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Glucosylsphingosine Promotes Α-Synuclein Pathology in Mutant GBA- Associated Parkinson's Disease

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Glucosylsphingosine Promotes Α-Synuclein Pathology in Mutant GBA- Associated Parkinson's Disease This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease Glucosylsphingosine promotes α-synuclein pathology in mutant GBA- associated Parkinson's disease Yumiko V. Taguchia,b, Jun Liuc, Jiapeng Ruanc, Joshua Pachecod, Xiaokui Zhangd, Justin Abbasib,e, Joan Keutzerd, Pramod K. Mistryc and Sreeganga S. Chandrab,e,f aDepartment of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06519. bProgram in Cellular Neuroscience, Neurodegeneration and Repair, Yale University, New Haven, Connecticut 06519. cDepartment of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06519. dSanofi Genzyme, Framingham, Massachusetts 01702. eDepartment of Neurology, Yale University School of Medicine, New Haven, Connecticut 06519. fDepartment of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06519. DOI: 10.1523/JNEUROSCI.1525-17.2017 Received: 1 June 2017 Revised: 17 August 2017 Accepted: 17 August 2017 Published: 28 August 2017 AUTHOR CONTRIBUTION: Y.V.T, J.L., J.R., J.P., X.Z., J.A., and J.K. conducted experiments. P.K.M. and S.S.C. designed and supervised experiments. S.S.C., Y.V.T., and P.K.M wrote manuscript. All authors read and edited text. Conflict of Interest: The authors declare no competing financial interests. We would like to thank Dr. Arthur Horwich, Yale University, and members of our labs for reading the manuscript and giving us suggestions. Special thanks to Dr. Li Gan (Gladstone/UCSF) for generously providing us with the neurogenin-2 knock-in iPSC line, and Dr. David Russell (University of Texas Southwestern Medical Center) for generously sending us the GBA2 S62 antibody. This work was supported by NINDS R01 NS064963, NS083846 and Regenerative Medicine Research Fund Grant 14-SCA-YALE-38 (SSC) and Center of Excellence in Clinical Translational Research Grant from Sanofi Genzyme and R01 AR 065932 from NIAMS (PKM). YVT was supported by the Cellular and Molecular Biology T32 GM007223 training grant and is presently a recipient of the NINDS T32 NS007224 training grant slot and the Lo Graduate Fellowship for Excellence in Stem Cell Research. Correspondence should be addressed to To whom correspondence should be addressed. S.S.C. can be reached at [email protected] or 203-785-6172, and P.K.M. can be reached at [email protected] or 203-785-3412. Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1525-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/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 © 2017 the authors 1 Glucosylsphingosine promotes α-synuclein pathology in mutant GBA-associated Parkinson’s disease 2 3 Abbreviated Title: Glucosylsphingosine promotes α-synuclein pathology 4 5 Yumiko V. Taguchia,b, Jun Liuc, Jiapeng Ruanc, Joshua Pachecod, Xiaokui Zhangd, Justin Abbasib,e, Joan 6 Keutzerd, Pramod K. Mistryc*, Sreeganga S. Chandrab,e,f*. 7 a Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06519. 8 b Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University, New Haven, Connecticut 9 06519. 10 c Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06519. 11 d Sanofi Genzyme, Framingham, Massachusetts 01702. 12 e Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06519. 13 f Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06519. 14 15 *To whom correspondence should be addressed. S.S.C. can be reached at [email protected] or 16 203-785-6172, and P.K.M. can be reached at [email protected] or 203-785-3412. 17 18 AUTHOR CONTRIBUTION 19 Y.V.T, J.L., J.R., J.P., X.Z., J.A., and J.K. conducted experiments. P.K.M. and S.S.C. designed and supervised 20 experiments. S.S.C., Y.V.T., and P.K.M wrote manuscript. All authors read and edited text. 21 22 CONFLICT OF INTEREST 23 The authors declare no competing financial interests. 24 25 ACKNOWLEDGMENTS 26 We would like to thank Dr. Arthur Horwich, Yale University, and members of our labs for reading the 27 manuscript and giving us suggestions. Special thanks to Dr. Li Gan (Gladstone/UCSF) for generously 28 providing us with the neurogenin-2 knock-in iPSC line, and Dr. David Russell (University of Texas 29 Southwestern Medical Center) for generously sending us the GBA2 S62 antibody. This work was supported by 30 NINDS R01 NS064963, NS083846 and Regenerative Medicine Research Fund Grant 14-SCA-YALE-38 (SSC) 31 and Center of Excellence in Clinical Translational Research Grant from Sanofi Genzyme and R01 AR 065932 32 from NIAMS (PKM). YVT was supported by the Cellular and Molecular Biology T32 GM007223 training grant 33 and is presently a recipient of the NINDS T32 NS007224 training grant slot and the Lo Graduate Fellowship for 34 Excellence in Stem Cell Research. 