Therapeutic Interventions Tested in

APBD Models

Or Kakhlon, Alexander Lossos, H. Orhan Akman, Salvatore DiMauro

Department of Neurology

Hadassah University Hospital

06 July 2011

Vilchez et al (2007) Nat Neurosc

Glycogen build up is suppressed in neurons by a

well-regulated systemNevertheless, 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

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.

Neuronal Model validation:

GFP

GBE1

GBE1 merge

nt

shGBE1

(RT-PCR data pending)

1) Reduced GBE1 levels:

Neuronal Model validation (2):

2) Glycogen accumulation:

GFP

nt

shGBE1

Glycogen merge

GBE1 knockdown increases apoptosis in the

neuronal model

Necrotic

Undergoing apoptosis

End stage apoptosis

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

shGBE1

shGBE1/Rap

shGBE1/Rap/3-MA

GFP

LC3

Glycogen

Stimulation of autophagy can reversibly rescue PB

accumulation in GBE1 knocked down neurons

Stimulation of autophagy can reduce apoptosis in GBE1 knocked down neurons

Vinblastin reduces autophagosome maturation into autolysosmes and increases

autophagosome biogenesis. Vinblastin’s effects on autophagy were confirmed in

GBE1 knocked down neurons

LC3

Untr. Rap Rap+Vin

-LC3 I-LC3 II

Still need to confirm that vinblastin reduces sensitivity of LC3

degradation to lysosomal protease inhibitors

PB clearance and antiapoptotic effects of rapamycin in GBE1 knocked down

neurons are unaffected by vinblastine

Rap+Vin

mTOR

4

4. Inhibition of autophagosome biogenesis

Rapamycin

5

5. Rap. inhibits mTOR and induces autophagy. Rap was able to reduce PB and apoptosis.

Vin6

6. Vinblastin inhibits autophagosome maturation into Autolysosome,

but doesn’t counteract rapamycin’s effects.

DGKαIM

Autolysosome

GSK3β

PPactive

GS

Autoph.

1

1. PB Engulfment by the isolation membrane (IM)

Amphisome

2

2. Amphisome formation from Autophagosome and Multivesicular Body

MVB

7. Exosome release clears PB?

?7

R59949

8. Rapamycin works by GS inhibition

?8

=LC, autoph. marker

3. Autophagosome maturation to Autolysosome

3’

3

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.

Control

GBE1 KD

GBE1 KD/CoCl2

GBE1 KD/Rap.

GBE1 KD/LiCl

Fortuitously it was

found that cobalt

reduces PB levels

in the hearts of

GBE1 KD mice.

Cobalt and iron

chelation can

activate Hypoxia

Inducible Factor

(HIF) – a GBE1

inducer.

Test iron

chelation as a

therapeutic

strategy

Y329S Ctrl Y329S

DFOY329S

Preliminary data: Iron chelators

(especially DFP) reduce PB

accumulation in patient-derived

fibroblasts

0

10

20

30

40

50

60

Ctrl Y329S Y329S Rap

Y329S DFO

Y329S DFP

% o

f P

G p

osi

tive

fi

bro

bla

sts

Y329S

DFPY329S

Rap

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.