U.S. patent application number 16/616748 was filed with the patent office on 2021-05-20 for detection of par in the csf of patients with parkinson's disease.
The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to Shaida Andrabi, Ted Dawson, Valina Dawson, Tae-In Kam, Liana Rosenthal.
Application Number | 20210148898 16/616748 |
Document ID | / |
Family ID | 64455128 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210148898 |
Kind Code |
A1 |
Dawson; Ted ; et
al. |
May 20, 2021 |
DETECTION OF PAR IN THE CSF OF PATIENTS WITH PARKINSON'S
DISEASE
Abstract
Poly(ADP-ribose) (PAR) is a protein implicated in numerous
neurodegenerative disease states including Parkinson's disease
(PD). To date, no routine laboratory test has been developed to
diagnosis, assess or monitor patients suffering from PD. Disclosed
herein a novel method to assess the PAR concentration in the
cerebral spinal fluid (CSF) of a patient and correlate that
concentration to the medical condition of a patient with PD. Also
disclosed is the use of PAR as a biomarker for PD.
Inventors: |
Dawson; Ted; (Baltimore,
MD) ; Dawson; Valina; (Baltimore, MD) ; Kam;
Tae-In; (Baltimore, MD) ; Rosenthal; Liana;
(Lutherville, MD) ; Andrabi; Shaida; (Birmingham,
AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
Baltimore |
MD |
US |
|
|
Family ID: |
64455128 |
Appl. No.: |
16/616748 |
Filed: |
June 1, 2018 |
PCT Filed: |
June 1, 2018 |
PCT NO: |
PCT/US2018/035614 |
371 Date: |
November 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62514316 |
Jun 2, 2017 |
|
|
|
62679161 |
Jun 1, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/2835 20130101;
G01N 2500/20 20130101; G01N 2800/52 20130101; G01N 2333/91142
20130101; G01N 33/6896 20130101; C07K 16/44 20130101; C07K 2317/21
20130101; G01N 33/5308 20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH &
DEVELOPMENT
[0002] This invention was made with government support under grant
no. NS38377 and U01NS082133 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method for determining a poly(ADP-ribose) (PAR) concentration
in cerebral spinal fluid (CSF), said method comprising collecting a
sample of cerebrospinal fluid from a patient, performing a
PAR-sandwich ELISA on said CSF sample, thereby determining the PAR
concentration in the CSF.
2. The method according to claim 1, further comprising comparing
the PAR concentration in said CSF sample to at least one control
sample.
3. The method according to claim 1, wherein the sandwich ELISA
comprises at least at capture antibody and a detection antibody,
and said capture antibody, said detection antibody or both are an
anti-PAR monoclonal antibody.
4. The method according to claim 3, wherein the capture antibody is
attached to a solid support.
5. The method according to claim 3, wherein the anti-PAR monoclonal
antibody is fully human.
6. The method according to claim 1, wherein comparing the PAR
concentration in the CSF sample to at least one control sample is
done by colorimetric assay.
7. The method according to claim 3, wherein the detection antibody
is conjugated to biotin.
8. A method for determining the therapeutic efficacy of a medical
treatment for Parkinson's disease, said method comprising
collecting a sample of cerebrospinal fluid (CSF) from a patient
receiving at least one medical treatment for PD, measuring a
poly(ADP-ribose) (PAR) concentration in said CSF sample, and
comparing the PAR concentration in said patient to a PAR
concentration in at least one control sample.
9. The method according to claim 8, wherein the control sample is a
CSF sample collected from said patient at an earlier time
period.
10. The method according to claim 8, wherein the control sample is
a CSF sample collected from a healthy donor.
11. The method according to claim 9, wherein if the PAR
concentration in said patient is higher than the PAR concentration
in the control sample, then the medical treatment is not effective
for said patient, and if the PAR concentration in said patient is
the same or lower than the PAR concentration in the control sample,
then the medical treatment is effective for said patient.
12. The method according to claim 8, wherein the medical treatment
is administering to said patient one or more medicaments,
performing surgery on said patient or a combination thereof.
13. The method according to claim 8, wherein measuring the PAR
concentration in said CSF sample is by sandwich ELISA.
14. A method for monitoring the disease progression of a patient
with Parkinson's disease (PD), said method comprising collecting a
sample of cerebrospinal fluid (CSF) from a patient receiving at
least one medical treatment for PD, measuring a poly(ADP-ribose)
(PAR) concentration in said CSF sample, and comparing the PAR
concentration in said patient to a PAR concentration in at least
one control sample.
15. The method according to claim 14, wherein the control sample is
a CSF sample collected from said patient at an earlier time
period.
16. The method according to claim 14, wherein the control sample is
a CSF sample collected from a healthy donor.
17. The method according to claim 15, wherein if the PAR
concentration in said patient is higher than the PAR concentration
in the control sample, then the medical treatment is not effective
for said patient, and if the PAR concentration in said patient is
the same or lower than the PAR concentration in the control sample,
then the medical treatment is effective for said patient.
18. The method according to claim 14, wherein the medical treatment
is administering to said patient one or more pharmaceutical agents,
performing surgery on said patient or a combination thereof.
19. The method according to claim 14, wherein measuring the PAR
concentration in said CSF sample is by sandwich ELISA.
20. A method of diagnosing a patient with Parkinson's disease, said
method comprising collecting a sample of cerebrospinal fluid (CSF)
from a patient, measuring a poly(ADP-ribose) (PAR) concentration in
said CSF sample, and comparing the PAR concentration in said
patient to a PAR concentration in at least one control sample.
21. The method according to claim 20, wherein the control sample is
a CSF sample collected from said patient at an earlier time
period.
22. The method according to claim 20, wherein the control sample is
a CSF sample collected from a healthy donor.
23. The method according to claim 21, wherein if the PAR
concentration in said patient is higher than the PAR concentration
in the control sample, then said patient is at high risk for
developing PD or has PD, and if the PAR concentration in said
patient is the same or lower than the PAR concentration in the
control sample, then said patient is not at risk for developing PD
or does not have PD.
24. A theranostic method for Parkinson's disease, said method
comprising collecting a sample of cerebrospinal fluid (CSF) from a
patient who is receiving at least one medical treatment for PD,
measuring a poly(ADP-ribose) (PAR) concentration in said CSF
sample, comparing the PAR concentration in said patient to a PAR
concentration in at least one control sample.
25. The theranostic method according to claim 24, wherein the
control sample is a CSF sample collected from said patient at an
earlier time period.
26. The theranostic method according to claim 24, wherein the
control sample is a CSF sample collected from a healthy donor.
27. The theranostic method according to claim 25 wherein if the PAR
concentration in said patient is higher than the PAR concentration
in the control sample, then the method further comprises altering
at least one medical treatment received by said patient, and if the
PAR concentration in said patient is the same or lower than the PAR
concentration in the control sample, then the method further
comprises not altering the medical treatment received by said
patient.
28. The method according to claim 24, wherein measuring the PAR
concentration in said CSF sample is by sandwich ELISA.
Description
[0001] This application claims priority to U.S. Provisional
Application 62/514,316 filed on Jun. 2, 2017 and U.S. Provisional
Application 62/679,161 filed on Jun. 1, 2018, both of which are
incorporated by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] Poly(ADP-ribose) ("PAR") polymerase-1 ("PARP-1") is an
important nuclear enzyme that responds to DNA damage and is
required for DNA repair. Upon activation, PARP-1 catalyzes the
transfer of ADP-ribose from nicotinamide adenine dinucleotide
("NAD+") and conjugates PAR onto a variety of nuclear proteins such
as histones, DNA polymerases, topoisomerases, and transcription
factors as well as auto-modification of PARP-1 itself, thus
regulating a variety of physiologic processes. Excessive activation
of PARP-1 leads to an intrinsic cell death program, which has been
designated parthanatos (or, alternatively, PARP-1-dependent cell
death) to distinguish it from necrosis and apoptosis. Parthanatos
is known to occur in many diseases and conditions such as stroke,
Parkinson's disease, heart attack, diabetes, and ischemia
reperfusion injury. PARP-1 inhibition or PARP-1 gene deletion is
markedly protective in models of many cell injury paradigms,
including stroke, trauma, ischemia-reperfusion injury, diabetes,
and neurodegenerative diseases, indicating that parthanatos plays a
prominent role in these disorders.
[0004] The mitochondrial protein apoptosis-inducing factor ("AIF")
plays a pivotal role in parthanatos, during which AIF is released
from the mitochondria and translocates to the nucleus. AIF is a
mitochondrial oxidoreductase that, like cytochrome C, has two
independent functions. The first is within mitochondria, involving
cell survival, thought to be through assembly or stabilization of
respiratory complex I. The second is as a promoter of parthanatos
cell death. AIF is released into the cytoplasm following PARP-1
activation, ultimately entering the nucleus to induce cell
death.
[0005] Parkinson's disease (PD) is an age-related neurodegenerative
disease in which .alpha.-syn deposits as fibrils in
intracytoplasmic inclusions in structures termed Lewy bodies and
neurites. Recombinant .alpha.-syn can be aggregated in vitro to
form fibrils similar in structure to those found in vivo, and these
.alpha.-syn pre-formed fibrils (.alpha.-syn PFF) can spread in a
prion-like manner: both in in vitro neuronal cultures and in vivo
when injected into the mouse brain with accompanying
phosphorylation of .alpha.-syn on serine 129, a marker of
pathologic .alpha.-syn and neurotoxicity. While it is clear that
aggregated .alpha.-syn underlies the pathology of PD, what drives
abnormal aggregation of .alpha.-syn as well as the cell injury and
death mechanisms that are activated by this aggregation are not yet
known. Since poly (ADP-ribose) (PAR) polymerase-1 (PARP-1) and PAR
play a major contributing role in cell death relevant to neurologic
disorders, here the role for PARP-1 and PAR in pathologic
.alpha.-syn induced neurodegeneration was evaluated.
[0006] Additionally, PD is a slowly progressive, neurodegenerative
CNS disorder characterized by slow and decreased movement, muscular
rigidity, resting tremor, postural instability, cognitive
impairment and dementia. The major pathological feature of PD is
selective degeneration of dopaminergic neurons in the substantia
nigra pars compacta (SNpc) and loss of their terminals in the
caudate and putamen. Loss of substantia nigra neurons, which
project into the caudate nucleus and putamen, depletes dopamine in
these areas. Evidence suggests that multiple factors, including
genetic and environmental ones, contribute to the dopaminergic
neurodegeneration in this neurodegenerative disease.
[0007] The medical treatment of PD is directed to stopping, slowing
or reducing the extent of or minimizing the neurodegenerative
process in nigrostriatal neurons (neuroprotective therapy) and
eliminating the biochemical imbalance (symptomatic therapy). The
main directions of symptomatic therapy in PD are to increase
dopamine synthesis, or stimulate dopamine receptors activity and
dopamine release from the presynaptic space, and to inhibit
dopamine reuptake by presynaptic receptors and dopamine
catabolism.
[0008] Because there is no known cure for PD at this time and
different patients respond to treatment methods differently, it is
important for a medical professional to carefully monitor disease
progression in a patient. This allows the medical professional to
adjust or alter the medical treatment the patient receives in the
event that the current treatment is ineffective. The current method
for monitoring PD progress is a subjective scale based on the level
of disability and impairment experienced by a patient.
[0009] Thus there remains a need to more specifically to assess and
monitor the treatment and progression of PD patients in order fine
tune medical treatment. A need also exists for a quantitative as
well as qualitative scale that eliminates or reduces the subjective
component of the assessment.
BRIEF DESCRIPTION OF THE INVENTION
[0010] In a first aspect, disclosed herein is a method for
determining a poly(ADP-ribose) (PAR) concentration in cerebral
spinal fluid, the method comprising collecting a sample of
cerebrospinal fluid (CSF) from a patient, performing a PAR-sandwich
ELISA on the CSF sample, thereby determining the PAR concentration
in the CSF.
