Advances in APBD Research: High Content Screening, Antisense
Oligonucleotides and Computational Drug Design
Or Kakhlon
Department of Neurology Hadassah University Hospital
20 June 2012 APBD Research Foundation Annual Meeting, New York, NY
Funding: APBD Research Foundation
Glycogen biosynthesis involves chain elongation by Glycogen Synthase (GS) and chain branching by Glycogen Branching Enzyme (GBE). If chain elongation outbalances chain branching, glycogen could form starch-like precipitates made up of long, non-branched chains called polyglucosans.
Normal glycogen, branchedPolyglucosan, non-branched
GS/GBE activity ratio
Glycogen build up is normally suppressed in neurons by a well-regulated system, which inactivates (phosphorylates) and degrades Glycogen Synthase (GS)
What causes Adult Polyglucosan Body Disease (APBD)?
Vilchez et al (2007) Nat Neurosc
Glycogen build up is suppressed in neurons by a well-regulated system
Nevertheless, over time glycogen could precipitate as polyglucosan bodies if chain elongation is not adequately balanced by its branching.
Striano et al (2008) Nat Clin Pract NeurolWierzba-Bobrowicz et al (2008) Pholia Neuropathol
APBD
Two experimental approaches for curing APBD:
1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.
2. Clearance of polyglucosan bodies.
1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.
There are pre-existing PG which cannot be removed by any GS/GBE modulating strategy.
Y329S
Keto 3d
Y329S
Gluc/Rap 3d
PG In
tens
ity/c
ell
Gluc/Rap
Gluc/Co
Y329S
Gluc/Co 3d
GFP LC3 Glysh
GBE
1sh
GBE
1/Ra
psh
GBE
1/Ra
p/3-
MA
Rapamycin can induce autophagy and also reverse polyglucosan accumulation. However, only in neurons transduced with shGBE1 lentiviruses.
Therefore, rapamycin could only suppress de novo PG synthesis, not degrade pre-existing PG
Nevertheless, there are three therapeutic strategies for reducing the GS/GBE ratio:
1A. Injection of Antisense Oligonucleotides against PTG & GS in collaboration with ISIS Pharmaceuticals. This approach is already in Phase I clinical trials for treating other disorders such as Spinal Muscular Atrophy (SMA)
1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.
Plan: 1. In vitro screening for the identification of antisense oligonucleotides (ASO) to PTG is currently in progress (expected to be completed by July 2012).
2. Scaling up the drug and screening it in vivo both by inntracerebroventricular and systemically by subcutaneous injection (a few months).
3. Lead ASOs identified by the in vivo screens will be tested in the APBD mouse model (expected by fall of 2012). Hua et al (2010) Genes Dev
1B. Candidate Testing
1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.
Testing three types of compounds known to reduce the GS/GBE ratio:
1. GS inhibitors (AMP Kinase (AMPK) and GSK3β activators). Examples: 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), PI3K inhibitors (e.g., wortmanin, Akt inhibitor IV), Berberine (herbal drug) etc.
2. GBE stabilizers.
3. Compounds predicted by solvent mapping to replace mutated Tyr329 in GBE1, or to destabilize GS and PTG.
Candidate testing
Compounds predicted by solvent mapping (in collaboration with Dima Kozakov, Boston University) to replace mutated Tyr329 in GBE1, or to destabilize GS and PTG.
Candidate testing
Designing drugs using protein solvent mapping GBE, GS and PTG were checked for "druggability" (activators for GBE and inhibitors for GS and PTG).
Kozakov et al (2012) PNAS
The Y329S site is druggable, as it is not conserved (less off target binding) and is concave and with a hydrophobic functional group.
Plan:
a. Testing candidate binding using calorimetry.
b. Testing the effect of bound drugs on GBE and GS activity using our established biochemical assays.
c. Testing library compounds for binding and activity modulation.
1C. Screening both for compounds which reduce GS/GBE ratio, or clear PG: High Content Screening using the IN CELL 2000 Analyzer
1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.
Ctrl
Y329S
keto keto/Rap
Gluc Gluc/Rap Gluc/Co
Optimization by epifluorescence and confocal microscopes: Some representative photos
Integrated intensity of punctate PAS fluorescence (PAS/IP) is significantly higher in APBD patient (Y329Shomozygous) skin fibroblasts than in control fibroblasts.
