U.S. patent application number 16/932941 was filed with the patent office on 2020-11-05 for novel pharmacological therapy for neuronopathic gaucher disease.
The applicant listed for this patent is Children's Hospital Medical Center. Invention is credited to Benjamin Liou, Ying Sun.
Application Number | 20200345808 16/932941 |
Document ID | / |
Family ID | 1000004969759 |
Filed Date | 2020-11-05 |
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United States Patent
Application |
20200345808 |
Kind Code |
A1 |
Sun; Ying ; et al. |
November 5, 2020 |
NOVEL PHARMACOLOGICAL THERAPY FOR NEURONOPATHIC GAUCHER DISEASE
Abstract
The instant disclosure relates to methods and compositions for
the treatment of Gaucher disease, particularly type II and III
neuronopathic Gaucher disease (nGD). The methods include the step
of administering to an individual in need thereof an effective
amount of a ryanodine receptor inhibitor or a pharmaceutically
acceptable salt thereof.
Inventors: |
Sun; Ying; (Mason, OH)
; Liou; Benjamin; (Cincinnati, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital Medical Center |
Cincinnati |
OH |
US |
|
|
Family ID: |
1000004969759 |
Appl. No.: |
16/932941 |
Filed: |
July 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15497789 |
Apr 26, 2017 |
10751387 |
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16932941 |
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62332178 |
May 5, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/7085 20130101;
A61K 31/395 20130101; G01N 2800/04 20130101; A61K 38/177
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 31/395 20060101 A61K031/395 |
Claims
1. A method of treating a condition associated with neuronopathic
Gaucher disease (nGD), in an individual in need thereof, comprising
administering an effective amount of dantrolene or pharmaceutically
acceptable salt thereof to said individual wherein said condition
is selected from one or more of brain inflammation, neuron loss,
and neurodegeneration.
2. The method of claim 1, wherein said Gaucher disease is type
II.
3. The method of claim 1, wherein said Gaucher disease is type
III.
4-13. (canceled)
14. A method for a neurological sign in an individual having nGD,
comprising administering dantrolene, or pharmaceutically salt
thereof, to said individual.
15. The method of claim 1, wherein said improvement in
neurodegeneration is an improvement in gait impairment.
16. The method of claim 1, wherein said administration is in an
amount sufficient to increase GCase activity.
17. The method of claim 1, wherein said administration reduces
substrate accumulation.
18. The method of claim 1, wherein said administration protects
calmodulin and CAMKIV expression.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
62/332,178, to Sun et al., filed on May 5, 2016, the contents of
which are incorporated by reference in their entirety for all
purposes.
BACKGROUND
[0002] Treatment of genetic brain disorders including
neurodegenerative diseases are an unmet medical need. These include
rare, lysosomal storage diseases, and more common diseases such as
Parkinson's disease. Neurodegenerative diseases strike about 50
million Americans each year, demanding enormous cost in medical
expenses and lost productivity. Currently, there is no effective
treatment for neurodegenerative diseases and there is a need for
such in the art. The instant disclosure seeks to address one or
more of the aforementioned needs in the art.
BRIEF SUMMARY
[0003] Disclosed are methods and compositions for the treatment of
Gaucher disease, particularly type II and III neuronopathic Gaucher
disease (nGD). The methods include the step of administering to an
individual in need thereof an effective amount of a ryanodine
receptor inhibitor or a pharmaceutically acceptable salt
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1. Ryrs expression in 4L;C* mouse brains. (A) Decreased
mRNA expression (fold change of WT level) of three Ryrs in 4L;C*
brain regions determined by RNASeq analysis (n=3 mice/group). (B)
Immunoblot of brain lysate showed Ryr3 was expressed at normal
levels at 13 days of age and significantly decreased by 44 days of
age in 4L;C* brain (27% of WT level) compared to WT mice. P-values
were from Student's t-test, n=3 mice/group, 2-4 replicates of the
experiment. (C) Immunofluorescence staining of brain sections by
anti-Ryr1 and anti-Ryr3 antibodies (green) showed decreased Ryr
signals in 4L;C* brain including cortex and midbrain regions. Scale
bar is 20 .mu.m for all the images. (D) Co-staining of Ryr3 (green)
with neural cell markers. NeuN (red) is for neurons. GFAP (red) is
for astrocytes. 04 (red) is for oligodendrocytes. Ryr3 signals were
detected in neurons, astrocytes and oligodendrocytes in WT
midbrains. 4L;C* midbrain had low level of Ryr 3 signals in those
cells compared to WT. Scale bar is 20 .mu.m for all the images.
DAPI (blue) stained cell nuclei. Images are representative from n=3
mice for each genotype.
[0005] FIG. 2. Substrates, calcium levels and mitochondrial
function in nGD (CBE-N2a) cells. (A) Substrates levels. GC and GS
concentration was significantly increased in CBE-N2a cells compared
to N2a cells. Treatment with G6 led to a significant reduction of
GC and GS in N2a and CBE-N2a cells, respectively. Dantrolene alone
did not significantly affect GC and GS levels in CBE-N2a cells.
CBE-N2a cells treated with both G6 and dantrolene showed similar
level of GC and GS reduction to G6 alone. (B) Cytosolic calcium
levels. Differentiated N2a cells were treated with CBE (2 mM) for 5
days. CBE-N2a cells were further incubated for 5 days with
ryanodine (Ryan, 10 dantrolene (Dan, 12.5 AM) or G6 (0.8 I.LM), or
combinations of Dan/G6 or Ryan/G6. Cytosolic calcium level was
measured using Fura-2 as reporter after adding caffeine. Relative
cytosolic calcium level [Fura-2 AF/F (340/380-510 nm)] normalized
to mg protein in cells shows the magnitude of cytosolic calcium
above baseline (see method). CBE-N2a cells had significant higher
cytosolic calcium levels than N2a cells. Ryanodine, dantrolene, and
G6 significantly reduced cytosolic calcium levels in CBE-N2a cells.
Combinations of Dan/G6 or Ryan/G6 in CBE-N2a cells also caused
significant decrease in cytosolic calcium levels. (C) Mitochondrial
function in N2a cells. Reduced oxygen consumption rate (OCR,
pmol/min/mg mitochondrial protein) parameters (ATP production,
basal respiration and maximal respiration) in CBE-N2a cells were
reduced compared to N2a cells. Dantrolene or G6 treatment
significantly improved OCR in CBE-N2a cells compared to untreated
CBE-N2a cells. Relative levels of OCR in treated cells compared to
N2a were indicated below the graph. Each data are from 2 to 4
experiments (n=3 cell or cell lysate samples/treatment group, six
replicates/sample/experiment) and reported as mean.+-.SEM. One-way
ANOVA with post-hoc Tukey test (P<0.05).
[0006] FIG. 3. Dantrolene treatment in 4L;C* mice. (A) Gait
analysis. Left stride (Left panel), right stride (Middle panel),
base width (Right panel). The mice were subjected to two to three
tests at 30 and 40 days of age. Dantrolene (Dan) treatment
significantly increased left and right strides in 4L;C* mice at 40
days of age, and reduced base width at both 30 and 40 days of age
compared to untreated 4L;C* mice. Littermate (4L;WT) mice that have
no phenotype were used as normal controls in the analysis. Data
were analysed by Student's t-test. (B) Life span. The survival rate
of dantrolene treated 4L;C* mice (blue) was significantly increased
compared to untreated 4L;C* mice (orange). Median survival days is
50 days or 44 days for treated or untreated 4L;C* mice,
respectively. The life span of dantrolene treated 4L;C* mice was
prolonged by 12.3%. Littermate (4L;WT) control mice (black) had
normal life span. Data are presented as Kaplan-Meier curve analysed
by Mantel-Cox test. (C) CNS-inflammation. Positive CD68 staining
(brown) in microglial cells indicate inflammation in 4L;C* brain.
Compared to untreated 4L;C, the CD68 signal was significantly
reduced in dan-trolene treated 4L;C* brains. The representative
image for each group is shown. CD68 signal intensity in brain
sections was quantitated by NIH image J and presented as % of
untreated 4L;C* level. P-value was from Student's t-test (n=2-3
mice/group). (D) Mitochondrial ATP production rate. 4L;C brain had
37% of ATP production rate (pmol/min/mg mitochondrial protein)
compared to WT brains. Dantrolene treatment on 4L;C* mice improved
ATP production to 77% of WT level. One-way ANOVA with post-hoc
Tukey test (P<0.05), n=3 mice/group, 6 replicates/sample/assay,
duplicate assays. (E) Immunoblot of LC3. LC3-II is barely
detectable in WT brain. LC3-II levels were increased in 4L;C
cerebrum compared to WT. Dantrolene treated 4L;C* cerebrum showed
significantly reduced level of LC3-II compared to untreated 4L;C*.
One-way ANOVA with post-hoc Tukey test (P<0.05), n=3 mice/group,
duplicate experiments.
[0007] FIG. 4. NeuN positive neurons in brain regions. WT,
dantrolene (Dan) treated 4L;C* and untreated 4L;C* brain sagittal
sections from 44 day old mice were stained with anti-NeuN antibody.
(A) Compared to WT cortex, cerebellum, midbrain and brain stem,
4L;C* mice had reduced NeuN positive cells (green) in those
regions. Representative images from each group are shown. (B)
Dantrolene treated 4L;C* mice had significantly more NeuN positive
cells than untreated 4L;C* in each region. In the graph, NeuN
positive cell counts in each group are shown as a percentage of WT
for each brain region. Data were analysed by One-way ANOVA with
post-hoc Tukey test (P<0.05), n=4 images/section, 2
sections/mouse, 3 mice/group.
[0008] FIG. 5. Ryr expression in dantrolene treated 4L;C* brain.
(A) Immunoblot of Ryr3 in CBE-N2a cells. Ryr3 protein level was
lower in CBE-N2a than N2a cells, and increased in dantrolene
treated CBE-N2a cells. (B) 4L;C* cerebrum showed significantly
reduced Ryr3 protein at 9% of WT level. In dantrolene treated 4L;C*
cerebrum, Ryr3 protein level was significantly increased compared
to untreated 4L;C*. 4L;C* panel was spliced to make panel layout
consistent with other graphs. A dotted line shows splice area. (C)
Immunofluorescence staining of Ryr3.4L;C*midbrain and brain stem
showed reduced Ryr3 (green) signal at 49% or 34% of WT level,
respectively. In dantrolene treated 4L;C* brain, Ryr3 signal was
increased to 94% in midbrain and 79% in brain stem of WT level.