[0011] In a second aspect, disclosed herein is a method for
determining the therapeutic efficacy of a medical treatment for
Parkinson's disease, the method comprising collecting a sample of
cerebrospinal fluid (CSF) from a patient, measuring a
poly(ADP-ribose) (PAR) concentration in the CSF sample, and
comparing the PAR concentration in the patient to a PAR
concentration in at least one control sample.
[0012] In yet another aspect, disclosed herein is a method for
monitoring the disease progression of a patient with Parkinson's
disease (PD), the method comprising collecting a sample of
cerebrospinal fluid (CSF) from a patient, measuring a
poly(ADP-ribose) (PAR) concentration in the CSF sample, and
comparing the PAR concentration in the patient to a PAR
concentration in at least one control sample, wherein the patient
is receiving at least one medical treatment for PD.
[0013] In still yet another aspect, disclosed herein is a method of
diagnosing a patient with Parkinson's disease, the method
comprising collecting a sample of cerebrospinal fluid (CSF) from a
patient, measuring a poly(ADP-ribose) (PAR) concentration in the
CSF sample, and comparing the PAR concentration in the patient to a
PAR concentration in at least one control sample.
[0014] In still yet another aspect, disclosed herein is a
theranostic method for Parkinson's disease, the method comprising
collecting a sample of cerebrospinal fluid (CSF) from a patient who
is receiving at least one medical treatment for PD, measuring a
poly(ADP-ribose) (PAR) concentration in the CSF sample, comparing
the PAR concentration in the patient to a PAR concentration in at
least one control sample.
[0015] In still yet another aspect, disclosed herein is the use of
PAR in the CSF of a patient as a biomarker for PD.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic drawing of the processes in a cell
illustrating the model of PAR-dependent AIF release in
parthanatos.
[0017] FIG. 2 is a schematic drawing of the sandwich ELISA assay
with an anti-PAR antibody and a concentration curve.
[0018] FIG. 3 is a bar graph of the concentration of PAR in the CSF
of a PD patient versus healthy controls.
[0019] FIG. 4 is a bar graph of the concentration of PAR in
PD-normal patients vs PD-cognitively impaired patients.
[0020] FIG. 5 shows activation of PARP-1 in .alpha.-syn PFF-treated
primary cortical neurons. The representative western blot analysis
(top) and quantification (bottom) of the levels of PAR
accumulation. Bars represent mean.+-.s.e.m. One-way ANOVA followed
by Tukey's post hoc test (n=3-4).
[0021] FIG. 6 shows representative images of Hoechst and propidium
iodide (PI) staining from primary cortical neurons pre-incubated
with either ABT-888 (10 .mu.M), AG-014699 (1 .mu.M) or BMN 673 (10
.mu.M) for 1 h, and further incubated with .alpha.-syn PFF (5
.mu.g/ml) for 14 days. Scale bar, 20 .mu.m.
[0022] FIG. 7 shows quantification of cell death. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3).
[0023] FIG. 8 shows inhibition of .alpha.-syn PFF-induced PAR
accumulation was determined by western blot analysis.
[0024] FIG. 9 shows representative images of Hoechst and propidium
iodide (PI) staining from primary cortical neurons transduced with
AAV-sgCon or AAV-sgPARP-1, and further incubated with .alpha.-syn
PFF for 14 days. Scale bar, 20 .mu.m.
[0025] FIG. 10 shows quantification of cell death. Bars represent
mean.+-.s.e.m. (n=3).
[0026] FIG. 11 shows representative images of Hoechst and propidium
iodide (PI) staining from WT or PARP-1 KO primary cortical neurons,
and further incubated with .alpha.-syn PFF for 14 days. Scale bar,
20 .mu.m.
[0027] FIG. 12 shows quantification of cell death. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3). *P<0.05, **P<0.005, ***P<0.0005.
[0028] FIG. 13 shows representative immunoblots and quantification
of the levels of PAR accumulation in the striatum of .alpha.-syn
PFF injected mice. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test (n=4).
[0029] FIG. 14 shows representative TH and Nissl staining of SNpc
DA neurons of .alpha.-syn PFF injected WT, PARP-1KO, and WT mice
fed with ABT-888 at 6 months after intrastriatal .alpha.-syn PFF or
PBS injection.
[0030] FIG. 15 shows stereological counts. Data are mean.+-.s.e.m.
One-way ANOVA followed by Tukey's post hoc test (n=5 to 7 mice per
group).
[0031] FIG. 16 shows DA concentrations in the striatum of WT,
PARP-1 KO, and WT mice fed with ABT-888 at 6 months after
intrastriatal .alpha.-syn PFF or PBS injection measured by HPLC.
Bars represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's
post hoc test. (n=6 to 30 mice per group).
[0032] FIG. 17 shows pole test results 180 days after .alpha.-syn
PFF injection. The pole test was performed in WT, PARP-1 KO, or WT
mice fed with ABT-888. Data are the means s.e.m. One-way ANOVA
followed by Tukey's post hoc test (n=7 to 9 mice per group).
*P<0.05, **P<0.005, ***P<0.001.
[0033] FIG. 18 shows grip strength results 180 days after
.alpha.-syn PFF injection. The grip strength test was performed in
WT, PARP-1 KO, or WT mice fed with ABT-888. Data are the
means.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=7 to 9 mice per group). *P<0.05, **P<0.005,
***P<0.001.
[0034] FIG. 19 shows acceleration of .alpha.-syn fibrillization by
PAR. Monomeric .alpha.-syn either with or without 5 nM purified PAR
was incubated at 37.degree. C. for indicated times. Fibrillization
of .alpha.-syn was detected by immunoblotting using .alpha.-syn
antibody (top). Data are mean.+-.s.e.m. (bottom). One-way ANOVA
followed by Tukey's post hoc test (n=3).
[0035] FIG. 20 shows the rate of formation of .alpha.-syn fibrils
either with or without PAR was monitored by thioflavin T
fluorescence (n=3).
[0036] FIG. 21 shows representative transmission electron
microscopy (TEM) images for .alpha.-syn fibrils. Scale bar, 200
nm.
[0037] FIG. 22 shows suppression of NMDA-induced .alpha.-syn
fibrillization in PARP-1 KO neurons. Primary cortical neurons from
WT or PARP-1 KO embryos were transduced with AAV-.alpha.-syn and
then further incubated with 500 .mu.M NMDA for 5 min. The
.alpha.-syn fibrillization was detected by western blot analysis 6
h after NMDA treatment.
[0038] FIG. 23 shows prevention of NMDA-induced .alpha.-syn
fibrillization by PARP1 inhibitors. The primary cortical neurons
transduced with AAV-.alpha.-syn were pre-treated with 10 .mu.M
ABT-888 or 1 .mu.M AG-014699 for 1 h, and further incubated with
500 .mu.M NMDA for 5 min. The .alpha.-syn fibrillization was
detected by western blot analysis 6 h after NMDA treatment.
[0039] FIG. 24 shows .alpha.-syn PFF or PAR-.alpha.-syn PFF
incubated with increasing concentration of PK (0-2.5 .mu.g/ml) and
immunoblotted with .alpha.-syn antibody (top). Quantification
represents the ratio of cleaved to uncleaved .alpha.-syn (bottom).
Data are mean.+-.s.e.m. (bottom). One-way ANOVA followed by Tukey's
post hoc test (n=3).
[0040] FIG. 25 shows representative immunostaining of p-.alpha.-syn
(red) in primary cortical neurons treated with .alpha.-syn PFF or
PAR-.alpha.-syn PFF for 1, 4 and 7 days. Quantification of
p-.alpha.-syn signals normalized with DAPI (right). Bars represent
mean.+-.s.e.m. (bottom). One-way ANOVA followed by Tukey's post hoc
test (n=5).
[0041] FIG. 26 shows primary cortical neurons treated with
.alpha.-syn PFF or PAR-.alpha.-syn PFF were sequentially extracted
with 1% TX-100 (TX soluble) and 2% SDS (TX insoluble). Lysates were
subjected to immunoblotting using .alpha.-syn, p-.alpha.-syn, and
GAPDH antibodies. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test (n=3). ND, not detected.
*P<0.05, **P<0.005, ***P<0.0005 as compared to .alpha.-syn
PFF for 1 day. .sup.#P<0.05, .sup.##P<0.005,
.sup.###P<0.0005.
[0042] FIG. 27 shows representative TH and Nissl staining of SNpc
DA neurons of WT mice at 1, 3, and 6 months after instrastriatal
PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or PAR injection.
[0043] FIG. 28 shows stereological counts. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=5
to 8 mice per group).
[0044] FIG. 29 shows DA concentrations in the striatum of PBS,
.alpha.-syn PFF, PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3,
and 6 months measured by HPLC. Bars represent mean.+-.s.e.m.
One-way ANOVA followed by Tukey's post hoc test (n=4 to 6 mice per
group).
[0045] FIG. 30 shows representative p-.alpha.-syn immunostaining in
the SNpc of WT mice at 1, 3, and 6 months after instrastriatal PBS,
.alpha.-syn PFF, PAR-.alpha.-syn PFF, or PAR injection. Scale bar,
100 .mu.m.
[0046] FIG. 31 shows quantification of p-.alpha.-syn levels. Bars
represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc
test (n=5 to 8 mice per group).
[0047] FIG. 32 shows behavioral abnormalities of PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3, and 6
months measured by pole test. Data are the means s.e.m. One-way
ANOVA followed by Tukey's post hoc test (n=9 to 14 mice per
group).
[0048] FIG. 33 shows behavioral abnormalities of PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3, and 6
months measured by grip strength test. Data are the means s.e.m.
One-way ANOVA followed by Tukey's post hoc test (n=9 to 14 mice per
group).
[0049] FIG. 34 shows increase of PAR in CSF of PD patients. The
levels of PAR in CSF of healthy controls (n=31) and PD patients
(n=80) were determined by PAR ELISA. Bars represent mean.+-.s.e.m.
Student t test with Welch's correction.
[0050] FIG. 35 shows increase of PAR in CSF of PD patients. The
levels of PAR in CSF of healthy controls (n=33) and PD patients
(n=21) were determined by PAR ELISA. Bars represent mean.+-.s.e.m.
Student t test with Welch's correction.
[0051] FIG. 36 shows increase and co-localization of PAR in Lewy
body of PD patients. Representative .alpha.-syn (red) and PAR
(green) immunostaining in the SNpc of PD patients.
[0052] FIG. 37 shows representative images of p-.alpha.-syn (red)
in primary cortical neurons pre-incubated with either ABT-888 (10
.mu.M), AG-014699 (1 .mu.M) or BMN 673 (10 .mu.M) for 1 h, and
further incubated with .alpha.-syn PFF for 7 days. Nuclei are
stained with DAPI (blue).
[0053] FIG. 38 shows quantification of p-.alpha.-syn signals
normalized with DAPI. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test (n=6).
[0054] FIG. 39 shows representative immunoblots of .alpha.-syn in
the detergent-soluble and insoluble fraction from primary cortical
neurons pre-treated with PARP inhibitors for 1 h followed by
incubated with .alpha.-syn PFF for 7 days.
[0055] FIG. 40 shows quantification of .alpha.-syn levels in the
detergent-insoluble fraction normalized to b-actin. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3).
[0056] FIG. 41 shows inhibition of .alpha.-syn PFF-induced PAR
accumulation in primary cortical neurons infected with
AAV-sgPARP1.
[0057] FIG. 42 shows inhibition of .alpha.-syn PFF-induced PAR
accumulation in primary cortical neurons from PARP1 KO embryos.
