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Disease-Associated Mutations Hyperactivate KIF1A Motility and Anterograde Axonal Transport of Synaptic Vesicle Precursors

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Disease-Associated Mutations Hyperactivate KIF1A Motility and Anterograde Axonal Transport of Synaptic Vesicle Precursors Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors Kyoko Chibaa, Hironori Takahashib, Min Chenc, Hiroyuki Obinatad, Shogo Araie, Koichi Hashimotoc, Toshiyuki Odab, Richard J. McKenneya,1, and Shinsuke Niwad,1 aDepartment of Molecular and Cellular Biology, University of California, Davis, CA 95616; bDepartment of Anatomy and Structural Biology, Graduate School of Medicine, University of Yamanashi, Chuo, 409-3898 Yamanashi, Japan; cDepartment of System Information Sciences, Graduate School of Information Sciences, Tohoku University, Sendai, 980-8579 Miyagi, Japan; dFrontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, 980-0845 Miyagi, Japan; and eDepartment of Robotics, Graduate School of Engineering, Tohoku University, Sendai, 980-8579 Miyagi, Japan Edited by Iva Greenwald, Columbia University, New York, NY, and approved August 5, 2019 (received for review April 4, 2019) KIF1A is a kinesin family motor involved in the axonal transport Hereditary spastic paraplegia (SPG) is a human motor neuron of synaptic vesicle precursors (SVPs) along microtubules (MTs). disease associated with mutations in more than 60 genes (13). In humans, more than 10 point mutations in KIF1A are associ- Familial mutations in KIF1A have been identified as causes of ated with the motor neuron disease hereditary spastic paraplegia SPG (14). In addition to familial SPG, de novo mutations in (SPG). However, not all of these mutations appear to inhibit the KIF1A cause SPG associated with intellectual disabilities (15). motility of the KIF1A motor, and thus a cogent molecular expla- Most KIF1A mutations causing these neuropathies are located nation for how KIF1A mutations lead to neuropathy is not avail- within the conserved motor domain. Because the motor domains able. In this study, we established in vitro motility assays with of KIFs convert the energy of ATP hydrolysis into directional purified full-length human KIF1A and found that KIF1A mutations motility along the MT (2), it is thought that disease-associated associated with the hereditary SPG lead to hyperactivation of mutations in the motor domain most likely disrupt the motile KIF1A motility. Introduction of the corresponding mutations into mechanism of KIF1A and consequently anterograde axonal CELL BIOLOGY the Caenorhabditis elegans KIF1A homolog unc-104 revealed ab- transport of SVPs (13, 14, 16). Here we show that, in contrast to normal accumulation of SVPs at the tips of axons and increased this concept, some disease-associated mutations in KIF1A ac- anterograde axonal transport of SVPs. Our data reveal that hyper- tually lead to hyperactivation of motor activity, resulting in activation of kinesin motor activity, rather than its loss of function, overactive anterograde transport of SVPs. Our results highlight is a cause of motor neuron disease in humans. how proper cellular control over the motile activity of MT-based motors is essential for neuronal homeostasis in humans. axonal transport | hereditary spastic paraplegia | kinesin | KIF1A | UNC-104 Results xonal transport along microtubules (MTs) is fundamental Not All Disease-Associated Mutations in KIF1A Are Loss of Function. Afor the development and maintenance of neuronal cells. We noticed that the gain-of-function V6I mutation in unc-104, Kinesin superfamily proteins (KIFs) are a large family of MT- identified in our previous C. elegans genetic screen, is equiva- dependent molecular motors, some of which are integral in lent to the dominant SPG-associated mutation, V8M, in human driving anterograde axonal transport (1, 2). The kinesin-3 family KIF1A (11, 17) (Fig. 1A and SI Appendix, Fig. S1 A and B). We has been shown to transport a wide variety of cargoes in axons thus reasoned that not all disease-associated mutations in human (3–6). The constituents of synaptic vesicles (SVs) are synthesized in the cell body and transported to synapses by vesicle carriers Significance called synaptic vesicle precursors (SVPs) by the kinesin-3 family β motors KIF1A and KIF1B (5, 6). Genetic and cell biological Anterograde axonal transport supplies organelles and protein studies in Caenorhabditis elegans have revealed the basic molecular complexes throughout axonal processes to support neuronal mechanism of SVP axonal transport. UNC-104, a founding member morphology and function. It has been observed that reduced of the kinesin-3 family and a C. elegans ortholog of KIF1A and anterograde axonal transport is associated with neuronal dis- KIF1Bβ, was originally discovered in C. elegans through genetic eases. In contrast, here we show that particular disease- screening (7, 8). In loss-of-function unc-104 mutants, SVPs are not associated mutations in KIF1A, an anterograde axonal motor properly transported to synapses, leading to abnormal