DAPI (blue) stained cell nuclei. Scale bar is 20 1.un for all the
images. (D and E) CAMK IV and calmodulin (CAM). 4L;C* cerebrum
showed decreased level of CAMK IV (D) and increased level of CAM
(E) compared to WT. Dantrolene treatment normalized expression of
CAMK IV and CAM to nearly WT level One-way ANOVA with post-hoc
Tukey test (P<0.05), n=2-3 mice, 2-4 replicates of the
experiment
[0009] FIG. 6. Scheme for generating nGD cell model. A) N2a cells
were differentiated in the presence of retinoic acid (RA) and cAMP.
The differentiated N2a cells were treated with CBE, a Gcase
inhibitor, to generate nGD cells. B) Undifferentiated N2a Cells
were stained positive with anti-nestin antibody (green). The
differentiated CBE-N2a cells were positive for Map2 (red) a mature
neuron marker. Scale bar is same for all images.
[0010] FIG. 7. Recording of cytosolic calcium levels.
Differentiated N2a cells were treated with CBE (2 mM) for 5 days.
CBE-N2a cells were further incubated for 5 days with ryanodine
(Ryan, 10 uM), dantrolene (Dan, 12. uM) or G6 (0.8 uM), or
combinations of Dan/G6 or Ryan/G6. Cytosolic calcium levels were
measured using Fura-2 as a reporter. Baseline calcium levels were
measured prior to caffeine addition (-30 seconds). Cytosolic
calcium levels at the baseline (0 second) are the following:
(N2a/CBE)>(N2a/CBE/G6)>(N2a/CBE/Dan).about.(N2a/CBE/Dan/G6)>(N2a-
/CBE/Ryr/G6)>(N2a/CBE/Ryr)>(N2a). Calcium levels were
recorded every 30 seconds for a duration of 300 seconds after
addition of caffeine. CBE-N2a cells treated showed reduced
cytosolic calcium levels compared to CBE-N2a cells. Relative
cytosolic calcium level (Fura-2 .DELTA.F/F (340/380-510 nm) for
each time point were normalized to mg of protein in the cells. A
representative recording of three experiments is shown. Each data
point repeated 3 times. Mean.+-.SEM was plotted in the graph.
[0011] FIG. 8. Gait analysis with mannitol-vehicle group. Left
stride (Left panel), right stride (Middle panel), base width (Right
panel). The mice were subjected to two to three tests at 30 and 40
days of age. Compared to untreated 4L;C*, mannitol (Man)-4L;C* mice
did not show changes in base width. Dantrolene (Dan) treatment
significantly reduced base width at both 30 and 40 days of age
compared to untreated 4L;C* and Man-4L;C* mice. Both Dan and Man
improved left and right strides in 4L;C* mice at 40 days of age,
however, Dan-4L;C*mice (10%). Littermate (4L; WT) mice that have no
phenotype were used as controls in the analysis. Data were analyzed
by Student's t-test.
[0012] FIG. 9. NeuN positive neurons (green) in brain. (A) WT,
dantrolene treated 4L;C* and untreated 4L;c* brains were stained
with anti-NeuN antibody. (B) In the graph, NeuN positive cells were
quantitated from images of sagittal brain sections by Fiji for
Image J and presented as percentage of NeuN positive cells in WT
brains. One-way ANOVA with post-hoc Tukey test (p<0.05), (n=6
sections, 2 sections/mouse, 3 mice/group). Scale bar is same for
all images.
[0013] FIG. 10. (A) Increased GCase activity in dantrolene treated
fibroblasts. The cells were treated with 12.5 uM dantrolene for 5
days. Dantrolene treatment significantly increased GCase activity
in WT, 4L/4L, 4L;C* and 9H/9H fibroblasts, but did not affect GCase
in 9V/9V fibroblasts. Student's t-test (n=3 cell lysate
samples/genotype, each sample assayed in triplicates). (B) In
dantrolene treated mice, brain GCase activity was significantly
increased compared to untreated mice at 44 days of age. Student's
t-test (n=6 mice/group). (C) Glucosylceramide (GC) concentration
was decreased in dantrolene treated 4L;C* brain, but did not reach
significance, compared to the untreated 4L;C*. Glucosylsphingosine
(GS) concentrations in the dantrolene treated 4L;C* brains were not
significantly different from that in the untreated 4L;C* brain.
Student's t-test (n=6 mice/group). (D) Co-staining of GCase (green)
and Lamp1 (red). A bar graph is plotted at Y-axis of Pearson
Correlation Coefficient (PCC, r) calculated from the fluorescence
signals of captured images (n=8-13 cells/group) showing the degrees
of GCase co-localization with lysosomal marker Lamp 1. PCC was
increased 1.7-fold in dantrolene treated 4L;C* compared to
untreated 4L;C* brain. One representative image per group is shown.
One-way ANOVA with post-hoc Tukey test (p<0.05). Scale bar is
same for all images. (E). GC (left panel) and GS (right panel)
levels in 4L;C* brain with age. Student's t-test (n=2-10 mice per
age group).
DETAILED DESCRIPTION OF THE INVENTION
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
document, including definitions, will control. Preferred methods
and materials are described below, although methods and materials
similar or equivalent to those described herein can be used in
practice or testing of the present invention. All publications,
patent applications, patents and other references mentioned herein
are incorporated by reference in their entirety. The materials,
methods, and examples disclosed herein are illustrative only and
not intended to be limiting.
[0015] The terms and expressions used herein have the ordinary
meaning as is accorded to such terms and expressions with respect
to their corresponding respective areas of inquiry and study except
where specific meanings have otherwise been set forth herein.
[0016] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a method" includes a plurality of such methods and reference to "a
dose" includes reference to one or more doses and equivalents
thereof known to those skilled in the art, and so forth.
[0017] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, e.g., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, or up to 10%,
or up to 5%, or up to 1% of a given value. Alternatively,
particularly with respect to biological systems or processes, the
term can mean within an order of magnitude, preferably within
5-fold, and more preferably within 2-fold, of a value. Where
particular values are described in the application and claims,
unless otherwise stated the term "about" meaning within an
acceptable error range for the particular value should be
assumed.
[0018] The terms "individual," "host," "subject," and "patient" are
used interchangeably to refer to an animal that is the object of
treatment, observation and/or experiment. Generally, the term
refers to a human patient, but the methods and compositions may be
equally applicable to non-human subjects such as other mammals. In
some embodiments, the terms refer to humans. In further
embodiments, the terms may refer to children.
[0019] The term "therapeutically effective amount," as used herein,
refers to any amount of a compound which, as compared to a
corresponding subject who has not received such amount, results in
improved treatment, healing, prevention, or amelioration of a
disease, disorder, or side effect, or a decrease in the rate of
advancement of a disease or disorder. The term also includes within
its scope amounts effective to enhance normal physiological
function.
[0020] The terms "treat," "treating" or "treatment," as used
herein, refers to methods of alleviating, abating or ameliorating a
disease or condition symptoms, preventing additional symptoms,
ameliorating or preventing the underlying metabolic causes of
symptoms, inhibiting the disease or condition, arresting the
development of the disease or condition, relieving the disease or
condition, causing regression of the disease or condition,
relieving a condition caused by the disease or condition, or
stopping the symptoms of the disease or condition either
prophylactically and/or therapeutically.
[0021] The term "pharmaceutically acceptable," as used herein,
refers a material, such as a carrier or diluent, which does not
abrogate the biological activity or properties of the compounds
described herein. Such materials are administered to an individual
without causing undesirable biological effects or interacting in a
deleterious manner with any of the components of the composition in
which it is contained.
[0022] The term "pharmaceutically acceptable salt," as used herein,
refers to a formulation of a compound that does not cause
significant irritation to an organism to which it is administered
and does not abrogate the biological activity and properties of the
compounds described herein.
[0023] The terms "composition" or "pharmaceutical composition," as
used herein, refers to a mixture of at least one compound, such as
the compounds of Formula (I) provided herein, with at least one and
optionally more than one other pharmaceutically acceptable chemical
components, such as carriers, stabilizers, diluents, dispersing
agents, suspending agents, thickening agents, and/or
excipients.
[0024] The term "carrier" applied to pharmaceutical compositions of
the disclosure refers to a diluent, excipient, or vehicle with
which an active compound (e.g., dextromethorphan) is administered.
Such pharmaceutical carriers can be sterile liquids, such as water,
saline solutions, aqueous dextrose solutions, aqueous glycerol
solutions, and oils, including those of petroleum, animal,
vegetable, or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin, 18th Edition.
[0025] Disclosed are new treatment strategies for the management of
Gaucher disease, which may be similarly applied to treatment of
other neurodegenerative disorders such as Parkinson's disease. The
disclosed approaches include targeting the disease pathway,
balancing glycosphingolipid substrate production and/or
degradation, and restoring enzyme folding and trafficking to the
lysosome by chaperones.
[0026] Gaucher Disease
[0027] Gaucher Disease is an autosomal recessively inherited
lysosomal storage disease (LSD), caused by mutations on the GBA1
locus. First reported by Dr. Philippe Gaucher in 1882, the primary
biochemical defect is enzymatic deficiency of lysosomal
acid--.beta.-glucosidase, and is the most common lysosomal storage
disease. 90% of LSDs are neuronopathic.
[0028] Gaucher disease is caused by mutations in GBA1 that encodes
lysosomal acid 11-glucosidase (GCase) that has glucosylceramide
(GC) and its un-acylated form, glucosylsphingosine (GS) as
substrates (1-3). Gaucher disease is a common lysosomal storage
disease with a frequency of 1/57,000 live births (1). Based on
neuronopathic involvement, Gaucher disease is classified as type 1
(non-neuronopathic variant) and types 2 and 3 (neuronopathic
variants) (1). Type 2 patients present with acute neurological
signs and pathology within the first 3 to 6 months of life and with
death before 2 years of age (1,4). Type 3 patients exhibit
sub-acute neurological signs with a later onset and survival into
the 2nd to 4th decade (1,5,6). Two therapeutic strategies have
shown clinical efficacy in treating non-neuronopathic Type 1
Gaucher disease and include: 1) enzyme replacement therapy (ERT)
and 2) substrate reduction therapy (SRT). However, the enzyme in
ERT cannot cross the blood brain bather and the FDA approved SRT
compounds, miglustat and eliglustat, do not show effective central
nervous system (CNS) rescue (7-9). Thus, nGDs are not amenable to
current ERT and SRT. More recently, pharmaceutical chaperones and
newly developed small molecule substrate reduction agents have been
shown to penetrate into the brain. However, these have limited
efficacy in slowing disease progression and they do not alter the
disease course or prevent death in animal models (10-15). New
therapeutic approaches are needed to protect neuronal function as a
crucial goal for nGD intervention as has been a recent focus to
manage the CNS disease progression.