[0058] FIG. 43 shows representative images of p-.alpha.-syn (red)
in primary cortical neurons pre-incubated with either ABT-888 (10
.mu.M), AG-014699 (1 .mu.M) or BMN 673 (10 .mu.M) for 1 h, and
further incubated with .alpha.-syn PFF for 7 days. Nuclei are
stained with DAPI (blue).
[0059] FIG. 44 shows quantification of p-.alpha.-syn signals
normalized with DAPI. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test (n=6).
[0060] FIG. 45 shows representative immunoblots of .alpha.-syn in
the detergent-soluble and insoluble fraction from WT or PARP1 KO
primary cortical neurons incubated with .alpha.-syn PFF for 7
days.
[0061] FIG. 46 shows quantification of .alpha.-syn levels in the
detergent-insoluble fraction normalized to b-actin. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3).
[0062] FIG. 47 shows representative images of Hoechst and propidium
iodide (PI) staining from primary cortical neurons pre-treated with
ABT-888, Z-VAD, NEC-1, or 3-MA for 1 h, followed by further
incubation with .alpha.-syn PFF for 14 days. Scale bar, 20
.mu.m.
[0063] FIG. 48 shows quantification of cell death. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3). *P<0.05, ***P<0.001.
[0064] FIG. 49 shows representative images of .alpha.-syn PFF
transmission. .alpha.-syn PFF was added to chamber 1 (C1) of the
microfluidic device. On day 14, p-.alpha.-syn was detected in
chamber 2 (C2) and chamber 3 (C3) when WT neurons were present in
all three chambers, but very limited intensity of p-.alpha.-syn in
PARP-1 KO neurons in C2 and WT neurons in C3. Scale bar, 100
.mu.m.
[0065] FIG. 50 shows the p-.alpha.-syn signal in high resolution
images. Scale bar, 10 .mu.m.
[0066] FIG. 51 shows quantification of p-.alpha.-syn levels in each
chamber. Values are means.+-.s.e.m. Unpaired student's t test
(n=3). *P<0.05, **P<0.005.
[0067] FIG. 52 shows representative immunoblots of the midbrain
lysates from WT, PARP-1 KO, and WT mice fed with ABT-888 with
misfolded .alpha.-syn, p-.alpha.-syn, TH, DAT, PAR, and PARP-1
antibodies.
[0068] FIG. 53 shows quantification of TH levels. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3). *P<0.05, **P<0.005, ***P<0.001.
[0069] FIG. 54 shows quantification of DAT levels. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3). *P<0.05, **P<0.005, ***P<0.001.
[0070] FIG. 55 shows quantification of insoluble .alpha.-syn
levels. Bars represent mean.+-.s.e.m. One-way ANOVA followed by
Tukey's post hoc test (n=3). *P<0.05, **P<0.005,
***P<0.001.
[0071] FIG. 56 shows quantification of insoluble p-.alpha.-syn
levels. Bars represent mean.+-.s.e.m. One-way ANOVA followed by
Tukey's post hoc test (n=3). *P<0.05, **P<0.005,
***P<0.001.
[0072] FIG. 57 shows DA metabolite DOPAC concentrations in the
striatum of WT, PARP-1 KO, and WT mice fed with ABT-888 at 6 months
after intrastriatal .alpha.-syn PFF or PBS injection measured by
HPLC. Bars represent mean.+-.s.e.m. One-way ANOVA followed by
Tukey's post hoc test. (n=5 to 10 mice per group).
[0073] FIG. 58 shows DA metabolite 3-MT concentrations in the
striatum of WT, PARP-1 KO, and WT mice fed with ABT-888 at 6 months
after intrastriatal .alpha.-syn PFF or PBS injection measured by
HPLC. Bars represent mean.+-.s.e.m. One-way ANOVA followed by
Tukey's post hoc test. (n=5 to 10 mice per group).
[0074] FIG. 59 shows DA metabolite HVA concentrations in the
striatum of WT, PARP-1 KO, and WT mice fed with ABT-888 at 6 months
after intrastriatal .alpha.-syn PFF or PBS injection measured by
HPLC. Bars represent mean.+-.s.e.m. One-way ANOVA followed by
Tukey's post hoc test. (n=5 to 10 mice per group).
[0075] FIG. 60 shows representative images of p-.alpha.-syn
immunostaining from WT, PARP-1 KO, and WT mice fed with
ABT-888.
[0076] FIG. 61 shows quantification of p-.alpha.-syn intensity in
amygdala region. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test. (n=4 to 5 mice per group).
[0077] FIG. 62 shows quantification of p-.alpha.-syn intensity in
cortex region. Bars represent mean.+-.s.e.m. One-way ANOVA followed
by Tukey's post hoc test. (n=4 to 5 mice per group).
[0078] FIG. 63 shows quantification of p-.alpha.-syn intensity in
SNpc region. Bars represent mean.+-.s.e.m. One-way ANOVA followed
by Tukey's post hoc test. (n=4 to 5 mice per group).
[0079] FIG. 64 shows pole test results 180 days after .alpha.-syn
PFF injection. The pole test was performed in WT, PARP-1 KO, or WT
mice fed with ABT-888. Data are the means s.e.m. One-way ANOVA
followed by Tukey's post hoc test (n=6 to 30 mice per group).
*P<0.05, **P<0.005, ***P<0.001.
[0080] FIG. 65 shows grip strength test results 180 days after
.alpha.-syn PFF injection. The grip strength test was performed in
WT, PARP-1 KO, or WT mice fed with ABT-888. Data are the
means.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=6 to 30 mice per group). *P<0.05, **P<0.005,
***P<0.001.
[0081] FIG. 66 shows the effect of temperature on PAR-mediated
acceleration of .alpha.-syn fibrillization. Monomeric .alpha.-syn
either with or without 5 nM PAR was incubated at indicated
temperatures for 72 h. Fibrillization of .alpha.-syn was detected
by immunoblotting using .alpha.-syn antibody.
[0082] FIG. 67 shows the concentration dependence of PAR-mediated
.alpha.-syn fibrillization. The .alpha.-syn fibrillization was
detected by western blot analysis 36 h after incubation.
[0083] FIG. 68 shows quantification of .alpha.-syn fibrillization.
Bars represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's
post hoc test.
[0084] FIG. 69 shows representative immunoblot of .alpha.-syn
fibrillization generated by adding 5 nM of PAR, Poly (A), or ADPr
at 37.degree. C. for 36 h.
[0085] FIG. 70 shows quantification of .alpha.-syn fibrillization.
Bars represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's
post hoc test.
[0086] FIG. 71 shows primary cortical neurons from WT or PARP1 KO
embryos were transduced with AAV-.alpha.-syn for 5 days and then
PAR polymer was delivered with BioPorter for 6 h. The .alpha.-syn
fibrillization was detected by western blot analysis.
[0087] FIG. 72 shows WT or PARP1 KO SH-SY5Y cells transfected with
.alpha.-syn WT for 24 h and then further incubated with 50 .mu.M
MNNG for 15 min. The .alpha.-syn fibrillization was detected by
western blot analysis 6 h after MNNG treatment.
[0088] FIG. 73 shows WT or PARP1 KO SH-SY5Y cells transfected with
.alpha.-syn A53T for 24 h and then further incubated with 50 .mu.M
MNNG for 15 min. The .alpha.-syn fibrillization was detected by
western blot analysis 6 h after MNNG treatment.
[0089] FIG. 74 shows WT or PARP1 KO SH-SY5Y cells were transfected
with .alpha.-syn WT for 24 h and then PAR polymer was delivered
with BioPorter for 6 h. The .alpha.-syn fibrillization was detected
by western blot analysis.
[0090] FIG. 75 shows SH-SY5Y cells transfected with .alpha.-syn WT
for 24 h were pretreated with 10 .mu.M ABT-888 or 1 .mu.M AG-014699
for 1 h, and further incubated with 50 .mu.M MNNG for 15 min. The
.alpha.-syn fibrillization was detected by western blot analysis 6
h after MNNG treatment. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test. *P<0.05, **P<0.005,
***P<0.001.
[0091] FIG. 76 shows .alpha.-syn PFF or PAR-.alpha.-syn PFF was
incubated with 0.5 .mu.g/ml of PK and immunoblotted with
epitope-specific antibodies to .alpha.-syn.
[0092] FIG. 77 shows PAR-.alpha.-syn PFF was generated with
increasing dose of purified PAR polymer. Primary cortical neurons
were treated with the same amount of .alpha.-syn PFF or
PAR-.alpha.-syn PFF for 14 days. Cell death was determined by
Hoechst and propidium iodide (PI) staining. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3-4).
[0093] FIG. 78 shows representative immunostaining of p-.alpha.-syn
(red) in primary cortical neurons treated with increasing amount of
.alpha.-syn PFF or PAR-.alpha.-syn PFF for 4 days. Nuclei are
stained with DAPI (blue).
[0094] FIG. 79 shows quantification of p-.alpha.-syn signals
normalized with DAPI (n=5). One-way ANOVA with Tukey's post hoc
test. *P<0.05, **P<0.005, ***P<0.001.
[0095] FIG. 80 shows sterological counts of SNpc DA neurons of WT
mice at 1, 3, and 6 months after instrastriatal PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR injection. (A) Ipsilateral
Nissl-neurons were counted. Bars represent mean.+-.s.e.m. One-way
ANOVA followed by Tukey's post hoc test (n=5 to 8 mice per group).
*P<0.05, ***P<0.001.
[0096] FIG. 81 shows sterological counts of SNpc DA neurons of WT
mice at 1, 3, and 6 months after instrastriatal PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR injection. contralateral
TH-neurons were counted. Bars represent mean.+-.s.e.m. One-way
ANOVA followed by Tukey's post hoc test (n=5 to 8 mice per group).
*P<0.05, ***P<0.001.
[0097] FIG. 82 shows sterological counts of SNpc DA neurons of WT
mice at 1, 3, and 6 months after instrastriatal PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR injection. Nissl-positive neurons
were counted. Bars represent mean.+-.s.e.m. One-way ANOVA followed
by Tukey's post hoc test (n=5 to 8 mice per group). *P<0.05,
***P<0.001.
[0098] FIG. 83 shows a schematic diagram of dopamine
metabolism.
[0099] FIG. 84 shows no significant difference of DA concentration
in the contralateral striatum of PBS, .alpha.-syn PFF,
PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3, and 6 months
measured by HPLC. Bars represent mean.+-.s.e.m. One-way ANOVA
followed by Tukey's post hoc test.
[0100] FIG. 85 shows ipsilateral DOPAC concentration in the
striatum of PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or
PAR-injected mice at 1, 3, and 6 months measured by HPLC. Bars
represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc
test (n=4 to 6 mice per group). *P<0.05, **P<0.005,
***P<0.001.
[0101] FIG. 86 shows contralateral DOPAC concentration in the
striatum of PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or
PAR-injected mice at 1, 3, and 6 months measured by HPLC. Bars
represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc
test (n=4 to 6 mice per group). *P<0.05, **P<0.005,
***P<0.001.
[0102] FIG. 87 shows ipsilateral 3-MT concentration in the striatum
of PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or PAR-injected mice
at 1, 3, and 6 months measured by HPLC. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=4
to 6 mice per group). *P<0.05, **P<0.005, ***P<0.001.
[0103] FIG. 88 shows contralateral 3-MT concentration in the
striatum of PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or
PAR-injected mice at 1, 3, and 6 months measured by HPLC. Bars
represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc
test (n=4 to 6 mice per group). *P<0.05, **P<0.005,
***P<0.001.
[0104] FIG. 89 shows ipsilateral HVA concentration in the striatum
of PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or PAR-injected mice
at 1, 3, and 6 months measured by HPLC. Bars represent
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=4
to 6 mice per group). *P<0.05, **P<0.005, ***P<0.001.
