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Giant Axonal Neuropathy
A Disorder of Intermediate Filament Organization
By: Emily Gilles
Alberts, et al. Molecular Biology of the Cell, 4th Edition
Normal Intermediate Filaments
Alberts, et al. Molecular Biology of the Cell, 4th Edition
Provide mechanical strength
Not all cells have IFs.
Different cell types have different types of IFs.




Keratin is the IF in endothelial cells
Neurofilaments are found in the axons of neurons
Lamins are found lining the nuclear lamina of some cells
Vimentin is found in cells of mesenchymal origin
Disease vs. Control Axon
Timmerman, et al. Nature Genetics 26(3):254-255 (2000).
(a) = GAN patient

Closely packed
neurofilaments,
peripheral cluster of
microtubules
(b) = control

Homogenous distribution of
neurofilaments and
microtubules
Diagnosis = Sural Nerve Biopsy
Control
GAN
The pathological hallmark is the disorganization of the
intermediate filament network of the cytoskeleton,
with axons being predominantly affected.
The typical clinical case:
Maia, et al. Neuropediatrics 19(1):10-15 (1988).
Treiber-Held, et al. Neuropediatrics 25(2):89-93 (1994).
Disease onset typically occurs
between ages 4-7.
Death occurs before the age of 30.
Symptoms begin as clumsiness of
gait and progressive weakness
starting at the lower limbs.
Dysarthria, nystagmas, facial
weakness, and mental retardation
soon become apparent.
Kinky hair may or may not be
present.
Rare, autosomal recessive
Homozygosity Mapping
What is homozygosity mapping?
• Exactly like positional cloning
• Fewer affected individuals can be used
• Offspring must be from consanguineous
parents
In addition to being homozygous at
the disease allele, there is a
greatly increased likelihood of
being homozygous by descent in
adjacent regions of the genome.
Ben Hamida, et al. Neurogenetics 1(2):129-133 (1997).
Flanigan, et al. Annals of Neurology 43(1):143-148 (1998).
Refinement of the GAN locus
Cavalier, et al. European Journal of Human Genetics 8(7):527-534 (2000).
The Gene: Gigaxonin
Bomont, et al. Nature Genetics 26(3):370-374 (2000).
Expression is Ubiquitous
Bomont, et al. Nature Genetics 26(3):370-374 (2000).
RT-PCR was used to amplify RNAs from mouse tissues.
The probe used to detect was the EST aa726805.
Disease-Causing Mutations
Bomont, et al. Nature Genetics 26(3):370-374 (2000).
Additional Mutations Discovered
Bomont, et al. Human Mutations 21(4):446-451 (2003).
Kuhlenbaumer, et al Neurology 58(8):1273-1276 (2002).
Cytoskeletal Elements are Cross-Linked
Actin filaments, microtubules, and intermediate
filaments interact via cross-linking proteins
Picture below shows intermediate filaments
(blue) linked to microtubules (red) via plectin
(green)
Alberts, et al. Molecular Biology of the Cell, 4th Edition
Yeast Two-Hybrid Setup
Blue circle = DNA
binding domain
Yellow square =
gigaxonin
Red square = protein
from human brain
cDNA
Blue semi-circle =
activator domain
Criekinge WV and Beyaert R. Biological Proceedures Online 2:1-38 (1999)
Gigaxonin colocalizes with MAP1B-LC
Ding, et al. Journal of Cell Biology 158(3):427-433 (2002).
HA-Gig and flagMAP1B-LC were
cotransfected into cos7
cells
Gigaxonin and MAP1BLC colocalize together
D shows diffuse
accumulation of
gigaxonin in cytoplasm
when transfected alone
In vivo Results Similar to in vitro
Ding, et al. Journal of Cell Biology 158(3):427-433 (2002).
Gigaxonin colocalizes with MAP1B-LC in vivo.
Functional Significance
A = depolymerized
microtubules within 15
min of colchicine
treatment (untransfected
cells)
B = depolymerized
microtubules within 60
min of colchicine
treatment (single
transfected cells)
C and D = Intact network
after 2 hr colchicine
treatment (double
transfected cells)
Ding, et al. Journal of Cell Biology 158(3):427-433 (2002).
Molecular Diagnosis/Treatment
Molecular Diagnosis is only available on a
research basis.
There is no cure and treatment is based
on alleviating treatable symptoms.
Current diagnosis based on:
• nerve biopsy showing thinly myelnated, enlarged axons
• nerve conduction studies showing reduced nerve conduction
velocity (NCV), severely reduced compound motor action potentials
(CMAP) and absent sensory nerve action potentials (SNAP)
• abnormal visual evoked responses
• EEG showing increased slow wave activity
• MRI showing cerebellar and white matter abnormalities
Summary
Giant Axonal Neuropathy is a rare, autosomal recessive
disease. Diagnosis is mainly based on the appearance of
giant axons containing aggregated neurofilaments in a sural
nerve biopsy.
The disease locus was mapped to chromosome 16q21 by
homozygosity mapping.
Using additional markers the disease locus was refined to a
smaller, 590kb region.
Gigaxonin was identified by searching an EST database.
cDNA of the gene was cloned from a cDNA human brain
library.
Several different point mutations throughout the gene can
result in the disease phenotype.
Gigaxonin binds to MAP1B-LC, as determined fluorescently
tagged antibodies and immunoprecipitations. This interaction
improves the stability of the microtubule network.
Molecular treatment cannot be addressed until more is known
about the gigaxonin gene product.
References
Alberts, et al. Molecular Biology of the Cell, 4th Edition
Ben Hamida, et al. Neurogenetics 1(2):129-133 (1997).
Bomont, et al. Human Mutations 21(4):446-451 (2003).
Bomont, et al. Nature Genetics 26(3):370-374 (2000).
Cavalier, et al. European Journal of Human Genetics 8(7):527-534 (2000).
Criekinge WV and Beyaert R. Biological Procedures Online 2:1-38 (1999)
Ding, et al. Journal of Cell Biology 158(3):427-433 (2002).
Flanigan, et al. Annals of Neurology 43(1):143-148 (1998).
Kuhlenbaumer, et al Neurology 58(8):1273-1276 (2002).
Maia, et al. Neuropediatrics 19(1):10-15 1988.
Timmerman, et al. Nature Genetics 26(3):254-255 (2000).
Treiber-Held, et al. Neuropediatrics 25(2):89-93 (1994).
Identifying Gigaxonin Binding Partners
Ubiquitous expression
was confirmed with
immunoblot
Transfected cos7
cells expressed
gigaxonin
HA epitope tag at Cand N- terminal was
recognized by α-HA
antibody
Ding, et al. Journal of Cell Biology 158(3):427-433 (2002).