Inhibition of sphingolipid synthesis improves outcomes and survival in GARP mutant wobbler mice, a model of motor neuron degeneration Constance S. Petita,b,1, Jane J. Leea,b,1, Sebastian Bolanda,b, Sharan Swarupb, Romain Christianoa,b, Zon Weng Laia,b, Niklas Mejherta,b, Shane D. Elliotta,b, David McFallc, Sara Haquea,b, Eric J. Huangc, Roderick T. Bronsond, J. Wade Harperb, Robert V. Farese Jra,b,e,2,3, and Tobias C. Walthera,b,e,f,2,3 aDepartment of Molecular Metabolism, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA 02115; bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; cDepartment of Pathology, University of California, San Francisco, CA 94143; dDepartment of Immunology, Harvard Medical School, Boston, MA 02115; eBroad Institute of MIT and Harvard University, Cambridge, MA 02124; and fHoward Hughes Medical Institute, Harvard University, Boston, MA 02115 Edited by Pietro De Camilli, Yale University, New Haven, CT, and approved March 16, 2020 (received for review August 12, 2019) Numerous mutations that impair retrograde membrane trafficking wobbler mice, a partial loss-of-function mutation (L967Q) in the between endosomes and the Golgi apparatus lead to neurode- Vps54-encoded subunit of GARP is responsible for motor neuron generative diseases. For example, mutations in the endosomal loss with features similar to amyotrophic lateral sclerosis (ALS) retromer complex are implicated in Alzheimer’s and Parkinson’s (9–13). diseases, and mutations of the Golgi-associated retrograde protein Previously, we and others showed that mutations of GARP (GARP) complex cause progressive cerebello-cerebral atrophy type complex proteins result in abnormalities in sterol and sphingo- 2 (PCCA2). However, how these mutations cause neurodegenera- lipid metabolism, as well as vacuolar or lysosomal morphology, in tion is unknown. GARP mutations in yeast, including one causing cultured yeast or human HeLa cells (14, 15). In particular, PCCA2, result in sphingolipid abnormalities and impaired cell GARP mutations result in accompanying increases in the growth that are corrected by treatment with myriocin, a sphingo- lipid synthesis inhibitor, suggesting that alterations in sphingoli- sphingoid long-chain bases, sphinganine and sphingosine, that NEUROSCIENCE pid metabolism contribute to cell dysfunction and death. Here we are intermediates in sphingolipid metabolism. The accumulation tested this hypothesis in wobbler mice, a murine model with a of these lipids can lead to cell dysfunction or cell death (16, 17), homozygous partial loss-of-function mutation in Vps54 (GARP pro- and sphingoid base accumulation in GARP mutant cells correlates tein) that causes motor neuron disease. Cytotoxic sphingoid long- with impaired growth (14). Further, treatment of GARP-deficient chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler Significance mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of Neurodegenerative diseases, including Alzheimer’s and Par- wobbler fibroblasts revealed extensive missorting of lysosomal kinson’s diseases, are major public health problems. Analysis of proteins, including sphingolipid catabolism enzymes, to the Golgi gene mutations that cause these diseases points to an impor- compartment, which may contribute to the sphingolipid abnor- tant role of membrane trafficking within cells in disease malities. Our findings establish that altered sphingolipid metabo- development, but how such processes participate in the pa- lism due to GARP mutations contributes to neurodegeneration and thology is unclear. Here, we analyze a murine model of com- suggest that inhibiting sphingolipid synthesis might provide a use- promised membrane trafficking (due to mutation of a protein ful strategy for treating these disorders. complex at the Golgi apparatus) and find metabolites of sphingolipids, molecules enriched particularly in the brain, ac- sphingolipid | neurodegeneration | myriocin | amyotrophic lateral cumulate in cells and tissues of this model. Preventing this sclerosis | wobbler mice buildup pharmacologically improved the symptoms of neuro- degeneration and survival in these mice. Thus, our data provide eurodegenerative diseases are a major health challenge in evidence that modulating sphingolipid metabolism will provide Naging populations (1, 2). Despite intensive investigation, a therapeutic avenue to treat some forms of neurodegenerative effective therapies for these diseases are lacking, in part because disease. the most proximate causes of neurodegeneration are not well understood. Genetic evidence from humans or animal models Author contributions: C.S.P., J.J.L., R.V.F., and T.C.W. designed