[0105] FIG. 90 shows contralateral HVA concentration in the
striatum of PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF, or
PAR-injected mice at 1, 3, and 6 months measured by HPLC. Bars
represent mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc
test (n=4 to 6 mice per group). *P<0.05, **P<0.005,
***P<0.001.
[0106] FIG. 91 shows representative immunoblots of TH, DAT, and
.beta.-actin in the striatum of PBS, .alpha.-syn PFF,
PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3, and 6
months.
[0107] FIG. 92 shows quantification of TH and DAT levels in the
striatum normalized to .beta.-actin. Error bars represent the
mean.+-.s.e.m. One-way ANOVA followed by Tukey's post hoc test
(n=3-4).
[0108] FIG. 93 shows behavioral abnormalities of PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3, and 6
months measured by pole test. Data are the means s.e.m. One-way
ANOVA followed by Tukey's post hoc test. *P<0.05, **P<0.005,
***P<0.001.
[0109] FIG. 94 shows behavioral abnormalities of PBS, .alpha.-syn
PFF, PAR-.alpha.-syn PFF, or PAR-injected mice at 1, 3, and 6
months measured by grip strength test. Data are the means s.e.m.
One-way ANOVA followed by Tukey's post hoc test. *P<0.05,
**P<0.005, ***P<0.001.
[0110] FIG. 95 shows establishment of PAR ELISA. ELISA detected the
PAR as low as 3 pM and was saturated at 50 nM.
[0111] FIG. 96 shows representative immunoblots of PAR, TH, and
.beta.-actin in the substantia nigra of control and PD
patients.
[0112] FIG. 97 shows quantification of PAR and TH levels normalized
to .beta.-actin. Error bars represent the mean.+-.s.e.m. One-way
ANOVA followed by Tukey's post hoc test (n=5). *P<0.05,
***P<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0113] Previously, it was discovered that a novel and useful
mechanism for preventing cell death following activation of PARP-1
by administering agents that interfere with the PAR-AIF interaction
(see US 20120122765 which is incorporated by reference in its
entirety). Such mechanisms are useful in treating patients
suffering from Parkinson's disease. In this regard, the
identification of AIF as a PAR polymer-binding protein establishes
that therapeutic compounds which inhibit the interaction of PAR
polymer with AIF may be useful in mono or combination therapies as
protective compounds against stressors which activate PARP-1.
[0114] In continuing this work, disclosed herein is a novel method
for monitoring and assessing patients suffering from PD. Also
disclosed is a novel method for monitoring and assessing the
effectiveness of medical treatment administered to a patient
suffering from PD. The theranostic methods disclosed herein enable
a medical professional to better develop a treatment plan for a
patient suffering from PD that has the best opportunity to slow or
arrest the progression of the disease.
[0115] In one aspect, disclosed herein is a method for determining
the poly(ADP-ribose) (PAR) concentration in cerebral spinal fluid,
the method comprising: collecting a sample of cerebrospinal fluid
(CSF) from a patient, performing a PAR-sandwich ELISA on the CSF
sample, thereby determining the PAR concentration in the CSF.
[0116] In some embodiments, the method further comprises comparing
the PAR concentration in the CSF sample to the PAR concentration in
at least one control sample. The control sample can be a previously
collected CSF sample from the same patient or from a different
patient. In some aspects, more than one control sample is used.
Each control sample can be from the same patient or a different
patient selected independently from every other control sample.
[0117] In some aspects, the control sample is a prepared standard
with a known concentration of PAR such that it would enable a
comparison of the tested sample to the control sample, thereby
allowing a determination of the PAR concentration in the tested
sample. Prepared control standards, such as those shown in FIG. 2,
may be used thereby allowing for a comparison of the tested sample
to a generated control curve thus enabling quantification of the
PAR concentration in the tested sample.
[0118] A description of the sandwich-ELISA is provided elsewhere
herein. In some aspects, the capture antibody, the detection
antibody, or both are anti-PAR antibodies. They may be monoclonal
or polyclonal, and they may or may not be humanized. In some
aspects, the antibody is an anti-PAR antibody prepared from a human
combinatorial antibody library. In some aspects the human
combinatorial library is the HuCAL.RTM. Technology from
Bio-RAD.RTM.. For examples of the HuCAL.RTM. Technology, see
Knappik, A. et al., (2000). "Fully synthetic human combinatorial
antibody libraries (HuCAL) based on modular consensus frameworks
and CDRs randomized with trinucleotides," J Mol Biol., 296:57-86,
and Prassler, J. et al., (2011). "HuCAL PLATINUM, a synthetic Fab
library optimized for sequence diversity and superior performance
in mammalian expression systems," J Mol Biol., 413:261-78, both of
which are incorporated by reference for their teachings
thereof.
[0119] The detection antibody is conjugated to at least one agent
suitable for detection by colorimetric or other assay. In some
aspects, a second detection antibody specific for the first
detection antibody is used, and the second detection antibody is
conjugated to the agent that is suitable for detection by
colorimetric or other assay. In some aspects the agent suitable for
detection by colorimetric assay is biotin. Horseradish peroxidase
(HRP) strongly binds to biotin and reacts with
3,3',5,5'-tetramethylbenzidine (TMB) to form a colored product. In
some aspects, the colorimetric assay uses HRP and TMB to measure
the concentration of PAR in a CSF sample of a patient.
[0120] In another aspect, disclosed herein is a method for
determining the therapeutic efficacy of a medical treatment for
Parkinson's disease, the method comprising: collecting a sample of
cerebrospinal fluid (CSF) from a patient, measuring the
poly(ADP-ribose) (PAR) concentration in the CSF sample, and
comparing the PAR concentration in the patient to a PAR
concentration in at least one control sample.
[0121] In some aspects, the control sample is a previously
collected sample of CSF from the same patient. The previously
collected sample of CSF may have been collected at any time in the
past. For example the sample may have been collected 1 month, 2
months, 3 months, 4 months, 6 months, 9 months, 12 months, 15
months, 18 months, 21 months, 24 months, 27 months, 30 months, 33
months or 36 months prior to the currently collected sample. In
some aspects, the sample is collected at any point during the
lifetime of the patient. This sample collection can be done on
regular or repeated basis as part of routine monitoring of the
health of a patient. In some aspects, the control sample is taken
from a healthy patient. Healthy as used herein means that the
patient is not exhibiting any symptoms of a medical condition that
might affect the results of the test. In some aspects, the healthy
patient is not exhibiting any symptoms of PD or similar
neurological condition.
[0122] It has been previously demonstrated that activation of
PARP-1 and the accumulation of PAR are implicated in the
neuropathologies of PD, so in some aspects disclosed herein, an
increase in the PAR concentration in the CSF of a patient indicates
that the patient either has PD or is at risk of developing PD. In
some aspects, if the PAR concentration in a patient is higher than
the control sample that was previously collected from the same
patient, it is indicative that the PD in the patient is worsening.
In some aspects, an increase in the PAR concentration in the
patient compared to the control sample that was previously
collected from the same patient, it is indicative that the medical
treatment received by the patient is not arresting the progression
of the disease.
[0123] In a patient previously diagnosed with PD, either using the
methods and techniques disclosed herein or by another method, and
the patient is receiving medical treatment for PD, when the PAR
concentration in the patient increases as compared to the PAR
concentration of a previously collected sample from the same
patient, it is indicative that the medical treatment is not
arresting the disease. In these instances a medical professional
will make a decision as to whether or not to continue the same
treatment or alter the treatment received by the patient.
[0124] In yet another aspect, disclosed herein is a method of
monitoring the disease progression of a patient with PD, the method
comprising: collecting a sample of cerebrospinal fluid (CSF) from
the patient, measuring the poly(ADP-ribose) (PAR) concentration in
the CSF sample, and comparing the PAR concentration in the patient
to a PAR concentration in at least one control sample.
[0125] In some aspects, the control sample is a previously
collected sample of CSF from the same patient. The previously
collected sample of CSF may have been collected at any time in the
past. For example the sample may have been collected 1 month, 2
months, 3 months, 4 months, 6 months, 9 months, 12 months, 15
months, 18 months, 21 months, 24 months, 27 months, 30 months, 33
months or 36 months prior to the currently collected sample. In
some aspects, the sample is collected at any point during the
lifetime of the patient. This sample collection can be done on
regular or repeated basis as part of routine monitoring of the
health and/or treatment of a patient.
[0126] When comparing the PAR concentration to a sample taken from
the same patient at an earlier time period, an increase of the PAR
concentration in the CSF is indicative that the condition of the
patient is worsening. If the patient is receiving medical
treatment, then an increased PAR concentration would indicate that
the treatment is not effective in the patient. If the PAR
concentration is the same or lower, it would indicate that the
treatment the patient is receiving is effective in either slowing
or arresting the progression of the disease. The PAR concentration
is measured using a sandwich-ELISA as disclosed elsewhere
herein.
[0127] In still yet another aspect, disclosed herein is a method
for diagnosing, or determining that a patient is at risk of
developing, PD, the method comprises: collecting a sample of
cerebrospinal fluid (CSF) from the patient, measuring the
poly(ADP-ribose) (PAR) concentration in the CSF sample, and
comparing the PAR concentration in the patient to a PAR
concentration in at least one control sample.
[0128] According to this method, in some aspects, a patient is
diagnosed with PD, or is at risk of developing PD, if the PAR
concentration in the CSF of the patient is greater than a
predetermined concentration. In some aspects, that predetermined
concentration is the PAR concentration in the CSF of a healthy
patient. In some aspects, measuring the PAR concentration in the
CSF of the patient is done using a sandwich-ELISA as disclosed
elsewhere herein.
[0129] According to this method, if a patient has a PAR
concentration higher than a control sample from either a healthy
patient or a control sample previously taken from the same patient,
then the patient is diagnosed with PD or is determined to be at
risk of developing PD. If the PAR concentration in the CSF of the
patient is lower than or equal to the PAR concentration in a
negative control or a control sample of a healthy patient, then the
patient does not currently have PD or the risk of developing PD is
low at that time. Because the ultimate cause of PD is as yet
unknown, it is not possible to conclude that a patient will never
develop PD.
[0130] The term "theranostics" as used herein refers to the process
by which an individualized treatment plan or therapy is developed
for a specific patient. The word defines the ongoing clinical
efforts to develop more specific, individualized treatments for
various diseases, and to combine diagnostic and therapeutic
capabilities into a single agent. The rationale arose from the fact
that many diseases, such as PD and cancer, are immensely
heterogeneous, and current treatments are effective for only
limited patient populations or subpopulations and/or at only
specific stages of disease progression. The hope was that a close
combination of diagnosis and therapeutics could provide therapeutic
protocols that are more specific to individuals and, therefore,
more likely to offer improved prognoses.
[0131] There are no standard treatments for PD. An individualized
treatment plan is developed for each patient based on their
symptoms and overall health. Treatment options include medication,
surgery, and life style modifications. Examples of medicaments used
to treat PD include, but are not limited to, levodopa, dopamine
agonists, amantadine, anticholinergics, COMT inhibitors, and MAO-B
inhibitors. Surgical treatments include, but are not limited to,
deep brain stimulation, thalamotomy, pallidotomy, and
subthalamotomy. Lifestyle treatments include, but are not limited
to, exercise and diet. Many patients often explore so-called
"alternative medicine" which can include herbal and vitamin
supplements. Additional treatments are always being studied in
clinical trials. In some aspects, a patient is receiving at least
one type of medical treatment specifically for PD. It is not
uncommon for a patient to receive multiple different types of
medical treatment in order to determine the optimal combination for
the patient. Regardless of how many different types of medical
treatment are being administered to the patient, the methods and
techniques disclosed herein are suitable for assessing the PAR
concentrations in the patient and determining the therapeutic
efficiency of the medical treatment or combination of those
treatments.