35 36 Number of Figures: 9 37 Abstract Word Count: 154 38 Introduction Word Count: 648 1 39 Discussion Word Count: 1,145 40 2 41 ABSTRACT 42 Glucocerebrosidase 1 (GBA) mutations responsible for Gaucher disease (GD) are the most common genetic 43 risk factor for Parkinson’s disease (PD). While the genetic link between GD and PD is well-established, the 44 underlying molecular mechanism(s) are not well understood. We propose that glucosylsphingosine, a 45 sphingolipid accumulating in GD, mediates PD pathology in GBA-associated PD. We show that while GD- 46 related sphingolipids (glucosylceramide, glucosylsphingosine, sphingosine, sphingosine-1-phosphate) promote 47 α-synuclein aggregation in vitro, glucosylsphingosine triggers the formation of oligomeric α-synuclein species 48 capable of templating in human cells and neurons. Using newly generated GD/PD mouse lines of either sex 49 [Gba mutant (N370S, L444P, knockout) crossed to α-synuclein transgenics], we show that Gba mutations 50 predispose to PD through a loss-of-function mechanism. We further demonstrate that glucosylsphingosine 51 specifically accumulates in young GD/PD mouse brain. With age, brains exhibit glucosylceramide 52 accumulations co-localized with α-synuclein pathology. These findings indicate that glucosylsphingosine 53 promotes pathological aggregation of α-synuclein, increasing PD risk in GD patients and carriers. 54 55 SIGNIFICANCE STATEMENT 56 Parkinson’s disease (PD) is a prevalent neurodegenerative disorder in the ageing population. 57 Glucocerebrosidase 1 mutations which cause Gaucher disease (GD) are the most common genetic risk factor 58 for PD, underscoring the importance of delineating the mechanisms underlying mutant GBA-associated PD. 59 We show that lipids accumulating in GD, especially glucosylsphingosine, play a key role in PD pathology in the 60 brain. These data indicate that ASAH1 (acid ceramidase1) and GBA2 (Glucocerebrosidase 2) enzymes that 61 mediate glucosylsphingsoine production and metabolism are attractive therapeutic targets for treating mutant 62 GBA-associated PD. 3 63 INTRODUCTION 64 Parkinson’s disease (PD) is a progressive neurodegenerative disease affecting ~1% of the ageing population 65 (Nussbaum and Ellis, 2003). A key component of PD pathogenesis is α-synuclein, a presynaptic protein 66 normally functioning to regulate synaptic vesicle cycling (Chandra et al., 2003; Vargas et al., 2014). Point 67 mutations in the SNCA gene, encoding α-synuclein, cause autosomal dominant PD (A30P, A53T, A53E, E46K, 68 H50Q, and G51D) (Polymeropoulos et al., 1997; Kruger et al., 1998; Zarranz et al., 2004; Lesage et al., 2013; 69 Proukakis et al., 2013). Duplication and triplication of SNCA also cause PD (Singleton et al., 2003; Chartier- 70 Harlin et al., 2004), highlighting the contribution of increased wild type (WT) α-synuclein levels to disease risk. 71 The importance of α-synuclein to PD was validated by the discovery that Lewy bodies, the pathological 72 hallmarks of PD, consist mainly of aggregated α-synuclein (Spillantini et al., 1997). While aggregated α- 73 synuclein present in Lewy bodies is fibrillar, recent studies have suggested that soluble oligomers are more 74 toxic, largely contributing to PD-related neurodegeneration (Winner et al., 2011). 75 Mutations in GBA, encoding lysosomal glucocerebrosidase 1 (GCase1), are the most common genetic risk 76 factor for PD (Sidransky et al., 2009). Biallelic mutations in GBA cause Gaucher disease (GD), a lysosomal 77 storage disorder (Tsuji et al., 1987; Tsuji et al., 1988). The most common GCase1 mutations are N370S and 78 L444P, accounting for 70% of disease alleles (Charrow et al., 2000; Grabowski et al., 2014). Both GD patients 79 and heterozygous carriers are at increased risk for PD, with higher risk in homozygous individuals (~20 vs. 5 80 fold, respectively) (Bultron et al., 2010; Alcalay et al., 2014). Hence, understanding the mechanistic links 81 between Gcase1 mutations and PD is of importance. GD patients develop Lewy body pathology, underscoring 82 the importance of α-synuclein aggregation in GD-associated PD (Wong et al., 2004). However, it is unclear 83 how GCase1 mutations contribute to PD pathology. Indeed, several contradictory mechanisms have been 84 proposed. It has been suggested that mutant GCase1 physically interacts with and induces acceleration of α- 85 synuclein aggregation in lysosomes (Yap et al., 2011), while another study showed GCase1 LOF-induced 86 lysosomal dysfunction causes α-synuclein aggregation in lysosomes, further perturbing its function (Mazzulli et 87 al., 2011). 88 Gcase1 catalyzes the conversion of glucosylceramide (GlcCer) to glucose and ceramide. The primary defect in 89 GD is the accumulation of GlcCer in lysosomes and is seen most prominently in macrophages. GlcCer is 90 alternatively processed to glucosylsphingosine (GlcSph) via lysosomal acid ceramidase, which can readily exit 91 the lysosome (Fig. 1A) (Elleder, 2006; Hein et al., 2007; Ferraz et al., 2016). As GlcCer builds up, it spills over 92 into the cytosol (Hein et al., 2007).
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