phospho-parkin was detected in the insoluble 4M urea or 30% formic
acid fractions. To determine parkin levels, quantitative ELISA was
performed for human parkin in cortical lysates. Human parkin was
significantly increased in parkin (34%, N=8) or
parkin+A.beta..sub.1-42 (38%) injected animals (FIG. 53B) compared
to LacZ or A.beta..sub.1-42 alone. Independent studies were then
performed to determine the effects of parkin activity on
A.beta..sub.1-42 levels in the cortex, using human specific
A.beta..sub.1-42 ELISA. A significant increase (FIG. 53C, 7.8-fold,
N=8, P<0.05, ANOVA, Neumann Keuls with multiple comparison) in
the level of A.beta..sub.1-42 was observed 2 weeks post-injection
with lentiviral A.beta..sub.1-42 into the cortex. Co-expression of
parkin significantly decreased (6-fold) A.beta..sub.1-42 levels,
but A.beta..sub.1-42 remained significantly higher (89%) compared
to parkin or LacZ injected animals (FIG. 53E).
[0187] The effects of parkin on amyloidogenic proteins in animals
expressing A.beta..sub.1-42 were then determined. ELISA was
performed and a significant increase in rat p-Tau (AT8) was
observed in the cortex at 4 weeks but not 2 weeks post-injection
(FIG. 53D). Thioflavin-S staining was performed to examine whether
lentiviral A.beta..sub.1-42 and p-Tau accumulation lead to
formation of amyloidogenic proteins. Cortical brain sections showed
thioflavin-S positive staining in A.beta..sub.1-42-expressing
animals (FIG. 53H) compared to lentiviral LacZ injected controls
(FIG. 53E). Co-expression of parkin with A.beta..sub.1-42
eliminated thioflavin-S positive staining in the cortex 4 weeks
post-injection (FIG. 53G). These data show that parkin counteracts
A.beta..sub.1-42 induced amyloidogenic proteins.
[0188] Parkin mediates clearance of autophagic vacuoles containing
p-Tau and A.beta..sub.1-42. Whether parkin expression can mediate
the clearance of A.beta..sub.1-42-induced accumulation of
autophagic vacuoles was determined. Electron microscopy scanning of
cortical sections showed no accumulation of Insoluble parkin
co-localizes with intracellular A.beta. vacuoles in the cytosol of
lentiviral LacZ (FIG. 54A) or lentiviral parkin (FIG. 54B) injected
animals. Lentiviral expression of A.beta..sub.1-42 led to cytosolic
accumulation of autophagic vacuoles (FIG. 54C, arrows), suggesting
induction of autophagy 2-week post-injection with lentiviral
A.beta..sub.1-42. Co-expression of lentiviral parkin with
lentiviral A.beta..sub.1-42 led to formation of double membrane
vacuoles containing debris (FIG. 54D). These data suggest that
parkin leads to autophagic clearance of lentiviral
A.beta..sub.1-42-induced vacuoles. Sub-cellular fractionation was
performed and levels of A.beta..sub.1-42 and p-Tau were measured
using quantitative ELISA in these fractions. A significant increase
(61%, P<0.05, N=5) in the level of A.beta..sub.1-42 was detected
in AV-10 (FIG. 54E) compared to LacZ, parkin or
parkin+A.beta..sub.1-42 injected animals, indicating that
A.beta..sub.1-42 alters normal autophagy, leading to accumulation
of autophagosomes. However, co-expression of parkin led to
clearance of A.beta..sub.1-42 from AV-10 and significantly
increased A.beta..sub.1-42 levels in AV-20 (42%) and lysosomes
(35%) compared to LacZ and parkin alone (FIG. 54E). Because
A.beta..sub.1-42 expression induced p-Tau at 4 weeks
post-injection, levels of p-Tau (AT8) were also measured. Animals
injected with A.beta..sub.1-42 had a significant increase (31%) in
p-Tau levels in AV-10 compared to LacZ, parkin and
parkin-A.beta..sub.1-42 (FIG. 54F). However,
parkin+A.beta..sub.1-42 expression led to clearance from AV-10 and
significantly increased p-Tau levels in AV-20 (18%) and lysosomes
(20%).
[0189] Parkin regulates autophagosome clearance in
A.beta..sub.1-42-expressing animals. To determine the mechanisms by
which parkin can mediate clearance of autophagic vacuoles,
molecular markers of the autophagic pathway, leading to
autophagosomal clearance were examined. WB analysis showed no
difference in beclin levels in animals injected with lentiviral
LacZ, parkin or A.beta..sub.1-42 (FIG. 55A). However, a significant
increase in beclin levels (48% by densitometry, N=8, P<0.05)
were detected when parkin was co-expressed with A.beta..sub.1-42,
suggesting that parkin responds to A.beta..sub.1-42-induced stress.
The levels of autophagy-related genes (Atgs) including Atg7 and
Atg12 were also increased by 34% and 29%, respectively, in
parkin+A.beta..sub.1-42 injected animals (FIG. 55A) compared to
animals injected with LacZ, parkin or A.beta..sub.1-42 alone. Other
markers of the autophagic cascade LC3 were examined. No changes in
LC3-B levels were detected in animals injected with lentiviral LacZ
or parkin alone (FIG. 55B). Lentiviral A.beta..sub.1-42 expression
lead to a significant increase (32%, N=8, P<0.05) in LC3-B
levels, but parkin co-expression reversed the increase in LC3-B
(FIG. 55B). A significant increase in histone deacetylase 6 (HADC6)
levels (44%) were observed in animals injected with lentiviral
parkin+A.beta..sub.1-42 (FIG. 55B) compared to all other
conditions. These data suggest that parkin responds to
A.beta..sub.1-42 stress via up-regulation of molecular markers of
autophagy.
[0190] The EM and WB data was supplemented with
immunohistochemistry to evaluate the appearance of LC3-B staining.
Staining with anti-LC3-B antibody showed no reactivity in the
cortex of animals injected with LacZ (FIG. 55C) or parkin (FIG.
55D). However, lentiviral injection of A.beta..sub.1-42 increased
LC3-B staining (FIG. 55E), in agreement with WB data. Co-injection
of lentiviral parkin with A.beta..sub.1-42 (FIG. 55F) led to
disappearance of LC3-B staining. Stereological counting of LC3-B
positive cells revealed a significant increase (FIG. 55G. 52%,
P<0.05, N=8) in cortices co-injected with A.beta..sub.1-42
compared to other treatments, indicating that parkin activity
regulates autophagosome clearance in A.beta..sub.1-42 expressing
animals. To further determine whether parkin leads to clearance of
ubiquitinated proteins via autophagy anti-P62 antibody was used as
a probe. The levels of P62 were significantly (P<0.05, N=8)
increased when A.beta..sub.1-42 (21% by densitometry relative to
actin) was expressed compared to LacZ (FIG. 55F). However, parkin
co-expression led to complete disappearance of P62 staining,
suggesting autophagic degradation of ubiquitinated proteins.
[0191] These studies provide the first evidence of parkin
inactivity and decreased solubility in AD. The present data show
that parkin is inactivated and accumulates with A.beta..sub.1-42
and p-Tau in autophagosomes in AD. This novel finding shows that
decreased parkin E3 ubiquitin ligase activity can result in lack of
autophagic clearance leading to accumulation of the autophagic
vacuoles observed in AD brains. The gene transfer animal studies
provide evidence that lentiviral A.beta..sub.1-42 could inhibit
autophagosome maturation similar to AD. In conclusion, these data
demonstrate an association between parkin inactivation and
co-localization with intraneuronal A.beta..sub.1-42 with autophagic
dysfunction, indicating a beneficial role for parkin in autophagic
clearance. Parkin inactivation could lead to decreased autophagic
clearance and accumulation of un-degraded amyloidogenic proteins in
autophagosomes. Lentiviral expression of A.beta..sub.1-42 leads to
p-Tau and accumulation of autophagic vacuoles via inhibition of
autophagosome maturation and/or impairment of transport of
autophagic organelles. Parkin E3 ubiquitin ligase activity enhances
autophagic flux and amyloid clearance, possibly through increased
autophagosome maturation and recognition with lysosomes. Parkin's
role in autophagic clearance could be exploited as a therapeutic
strategy in neurodegenerative diseases.
TABLE-US-00003 TABLE 3 Summary and clinical diagnoses of AD
patients and control subjects used for biochemistry studies. BRC #
FDX Age Sex Race PMD FR Area 399 Control 79 F W 24 Motor 417
Control 80 F W 6 Motor 487 Control 73 M W 22 Motor 515 Control 62 M
W 19 Motor 705 Control 73 M W 9 Motor 1277 Control 80 F W 8 Motor
2052 Control 79 M W 16 Motor 1390 AD 75 M 12 Motor 1336 AD 82 M W
10 Motor 1652 AD 85 M W 18 Motor 1657 AD 82 M W 15 Motor 1697
AD/Infarcts 86 M 6 Motor 1671 AD 77 M W Motor 1801 AD 75 M 15 Motor
1870 AD 85 M W 3.5 Motor 1997 AD 85 M W 5.5 Motor 2070 AD 82 M W 19
Motor 2076 AD 84 F W 16 Motor 2078 AD, cerebrovas. 80 M 16 Motor
dis (NC)
TABLE-US-00004 TABLE 4 Summary and clinical diagnoses of AD
patients and control subjects used for immuno-histochemistry
studies. BRC # FDX CERAD BRAAK Age Sex Race PMD FX 1062 Control 58
M B 14 Hippocampus, MB 1252 Control 70 M W Hippocampus, MB 1277
Control 0 80 F W 8 Caudate, hippocampus, MB 1352 Control 78 F 14
Caudate, hippocampus, MB 1615 Control 72 M W 20 Caudate 1683
Control 1 91 F W 8 Caudate 1855 Control 77 M W Caudate,
hippocampus, MB 2201 Control 0 2 85 F W 27 Caudate, hippocampus, MB
1774 AD C 6 87 F W 17.5 Hippocampus 1778 AD C 6 80 F W 6.5
Hippocampus, MB 1782 AD 86 M W 19.5 Hippocampus, MB 1788 AD C 62 F
W 36.5 Hippocampus 1833 AD 79 F W 4.5 Entorhinal/Hippo 1851 AD 86 F
Entorhinal/Hippo 1854 AD C 6 89 M W 9.5 Hippocampus 1861 AD 85 M W
29 Hippocampus 2291 AD B 4 77 M W 8 Neocrotex
Example 4
[0192] Parkinson's disease is a movement disorder characterized by
death of dopaminergic (DA) substantia nigra (SN) neurons and brain
accumulation of .alpha.-Synuclein. The tyrosine kinase c-Abl is
activated in neurodegeneration. Lentiviral expression of
.alpha.-Synuclein leads to c-Abl activation (phosphorylation) and
c-Abl expression increases .alpha.-Synuclein levels in mouse SN, in
agreement with c-Abl activation in PD brains. Lentiviral
.alpha.-Synuclein induces accumulation of autophagosomes, and
boosting autophagy with the c-Abl inhibitor Nilotinib increases
autophagic clearance. Nilotinib is used for adult leukemia
treatment and it enters the brain within FDA approved doses,
leading to autophagic degradation of .alpha.-Synuclein and
limitation of cell death, including SN neurons. Nilotinib enhances
motor behavior in lentiviral PD models, increases DA levels and
induces hyper-activity in transgenic A53T mice. These data show
that Nilotinib can be a therapeutic strategy to degrade
.alpha.-Synuclein in PD and other Synucleinopathies.