[0132] Also disclosed herein is a theranostic method that will aid
medical professionals in developing an individualized treatment
plan for a patient with PD. The method comprises collecting a
sample of cerebrospinal fluid (CSF) from a patient who is receiving
at least one medical treatment for PD, measuring the
poly(ADP-ribose) (PAR) concentration in the CSF sample, and
comparing the PAR concentration in the patient to a PAR
concentration in at least one control sample.
[0133] The medical treatment may be any treatment disclosed
elsewhere herein or it may be any other treatment administered by,
or under the supervision of, a medical professional. In some
aspects, if a patient has been administered a medicament for at
least a time period where the medicament is expected to have an
effect, and the PAR concentration in the patient has increased,
then the method further comprises altering the manner in which the
medicament is administered to the patient. Altering the manner in
which the medicament is administered may comprise increasing or
decreasing the dose of the medicament; it may comprise increasing
or decreasing the frequency that the medicament is taken by or
administered to the patient; it may include changing or eliminating
a specific medicament being administered to the patient.
[0134] In another aspect, if the PAR concentration in the CSF of
the patient has increased compared to a previously collected sample
of CSF from the same patient, the medical professional may alter
then medical treatment received by the patient to add in an
additional form of treatment. For example, if the patient has only
received one or more medicaments as treatment, then the medical
professional may recommend a surgical alternative treatment.
Alternatively, if the patient has received one medicament (e.g.,
levodopa), then the medical professional may recommend the addition
of a second medicament (e.g., a dopamine agonist). The exact nature
in which the medical professional alters the medical treatment of
the patient will be individualized based on the medical needs of
the patient and the professional judgment of the medical
professional.
[0135] As used herein, the term "biomarker" refers to the
definition established by the National Institutes of Health
Biomarkers Definitions Working Group. It is "a characteristic that
is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention." Also disclosed herein is
the use of PAR in the CSF of a patient as a biomarker for PD. The
methods disclosed elsewhere herein are suitable for either
diagnosing a patient with PD or determining if a patient is at risk
for PD. An elevated concentration of PAR in the CSF of a patient is
indicative of PD or indicative that a patient is at risk of
developing PD. If a patient is already exhibiting symptoms of PD,
then an elevated concentration of PAR in the CSF of the patient
suggests that the patient has PD. Symptoms of PD include, but are
not limited to, tremors or shaking, changes in writing or speech
patterns, loss of smell, difficulty sleeping, difficulty walking or
moving, slowed movements, constipation, masked face, dizziness or
fainting, or stooping or hunching over. These early warning signs
of PD coupled with an elevated PAR concentration in the CSF of a
patient suggest the patient has PD. Further testing may be
necessary to confirm this diagnosis. Also disclosed herein is the
use of PAR as a biomarker for PD. Detection of an elevated
concentration of PD in the CSF of a patient indicates that the
patient either has or is at risk of developing PD.
[0136] As used herein, "sandwich ELISA," and illustrated in FIG. 2,
is a variant of the traditional ELISA that is highly specific for
sample antigen detection and quantification. The sandwich ELISA
quantifies antigens between two layers of antibodies (i.e., a
capture antibody and a detection antibody). The antigen to be
measured must contain at least two antigenic epitopes capable of
binding to the antibody, since at least two antibodies act in the
sandwich. Either monoclonal or polyclonal antibodies can be used as
the capture and detection antibodies in a sandwich ELISA system.
Monoclonal antibodies recognize a single epitope that allows for
the fine detection and quantification of small differences between
antigens while polyclonal antibodies are often used as the capture
antibody to trap as much of the antigen as possible. One advantage
of sandwich ELISA is in the ease of sample preparation, meaning the
sample does not have to be purified before analysis. Another
advantage of sandwich ELISA is the sensitivity of the technique to
detect and quantify specific antigens.
[0137] In some aspects of the sandwich ELISA as disclosed herein,
the capture antibody is immobilized on a plate, chip or other
physical structure. In the second step, the immobilized capture
antibody is exposed to the sample that includes the target protein.
As described herein, the target protein is PAR. After a
predetermined time period, a first detection antibody is added to
bind to the antigen that is bound to the immobilized capture
antibody. In some embodiments, a second detection antibody is added
to bind to the first detection antibody. In all embodiments, at
least one of the detection antibodies will comprise a detectable
substrate. The detectable substrate may be an enzyme that reacts
with an additional reagent to form a detectable, and quantifiable,
product, or detectable substrate may be detectable without further
reaction. In some embodiments, the detectable substrate is biotin.
In all embodiments, the detectable substrate will be detectable and
quantifiable. In some embodiments, the detection of the substrate
will be colorimetric.
[0138] Human antibodies suitable for use in the sandwich ELISA
disclosed herein are known in the art. For example, human myeloma
and mouse-human heteromyeloma cell lines for the production of
human monoclonal antibodies have been described in the art (Kozbor
J., Immunol. 133:3001, 1984; Brodeur et al., "Monoclonal Antibody
Production Techniques and Applications," Marcel Dekker, Inc., New
York, 1987, both of which are incorporated by reference for their
teachings thereof). In some aspects, the antibody is an anti-PAR
antibody prepared from a human combinatorial antibody library
(e.g., HuCAL.RTM. Technology from Bio-RAD.RTM.).
[0139] To determine whether .alpha.-syn PFF induces the activation
of PARP, levels of PAR are measured using a highly sensitive and
specific PAR monoclonal antibody after administration of
.alpha.-syn PFF to primary mouse cortical neurons (FIGS. 5 to 12).
.alpha.-syn PFF (1 .mu.g/ml) induced PARP activation peaks between
3 to 7 days and remained elevated for up to 14 days (FIG. 5).
Accompanying the elevation of PAR is the death of neurons as
assessed by propidium iodide (PI) staining (FIGS. 6 and 7).
Treatment of cortical neurons with the PARP inhibitors, ABT-888
(veliparib) (10 .mu.M), or AG-014699 (Rucaparib) (1 .mu.M) or BMN
673 (Talazoparib) (10 .mu.M) prevents the .alpha.-syn PFF-mediated
PARP activation and cell death (FIGS. 6, 7, and 8). These PARP
inhibitors also reduce .alpha.-syn PFF-mediated phosphorylation of
.alpha.-syn at serine 129 (p-.alpha.-syn) as assessed by
immunostaining (FIGS. 37 and 38), and .alpha.-syn aggregation as
indicated by immunoblot analysis (FIGS. 39 and 40), both of which
are associated with pathology in .alpha.-synucleinopathies. Since
PARP-1 plays a major role in parthanatos, PARP-1 was deleted from
cortical neurons using CRISPR/Cas9 via adeno associated virus (AAV)
transduction carrying a guide RNA against PARP-1 (FIGS. 9. 10 and
41) or used cortical cultures from PARP-1 knockouts (FIGS. 11, 12,
and 42 to 46). Deletion or knockout of PARP-1 prevents .alpha.-syn
PFF-mediated PARP activation and cell death (FIGS. 9 to 12, 41 and
42). Knockout of PARP1 also reduces p-.alpha.-syn immunostaining
and .alpha.-syn aggregation as indicated by immunoblot analysis
(FIGS. 43 to 46). Treatment of cortical neurons with the broad
spectrum caspase inhibitor Z-VAD-FMK (Z-VAD) partially reduces
.alpha.-syn PFF toxicity. The necroptosis inhibitor Necrostatin-1
(Nec-1) and the autophagy inhibitor 3-Methyladenine (3-MA) had no
effect, while the PARP inhibitor ABT-888 prevents .alpha.-syn PFF
toxicity (FIGS. 47 and 48). Since PARP inhibition and knockout of
PARP-1 reduces the accumulation of pathologic .alpha.-syn as
indicated by a reduction of p-.alpha.-syn immunostaining,
cell-to-cell transmission of .alpha.-syn was assessed as previously
described. Knockout of PARP-1 reduces the cell-to-cell transmission
of pathologic .alpha.-syn (FIGS. 49 to 51). These results taken
together suggest that .alpha.-syn PFF primarily kills neurons
through parthanatos and that PARP-1 contributes to generation of
pathologic .alpha.-syn.
[0140] Since synthetic .alpha.-syn PFF kill primary cortical
neurons via parthanatos, experiments were done to determine if
parthanatos plays a role in the loss of DA neurons following the
intrastriatal injection of .alpha.-syn PFF using a standard and
validated protocol (FIGS. 13 to 18). A single intrastriatal
injection of .alpha.-syn PFF (5 .mu.g) induces PARP activation as
determined by assessing PAR levels (FIG. 13). Intrastriatal
injection of .alpha.-syn PFF into PARP-1 knockout mice fails to
increase PAR levels (FIG. 13). A single intrastriatal injection of
.alpha.-syn PFF leads to an approximate 50% loss of DA neurons 6
months following the injection in WT mice (FIGS. 14 and 15). In
contrast, a single intrastriatal injection of .alpha.-syn PFF into
PARP-1 knockout mice fails to induce DA cell loss (FIGS. 14 and
15). WT mice were also fed a diet containing the PARP inhibitor
ABT-888 (125 mg/kg) and compared with mice given a control diet
(FIGS. 14 and 15). Mice treated with ABT-888 exhibit significantly
less loss of DA neurons after an intrastriatal injection of
.alpha.-syn PFF compared to mice on the control diet (FIGS. 14 and
15). ABT-888 also reduces the formation of .alpha.-syn PFF induced
increase in PAR levels (FIG. 52). Tyrosine hydroxylase (TH) and
dopamine transporter (DAT) levels are also reduced in WT mice in
response to .alpha.-syn PFF as determined by western blot analysis,
while the reduction in TH and DAT levels is prevented in PARP-1
knockout more or ABT-888 treated ST mice (FIGS. 52 to 54).
Accompanying the loss of DA neurons is a reduction in striatal DA
and its metabolites as determined by HPLC in WT mice, but not
PARP-1 knockout mice or ABT-888 treated mice (FIGS. 16, 57, 58 and
59). An injection of intrastriatal .alpha.-syn PFF leads to
.alpha.-syn pathology as assessed by western blotting (FIGS. 52, 55
and 56) and immunostaining for p-.alpha.-syn in DA neurons of WT
mice (FIGS. 60 to 63). .alpha.-syn pathology is markedly reduced in
PARP-1 knockout mice and ABT-888 treated WT mice consistent with
the absence and reduction of neurodegeneration, respectively.
Intrastriatal injection of .alpha.-syn PFF causes deficits on the
pole test, a sensitive behavioral measurement of dopaminergic
function in WT mice, whereas there are no deficits in PARP-1
knockout mice and ABT-888 WT treated mice (FIGS. 17 and 64). Both
forelimb plus hindlimb and forelimb grip strength are also reduced
in WT mice after .alpha.-syn PFF injection, but not in PARP-1
knockout or ABT-888 treated WT mice (FIGS. 18 and 65). Taken
together, these results indicate that the striatal .alpha.-syn
PFF-induced loss of DA neurons is dependent on PARP-1.
