1. Giant Axonal Neuropathy
A Disorder of Intermediate Filament Organization
By: Emily Gilles
Alberts, et al. Molecular Biology of the Cell, 4th Edition

2. Normal Intermediate Filaments
Alberts, et al. Molecular Biology of the Cell, 4th Edition
Intermediate filaments provide mechanical strength and resistant to shear stress. Ropelike fibers with a diameter of 10 nm.
Microtubules = bones, Actin filaments = muscles, Intermediate filaments = ligaments
Keratin = endothelial cells
Neurofilaments = in axons of neurons
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

3. Disease vs. Control Axon
Timmerman, et al. Nature Genetics 26(3): (2000).
(a) = GAN patient
Closely packed neurofilaments, peripheral cluster of microtubules
(b) = control
Homogenous distribution of neurofilaments and microtubules

4. 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.

5. The typical clinical case:
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
There isn’t a prevalence estimate, but fewer than 100 patients have been reported since 1972.
Dysarthria = difficulty in articulating words
Nystagmas = rapid, involuntary oscillating motion of the eye
Maia, et al. Neuropediatrics 19(1):10-15 (1988).
Treiber-Held, et al. Neuropediatrics 25(2):89-93 (1994).

6. Homozygosity Mapping What is homozygosity mapping?
Exactly like positional cloning
Fewer affected individuals can be used
Offspring must be from consanguineous parents
Homozygosity mapping is much like positional cloning, except a much smaller number of affected of individuals can be used. It is a strategy for mapping gene loci in offspring of consanguineous parents that derives its utility from the fact that affected children in addition to being homozygous at the disease allele, have a greatly increased likelihood of being homozygous by descent in adjacent regions of the genome. It allows gene mapping with a relatively small number of affected individuals.
Neurofilaments are composed of three subunits, NEF-H (chr 22q), NEF-M (chr 8p21), and NEF-L (chr 8p21). Because they found packed neurofilaments in dilated axons of the peripheral nerve in all patients, they first tested for linkage to mapped NF subunit genes. But these loci were not linked to the phenotype, so a genome wide screen was performed.
187 markers were used in the genomewide screen. At loci for which 12 or more of the total of 20 alleles were homozygous, nearby markers were analyzed. There was only one locus there was a flanking marker homozygous to a similar degree. (marker D16518) Genotyping revealed a region of homozygosity shared only by affected individuals in all 5 families. LOD scores for this locus was 4.18 for D16S Only two markers were shared by affected individuals---D16S3098 and D16S505. These markers surround a gene locus contained within 5.3 cM.
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): (1997).
Flanigan, et al. Annals of Neurology 43(1): (1998).

7. Refinement of the GAN locus
Additional YACs and BACs covering the region were identified by probing with markers from the 5 cM region. Used existing genes to determine the overlap of YACs and BACs. Eight polymorphic microsatellite markers and six biallelic markers were identified and ordered on the YAC and BAC contig. These markers were used to analyze linkage.
Analysis of phase known recombination events allowed them to define LC8 and D16S505 as new proximal end and distal recombinant marker. Families shared close geographic origin and an extended haplotype overlapping with the critical region. Haplotype sharing is good evidence for a common ancestor who passed the same mutation to both families. Divergence of the haplotype at LC3 and beyond indicates the occurrence of a historical distal recombination. The GAN critical interval is therefore delineated by the LC8 and LC3 markers. This region is less than 590 kb.
Haplotype analysis = Identify chromosomal segment shared by all affected individuals, but none of the unaffected individuals
Cavalier, et al. European Journal of Human Genetics 8(7): (2000).

8. The Gene: Gigaxonin
Four ESTs in the GAN locus presented an intron-exon structure. Only one of these ESTs were present in its entirety in the GAN critical region. They cloned the cDNA corresponding to EST aa by screening a human brain cDNA library. The cDNA and BAC sequences were aligned and 11 exons were identified. Mapping of the exons on a BAC contig indicates that they are in the same interval as EST and the D16S3098 marker.
Cloned DNA = 4,667 bp.
BTB domain mediates homomeric and heteromeric dimerization. Kelch repeats are predicted to adopt a beta-propellar structure, which are important for protein-protein interactions.
Most BTB/kelch proteins seem to be cytoskeletal protiens implicated in various cellular processes, such as actin cytoskeleton interaction and cytoplasmic sequestration of transcription factors.
Bomont, et al. Nature Genetics 26(3): (2000).

