Altered molecular and cellular mechanisms in KIF5A-associated neurodegenerative or neurodevelopmental disorders

Abstract

Mutations targeting distinct domains of the neuron-specific kinesin KIF5A associate with different neurodegenerative/neurodevelopmental disorders, but the molecular bases of this clinical heterogeneity are unknown. We characterised five key mutants covering the whole spectrum of KIF5A-related phenotypes: spastic paraplegia (SPG, R17Q and R280C), Charcot-Marie-Tooth disease (CMT, R864*), amyotrophic lateral sclerosis (ALS, N999Vfs*40), and neonatal intractable myoclonus (NEIMY, C975Vfs*73) KIF5A mutants. CMT-R864*-KIF5A and ALS-N999Vfs*40-KIF5A showed impaired autoinhibition and peripheral localisation accompanied by altered mitochondrial distribution, suggesting transport competence disruption. ALS-N999Vfs*40-KIF5A formed SQSTM1/p62-positive inclusions sequestering WT-KIF5A, indicating a gain of toxic function. SPG-R17Q-KIF5A and ALS-N999Vfs*40-KIF5A evidenced a shorter half-life compared to WT-KIF5A, and proteasomal blockage determined their accumulation into detergent-insoluble inclusions. Interestingly, SPG-R280C-KIF5A and ALS-N999Vfs*40-KIF5A both competed for degradation with proteasomal substrates. Finally, NEIMY-C975Vfs*73-KIF5A displayed a similar, but more severe aberrant behaviour compared to ALS-N999Vfs*40-KIF5A; these two mutants share an abnormal tail but cause disorders on the opposite end of KIF5A-linked phenotypic spectrum. Thus, our observations support the pathogenicity of novel KIF5A mutants, highlight abnormalities of recurrent variants, and demonstrate that both unique and shared mechanisms underpin KIF5A-related diseases.

Fig. 1. KIF5A structure and distribution of pathogenic variants.

A Schematic representation of KIF5A structure, including KIF5A domains and their main functions. B Distribution of KIF5A pathogenic variants associated with HSP/SPG10 (black), CMT (blue), ALS (red), and NEIMY (green) phenotypes. The p.E237V and p.K907M variants (purple) are associated with West syndrome and severe global developmental delay, and Leber optic neuropathy, respectively. Variants associated with more than one disease are indicated in italics. The variants investigated in this study are indicated in bold and boxed. See Supplementary Table 1 for a detailed list of the variants and the associated phenotypes.

Fig. 2. Predicted effects of the novel variants p.R17Q and c.3017 A > G.

A p.R17Q KIF5A motor domain structure modelled by SWISS-MODEL (https://swissmodel.expasy.org) using wild-type (WT) KIF5A head as template (SMTL ID: 1mkj.1). Modelling predicts the loss of the ATP/ADP binding site in KIF5A motor domain as a consequence of the R17Q substitution. B Splicing prediction of variants identified in exon 27 and intron flanking regions. Diagram of the effect (either predicted or experimentally proven) of different splicing variants. The dotted lines indicate the probable aberrant effect on splicing based on the algorithms used. Variants that cause skipping of exon 27 (p.N999Vfs*40, ΔExon27) are indicated in black, the c.2993-1 G > A predicted to result in p.G998Efs*50 is in green, and the c.3005 A > G predicted to cause p.D1002Gfs*41 is in blue. The novel variant c.3017 A > G reported in this study is indicated in red.

Fig. 3. Mutant KIF5A localisation, levels, and solubility.

A Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently transfected with WT or mutant pGFP-KIF5A constructs. Endogenous β3-tubulin was stained in red. Nuclei were stained with DAPI. Scale bar 20 µm. B Western blot analysis of KIF5A protein levels in SH-SY5Y cells transiently transfected with WT or mutant pKIF5A constructs (N = 4). An empty vector (EV) was used as a transfection mock. GAPDH protein levels were used for normalisation. The graph represents mean optical densities relative to samples overexpressing WT KIF5A ± SD. One-way ANOVA with Fisher’s LSD post-test was performed. ns not significant; *P < 0.05; **P < 0.01. C Western blot analysis of KIF5A fractionation between the NP-40–soluble and the NP-40–insoluble protein fractions deriving from the same whole cell lysate of SH-SY5Y cells transiently transfected with WT or mutant pKIF5A constructs. D Western blot analysis of KIF5A protein levels in SH-SY5Y cells transiently transfected with WT or mutant pKIF5A constructs and treated with 10 µg/ml CHX for 1-2-4-6 h (N = 3). GAPDH protein levels were used for normalisation. The graph represents mean optical densities expressed as percentages of either WT or mutant KIF5A baseline (i.e., 0 h) levels ± SD. Two-way ANOVA with Sidak’s post-test was performed comparing WT and mutant KIF5A protein levels at each time point. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. Reciprocal distribution between mutant KIF5A and the WT protein or KIF5A cargoes.

A Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently co-transfected with equal amounts of WT or mutant pGFP-KIF5A and WT pmRFP-KIF5A constructs. Nuclei were stained with DAPI. Scale bar 20 µm. B Confocal microscopy analysis (63× magnification) of NSC-34 transiently co-transfected with WT or mutant pGFP-KIF5A constructs and pDsRed2-Mito. Nuclei were stained with DAPI. Scale bar 20 µm.

Fig. 5. Interplay between mutant KIF5A and autophagy.