[0193] Stereotaxic injection. Six months old C57BL/6 mice were
stereotaxically injected with 1.times.104 m.o.i lentiviral c-Abl,
.alpha.-Synuclein (or LacZ control) bilaterally into the SN using
co-ordinates: lateral: 1.5 mm, ventral: 4.1 mm and horizontal:
-3.64. Viral stocks were injected through a microsyringe pump
controller (Micro4) using total pump (World Precision Instruments,
Inc.) delivery of 2 .mu.l at a rate of 0.2 .mu.l/min as previously
described (54-56). All animal experiments will be conducted in full
compliance with the recommendations of Georgetown University Animal
Care and Use Committee (GUAUC).
[0194] Nilotinib treatment. Three weeks post-injection with the
lentivirus, half the animals were IP treated daily with 10 mg/Kg
Nilotinib dissolved in DMSO and the other half received DMSO
treatments (3 .mu.l total) for an additional 3 weeks. Half of 6-8
months old A53T transgenic mice were IP treated daily with 10 mg/Kg
Nilotinib and the other half DMSO.
[0195] WB analysis. The nigrostriatal region was isolated from
.alpha.-Synuclein or c-Abl expressing mice and compared with LacZ
or total brain extracts from A53T mice. Tissues were homogenized in
1.times.STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA,
0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease cocktail inhibitor),
centrifuged at 10,000.times.g for 20 min at 40 C and the
supernatant containing the soluble protein fraction was collected.
The supernatant was analyzed by WB on SDS NuPAGE Bis-Tris gel
(Invitrogen). .alpha.-Synuclein was probed with (1:1000) mouse
anti-.alpha.-Synuclein antibody (BD Transduction Laboratories, USA)
or (1:1500) human antibodies (ThermoScientific). Total c-Abl was
probed with (1:500) rabbit polyclonal antibody (Thermo Fisher) and
p-c-Abl (Tyr-214) with (1:500) rabbit polyclonal antibody
(Millipore). .beta.-actin was probed (1:1000) with polyclonal
antibody (Cell Signaling Technology, Beverly, Mass., USA).
Autophagy antibodies, including beclin-1 (1:1000), Atg5 (1:1000),
Atg12 (1:1000) were used to probe according to autophagy antibody
sampler kit 4445 (Cell Signaling, Inc). A rabbit polyclonal
(Pierce) anti-LC3 (1:1000) and rabbit polyclonal (Thermo
Scientific) anti-actin (1:1000) were used.). Rabbit polyclonal
(1:1000) tubulin (Thermo Scientific) was used. Map 2 was probed
(1:1000) mouse monoclonal antibody (Pierce). Rabbit polyclonal
anti-SQSTM1/p62 (Cell Signaling Technology) was used (1:500). WBs
were quantified by densitometry using Quantity One 4.6.3 software
(Bio Rad).
[0196] IHC of brain sections. Animals were deeply anesthetized with
a mixture of Xylazine and Ketamine (1:8), washed with 1.times.
saline for 1 min and then perfused with 4% paraformaldehyde (PFA)
for 15-20 min. Brains were quickly dissected out and immediately
stored in 4% PFA for 24 h at 40 C, and then transferred to 30%
sucrose at 40 C for 48 h. Tyrosine Hydroxylase (TH) was probed
(1:100) with rabbit polyclonal (AB152) antibody (Millipore) and
human .alpha.-Synuclein was probed (1:100) with mouse monoclonal
antibodies (Thermo Scientific) and DAB counterstained.
[0197] Stereological methods. These were applied by a blinded
investigator using unbiased stereology analysis (Stereologer,
Systems Planning and Analysis, Chester, Md.) to determine the total
positive cell counts in 20 cortical fields on at least 10 brain
sections (.about.400 positive cells per animal) from each
animal.
[0198] Cell culture and transfection. Human neuroblastoma M17 cells
were grown in 24 well dishes (Falcon) as previously described (57,
58). Transient transfection was performed with 3 .mu.g
.alpha.-Synuclein, or c-Abl or beclin-1 shRNA cDNA (Open
Biosystems), or 3 .mu.g LacZ cDNA for 24 hr. Cells were treated
with 10 .mu.M Nilotinib for 24 hr. Cells were harvested 48 hr after
transfection. Cells were harvested one time with STEN buffer and
centrifuged at 10,000.times.g for 20 min at 4.degree. C., and the
supernatant was collected.
[0199] Human .alpha.-Synuclein enzyme-linked immunosorbent assay
(ELISA) These were performed using 50 .mu.l (1 .mu.g/.mu.l) of
brain lysates (in STEN buffer) detected with 50 .mu.l primary
antibody (3 h) and 100 .mu.l anti-rabbit secondary antibody (30
min) at RT. .alpha.-Synuclein levels were measured using human
specific ELISA (Invitrogen) according to manufacturers'
protocols.
[0200] Caspase-3 fluorometric activity assay--To measure caspase-3
activity in the animal models, we used EnzChek.RTM. caspase-3 assay
kit #1 (Invitrogen) on cortical extracts and Z-DEVD-AMC substrate
and the absorbance was read according to manufacturer's
protocol.
[0201] ELISA Dopamine and HVA. Total brain or mesencephalon were
collected and fresh 50 .mu.l (1 .mu.g/.mu.l) brain lysates (in STEN
buffer) were detected with 50 .mu.l primary antibody (1 h) and 100
.mu.l anti-rabbit secondary antibody (30 min) at RT according to
manufacturer's protocols (Abnova, Cat# BOLD01090J00011) for DA and
(Eagle Biosciences, Cat# HVA34-K01) for HVA.
[0202] Transmission EM. Brain tissues are fixed in (1:4, v:v) 4%
PFA-picric acid solution and 25% glutaraldehyde overnight, then
washed 3.times. in 0.1 M cacodylate buffer and osmicated in 1%
osmium tetroxide/1.5% potassium ferrocyanide for 3 h, followed by
another 3.times. wash in distilled water. Samples will be treated
with 1% uranyl acetate in maleate buffer for 1 h, washed 3.times.
in maleate buffer (pH 5.2), then exposed to a graded cold ethanol
series up to 100% and ending with a propylene oxide treatment.
Samples are embedded in pure plastic and incubated at 60.degree. C.
for 1-2 days. Blocks are sectioned on a Leica ultracut microtome at
95 nm, picked up onto 100 nm formvar-coated copper grids and
analyzed using a Philips Technai Spirit transmission EM. For
immuno-EM studies, sections with be incubated overnight with the
primary antibodies and Gold impregnated for EM analysis.
[0203] MALDI-TOF Mass Spec. Brain extracts are freeze dried (in
DMSO) and re-suspended in acetonitrile. Nilotinib quantification
will carried out on a 4800 MALDI-TOF-TOF Analyzer (Applied
Biosystems, CA, USA) in reflector-positive mode and then validated
in MS/MS mode as previously described (54, 59). Detected fragment
masses will be identified in SWISS-PROT databases using MASCOT.
[0204] Rotarod tests. Mice were placed on an accelerating rotarod
(Columbus Instruments) equipped with individual timers for each
mouse. Mice were trained to stay on the rod at a constant 5 rpm
rotation for at least 2 minutes, then the speed was gradually
increased by 0.2 rpm/min and the latency to fall was measured. All
values were converted to % control.
[0205] c-Abl activation is associated with accumulation of
.alpha.-Synuclein. To examine the relationship between c-Abl and
.alpha.-Synuclein, male C57BL/6 mice were stereotaxically injected
with 1.times.10.sup.4 m.o.i lentiviral c-Abl, or .alpha.-Synuclein
(or LacZ) bilaterally into the SN. Lentiviral .alpha.-Synuclein
expression for 6 weeks (FIG. 56A, 1st blot, 42% over LacZ level,
N=9) led to an increase in total c-Abl (110%) and tyrosine 412
(T412) phosphorylated (82%) c-Abl (FIG. 56A, p<0.05, N=9)
compared to actin, indicating c-Abl activation. Human post-mortem
PD striatal extracts also showed an increase in total (220%) and
T412 (150%) c-Abl (FIG. 56B&C, N=9) compared to control
subjects (N=7, p<0.02, two-tailed t-test). Conversely,
lentiviral expression of c-Abl in the mouse SN for 6 weeks led to
an increase (132%) in total c-Abl (FIG. 56D, p<0.05, N=9) and
T412 phosphorylation (71%) compared to actin and resulted in
increased levels of monomeric (51%) and high molecular weight (30%)
.alpha.-Synuclein, further confirming the relationship between
c-Abl and .alpha.-Synuclein accumulation.
[0206] Nilotinib is a second-generation c-Abl tyrosine kinase
inhibitor (TKI) formerly known as AMN107 (35-37). Mass spectroscopy
analysis revealed that intraperotenial (IP) injection of 10 or 20
mg/kg Nilotinib into wild type mice (N=5/time point), led to
detection of up to 30 ng Nilotinib per mg brain tissue 3-4 hr after
injection (FIG. 56E). The level of Nilotinib decreased to 3.4 ng/mg
7-8 hr post-injection, indicating that Nilotinib enters the brain
and is washed out within a few hours. Caspase-3 activity was then
evaluated as a measure of cell death 3 weeks post-injection with
lentiviral .alpha.-Synuclein followed by 3 weeks treatment with
either DMSO or Nilotinib (total 6 weeks). Daily IP injection of 10
mg/kg Nilotinib or DMSO (30 .mu.l) for 6 weeks did not result in
any difference in caspase-3 activity in LacZ injected mice (FIG.
56F, N=32), but lentiviral .alpha.-Synuclein expression increased
caspase-3 activity (FIG. 56F, 740%, p<0.05, N=14) and Nilotinib
reversed this increase to 140% of LacZ levels (p<0.05, N=14).
Similarly, daily IP injection of 10 mg/kg Nilotinib or DMSO (30
.mu.l) for 3 weeks into 7-8 months old transgenic model that
harbors the A53T mutation of .alpha.-Synuclein, showed an increase
in caspase-3 activity (FIG. 56G, 670%, 5 p<0.05, N=15) and
Nilotinib reversed this increase to 101% of wild type age-matched
controls with and without Nilotinib (N=64).
[0207] c-Ab1 inhibition via Nilotinib promotes autophagic
degradation of .alpha.-Synuclein. All animals were treated daily
with IP injection of 10 mg/kg Nilotinib or DMSO (A53T mice) for 3
weeks and lentiviral models were Nilotinib (or DMSO) treated 3
weeks post-injection with lentiviral .alpha.-Synuclein or LacZ.
Western blot (WB) showed significant decrease in total c-Abl (78%)
and T412 phosphorylated (52%) c-Abl compared to tubulin in
mesencephalon neurons in lentiviral .alpha.-Synuclein mice treated
daily with 10 mg/Kg Nilotinib compared to DMSO. Human
.alpha.-Synuclein levels increased to 202 ng/ml in lentiviral
.alpha.-Synuclein mice treated with DMSO, and Nilotinib reversed
this increase to 31 ng/ml compared to LacZ with and without
Nilotinib. Nilotinib treatment resulted in decreased levels of
monomeric (42%) and high molecular weight .alpha.-Synuclein
compared to actin level. An increase in several molecular markers
of autophagy including beclin-1 (62%), Atg-5 (43%) and Atg-12 (58%)
were observed compared to actin. Further analysis of autophagic
markers showed significant decreases in P62 (69%) and LC3-II
compared to both LC3-I (39%) and MAP-2 (41%) with Nilotinib
treatment, suggesting autophagic clearance of .alpha.-Synuclein.