PAS/IP in fibroblasts treated for 3 days with a ketogenic and diastase pre-digested is higher in patient-derived fibroblasts as compared to control fibroblasts. This means that patient-derived fibroblasts have pre-existing polyglucosans.
Patient-derived fibroblasts have pre-existing polyglucosans.
Rapamycin decreases PAS/PI. This reduction also only becomes statistically significant after diastase digestion.
Fibroblasts treated with glucose, rapamycin and cobalt have lower PAS/PI than fibroblasts treated with glucose and cobalt only, but equal PAS/PI to fibroblasts treated with glucose and rapamycin only.
The effect of rapamycin in reducing PAS/PI overrides the effect of cobalt in increasing it.
There is no significant difference in PAS/PI between fibroblasts treated with ketogenic medium and ketogenic medium supplemented with rapamycin.
Rapamycin couldn't degrade pre-existing polyglucosans (but only reduce de novo synthesized).
Cobalt increases PAS/PI staining in glucose fed fibroblasts with or without diastase digestion.
Diastase digestion amplifies the difference between fibroblasts treated with glucose and ketogenic medium.
Most PAS/PI staining is attributable to polyglucosans.
PAS/PI intensity in glucose fed fibroblasts is not significantly different between diastase digested and not digested conditions.
Polyglucosan analysis in control and Y329S fibroblasts by HCA using the IN CELL 2000
Analyser
Leonardo J. Solmesky, Ph.D.Cell Screening Facility for Personalized Medicine,
Lab for Neurodegenerative Diseases and Personalized Medicine,Department of Cell Research and Immunology,
Wise Faculty of Life Sciences,Tel Aviv University, Israel
Control Y329S/Gluc Y329S/Gluc/RapY329S/Gluc/Co
19Leonardo J. Solmesky, Ph.D. Cell Screening Facility for Personalized
Medicine
• High level of diffuse, non granular staining in control. Need more aggressive diastase treatment.
• Co increases PG intensity and rapamycin decreases it, reverting PAS staining to the diffuse pattern observed in control cells
ctrl Y329S
An example of an improved diastase treatment:
Polyglucosan granules count/cell. Populational distribution in different treatments
21
Glu
cG
luc/
Co
Glu
c/R
ap
• Rapamycin skews distribution to the left, i.e., reduces PG number.• Rapamycin increases area under the curve, i.e., increases cell count suggesting rescue against PG
toxicity.• Co appears to be toxic in this experiment.
• We need to analyze PG integrated intensity!
Polyglucosan granules mean area populational distribution in different treatments
22
Glu
cG
luc/
Co
Glu
c/R
ap
• Conclusion: Treatments do not affect mean PG size. Consistent with rap blocking de novo synthesis
Polyglucosan granules total area populational distribution in different treatments
23
Glu
cG
luc/
Co
Glu
c/R
ap
• As compared to mean PG size, total PG size correlates better with PG number
Nuclear area populational distribution in different treatments
24
Glu
cG
luc/
Co
Glu
c/R
ap
• Treatments did not affect nuclear size
Nuclear IxA populational distribution in different treatments
25
Glu
cG
luc/
Co
Glu
c/R
ap
• Treatments did not affect nuclear size
26
Population distribution among different phases of cell cycle under different treatments
Gluc Gluc/Co Gluc/Rap• Gluc/Co treatment led to growth arrest, while rapamycin slightly accelerated growth
High Throughput Screening
Readouts: PG integrated intensity (PAS/PI).
Positive hits analysis
1. If positive hits are suspected activators of AMPK, or GSK3β, testing activation of purified enzymes.
2. Testing for undesirable chemical liability (covalent protein binding, thioether adduct formation) consequent to metabolic processing.
3. Ranking compounds according to their effectiveness. Clusters of highest and lowest scorers will be aligned to predict a pharmacophore and toxicophore, respectively.
pharmacophore
in silico screening of pharmacophore and toxicophore (using Similarity Ensembl Approach (SEA))
Similar structures with known mechanism of action (future lead compounds)
Phar
mac
opho
re
Molecules with liability
Toxicophore
Possible targets
Candidates for therapeutic modulation.
Summary
• APBD can be ameliorated by reduction of the GS/GBE activity ratio.
• This approach is tested by a. Antisense Oligonucleotide injection; b. Solvent mapping of GBE, GS and PTG in order to find binding drugs; c. High Throughput Screening.
