An ALS-associated KIF5A mutant forms oligomers and aggregates and induces neuronal toxicity

Nakano, Juri; Chiba, Kyoko; Niwa, Shinsuke

doi:10.1101/2022.03.28.486133

Cited by 5 publications

(9 citation statements)

References 59 publications

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“…We expressed mScarlet-fused KIF5A(∆exon27) in cells and stained them with recombinant H2 mAb. KIF5A(∆exon27) formed aggregates in the cytoplasm as described previously (Fig 2G) (Nakano et al, 2022;Pant et al, 2022). Immunofluorescence microscopy showed that the recombinant H2 mAb recognized the KIF5A(∆exon27) aggregates in cells (Fig 2H and I).…”

Section: Use Of Recombinant H2supporting

confidence: 82%

Diversification of the recombinant anti-kinesin monoclonal antibody H2

Niwa

Chiba

2022

Preprint

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Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is fundamental to cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we generated vectors that expressed recombinant H2 in mammalian cells. We demonstrated that the recombinant H2 performed as well as the hybridoma-derived H2 in western blotting and immunofluorescence assays. The recombinant H2 could detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis (ALS)-associated KIF5A aggregates in the cell. Immunofluorescence microscopy showed that the single chain variable fragment (scFv) derived from the H2 mAb could specifically recognize KHCs in cells. In addition, we developed a chickenized anti-KHC scFv(H2), which broadens the application of H2 in immunofluorescence microscopy. Collectively, our findings validate recombinant H2 as useful for studying the function of KHCs.

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Section: Use Of Recombinant H2supporting

confidence: 82%

Diversification of the recombinant anti-kinesin monoclonal antibody H2

Niwa

Chiba

2022

Preprint

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“…While our manuscript was under review, two independent groups further supported that DExon27 causes neuronal toxicity through gain-of-function in different cellular and animal systems (Baron et al, 2022;Nakano et al, 2022).…”

Section: Discussionmentioning

confidence: 87%

ALS ‐linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain‐of‐function

et al. 2022

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Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C‐terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC‐derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single‐molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self‐association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain‐of‐function as an underlying disease mechanism in KIF5A‐associated ALS.

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“…It may also be the case that inactive motors can associate directly with membranous cargo (66), in prime position for cargo adaptor-driven activation at the right place and time. Recent studies showing that amyotrophic lateral sclerosis (ALS)-linked mutations in KIF5A disrupt autoinhibition underscore the broad pathophysiological significance of these questions (67)(68)(69).…”

Section: Discussionmentioning

confidence: 99%

Molecular architecture of the autoinhibited kinesin-1 lambda particle

et al. 2022

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Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the -helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.

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An ALS-associated KIF5A mutant forms oligomers and aggregates and induces neuronal toxicity

Cited by 5 publications

References 59 publications

Diversification of the recombinant anti-kinesin monoclonal antibody H2

Diversification of the recombinant anti-kinesin monoclonal antibody H2

ALS ‐linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain‐of‐function

Molecular architecture of the autoinhibited kinesin-1 lambda particle

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