Similarly, daily IP injection of Nilotinib for 3 weeks into 7-8
months A53T mice, which do not express .alpha.-Synuclein in the SN,
showed significant decrease in total c-Abl (64%) and T412
phosphorylated (70%) c-Abl compared to MAP-2 in total brain
extracts compared to DMSO treated mice. An increase in the level of
total (109%) and T412 (76%) c-Abl were observed in A53T mice
treated with DMSO compared to age-matched controls. A significant
increase in LC3-II level was observed in A53T+DMSO mice compared to
control and LC3-II completely disappeared in A53T mice treated with
Nilotinib, suggesting autophagic clearance. Human .alpha.-Synuclein
levels were increased to 785 ng/ml in A53T mice treated with DMSO,
and Nilotinib reversed this increase to 467 ng/ml compared to
control. Nilotinib treatment resulted in decreased levels of
monomeric (41%) and high molecular weight human .alpha.-Synuclein
compared to actin level. No differences in beclin-1 and Atg5 levels
were observed between A53T+DMSO mice and wild type control, but an
increase in Atg12 (24%) was noted compared to actin. However,
Nilotinib increased beclin-1 (69%) and Atg-5 (29%) compared to DMSO
treatment in A53T mice.
[0208] To further determine whether autophagy mediates
.alpha.-Synuclein clearance, human M17 neuroblastoma cells were
transfected with 3 .mu.g lacZ, .alpha.-Synuclein or shRNA beclin-1
for 24 hr and then treated with 10 .mu.M Nilotinib for additional
24 hr. An increase in .alpha.-Synuclein (264 ng/ml) was observed in
.alpha.-Synuclein transfected cells compared to LacZ (FIG. 57H,
p<0.05, N=12) treated with 1 .mu.l DMSO. Nilotinib reversed
.alpha.-Synuclein to 35 ng/ml (p<0.05) but blocking beclin-1
expression with shRNA attenuated Nilotinib clearance of
.alpha.-Synuclein (150 ng/ml) compared to DMSO (251 ng/ml),
suggesting autophagic involvement in .alpha.-Synuclein
clearance.
[0209] Nilotinib clears .alpha.-Synuclein and protects SN Tyrosine
hydroxylase (TH) neurons. Immunohistochemical staining of 20 .mu.m
thick brain sections showed human .alpha.-Synuclein expression in
mice injected with lentiviral .alpha.-Synuclein into the SN and
treated with DMSO (FIG. 57B) compared to LacZ+Nilotinib (or DMSO)
mice (FIG. 57A, N=12) and Nilotinib led to 84% (by stereology)
decrease of human .alpha.-Synuclein (FIG. 57C, p<0.05, N=12) in
SN neurons. A significant decrease in TH+ neurons (89% by
stereology) was observed in lentiviral .alpha.-Synuclein+DMSO (FIG.
57E&H) compared to LacZ+Nilotinib (FIG. 57D&G) mice, and
Nilotinib treatment of .alpha.-Synuclein expressing mice reversed
TH+ neuron loss back to 82% (FIG. 57F&I, by stereology) of LacZ
level (p<0.05, N=12). Stereological counting showed a similar
decrease (72%) of Nissl counter-stained TH+ cells in
.alpha.-Synuclein+DMSO (FIG. 57K) compared to LacZ (FIG. 57J) and
64% of .alpha.-Synuclein+Nilotinib (FIG. 57L, p<0.05, N=12),
suggesting that .alpha.-Synuclein causes cell death and not
down-regulation of TH. Transmission electron microscopy of SN
neurons showed accumulation of cytosolic debris (FIG. 58A) and
autophagic vacuoles (AV) in Lentiviral .alpha.-Synuclein expressing
mice with DMSO treatment. Accumulation of cytosolic AVs containing
debris was observed in these animals (FIG. 58C&E), suggesting
autophagosome accumulation, consistent with increased LC3-II by WB.
Nilotinib treatment led to appearance of larger AVs that seemed to
be derived from fusion of multiple autophagic compartments (FIG.
58B, D &F).
[0210] Nilotinib attenuates .alpha.-Synuclein levels in A53T mice.
Immunohistochemical staining of 20 .mu.m 7 thick brain sections
showed abundant expression of human .alpha.-Synuclein in the
striatum of 6-8 months old transgenic A53T mice treated with DMSO
(FIG. 59A), brainstem (FIG. 59B), cortex (FIG. 59C) and Hippocampus
(FIG. 59D). No .alpha.-Synuclein staining was detected in SN of
A53T mice. Daily IP injection of Nilotinib for 3 weeks led to
striatal decrease (72%) of human .alpha.-Synuclein (FIG. 59E),
completely eliminated .alpha.-Synuclein from brainstem (FIG. 59F),
and reduced cortical (FIG. 59G, 71%) and hippocampal (FIG. 59H,
81%) .alpha.-Synuclein (p<0.05, N=7) in transgenic A53T
mice.
[0211] Nilotinib increases DA level and improves motor performance.
To evaluate .alpha.-Synuclein and Nilotinib effects on DA
metabolism, DA and its metabolite Homovanilic acid (HVA) were
measured using ELISA. A significant decrease (p<0.05, N=8) in DA
(62%) and HVA (36%) were observed in brain mesencephalon extracts
of lentiviral .alpha.-Synuclein+DMSO compared to LacZ mice with and
without Nilotinib. However, Nilotinib injection significantly
(P<0.05, N=8) reversed DA and HVA loss back to control lacZ
levels Lentiviral .alpha.-Synuclein expression in SN decreased
rotarod motor performance to 39% of LacZ controls with and without
Nilotinib, but Nilotinib treatment of .alpha.-Synuclein mice
reversed motor performance to 86% of LacZ level, suggesting that
reversal of DA levels leads to improved motor performance. No loss
of DA or HVA were observed in transgenic A53T mice treated with
DMSO compared to age-matched control with and without Nilotinib,
but Nilotinib dramatically increased both DA (174%) and HVA (50%)
levels in A53T mice. No noticeable differences of rotarod
performance were observed between 6-8 months old A53T mice treated
with DMSO and wild type controls. However, Nilotinib increased
rotarod motor performance (45%) above control levels, suggesting
hyperactivity in A53T mice.
Example 5
[0212] The tyrosine kinase c-Abl is activated in neurodegenerative
disorders, including Alzheimer's disease (AD). Nilotinib is a c-Abl
inhibitor approved by the U.S. Food and Drug Administration (FDA)
for treatment of adult leukemia. These studies show that
Nilotinib-mediated parkin activation stimulated the autophagic
clearance pathway, leading to amyloid degradation and cognitive
improvement in a parkin-dependent manner. Nilotinib failed to clear
autophagic vacuoles and amyloid proteins in parkin-/- mice, despite
an increase in beclin-1 levels, whereas beclin-1 knockdown
attenuated A.beta. clearance, underscoring an indispensable role
for endogenous parkin in autophagy. These data showed that
Nilotinib-mediated c-Abl inhibition is a therapeutic strategy to
rescue cells from intraneuronal amyloid toxicity and prevent both
plaque deposition and progression from mild cognitive impairment to
AD.
[0213] Human postmortem brain tissues. Human postmortem samples
were obtained from John's Hopkins University brain bank. Patients'
description and sample preparation are summarized in Example 1.
Data were analyzed as mean.+-.Standard deviation, using Two-tailed
t-test (P<0.05).
[0214] Stereotaxic injection. Lentiviral constructs encoding LacZ,
or A.beta..sub.1-42 were stereotaxically injected 1.times.106
multiplicity of infection (m.o.i) bilaterally into the CA1
hippocampus of 1 year old C57BL/6 or parkin-/-. A Total of 6W
lentiviral stocks were delivered at a rate of 0.2 .mu.l/min and.
All procedures were approved by the Georgetown University Animal
Care and Use Committee (GUACUC).
[0215] Nilotinib treatment. Nilotinib was dissolved in DMSO and a
total volume of 30 .mu.l were intra-peroteneally (IP) injected once
a day for 3 weeks. Half the animals received DMSO and the other
half received Nilotinib in DMSO.
[0216] Western blot analysis. Brain tissues were homogenized in
1.times.STEN buffer, centrifuged at 10,000.times.g for 20 min at 40
C, and the supernatants containing the soluble fraction of proteins
were collected. The pellet was re-suspended in either 4M urea or
30% formic acid and adjusted to pH 7 with 1N NaOH and centrifuged
at 10,000.times.g for 20 min at 4.degree. C., and the supernatant
containing the insoluble fraction was collected. Total parkin was
immunoprobed (1:1000) with PRK8 antibody. Rabbit polyclonal
antibodies anti-beclin-1 (1:1000), autophagy like gene (Atg)-5
(1:1000), Atg12 (1:1000) and LC3-B (1:1000) were used to probe
autophagy proteins using antibody sampler kit 4445 (Cell Signaling,
Inc). A rabbit polyclonal (Pierce) anti-LC3 (1:1000) and rabbit
polyclonal (Thermo Scientific) anti-actin (1:1000) were used.
Rabbit polyclonal (1:1000) tubulin (Thermo Scientific) and mouse
monoclonal (1:500) anti-ubiquitin (Santa Cruz Biotechnology) were
used. Map 2 was probed (1:1000) mouse monoclonal antibody
(Pierce).
[0217] Immunohistochemistry. Immunohistochemistry was performed on
20 micron-thick 4% paraformaldehyde (PFA) fixed cortical brain
sections. A.beta..sub.1-42 was probed (1:200) with rabbit
polyclonal specific anti-A.beta..sub.1-42 antibody (Zymed) that
recognizes a.a.1-42, and (1:200) mouse monoclonal antibody (4G8)
that recognizes amino acid 17-24 (Covance) and counterstained with
DAPI. Parkin was immunoprobed (1:200) with mouse anti-parkin (PRK8)
antibody that recognizes amino acid 399-465 (Signet Labs, Dedham,
Mass.) and rabbit polyclonal (1:200) anti-parkin (AB5112) antibody
that recognizes amino acid 305-622 (Millipore) and counterstained
with DAPI. Mouse monoclonal (6E10) antibody (1:100) with DAB were
used (Covance) and thioflavin-S was performed according to
manufacturer's instructions (Sigma).
[0218] Stereological methods. Stereological methods were applied by
a blinded investigator using unbiased stereology analysis
(Stereologer, Systems Planning and Analysis, Chester, Md.) as
described in (20,36).
[0219] ELISA. A.beta. and p-Tau enzyme-linked immunosorbent assay
(ELISA) using specific p-Tau, A.beta.1-40 and A.beta..sub.1-42
ELISA and caspase-3 activity were performed according to
manufacturer's protocol.
[0220] Transmission Electron Microscopy. Brain tissue were fixed in
(1:4, v:v) 4% paraformaldehyde-picric acid solution and 25%
glutaraldehyde and analyzed by a blind investigator as described in
(20,36).
