Niwa S (2016) Sci Rep "The nephronophthisis-related gene ift-139 is required for ciliogenesis in Caenorhabditis elegans."

Niwa S

[Sci Rep, 2016]

Defects in cilia cause a spectrum of diseases known as ciliopathies. Nephronophthisis, a ciliopathy, is the most common genetic cause of renal disease. Here, I cloned and analysed a nephronophthisis-related gene ift-139 in Caenorhabditis elegans. ift-139 was exclusively expressed in ciliated neurons in C. elegans. Genetic and cellular analyses suggest that ift-139 plays a role in retrograde intraflagellar transport and is required for cilia formation. A homologous point mutation that causes ciliopathy disrupted the function of ift-139 in C. elegans. ift-139 is an orthologue of human TTC21B, mutations in which are known to cause nephronophthisis 12 and short-rib thoracic dysplasia 4. These results suggest that ift-139 is evolutionarily conserved and fundamental to the formation of cilia.

Niwa S (2017) J Vis Exp "Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons."

Niwa S

[J Vis Exp, 2017]

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Niwa S et al. (2017) Sci Rep "Structural basis for CRMP2-induced axonal microtubule formation."

Shigematsu H, Shirouzu M, Nakamura F, Fukai S, Aoki M, Goshima Y, Nitta R, Niwa S, Matsumoto T, Yamagata A, Tomabechi Y, Hirokawa N

[Sci Rep, 2017]

Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2-tubulin/microtubule interaction in the course of axonogenesis. The X-ray structural studies indicated that the main interface to the soluble tubulin-dimer is the last helix H19 of CRMP2 that is distinct from the known C-terminal tail-mediated interaction with assembled microtubules. In vitro structural and functional studies also suggested that the H19-mediated interaction promoted the rapid formation of GTP-state microtubules directly, which is an important feature of the axon. Consistently, the H19 mutants disturbed axon elongation in chick neurons, and failed to authorize the structural features for axonal microtubules in Caenorhabditis elegans. Thus, CRMP2 induces effective axonal microtubule formation through H19-mediated interactions with a soluble tubulin-dimer allowing axonogenesis to proceed.

Niwa S et al. (2016) Cell Rep "Autoinhibition of a Neuronal Kinesin UNC-104/KIF1A Regulates the Size and Density of Synapses."

Zhao C, Morikawa M, Lu H, Hirokawa N, Niwa S, Lipton DM, Shen K

[Cell Rep, 2016]

Kinesin motor proteins transport intracellular cargoes throughout cells by hydrolyzing ATP and moving along microtubule tracks. Intramolecular autoinhibitory interactions have been shown for several kinesins invitro; however, the physiological significance of autoinhibition remains poorly understood. Here, we identified four mutations in the stalkregion and motor domain of the synaptic vesicle(SV) kinesin UNC-104/KIF1A that specifically disrupt autoinhibition. These mutations augment both microtubule and cargo vesicle binding invitro. Invivo, these mutations cause excessive activation of UNC-104, leading to decreased synaptic density, smaller synapses, and ectopic localization of SVs in the dendrite. We also show that the SV-bound small GTPase ARL-8 activates UNC-104 by unlocking the autoinhibition. These results demonstrate that the autoinhibitory mechanism is used to regulate the distribution of transport cargoes and is important for synaptogenesis invivo.

Niwa S et al. (2017) Curr Biol "BORC Regulates the Axonal Transport of Synaptic Vesicle Precursors by Activating ARL-8."

Feng W, Liew GM, Niwa S, Shen K, Lu SY, Tao L, Nachury MV

[Curr Biol, 2017]

Axonal transport of synaptic vesicle precursors (SVPs) is essential for synapse development and function. The conserved ARF-like small GTPase ARL-8 is localized to SVPs and directly activates UNC-104/KIF1A, the axonal-transport kinesin for SVPs in C.elegans. It is not clear how ARL-8 is activated in this process. Here we show that part of the BLOC-1-related complex (BORC), previously shown to regulate lysosomal transport, is required to recruit and activate ARL-8 on SVPs. We found mutations in six BORC subunits-blos-1/BLOS1, blos-2/BLOS2, snpn-1/Snapin, sam-4/Myrlysin, blos-7/Lyspersin, and blos-9/MEF2BNB-cause defects in axonal transport of SVPs, leading to ectopic accumulation of synaptic vesicles in the proximal axon. This phenotype is suppressed by constitutively active arl-8 or unc-104 mutants. Furthermore, SAM-4/Myrlysin,a subunit of BORC, promotes the GDP-to-GTP exchange of ARL-8 invitro and recruits ARL-8 onto SVPs invivo. Thus, BORC regulates the axonal transport of synaptic materials and synapse formation by controlling the nucleotide state of ARL-8. Interestingly, the other two subunits of BORC essential for lysosomal transport, kxd-1/KXD1 and blos-8/Diaskedin, are not required for the SVP transport, suggesting distinct subunit requirements for lysosomal and SVP trafficking.