research; C.S.P., J.J.L., S.B., S.S., R.C., Z.W.L., S.D.E., S.H., and J.W.H. performed research; J.J.L. and S.B. contributed has implicated impaired trafficking and cargo sorting by retro- new reagents/analytic tools; C.S.P., J.J.L., S.B., S.S., R.C., Z.W.L., N.M., D.M., E.J.H., R.T.B., mer or the Golgi-associated retrograde protein (GARP) com- and J.W.H. analyzed data; and C.S.P., J.J.L., R.V.F., and T.C.W. wrote the paper. plex as causes of neurodegenerative disease (3, 4). This The authors declare no competing interest. trafficking pathway involves the retromer complex at endosomes This article is a PNAS Direct Submission. for sorting endocytosed proteins into membrane carriers and the Published under the PNAS license. GARP complex for tethering retrograde carriers at the Golgi Data deposition: The mass spectrometry proteomics data have been deposited to the apparatus to receive those proteins. Defects in endosome-to-Golgi ProteomeXchange Consortium via the PRIDE partner repository with the dataset retrograde trafficking caused by a mutation in the retromer identifier PXD007981. component VPS35 (D620N) have been reported as causes of late- 1C.S.P. and J.J.L. contributed equally to this work. ’ and early-onset Parkinson s disease (5). Reduced expression of 2R.V.F. and T.C.W. contributed equally to this work. ’ retromer proteins has also been linked to Alzheimer sdisease 3To whom correspondence may be addressed. Email: [email protected] or (AD) (6). At the Golgi complex, mutations of GARP proteins [email protected]. (e.g., VPS51 and VPS53) have been identified as causes of severe This article contains supporting information online at https://www.pnas.org/lookup/suppl/ childhood neurological diseases, including progressive cerebello- doi:10.1073/pnas.1913956117/-/DCSupplemental. cerebral atrophy type 2 (PCCA2) in the case of VPS53 (7, 8). In First published April 28, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1913956117 PNAS | May 12, 2020 | vol. 117 | no. 19 | 10565–10574 Downloaded by guest on September 28, 2021 A wild type wild type wobbler wobbler **** ns 20 1 GOLGA2 100 (fold change) Golgi (µm) area covered by Golgi 10 μm 0 lysosome to nearest 0 wild type wobbler **** **** **** 0.20 1.0 1 nm) ) 2 LAMP1 0.10 0.5 (µm lysosomes ( area of lysosome distance between per cell (fold change) 10 μm number of lysosomes 0 0 0 BC 8 8 Neu1 Sptlc2 Smpdl3a Cers2 Neu3* 6 Lamp1 6 Gm2a* Asah2 Smpdl3b Sgpl1 Hexb* Galc* Npc1 Kdsr Asah1* Psap CtsD Smpd2 Psap* Gba* Cers6 Hexa Naga* Hexa* Smpd3 Cers5 Ugcg Sgms1 Smpd1* Golga2 Pdxdc1 4 4 Abca2 (p-value) Degs1 (p-value) 10 10 Sptlc1 B4galt5 Gla* -log -log Smpd4 2 2 Agk B3galnt1 B3galnt1 Gnptg Golgi proteins Golgi proteins Lysosomal proteins Sphk2* SL biosynthesis Golgi proteins (IF,WB) 0 0 Sgms2 SL degradation Lysosomal proteins (IF,WB) Lysosomal proteins -6 -4-20246 -6 -4 -2 0 2 4 6 -log fold change (wobbler / wild type) 2 -log2 fold change (wobbler / wild type) D Golgi fraction NPC1 150 kDa HEXA 50 kDa PSAP 50 kDa 50 kDa Pro-CTSD Prepro-CTSD 37 kDa Ponceau stain Mature CTSD 37 kDa ACTIN wild typewobbler wild typewobbler Fig. 1. The wobbler mutation of the GARP complex leads to missorting of proteins. (A) GOLGA2 staining in MEFs reveals no significant (ns) differences in the size of the Golgi compartment, whereas LAMP1 staining in wild-type and wobbler MEFs, followed by quantification of LAMP1 per cell (n ∼ 250 cells per condition), shows reduced numbers and larger size of lysosomes. Mean, SD ****P < 0.0001 by Mann–Whitney test. (Scale bar, 10 μm.) Analysis also revealed altered distribution of lysosomes depicted as distance from LAMP1 to GOLGA2 and between LAMP1 particles per cell area (n ∼ 20 cells per genotype) Mean, 95% CI ****P < 0.0001 by Mann–Whitney test. (B) TMT-based quantitative proteomics shows the enrichment of lysosomal proteins (orange) (P value = 2.98 × 10−35) and (C) enrichment of sphingolipid metabolism enzymes (P value = 6.85 × 10−11) in the Golgi-enriched fractions from wobbler cells, by Wilcoxon rank sum test. Lysosomal proteins (orange and green) are enriched in wobbler MEFs while Golgi proteins (purple) were relatively unchanged. Proteins labeled for imaging by immunofluorescence (A) or for Western blots (D and SI Appendix, Fig. S1F) are labeled (purple and green). Sphingolipid biosynthetic (blue) and catabolic (red) enzymes in the Golgi-enriched fraction shows many catabolic enzymes normally residing at lysosome (asterisk). (D) Western blot and Ponceau analysis of lysosomal proteins in Golgi-enriched fractions from wild-type and wobbler cells verifies enrichment of lysosomal proteins and reveals defects in CTSD processing. Actin is shown as a loading control in these fractions with enriched,