[0221] Cell culture and transfection. Human neuroblastoma M17 or
rat B35 cells were grown in 24 well dishes (Falcon). Transient
transfection was performed with 3 .mu.g A.beta..sub.1-42 cDNA, or 3
.mu.g LacZ cDNA for 24 hr. Cells were treated with 10 .mu.M
Nilotinib for 24 hr. Cells were harvested 48 hr after transfection.
Cells were harvested one time with STEN buffer and centrifuged at
10,000.times.g for 20 min at 4.degree. C., and the supernatant was
collected.
[0222] Parkin ELISA. ELISA was performed on brain soluble brain
lysates (in STEN buffer) or insoluble brain lysates (4M urea) using
mouse specific parkin kit (MYBioSource) in 50 .mu.l (1 .mu.g/.mu.l)
of brain lysates detected with 50 .mu.l primary parkin antibody (3
h) and 100 .mu.l anti-rabbit antibody (30 min) at RT. Extracts were
incubated with stabilized Chromogen for 30 minutes at RT and
solution was stopped and read at 450 nm, according to
manufacturer's protocol.
[0223] Parkin E3 ubiquitin ligase activity. To determine the
activity of parkin E3 ligase activity, E3LITE Customizable
Ubiquitin Ligase Kit (Life Sensors, UC#101), which measures the
mechanisms of E1-E2-E3 activity in the presence of different
ubiquitin chains was used. To measure parkin activity in the
presence or absence of substrates, parkin was immunoprecipitted
(1:100) with PRK8 antibodies. UbcH7 was used as an E2 that provides
maximum activity with parkin E3 ligase and added E1 and E2 in the
presence of recombinant ubiquitin, including wild type or no lysine
mutant (1(0), or K48 or K63 to determine the lysine-linked type of
ubiquitin. E3 was added as IP parkin to an ELISA microplate that
captures poly-ubiquitin chains formed in the E3-dependent reaction,
which was initiated with ATP at room temperature for 60 minutes.
Controls included, E1-E2-E3 and a poly-ubiquitin chain control in
addition to E1, E2 and A.beta..sub.1-42 without parkin and assay
buffer for background reading. The plates were washed 3 times and
incubated with streptavidin-HRP for 5 minutes and were read on a
chemiluminecense plate reader.
[0224] 20S proteasome activity assay. Brain extracts 100 .mu.g were
incubated with 250 .mu.M of the fluorescent 20S proteasome specific
substrate Succinyl-LLVY-AMC at 37.degree. C. for 2 h. The medium
was discarded and proteasome activity was measured in tissue
homogenates.
[0225] Morris water maze. All animals were pre-trained (trials) to
swim for 90 seconds in a water maze containing a platform submerged
in water (invisible) for 4 consecutive days once a day. The
pretraining trials "teach" the swimming animals that to "escape",
they must find the hidden platform, and stay on it. The water maze
"test" was performed on day 5, (40), when the platform was removed
and mice have to swin and find it, thus assessing acquisition and
retention. All parameters, including distance travelled to reach
platform, speed to get to the platform, latency or time spent on
platform, and platform entry were digitally recorded on a computer
and analyzed by a blind investigator.
[0226] Novel Object Recognition (NOR). Mice were placed
individually in a 22.times.32.times.30 cm testing chamber for a 5
min habituation interval and returned to their home cages. Thirty
minutes later mice were placed in the testing chamber for 10 min
with two identical objects (acquisition session), then returned to
their home cages and 90 later placed back in the testing chamber in
the presence with one of the original objects and one novel object
of the same size but of a different color and shape (recognition
session). Sessions were video recorded. Time spent exploring the
objects were scored by blind observer. The recognition index was
calculated as (time exploring one of the objects/time exploring
both objects).times.100 for acquisition session, and (time
exploring new object/time exploring both familiar and novel
objects).times.100 for the recognition session. Statistical
calculations to estimate differences between sessions were
performed with a pairwise t-test.
[0227] Nilotinib activates parkin and induces autophagic clearance
in a beclin-1-dependent manner. To test Nilotinib effects on
autophagic mechanisms, human M17 or rat B35 neuroblastoma cells
were transfected with 3 .mu.g of human cDNA A.beta..sub.1-42 (or
LacZ) for 24 hr, and then treated these cells with several
concentrations (1 nM, 100 nM, 1 .mu.M and 10 .mu.M) of Nilotinib
for 24 hr. No cell death (by MTT,
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
detected in LacZ cells treated with DMSO or Nilotinib (FIG. 60A).
Cells expressing A.beta..sub.1-42 had a significant level of cell
death (62%, p<0.05, N=12) that was reversed to 83% of control
level by Nilotinib (FIG. 60A, N=12), suggesting protective effects
for Nilotinib against A.beta..sub.1-42 toxicity. Parkin levels were
measured via ELISA using parkin-/- brain extracts as a specificity
control (FIG. 66A). A non-significant increase (17%) in parkin was
observed with 10 .mu.M Nilotinib but a significant increase (24%,
FIG. 78A, N=12, p<0.05) was reached in extracts treated with
A.beta..sub.1-42+Nilotinib, suggesting that parkin increases in
response to A.beta..sub.1-42 stress. To determine whether parkin
increase is associated with proteasome activity, the
Chymotrypsin-like assay was used with the 20S proteasome inhibitor
lactacystin as a specificity control (FIG. 60B). Nilotinib did not
affect proteasome activity in LacZ cells (FIG. 60B). Proteasome
activity was increased (43%, p<0.05, N=12) in A.beta..sub.1-42
cells, an effect that was reversed by treatment with Nilotinib.
Soluble (STEN buffer), insoluble (30% formic acid) and cell culture
medium levels of A.beta..sub.1-42 were measured after Nilotinib
treatment. The level of secreted A.beta..sub.1-42 was (6-fold)
higher than LacZ cells; and Nilotinib decreased this by 24% (FIG.
60C, p<0.05, N=12). Nilotinib completely reversed the 2-fold
increase in soluble and 3.5-fold increase in insoluble
A.beta..sub.1-42.
[0228] Lentiviral parkin injected into AD mice increases beclin-1
levels and autophagic clearance of AD. Blocking beclin-1 expression
using shRNA (FIG. 60D, top blot) resulted in a significant increase
(FIG. 60D, 28%, N=12) in Nilotinib-induced parkin. A.beta..sub.1-42
levels were unaffected in the media with Nilotinib treatment
compared to A.beta..sub.1-42 expressing cells, and were
significantly higher than A.beta..sub.1-42+Nilotinib (FIG. 60C).
Soluble and insoluble A.beta..sub.1-42 were partially (42% and 21%,
respectively) decreased compared to A.beta..sub.1-42 transfected
cells, but remained 2-fold higher compared to
A.beta..sub.1-42+Nilotinib (FIG. 60C, p<0.05, N=12), indicating
that beclin-1 is required for complete A.beta..sub.1-42 clearance.
Secreted A.beta..sub.1-42 (media) may have accumulated in the first
24 hr after transfection, prior to Nilotinib treatment. To verify
whether autophagy is involved in A.beta..sub.1-42 clearance, LC3
(Light Chain Protein-3) levels (FIG. 60D) were examined and LC3-II
with expression of A.beta..sub.1-42 alone, or with shRNA
beclin-1+Nilotinib (N=12) was detected, indicating autophagosome
formation, but LC3-II completely disappeared with Nilotinib,
suggesting autophagic clearance (FIG. 60D). To determine Nilotinib
effects on parkin function, parkin (E3) was immunoprecipitated and
E1, E2 (UbcH7) was added and either wild type ubiquitin containing
all seven lysines or the no-lysine mutant ubiquitin (KO). Nilotinib
(24 hr) significantly increased parkin self poly-ubiquitination
compared to DMSO and specificity controls (FIG. 60E, 170%, N=5,
P<0.05), including recombinant E1-E2-E3 (positive) or KO
(negative) (FIG. 60E), suggesting that Nilotinib increases parkin
E3 ubiquitin ligase activity.
[0229] Nilotinib crosses the blood brain barrier. To determine
whether Nilotinib crosses the blood brain barrier 2-month old
C57BL/6 mice were intraperitoneally (IP) injected with 10 mg/kg, 20
mg/kg or 50 mg/kg Nilotinib (304, in DMSO) and the animals were
sacrificed 4-6 hr after injection. Mass spectroscopy analysis of
total brain lysates showed up to 30 ng/ml Nilotinib in the brain
with 10 mg/kg. Nilotinib treatment (N=35) daily for 9 consecutive
days significantly decreased (44%) total c-Abl levels and T412
(50%), suggesting c-Abl inhibition. This treatment with 10 mg/kg
Nilotinib decreased pan-tyrosine phosphorylation, increased (29%,
p<0.05, N=35) parkin and decreased ubiquitinated protein
smear.
[0230] To determine Nilotinib effects on neuronal death,
1.times.10.sup.6 m.o.i lentiviral A.beta..sub.1-42 was
stereotaxicaly injected bilaterally into the hippocampus of 1 year
old C57BL/6 (wild type) or parkin-/- mice and 3 weeks later 10
mg/kg as injected once a day for 3 additional weeks. No differences
in caspase-3 activation were observed between DMSO and Nilotinib
treated wild type mice (FIG. 60F, N=64); however, a significant
increase (165%) in caspase-3 activation was observed in lentiviral
A.beta..sub.1-42 age-matched mice (FIG. 60F, p<0.05, N=35),
while Nilotinib significantly reversed (45% above control) the
effects of A.beta..sub.1-42. Similarly, no differences in caspase-3
activation were observed between DMSO and Nilotinib treated
parkin-/- mice (FIG. 60F, N=16), but a significant increase was
observed in lentiviral A.beta..sub.1-42 mice with (165%) or without
(180%) Nilotinib (P<0.05, N=19), suggesting that Nilotinib
depends on parkin to protect against A.beta..sub.1-42.
[0231] Nilotinib clearance of brain amyloid is associated with
parkin activation. To determine whether Nilotinib affects A.beta.
level in vivo, 8-12 months old AD transgenic mice which express
neuronally derived human APP gene, 770 isoform, containing the
Swedish K670N/M671L, Dutch E693Q and Iowa D694N mutations (Tg-APP)
under the control of the mouse thymus cell antigen 1, theta, Thy1,
promoter were treated (10 mg/kg IP injection) for 3 weeks. These
mice expressed significantly higher levels of soluble (156 ng/ml)
and insoluble (173 ng/ml) A.beta..sub.1-42 compared to 1-year old
control with and without Nilotinib (FIG. 61A, p<0.05, N=9) while
Nilotinib greatly reduced soluble A.beta..sub.1-42 (35 ng/ml, which
remained significantly higher than control) and reversed the
increase in insoluble A.beta..sub.1-42. Significant increases in
soluble (281 ng/ml) and insoluble (250 ng/ml) A.beta..sub.1-40 were
also detected in Tg-APP mice compared to 1-year old control (FIG.
61B, p<0.05, N=9), and were reversed by Nilotinib. p-Tau was
also increased at ser 396 (109 ng/ml) and AT8 (288 ng/ml) compared
to 1-year old control (FIG. 61C, p<0.05, N=9). Nilotinib
significantly reduced but did not completely reverse these
increases.