Anazawa Y et al. (2022) Methods Mol Biol "Analyzing the Impact of Gene Mutations on Axonal Transport in Caenorhabditis Elegans."

Niwa S, Anazawa Y

[Methods Mol Biol, 2022]

The development and functions of neurons are supported by axonal transport. Axonal transport is a complex process whose regulation involves multiple molecules, such as microtubules, microtubule-associated proteins, kinases, molecular motors, and motor binding proteins. Gain of function and loss of function mutations of genes that encode these proteins often lead to human axonal neuropathy. Caenorhabditis elegans provides a powerful genetic system to study the consequences of gene mutations for axonal transport. Here, we discuss advantages and limitations of using C. elegans, propose standard criteria, and describe methods to analyze the impact of gene mutations on axonal transport in C. elegans. To obtain solid conclusions, it is necessary to image single neurons in vivo labeled by a specific promoter and to confirm that a mutation changes the localization of a cargo. The motility parameters of the transported cargo should then be analyzed in the mutant. This method enables the axonal transport of proteins and organelles, such as synaptic vesicle precursors and mitochondria, to be analyzed.

Higashida M et al. (2022) Genes Cells "Dynein intermediate chains DYCI-1 and WDR-60 have specific functions in Caenorhabditis elegans."

Higashida M, Niwa S

[Genes Cells, 2022]

Dynein is a microtubule-dependent motor protein required for cell division, retrograde intracellular transport, and intraflagellar transport (IFT). Dynein 1 and dynein 2 serve as molecular motors in the cytoplasm and cilia, respectively. Each dynein consists of multiple subunits. Although the components of dynein 1 and dynein 2 are different and specific in most species, a previous study has suggested that dynein intermediate chain subunit DYCI-1 is shared by both dynein 1 and 2 in Caenorhabditis elegans (C. elegans). Here, we show that C. elegans has two dynein intermediate chains-DYCI-1 and WDR-60-and their functions are different. Mutational analysis showed that dyci-1 is essential for the retrograde axonal transport of synaptic vesicles. In the same mutant allele, IFT is not affected at all. Instead, wdr-60 is essential for IFT. Thus, we suggest that dynein 1 and dynein 2 have specific intermediate chains in C. elegans as in other organisms. This article is protected by copyright. All rights reserved.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Nakano J et al. (2022) Genes Cells "An ALS-associated KIF5A mutant forms oligomers and aggregates and induces neuronal toxicity."

Niwa S, Nakano J, Chiba K

[Genes Cells, 2022]

KIF5A is a kinesin superfamily motor protein that transports various cargos in neurons. Mutations in Kif5a cause familial amyotrophic lateral sclerosis (ALS). These ALS mutations are in the intron of Kif5a and induce mis-splicing of KIF5A mRNA, leading to splicing out of exon 27, which in human KIF5A encodes the cargo-binding tail domain of KIF5A. Therefore, it has been suggested that ALS is caused by loss of function of KIF5A. However, the precise mechanisms regarding how mutations in KIF5A cause ALS remain unclear. Here, we show that an ALS-associated mutant of KIF5A, KIF5A(Δexon27), is predisposed to form oligomers and aggregates in cultured mouse cell lines. Interestingly, purified KIF5A(Δexon27) oligomers showed more active movement on microtubules than wild-type KIF5A in vitro. Purified KIF5A(∆exon27) was prone to form aggregates in vitro. Moreover, KIF5A(Δexon27)-expressing Caenorhabditis elegans neurons showed morphological defects. These data collectively suggest that ALS-associated mutations of KIF5A are toxic gain-of-function mutations rather than simple loss-of-function mutations.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.