[0141] Since PAR causes liquid demixing of intrinsically disordered
proteins leading to their aggregation, experiments were performed
to determine whether PAR could seed and accelerate .alpha.-syn
aggregation. Recombinant .alpha.-syn was incubated at 37.degree. C.
and agitated in the presence and absence of 5 nM PAR,
concentrations that are observed in brain tissue. High molecular
weight forms of .alpha.-syn in the absence of PAR are observed as
early as 4 hours of incubation and .alpha.-syn continues to
fibrillize with time (FIG. 19). Different molecular weight forms of
.alpha.-syn are observed at 72 hours. In the presence of PAR, the
fibrillization of .alpha.-syn is markedly accelerated with
different molecular weight forms of .alpha.-syn being observed as
early as 24 h of incubation (FIG. 19). Thioflavin T fluorescence
also indicates that PAR accelerates the fibrillization of
.alpha.-syn, while PAR alone has no effect on thioflavin T
fluorescence (FIG. 20). The effect of temperature on PAR mediated
acceleration of the fibrillization of .alpha.-syn was also assessed
(FIG. 66). PAR causes the fibrillization of .alpha.-syn at
temperatures that are not permissive for .alpha.-syn fibrillization
in the absence of PAR (FIG. 66). Transmission electron microscopy
(TEM) was used to monitor the formation of .alpha.-syn fibrils. At
12 hours .alpha.-syn fibrils begin to form in the absence of PAR
and become fully formed and extensive after 72 hours of agitation
and incubation at 37.degree. C. (FIG. 3C). In contrast, in the
presence of PAR, .alpha.-syn is extensively fibrillated at 12 hours
and becomes more extensively fibrillated at 24 and 72 hours (FIG.
21). At 36 hours the concentration dependence of .alpha.-syn
fibrillization in the presence of PAR was monitored. 1 nM PAR is
capable of enhancing .alpha.-syn fibrillization with peak
aggregation occurring at 5 nM PAR while 20 nM PAR does not
appreciably increase .alpha.-syn fibrillization (FIGS. 67 and 68).
Since PAR is a highly negatively charged molecule, the effects of
another highly charged polymer were tested, PolyA, and which had no
effects on .alpha.-syn fibrillization (FIGS. 69 and 70). ADP ribose
monomer also had no effect on .alpha.-syn fibrillization (FIGS. 69
and 70). To determine whether endogenous PAR formation can
accelerate .alpha.-syn fibrillization, primary mouse cortical
neurons overexpressing WT human .alpha.-syn following
AAV-.alpha.-syn transduction were treated with a toxic dose of
N-methyl-D-aspartate (NMDA). In WT cultures NMDA treatment leads to
activation of PARP as assessed by PAR immunoblot and a concomitant
aggregation of .alpha.-syn, while .alpha.-syn did not aggregate in
PARP-1 KO cultures treated with NMDA (FIG. 22). Exogenous
administration of PAR via Bioporter increases the aggregation of
.alpha.-syn in both WT and PARP-1 KO cultures transduced with
AAV-.alpha.-syn (FIG. 71), indicating that PAR, not PARP-1, can
directly increase .alpha.-syn aggregation. Two different PARP
inhibitors, ABT888 and AG014699, prevent .alpha.-syn aggregation
and PARP activation in response to NMDA administration (FIG. 23).
In SH-SY5Y cells the potent PARP activator
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhances the
aggregation of overexpressed WT or A53T .alpha.-syn in SH-SY5Y
cells, while MNNG has no effect in PARP-1 KO SH-SY5Y cells (FIGS.
72 and 73). Exogenous administration of PAR increased the
aggregation of .alpha.-syn in both SH-SY5Y WT and SH-SY5Y PARP-1 KO
cultures (FIG. 74). Two different PARP inhibitors, ABT888 and
AG014699, prevent .alpha.-syn aggregation and PARP activation in
response to MNNG administration in SH-SY5Y cells (FIG. 75).
[0142] To determine whether PAR changes the biophysical properties
of .alpha.-syn PFF, a series of biochemical analysis were conducted
using .alpha.-syn PFF and .alpha.-syn PFF in the presence of PAR
(PAR-.alpha.-syn PFF). First, proteinase K (PK) digestion of
.alpha.-syn PFF was performed and monitored by .alpha.-syn
immunoblots. .alpha.-syn PFF and PAR-.alpha.-syn PFF showed very
distinct banding patterns after PK digestion, with PAR-.alpha.-syn
PFF's being more resistant to increasing concentrations of PK (FIG.
24). PAR-.alpha.-syn PFF showed predominantly an undigested band of
.alpha.-syn (1.sup.st band) with comparable digested bands only at
higher concentration of PK, while .alpha.-syn PFF degraded into
smaller fragments (2.sup.nd to 5.sup.th band) at lower
concentration of PK (0.5 and 1 .mu.g/ml) and these bands became
predominant at higher concentration of PK (1.5-2.5 .mu.g/ml) (FIG.
24). Using epitope specific antibodies to .alpha.-syn, it was found
that PAR renders the majority of the .alpha.-syn regions resistant
to PK digestion (FIG. 76). The resistance to PK digestion of
PAR-.alpha.-syn PFF suggests that PAR induces the formation of
distinct .alpha.-syn PFF strains with more misfolded and compact
structure than .alpha.-syn PFF. .alpha.-syn PFF and PAR-.alpha.-syn
PFF-induced neuronal cell death were then compared in cultured
neurons. After 14 days of treatment, there is enhanced cell death
in cultures treated with PAR-.alpha.-syn PFF as compared to that
with .alpha.-syn PFF (FIG. 77). PAR itself does not cause
significant cell death even at higher concentration (20 nM) (FIG.
77). To further confirm the potencies of neuropathology,
p-.alpha.-syn immunoreactivity was monitored after treatment of
varying concentration of .alpha.-syn PFF or PAR-.alpha.-syn PFF.
P-.alpha.-syn immunoreactivity was observed at 20 ng of
PAR-.alpha.-syn PFF at an equivalent level to 500 ng of .alpha.-syn
PFF, suggesting the PAR modification of .alpha.-syn PFF increases
toxicity by 25 fold (FIGS. 78 and 79). PAR-.alpha.-syn PFF
significantly increased p-.alpha.-syn immunoreactivity at higher
concentrations (FIGS. 78 and 79). Phospho-.alpha.-syn
immunoreactivity at different time points was also monitored in
cultured neurons exposed to .alpha.-syn PFF or PAR-.alpha.-syn PFF.
In the absence of PAR, .alpha.-syn PFF treatment leads to barely
detectable p-.alpha.-syn immunoreactivity 1 day post treatment,
while PAR-.alpha.-syn PFF treatment leads to detectable levels of
p-.alpha.-syn immunoreactivity as early as 1 day post treatment and
markedly enhances immunoreactivity at 7 days (FIG. 25). Immunoblot
analysis of .alpha.-syn indicates that aggregated and
phosphorylated .alpha.-syn are detectable at 4 days, while in the
absence of PAR these species of .alpha.-syn are only detectable
after 7 days of treatment. In the presence of PAR there is an
increase in the aggregated form of .alpha.-syn (FIG. 26).
[0143] To determine whether the PAR-.alpha.-syn PFF strain exhibits
enhanced neurotoxicity in vivo, a single intrastriatal injection of
PAR-.alpha.-syn PFF (5 .mu.g) was compared to that of .alpha.-syn
PFF (5 .mu.g). There is trend toward the loss of DA neurons
ipsilateral to the injection side of SNpc after one month and a
significant loss of DA neurons after 3 months following
PAR-.alpha.-syn PFF injection, while .alpha.-syn PFF injection has
no effect at these time points (FIGS. 27, 28 and 80). Six months
after PAR-.alpha.-syn PFF or .alpha.-syn PFF injection there is no
significant difference in the loss of DA neurons (FIGS. 27, 28 and
80). There is no significant loss of DA neuron contralateral to the
injection side at any time point (FIGS. 81 and 82). PAR injection
by itself has no effect on DA neuron number (FIGS. 27, 28, and 80).
PAR-.alpha.-syn PFF also accelerate the loss of striatal DA and its
metabolites as determined by HPLC with significant reductions in DA
and its metabolites being observed 1 month after the
PAR-.alpha.-syn PFF injection in contrast to .alpha.-syn PFF (FIGS.
29 and 83 to 90). Three and 6 months after PAR-.alpha.-syn PFF or
.alpha.-syn PFF injection there is no significant difference in the
loss of DA and its metabolites (FIGS. 29 and 83 to 90). TH and DAT
levels are also reduced after PAR-.alpha.-syn PFF compared to
.alpha.-syn PFF as determined by western blot analysis three months
after the injection, while there is no difference in the degree of
loss at 6 months (FIGS. 91 to 94). .alpha.-syn pathology as
assessed by immunostaining for p-.alpha.-syn in DA neurons was
increased by PAR-.alpha.-syn PFF compared to .alpha.-syn PFF at 3
months and 6 months after injection (FIGS. 30 and 31).
PAR-.alpha.-syn PFF caused a deficit on the pole test at 3 months
consistent with loss of DA neurons and DA deficits at 3 months,
whereas there are no significant deficits in .alpha.-syn PFF or PAR
injected mice (FIGS. 32 and 93). Both forelimb plus hindlimb and
forelimb grip strength were also reduced in PAR-.alpha.-syn PFF,
but not .alpha.-syn PFF or PAR inject mice at 3 months (FIGS. 33
and 94). At 6 months there is no significant difference in the
behavioral deficits induced by PAR-.alpha.-syn PFF or .alpha.-syn
PFF (FIGS. 32, 33, 92 and 94). Taken together, these results
indicate that PAR-.alpha.-syn PFF are substantially more neurotoxic
then .alpha.-syn PFF in vivo.
[0144] To determine whether PAR plays a role in patients with PD,
PAR levels were monitored in the CSF of patients with PD versus
controls (Table 1) using a sensitive ELISA for PAR (FIG. 95). PAR
levels are elevated in PD patients compared to controls in two
independent patient cohorts (FIGS. 34 and 35). As previously
reported, PAR levels are increased in the substantia nigra of
patients with PD compared to controls (FIGS. 96 and 97, and Table
2). PAR immunoreactivity is colocalized with .alpha.-syn in Lewy
Bodies of PD patients (FIG. 36).
[0145] The results indicate that .alpha.-syn PFF kills neurons both
in vitro and in vivo via activation of PARP-1 in a cell death
process designated parthanatos. Knockout of PARP-1 and inhibition
of PARP prevents the neurodegeneration and behavioral deficits
initiated by an intrastriatal .alpha.-syn PFF injection. Activation
of parthanatos seems to be the primary driver of .alpha.-syn PFF
neurodegeneration since necroptosis and autophagy inhibitors have
no effect on .alpha.-syn PFF neurotoxicity and there is only modest
protection by caspase inhibition. It is known that .alpha.-syn PFF
induce inflammatory mediator activation, which likely contributes,
in part, to cell death and accounts for the modest neuroprotection
by the broad spectrum caspase inhibitor, ZVAD.
[0146] Recent studies have identified conformational variants of
.alpha.-syn strains that exhibit distinct neurotoxicity, seeding
abilities and propagation, which contribute to different properties
of .alpha.-synucleinopathies. Given that .alpha.-syn PFF induces
PARP activation and PAR accumulation, PAR then accelerates
.alpha.-syn fibrillization and changes the biochemical properties
of .alpha.-syn PFF converting it to a more toxic strain. Consistent
with this notion are observations that PAR-.alpha.-syn PFF shows an
approximate 25 fold increase in .alpha.-syn aggregation and
neurotoxicity compared to the parental .alpha.-syn PFF. Moreover
PAR-.alpha.-syn PFF-injected mice show an accelerated disease
progression and phenotype compared to .alpha.-syn PFF injected
mice. In addition to PAR levels being increased in cultured neurons
and mouse brain, it was observed that PAR levels in PD are elevated
not only in the substantia nigra, but also the CSF. The elevation
of PAR in the CSF and brains of PD patients and evidence of PARP
activation in the substantia nigra of PD patients suggests that
PARP activation contributes to the pathogenesis of PD through
parthanatos and conversion of .alpha.-syn to a more toxic
strain.
