Or Kahlon, Ph.D.

1. 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

2. 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.
Polyglucosan, non-branched Normal glycogen, branched
GS/GBE activity ratio

3. What causes Adult Polyglucosan Body Disease (APBD)?
Glycogen build up is normally suppressed in neurons by a well-regulated system,
which inactivates (phosphorylates) and degrades Glycogen Synthase (GS)

4. Glycogen build up is suppressed in neurons by a Nevertheless, over
well-regulated system time glycogen could
precipitate as
polyglucosan bodies
if chain elongation is
not adequately
balanced by its
branching.
Vilchez et al (2007) Nat Neurosc
APBD
Striano et al
(2008) Nat Clin
Wierzba-Bobrowicz et al (2008) Pholia Neuropathol Pract Neurol

5. 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.

6. 1. Ameliorating APBD, or slowing down the progress of the disease by
reducing the GS/GBE activity ratio.
There are pre-existing PG which cannot be removed by any GS/GBE
modulating strategy.
Y329S
Gluc/Co
Y329S
PG Intensity/cell
Gluc/Co 3d
Gluc/Rap
Y329S
Keto 3d
Gluc/Rap 3d

7. 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
GFP LC3 Gly
shGBE1
hGBE1/Rap/3-MA shGBE1/Rap

8. 1. Ameliorating APBD, or slowing down the progress of the disease by
reducing the GS/GBE activity ratio.
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)
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

9. 1. Ameliorating APBD, or slowing down the progress of the disease by
reducing the GS/GBE activity ratio.
1B. Candidate Testing

10. Candidate testing
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.

11. 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.
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

12. The Y329S site is druggable, as it is not conserved (less off target binding) and is
concave and with a hydrophobic functional group.

13. 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.

14. 1. Ameliorating APBD, or slowing down the progress of the disease by
reducing the GS/GBE activity ratio.
2. Clearance of polyglucosan bodies.
1C. Screening both for compounds which reduce GS/GBE ratio, or clear
PG: High Content Screening using the IN CELL 2000 Analyzer

15. Ctrl Optimization by epifluorescence
and confocal microscopes: Some
representative photos
Y329S
keto keto/Rap
Gluc Gluc/Rap Gluc/Co

16. 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.

17. 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).
Diastase digestion amplifies the difference between
fibroblasts treated with glucose and ketogenic
medium.
PAS/PI intensity in glucose fed fibroblasts is not
significantly different between diastase digested and
not digested conditions.
Most PAS/PI staining is attributable to polyglucosans.
Cobalt increases PAS/PI staining in glucose fed fibroblasts with or without diastase digestion.
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.

18. 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

19. Control Y329S/Gluc Y329S/Gluc/Co Y329S/Gluc/Rap
•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 Leonardo J. Solmesky, Ph.D. Cell
Screening Facility for Personalized 19
Medicine

20. An example of an improved diastase treatment:
ctrl Y329S

21. Polyglucosan granules count/cell. Populational distribution in different
treatments
Gluc
Gluc/Co
Gluc/Rap
•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. •We need to analyze PG integrated intensity! 21
•Co appears to be toxic in this experiment.

22. Polyglucosan granules mean area populational distribution in different
treatments
Gluc
Gluc/Co
Gluc/Rap
•Conclusion: Treatments do not affect mean PG size. Consistent with rap blocking de novo synthesis
22

23. Polyglucosan granules total area populational distribution in different
treatments
Gluc
Gluc/Co
Gluc/Rap
•As compared to mean PG size, total PG size correlates better with PG number
23

24. Nuclear area populational distribution in different treatments
Gluc
Gluc/Co
Gluc/Rap
•Treatments did not affect nuclear size
24

25. Nuclear IxA populational distribution in different treatments
Gluc
Gluc/Co
Gluc/Rap
•Treatments did not affect nuclear size
25

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
26

27. High Throughput Screening pharmacophore
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.
in silico screening of pharmacophore
and toxicophore (using Similarity
Ensembl Approach (SEA))
Similar structures with known
mechanism of action (future lead Molecules with liability
compounds)
Possible targets
Candidates for therapeutic modulation.

28. 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.

29. Manifesting heterozygotes conundrum

30. Genomic DNA indicates a heterozygous p.Y329S mutation. However, cDNA reveals only one
mutated allele, indicating that the second allele is missing.
cDNA sequencing:
Manifesting Manifesting
heterozygote heterozygote
Wt ctrl Homozygote whole blood lymphoblasts

31. Manifesting hetreozygotes (MH) phenomenon – is it due to differential allelic expression?
First and foremost need control: Test MH carrier parents for mRNA homozigosity of cDNA v heterozygosity of gDNA
Yes No
Need to explain the manifesting
Is there reduced/no expression of WT allele?
heterozygotes phenomenon by
other ways – alternative to
differential allelic expression
Quantitate GBE expression by RT-PCR
GBE expression in MH is roughly 50% GBE expression in MH is
of normal roughly the same as normal
Suggests WT allele is suppressed Suggests compensation by
overexpression
Polymorphism: Check: Methylation
Collaboration: Corroborate results by (Bisulfite sequencing), compare
haplotypes between MH and Look for post transcriptional
PAS-based genome-wide siRNA
WT, Linkage analysis to MH modifications of GBE in MH
screen in wt MEFs
trait, whole gene analysis, exome
analysis, CGH to check copy number
differences, exome analysis, whole
genome analysis, transcription factor
binding, (exclude small deletions?)

32. Thanks
Tel Aviv University Hadassah Medical Center Boston University
Leonardo Solmesky Alexander Lossos Dima Kozakov
Miguel Weil
Columbia University ISIS Pharmaceuticals
Orhan Akman Tamar Grossman
Salvatore DiMauro

33. Apoptosis is also reported in glycogen synthase-activated
neurons
Vilchez et al (2007) Nat Neurosc
Suggestion: Polyglucosan accumulation induces apoptosis

34. Using the model to test therapeutic approaches:
Induction of autophagy
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
Sarkar et al (2009) Cell Death Differ
Test autophagy enhancers as a therapeutic strategy against APBD

35. Autophagy can be stimulated and inhibited in neurons by
rapamycin and 3-methyl adenine, respectively
Jaeger & Wyss-Coray (2009) Mol Neurodegen

36. How can PB be cleared by autophagy? Classical mode of action –
autophagic engulfment
followed by autolysosomal
degradation (by acid
Rapamycin maltase?)
mTOR
(through
Ulk1/2
IM inhibition)
MVB Autolysosome
Autoph.
Amphisome
=LC3

37. Can the effects of
rapamycin be reproduced if
autophagosome maturation
to autolysosomes is
inhibited?
Rapamycin
mTOR
(through
Ulk1/2
IM inhibition)
MVB Autolysosome
Autoph.
Amphisome
Vin

38. Saponized neurons: Autophagic flux is fast.
Rapamycin induces autopahgy and thus slows down autophagic flux.
Vinblastine blocks autophagic maturation, further slowing down autophagic flux
DMSO Rap Rap+Vin
300
200
100
0
LC3 LC3 LC3
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.
*
Fibroblasts
Rap *
Rap
Rap
Vin Starved
DMSO Rap PI Vin PI Vin
-LC3 I
-LC3 II
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

39. Blocking autophagic maturation by vinblastine did not
reverse down-modulation of polyglucosan E
accumulation and apoptosis by rapamycin.
Rapamycin protection of GBE1-knocked-down
neurons did not depend on autophagic maturation
and polyglucosan degradation in autolysosomes
Q: Why does 3-MA antagonize Rap?
A: Probably not via activation of autophagy or GSK3.
Perhaps via PFK inhibition
C GFP Gly merge
Rap+Vin
D
nt/Rap shGBE1/Rap shGBE1/Rap/3-MA shGBE1/Rap/Vin
FL-2 PI
FL-1 Annexin V Fig. 5

40. 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

41. Suggestion: Rap-stimulated PB
clearance is mediated by
exosome release, bypassing
autophagosomal maturation
Rapamycin
mTOR
(through DGKα ?
Ulk1/2
IM inhibition)
MVB Autolysosome
Autoph.
Amphisome
Vin

42. 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.
A B
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
shGBE/Rap shGBE/Rap glycogen deposits appear
Ca b D
shGBE/Rap
shGBE

43. ?
Rapamycin P active
GSK3β GS
mTOR
X
IM Autolysosome
Autoph. MVB
X
Amphisome
X
Vin
=LC3
=Polyglucosan Body

44. 1.2
Main conclusion: -G6P
Rap positive +G6P
GS activity (nmol/min/mg Protein)
1
effect was
probably 0.8
mediated by GS
inhibition. 0.6
0.4
None of the
treatments affected 0.2
G-6-P-stimulated GS
activity, suggesting it
0
overrode GS nt shGBE1 shGBE1 nt Rap shGBE1
phosphorylation Rap Rap 3-MA
state.
GS
-75
-50
Tubulin

45. Candidate testing
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.

46. 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
LOD Score
mediate PB clearance?

47. 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.

48. 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.

49. APBD Neuronal Model Produced by transduction with
lentiviral particles encoding for shRNA against GBE1
Demonstration of GBE1 knockdown:
A
RT-PCR & activity
300 1.2
GBE1 mRNA (relative values)
GBE1 activity
250 1
(nmol/min/mg protein)
GBE1 mRNA Indirect
GBE1 Activity
200 0.8
immunofluorescence
150 0.6
C GFP GBE1 merge
100 0.4
50 0.2
0 0
nt
nt shGBE1
Western: Reduction comparable
to that found in patients
homozygous
shGBE1
shGBE1
B
Control
Y329S
nt
GBE1 -75 kD
Tubulin -50 kD

50. Polyglucosan accumulates in GBE1 knocked down neurons
Glycogen detected as polyglucosan
punctae similar to GSK3 inhibition or
PTG over expression.
Lower expressers of shGBE1
lentiviruses are less affected:
Polyglucosan is the culprit
GFP Gly merge
A
nt
B
shGBE1
C
shGBE1
GFP DIC Gly merge
Vilchez et al (2007) Nat Neurosc

51. A GBE1 knockdown increases apoptosis
B
nt
C
FL-2 PI
shGBE1
D
SNP
FL-1 Annexin V