- Title
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Peripheral Neuropathy and Decreased Locomotion of a RAB40B Mutation in Human and Model Animals
- Authors
- Son, W., Jeong, H.S., Nam, D.E., Lee, A.J., Nam, S.H., Lee, J.E., Choi, B.O., Chung, K.W.
- Source
- Full text @ Exp. Neurobiol.
Identification of RAB40B mutation in family with axonal neuropathic phenotype. (A) Pedigree of a CMT family (FC162) in which nonsense mutation of RAB40B was identified. Affected members are indicated by black boxes and circles. Genotypes of RAB40B c.249C>A are provided at the bottom of all the examined individuals. (B) Chromatograms of mutation sites. Black and red arrows indicate mutation site of rs781464100 (c.249C>A, p.Y83X). (C) Amino acid sequence alignment between human RAB40B and zebrafish and Drosophila homologs. The Rab40-characteristic SOCS domain is highlighted by yellow boxes. Position of the nonsense mutation (p.Y83X) is indicated by red letters. |
Zebrafish transformed by mutant hRAB40B showed motility defects. (A) Developmental morphologies of control, Mock or exogenous hRAB40B-Y83X mRNA-expressed larvae at 84 hpf. Scale bars, 2,000 μm. (B) Heatmap images showing swimming patterns between the control and hRAB40B-Y83X mRNA-injected zebrafish larvae. (C) Quantification of velocity analyzed in control zebrafish (n=65), Mock-expressing zebrafish (n=70), and hRAB40B-Y83X-transformant zebrafish (n=76). (D) Lateral view images after staining with α-SV2 (presynaptic region) and α-BTX (postsynaptic region) of the whole-mounted zebrafish at 84 hpf. Scale bars, 50 μm. (E) Comparison of NMJ signal ratios within ROI among control (n=43), Mock-expressing zebrafish (n=20), and hRAB40B-Y83X-transformant zebrafish (n=22). Comparisons of NMJ innervation in the presynaptic area (F) and postsynaptic area (G) among control (n=40), Mock-expressing zebrafish (n=23) and hRAB40B-Y83X-transformant zebrafish (n=20). Statistical significance was determined using an unpaired Student’s t-test (**p<0.01, ***p<0.001). |
Expressions of the hRAB40B in the transgenic embryos. After establishment of hRAB40B transgenic strains, expressions of the transformed genes were confirmed by RT-PCR in embryos from crosses of the transgenic flies with GAL4 drivers. Act5c-GAL4 is used for the control (Wt: Act5c>hRAB40B-wt, Y83X: Act5c>hRAB40B-Y83X). |
Reduced locomotion ability by ectopic expression of the C-terminal truncation mutation of hRAB40B. The locomotion ability of adult flies was quantified in a climbing assay. Progressive deterioration of motor performance was shown when the mutant hRAB40B was expressed ubiquitously (A, B, tub-GAL4) or under control of the pan-glial repo-GAL4 (B), but not with OK371 (A) or other neuron-specific GAL4 drivers. (C) The Drosophila Cul5 knockdown enhanced the locomotion defect with expression of the mutant hRAB40B, showing a genetic interaction of the defective motor phenotype with the CRL5-mediated proteolytic regulation (tested groups of each genotype, n=6; *p<0.05, **p<0.01, ***p<0.001). |
Locomotion abilities after ectopic expression of the mutant hRAB40B under control of tub-GAL4 or neuron-specific GAL4 drivers. The locomotion ability of adult flies was quantified in a climbing assay. A ubiquitous expression of the mutant hRAB40B reduced climbing ability (A, tub-GAL4). Repetition of the defective motor phenotype was assessed with neuron-specific GAL4 drivers: (A) elav-GAL4 or nSyb-GAL4 and (B) OK371, or OK307, ppk-GAL4, D42-GAL4 (tested groups of each genotype, n=4; *p<0.05). |