• High Throughput Screening is also used to find potential compounds which would clear PG and thus could not only ameliorate, but alleviate APBD pathogenesis.
Manifesting heterozygotes conundrum
Wt ctrl
Manifesting heterozygote lymphoblastsHomozygote
Manifesting heterozygote whole blood
Genomic DNA indicates a heterozygous p.Y329S mutation. However, cDNA reveals only one mutated allele, indicating that the second allele is missing.
cDNA sequencing:
Test MH carrier parents for mRNA homozigosity of cDNA v heterozygosity of gDNA
Yes
Need to explain the manifesting heterozygotes phenomenon by other ways – alternative to differential allelic expression
No
Manifesting hetreozygotes (MH) phenomenon – is it due to differential allelic expression?
First and foremost need control:
Is there reduced/no expression of WT allele?
Quantitate GBE expression by RT-PCR
GBE expression in MH is roughly 50% of normal
Suggests WT allele is suppressed
Polymorphism: Check: Methylation (Bisulfite sequencing), compare haplotypes between MH and WT, Linkage analysis to MH trait, whole gene analysis, exome analysis, CGH to check copy number differences, exome analysis, whole genome analysis, transcription factor binding, (exclude small deletions?)
GBE expression in MH is roughly the same as normal
Suggests compensation by overexpression
Look for post transcriptional modifications of GBE in MH
Collaboration: Corroborate results by PAS-based genome-wide siRNA screen in wt MEFs
Thanks
Hadassah Medical Center
Alexander Lossos
Tel Aviv University
Leonardo SolmeskyMiguel Weil
Boston University
Dima Kozakov
Columbia University
Orhan AkmanSalvatore DiMauro
ISIS Pharmaceuticals
Tamar Grossman
Apoptosis is also reported in glycogen synthase-activated neurons
Stauro
sporin
e
Prote
in ta
rget
ing
to G
lyco
gen
Suggestion: Polyglucosan accumulation induces apoptosis
Vilchez et al (2007) Nat Neurosc
Sarkar et al (2009) Cell Death Differ
Inclusion bodies (PBs) formed. Can induction of autophagy facilitate their clearance?
Can autophagy be cytoprotective against apoptosis?
Maiuri et al (2007) Nat Rev Mol Cell Biol
Test autophagy enhancers as a therapeutic strategy against APBD
Using the model to test therapeutic approaches: Induction of autophagy
Jaeger & Wyss-Coray (2009) Mol Neurodegen
Autophagy can be stimulated and inhibited in neurons by rapamycin and 3-methyl adenine, respectively
Rapamycin
mTOR
(through Ulk1/2 inhibition)IM
Autoph. MVB
Amphisome
Autolysosome
=LC3
Classical mode of action – autophagic engulfment followed by autolysosomal degradation (by acid maltase?)
How can PB be cleared by autophagy?
Vin
Rapamycin
mTOR
(through Ulk1/2 inhibition)IM
Autoph. MVB
Amphisome
Autolysosome
Can the effects of rapamycin be reproduced if autophagosome maturation to autolysosomes is inhibited?
DMSO RapRapPI
RapVin
RapVinPI Vin St
arve
dFi
brob
last
s-LC3 I-LC3 II
LC3 LC3 LC3
DMSO Rap Rap+Vin
Untr Rap Rap/Vin
0
50
100
150
200
1.1±0.2 1.5±0.1 2.0±0.2 2.2±0.1 1.9±0.3 1.7±0.2 6.2±0.5
* *
Saponized neurons: Autophagic flux is fast.
Rapamycin induces autopahgy and thus slows down autophagic flux.Vinblastine blocks autophagic maturation, further slowing down autophagic flux
Confirmation of vinblastine’s effect:
1. Reproduction of the block in autophagic flux induced by lysosomal protease inhibitors (PI).
2. Blunting the sensitivity of rapamycin induced neurons to PI.
GFP Gly mergeC
nt/Rap shGBE1/Rap shGBE1/Rap/3-MA shGBE1/Rap/Vin
FL-2
PI
FL-1 Annexin V
D
E
Fig. 5
Blocking autophagic maturation by vinblastine did not reverse down-modulation of polyglucosan accumulation and apoptosis by rapamycin.
Rapamycin protection of GBE1-knocked-down neurons did not depend on autophagic maturation and polyglucosan degradation in autolysosomes.