A Western blot analysis of basal SQSTM1/p62 and MAP1LC3 protein levels in SH-SY5Y cells transiently transfected with WT or mutant pKIF5A constructs (N = 3). An empty vector (EV) was used as a transfection mock. GAPDH protein levels were used for normalisation. Graphs represent mean SQSTM1/p62 (left graph) and MAP1LC3 (right graph) optical densities relative to samples overexpressing WT KIF5A ± SD. One-way ANOVA with Fisher’s LSD post-test was performed. B Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently transfected with WT or mutant pGFP-KIF5A constructs. Endogenous MAP1LC3 was stained in red. Nuclei were stained with DAPI. Scale bar 20 µm. C Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently transfected with WT or mutant pGFP-KIF5A constructs. Endogenous SQSTM1/p62 was stained in red. Nuclei were stained with DAPI. Arrows highlight co-localisation between N999Vfs*40 KIF5A and SQSTM1/p62. Scale bar 20 µm. D Western blot analysis of KIF5A protein levels in SH-SY5Y cells transiently transfected with WT or mutant pKIF5A constructs and treated with 20 mM NH₄Cl for 16 h (N = 3). The graph represents the mean fold-change of GAPDH-normalised WT or mutant KIF5A protein levels induced by the treatment ± SD. One-way ANOVA with Fisher’s LSD post-test was performed.

Fig. 6. Interplay between mutant KIF5A and the UPS.

A Western blot analysis of KIF5A protein levels in SH-SY5Y cells transiently transfected with WT or mutant pKIF5A constructs and treated with 10 µM MG132 for 16 h (N = 3). An empty vector (EV) was used as a transfection mock. The graph represents the mean fold-change of GAPDH-normalised WT or mutant KIF5A protein levels induced by the treatment ± SD. One-way ANOVA with Fisher’s LSD post-test was performed. *P < 0.05; ***P < 0.001. B Western blot analysis of KIF5A fractionation between the NP-40–soluble and the NP-40–insoluble protein fractions deriving from the same whole cell lysate of SH-SY5Y transiently transfected with WT or mutant pKIF5A constructs and treated with 10 µM MG132 for 16 h (N = 3). The graph represents mean ratio between KIF5A insoluble and soluble protein levels in each experimental condition ± SD. Two-way ANOVA with Fisher’s LSD post-test was performed comparing insoluble/soluble KIF5A ratio between untreated (NT) and treated (MG132) samples for each KIF5A condition. *P < 0.05; ***P < 0.001. C Western blot analysis of Ub-R-YFP protein levels in SH-SY5Y transiently co-transfected with WT or mutant pKIF5A constructs and Ub-R-YFP (N = 3). GAPDH protein levels were used for normalisation. The graph represents mean Ub-R-YFP optical densities relative to samples overexpressing WT KIF5A ± SD. One-way ANOVA with Fisher’s LSD post-test was performed. **P < 0.01. D Proteasome activity analysis in SH-SY5Y cells overexpressing WT or mutant pKIF5A constructs (N = 3). Specific AMC-conjugated peptides (N-Suc-LLVY-AMC and Z-LLE-AMC) were used to evaluate chymotrypsin-like (top graph) and caspase-like (bottom graph) proteasome activities. Samples treated with 1 µM MG132 for 16 h were used as control. Graphs represent mean fluorescence levels relative to samples overexpressing WT KIF5A ± SD. One-way ANOVA with Fisher’s LSD post-test was performed. *P < 0.05; ***P < 0.001.

Fig. 7. ALS- and NEIMY-KIF5A.

A Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently transfected with WT or frameshift pGFP-KIF5A constructs. Endogenous β3-tubulin was stained in red. Nuclei were stained with DAPI. Scale bar 20 µm. B Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently co-transfected with equal amounts of WT or frameshift pGFP-KIF5A and WT pmRFP-KIF5A constructs. Nuclei were stained with DAPI. Scale bar 20 µm. C Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently co-transfected with WT or frameshift pGFP-KIF5A constructs and pDsRed2-Mito. Nuclei were stained with DAPI. Scale bar 20 µm. D Western blot analysis of KIF5A and SQSTM1/p62 fractionation between the NP-40–soluble and the NP-40–insoluble protein fraction in SH-SY5Y cells transiently transfected with WT or frameshift pKIF5A constructs (N = 3). An empty vector (EV) was used as a transfection mock. The graph represents mean ratio between SQSTM1/p62 insoluble and soluble protein levels in each experimental condition ± SD. Student’s t-test was performed comparing SQSTM1/p62 insoluble/soluble ratio in samples overexpressing N999Vfs*40 or C975Vfs*73 KIF5A. *P < 0.05. E Confocal microscopy analysis (63× magnification) of NSC-34 cells transiently transfected with WT or frameshift pGFP-KIF5A constructs. Endogenous HDAC6 was stained in red. Nuclei were stained with DAPI. Scale bar 20 µm. F Fluorescence recovery after photobleaching analysis of frameshift KIF5A aggregates in NSC-34 cells 48 h after transient transfection with pGFP-KIF5A constructs (N = 3). Scale bar 5 µm. The graph represents the percentage of fluorescence recovery of each aggregate over time compared to the baseline (i.e. 0 s) ± SD. Two-way ANOVA with Sidak’s post-test was performed comparing N999Vfs*40 and C975Vfs*73 KIF5A fluorescence recovery levels at each time point. *P < 0.05; ***P < 0.001.