accumulation for synaptic vesicle precursors, induce hyperactivation of KIF1A of SVs in cell bodies and dendrites (8). motor activity and increased axonal transport of synaptic ves- Several kinesin motors are known to be regulated through icle precursors. Our results advance the existing knowledge of autoinhibitory mechanisms (9), and the axonal transport of SVPs the regulation of motor proteins in axonal transport and pro- is known to be controlled through autoinhibition of UNC-104/ vide insight into the cell biology of motor neuron diseases. KIF1A motor activity (10, 11). We previously obtained gain-of- function unc-104 mutants via suppressor screening of arl-8 mu- Author contributions: R.J.M. and S.N. designed research; K.C., H.T., H.O., T.O., and S.N. performed research; M.C., S.A., and K.H. contributed new reagents/analytic tools; S.N. tants (11). These gain-of-function mutations were mapped to the analyzed data; and K.C., H.T., K.H., R.J.M., and S.N. wrote the paper. motor domain, coiled-coil 1 (CC1) domain, and coiled-coil 2 The authors declare no conflict of interest. (CC2) domain (10, 12) and appear to lead to constitutive acti- This article is a PNAS Direct Submission. vation of UNC-104 motor activity (11). In mutant C. elegans that Published under the PNAS license. have gain-of-function unc-104 mutations, the axonal transport of 1To whom correspondence may be addressed. Email: [email protected] or SVPs is significantly increased. The mutated amino acid residues [email protected]. that lead to hyperactivation of UNC-104 are well conserved in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mammalian KIF1A, and analogous mutations disrupt the auto- 1073/pnas.1905690116/-/DCSupplemental. inhibition of mammalian KIF1A expressed in COS-7 cells (11). Published online August 27, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1905690116 PNAS | September 10, 2019 | vol. 116 | no. 37 | 18429–18434 Downloaded by guest on October 1, 2021 A S58L T99M G199R E253K or not loss of function. To discriminate among these possibilities, (ID, SPG, (ID, SPG, (ID, SPG, (ID, SPG, R307Q we next performed in vitro single molecule assays (Fig. 2). Pre- de novo) de novo) de novo) de novo) (ID, SPG, vious studies have shown that CC1 and CC2 bind to the neck de novo) coiled-coil domain and motor domain of KIF1A and inhibit its Human – — KIF1A Motor PH binding to MTs (10 12). The landing rate the number of mole- cules that bind to MTs normalized by time and MT length—is V8M A255V R350G (no ID, SPG, (no ID, SPG, (no ID, SPG, related to the ability of the motor domains to have productive AD) AR) AR) interactions with the MT (10). Thus, if the autoinhibition of KIF1A B D is disrupted by disease-associated mutations, the landing rate of mutant motors on MTs should increase in vitro. Full-length human KIF1A, KIF1A(V8M), KIF1A(A255V), and KIF1A(R350G) fused to a C-terminal mScarlet-strepII tag were expressed in sf9 cells using baculovirus and purified (Fig. 2A). Purified motors were diluted to approximately 1 nM, and unc-104(e1265) their behavior on MTs was observed using total internal reflec- tion fluorescence microscopy. We observed processive movement C of purified KIF1A on MTs, but with a relatively low landing rate (0.002 ± 0.004/μm/s at 1 nM) compared with previous data obtained +R307Q +A255V +KIF1A wild type +S58L +E253K +R350G control +V8M +T99M +G199R by the analysis of purified tail-truncated and constitutively dimer- ized constructs of KIF1A (10, 19) (Fig. 2 B and C and SI Appendix, Fig. S2), consistent with the motor existing predominantly in an unc-104(e1265) +hs KIF1A unc-104(e1265) mutant Fig. 1. Complementation of an unc-104 mutant worm by human KIF1A AB M 1 nM KIF1A 10 nM KIF1A 1 nM V8M cDNA. (A) Disease-associated mutations analyzed in this study. Mutations (kDa) wt V8MA255VR350G 250 shown above and below in the KIF1A schematic indicate de novo and fa- 150 100 milial mutations, respectively. ID, patients with intellectual disability; no ID, 75 patients without intellectual disability; SPG, patients with SPG. AD and AR 55 indicate autosomal dominant and autosomal recessive mutations, respectively. 35 1 nM A255V 1 nM R350G (See also SI Appendix,Fig.S1.) (B and C) The phenotypes of unc-104(e1265) C wt (B)andunc-104(e1265) expressing human KIF1A cDNA with the unc-104 25 V8M 20 promoter (C). (Scale bars: 1 mm.) (D) Bar graph showing worm movements. A255V unc-104(e1265) mutant worms without KIF1A expression (control) exhibit 15 R350G severe movement defects compared with the WT N2 strain. Ectopic ex- 10 pression of WT KIF1A, KIF1A(V8M), KIF1A(A255V), and KIF1A(R350G) using the DEwild type KIF1A KIF1A(V8M) (****) unc-104 promoter in the unc-104(e1265) background results in WT movement. 0.77±0.49 µm/s 1.6±0.71 µm/s Expression of other KIF1A with disease mutations (S58L, T99M, G199R, E253K, and R307Q) do not fully rescue unc-104(e1265) mutant worms. Asterisks indi- Number cate that the velocity is statistically not different from that of WT, indi- Number cating complete rescue (Tukey’s multiple comparison test).
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