[0232] Nilotinib abrogates alteration of parkin solubility in AD
mice. To determine whether parkin level is affected in AD models,
the level of parkin was measured in Tg-APP mice in both the soluble
(STEN) and insoluble (4M urea) fractions. Brain lysates from
parkin-/- mice were used as specificity controls. No changes in
soluble or insoluble parkin were detected in control mice with and
without Nilotinib (FIG. 61D, N=9). However, Nilotinib significantly
increased the level of soluble parkin from 64 ng/ml in Tg-APP+DMSO
to 119 ng/ml (FIG. 61D, N=11, p<0.05) while it significantly
decreased insoluble parkin level from 54 ng/ml to 31 ng/ml in
Nilotinib treated mice (FIG. 61D, p<0.05, N=11). These data
suggest increased levels of insoluble parkin in Tg-APP. Western
blot revealed increased levels of total (51%) and T412 c-Abl (64%)
in Tg-APP compared to control (FIG. 61E, p<0.05, N=11), while
Nilotinib again reversed these increases (FIG. 61E, p<0.05,
N=11). c-Abl inhibition with Nilotinib reduced the level of CTFs
(44%, p<0.05, N=11) relative to MAP-2.
[0233] c-Abl activation is associated with decreased parkin level
in post-mortem AD cortex. Whether c-Abl is altered in human
post-mortem AD cortex was examined (N=12 AD and 7 control).
Significantly increased levels (90%) of total (FIG. 61F) and T412
(184%) c-Abl were detected in AD brains (FIG. 61F). The ratio of
p-cAbl over total c-Abl (FIG. 61G) was also increased (102%). In
contrast, parkin was decreased (70%) in AD cortex (FIG. 61F&G)
compared to actin. Parkin insolubility may be associated with loss
of E3 ligase function, so it was determined whether endogenous
parkin can mediate A.beta..sub.1-42 clearance. Significant
increases (p<0.05, N=12) in soluble (180 ng/ml) and insoluble
(209 ng/ml) A.beta..sub.1-42 were observed in wild type lentiviral
A.beta..sub.1-42 mice (FIG. 61H), but Nilotinib completely reversed
A.beta..sub.1-42 back to control level. Lentiviral A.beta..sub.1-42
in parkin-/- mice (FIG. 61H) significantly increased soluble (241
ng/ml) and insoluble (246 ng/ml) A.beta..sub.1-42 compared to
lentiviral A.beta..sub.1-42 in wild type mice (N=12).
Interestingly, Nilotinib failed to clear soluble (297 ng/ml) and
insoluble (274 mg/ml) A.beta..sub.1-42 in parkin-/- mice,
suggesting that endogenous parkin is required for A.beta..sub.1-42
clearance. Similarly, Nilotinib decreased p-Tau ser 396 (FIG. 61I)
in wild type mice (68 ng/ml) compared to A.beta..sub.1-42
expression (124 ng/ml) while p-Tau was increased (264 ng/ml) in
parkin-/- mice and Nilotinib did not lower p-Tau (189 ng/ml) level
(FIG. 61I, p<0.05, N=11).
[0234] Nilotinib promotes autophagic clearance of amyloid in a
parkin-dependent manner. Western blot (WB) of total brain lysates
in 1 year old wild type mice injected with lentiviral
A.beta..sub.1-42 showed a significant decrease in total (55%) and
T412 (45%) c-Abl following daily treatment with 10 mg/kg Nilotinib
for 3 weeks (FIG. 62A, p<0.05, N=9). A significant decrease
(38%) in LC3-B and disappearance of LC3-II (which indicates
autophagosome accumulation) were observed in Nilotinib compared to
DMSO treated mice (FIG. 62A, p<0.05, N=9). No changes in the
neuronal marker MAP-2 (loading control) were detected. A
significant increase in parkin level (62%) was associated with a
similar increase in beclin-1 (53%) and other molecular markers of
autophagy, including Atg5 (34%) and Atg12 (41%) relative to tubulin
(FIG. 62B, p<0.05, N=9), consistent with the hypothesis that
c-Abl inhibition may mediate autophagic clearance via increased
parkin activity. Nilotinib treatment of A.beta..sub.1-42 mice also
increased parkin, decreased autophagic markers LC3-B and LC3-II
(FIG. 62C, p<0.05, N=12), and increased beclin-1 (53%) and Atg5
(62%) compared to DMSO (p<0.05). Total Tau was unaffected in
Tg-APP mice between DMSO and Nilotinib groups (FIG. 62D, N=12). A
significant decrease in AT8 (71%), AT180 (34%) and Ser 396 (64%)
with no change in p-Tau Ser 262 compared to actin (FIG. 62D,
P<0.05) were observed in Nilotinib treated A.beta..sub.1-42
mice.
[0235] Nilotinib effects also were examined in lentiviral
A.beta..sub.1-42 treated parkin-/- and wild type mice (FIG.
62E&F). Interestingly, parkin-/- mice had significantly higher
levels of autophagic markers, including beclin-1 (FIG. 62E, 120%,
N=9) compared to control. Nilotinib did not clear LC3-II in
parkin-/- mice and no difference was observed in LC3-A between
parkin-/- and control mice (FIG. 62E). Significant increases in
Atg12 (FIG. 62F, 64%) and Atg5 (FIG. 62F, 74%) were observed in
parkin-/- compared to control and the levels of these markers also
were not changed in response to Nilotinib. These data indicate that
despite the compensatory increase in autophagic markers, Nilotinib
cannot clear autophagosomes in parkin-/- mice, further suggesting
that parkin is essential for autophagosome maturation.
[0236] Nilotinib increases parkin level and decreases plaque load
in Tg-APP mice. Staining of 20 .mu.m brain sections shows plaque
formation within various brain regions in Tg-APP mice treated with
DMSO (FIG. 63A-D representing different animals), though plaque
staining disappeared in the Nilotinib group after 3-week treatment
(FIG. 63E-H). These results were confirmed by thioflavin-S staining
(FIG. 67). Higher magnification shows endogenous parkin associated
with Tg-APP (FIG. 631) and plaque deposition (FIG. 631 &K) in
the hippocampus. Nilotinib increases endogenous parkin (FIG. 63L)
and results in plaque disappearance (FIG. 63M&N). Using
different parkin antibodies to show parkin (FIG. 630) and plaque
(FIG. 63P&Q), Nilotinib increased parkin levels (FIG. 63R) and
dissolved plaques (FIG. 63S&T). To determine whether parkin
targets intracellular A.beta. to decrease extracellular plaque
load, lentiviral injection was used to show intracellular
A.beta..sub.1-42 within the hippocampus (FIG. 63U, inset higher
magnification) and Nilotinib clearance of intracellular
A.beta..sub.1-42 (FIG. 63V, inset is higher magnification).
Lentiviral injection into the hippocampus led to intracellular
A.beta..sub.1-42 expression throughout the cortex (FIG. 63W, inset
higher magnification) and, again, Nilotinib eliminated
A.beta..sub.1-42 accumulation (FIG. 63X, inset higher
magnification). Lower magnification images show formation of
plaques in A.beta..sub.1-42 expressing mice 6 weeks post-injection
(FIG. 64A-C). Nilotinib (daily for 3 weeks) eliminates plaque
formation in A.beta..sub.1-42 wild type mice (FIG. 64D-F).
A.beta..sub.1-42 expression in parkin-/- mice showed more plaque
formation (FIG. 64G-I) and Nilotinib did not reduce plaque load in
these mice (FIG. 64J-L). Quantification of plaque size using Image
J to delineate boundaries around individual plaques (N=15-25
plaques, 2 plaques per animal) (FIG. 68A-D) showed an average
plaque size around 48 .mu.m (FIG. 68A&I, N=12) in
A.beta..sub.1-42 wild type mice, while Nilotinib reduced plaque
size to 5 .mu.m (FIG. 68B&I, p<0.05,). In contrast, plaque
size was larger in parkin-/- mice (FIG. 68C&I, 85 .mu.m, N=6),
and Nilotinib did not reduce plaque size (FIG. 68D&I, 79
.mu.m). Stereological counting of A.beta.-42 positive cells showed
significantly reduced (N=5200 cells) staining in Nilotinib treated
(FIG. 68F&J) compared to DMSO treated A.beta..sub.1-42
expressing wild type mice (FIG. 68E&J, p<0.05). However,
parkin-/- mice had significantly fewer A.beta..sub.1-42 positive
cells (FIG. 68G&J, N=14566) and Nilotinib did not alter
intracellular staining (FIG. 68H&J, N=13250), raising the
possibility that endogenous parkin can modify intracellular
A.beta..sub.1-42, leading to intraneuronal degradation, thus
limiting its secretion.
[0237] Parkin mediates K63-linked ubiquitination of
A.beta..sub.1-42. To determine whether parkin mediates any specific
poly-ubiquitin linkages of A.beta..sub.1-42 that would facilitate
its degradation, parkin was immunoprecipitated and synthetic
A.beta..sub.1-42 was used as a substrate. A cocktail of recombinant
E1-E2-E3 and poly-ubiquitin chains were used as positive controls
(FIG. 68K). No activity was detected with lysine null ubiquitin
(KO), and parkin activity was not significantly altered with K48
ubiquitin mutant. However, poly-ubiquitin signals were
significantly increased (89%) in the presence of A.beta..sub.1-42
compared to parkin alone (FIG. 68K, p<0.05, N=6) with K63
ubiquitin, suggesting that parkin promotes K63-linked
poly-ubiquitination of A.beta..sub.1-42. Poly-ubiquitin signals
were also significantly higher with wild type ubiquitin in the
presence of A.beta..sub.1-42 (43%).
[0238] Impairment of autophagic clearance in the absence of parkin.
Transmission electron microscopy revealed (N=6 animals per
treatment) autophagic defects in lentiviral A.beta..sub.1-42
expressing mice, manifested in hippocampal appearance of dystrophic
neurons (FIG. 64M), accumulation of undigested vacuoles in the
cortex (FIG. 64N) and enlargement of hippocampal lysosomes (FIG.
64O), suggesting deficits in proteolytic degradation. Nilotinib
reversed these effects in the hippocampus (FIG. 64P&R), where
no dystrophic neurons or lysosomal enlargement were detected, and
contributed to cortical clearance of vacuoles (FIG. 64Q). In
contrast, Nilotinib failed to eliminate dystrophic neurons in the
hippocampus of parkin-/- mice (FIG. 64S&V), and was unable to
clear vacuoles in cortex and hippocampus (FIG. 64T-X).
[0239] Nilotinib improves cognitive performance in a
parkin-dependent manner. The Morris water maze test was performed
after 4 days of training trials in which the platform was placed in
the SE corner and mice were initially placed in the NW corner of
the pool. A.beta..sub.1-42-injected (+DMSO) mice remained longer
(24%) in the NW quadrant compared to control (LacZ+Nilo) (FIG. 65A,
N=14), while Nilotinib reversed time (in seconds) spent in NW back
to the level observed in control mice. A.beta..sub.1-42 parkin-/-
mice (N=7) with and without Nilotinib remained significantly more
in the NW quadrant (FIG. 65A). A.beta..sub.1-42 expressing wild
type and parkin-/- spent significantly less time in SE (FIG. 65A,
47%, p<0.05) compared to control, but Nilotinib significantly
improved time spent in SE in wild type but not parkin-/- compared
to control (26%) and DMSO (61%). A heat map for each group showed
that controls learned quickly to find (SE) platform area, and
A.beta..sub.1-42 (DMSO) animals spent more time roaming, while
Nilotinib improved platform search. In contrast,
parkin-/-.+-.Nilotinib wandered aimlessly in the maze.
