Genetic Analysis of a Novel Tubulin Mutation That Redirects Synaptic Vesicle Targeting and Causes Neurite Degeneration in C. elegans

et al. (2014) Genetic Analysis of a Novel Tubulin Mutation That Redirects Synaptic Vesicle Targeting and Causes Neurite Degeneration in C. elegans. PLoS Genet 10(11): e1004715. doi:10.1371/journal.pgen.1004715 Genetic Analysis of a Novel Tubulin Mutation That Redirects Synaptic Vesicle Targeting and Causes Neurite Degeneration in C. elegans Jiun-Min Hsu 0 1 Chun-Hao Chen 0 1 Yen-Chih Chen 0 1 Kent L. McDonald 0 1 Mark Gurling 0 1 Albert Lee 0 1 Gian Garriga 0 1 Chun-Liang Pan 0 1 Andrew D. Chisholm, University of California, San Diego, United States of America 0 Current address: Department of Biochemistry, University of Utah , Salt Lake City, Utah , United States of America 1 1 Institute of Molecular Medicine, College of Medicine, National Taiwan University , Taipei, Taiwan, 2 Electron Microscope Laboratory , University of California, Berkeley, Berkeley, California, United States of America, 3 Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California, United States of America, 4 Department of Chemistry, National Taiwan University , Taipei , Taiwan 2 www.plosgenetics.org Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gainof-function mutation in the C. elegans a-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of a-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This study was funded by National Science Council (NSC99-2320-B-002-080 and NSC100-2320-B-002-095-MY3), National Health Research Institute (NHRI-EX101-10119NC), and National Taiwan University (NTU-CDP-102R7810), to CLP, and National Institutes of Health grant (NS32057) to GG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Microtubule and molecular motors mediate polarized transport of neuronal proteins to either axons or dendrites [1]. Microtubules are oriented uniformly with their plus ends towards the distal end of the axon, which facilitates kinesin-dependent targeting of presynaptic proteins [2]. By contrast, targeting of postsynaptic molecules to the dendrite, such as glutamate receptors, requires the minus end-oriented dynein motors [3], consistent with the fact that many microtubules in the dendrites orient the minus end distally [4]. Mislocalization of presynaptic proteins to the dendrite occurs when this polarized pattern of axon-dendritic microtubule arrays is disrupted [5,6], when kinesin function is compromised [7,8], or when dynein activity is inadvertently increased [3,7,8]. Synaptic vesicle (SV) precursors are generated in the neuronal cell body and transported to the synapses by the unidirectional motor Kinesin 3/KIF1A [1]. On the other hand, the dynein motor complex mediates retrograde SV transport in the axon [1,9]. Since SVs are cargos for both KIF1A and dynein, it is intriguing that they are exclusively targeted to the axon and prevented from entering the dendrites. Previous biochemical and structural studies suggest that kinesin and dynein share an overlapping binding region at the C-terminus of a-tubulin [10]. The N-terminus of the H12 helix of the atubulin contains a stretch of absolutely conserved acidic residues (414EEGE, equivalent to 415EEGE in the yeast a-tubulin) and interacts with ATP-bound KIF1A [11]. A recent study on dynein structures also implicates this region in the interaction between microtubule and the microtubule-binding domain (MTBD) of dynein [12], although validation of this model in the context of in vivo, eukaryotic system is still lacking. Mutations of any of the three glutamic acids in the yeast a-tubulin to alanine dramatically reduced the frequency of kinesin binding to the microtubules [13]. Mutations of several conserved, acidic residues in the H12 helix of Axons and dendrites are two classes of neuronal process that differ in their functions and molecular compositions. Proteins important for synaptic functions are mostly synthesized in the cell body and sorted differentially into the axon or dendrites. Microtubules in the axon and dendrite maintain their structural integrity and regulate polarized protein transport into these compartments. We identified a novel a-tubulin mutation in C. elegans that caused mistargeting of synaptic vesicles and induced progressive neurite swelling, which resulted in late-onset neurodegeneration. We showed that this tubulin mutation weakened microtubule network and abnormally increased microtubule affinity for dynein, a motor protein responsible for cargo sorting to the dendrite. This enhanced microtubule-dynein affinity is due to augmented negative charge at the carboxyl terminus of a-tubulin. Neurite swelling and neurodegeneration could be ameliorated by reduced physical activity, suggesting that recurrent mechanical strain from muscle contraction jeopardized neurite integrity in the long run. Mutations in a- and btubulins are found in human neurological diseases; our findings therefore contribute to understanding the pathogenic mechanism of human neurological diseases associated with tubulin mutations. b-tubulin (E410, E412, D417) to alanine similarly reduced microtubule affinity for kinesins. Interestingly, E410K, D417H and D417N in the human b-tubulin TUBB3, among other point mutations, had been found in patients with congenital neurological syndro (...truncated)