[0029] Accumulated substrates due to defective GCase function cause
pathology in the CNS of Gaucher disease. Studies from human
patients, animal models and cell models show involvement of
multiple pathological pathways in nGD pathogenesis including,
inflammation, mitochondrial dysfunction, disrupted calcium
homeostasis, altered autophagy/protease function and necrosis
(16-25). Disrupted calcium homeostasis, in particular, is a major
pathological factor contributing to many neurodegenerative diseases
and may lead to neurological deterioration in GD (18,19,25).
Dantrolene is an antagonist of ryanodine receptors (Ryrs) and
clinically used for the treatment of malignant hyperthermia and
neuroleptic malignant syndrome (26). Ryrs are a class of
intracellular calcium channels, expressed in muscles, neurons and
other cell types that mediate the release of calcium ions from
intracellular organelles, sarcoplasmic reticulum and endoplasmic
reticulum (ER). These are essential to a variety of signalling
pathways (27). The unique mechanism of dantrolene in blocking
intracellular calcium release through Ryrs makes it an attractive
potential approach to prevent neuronal dysfunction. Indeed,
Applicant has discovered that potential clinical utility for nGD
may be possible in view of findings that modulating calcium with
dantrolene improves neuronal function in several neurodegenerative
diseases including Huntington disease, Alzheimer diseases and
kinate-injury model (28-32).
[0030] There are three types of Gaucher Disease, as shown in Table
1.
TABLE-US-00001 TABLE 1 Types of Gaucher Disease Type 1 Type 2 Type
3 Non-neuronopathic Acute neuronopathic Sub-acute neuronopathic
Hepatosplenomegaly Neurodegeneration Neurodegeneration Bone Disease
Visceral involvement 6-80+ years <2 years 10-40 years 1/40,000
1/100,000 1/100,000 Ashkenazi Jews 1/850
[0031] 95% are Type 1, 1% are Type 2, and 5% are Type 3.
Approximately 400 patients were reported to the International
Collaborative Gaucher Registry (2015). Types 2 and 3 are
neuronopathic.
[0032] Symptoms of neuronopathic Gaucher (nGD) disease include
ataxia, dementia, seizures (progressive myoclonic), progressive
spasticity, abnormal brainstem auditory evoked potentials;
cognitive impairment and pathologic reflexes, dysphagia or apnea.
Histologically, neuronal loss, lipid laden macrophages in
periadventitial region, gliosis and microglial proliferation are
observed. Biochemically, increased levels of glucosylceramide and
glucosylsphingosine in the brain are observed. GBA1 mutations
confer a 20-30-fold increased risk for development of Parkinson's
disease (PD). Approximately 7-10% of PD patients have a GBA1
mutation, and GBA1 is listed as one of the target genes for disease
modifying strategies for Parkinson's disease by The Michael J. Fox
Foundation. The proposed mechanism of Gaucher disease linked
Parkinson's disease. See Mazulli et al., Cell V146:37-52, 8 Jul.
2011.
[0033] Currently, clinical therapies for Gaucher disease include
Enzyme replacement therapy (ERT) and Substrate reduction therapy
(SRT). ERT treatments include Cerezyme (imiglucerase) (Genzyme),
VPRIV (velaglucerase alfa) (Shire), and ELELYSO (taliglucerase
alfa) (Pfizer). SRT treatments include Cerdelga (eliglustat)
(Genzyme), and Zavesca (miglustat) (Actelion). ERT increases
Acid-beta-glucosidase (GCase), whereas SRT decreases
glucosylceramide synthase (GCS).
[0034] Applicant has discovered a novel treatment method for
Gaucher disease, particularly type II and III neuronopathic Gaucher
disease (nGD).
[0035] In one aspect, the instant disclosure provides a method of
treating an individual having Gaucher disease (nGD), in particular
type II or type III, comprising the step of administering an
effective amount of a ryanodine receptor inhibitor or a
pharmaceutically acceptable salt thereof. In one aspect, the
ryanodine receptor antagonist can be any ryanodine receptor
antagonist known in the art.
[0036] In one aspect, the ryanodine receptor inhibitor may be
selected from dantrolene, JTV-519, Flecainide-d3, Flecainide,
4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365), DHBP
(1,1'-diheptyl-4,4'-bipyridium), Ruthenium red (R2751), Ryanodine,
or a combination thereof.
[0037] JTV-519 is a potent inhibitor or the ryanodine receptor 2
(RYR2) blocker. RYR2 is a cardiac calcium channel that regulates
calcium levels in the sarcoplasmic reticulum. JTV-519 stabilizes
RYR2 in the closed state. (See, e.g., Science. 2004 Apr. 9;
304(5668):292-6. Protection from cardiac arrhythmia through
ryanodine receptor-stabilizing protein calstabin2. Wehrens XH1,
Lehnart S E, Reiken S R, Deng S X, Vest J A, Cervantes D, Coromilas
J, Landry D W, Marks A R.)
[0038] Flecainide-d3 is a deuterated version of the antiarrythmic,
Flecainide. (See, e.g., Mol Pharmacol. 2014 December;
86(6):696-706. doi: 10.1124/mo1.114.094623. Epub 2014 Oct. 1.
Multiple modes of ryanodine receptor 2 inhibition by flecainide.
Mehra D1, Imtiaz MS1, van Helden DF1, Knollmann BC1, layer
DR2.)
[0039] Flecainide is an antiarrythmic RyR-2 inhibitor (See, e.g.,
Mol Pharmacol. 2014 December; 86(6):696-706. doi:
10.1124/mo1.114.094623. Epub 2014 Oct. 1. Multiple modes of
ryanodine receptor 2 inhibition by flecainide. Mehra D1, Imtiaz
MS1, van Helden DF1, Knollmann BC1, layer DR2.)
[0040] 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA
365)--See, e.g., Inhibition of Ryanodine Receptors by
4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in
Canine Pulmonary Arterial Smooth Muscle Cells, Olga Ostrovskaya,
Ravi Goyal, Noah Osman, Claire E. McAllister, Isaac N. Pessah,
Joseph R. Hume and Sean M. Wilson, Journal of Pharmacology and
Experimental Therapeutics October 2007, 323 (1) 381-390; DOI:
https://doi.org/10.1124/jpet.107.122119.
[0041] DHBP (1,1'-diheptyl-4,4'-bipyridium)--See, e.g., J Neurosci
Res. 2007 Aug. 1; 85(10):2207-15. Functional ryanodine receptors
are expressed by human microglia and THP-1 cells: Their possible
involvement in modulation of neurotoxicity. Klegeris A1, Choi H B,
McLarnon J G, McGeer P L
[0042] Additional ryanodine receptor antagonists include Ruthenium
red (.mu.M) (R2751), Ryanodine (>10 .mu.M), Imperatoxin (nM)
(I148), and Dantrolene (.mu.M) (D9175), all of which are available
from Sigma.
[0043] In one aspect, the ryanodine receptor inhibitor may be
administered in an amount sufficient to reduce nGD associated
autophagy. Reduced autophagy may be determined by reduced LC3-II
levels as compared to pre-treatment levels in an individual.
[0044] In one aspect, the ryanodine receptor may be administered in
an amount sufficient to improve mitochondrial function.
[0045] In one aspect, the ryanodine receptor may be administered in
an amount sufficient to improve sensory motor function.
[0046] In one aspect, the ryanodine receptor may be administered in
an amount sufficient to increase or maintain Ryrs expression.
[0047] Further disclosed are methods of improving survival in an
individual having nGD Type II or Type III. The method may comprise
the step of administering an effective amount of a ryanodine
receptor inhibitor.
[0048] Further disclosed is an article of manufacture. The article
of manufacture may comprise a container comprising a label; and a
composition comprising a ryanodine receptor inhibitor or a
pharmaceutically acceptable salt thereof and a pharmaceutically
acceptable carrier, wherein the label indicates that the
composition for the treatment of an individual having nGD Type II
or Type III. The article of manufacture may further comprise a
means for delivery of said composition to an individual in need
thereof
[0049] Dosage
[0050] As will be apparent to those skilled in the art, dosages
outside of these disclosed ranges may be administered in some
cases. Further, it is noted that the ordinary skilled clinician or
treating physician will know how and when to interrupt, adjust, or
terminate therapy in consideration of individual patient
response.
[0051] In one aspect, the dosage of an agent disclosed herein,
based on weight of the active compound, administered to an
individual in need thereof may be about 0.25 mg/kg, 0.5 mg/kg, 0.1
mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, or
more of a subject's body weight. In another embodiment, the dosage
may be a unit dose of about 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1
mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1
mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25
mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg
to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg
to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5
mg, or 1 mg to 2.5 mg.
[0052] In one aspect, an agent disclosed herein may be present in
an amount of from about 0.5% to about 95%, or from about 1% to
about 90%, or from about 2% to about 85%, or from about 3% to about
80%, or from about 4%, about 75%, or from about 5% to about 70%, or
from about 6%, about 65%, or from about 7% to about 60%, or from
about 8% to about 55%, or from about 9% to about 50%, or from about
10% to about 40%, by weight of the composition.
[0053] The compositions may be administered in oral dosage forms
such as tablets, capsules (each of which includes sustained release
or timed release formulations), pills, powders, granules, elixirs,
tinctures, suspensions, syrups, and emulsions. They may also be
administered in intravenous (bolus or infusion), intraperitoneal,
subcutaneous, or intramuscular forms all utilizing dosage forms
well known to those of ordinary skill in the pharmaceutical arts.
The compositions may be administered by intranasal route via
topical use of suitable intranasal vehicles, or via a transdermal
route, for example using conventional transdermal skin patches. A
dosage protocol for administration using a transdermal delivery
system may be continuous rather than intermittent throughout the
dosage regimen.