A.beta..sub.1-42 animals entered the SE (platform entry, clear
bars) less (FIG. 77B, 37%) than control, but Nilotinib reversed the
number of entries back to control, while parkin-/- entered
significantly less (34%, P<0.05, N=7), suggesting that Nilotinib
enhanced memory in a parkin-dependent manner. However, the distance
travelled (FIG. 77B, back bars) by A.beta..sub.1-42
parkin-/-.+-.Nilotinib was significantly decreased (80% and 75%,
respectively) compared to control and wild type (P<0.05).
[0240] These experiments were repeated in 1 year old Tg-APP mice
and age-matched control (C57BL/6). Tg-APP (+DMSO) mice remained
less (24%) in NW (FIG. 77C, 28%, N=12) and spent significantly less
time in SE (FIG. 77C, 28%, p<0.05). Nilotinib treatment (10
mg/kg daily for 3 weeks) significantly reversed time spent in SE
back to control level. A heat map for each group shows that Tg-APP
did better in finding the platform with Nilotinib (FIG. 77C), and
Tg-APP+Nilotinib entered SE (platform entry, clear bars)
significantly more times than did control mice (FIG. 77D, 30%
higher than control), while Tg-APP+DMSO did significantly worse
than control (FIG. 77D, 25%). The distance traveled (FIG. 77D,
black bars) was also significantly reduced in DMSO (86%) compared
to Nilotinib treated Tg-APP mice, which had values 30% above
control levels (FIG. 77D, P<0.05, N=12). Novel object
recognition was also tested and showed that Tg-APP+Nilotinib
performed significantly better at finding new objects (FIG. 77E,
31%, p<0.001, N=17) than DMSO mice, while A.beta..sub.1-42
parkin-/- mice did not learn with or without Nilotinib (FIG. 77E,
N=5).
Example 6
[0241] These studies shows that parkin ubiquitinates TDP-43 and
facilitates its cytosolic accumulation through a multi-protein
complex with HDAC6.
Experimental Procedures
[0242] Stereotaxic injection--Stereotaxic surgery was performed to
inject the lentiviral (Lv) constructs encoding either LacZ, parkin
and/or TDP-43 into the primary motor cortex of two-month-old male
Sprague-Dawley rats weighing between 170-220 g. Animals were
injected into left side of the motor cortex with 2.times.10.sup.9
m.o.i Lv-LacZ and into the right side with 1.times.10.sup.9 m.o.i
Lv-parkin+1.times.10.sup.9 m.o.i Lv-LacZ; or 1.times.10.sup.9 m.o.i
Lv-TDP-43+1.times.10.sup.9 m.o.i Lv-LacZ; or 1.times.10.sup.9 m.o.i
Lv-parkin+1.times.10.sup.9 m.o.i Lv-TDP-43. All animals were
sacrificed two weeks post-injection and the left cortex was
compared to the right cortex. A total of 8 animals of each
treatment (32 animals) were used for WB, ELISA and
immuno-precipitation and 8 animals of each treatment (32 animals)
for immunohistochemistry. A total N=64 animals were used.
Transgenic hemizygous mice harboring human TDP-43 with the A315T
mutation under the control of prion promoter and C57BL6/J mice
controls were used. The colony was obtained from Jackson Laboratory
Repository (JAX Stock No. 010700) and displayed a lifespan
considerably shorter than previous reports, with almost 90% of all
pups, including males and females manifesting motor symptoms around
21-30 days. Hemizygous mice were bred via mating of hemizygous with
non-carrier wild type C57BL/6, and upon genotyping, half were
identified as transgenic and the other half was non-transgenic
control. All mice used are F1 generation from direct mating between
hemizygous and C57BL/6 mice. These studies were approved and
conducted according to Georgetown University Animal Care and Use
Committee (GUACAC).
[0243] Cell culture and transfection. Human neuroblastoma M17 cells
(seeding density 2.times.10.sup.5 cells) were grown in 24 well
dishes (Falcon) to 70% confluence in Dulbecco's Modified Eagle
Medium (DMEM; Invitrogen) plus 10% (v/v) heat-inactivated fetal
bovine serum (Invitrogen), penicillin/streptomycin, and 2 mM
L-glutamine at 37.degree. C. and 5% CO2, washed twice in
phosphate-buffered saline (PBS). Transient transfection was
performed with 3 .mu.g parkin cDNA or 3 .mu.g TDP-43 cDNA, or 3
.mu.g LacZ cDNA. Cells were treated with 5 .mu.M tubacin for 24
hours and DAPI stained in 12 well dishes. Cells were harvested 24
hours after transfection. Transfection was performed in DMEM
without serum using Lipofectamine 2000 (Invitrogen) according to
the manufacturer's protocol. Cells were harvested one time with
lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM
ethylenediaminetetraacetic acid, 1 mM ethyleneglycoltetraacetic
acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
3-glycerophosphate, 1 mM sodium orthovanadate, 1 .mu.g/ml
leupeptin, and 0.1 mM PMSF) and centrifuged at 10,000.times.g for
20 min at 4.degree. C., and the supernatant was collected. Western
blot was performed on NuPAGE 4-12% Bis-Tris gel (Invitrogen).
Protein estimation was performed using the microscale BioRad
protein assay (BioRad Laboratories Inc, Hercules, Calif., USA).
[0244] Western blot analysis--The cortex was dissected out and
homogenized in 1.times.STEN buffer (50 mM Tris (pH 7.6), 150 mM
NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease
cocktail inhibitor). The pellet was then re-suspended in 4M urea
and homogenized and centrifuged at 5.000 g and the supernatant
containing the insoluble protein fraction was collected. Total
TDP-43 was probed either with (1:1000) mouse monoclonal (2E2-D3)
antibody generated against N-terminal 261 amino acids of the full
length protein (Abnova) or (1:1000) Rabbit polyclonal (ALS10)
antibody (ProteinTech, Cat#10782-2-AP). Rabbit polyclonal
anti-ubiquitin (Chemicon International) was used (1:1000), and
rabbit polyclonal anti-parkin (Millipore) antibody was used
(1:1000) for WB. Rabbit polyclonal anti-actin (Thermo Scientific)
was used (1:1000). Rabbit polyclonal anti-SQSTM1/p62 (Cell
Signaling Technology) was used (1:500). Rabbit monoclonal (1:1000)
HDAC6 (Cell Signaling Technology) was used. SIAH2 was probed
(1:400) with mouse monoclonal antibody (Novus Biologicals) and
HIF-1.alpha. with (1:1000) mouse monoclonal antibody (Novus
Biologicals). Immuno-precipitation was performed on a total of 100
mg protein with (1:100) rabbit polyclonal TDP-43 antibody
(ProteinTech), or rabbit monoclonal (1:100) parkin antibody
(Invitrogen) and then compared with the input samples. Western
blots were quantified by densitometry using Quantity One 4.6.3
software (Bio Rad). Densitometry was obtained as arbitrary numbers
measuring band intensity. Data were analyzed as mean.+-.St.Dev,
using ANOVA, with Neumann Keuls multiple comparison between
treatment groups.
[0245] Parkin enzyme-linked immunosorbent assay (ELISA)--was
performed on brain soluble brain lysates (in STEN buffer) or
insoluble brain lysates (4M urea) using mouse specific parkin kit
(MYBioSource) in 50 .mu.l (1 .mu.g/.mu.l) of brain lysates detected
with 50 .mu.l primary antibody (3 h) and 100 .mu.l anti-rabbit
antibody (30 min) at RT. Extracts were incubated with stabilized
Chromogen for 30 minutes at RT and solution was stopped and read at
450 nm, according to manufacturer's protocol.
[0246] Parkin E3 ubiquitin ligase activity. To determine the
activity of parkin E3 ligase activity E3LITE Customizable Ubiquitin
Ligase Kit (Life Sensors, UC#101), which measures the mechanisms of
E1-E2-E3 activity in the presence of different ubiquitin chain, was
used. To measure parkin activity in the presence or absence of
substrates, parkin (1:100) was immunoprecipitated with PRK8
antibodies and TDP-43 (1:100) was immunoprecipitated with human
TDP-43 (Abnova) from 100 mg TDP43-Tg brain lysates. UbcH7 was used
as an E2 that provides maximum activity with parkin E3 ligase and
added E1 and E2 in the presence of recombinant ubiquitin, including
wild type containing all seven possible surface lysines, no lysine
mutant (KO), or K48 or K63 to determine the lysine-linked type of
ubiquitin. E3 was added as IP parkin or recombinant parkin (Novus
Biologicals) to an ELISA microplate that captures polyubiquitin
chains formed in the E3-dependent reaction, which was initiated
with ATP at room temperature for 60 minutes. Also included were an
E1-E2-E3 and a polyubiquitin chain control in addition to E1, E2
and TDP-43 without parkin and assay buffer for background reading.
The plates were washed 3 times and incubated with detection reagent
and streptavidin-HRP for 5 minutes and the polyubiquitin chains
generated by E1-E2-E3 machinery were read on a chemiluminecense
plate reader.
[0247] Immunoprecipitation and ubiquitination assay. Either TDP-43
or parkin were separately immunoprecipitated in 100 .mu.l (100
.mu.g of proteins) 1.times.STEN buffer using (1:100) human specific
anti-TDP-43 monoclonal antibody (Abnova) or (1:100) anti-parkin
mouse monoclonal antibody (PRK8; Signet Labs; Dedham, Mass.),
respectively. Following immunoprecipitation, 300 ng of each
substrate protein (parkin and TDP-43) were mixed in the presence of
1 .mu.g recombinant human ubiquitin (Boston Biochem, Mass.), 100 mm
ATP, 1 .mu.g recombinant UbcH7 (Boston Biochem), 40 ng E1
recombinant enzyme (Boston Biochem) and incubated at 37.degree. C.
in an incubator for 20 min. The reaction was heat inactivated by
boiling for 5 min and the substrates were analyzed by western
blot.
[0248] Immunohistology--Immunohistochemistry was performed on 20
.mu.m-thick sections of brain or cervical spinal cord. TDP-43 was
probed (1:200) with rabbit polyclonal (ALS10) antibody
(ProteinTech, Cat#10782-2-AP). Rabbit polyclonal anti-ubiquitin
(Chemicon International) was used (1:100), and mouse monoclonal
anti-parkin (Millipore) antibody was used (1:200) for
immunohistochemistry. Toluidine blue and DAPI staining were
performed according to manufacturer's instructions (Sigma).
Counting of Toluidine blue staining of centric axons within 10
random fields of each slide was performed by a blind investigator
in N=8 animals of each treatment. All staining experiments were
scored by a blind investigator to the treatments.
[0249] 20S proteasome activity assay--Brain extracts 100 .mu.g were
incubated with 250 .mu.M of the fluorescent 20S proteasome specific
substrate Succinyl-LLVY-AMC at 37.degree. C. for 2 h. The medium
was discarded and homogenates were lysed in 50 mM HEPES, pH 7.5, 5
mM EDTA, 150 mM NaCl and 1% Triton X-100, containing 2 mM ATP. The
fluoropore 7-Amino-4-methylcoumarin (AMC), which is released after
cleavage from the labeled substrate Succinyl-LLVY-AMC (Chemicon
International, Inc.), is detected and free AMC fluorescence is
quantified using a 380/460 nm filter set in a fluorometer
(absorption at 351 nm and emission at 430 nm). Non-proteasomal side
reactivity was measured by adding lactacystin as a specific
proteasome inhibitor to the reaction mix and subtracted these
values from total for an accurate measure of specific proteasome
activity.