[0147] This written description uses examples to disclose the
invention, including the best mode, and also to enable any person
skilled in the art to practice the invention, including making and
using any devices or systems and performing any incorporated
methods. The patentable scope of the invention is defined by the
claims, and may include other examples that occur to those skilled
in the art. Such other examples are intended to be within the scope
of the claims if they have structural elements that do not differ
from the literal language of the claims, or if they include
equivalent structural elements with insubstantial differences from
the literal languages of the claims.
EXAMPLES
[0148] Animals
[0149] C57BL/6 mice were obtained from the Jackson Laboratories
(Bar Harbor, Me.). PARP-1 KO mice were obtained from The Jackson
Laboratory (Bar Harbor, Me., USA). The littermates of WT and PARP1
KO mice were used in experiments. All housing, breeding, and
procedures were performed according to the NIH Guide for the Care
and Use of Experimental Animals and approved by Johns Hopkins
University Animal Care and Use Committee.
[0150] Preparation of .alpha.-syn PFF and PAR-.alpha.-syn PFF
[0151] Recombinant mouse .alpha.-syn proteins were purified.
.alpha.-syn PFF were prepared in PBS by constantly agitating
.alpha.-syn with a thermomixer (1,000 rpm at 37.degree. C.)
(Eppendorf). After 7 days of incubation, the .alpha.-syn aggregates
were diluted to 0.1 mg/ml with PBS and sonicated for 30 s (0.5 sec
pulse on/off) at 10% amplitude (Branson Digital Sonifier, Danbury,
Conn., USA). The .alpha.-syn PFF were kept at -80.degree. C. until
use. Synthesis and purification of PAR polymer were performed as
described elsewhere. PAR-.alpha.-syn PFF was prepared by adding 5
nM or indicated dose of PAR in .alpha.-syn fibrillization
reaction.
[0152] Stereotaxic Injection of .alpha.-Syn PFF
[0153] Two to 3-month-old WT and PARP1 KO mice were deeply
anesthetized with a mixture of ketamine (100 mg/kg) and xylazine
(10 mg/kg). PBS, .alpha.-syn PFF, PAR-.alpha.-syn PFF or PAR was
unilaterally injected into striatum (2 .mu.l per hemisphere at 0.4
.mu.l/min) with the following coordinates: anteroposterior
(AP)=+2.0 mm, mediolateral (ML)=.+-.2.0 mm, dorsoventral (DV)=+2.8
mm from bregma. After the injection, the needle was maintained for
an additional 5 min for a complete absorption of the solution.
After surgery, animals were monitored and post-surgical care was
provided. Behavioral tests were performed 1, 3 and 6 months after
injection and mice were euthanized for biochemical and histological
analysis. For biochemical studies, tissues were immediately
dissected and frozen at -80.degree. C. For histological studies,
mice were perfused with PBS and 4% PFA and brains were removed,
followed by fixation in 4% PFA overnight and transfer to 30%
sucrose for cryoprotection.
[0154] Thioflavin T (ThT) Binding Assay
[0155] .alpha.-syn fibrillization with or without PAR was monitored
with ThT fluorescence. Aliquots of 5 .mu.L from the incubation
mixture were taken at various time points, diluted to 100 .mu.L
with 25 .mu.M ThT in PBS, and incubate for 10 min at room
temperature. The fluorescence was recorded at 450 nm excitation and
510 nm emission using SpectraMax plate reader (Molecular Devices,
Sunnyvale, Calif.). The experiments were performed in
triplicate.
[0156] Transmission Electron Microscopy (TEM) Measurements
[0157] .alpha.-syn PFF or PAR-.alpha.-syn PFF were adsorbed to glow
discharged 400 meshed carbon coated copper grids (Electron
Microscopy Sciences) for 2 min, quickly washed twice with Tris-HCl
(50 mM, pH 7.4), and floated upon two drops of 0.75% uranyl formate
for 30 s each. The grids were allowed to dry before imaging on a
Phillips CM 120 TEM operating at 80 kV. The images were captured
and digitized with an ER-80 CCD (8 megapixel) by advanced
microscopy techniques.
[0158] Intracellular Delivery of PAR
[0159] Purified PAR was intracellularly delivered using BioPORTER
(Genelnatis, San Diego, Calif.) according to the manufacturer's
instructions. PAR polymer was diluted to desired concentration with
PBS. The diluted solution was added to the dried BioPORTER reagent
and mixed gently, followed by incubation at room temperature for 5
min. The BioPORTER-PAR complex was added to cell culture after a
wash in serum-free media and incubated for 3-4 h at 37.degree. C.
Cultures were subsequently used for experiments.
[0160] Tissue Lysate Preparation and Western Blot Analysis
[0161] Human post mortem brain (Table 1) or mouse brain tissue were
homogenized and prepared in lysis buffer [50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 0.5% SDS, 0.5%
sodium-deoxycholate, phosphatase inhibitor mixture I and II
(Sigma-Aldrich, St. Louis, Mo., USA), and complete protease
inhibitor mixture (Roche, Indianapolis, Ind., USA)], using a Diax
900 homogenizer (Sigma-Aldrich). After homogenization, samples were
rotated at 4.degree. C. for 30 min for complete lysis, the
homogenate was centrifuged at 15,000 g for 20 min and the
supernatants were used for further analysis. Protein levels were
quantified using the BCA assay (Pierce, Rockford, Ill., USA),
samples were separated using SDS-polyacrylamide gels and
transferred onto nitrocellulose membranes. The membranes were
blocked with 5% non-fat milk in TBS-T (Tris-buffered saline with
0.1% Tween-20) for 1 h, probed using primary antibodies (Table 3)
and incubated with appropriate HRP-conjugated secondary antibodies.
The bands were visualized by ECL substrate.
TABLE-US-00001 TABLE 1 Clinical Information of Control and PD CSF
used in FIG. 34 Group Age Sex Control 72 F (n = 31) 82 F 82 M 56 F
70 F 69 F 67 F 57 F 70 F 66 F 78 M 46 F 61 F 68 M 54 F 66 F 46 F 76
M 68 M 74 M 57 F 64 F 65 F 69 F 82 F 71 M 55 M 67 F 60 M 74 F 69 M
PD 66 M (n = 80) 70 M 87 M 56 F 69 F 55 F 72 M 66 M 56 F 78 M 70 F
59 F 57 M 61 M 61 M 73 M 71 M 57 M 69 F 62 M 51 F 68 F 59 F 61 M 70
M 64 F 74 M 63 M 70 M 67 M 67 M 74 F 67 M 57 M 72 M 56 F 77 F 68 M
75 F 67 M 52 M 55 M 75 M 67 M 63 M 65 F 74 F 69 M 76 M 64 M 56 M 60
F 58 F 49 M 49 M 66 M 54 M 74 F 64 M 64 M 68 M 68 F 67 F 68 M 70 M
63 M 64 M 78 M 69 M 58 F 61 M 68 M 79 M 66 M 56 F 66 M 68 M 74 M 75
M 64 F Summarized information Group Mean age .+-. s.e.m. F/M ratio
(%) Control 66.9 .+-. 1.69 67.7/32.3 PD 66.0 .+-. 0.85
31.2/68.8
[0162] Cell Culture, Transfection, Primary Neuronal Culture and
Treatment
[0163] SH-SY5Y cells (ATCC) were cultured in DMEM containing 10%
fetal bovine serum and penicillin/streptomycin at 37.degree. C.
under 5% CO.sub.2. The cells were transfected using PolyFect
reagent (Qiagen) according to the manufacturer's instructions.
Primary cortical neurons from WT or PARP1 KO embryos were prepared.
Briefly, the primary cortical neurons were cultured at embryonic
day 16 in neurobasal media supplemented with B-27, 0.5 mM
L-glutamine, penicillin and streptomycin (Invitrogen, Grand Island,
N.Y.). The neurons were replaced to fresh medium every 3-4 days.
ABT-888 (10 .mu.M), AG-014699 (1 .mu.M), BMN 673 (10 .mu.M), Z-VAD
(20 .mu.M), Nec-1 (20 .mu.M) or 3-MA (500 .mu.M) was pretreated to
neurons 1 h before .alpha.-syn PFF treatment. .alpha.-syn PFF were
added at 7 days in vitro (DIV) and further incubated for indicated
times followed by cell death assay or biochemical experiments.
Primary neurons were infected with AAV2-control sgRNA or AAV2-PARP1
sgRNA (ViGene Bioscineces, Rockville, Md., USA), and
AAV-.alpha.-syn WT or AAV-.alpha.-syn A53T at DIV 4-5.
[0164] Cell Death Assessment
[0165] Primary cultured cortical neurons were treated with
.alpha.-syn PFF or PAR-.alpha.-syn PFF for 14 days. Percent of cell
death was determined by staining with 7 .mu.M Hoechst 33342 and 2
.mu.M propidium iodide (PI) (Invitrogen, Carlsbas, Calif.). Images
were taken and counted by Zeiss microscope equipped with automated
computer assisted software (Axiovision 4.6, Carl Zeiss, Dublin,
Calif., USA).
[0166] Microfluidic Chambers
[0167] Triple compartment microfluidic devices (TCND1000) were
obtained from Xona Microfluidic, LLC (Temecula, Calif., USA). Glass
coverslips were prepared and coated before being affixed to the
microfluidic device. Approximately 100,000 WT or PARP1 KO neurons
were plated per chamber individually. At 7 DIV, 0.5 mg .alpha.-syn
PFF were added into chamber 1. To control for direction of flow, a
50 .mu.l difference in media volume was maintained between chamber
1 and chamber 2 and chamber 2 and chamber 3. Neurons were fixed on
day 14 after .alpha.-syn PFF treatment using 4% paraformaldehyde in
PBS. The chambers were then processed for immunofluorescence
staining with p-.alpha.-syn antibody.
[0168] Behavioral Tests
[0169] The behavioral deficits in .alpha.-syn PFF injected WT or
PARP-1 KO mice, .alpha.-syn PFF injected mice fed ABT-888, and
.alpha.-syn PFF or PAR-.alpha.-syn PFF injected mice were assessed
by the pole test and the grip strength test 1 week prior to
sacrifice. All the experiments were performed in a blind
fashion.
[0170] Pole Test.
[0171] A metal rod (75 cm long with a 9 mm diameter) wrapped with
bandage gauze was used as the pole. Before the actual test, the
mice were trained for two consecutive days and each training
session consisted of three test trials. Mice were placed on 7.5 cm
from the top of the pole and the time to turn and total time to
reach the base of the pole were recorded. The end of test was
defined as placing all 4 paws on the base. The maximum cutoff time
to stop the test and recording was 60 s. After each trial, the maze
was cleaned with 70% ethanol.
[0172] Grip Strength Test.
[0173] Neuromuscular function was measured by determining the
maximal peak force developed by the mice using an apparatus
(Bioseb, USA). Mice were placed onto a metal grid to grasp with
either fore or both limbs that are recorded as `fore limb` and
`fore and hindlimb`, respectively. The tail was gently pulled and
the force applied to the grid before the mice lose grip was
recorded as the peak tension displayed in grams (g).
[0174] Dopamine and Derivatives Measurement Using HPLC
[0175] Biogenic amine concentrations were measured by
high-performance liquid chromatography with electrochemical
detection (HPLC-ECD). The striatum was rapidly removed from the
brain, weighted and sonicated in ice cold 0.01 mM of perchloric
acid containing 0.01% EDTA. The 60 ng of 3,4-dihydroxybenzylamine
(DHBA) was used as an internal standard. After centrifugation at
15,000 g for 30 min at 4.degree. C., the supernatant was cleaned
using a 0.2 .mu.m filter and 20 .mu.M of supernatant was analyzed
in the HPLC column (3 mm.times.150 mm, C-18 reverse phase column,
Acclaim.TM. Polar Advantage II, Thermo Scientific, USA) by a dual
channel coulochem III electrochemical detector (Model 5300, ESA,
Inc. Chelmsford, Mass., USA). The protein concentrations of tissue
homogenates were measured using the BCA protein assay kit (Pierce,
Rockford, Ill., USA). Data were normalized to protein
concentrations and expressed in ng/mg protein.
