CMT2-associated KIF5AR280H disrupts SFPQ binding and alters localization of its mRNA cargo. (A) A schematic model for axon survival mediated by preferential transport of SFPQ by the KIF5A motor. bclw is an mRNA bound to SFPQ and its local translation in axons mediates axon survival (Cosker et al., 2016; Cosker et al., 2013). (B) A schematic representation of KIF5A, KLC1, and SFPQ. Red closed arrowhead indicates the location of KIF5AR280H CMT2 mutation, and open arrowhead indicates the location of SFPQY527A (Y-acidic motif). (C) Top: HEK 293T cells transfected with HA-SFPQ, and with either FLAG-tagged WT KIF5A or KIF5AR280H. HA was immunoprecipitated and blotted against FLAG and HA. Input represents 0.5% of 0.5 mg of protein lysate used for HA IP; total elute from the bead was run on the gel. Bottom: Quantification of precipitated protein normalized to input. **P = 0.0019 by unpaired two-tailed t test; n = 7; data represent mean ± SEM, (D) Examples of TUJ1 immunostaining. Magnified view depicts individual axon morphology, and corresponding binarized images show axons of DRG neurons grown in compartmented cultures expressing control, WT KIF5A, KIF5AR280H, and KIF5AR280H rescued with SFPQ. Red boxes outline regions shown for the higher magnification view of axons. Asterisks indicate interruptions in TUJ1 continuity. Scale bar: 100 µm and 10 µm (magnified image). (E) Quantification of axonal degeneration from E, which is calculated by using the ratio of the region of fragmented axons to total axon area (degeneration index [Sasaki et al., 2009]). Control (n = 36 captured images), WT KIF5A (n = 44), KIF5AR280H (n = 43), or KIF5AR280H + SFPQ (n = 44) from three independent experiments; ****P < 0.0001 by one-way ANOVA; data represent mean ± SEM. (F)bclw and β actin mRNA quantified by quantitative RT-PCR from CB and distal axons (DA) from compartmented cultures for control (Ctrl), WT KIF5A, KIF5AR280H-expressing neurons. Data normalized to gapdh; **P = 0.0068; *P = 0.0342 by one-way ANOVA; data from three independent experiments; data represent mean ± SEM.
Figure 6.

CMT2-associated KIF5AR280H disrupts SFPQ binding and alters localization of its mRNA cargo. (A) A schematic model for axon survival mediated by preferential transport of SFPQ by the KIF5A motor. bclw is an mRNA bound to SFPQ and its local translation in axons mediates axon survival (Cosker et al., 2016; Cosker et al., 2013). (B) A schematic representation of KIF5A, KLC1, and SFPQ. Red closed arrowhead indicates the location of KIF5AR280H CMT2 mutation, and open arrowhead indicates the location of SFPQY527A (Y-acidic motif). (C) Top: HEK 293T cells transfected with HA-SFPQ, and with either FLAG-tagged WT KIF5A or KIF5AR280H. HA was immunoprecipitated and blotted against FLAG and HA. Input represents 0.5% of 0.5 mg of protein lysate used for HA IP; total elute from the bead was run on the gel. Bottom: Quantification of precipitated protein normalized to input. **P = 0.0019 by unpaired two-tailed t test; n = 7; data represent mean ± SEM, (D) Examples of TUJ1 immunostaining. Magnified view depicts individual axon morphology, and corresponding binarized images show axons of DRG neurons grown in compartmented cultures expressing control, WT KIF5A, KIF5AR280H, and KIF5AR280H rescued with SFPQ. Red boxes outline regions shown for the higher magnification view of axons. Asterisks indicate interruptions in TUJ1 continuity. Scale bar: 100 µm and 10 µm (magnified image). (E) Quantification of axonal degeneration from E, which is calculated by using the ratio of the region of fragmented axons to total axon area (degeneration index [Sasaki et al., 2009]). Control (n = 36 captured images), WT KIF5A (n = 44), KIF5AR280H (n = 43), or KIF5AR280H + SFPQ (n = 44) from three independent experiments; ****P < 0.0001 by one-way ANOVA; data represent mean ± SEM. (F)bclw and β actin mRNA quantified by quantitative RT-PCR from CB and distal axons (DA) from compartmented cultures for control (Ctrl), WT KIF5A, KIF5AR280H-expressing neurons. Data normalized to gapdh; **P = 0.0068; *P = 0.0342 by one-way ANOVA; data from three independent experiments; data represent mean ± SEM.

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