[0250] qRT-PCR in neuronal tissues. qRT-PCR was performed on
Real-time OCR system (Applied Biosystems) with Fast SYBR-Green PCR
master Mix (Applied Biosystems) in triplicate from reverse
transcribed cDNA from control un-injected, or lentiviral LacZ,
parkin, TDP-43 and TDP-43+parkin injected rat cortical brain
tissues. These experiments were repeated in human neuroblastoma M17
cells and A315T-Tg compared to non-transgenic C57BL/6 controls.
Human wild-type parkin forward primer CCA TGA TAG TGT TTG TCA GGT
TC and a reverse primer GTT GTA CTT TCT CTT CTG CGT AGT GT were
used. Gene expression values were normalized using GADPH
levels.
Results
[0251] TDP-43 inhibits proteasome activity and alters parkin
protein levels. To determine the effects of TDP-43 on parkin in
transgenic animals, the A315T mutant TDP-43 transgenic mice
(TDP43-Tg), which were reported to have aggregates of ubiquitinated
proteins in layer 5 pyramidal neurons in frontal cortex, as well as
spinal motor neurons, without cytoplasmic TDP-43, was used. This
model is relevant to these studies because it shows nuclear TDP-43
driven pathology, independent of cytoplasmic TDP-43 inclusions.
Western blot analysis showed accumulation of full length and TDP-43
fragments (.about.35 kDA) as well as higher molecular weight
species with human TDP-43 antibody (FIG. 69A, 1st blot) compared to
non-transgenic controls, suggesting TDP-43 pathology. Further
analysis of the soluble brain lysate (STEN extract) showed
increased parkin levels by Western blot (FIG. 69A, 2nd blot, 82%,
P<0.05, N=8) and appearance of a lower molecular weight band,
perhaps indicating parkin cleavage. Increased levels of ubiquitin
smears (FIG. 69A, 3rd blot) were also observed using anti-ubiquitin
antibodies, suggesting accumulation of ubiquitinated proteins. It
was determined whether parkin solubility was altered in TDP43-Tg
mice. The protein pellet was resuspended after STEN extraction in
4M urea to detect the insoluble fraction and we detected a
significant increase (FIG. 69B, 95% by densitometry, P<0.05,
N=8) in insoluble parkin in 1-month old TDP43-Tg mice compared to
control (FIG. 69B&C, P<0.05, N=8), suggesting that TDP-43
aggregates are associated with altered parkin solubility. The ratio
of soluble over insoluble parkin was not significantly changed
(FIG. 69C, P<0.05), suggesting that TDP-43 accumulation
increases soluble and insoluble parkin levels. Probing for TDP-43
in 4M urea extracts was also performed and increased levels of
insoluble TDP-43 (FIG. 69B, 2nd blot) were detected in TDP43-Tg
compared to control. To verify the changes in parkin level observed
by WB, quantitative parkin ELISA was performed to determine the
levels of both soluble (STEN extract) and insoluble (4M urea)
parkin, using brain extracts from parkin.sup.-/- mice as control
for ELISA specificity (FIG. 69D, N=8). A significant increase in
both soluble (46%, P<0.05) and insoluble (64%) parkin was
detected in TDP43-Tg mice compared to control level (FIG. 69D,
P<0.05, N=8), further suggesting an increase in parkin level and
insolubility in TDP43-Tg mice.
[0252] The seven in absentia homolog (SIAH) protein is another E3
ligase involved in ubiquitination and proteasomal degradation of
specific proteins. SIAH is rapidly degraded via the proteasome.
SIAH2 was used as an E3 ligase control to determine whether TDP-43
decreases parkin solubility, leading to alteration of its E3 ligase
function independent of other E3 ligases. Western blot analysis
showed a significant increase (215%) in soluble SIAH2 levels (FIG.
69E&F, P<0.05, N=8) in TDP43-Tg mice compared to control,
indicating lack of degradation of SIAH2 perhaps due to proteasomal
impairment. However, SIAH2 was not detected in the insoluble
fraction. A lower molecular weight band was also observed at 17 kDa
(FIG. 69E) in transgenic mice, suggesting possible cleavage of
SIAH2 dimeric structure. Further examination of the level of SIAH2
target molecule HIF-1.alpha. showed a significant increase (76%,
P<0.05) in protein level (FIG. 69E&F), suggesting lack of
proteasomal degradation.
[0253] To ascertain the effect of TDP-43 on parkin level and
proteasome activity wild type TDP-43, (FIG. 69G, 1st blot) was
expressed in the presence or absence of parkin (FIG. 69G, 2nd blot)
in human M17 neuroblastoma cells. Expression of TDP-43 alone led to
appearance of endogenous parkin protein (FIG. 69G, 2nd blot),
suggesting that TDP-43 regulates parkin mRNA to induce protein
expression. Co-expression of exogenous parkin and TDP-43 led to a
slight decrease in TDP-43 levels (FIG. 69G, 1st blot) and a
noticeable decrease in ubiquitinated proteins (FIG. 69G, 3rd blot)
compared to TDP-43 alone. SIAH2 was difficult to detect in control
M17 cells (FIG. 69G, 4.sup.th blot), but accumulated when TDP-43
was expressed despite the increase in endogenous parkin, however,
exogenous parkin co-expression with TDP-43 led to disappearance of
SIAH2 (FIG. 69G, 4.sup.th blot). The effects of parkin expression
alone (FIG. 69G, 2nd blot) were further compared to LacZ on TDP-43
and SIAH2 levels. No differences were observed between control
(FIG. 69F), LacZ and parkin transfected M17 cells (FIG. 69H) on
endogenous TDP-43 expression level (FIG. 69H, 1st blot). A higher
level of ubiquitinated protein smears were observed with parkin
expression (FIG. 69H, 3.sup.rd blot), consistent with parkin role
as an E3 ubiquitin ligase, but the level of SIAH2 was significantly
decreased (FIG. 69H, 4.sup.th blot, 74%, P<0.05) compared to
actin control. To determine whether SIAH2 accumulation is due to
decreased E3 ligase activity or proteasomal function, we measured
proteasome activity (FIG. 69I) and found that TDP-43 significantly
decreased (66%) proteasome activity (P<0.05, N=12), while parkin
co-expression significantly reversed proteasome activity to 74% of
control or parkin levels, but remained significantly less (26%)
than control. These data show that TDP-43 increases parkin
expression levels, while proteasomal inhibition leads to decreased
degradation of proteins, including the rapidly degrading SIAH2.
[0254] Lentiviral expression of TDP-43 in rat motor cortex results
in increased protein levels in preganglionic cervical spinal cord
inter-neurons. Wild type TDP-43 was expressed using lentiviral gene
delivery into the motor cortex of 2-month old Sprague Dawley rats.
Immunohistochemistry using rabbit polyclonal antibody that
recognizes human and rat TDP-43 (ALS10, ProteinTech) showed
increased TDP-43 protein levels and cytosolic accumulation 2 weeks
post-injection (FIG. 70B) compared to LacZ injected contralateral
(FIG. 70A) hemisphere. To ascertain specificity of gene expression,
human specific (hTDP-43) mouse monoclonal antibody that recognizes
a.a.1-261 (Abcam) was used and positive human TDP-43 staining was
observed within 4 mm radius in 38% (by stereology, N=8) of cortical
neurons (FIG. 70D) compared to LacZ injected (FIG. 82C) hemisphere.
Further examination of cervical spinal cord revealed 13% increase
in immunoreactivity to hTDP-43 (FIG. 70F) and increased reactivity
to TDP-43 antibody (FIG. 70G) in preganglionic inter-neurons, which
were morphologically identified in the contralateral side of TDP-43
injected motor cortex (FIG. 70E) compared to the contralateral
spinal cord injected with LacZ (FIG. 70H&I), suggesting that
hTDP-43 expression in the motor cortex leads to increased protein
levels in the contralateral spinal cord. Furthermore, stereological
counting revealed 46% (by stereology, N=8) increase in the levels
of hTDP-43 (FIG. 70J) and increased immunoreactivity to TDP-43
antibody (FIG. 70K) in the dorso-cortical spinal tract (DCST) of
cervical spinal cord contralateral to cortical TDP-43 expression
compared to LacZ injected side (FIG. 70L&M). Toluidine blue
staining and quantification by a blind investigator of centric
axons within 10 random fields of each slide showed increased number
(18%, N=8) of axons (FIG. 70N, arrows) in enlarged circles,
suggesting axonal degeneration compared to the contralateral DCST
(FIG. 70O). Some centric axons were detected in all treatments.
[0255] Lentiviral parkin expression increases cytosolic
co-localization of TDP-43 with ubiquitin. Because TDP-43 is
detected in ubiquitinated forms within the cytosol in human
disease, it was sought to determine whether ubiquitination is
beneficial or detrimental to TDP-43 using parkin as a ubiquitous
E3-ubiquitin ligase in the human brain. TDP-43 was co-expressed
with parkin and animals were sacrificed 2 weeks post-injection.
Staining of 20 .mu.m thick brain sections showed endogenous parkin
expression (FIG. 71A) and TDP-43 (FIG. 71B), which was
predominantly localized to DAPI stained nuclei (FIG. 71C) in
LacZ-injected rat motor cortex. Staining with anti-ubiquitin
antibodies (FIG. 71D) in rats expressing TDP-43 in the motor cortex
(FIG. 71E) did not result in any noticeable co-localization between
TDP-43 and ubiquitin (FIG. 71F). Stereological counting showed 38%
increase in hTDP-43 stained cells (FIG. 71D). However, cytosolic
TDP-43 was observed in cortical neurons expressing TDP-43 (FIG.
71F) compared to nuclear TDP-43 in LacZ injected animals (FIG.
71C). We expressed parkin in the rat motor cortex (FIG. 71G)
together with TDP-43 (FIG. 71H) and observed cytosolic
co-localization of parkin and TDP-43 (FIG. 71I, 35% by stereology).
We further stained with anti-ubiquitin antibodies and observed
increased levels of ubiquitin (FIG. 71J, 35% by stereology) in
animals injected with parkin and TDP-43 (FIG. 71K). Interestingly,
enhanced ubiquitin signals co-localized with TDP-43 in the cytosol,
suggesting that ubiquitination may result in cytosolic
sequestration of TDP-43. To determine whether exogenous parkin
expression affects endogenous TDP-43 protein localization, we
stained with parkin (FIG. 71M, 28% by stereology) and TDP-43 (FIG.
71N) antibodies, but we did not observe any changes in the pattern
of TDP-43 staining (FIG. 71O).
[0256] Parkin promotes K48 and K63-linked ubiquitin to TDP-43. To
demonstrate whether parkin mediates TDP-43 ubiquitination
immuno-precipitation was performed to show ubiquitinated TDP-43 in
the presence of parkin expression. Western blot analysis of the
input showed that increased exogenous parkin (FIG. 72A, 1st blot,
N=8, P<0.05, 42%) in the rat motor cortex, increases the levels
of ubiquitinated proteins (FIG. 72A, 2nd blot). Densitometry
analysis of TDP-43 blots (FIG. 72A, 3rd blot) showed a significant
increase (48%, N=8) in TDP-43 levels in brains injected with
lentiviral TDP-43 (consistent with our previous work compared to
LacZ or parkin injected brains. However, co-injection of TDP-43 and
parkin did not result in any significant changes in TDP-43 levels
(P<0.05, N=8), suggesting that parkin mediates TPD-43
ubiquitination, which may not lead to protein degradation. A
non-functional parkin mutant (T240R, threonine to arginine
mutation), which was co-expressed with TDP-43 (FIG. 72A, top blot)
was also used and no changes in ubiquitinated proteins (FIG. 72A,
2nd blot) or TDP-43 levels (FIG. 72A, 3rd blot) were detected.