[0054] A dosage regimen will vary depending upon known factors such
as the pharmacodynamic characteristics of the agents and their mode
and route of administration; the species, age, sex, health, medical
condition, and weight of the patient, the nature and extent of the
symptoms, the kind of concurrent treatment, the frequency of
treatment, the route of administration, the renal and hepatic
function of the patient, and the desired effect. The effective
amount of a drug required to prevent, counter, or arrest
progression of a symptom or effect of Gaucher disease can be
readily determined by an ordinarily skilled physician
[0055] Compositions may include suitable dosage forms for oral,
parenteral (including subcutaneous, intramuscular, intradermal and
intravenous), transdermal, sublingual, bronchial or nasal
administration. Thus, if a solid carrier is used, the preparation
may be tableted, placed in a hard gelatin capsule in powder or
pellet form, or in the form of a troche or lozenge. The solid
carrier may contain conventional excipients such as binding agents,
fillers, tableting lubricants, disintegrants, wetting agents and
the like. The tablet may, if desired, be film coated by
conventional techniques. Oral preparations include push-fit
capsules made of gelatin, as well as soft, scaled capsules made of
gelatin and a coating, such as glycerol or sorbitol. Push-fit
capsules can contain active ingredients mixed with a filler or
binders, such as lactose or starches, lubricants, such as talc or
magnesium stearate, and, optionally, stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable
liquids, such as fatty oils, liquid, or liquid polyethylene glycol
with or without stabilizers. If a liquid carrier is employed, the
preparation may be in the form of a syrup, emulsion, soft gelatin
capsule, sterile vehicle for injection, an aqueous or non-aqueous
liquid suspension, or may be a dry product for reconstitution with
water or other suitable vehicle before use. Liquid preparations may
contain conventional additives such as suspending agents,
emulsifying agents, wetting agents, non-aqueous vehicle (including
edible oils), preservatives, as well as flavoring and/or coloring
agents. For parenteral administration, a vehicle normally will
comprise sterile water, at least in large part, although saline
solutions, glucose solutions and like may be utilized. Injectable
suspensions also may be used, in which case conventional suspending
agents may be employed. Conventional preservatives, buffering
agents and the like also may be added to the parenteral dosage
forms. For topical or nasal administration, penetrants or
permeation agents that are appropriate to the particular barrier to
be permeated are used in the formulation. Such penetrants are
generally known in the art. The pharmaceutical compositions are
prepared by conventional techniques appropriate to the desired
preparation containing appropriate amounts of the active
ingredient, that is, one or more of the disclosed active agents or
a pharmaceutically acceptable salt thereof according to the
invention.
[0056] The dosage of an agent disclosed herein used to achieve a
therapeutic effect will depend not only on such factors as the age,
weight and sex of the patient and mode of administration, but also
on the degree of inhibition desired and the potency of an agent
disclosed herein for the particular disorder or disease concerned.
It is also contemplated that the treatment and dosage of an agent
disclosed herein may be administered in unit dosage form and that
the unit dosage form would be adjusted accordingly by one skilled
in the art to reflect the relative level of activity. The decision
as to the particular dosage to be employed (and the number of times
to be administered per day) is within the discretion of the
physician, and may be varied by titration of the dosage to the
particular circumstances of this invention to produce the desired
therapeutic effect.
[0057] Kits
[0058] Kits are also provided. In one aspect, a kit may comprise or
consist essentially of agents or compositions described herein. The
kit may be a package that houses a container which may contain a
composition comprising an oxime or pharmaceutically acceptable salt
thereof as disclosed herein, and also houses instructions for
administering the agent or composition to a subject. In one aspect,
a pharmaceutical pack or kit is provided comprising one or more
containers filled with one or more composition as disclosed herein.
Associated with such container(s) can be various written materials
such as instructions for use, or a notice in the form prescribed by
a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use, or sale for human
administration.
[0059] As there may be advantages to mixing a component of a
composition described herein and a pharmaceutically acceptable
carrier, excipient or vehicle near the time of use, kits in which
components of the compositions are packaged separately are
disclosed. For example, the kit can contain an active ingredient in
a powdered or other dry form in, for example, a sterile vial or
ampule and, in a separate container within the kit, a carrier,
excipient, or vehicle, or a component of a carrier, excipient, or
vehicle (in liquid or dry form). In one aspect, the kit can contain
a component in a dry form, typically as a powder, often in a
lyophilized form in, for example, a sterile vial or ampule and, in
a separate container within the kit, a carrier, excipient, or
vehicle, or a component of a carrier, excipient, or vehicle.
Alternatively, the kit may contain a component in the form of a
concentrated solution that is diluted prior to administration. Any
of the components described herein, any of the carriers, excipients
or vehicles described herein, and any combination of components and
carriers, excipients or vehicles can be included in a kit.
[0060] Optionally, a kit may also contain instructions for
preparation or use (e.g., written instructions printed on the outer
container or on a leaflet placed therein) and one or more devices
to aid the preparation of the solution and/or its administration to
a patient (e.g., one or a plurality of syringes, needles, filters,
tape, tubing (e.g., tubing to facilitate intravenous
administration) alcohol swabs and/or the Band-Aid.RTM. applicator).
Compositions which are more concentrated than those administered to
a subject can be prepared. Accordingly, such compositions can be
included in the kits with, optionally, suitable materials (e.g.,
water, saline, or other physiologically acceptable solutions) for
dilution. Instructions included with the kit can include, where
appropriate, instructions for dilution.
[0061] In other embodiments, the kits can include pre-mixed
compositions and instructions for solubilizing any precipitate that
may have formed during shipping or storage. Kits containing
solutions of Compound I, or a pharmaceutically acceptable salt
thereof, and one or more carriers, excipients or vehicles may also
contain any of the materials mentioned above (e.g., any device to
aid in preparing the composition for administration or in the
administration per se). The instructions in these kits may describe
suitable indications (e.g., a description of patients amenable to
treatment) and instructions for administering the solution to a
patient.
EXAMPLES
[0062] Neuronopathic Gaucher disease (nGD) manifests as severe
neurological symptoms in patients with no effective treatment
available. Ryanodine receptors (Ryrs) are a family of calcium
release channels on intracellular stores. The following data was
used to to determine if Ryrs are potential targets for nGD
treatment. A nGD cell model (CBE-N2a) was created by inhibiting
acid/3-glucosidase (GCase) in N2a cells with conduritol B epoxide
(CBE). Enhanced cytosolic calcium in CBE-N2a cells was blocked by
either ryanodine or dantrolene, antagonists of Ryrs and by
Genz-161, a glucosylceramide synthase inhibitor, suggesting
substrate-mediated ER-calcium efflux occurs through ryanodine
receptors. In the brain of a nGD (4L;C) mouse model, expression of
Ryrs was normal at 13 days of age, but significantly decreased
below the wild type level in end-stage 4L;C* brains at 40 days.
Treatment with dantrolene in 4L;C* mice starting at postnatal day 5
delayed neurological pathology and prolonged survival. Compared to
untreated 4L;C* mice, dantrolene treatment significantly improved
gait, reduced LC3-II levels, improved mitochondrial ATP production
and reduced inflammation in the brain. Dantrolene treatment
partially normalized Ryr expression and its potential regulators,
CAMK IV and calmodulin. Furthermore, dantrolene treatment increased
residual mutant GCase activity in 4L;C* brains. These data
demonstrate that modulating Ryrs has neuroprotective effects in nGD
through mechanisms that protect the mitochondria, autophagy, Ryr
expression and enhance GCase activity. This study suggests that
calcium signalling stabilization, e.g. with dantrolene, could be a
potential disease modifying therapy for nGD.
[0063] Here, nGD cell (CBE-N2a) and mouse (4L;C*) models were used
to determine the biochemical, histological, and behavioural effects
of dantrolene in nGD. The 4L;C* model is a viable analog of human
nGD that develops progressive accumulation of substrates and CNS
pathology and symptoms (4,18,33,34). 4L;C* mice have been used to
investigate pathological mechanisms and test potential therapeutics
for nGD (14,18,35). The present study shows dantrolene treatment
improves mitochondrial function and protects Ryrs expression in nGD
cell and mouse models. Furthermore, dantrolene treatment improved
gait, reduced inflammation and prolonged survival in 4L;C* mice,
establishing therapeutic potential for dantrolene in nGD.
[0064] Results
[0065] Ryrs Expression in Brain of the nGD Mouse Model
[0066] The nGD mouse model (4L;C*) has the homozygous Gba1 mutation
V394L/V394L and a lack of saposin C (33). The 4L;C* mice have
substrate (GC and GS) accumulation in the brain that increases with
age and is associated with progressive gait impairment, brain
pathology, and survival limited to approximately 45 days
(14,18,33). Transcriptome analysis by RNAseq of cortex, brain stem,
midbrain and cerebellum of 4L;C* mice brains showed a reduction of
Ryr1, Ryr2 and Ryr3 mRNAs at 44 days of age (FIG. 1A) (18).
Although all three Ryrs are expressed in the brain, Ryr2 is
predominantly seen in cardiac muscle (27,36). Levels of Ryrs
protein in neuronal cells and mouse brains were determined for Ryr3
or Ryr1 Immunoblot analysis showed Ryr3 protein in 4L;C* brains was
maintained at wild-type (WT) levels at 13 days of age, but was
significantly reduced to 27% of WT level at 44 days of age, the
end-stage of the disease (FIG. 1B). Immunofluorescence staining
with anti-Ryr1 and anti-Ryr3 antibodies showed that Ryrs signal at
44-days in 4L;C* cortex and midbrain was reduced below WT levels
(FIG. 1C). Neural cells expressing Ryrs were characterized by
co-staining of anti-Ryr3 antibody with either anti-NeuN (neuron),
anti-GFAP (astro-cyte), or anti-04 (oligodendrocyte) antibodies,
respectively. The results showed that Ryr3 was expressed in
neurons, astrocytes and oligodendrocytes (FIG. 1D). 4L;C* brains
had reduced Ryr3 levels in all three cell types (FIG. 1D). These
data demonstrate a reduction of Ryrs expression in 4L;C* brains at
the disease end-stage implicating Ryrs in nGD pathogenesis.
[0067] Dantrolene Blocks Substrate-Mediated Calcium Efflux from
Ryrs in nGD Cells
[0068] GC mediates ER calcium release (25). To determine if Ryrs
are involved in substrate-mediated ER calcium release, a new nGD
cell model (CBE-N2a) was generated using N2a cells. N2a is a mouse
neuroblastoma cell line that can be differentiated into mature
neurons by cAMP and retinoic acid (FIG. 6) (37). Differentiated N2a
cells were treated with conduritol B epoxide (CBE), an irreversible
covalently bound GCase inhibitor. This resulted in significant GC
and GS accumulation in CBE-N2a cells, thereby creating an nGD model
(FIG. 2A). The effects of Genz-161 (G6), an inhibitor of
glucosylceramide synthase to reduce the production of the
substrates GC and GS, were evaluated in CBE-N2a cells (14).
