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).