9. Expression is Ubiquitous
Ubiquitous expression confirmed by RT-PCR on multiple mouse tissues for the mouse orthologue (EST aa726085). Possible slightly higher levels of expression in the brain.
Bomont, et al. Nature Genetics 26(3): (2000).
RT-PCR was used to amplify RNAs from mouse tissues.
The probe used to detect was the EST aa

10. Disease-Causing Mutations
Mutation Analysis = screened for mutations at the genomic level. Intron primers were available. PCR products were analyzed by SSCP and silver stained. Missense mutations were searched for on control DNAs by restriction endonuclease digestion.
ASO = allele specific oligonucleotide hybridization.
SSCP = Single-Strand Conformation Polymorphism Analysis PCR is used to amplify the region of interest and the resultant DNA is separated as single-stranded molecules by electrophoresis in a non-denaturing polyacrylamide gel (Orita et al, 1989). A strand of single-stranded DNA folds differently from another if it differs by a single base
Mutations were discovered in 12/15 families. There is no evidence for non-allelic heterogeneity. PCR amplification excludes a deletion mutation in the three homozygous families. Or, mutations could be inversions, partial duplications, activation of a cryptic splice site, regulatory mutations, or were not detected by the SSCP method.
Bomont, et al. Nature Genetics 26(3): (2000).

11. Additional Mutations Discovered
One confirmed by RE digest with Taq1 and the other by SSCP analysis.
Determined by RE digest and ASO hybridization.
Bomont, et al. Human Mutations 21(4): (2003).
Kuhlenbaumer, et al Neurology 58(8): (2002).

12. 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)
Electron micrograph
Alberts, et al. Molecular Biology of the Cell, 4th Edition

13. Yeast Two-Hybrid Setup
Blue circle = DNA binding domain
Yellow square = gigaxonin
Red square = protein from human brain cDNA
Blue semi-circle = activator domain
Yeast two-hybrid system was used to identify protein with which gigaxonin interacts. The full-length gigaxonin was fused to a GAL4 DNA-binding domain and used as the bait to screen a human brain cDNA library. Identification of multiple positive clones suggested that microtubule-associated protein 1B light chain is a neuronal binding parner for gigaxonin
Gal4 = DNA binding domain and activator domain
Criekinge WV and Beyaert R. Biological Proceedures Online 2:1-38 (1999)

14. Gigaxonin colocalizes with MAP1B-LC
HA-Gig and flag-MAP1B-LC were cotransfected into cos7 cells
Gigaxonin and MAP1B-LC colocalize together
D shows diffuse accumulation of gigaxonin in cytoplasm when transfected alone
Diffuse distribution throughout cytoplasm suggests that gigaxonin lacks a direct cytoskeleton-binding site.
They also did an immunoprecipitation on the cotransfected cells and found that the two proteins coimmunoprecipitated together, confirming the direct association.
Ding, et al. Journal of Cell Biology 158(3): (2002).

15. In vivo Results Similar to in vitro
Double fluorescence shows colocalization in cultured mouse DRG neurons (dorsal root ganglion)
Arrows denote colocaliation
Coimmuniprecipitation assay of mouse brain tissue also confirmed the association of gigaxonin and MAP1B-LC
Ding, et al. Journal of Cell Biology 158(3): (2002).
Gigaxonin colocalizes with MAP1B-LC in vivo.

16. 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)
Examined microtubule stability in cells transfected with either MAP1B-LC alone or gigaxonin + MAP1B-LC. Cells were treated with colchicine, which will normally depolymerize microtubules within 15 minutes.
Cotransfected cells, the microtubule network remained intact even after a 90-minute treatment with colchicine.
Ding, et al. Journal of Cell Biology 158(3): (2002).

17. 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

18. 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.

19. References Alberts, et al. Molecular Biology of the Cell, 4th Edition
Ben Hamida, et al. Neurogenetics 1(2): (1997).
Bomont, et al. Human Mutations 21(4): (2003).
Bomont, et al. Nature Genetics 26(3): (2000).
Cavalier, et al. European Journal of Human Genetics 8(7): (2000).
Criekinge WV and Beyaert R. Biological Procedures Online 2:1-38 (1999)
Ding, et al. Journal of Cell Biology 158(3): (2002).
Flanigan, et al. Annals of Neurology 43(1): (1998).
Kuhlenbaumer, et al Neurology 58(8): (2002).
Maia, et al. Neuropediatrics 19(1):
Timmerman, et al. Nature Genetics 26(3): (2000).
Treiber-Held, et al. Neuropediatrics 25(2):89-93 (1994).

20. Identifying Gigaxonin Binding Partners
Ubiquitous expression was confirmed with immunoblot
Transfected cos7 cells expressed gigaxonin
HA epitope tag at C- and N- terminal was recognized by α-HA antibody
Ding, et al. Journal of Cell Biology 158(3): (2002).