Treating N2a and CBE-N2a cells with G6 gave GC reductions to 7% and
26% of untreated levels, respectively (FIG. 2A). G6 treated CBE-N2a
also had significantly decreased GS (FIG. 2A). Dantrolene is an
antagonist of Ryrs and blocks Ryr-mediated ER calcium release (17).
Dantrolene alone did not affect GC and GS levels in CBE-N2a cells
(FIG. 2A). Co-treatment of G6 and dantrolene of CBE-N2a cells led
to a similar degree of GC and GS reduction as G6 alone, indicating
a specific effect of G6 on inhibition of substrate accumulation
(FIG. 2A). These results demonstrate that G6 inhibits GC production
and reduces substrate accumulation in CBE-N2a cells.
[0069] Using CBE-N2a cells as an nGD cell model, the effects of
dan-trolene, ryanodine and G6 on substrate-mediated ER-calcium
release were evaluated (FIG. 2B). After CBE-N2a cells were treated
with those agents, baseline calcium levels were recorded prior
caffeine addition. CBE-N2a cells have higher calcium levels than in
N2a cells without caffeine at baseline (FIG. 7). Caffeine is a
stimulant or releaser of calcium from ER stores for monitoring
GC/GS-mediated ER-calcium levels and the Ryr response (38,39).
After adding caffeine, cytosolic calcium levels were measured every
30 s over the duration of 300 s using Fura-2 (FIG. 7). The
magnitude of cytosolic calcium above baseline in the cells with
each treatment is shown in FIG. 2B. CBE-N2a cells showed a
significant increase in cytosolic calcium levels compared to N2a
cells. When CBE-N2a cells were treated with G6, the calcium levels
were significantly lower than that in untreated CBE-N2a cells (FIG.
2B), suggesting substrate-mediated calcium release. In
dantrolene-treated CBE-N2a cells, calcium levels were significantly
reduced compared to untreated CBE-N2a and approached levels
observed in control N2a cells (FIG. 2B). Consistent with
dantrolene, CBE-N2a cells treated with ryanodine also showed a
significant reduction in calcium levels (FIG. 2B). Co-treatment of
G6 and dantrolene or G6 and ryanodine also showed a significant
decrease in calcium levels. These results demonstrate that
substrate-mediated calcium release occurs through Ryrs.
[0070] Dantrolene Protects Mitochondrial Function in nGD Cells
[0071] To determine if substrate-mediated ER calcium efflux
promotes mitochondrial dysfunction, the effects of dantrolene and
G6 on mitochondrial function were evaluated in CBE-N2a cells.
Treated cells were plated on a Seahorse plate to assay
mitochondrial function reported as Oxygen Consumption Rate (OCR)
that includes the following parameters: ATP production, basal
respiration, and maximal respiration. CBE-N2a cells showed a
significant reduction in OCR as evidenced by approximately 50% in
all the parameters, including rate of ATP production, basal
respiration, and maximal respiration, compared to N2a cells,
indicating reduced mitochondrial function in this nGD cell model
(FIG. 2C). Dantrolene treatment significantly improved OCR in
CBE-N2a cells (FIG. 2C). G6 treatment also resulted in significant
increases in OCR in CBE-N2a cells (FIG. 2C). The greatest increase
in OCR was achieved when CBE-N2a cells were treated with both
dantrolene and G6 (FIG. 2C). These results demonstrate that both
reduction of substrate levels by G6 or antagonizing Ryrs by
dantrolene have protective effects on mitochondrial function in nGD
cells. Combining G6 and dantrolene resulted in more improvement
than either dantrolene or G6 alone.
[0072] Dantrolene Treatment Mitigates the Neuropathic Phenotype in
the nGD Mouse Model
[0073] The in vivo efficacy of dantrolene on nGD was evaluated in
the 4L;C* mouse model. 4L;C* mice were treated with dantrolene by
intraperitoneal (IP) injection at 10 mg/kg on each of three days
per week, starting from postnatal day 5. Because dantrolene
formulation contains mannitol, two control groups were included in
the study: untreated 4L;C* mice and 4L;C* mice injected with
mannitol at 30 mg/kg (same level of mannitol in dantrolene
formulation) on each of three days per week. Dantrolene treated
littermates (4L;WT) were also included as controls. These
litter-mates do not show neurological impairments and live beyond
80 days.
[0074] Gait was measured at 30 and 40 days of age. 4L;C* mice
typically develop a duck-like walking with paralysis of the hind
limbs (18,33). Untreated 4L;C* mice made significantly shorter
strides and had a wider base width at 30 and 40 days of age
compared to 4L;WT littermates (FIG. 3A). Dantrolene treatment
significantly reduced base stride width at 30 and 40 days of age
compared to age-matched untreated 4L;C* mice (FIG. 3A).
[0075] Dantrolene treated 4L;C* mice made significantly longer
strides, a 30% increase compared to untreated 4L;C* mice at 40 days
of age (FIG. 3A). However, the gait in dantrolene treated 4L;C*
mice did begin to deteriorate after 50 days of age (data not
shown). The life span of dantrolene treated 4L;C* mice was also
significantly extended compared to untreated 4L;C* mice (P=0.0012)
as determined by Kaplan-Meier analyses and the Mantel-Cox test
(FIG. 3B). The treatment prolonged survival in 4L;C* mice by 12.7%
compared to untreated 4L;C* mice (FIG. 3B). To determine if
mannitol would affect the phenotype, one group of 4L;C* mice was
injected with mannitol and gait and survival were evaluated.
Mannitol-4L;C* mice showed no changes on base width, but they had a
10% increase (P<0.05) in stride length compared to untreated
4L;C* mice at 40 days of age. However, this improvement is
significantly less than the 30% improvement in dantrolene treated
4L;C* mice (FIG. 8). No differences were observed in life span
between untreated and mannitol-treated 4L;C* mice (data not shown),
indicating a specific effect of dantrolene on survival. In the
following biochemical and histological studies, untreated 4L;C*
brains were used as a control for dantrolene treatment effect.
Dantrolene treatment did not affect body or organ weights in
treated mice. These results demonstrate that dantrolene
significantly improved gait, delayed motor function decline, and
extended the life span of 4L;C* mice.
[0076] CNS inflammation was determined by staining tissue sections
with an anti-CD68 antibody. CD68 is a marker for activated
microglia and macrophages. Positive CD68 signals (brown) were
detected in most brain regions of untreated 4L;C* mice (FIG. 3C).
With dantrolene treatment, the CD68 signal showed a significant
decrease of 63% of that in untreated mice, indicating attenuation
of CNS inflammation by dantrolene (FIG. 3C).
[0077] Brain mitochondrial function was determined by Seahorse
assay using isolated mitochondria from WT, untreated and dantrolene
treated 4L;C* brains at 40 days of age. ATP production rate in
4L;C* brain mitochondria was significantly reduced compared to WT
(FIG. 3D). Dantrolene treated 4L;C* mice showed significantly
increased mitochondrial ATP production rate in the brain, 77% of WT
level compared to 37% in the untreated 4L;C* brain (FIG. 3D)
indicating a protection of mitochondrial function in nGD by
dantrolene.
[0078] Altered autophagy is also evident in nGD and 4L;C* mouse
brains (20,33,34). LC3-II is a marker for autophagy activity and
was evaluated in dantrolene treated 4L;C* brains compared to
untreated 4L;C* and WT brains to determine the effect of the
treatment on autophagy function. LC3-II is a membrane bound form of
LC3. Elevated LC3-II levels indicate altered autophagy. By
immunoblot analysis, LC3-II was undetectable in WT brain. Increased
LC3-II levels were observed in untreated 4L;C* brains whereas
dantrolene treated 4L;C* brains had significantly reduced LC3-II
levels at 50% of untreated level, but still more than WT level
(FIG. 3E). This result indicated that dantrolene treatment
partially prevented alterations in autophagy in 4L;C* brains.
[0079] Neurodegeneration in 4L;C* brains were evaluated by counting
NeuN positive mature neurons in multiple brain regions (cortex,
brain stem, midbrain and cerebellum) from whole sagittal sections
(FIG. 4 and FIG. 9). 4L;C* brains had significantly fewer NeuN
positive cells (60-80% of WT NeuN+ cells) than WT brains. Treatment
with dantrolene resulted in an increase in NeuN positive cells to
>80% of WT NeuN+ cells. Decreased NeuN positive cells were
observed in cortex, cerebellum, midbrain and brain stem of 4L;C*
mice. All of those regions showed an increase in neurons with
dantrolene treatment compared to the untreated 4L;C* mice (FIG. 4).
These results indicated dantrolene treatment reduced
neurodegeneration in 4L;C* mice.
[0080] Dantrolene Protects Ryr, Calmodulin and CAMK IV
Expression
[0081] The effect of dantrolene on Ryr protein expression was
determined in nGD cells and 4L;C* mouse brains (FIG. 5) Immunoblot
analysis showed that Ryr3 protein was reduced to 47% of the WT
level in CBE-N2a cells. In dantrolene-treated CBE-N2a cells, levels
of Ryr3 were increased to 76% of WT level (FIG. 5A). In 4L;C* mice,
Ryr3 was profoundly decreased to just 9% of WT levels in the
cerebrum at 44 days of age (FIG. 5B). Dantrolene treatment in 4L;C*
mice significantly increased Ryr3 levels to 77% of WT in the
cerebrum (FIG. 5B) Immunofluorescence using an anti-Ryr3 antibody
confirmed the increase in Ryr signals. In dantrolene treated 4L;C*
midbrain (94% of WT) and brain stem (79% of WT) regions (FIG. 5C).
Ryr 3 signals were increased in neurons, astrocytes and
oligodendrocytes in dantrolene treated 4L;C* mouse brains See Table
1.
TABLE-US-00002 TABLE 1 Ryr3 signal levels in brain cells Neuron
Astrocyte Oligodendrocyte WT ++++ +++ ++ 4L; C* + Dan ++ ++ + 4L;
C.sup.+ + .+-. .+-. .sup.+relative level of immunoflourescence
signal of Ryr3 in the mice brain co-stained with Ryr3 and NeuN for
neuron, GFAP for astrocyte, or O4 for oligodendrocyte. Dan,
dantrolene.
