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Giant Axonal Neuropathy

By: Emily Gilles. Giant Axonal Neuropathy. A Disorder of Intermediate Filament Organization. Alberts, et al . Molecular Biology of the Cell, 4 th Edition. Normal Intermediate Filaments. Provide mechanical strength Not all cells have IFs. Different cell types have different types of IFs.

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Giant Axonal Neuropathy

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

  2. Normal Intermediate Filaments • 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 Alberts, et al. Molecular Biology of the Cell, 4th Edition

  3. Disease vs. Control Axon • (a) = GAN patient • Closely packed neurofilaments, peripheral cluster of microtubules Timmerman, et al. Nature Genetics26(3):254-255 (2000). • (b) = control • Homogenous distribution of neurofilaments and microtubules

  4. Diagnosis = Sural Nerve Biopsy The pathological hallmark is the disorganization of the intermediate filament network of the cytoskeleton, with axons being predominantly affected. Control GAN

  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 Maia, et al. Neuropediatrics19(1):10-15 (1988). Treiber-Held, et al. Neuropediatrics25(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 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. Neurogenetics1(2):129-133 (1997). Flanigan, et al. Annals of Neurology43(1):143-148 (1998).

  7. Refinement of the GAN locus Cavalier, et al. European Journal of Human Genetics8(7):527-534 (2000).

  8. The Gene: Gigaxonin Bomont, et al. Nature Genetics26(3):370-374 (2000).

  9. Expression is Ubiquitous Bomont, et al. Nature Genetics26(3):370-374 (2000). RT-PCR was used to amplify RNAs from mouse tissues. The probe used to detect was the EST aa726805.

  10. Disease-Causing Mutations Bomont, et al. Nature Genetics26(3):370-374 (2000).

  11. Additional Mutations Discovered Bomont, et al. Human Mutations21(4):446-451 (2003). Kuhlenbaumer, et al Neurology58(8):1273-1276 (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) 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 Criekinge WV and Beyaert R. Biological Proceedures Online2:1-38 (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 Ding, et al. Journal of Cell Biology158(3):427-433 (2002).

  15. In vivo Results Similar to in vitro • Gigaxonin colocalizes with MAP1B-LC in vivo. Ding, et al. Journal of Cell Biology158(3):427-433 (2002).

  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) Ding, et al. Journal of Cell Biology158(3):427-433 (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. Neurogenetics1(2):129-133 (1997). Bomont, et al. Human Mutations21(4):446-451 (2003). Bomont, et al. Nature Genetics26(3):370-374 (2000). Cavalier, et al. European Journal of Human Genetics8(7):527-534 (2000). Criekinge WV and Beyaert R. Biological Procedures Online2:1-38 (1999) Ding, et al. Journal of Cell Biology158(3):427-433 (2002). Flanigan, et al. Annals of Neurology43(1):143-148 (1998). Kuhlenbaumer, et al Neurology58(8):1273-1276 (2002). Maia, et al. Neuropediatrics19(1):10-15 1988. Timmerman, et al. Nature Genetics26(3):254-255 (2000). Treiber-Held, et al. Neuropediatrics25(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 Biology158(3):427-433 (2002).

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