[0176] Immunohistochemistry and Immunofluorescence
[0177] Mice were perfused with PBS and 4% PFA and brains were
removed, followed by fixation in 4% PFA overnight and transfer to
30% sucrose for cryoprotection. Immunohistochemistry (IHC) and
immunofluorescence (IF) was performed on 40 .mu.m thick serial
brain sections. Primary antibodies and working dilutions are
detailed in Table 2. For histological studies, Free-floating
sections were blocked with 10% goat serum in PBS with 0.2% Triton
X-100 and incubated with TH or p-.alpha.-syn antibodies followed by
incubation with biotin-conjugated anti-rabbit or mouse antibody,
respectively. After three times of washing, ABC reagent (Vector
laboratories, CA, USA) was added and the sections were developed
using SigmaFast DAB peroxidase substrate (Sigma-Aldrich). Sections
were counterstained with Nissl (0.09% thionin). For the
quantification, both TH- and Nissl-positive DA neurons from the
SNpc region were counted through optical fractionators, the
unbiased method for cell counting, using a computer-assisted image
analysis system consisting of an Axiophot photomicroscope (Carl
Zeiss) equipped with a computer controlled motorized stage (Ludl
Electronics, Hawthorne, N.Y., USA), a Hitachi HV C20 camera, and
Stereo Investigator software (MicroBright-Field, Williston, Vt.,
USA). The total number of TH-stained neurons and Nissl counts were
analyzed. For immunofluorescent studies, double-labeled sections
with TH and p-.alpha.-syn antibodies were incubated with a mixture
of Alexa-fluor 488- and 594-conjugated secondary antibodies
(Invitrogen, Carlsbad, Calif., USA). The fluorescent images were
acquired by confocal scanning microscopy (LSM710, Carl Zeiss). All
the images were processed by the Zen software (Carl Zeiss). The
selected area in the signal intensity range of the threshold was
measured using ImageJ software.
TABLE-US-00002 TABLE 2 Clinical Information of Control and PD CSF
used in FIG. 35 Group Age Sex Control 71 M (n = 33) 62 F 63 M 63 F
74 F 65 M 62 M 64 M 79 M 66 M 27 M 60 F 55 M 60 M 62 F 60 F 65 F 66
F 27 M 63 F 62 M 62 F 60 F 74 M 61 M 66 M 76 F 65 F 61 F 71 M 71 F
63 F 74 M PD 50 M (n = 21) 67 F 76 M 57 F 54 M 51 F 56 F 50 M 66 M
53 M 56 F 50 M 43 M 75 M 58 F 69 M 62 F 62 M 85 M 73 F 82 F
Summarized information Group Mean age .+-. s.e.m. F/M ratio (%)
Control 62.76 .+-. 1.83 48.5/51.5 PD 61.67 .+-. 2.52 42.9/57.1
[0178] PK Digestion of .alpha.-Syn PFF
[0179] PK digestion was performed. Ten micrograms of .alpha.-syn
PFF or PAR-.alpha.-syn PFF were mixed with 0.5 to 2.5 ug/ml of PK
in PBS and incubated at 37.degree. C. for 30 min. The reaction was
stopped by adding 1 mM PMSF, boiled with SDS-sample buffer for 5
min. The bands of the PK digestion products were detected by
immunoblotting using epitope-specific .alpha.-syn antibodies (Table
3).
TABLE-US-00003 TABLE 3 List of Some of the Antibodies Tested in
this Study Antibody Source Identifier Dilution PAR Made in N/A
1:2,000 (WB) Dawson lab 1:500 (IF) PARP-1 BD Bioscience 611039
1:2,000 (WB for human) Cell Signaling 9532 1:1,000 (WB for mouse)
.alpha.-syn BD Bioscience 610787 1:3,000 (WB) 1:500 (IHC)
.alpha.-syn (121-125), Sigma S5566 1:3,000 (WB) Syn211 .alpha.-syn
(N-term) LSBio LS- 1:3,000 (WB) C352877 .alpha.-syn (115-122),
LB509 Abcam ab27766 1:3,000 (WB) .alpha.-syn (61-95), 5C2 Novus
NBP1- 1:3,000 (WB) 04321 p-.alpha.-syn (Ser129) Biolegend 825701
1:1,000 (WB) 1:500 (IF) TH Novus NB300-19 1:2,000 (WB) Biologicals
1:1,000 (IHC, IF) DAT Sigma D6944 1:1,000 (WB) GAPDH Abcam ab8245
1:5,000 (WB) .beta.-actin-HRP Sigma A3854 1:20,000 (WB)
[0180] Human Clinical Trials
Human CSF Samples and PAR ELISA
[0181] Participants at the Johns Hopkins University site of the
NINDS Parkinson's Disease Biomarker Program (PDBP) underwent
extensive clinical and cognitive testing and a lumbar puncture
annually. The CSF was centrifuged, aliquoted, and stored at
-80.degree. C. within one hour of acquisition. Two different clones
(#19 and #25) of monoclonal anti-PAR antibody were used for PAR
ELISA. Anti-PAR antibody (capture antibody, clone #19) (5 ug/ml)
was coated on 96-well microtiter plate (NUNC, Cat #46051), various
concentration of purified PAR (0-200 nM, positive control) and CSF
samples from either normal or PD patients were added to each well
and incubated for 1 h at room temperature (RT). After washing the
plates five times with PBST (0.05% Tween20 in PBS buffer), the
biotinylated PAR antibody (detection antibody, clone #25) was
incubated for 1 h at RT. The color change was detected via
HRP-conjugated streptavidin antibody (Thermo Scientific). The assay
can detect the PAR as low as 3 pM and is saturated at 50 nM.
Clinical Dementia Rating
[0182] The Clinical Dementia Rating (CDR) scale is a five-point
scale used to assess six different areas of cognitive and
functional performance applicable to patients with neurological
degeneration and dementia. See Hughes C P, Berg L, Danziger W L,
Coben L A, Martin R L, "A New Clinical Scale for the Staging of
Dementia," Br J Psychiatry, (1982) 140:566-72 which is incorporated
by reference for its teaching thereof. The six areas are memory,
orientation, judgment & problem solving, community affairs,
home & hobbies, and personal care. All patients were assessed
using the CDR scale and a personal interview to determine if they
had normal cognition, mild cognitive impairment, or dementia.
[0183] Student's t-test were used to compare the concentration of
PAR between controls and individuals with PD and those with normal
cognition and cognitive impairment. A generalized linear model then
evaluated the determinants of change in PAR concentration and
whether PAR concentration was associated with cognitive
changes.
Human Combinatorial Antibody Library
[0184] The HuCAL.RTM. technology presents an alternative method to
the conventional methods of obtaining custom antibodies. Whereas
the production of monoclonal antibodies requires immunizing a
mouse, rabbit, or goat, and subsequently extracting the B cells
from the spleen to recover the antibodies presented, HuCAL.RTM.
technology allows for faster production time. Through the use of
library of complementarity determining regions (CDR), 6 light chain
variable regions (V.sub.L) and 7 heavy chains (V.sub.H), it is
possible to generate billions of antibodies in vitro. This is
paired with a phage display that incorporates the antibody genes
into bacteriophages that presents the antibody on the coating via a
disulfide bond, thereby presenting a physical linkage of the
phenotype and genotype. Reductive cleavage of the disulfide bond
allows for the recovery of the antibodies following a screening,
independent of the affinity to the antigen. AbD Serotec was
provided with synthetic purified terminally-biotinylated PAR
polymer. It was synthesized through reductive amination of pure PAR
polymer (2-300 mer). The antibody clones were selected for binding
to only polymers and oligomers, and not the ADP-ribose monomer.
[0185] The HuCAL.RTM. antibody generation process begins with the
immobilization of the antigen (PAR) using a covalent coupling to
magnetic beads. These are subsequently incubated with the
HuCAL.RTM. library where nonspecific antibodies are washed out and
the specific antibody-phages are eluted. E. coli cultures are
subjected to infection from the specific antibody-phages to
generate an enriched antibody library for the subsequent round of
phage screening. The DNA from these enriched antibody-phases is
retrieved and subcloned into Fab expression vectors and plated into
E. coli colonies to produce the Fab fragments. Following this are
the colony picking, primary screening, where the colonies were
grown in a 384-microtiter plate. Antibody expression is induced and
collected after lysing the cultures. These cultures are screened by
ELISA with the terminally labelled-PAR antigen. The positive hits
from the primary screening are then sequenced to identify the
unique antibodies, which are stored for future synthesis for
reproducibility. Secondary screening was performed to select out
PAR monomer binding antibodies. Finally, expression and
purification through affinity chromatography was performed to
obtain antibody clones.
Sandwich-ELISA
[0186] Anti-PAR antibody (capture antibody, clone #19) (5 .mu.g/ml)
was coated on a 96-well microtiter plate (NUNC, Cat #46051).
Different concentrations of purified PAR (0-200 nM, positive
control) and CSF samples from either normal or PD patients were
added to each well, and incubated for 1 hr at room temperature
(RT). After washing the plates five times with PBST (0.05% Tween20
in PBS buffer), the biotinylated PAR antibody (detection antibody,
clone #25) was incubated for 1 hr at RT. The color change was
detected via HRP-conjugated streptavidin antibody. This assay
detects the PAR concentration as low as 3 pM and is saturated at 50
nM.
Results
[0187] One hundred ten individuals contributed CSF at baseline (80
PD, 30 control), 94 individuals contributed CSF at the first
follow-up (68 PD, 26 controls), 71 individuals contributed CSF at
the second follow-up (51 PD, 20 controls), and 36 individuals
contributed CSF at the third follow-up (28 PD, 8 controls). At
baseline, the average age for both PD and controls was
approximately 66 years (p=0.71) and 67% of PD patients were men
while 37% of controls were men (p<0.01). Mean PD duration was
6.7 years. There were differences in the mean concentration of PAR
between individuals with PD and controls at the first three visits,
with a trend toward a difference in the 4th visit (visit 1: PD mean
112.13, control mean 87.99 p=0.04; visit 2: PD mean 145.49, control
mean 110.63 p=0.04; visit 3: PD mean 132.29, control mean 86.06
p=0.01; visit 4: PD mean 151.88, control mean 111.07 p=0.08).
TABLE-US-00004 PAR Concentration (pM) PD Patients Healthy Controls
Visit 1 112.13 87.99 Visit 2 145.49 110.63 Visit 3 132.29 86.06
Visit 4 151.88 111.07
[0188] Disease status was a significant predictors of PAR
concentration (p<0.01), even after controlling for age, gender,
MDS-UPDRS Motor scores, levodopa equivalent dosing, and cognitive
impairment (see FIGS. 3 and 4). PAR concentration at visit 2 and
visit 4 were significantly different from PAR concentration at
visit 1 (p<0.01, p=0.01) Among only PD participants, PAR
concentration (p=0.03) and MDS-UPDRS Motor scores (p<0.01) were
significant predictors of cognitive decline.
[0189] Statistical Analysis
[0190] All data are represented as mean.+-.s.e.m. with at least 3
independent experiments. Statistical analysis was performed using
GraphPad Prism. Differences between 2 means and among multiple
means were assessed by unpaired two-tailed student t test and ANOVA
followed by Tukey's post hoc test, respectively. Significance was
assessed as *P<0.05, **P<0.005, and ***P<0.001.
[0191] In summary, there was a significant difference in the PAR
concentration between PD and control patients. There was also a
significant difference in the PAR concentration between patients on
subsequent sample collection visits.
* * * * *