[0082] In the calcium/calmodulin-dependent protein kinase pathway,
calmodulin, a calcium sensor protein, binds to calcium and promotes
a calcium-dependent kinase activity including CAMK IV
(Calcium/calmodulin-dependent protein kinase type IV) for gene
expression and biological functions (40). Calmodulin and CAMK IV
are potential modulators for Ryr expression. Decreased CAMK IV mRNA
and increased calmodulin 2 mRNA have been shown in 4L;C* brains
using RNAseq analyses (18). Expression levels of calmodulin and
CAMK IV in 4L;C* brains were determined by immunoblot. CAMK IV
protein level was reduced to 61% of WT level in untreated 4L;C*
cerebrum. With dantrolene treatment, the expression of CAMK IV was
increased by 28% (FIG. 5, D). In this study, calmodulin protein was
analysed using an anti-Cam antibody that reacts to a class of
calmodulins including calmodulin 2. Calmodulin levels were 2 fold
above WT levels in 4L;C* brains and were normalized to WT levels
with dantrolene treatment (FIG. 5, E). These data suggest that
modulating cytosolic calcium levels with dantrolene has protective
effects on Ryrs expression as well as proteins involved in the
calcium-calmodulin dependent signalling pathway.
[0083] Dantrolene Treatment Effects on GCase Function
[0084] Modulating ER calcium release promotes chaperone enhancement
of GCase stability and trafficking to the lysosome, thereby
improving GCase activity (17,41,42). The effect of dantrolene on
GCase activity was evaluated in mouse fibroblast cells from Gba1
mutants homozygous for V493L/V394L (4L/4L), D409H/D409H (9H/9H),
and D409V/D409V (9V/9V), 4L;C* and WT mice. Dantrolene-treated
4L/4L, 4L;C*, 9H/9H and WT fibroblasts showed significantly
increased GCase activity compared to the untreated cells (FIG. 10,
A). Dantrolene treated 9V/9V cells did not show an increase in
GCase activity.
[0085] This result showed that dantrolene promoted GCase activity
in select mutants, e.g. V394L and D409H, and WT enzyme.
[0086] In vivo effects of dantrolene on GCase function (activity
and substrate levels) were analysed in dantrolene treated mouse
brains at 44 days of age. The cerebrum was used for GCase activity
and cerebellum was used for substrates analysis. Compared to
untreated 4L;C*, dantrolene-treated 4L;C* cerebrum showed a 1.2
fold increase in GCase activity which is significantly higher than
untreated 4L;C* (FIG. 10, B). However, substrate (GC and GS)
concentrations were not reduced in the treated 4L;C* cerebellum
(FIG. 10, C). Co-staining for GCase and Lamp1 of mouse brain
sections showed a 1.7-fold increased colocalization of GCase within
the lysosomes of dantrolene-treated 4L;C* mice compared to
untreated 4L;C* brains. However, this level is <40% of Pearson
coefficient for WT GCase (FIG. 10, D), indicating <40% mature
mutant enzyme trafficked to the lysosomes in dantrolene-treated
brains. These results suggest that the effect of dantrolene on
GCase activity with the regimen used in this study was not
sufficient to translate into significant hydrolytic function for
clearance of excess substrates.
DISCUSSION
[0087] Currently, there are no effective treatments available for
nGD. The goal of the present study was to test the therapeutic
potential of a ryanodine receptor antagonist in cell and animal
models of nGD. Applicant has shown that dantrolene treatment
improves mitochondrial function, protects basal autophagy, delays
abnormal gait, and most importantly prolongs survival in nGD mice.
Dantrolene prevented the decreased expression of Ryrs in nGD cells
and mice, and normalized expression of calmodulin and CAMK IV, key
molecules in the calcium/calmodulin-dependent regulation pathway,
in mice (40,43,44). Furthermore, dantrolene reduced inflammation
and the loss of NeuN+ neurons in mice indicating a compelling
neuroprotective effect of treatment. This study shows that
modulating ER calcium efflux through Ryrs by dantrolene has
therapeutic value for nGD.
[0088] Neuroprotection by dantrolene is likely through several
mechanisms including protection of mitochondrial function, Ryrs
expression and autophagy, and enhancement of GCase activity.
[0089] Abnormal calcium homeostasis has been implicated in GD
(25,45,46). Accumulation of GC causes excess calcium efflux from
ER, specifically through Ryrs in neurons in GD (38). Sustained
increase in cytosolic calcium accounts for the abnormal cellular
function in many lysosomal storage diseases including GD (47),
although these changes are not fully understood. Using CBE-N2a
cells treated with G6 to inhibit glucosylceramide production, or
dantrolene to inhibit calcium efflux through Ryrs, Applicant
confirmed that increased cytosolic calcium efflux through Ryrs is
mediated by excess substrates, supporting previous findings (25).
Most importantly, this new nGD cell model allows further
investigation of altered cellular function resulting from disrupted
calcium homeostasis.
[0090] Ryr protein down-regulation in nGD cells and mouse brains is
a novel finding. All three Ryrs are expressed in brain. Ryr1 is
predominantly in the skeletal muscle and Ryr2 is mainly in cardiac
muscle, whereas Ryr3 is expressed at a low level in a variety of
tissues including brain (27,36,48). Decreased expression of Ryrs
has been reported in a mouse model of Alzheimer disease that also
shows aberrant cellular calcium homeostasis (49). Ryrs protein
levels in this study were determined primarily on Ryr3 with a
high-quality antibody. Ryr3 was shown to be ubiquitously expressed
in the mouse brain. Reduced staining for Ryr3 was found in neurons,
astrocytes and oligodendrocytes in 4L;C* mice at end-stage. Normal
levels of Ryr3 protein were expressed in 4L;C* brains at 13 days of
age, but were reduced to 27% of WT level by the end stage (44
days), suggesting Ryr down-regulation is associated with disease
progression and increased substrate accumulation. Thus, Ryr
down-regulation is likely caused by substrate accumulation. In
4L;C* mouse brain, substrate levels were higher than that in WT
even prior to 13 days, and continued to increase with age (FIG. 10,
E). Excess substrate can cause ER-calcium efflux and increases in
cytosolic calcium (25). The sustained increase of cytosolic calcium
could lead to a feed-back regulation, via calcium dependent
transcription regulation pathways, and reduce the expression of
Ryrs thereby preventing further calcium efflux from the ER. These
results show that blocking ER-calcium release through Ryrs with
dantrolene protects Ryr expression, supporting this feed-back
regulation mechanism. Early intervention before Ryr level reduction
would be critical to achieve maximal protection.
[0091] Expression of Ryrs can be regulated by the
calcium-signalling pathway. In this pathway, calcium and calmodulin
binding leads to basal CAMK IV activity which can regulate the
expression of Ryrs. Calmodulin bound to calcium is required for the
initiation of CAMK-CREB cascade for gene regulation (43,44,50,51).
Like many calcium binding proteins, Ryrs have CREB binding elements
(52), therefore, their expression is likely regulated by CAMK
IV-CREB signalling (53,54). Enhanced expression of calmodulin in
4L;C* brains may reflect increased cytosolic calcium. Reduced CAMK
IV mRNA and protein levels in 4L;C* brains suggest that less CAMK
IV is available for gene regulation, or decreased transcriptional
activity (18,52-54). Excess substrate-mediated calcium release in
nGD brains could hinder calcium/calmodulin kinase signalling
dependent CREB phosphorylation and lead to reduced Ryrs expression.
When nGD mice are treated at an earlier stage with dantrolene
(starting at postnatal 5 days) expression of CAMK IV and calmodulin
were normalized, highlighting their involvement in the modulating
Ryrs expression in nGD. Therefore, protection of Ryrs expression
with dantrolene should benefit nGD.
[0092] Ryrs down-regulation was concomitant with disease
progression. Importantly, the expression of Ryrs was protected by
dantrolene. This suggests a role for Ryr in nGD pathogenesis and an
attractive target for therapy. Ryrs on ER are key for the
regulation of intracellular calcium and an important calcium
modulator for neuronal function (49,55). Both ryanodine and
dantrolene are antagonists for Ryrs. Ryanodine is toxic to cells
making it unsuitable for in vivo studies. In contrast, dantrolene
is FDA approved for treatment of malignant hyperthermia (56),
readily available, and is distributed in tissues e.g. muscle and
liver. It also penetrates the blood-brain barrier to allow for
access into the brain (57). Dantrolene is specific to Ryr1 and Ryr3
(58). Blocking Ryrs with dantrolene is associated with improvement
in learning and memory performance and protection of synaptic
function in neurons in Alzheimer and Huntington disease models
(28,32). The significant attenuation of neuropathic phenotype in
4L;C* mice with dantrolene also suggests a clinical benefit for
nGD. Impaired calcium homeostasis is a common pathologic factor in
glycosphingolipid storage diseases. Specific substrate accumulation
causes reduced lysosomal calcium in Niemann-Pick C1 disease and
decreased ER calcium in Sandhoff and Niemann-Pick A diseases
(59-61). Thus, modulating Ryrs in order to normalize calcium
homeostasis could have therapeutic potential for glycosphingolipid
storage diseases.
[0093] Targeting Ryrs to modulate calcium homeostasis likely acts
through multiple mechanisms to achieve neuroprotection in nGD,
including protection of mitochondrial function and autophagy,
normalizing gene expression/regulation (Ryr), and rescuing mutant
GCase activity. Mitochondrial dysfunction is a well-documented
pathological feature in nGD (18,62). Inhibition of GCase function
by CBE leads to defective mitochondrial function in a human
dopaminergic cell line (23). In chronic nGD mouse brains decreased
mitochondrial ATP production and oxygen consumption are associated
with protein aggregation (a-synuclein and amyloid precursor
protein) on mitochondria (21). Impaired respiratory chain and
mitochondrial membrane potential has been reported in primary
neurons and astrocytes of acute nGD mice (23,62). Reduced
mitochondrial function is also a significant feature in 4L;C* mice
and in CBE-N2a cells (18). ER and mitochondria are physically and
functionally linked with altered calcium homeostasis promoting
mitochondrial dysfunction and disruption of the mitochondrial
membrane potential (63-65). Furthermore, the role of Ryr in ER
calcium and mitochondrial function is supported by a study of the
conditional knockout of cardiac Ryr2 in mice (66). Here, depletion
of Ryr2 leads to reduced mitochondrial calcium and oxidative
metabolism (66). Therefore, modulating calcium homeostasis through
Ryrs would have benefits for mitochondrial function. Indeed, in
both CBE-N2a cells and 4L;C* mice, dantrolene treatment
significantly improved the rate of mitochondrial oxygen consumption
and ATP production. These data support the notion that Ryr
antagonism may have protective effects on mitochondrial function in
nGD.
[0094] In many lysosomal storage diseases, defective lysosomal
enzyme/protein function interrupts the fusion of the lysosome with
the autophagosome for proper degradation of macromolecules (67-70).