TDP-43 was immune-precipitated and probed with ubiquitin (FIG. 72A,
4.sup.th blot) to ascertain that high molecular weight species are
ubiquitinated TDP-43 proteins and not some protein aggregates. An
increase in protein smear was observed when TDP-43 was co-injected
with parkin, compared to TDP-43, parkin or LacZ alone, suggesting
increased TDP-43 ubiquitination in the presence of wild type
parkin. However, no differences were observed in the levels of
ubiquitinated proteins (FIG. 72A, 4.sup.th blot) when TDP-43 was
immuno-precipitated with or without expression of T240R mutant
parkin, suggesting that functional parkin mediates TDP-43
ubiquitination.
[0257] To determine whether TDP-43 affects parkin E3 ubiquitin
ligase activity, parkin (FIG. 72B, left blot) and TDP-43 (FIG. 4B,
right blot) were immune-precipitated and an enzyme activity assay
was performed. Positive controls with E1-E2-E3 or poly-ubiquitin
chains or recombinant parkin (Novus Biologicals) were used to
measure E3 ubiquitin ligase activity and poly-ubiquitin chain
readings (FIG. 72C). No parkin activity was detected with the
lysine null (KO) ubiquitin, but either mutant K48 or K63-linked
ubiquitin showed an increase in parkin E3 ubiquitin ligase activity
compared to control KO (FIG. 72C, N=4). Parkin activity with K63
ubiquitin was significantly higher (83%, P<0.05, N=4) than
K48-linked ubiquitin, suggesting that parkin undergoes K48 and
K63-linked auto-ubiquitination. Parkin was also ubiquitinated using
wild type ubiquitin, which contains all 7 lysine residues. To
determine whether parkin activity is altered in the presence of
TDP-43, both parkin and TDP-43 were added to the enzyme mix. As
expected no activity was detected with lysine null ubiquitin (KO),
but parkin activity was significantly increased compared to parkin
alone (FIG. 72C, P<0.05, N=8) with K48 (154%) and K63 (156%)
ubiquitin, indicating that parkin activity is even higher in the
presence of a substrate. Parkin also showed a significantly higher
level of activity with wild type ubiquitin in the presence of
TDP-43 (279%) compared to parkin alone.
[0258] To ascertain that parkin mediates ubiquitination of TDP-43,
parkin and TDP-43 were immunoprecipitated separately and in vitro
ubiquitination assays were performed. Incubation of both parkin and
TDP-43 in the presence of either wild type (FIG. 72D, 2nd lane) or
K48 (7th lane) or K63 (8th lane) ubiquitin (FIG. 72D, N=3), showed
a protein smear upon WB analysis with TDP-43 antibodies compared to
lysine null (KO) ubiquitin (6th lane), or in the absence of E1 or
E2 or both (all other lanes, suggesting that parkin mediates K48
and K63-linked ubiquitination of TDP-43. Additionally, parkin
incubation in the presence of either wild type (FIG. 72E, 2nd lane)
or K48 (7th lane) or K63 (8th lane) ubiquitin (FIG. 72E, N=3),
showed a protein smear upon WB analysis with parkin antibodies
compared to lysine null (KO) ubiquitin (6th lane), or in the
absence of E1 or E2 or both (all other lanes, suggesting that
parkin undergoes K48 and K63-linked auto-ubiquitination.
[0259] The activity of the 20S proteasome (FIG. 72F), which was
significantly decreased (31%, P<0.05) when TDP-43 was expressed
alone (N=8, P<0.05), but co-expression of parkin significantly
reversed proteasome activity (48%, P<0.05) compared to TDP-43
alone, was measured. However, proteasome activity in parkin
expressing cortex remained significantly higher than LacZ (73%,
P<0.05) and parkin+TDP-43 (31%, P<0.05) injected animals,
indicating that parkin activity partially reverses proteasome
activity.
[0260] Parkin forms a multi-protein complex with HDAC6 to mediate
TDP-43 translocation from nucleus to cytosol. Lack of degradation
of ubiquitinated TDP-43 and cytosolic accumulation of parkin,
TDP-43 and ubiquitin in gene transfer animals led to examination of
possible mechanisms to translocate TDP-43 to the cytosol. Western
blot analysis showed a significant increase (41%, P<0.05) in
HDAC6 levels when TDP-43 was expressed compared to LacZ or parkin
injected animals (FIG. 72G&H, 1st blot, P<0.05, N=8).
However, further increases in HDAC6 levels (FIG. 72G&H, 112%,
P<0.05) were detected when parkin was co-expressed with TDP-43,
suggesting possible interaction between these proteins. Examination
of molecular markers of autophagy showed a significant increase in
P62 (28%, P<0.05) when parkin was co-expressed with TDP43 (FIG.
72G&H, 2nd blot) compared to all other treatments, suggesting
accumulation of ubiquitinated proteins. No changes in other markers
of autophagy (LC3, beclin, Atgs) or appearance of autophagic
vacuoles by EM were seen. Human TDP-43 was immunoprecipitated from
transgenic mice and TDP-43 was verified at 46 kDa using hTDP-43
antibody (FIG. 73A, 1st & 2nd blots). Stripping and re-probing
with parkin antibody showed a slightly higher band around 50 kDa,
suggesting presence of parkin protein (FIG. 73A, 3rd blot). Further
stripping and probing with HDAC6 antibody (FIG. 73A, 4.sup.th blot)
showed a higher molecular weight band around 120 kDa, indicating a
multi-protein complex between parkin, TDP43 and HDAC6. A reserve
experiment was performed via parkin immuno-precipitation and
verification of human TDP-43 presence (FIG. 73B, 1st & 2nd
blot). Stripping and probing with parkin antibody showed parkin
band in both transgenic and non-transgenic control mice (FIG. 73B,
3rd blot), indicating that parkin was successfully
immuno-precipitated. A higher molecular weight band representative
of HDAC6 (FIG. 73B, 4.sup.th blot) was detected in transgenic but
not control mice, further suggesting multi-protein complex
formation between TDP43, parkin and HDAC6.
[0261] To ascertain that both parkin and HDAC6 are required for
TDP-43 translocation, GFP-tagged TDP-43 was expressed in M17
neuroblastoma cells in the presence of wild type or
loss-of-function mutant (T240R) parkin, and treated with 5 .mu.M
selective HDAC6 inhibitor for 24 hours. GFP expression was
predominantly observed within DAPI-stained nuclei in live M17 cells
(FIG. 73C, insert is higher magnification), however parkin
co-expression led to significant GFP fluorescence within the
cytoplasm (FIG. 73D&E) and neuronal processes (FIG. 73D, insert
shows higher magnification of GFP fluorescence). Treatment with the
HDAC6 inhibitor, tubacin, did not lead to GFP fluorescence in the
cytosol in the presence (FIG. 73F) or absence (FIG. 73G) of parkin.
Loss of parkin E3 ubiquitin ligase function (T240R) did not lead to
TDP-43 accumulation in the cytosol (FIG. 73H), suggesting that the
E3 ubiquitin ligase function of parkin and HDAC6 activity are
required to facilitate TDP-43 accumulation within the cytosol.
[0262] To verify whether TDP-43 expression increases parkin mRNA
levels, performed qRT-PCR was performed in samples isolated from
rat cortex, human M17 cells and TDP43-Tg mice. Park2 mRNA levels in
M17 cells expressing parkin was significantly higher (FIG.
73I&J, 55%, P<0.05, N=4) than LacZ, but similar to TDP-43
injected brains (61%, P<0.05). Parkin co-expression with TDP-43
showed significantly higher levels of park2 mRNA (FIG. 73J, 74%,
P<0.05, N=4) compared to parkin alone. Similarly, Park2 mRNA
levels in rat brains expressing parkin was significantly higher
(FIG. 73K&L, 41%, P<0.05, N=4) than LacZ animals, as well as
TDP-43 injected brains (21%, P<0.05). However, parkin
co-expression with TDP-43 showed significantly higher levels of
park2 mRNA (FIG. 73J, 84%, P<0.05, N=4) compared to all other
treatments. Therefore, park2 mRNA levels between TDP43-Tg and
non-transgenic control littermates were compared. A significant
increase (FIG. 73M&N, 114%, N=4, P<0.05) in park2 mRNA was
observed in TDP43-Tg brains injected compared to C57BL/6 controls,
showing that parkin is a transcriptional target for TDP-43.
Example 7
Parkin Plays an Essential Role in Motor Neuron Survival Via
Modulation of Nuclear TDP-43 Transport to the Synapse
[0263] E3 ubiquitin ligase Parkin is important in
neurodegeneration. Parkin promotes specific ubiquitination of
TAR-DNA binding protein (TDP)-43, and could mediate its transport
via complex formation with histone deacetylase 6 (HDAC6). In
healthy neurons, TDP-43 is predominantly nuclear and could be
transported to the synapse for generation of synaptic proteins. As
shown in FIG. 74, 1). Parkin could ubiquitinate TDP-43 and
translocate it from the nucleus to the cytosol; 2). Parkin-HDAC6
complex is required for axoplasmic TDP-43 transport to the synapse;
and 3). TDP-43 availability at the synapse modulates expression of
synaptic proteins that maintain glutamate metabolism.
[0264] Long motor neurons, which degenerate in Amyotrophic Lateral
Sclerosis (ALS), could depend on axonal TDP-43 transport to distant
synapses, thus increasing their vulnerability to TDP-43
localization. In neurodegeneration, including ALS and
Frontotemporal Dementia (FTD-TDP), wild type and mutated TDP-43
aggregate, and neurons bearing TDP-43 aggregates express less
parkin. Data provided herein show that parkin alters TDP-43
localization, reverses TDP-43-induced alteration in glutamate
levels and improves motor performance. TDP-43 binds to mRNAs that
code for proteins involved in synaptic function, including
synaptotagmin and vesicular glutamate transporters. Glutamate
transport is defective in ALS, due to loss of glutamate
transporters that facilitate conversion of synaptic glutamate into
glutamine. Thus, nuclear TDP-43 translocation and axoplasmic
transport to the synapse could be particularly important for motor
neurons.
[0265] Parkin-mediated TDP-43 localization to the synapse could
affect synaptic proteins that maintain glutamate metabolism. Thus,
parkin could play an essential role in motor neuron survival via
modulation of nuclear TDP-43 transport to the synapse.
[0266] The following data support these conclusions. FIG. 75 shows
the distribution of GFP-tagged TDP-43 in M17 cells transfected with
3 mg cDNA for 24 hrs and then treated with Nilotinib (10 mM) or
Bosutinib (5 mM) and HDAC6 inhibitor Tubacin (5 mM) for additional
24 hrs. Inserts (B&D) represent higher magnification images
showing translocation of GFP-tagged TDP-43 from nucleus (A) into
the cytosol (B&D, and inserts), while tubacin impairs
translocation (C&E).
[0267] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *