Researchers have developed a neuronal model of APBD that recapitulates glycogen accumulation and increased apoptosis seen in patients. Treating the model with rapamycin reversed adverse phenotypes by possibly stimulating autophagosome formation and exosome release, or inhibiting glycogen synthase. Researchers are now testing rapamycin and other therapies like iron chelators in the neuronal model, mouse model, and patient cells to identify effective treatments for APBD.

1. Therapeutic Interventions Tested in
APBD Models
Or Kakhlon, Alexander Lossos, H. Orhan Akman, Salvatore DiMauro
Department of Neurology
Hadassah University Hospital
06 July 2011

2. 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

3. 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.
•Testing the methods also in brain cells derived from APBD mouse model and in
APBD patient-derived cells.

4. Neuronal Model validation:
1) Reduced GBE1 levels:
GFP GBE1 merge
GBE1
nt
(RT-PCR data pending)
shGBE1

5. Neuronal Model validation (2):
2) Glycogen accumulation:
GFP Glycogen merge
nt
shGBE1

6. GBE1 knockdown increases apoptosis in the
neuronal model
Necrotic
Undergoing apoptosis
End stage apoptosis

7. 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

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

9. Stimulation of autophagy can reversibly rescue PB
accumulation in GBE1 knocked down neurons
shGBE1
shGBE1/Rap
shGBE1/Rap/3-MA
GFP
LC3
Glycogen

10. Stimulation of autophagy can reduce apoptosis in GBE1 knocked down neurons

11. Vinblastin reduces autophagosome maturation into autolysosmes and increases
autophagosome biogenesis. Vinblastin’s effects on autophagy were confirmed in
GBE1 knocked down neurons
Untr. Rap Rap+Vin
LC3
-LC3 I
-LC3 II
Still need to confirm that vinblastin reduces sensitivity of LC3
degradation to lysosomal protease inhibitors

12. PB clearance and antiapoptotic effects of rapamycin in GBE1 knocked down
neurons are unaffected by vinblastine
Rap+Vin

13. 8 ?
P
P
Rapamycin active
GSK3β GS
5
mTOR
4 7 ?
DGKα
IM Autolysosome
Autoph. MVB R59949
3
1 2
Amphisome
3’
6 =LC, autoph. marker
Vin
1. PB Engulfment by the isolation membrane (IM)
2. Amphisome formation from Autophagosome and Multivesicular Body
3. Autophagosome maturation to Autolysosome
4. Inhibition of autophagosome biogenesis
5. Rap. inhibits mTOR and induces autophagy. Rap was able to reduce PB and apoptosis.
6. Vinblastin inhibits autophagosome maturation into Autolysosome,
but doesn’t counteract rapamycin’s effects.
7. Exosome release clears PB?
8. Rapamycin works by GS inhibition

14. The effect of rapamycin and other mTOR inhibitors (new combinations affecting
mTOR, PI3 Kinase and Akt together) are now being tested in the APBD mouse
model in close collaboration between Dr. Akman and Dr. Kakhlon.

15. Fortuitously it was
found that cobalt
reduces PB levels
in the hearts of Control
GBE1 KD mice.
Cobalt and iron
chelation can
activate Hypoxia GBE1 KD
Inducible Factor
(HIF) – a GBE1
inducer.
GBE1 KD/CoCl2
Test iron
chelation as a GBE1 KD/Rap.
therapeutic
strategy
GBE1 KD/LiCl

16. Preliminary data: Iron chelators 60
% of PG positive
50
(especially DFP) reduce PB
fibroblasts
40
accumulation in patient-derived 30
fibroblasts 20
10
0
Ctrl Y329S Y329S Y329S Y329S
Rap DFO DFP
Y329S
Y329S Ctrl Y329S
DFO
Y329S Y329S
DFP Rap

17. Summary
• We have generated a neuronal model of APBD, recapitulating the clinical finding
of glycogen (possibly polyglucosan bodies) accumulation and demonstrating
increased apoptosis.
•Treating model neurons with rapamycin was able to reverse the adverse
phenotypes associated with GBE1 deficiency.
•These effects of rapamycin are possibly attributable to
autophagosome/mulivesicular body-driven exosome release.
•Another possible mechanism explaining rapamycin effects is inhibition of
glycogen synthase. This mode of action will be assessed.
•All possible therapeutic modes (for now, especially mTOR inhibitors and iron
chelators) are now being tested in the neuronal model, the mouse model and
APBD patient-derived cells in close collaboration between Dr. Akman and Dr.
Kakhlon.