Abnormal autophagy, either increased or decreased, has been
demonstrated in nGD cell and animal models, and patient brain
samples (5,20,22). Little is known, however, about the mechanisms
regulating autophagy in nGD, although it has been shown that
autophagy in general is regulated by cytosolic calcium (71,72).
Increased calcium load induced by starvation is known to induce
autophagy (71). In this study, LC3-II was used to monitor
autophagy. LC3 (microtubule-associated protein 1 light chain 3) has
two forms, LC3-I is distributed within the cytoplasm and nucleus
and LC3-II conjugates to phosphatidylethanolamine on the
autophagosomal membrane. Increased LC3-II reflects an induction of
autophagy in response to cellular stress. In the nGD mice,
dantrolene treatment significantly reduced LC3-II to WT levels
suggesting that autophagy in nGD is regulated by the ER-calcium
flux through Ryrs. Thus, modulating cytosolic calcium levels could
be a target for maintaining basal autophagy and normal cell
function.
[0095] Modulation of ER-calcium can regulate calcium-dependent
chaperones for GCase protein folding control and proper degradation
(17,41,42,73). Inhibition of Ryrs reduces intraluminal calcium
levels and enhances the expression of ER chaperone proteins that
promote protein folding. Specifically, for mutated GCase L'i44P in
fibroblasts, this leads to increased folding and trafficking to the
lysosome (17,74). Modulating ER proteostasis by antagonizing Ryr
with dantrolene has shown improvement in Niemann-Pick disease by
enhanced type C1 protein level and attenuated the cholesterol and
sphingolipids in Niemann-Pick type C disease fibroblasts (73). In
addition to Ryrs, blocking L type calcium channels (LTCC) also
induces chaperone activity on mutant GCase (75). Although
diltiazem, an LTCC blocker, improves expression of cellular
chaperones and folding of the mutated enzymes in mouse and human
fibroblasts (75,76), it fails to translate to similar changes in
animal models of GD (76). In the present study, dantrolene was able
to enhance GCase activity in select homozygous Gba1 mutations
(V493L/V394L, D409H/D409H), 4L;C* and WT mice fibroblasts. In 4L;C*
mouse brains dantrolene enhanced mutant GCase activity and
lysosomal localization, however, the level was not sufficient to
prevent substrate accumulation. Increasing administration to a
daily regimen may achieve better efficacy on GCase hydrolytic
function.
[0096] This study highlights a profound pathogenic role for Ryrs in
nGD and supports a novel strategy to preserve calcium homeostasis
in the early stages of nGD and thereby slow disease progression,
and demonstrates that targeting Ryrs to modulate calcium
homeostasis has neuroprotective potential in nGD by protecting
mitochondrial function, normalizing expression of Ryrs, protecting
autophagy, enhancing mutant GCase activity and reducing
inflammation and neuron loss.
[0097] Materials and Methods
[0098] Materials
[0099] The following were from commercial sources: N2a
(ATCCIO-CCLim-131, VA); CBE (EMD Millipore, Bedford, Mass.);
Dantrolene, retinoic acid, dbcAMP and caffeine (Sigma, St. Louis,
Mo.); M-PER Mammalian Protein Extraction Reagent, SuperSignalm West
Dura ECL detection kit, Pierce BCA protein assay kit (ThermoFisher
Sci, Waltham, Mass.). Antibodies: Ryr1, Ryr3, Map2 and Nestin (EMD
Millipore, Bedford, Mass.); CAM (calmodulin) (Santa Cruz
Biotechnology, Dallas, Tex.); LC3 (Novus, Littleton, Colo.); CAMK
IV (calcium/calmodulin-dependent kinase IV) (Abcam, San Francisco,
Calif.); CD68 (BD Biosciences, Franklin Lakes, N.J.); NuPAGE Gels
(3-8%, 4-12%, 10%, 16%) and Bis-Tris Buffer, Sodium Acetate Buffer,
SeaBlue Protein Standard plus, PVDF membrane, iBlot transfer kit,
and iBind Cards (Life Technologies. Carlsbad, Calif.); AP colour
reagent and AP Conjugate Substrate Kit (Bio-Red, Hercules, Calif.);
VECTASHIELD mounting medium containing DAPI (Vector, Burlingame,
Calif.); Glass chamber slides (LAB-TEK, Rochester, N.Y.).
Genz-161(G6) was provided by Sanofi Genzyme (Cambridge, Mass.).
[0100] Cell Culture and Treatment
[0101] N2a cells were maintained in DMEM/10% FBS (Gibco) medium.
Neuronal differentiation of N2a was carried out in the
differentiation medium containing 0.5% FBS, 10 .mu.M retinoic acid,
50 ng/ml GDNF (Alomore lab) and 1 mM dbcAMP in DMEM for 3 days
(37). The differentiated N2a cells were treated with 2 mM CBE for 5
days in the differentiation medium. Dantrolene (12.5 .mu.M),
ryanodine (10 .mu.M) or G6 (0.8 .mu.M) was added to the CBE-N2a
cells and cultured for 5 days. The medium with the reagents was
changed every 2 days. The differentiation status of the neuronal
cells was confirmed by anti-Map2 (mature neuron) or anti-Nestin
(undifferentiated neuronal cells) antibody staining. Cell viability
after each drug treatment was measured by CellTiter-Fluoirm Cell
Viability assay (Promega). The dose for each compound was
determined from CellTiter-Fluor.TM. Cell Viability assay with
>95% viability and endotoxin test of <0.05 EU/ml (GenScript
ToxinSenor). The cells were from same passage and treated at the
same time for all the experiments.
[0102] Calcium Level Measurement
[0103] Intracellular calcium levels were determined using the
Fura-2 QBT Calcium Kit (Molecular Devices, Sunnyvale, Calif.) on 96
well plates by M5 SpectraMax plate reader (Molecular Devices). N2a
cells were seeded in each well (20,000/well) with differentiation
medium (0.5% FBS, DMEM, 10 AM retinoic acid, 1 mM dbcAMP) for 72 h
(37). The differentiated N2a cells were cultured with DMEM medium
with 10% FBS containing CBE (2 mM) for 5 days, followed by
treatment with different combinations of dantrolene (12.5 .mu.M),
ryanodine (10 .mu.M) and G6 (0.8 .mu.M) for a further 5 days. The
cell medium was replaced every two days. The treated cells were
incubated with Fura-2 QBT dye at 37 C for 1 h for Fura-2 QBT uptake
(Fura2 QBT.RTM. calcium kit protocol, Molecular Devices). Caffeine
(10 mM) was used as an ER calcium efflux stimulant in the assay
(38,39). The plate reader setting was 340/380 nm (excitation) and
510 nm (emission). Baseline calcium levels were measured prior to
caffeine addition (-30 s). Cytosolic calcium levels were recorded
every 30 s for duration of 300 s after addition of caffeine.
Relative cytosolic calcium levels were determined with the values
at 300 s endpoint minus Os baseline. Relative cytosolic calcium
level [Fura-2 AF/F (340/380510 nm] for control or treated cells was
calculated by Fura-2 fluorescence unit (FU) measured at 340 nm/510
nm (AF=RFU/340 at 300 s end point minus baseline RFU/340 at 0 s)
divided by FU at 380 nm/510 nm (F=RFU/380 at 300 s-baseline RFU/380
at 0 s) (Fura2 QBT.RTM. calcium kit protocol, Molecular Devices)
and normalized to protein level in cells. Data were collected and
analysed by SoftMax Pro software (Molecular Devices) and graphical
charts were generated by Graphpad Prism 6.0.
[0104] Mice and Treatment
[0105] 4L;C* mice were generated by backcrossing of V394L/V394L
Gba1 (4L) and saposin C-/- (C) homozygosity as described previously
(33). The strain background of 4L;C* mice was C57BL/6J:129SvEv.
Strain- and age-matched WT mice and non-4L;C* littermates (no
neurological phenotype) were included as controls. All mice were
housed under pathogen-free conditions in an animal facility
according to IACUC approved protocols at Cincinnati Children's
Hospital Medical Center (CCHMC).
[0106] The 4L;C* mice and non-4L;C* littermates were treated with
Dantrolene sodium (Revonto.RTM. US WorldMeds, Louisville, Ky.)
starting at postnatal day 5 by IP injection at 10 mg/kg, three days
per week. Mouse body weight was recorded before each injection.
Control mice received vehicle (mannitol, APP Pharmaceuticals,
Schaumburg, Ill.) injections or no injection at all. The mice were
monitored for survival and gait impairments during the
treatment.
[0107] Gait Analysis
[0108] Progression of the neurobehavioural phenotype was assessed
by gait analysis to determine sensorimotor function (77,78). Mice
were trained to walk through a narrow alley leading into their
home-cage. Once trained, paper was placed along the alley floor and
the hind paws of each mouse were brushed with nontoxic paint. The
mice were then placed at the beginning of the alley. As they walked
into their home-cage they left their paw prints on the paper
underneath. Stride length and width were determined by measuring
the distance between hind paw prints. The 4L;C* and control mice,
untreated and non-4L;C* littermates were tested for gait at 30 and
40 days of age.
[0109] RNAseq Analyses
[0110] Expression of Ryr mRNAs in 4L;C* brain regions (cortex,
cerebellum, midbrain and brain stem) was analysed by RNAseq as
described previously (18).
[0111] Immunohistochemistry and Immunofluorescence
[0112] Mouse brains were collected after transcardial perfusion
with saline. Half of the brain (sagittal cut) was fixed in 4%
paraformaldehyde (PFA) for processing as frozen blocks. CD68
monoclonal antibody staining was performed as previously described
(33) using The BenchMark XT IHC/ISH Staining Module (Ventana
Medical System, Tucson, Ariz.) at CCHMC Pathology Research Core.
Fixed sections were counterstained with hematoxylin. The sections
were then scanned by AperiolmageScope v2. The CD68 signal was
quantified using Image J FIJI (79).
[0113] Immunofluorescence staining was performed on PFA fixed
brains. The brain sections were incubated in 0.3% Triton X-100 for
30 min, and treated with 50 mM NH.sub.4Cl in 1.times.PBS for 15 min
followed by 1.times.PBS wash. The sections were blocked for 1 hr at
RT in Blocking buffer (10% goat serum and 0.4% Triton X-100 in
PBS). Rabbit anti-Ryr 1 antibody (1:100) was diluted in the
blocking solution and incubated overnight at 4.degree. C. After
washing in PBS (3.times.10 min), the secondary antibody goat
anti-rabbit conjugated with Alexa Fluor.RTM. 488 (1:1500) in
blocking buffer was applied to the sections and incubated for 2 hrs
at RT. Mouse anti-Ryr 3 antibody (1:100) and goat anti-mouse
conjugated with Alexa Fluor.RTM. 488 (1:1500) were used for Ryr 3
detection. For co-staining of neural cells with Ryr3, mouse
anti-NeuN antibody (1:500, Millipore), mouse anti-GFAP (1:100,
Millipore) or mouse anti-04 (1:100, Millipore) with goat anti-mouse
conjugated Alexa Fluor.RTM. 594 (1:1000) were used for NeuN, GFAP,
or 04 detection, respectively. Rabbit anti-Ryr 3 (1:250, Millipore)
with goat anti-rabbit conjugated Alexa Fluor.RTM. 488 (1:1000) were
used for Ryr3. Lysosomal localization of GCase was determined using
rabbit anti-mouse GCase (1:50) antibody (15) with goat anti-rabbit
conjugated Alexa Fluor.RTM. 488 (1:1000) for mouse GCase detection.
Rat anti-Lamp1 (1:100, RDI) and rabbit anti-rat IgG conjugated
Texas Red (1:1000, Abcam) were used for Lamp1 detection. Sections
were mounted with VECTASHIELD mounting medium containing DAPI
(Vector H1200). Fluorescence signals were visualized and captured
by Zeiss Axiovert 200 M microscopy equipped with an Apotome.
Pearson correlation coefficient software is incorporated in Apotome
microscope and used to analyse the co-localization of two signals
(GCase and Lamp1) in cells. The average Pearson correlation
coefficient number (from 0 to 1) was derived from the cells in
multiple images (21). For counting NeuN.sup.+ neurons, mouse
anti-NeuN antibody (1:500, Millipore) was applied to brain sections
followed by secondary antibody conjugated with FITC. The images of
NeuN.sup.+ cells in brain sections were acquired by Nikon C2 plus
confocal microscope with NIS image stitching function for large
image capture in ND acquisition. All NIS images in 32 bit were
converted into 16 bit format and analysed by ImageJ FIJI.
NeuN.sup.+ cells were counted from whole brain images and from
multiple images (3.5.times.2.0 mm) in each brain region (cortex,
midbrain, cerebellum and brain stem).
[0114] For cell culture immunostaining differentiated N2a cells
were seeded on chamber slides and stained with anti-Map2 antibody
(1:200) followed by the protocol described above except for using
3% BSA in PBS as blocking buffer. Fluorescent images were processed
by Zeiss Apotome 200M.
[0115] Immunoblot
[0116] Half of mice brain (sagittal cut) tissues were homogenized
in M-PER Mammalian Protein Extraction Reagent and subjected to
electrophoresis. Ryr proteins (-500 kD) in tissues lysate were
separated on 3-8% NuPAGE gel running at 90V for 12 h at 4.degree.
C. in acetate buffer. 13-actin was resolved on 4-12% Bis-Tris gel.
The proteins were transferred to PVDF membrane using iBlot 2 gel
transfer device following manufacturer's instructions. The blots
were incubated with anti-mouse Ryr3 monoclonal antibody (1/250) or
anti-13-actin (1:5000) overnight at 4.degree. C. in 1.5% BSA/1.5%
milk/PBS buffer. LC3, CAMK IV and calmodulin were separated on 16%
Tris-glycine gel (Novex) and detected by anti-LC3 (1:1000),
anti-CAMK IV (1:500) or anti-CAM (1:1000), respectively. The
signals were detected with either ECL detection reagent or AP
Conjugate Substrate Kit according to manufacturer's instructions.
Band intensities on immunoblots were quantitated by Image J (NIH,
Baltimore, Va.).
[0117] Seahorse Mitochondrial Function Assay
[0118] Cultured N2a cells treated with or without CBE, dantrolene
or G6 were transferred to a XF96 assay plate at 10,000 cells/well
and allowed to grow overnight in DMEM medium containing 10% FBS
with each compound as described above in Cell culture and treatment
section. Cell numbers were determined using a haemocytometer. Prior
to mitochondrial function assay on XF96 Extracellular Flux
Analyzers (Seahorse Biosciences), the cells in the wells of XF96
assay plates were washed by gently adding warm assay medium to the
side of each well. The plates were transferred to a 37.degree. C.
incubator without CO.sub.2 for 30-60 min before the addition of XF
assay medium (Seahorse Bioscience) containing 25 mM glucose and 1
mM pyruvate. After calibration of the Analyzer, the plate was
sequentially injected with A: 25 .mu.l of 8 .mu.M oligomycin, B: 25
.mu.I of 27 .mu.M cyanide-p-trifluoromethoxyphenylhydrazone (FCCP),
and C: 25 .mu.I of 50 .mu.M antimycin A. Oxygen consumption rate
(OCR) was measured, and the data were analysed using the XFe Wave
software. The respiration parameters were calculated by subtracting
the average respiration rates before and after the addition of the
electron transport inhibitors: oligomycin, FCCP and antimycin A.
The parameters include basal respiration (baseline respiration
minus antimycin A post injection respiration), ATP production
(baseline respiration minus oligomycin post injection respiration),
and maximal respiratory (FCCP stimulated respiration minus
antimycin A post injection respiration). Cell mitochondrial
function presented as respiration parameters that were normalized
by cell numbers (39).
[0119] Brain mitochondria were isolated from 4L;C, WT, and
dantrolene treated 4L;C mice as described with slight modifications
(21,80). Mouse brain tissues were digested with trypsin for 30 min
on ice followed by homogenization on the gentle MACS Dissociator
using Program m_mito_tissues_01 (MACS, Miltenyi Biotec).
Homogenates were suspended in 4 ml ice-cold buffer (1.0 mM KCl, 1.0
mM Tris-HCl, and 0.1 mM EDTA, pH 8.0) and mixed with 0.67 ml of 2 M
sucrose. The suspension was centrifuged at 1300.times.g for 5 min
to remove nuclei, unbroken cells and large membrane fragments. The
supernatant containing mitochondria were pelleted after
centrifugation at 9600.times.g for 10 min at 4.degree. C. The
mitochondrial pellet was resuspended in the Storage Buffer (MACS,
Miltenyi Biotec). Total mitochondrial protein was determined using
Bradford Assay reagent (Bio-Rad). ATP production rates were
determined with isolated mitochondria using XF96 Extracellular Flux
Analyzers. The mitochondria were diluted in cold 1.times.MAS
(Mitochondria` Assay Solution) and substrate (pyruvate/malate)
(Seahorse Biosciences). The mitochondrial suspension (20 .mu.g
mitochondrial proteins in 25 .mu.I) was aliquoted into each well
while the plate was on ice. The plate was then centrifuged using a
swinging bucket microplate adaptor at 2000.times.g for 20 min at
4.degree. C. To start the assay, 155 .mu.I of pre-warmed
(37.degree. C.) 1.times.MAS and substrate were added to each well
containing isolated mitochondria and incubated at 37.degree. C.
with no CO.sub.2 for 10 min. After calibration of the Analyzer, the
plate containing mitochondria was sequentially injected with A:
port A, 20 .mu.I of 40 mM ADP (4 mM, final); port B, 22 .mu.I of 25
.mu.g/ml oligomycin (2.5 .mu.g/ml, final); port C, 24 .mu.I of 40
.mu.M FCCP (4 .mu.M, final); and port D, 26 .mu.I of 40 .mu.M
antimycin A (4 .mu.M, final). OCR was measured and the data were
analysed using the XFe Wave software as described above. ATP
production rate in brain mitochondria was normalized to mg of
mitochondrial protein (18).
[0120] GCase Activity Analyses
[0121] Cells were homogenized in 1% sodium taurocholate/1% Triton
X-100. GCase activity was determined fluorometrically using
4MU-Glucose as the substrate in 0.25% sodium taurocholate, 0.25%
Triton X-100 and 0.1M citric-phosphate buffer (pH 5.6) as 5.
[0122] described previously (81). Brain tissues were homogenized in
1.times.PBS and incubated in 5 .mu.M brain phosphatidylserine and
0.1M citric-phosphate buffer (pH 5.6) for GCase activity assay
using 4MU-Glucose as substrate (82). Protein concentrations were
determined by BCA assay using BSA as standard.
[0123] Glycosphingolipid Analyses
[0124] Glycosphingolipids in mouse brains and N2a cells were
extracted with chloroform and methanol as described (83). GC and GS
content in the extracts was analysed by ESI-LC-MS/MS using a Waters
Quattro Micro API triple quadrupole mass spectrometer (Milford,
Mass.) interfaced with Acquity UPLC system as described (35). The
concentration of GC and GS in the brain was normalized to mg tissue
weight and in the cells was normalized by mg protein in the cell
lysate.
[0125] Statistical Analyses
[0126] The data are presented as mean.+-.SEM and were analysed by
Student's t-test or one-way ANOVA with post-hoc Tukey test using
GraphPad Prism 6. The level of significance was set at P<0.05.
Survival analysis was performed using Kaplan-Meier and the
Mantel-Cox tests.
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[0210] All percentages and ratios are calculated by weight unless
otherwise indicated.
[0211] All percentages and ratios are calculated based on the total
composition unless otherwise indicated.
[0212] It should be understood that every maximum numerical
limitation given throughout this specification includes every lower
numerical limitation, as if such lower numerical limitations were
expressly written herein. Every minimum numerical limitation given
throughout this specification will include every higher numerical
limitation, as if such higher numerical limitations were expressly
written herein. Every numerical range given throughout this
specification will include every narrower numerical range that
falls within such broader numerical range, as if such narrower
numerical ranges were all expressly written herein.
[0213] The dimensions and values disclosed herein are not to be
understood as being strictly limited to the exact numerical values
recited. Instead, unless otherwise specified, each such dimension
is intended to mean both the recited value and a functionally
equivalent range surrounding that value. For example, a dimension
disclosed as "20 mm" is intended to mean "about 20 mm."
[0214] Every document cited herein, including any cross referenced
or related patent or application, is hereby incorporated herein by
reference in its entirety unless expressly excluded or otherwise
limited. The citation of any document is not an admission that it
is prior art with respect to any invention disclosed or claimed
herein or that it alone, or in any combination with any other
reference or references, teaches, suggests or discloses any such
invention. Further, to the extent that any meaning or definition of
a term in this document conflicts with any meaning or definition of
the same term in a document incorporated by reference, the meaning
or definition assigned to that term in this document shall
govern.
[0215] While particular embodiments of the present invention have
been illustrated and described, it would be obvious to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the
invention. It is therefore intended to cover in the appended claims
all such changes and modifications that are within the scope of
this invention.
* * * * *
References