Rap+Vin
Q: Why does 3-MA antagonize Rap?A: Probably not via activation of autophagy or GSK3. Perhaps via PFK inhibition
As opposed to neurons, in APBD patient derived fibroblasts Rap probably does clears PB by autophagy
Control Gbe1Y329S/Y329S patient Gbe1Y329S/Y329S/Rap
Uranyl-Et-OH
Vin
Rapamycin
mTOR
(through Ulk1/2 inhibition)
?DGKα
IM
Autoph. MVB
Amphisome
Autolysosome
Suggestion: Rap-stimulated PB clearance is mediated by exosome release, bypassing autophagosomal maturation
A B
C a b
PBs were not found in either MVB (A), amphisomes (B &C), apparent exosomes (C), or cytosol (A-C) in GBE1-knocked down neurons treated with rapamycin.
shGBE/Rap shGBE/Rap
shGBE/Rap
Relatively small PBs were observed in untreated GBE1-knocked down neurons (D). Suggestion: Neurons not living with other cell types might succumb to cell death once glycogen deposits appear
D
shGBE
mTOR
Rapamycin
Vin
IM Autolysosome
GSK3β
PP active GS
Autoph.
Amphisome
MVB
X
XX
=LC3
=Polyglucosan Body
?
nt shGBE1 shGBE1 Rap
nt Rap shGBE1 Rap 3-MA
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1-G6P
+G6P
GS a
ctivi
ty (
nmol
/min
/mg
Prot
ein)
-75
-50
GS
Tubulin
Main conclusion: Rap positive effect was probably mediated by GS inhibition.
None of the treatments affected G-6-P-stimulated GS activity, suggesting it overrode GS phosphorylation state.
Testing three types of compounds known to reduce the GS/GBE ratio:
1. GS inhibitors (AMP Kinase (AMPK) and GSK3β activators). Examples: 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), PI3K inhibitors (e.g., wortmanin, Akt inhibitor IV), Berberine (herbal drug) etc.
2. GBE stabilizers.
3. Compounds predicted by solvent mapping to replace mutated Tyr329 in GBE1, or to destabilize GS.
Candidate testing
LO
D
Sco
re
Another direction: Microtubule-mediated transport :
A nerve biopsy from Hereditary Spastic Paraplegia reveals PB.
Linkage analysis along chromosome 2 shows the locus where markers for the diseased state co-segregated only in patients and not in healthy family members. This locus encodes the MT motor KIF1A
Do microtubule motors mediate PB clearance?
Summary
• We have shown that GBE1 knockdown causes polyglucosan accumulation and apoptosis in a pure neuronal model.
• These phenotypes can be rescued by rapamycin via inhibition of Glycogen Synthase and not via induction of autophagy
• We conclude that polyglucosan accumulation is causal for APBD. Therapeutic search should therefore focus on restricting polyglucosan accumulation.
Main objectives
• Establishing a neuronal model of APBD in which GBE1 is repressed and PB are observed.
• Using the model to test pharmacological and biochemical methods for correcting adverse phenotypes associated with GBE1 deficiency.
APBD Neuronal Model Produced by transduction with lentiviral particles encoding for shRNA against GBE1
Demonstration of GBE1 knockdown:
nt shGBE10
50
100
150
200
250
0
0.2
0.4
0.6
0.8
1
1.2
GBE1 activity
GBE1 mRNA
GB
E1
Ac
tiv
ity
(n
mo
l/min
/mg
pro
tein
)
GB
E1
mR
NA
(re
lati
ve
va
lue
s)
A RT-PCR & activity
Tubulin
GBE1
Con
trol
Y32
9S
hom
ozyg
ous
nt
shG
BE
1
-75 kD -50 kD
B
Western: Reduction comparable to that found in patients
GFP GBE1 mergeC
nt
shGBE1
Indirect immunofluorescence
GFP Gly merge
nt
shGBE1
shGBE1
GFP DIC Gly merge
A
B
C
Polyglucosan accumulates in GBE1 knocked down neurons
Lower expressers of shGBE1 lentiviruses are less affected:
Polyglucosan is the culprit
Vilchez et al (2007) Nat Neurosc
Glycogen detected as polyglucosan punctae similar to GSK3 inhibition or
PTG over expression.
nt
shGBE1
A
B
C
D
FL-1 Annexin V
SNP
FL-2
P
I
GBE1 knockdown increases apoptosis
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