Molecular Characterization of Polyglucosan Body, Cause or Consequence in the Disease

Hasan Orhan AKMAN , PhD. William J Craigen, Md PhD.

Salvatore DiMauro, MD

Polyglucosan Body (PGB)

• Polyglucosan (PG) is the polymer of glucose molecules, however, unlike glycogen due to none or poor branching it is insoluble showing starch like properties.

• PG accumulates in the cytosol and forms large aggregates known as PGB. They are visible after diastase digestion and PAS staining.

Polyglucosan Accumulates in the Axons and Cells.

Klein et al. Muscle Nerve. 2004 Feb;29(2):323-8

Molecular Mechanisms for Poor Branching;

• Skewed ratio of glycogen synthase vs.branching activity (GS/GB).

• PFK deficiency

Normal Glycogen

Polyglucosan

PGB Disease Caused By GBE Deficiency has Different Variants Based on Residual Enzyme Activity

Andersen Disease Complete Loss of Enzyme Activity– Lethal in infantile

– Decrease in fetal movements and hydromnios

– Liver and neuromuscular problems

Juvenile form with low Activity – Mainly Liver and Heart,

Adult PGB Disease (APBD) 20% or Less Residual Activity – Muscle weakness,

– Loss of sensation

– Muscles wasting in the arms and/or legs.

– Impaired bladder control (neurogenic bladder)

– Mental confusion (dementia very rare component of APBD)

Targeting Vector

5’ ARM 2.1 kb PGK-Neomycin 3’ARM 6.5 kb Exon 7

Y329S TAT to TCTTyr Ser

FRT

LoxP

Exon 7Intron 6 Intro n 7

Exon 7 PGK-Neo

Exon 7 PGK-Neo ?

Exon 7

Exon 7

Cre Recombination and Excision

Flpe Recombination and Excision of Exon 7 in mouse genome

Intron 6 Intron 7

Gbe1neo

Gbe1Y329S

Gbe1del

Effect of Selection cassette on the progress of the disease

Relative Activity of GBE in Gbe1Neo/Neo

Tissue Genotype % Value % SD n=5

BrainWT 100 2

Flox/Flox 2 42

HeartWT 100 16

Flox/Flox 16 11

LiverWT 100 6

Flox/Flox 6 28

MuscleWT 100 7

Flox/Flox 7 11

KidneyWT 100 1

Flox/Flox 0 NA

Physical Activity (4 months of age)

0.0

100.0

200.0

300.0

400.0

Control GBE1

Av

era

ge

dis

tan

ce

ru

n (

me

ter)

*

Dis

tance r

un (

mete

rs)

Heart

Muscle

Liver

3 weeks old liver

4 month old liver

Brain

PGB in Axons and Cell Body

Mouse Skin Fibroblasts have Polyglucosan

Heart

Skeletal MuscleC

ontr

ol

Gbe1

ne

o/n

eo

Gbe1

ne

o/n

eo

Gbe1

ne

o/n

eo

Glucose Metabolism is Altered in Gbe1neo/neo mice

0

50

100

150

200

250

300

350

400

450

0 20 40 60 80 100 120 140 160 180 200

Glu

cose

Co

nce

ntr

atio

n

mg/

dl

Time (minute)

Control

GBE1

*

*

*

Polyglucosan Can Be Used in The Liver During Fasting

*

*p< 0.002

Conclusion These two models are successful mouse models of APBD

These animal models can be used to study

• Molecular characterization of (PGB).(ii) Determine if the PGB cause or consequence in the disease? (iii)Is it possible to digest PGB in the cell or tissue? if possible how it

effects the course of the disease