A Model of Hereditary Sensory and Autonomic Neuropathy Type 1 Reveals a Role of Glycosphingolipids in Neuronal Polarity
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Research Articles: Neurobiology of Disease A model of hereditary sensory and autonomic neuropathy type 1 reveals a role of glycosphingolipids in neuronal polarity Mengqiao Cui1, Rong Ying1, Xue Jiang1, Gang Li1, Xuanjun Zhang1, Jun Zheng1, Kin Yip Tam1, Bin Liang2, Anbing Shi3, Verena Gobel4 and Hongjie Zhang1 1Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau SAR, China 2Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiao-Chang Dong Road, Kunming, Yunnan 650223, China 3Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China 4Mucosal Immunology and Biology Research Center, Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA https://doi.org/10.1523/JNEUROSCI.2541-18.2019 Received: 6 October 2018 Revised: 15 May 2019 Accepted: 19 May 2019 Published: 28 May 2019 Author contributions: M.C., V.G., and H.Z. designed research; M.C., R.Y., X.J., and H.Z. performed research; M.C., R.Y., X.J., B.L., A.S., V.G., and H.Z. analyzed data; M.C., G.L., X.Z., J.Z., K.Y.T., B.L., A.S., V.G., and H.Z. edited the paper; G.L., X.Z., J.Z., and K.Y.T. contributed unpublished reagents/analytic tools; V.G. and H.Z. wrote the paper; H.Z. wrote the first draft of the paper. Conflict of Interest: The authors declare no competing financial interests. We thank the C. elegans Genetic Center, funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and National Bioresource Project (Japan) for strains; Hong Zhang for assistance with CRISPR- based gene editing; and Garry G. Wong, and members of the Zhang and Wong laboratories for reagents and discussion. Work in our lab is supported by the Science and Technology Development Fund, Macao S.A.R. (FDCT) project 060/2015/A2, 018/2017/AMJ and 050/2018/A2, Multi-Year Research Grant, University of Macau (MYRG) projects 2016-00066-FHS and 2017-00082-FHS. Correspondence should be addressed to To whom correspondence should be addressed: H. Z. at [email protected]; V. G. at [email protected] Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.2541-18.2019 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 the authors 1 A model of hereditary sensory and autonomic neuropathy type 1 reveals a role 2 of glycosphingolipids in neuronal polarity 3 4 Abbreviated title: Sphingolipids in neuronal polarity 5 6 Mengqiao Cui1, Rong Ying1, Xue Jiang1, Gang Li1, Xuanjun Zhang1, Jun Zheng1, Kin Yip 7 Tam1, Bin Liang2, Anbing Shi3, Verena Gobel4*, Hongjie Zhang1* 8 1Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau 9 SAR, China 10 2Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of 11 Zoology, Chinese Academy of Sciences, 32 Jiao-Chang Dong Road, Kunming, Yunnan 12 650223, China 13 3Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji 14 Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, 15 China 16 4Mucosal Immunology and Biology Research Center, Department of Pediatrics, 17 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA 18 19 * To whom correspondence should be addressed: 20 H. Z. at [email protected]; V. G. at [email protected] 21 22 Number of pages: 51 23 Number of figures: 8 24 Number of tables: 1 25 Number of extended data figures: 3 26 Number of words in abstract: 250 27 Number of words in introduction: 597 28 Number of words in discussion: 1648 29 Conflict of interest: The authors declare no competing financial interests. 30 Acknowledgements: We thank the C. elegans Genetic Center, funded by NIH Office of 31 Research Infrastructure Programs (P40 OD010440), and National Bioresource Project 32 (Japan) for strains; Hong Zhang for assistance with CRISPR-based gene editing; and Garry 33 G. Wong, and members of the Zhang and Wong laboratories for reagents and discussion. 34 Work in our lab is supported by the Science and Technology Development Fund, Macao 35 S.A.R. (FDCT) project 060/2015/A2, 018/2017/AMJ and 050/2018/A2, Multi-Year Research 36 Grant, University of Macau (MYRG) projects 2016-00066-FHS and 2017-00082-FHS. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Abstract 58 Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal 59 dominantly inherited neuropathy, clinically characterized by a loss of distal peripheral 60 sensory and motoneuronal function. Mutations in subunits of serine palmitoyltransferase 61 (SPT) have been linked to the majority of HSAN1 cases. SPTs catalyze the condensation of 62 L-serine with palmitoyl-CoA, the first committed and rate-limiting step in de novo sphingolipid 63 biosynthesis. Despite extensive investigation, the molecular pathogenesis of HSAN1 64 remains controversial. Here, we established a C. elegans model of HSAN1 by generating 65 a sptl-1(c363g) mutation, encoding SPTL-1(C121W) and equivalent to human SPTLC1C133W, 66 at the C. elegans genomic locus through CRISPR. The sptl-1(c363g) homozygous mutants 67 exhibited the same larval lethality and epithelial polarity defect as observed in sptl- 68 1(RNAi) animals, suggesting a loss-of-function effect of the SPTL-1(C121W) mutation. sptl- 69 1(c363g)/+ heterozygous mutants displayed sensory dysfunction with concomitant neuronal 70 morphology and axon-dendrite polarity defects, demonstrating that the C. elegans model 71 recapitulates characteristics of the human disease. sptl-1(c363g)-derived neuronal defects 72 were copied in animals with defective sphingolipid biosynthetic enzymes downstream of 73 SPTL-1, including ceramide glucosyltransferases, suggesting that 74 SPTLC1C133W contributes to the HSAN1 pathogenesis by limiting the production of complex 75 sphingolipids, including glucosylceramide. Overexpression of SPTL-1(C121W) led to similar 76 epithelial and neuronal defects and to reduced levels of complex sphingolipids, 77 specifically glucosylceramide, consistent with a dominant-negative effect of SPTL- 78 1(C121W) that is mediated by loss of this downstream product. Genetic interactions 79 between SPTL-1(C121W) and components of directional trafficking in neurons suggest that 80 the neuronal polarity phenotype could be caused by GSL-dependent defects in polarized 81 vesicular trafficking. 82 83 84 85 Significance Statement 86 The symptoms of inherited metabolic diseases are often attributed to the accumulation of 87 toxic intermediates or byproducts, no matter whether the disease-causing enzyme 88 participates in a biosynthetic or a degradation pathway. By showing that the phenotypes 89 observed in a C. elegans model of HSAN1 disease could be caused by loss of a 90 downstream product (glucosylceramide) rather than the accumulation of a toxic byproduct, 91 our work provides new insights into the origins of the symptoms of inherited metabolic 92 diseases while expanding the repertoire of sphingolipid functions, specifically, of 93 glucosylceramides. Although these findings have their most immediate relevance 94 for neuroprotective treatments for HSAN1, they may have implications for a much broader 95 range of neurologic conditions. 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 Introduction 114 HSAN1 is a rare progressive neurological disorder, clinically characterized by distal 115 peripheral sensory loss and disturbances in motoneuronal function, genetically identified to 116 be a heterogeneous condition associated with mutations in several genes (Dawkins et al., 117 2001; Rotthier et al., 2012). Among these genes, mutations in the first (SPTLC1) and second 118 (SPTLC2) subunits of the enzyme serine palmitoyltransferase have been linked to the 119 majority of HSAN1 cases. SPTLC1C133W is the most frequently identified mutation in patients 120 (Bejaoui et al., 2001; Rotthier et al., 2009). 121 Despite extensive investigation, the molecular pathogenesis of HSAN1 remains 122 controversial. Initially, it was believed to be caused by a loss of SPT function, which is 123 supported by evidence for reduced SPT activity and decreased total sphingolipid (SL) levels 124 in the presence of a mutant SPTLC1 allele (Bejaoui et al., 2002; Gable et al., 2002). 125 However, this hypothesis is challenged by reports of unchanged or even increased SL 126 quantities in symptomatic HSAN1 patients and SPTLC1C133W transgenic mice (Dedov et al., 127 2004; McCampbell et al., 2005). Recently, arguments for a “gain-of-function” scenario have 128 been advanced, based on the identification of a common feature of almost all examined 129 HSAN1-causing SPT mutations: a shift in SPT substrate specificity for L-alanine and L- 130 glycine over L-serine to form deoxy-sphingoid bases (DSBs), with the accumulation of 131 deoxy-sphingolipids thought to be the toxic cause of the nervous system phenotype (Eichler 132 et al., 2009; Penno et al., 2010; Rotthier et al., 2010; Garofalo et al., 2011; Rotthier et al., 133 2011). 134 However, elevated deoxy-sphingolipid levels have also been reported in conditions 135 with intact SPT activity (Zitomer et al., 2009; Bertea et al., 2010). Moreover, the SPT 136 activity is more severely reduced in SPTLC1C133W transgenic mice carrying two wild-type 137 alleles than in heterozygous SPTLC1 knockout mice (Eichler et al., 2009). Therefore, it is 138 still possible that the observed phenotypic changes in HSAN1 are due to a dominant 139 negative loss-of-function scenario.