Inhibition of 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 , 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 Previously, we and others showed that mutations of GARP (GARP) complex cause progressive cerebello-cerebral atrophy type complex result in abnormalities in sterol and sphingo- 2 (PCCA2). However, how these mutations cause neurodegenera- , 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 , 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 , to the Golgi 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 , 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

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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, but not purified, Golgi membranes.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1913956117 Petit et al. Downloaded by guest on September 30, 2021 yeast or HeLa cells with myriocin, an inhibitor of the first step in proteins annotated as lysosomal ( term, cellular sphingolipid synthesis catalyzed by serine palmitoyl transferase, component “lysosome”), 143 were enriched in the Golgi-enriched corrects impaired cell growth, cell dysfunction, and cell death (14). fraction of wobbler MEFs. The wobbler Golgi-enriched fraction Treatment with myriocin similarly alleviates lysosomal dysfunction also contained many lysosomal enzymes involved in sphingolipid and sphingolipid accumulation in Drosophila melanogaster mutated degradation, including GBA, NEU3, ASAH1, ASAH2, and for the homolog of Parkinsonism-associated PARK14 that exhibit GALC (Fig. 1C). The levels of many of the sphingolipid bio- impaired retromer function (e.g., decreased punctae of VPS26) synthetic enzymes, such as KDSR, DEGS1, and SPTLC1, which (18). Additionally, myriocin treatment suppresses behavioral ab- are normally found in the Golgi, were found at levels close to normalities associated with aberrant synapses in the PARK14 mu- those of wild-type controls. However, the levels of several enzymes tants (18). These findings suggest that mutations in retrograde of sphingolipid biosynthesis normally found in the ER, such as trafficking and cargo significantly perturb sphingolipid metabolism, serine palmitoyl transferases (SPTCLC1 and SPTLC2) and several and sphingolipid accumulation may contribute to cellular dysfunc- synthases (CERS2, CERS5, and CERS6) were relatively tion and neurodegenerative . more abundant in the Golgi-enriched fraction of wobbler MEFs. In this study, we tested the hypothesis that inhibiting the ac- To confirm the mislocalization of lysosomal proteins to Golgi- cumulation of sphingolipid intermediates alleviates cellular ab- enriched fractions in wobbler MEFs, we performed immuno- normalities and slows neurodegeneration that is caused by blotting of these fractions. Indeed, the levels of Niemann-Pick defects in retrograde trafficking in wobbler mice. We examined disease, type C1 (NPC1), a protein critical for intracellular the cellular phenotypes in embryonic fibroblasts derived from cholesterol trafficking, β-hexsoaminidase subunits (HEXA), an the mice with respect to sphingolipid metabolism and to sorting critical for degradation of , and of lysosomal proteins, including enzymes of sphingolipid catab- (PSAP), a crucial factor in hydrolysis, all olism. We also tested whether treating the mice with myriocin appeared to be enriched in wobbler Golgi fractions (Fig. 1B and would slow the progression of motor neuron disease and improve D). Also, blotting for cathepsin D (CtsTSD) revealed impaired their health and lifespan. processing of the prepro-CTSD and pro-CTSD forms to its Results mature form, which normally occurs in the lysosome. Taken together, these results indicate that the Vps54 mutation Missorting of Lysosomal Enzymes of Sphingolipid Degradation to wobbler in wobbler fibroblasts results in a profound missorting of lyso- Golgi-Enriched Fraction in Fibroblasts. Because deletions somal proteins to the Golgi apparatus, including many enzymes of components of the GARP complex can perturb cell mor- of sphingolipid degradation. These data are consistent with im- phology and function (14), we first assessed whether the Vps54 NEUROSCIENCE paired trafficking of these lysosomal proteins from the Golgi to mutation L967Q affects morphology of organelles in murine the lysosome. embryonic fibroblasts (MEFs) derived from wobbler mice. Whereas the Golgi apparatus (detected by Golga2 staining) Accumulation of Sphingoid Long-Chain Bases in Embryonic Fibroblasts wobbler appeared normal in MEFs, the lysosomes (detected by and Spinal Cords of wobbler Mice. Given the mislocalization of many lysosome-associated membrane protein [LAMP1]) appeared sphingolipid metabolism enzymes in wobbler MEFs, we next ex- A larger and were reduced in numbers (Fig. 1 ). Further, lyso- amined the effects of the wobbler mutation on the lipidome. We somes appeared to be more concentrated near the nucleus of found that levels of the long-chain bases, sphingosine and sphin- cells and less dispersed to the periphery. Upon analyzing cells ganine, and were increased in wobbler MEFs (Fig. 2 A stained for both lysosomes and Golgi, we found that lysosomes and B, Top). Sphinganine is part of the biosynthetic pathway, were in closer proximity to the Golgi in wobbler cells than in whereas sphingosine is an intermediate in sphingolipid degrada- control cells. Other organelles, such as mitochondria (SI Ap- tion. Ceramide is an intermediate of both synthetic and degra- pendix, Fig. S1A), appeared to be unaffected in wobbler MEFs, and there was no evidence of activation of the ER stress re- dative pathways. In addition, wobbler MEFs exhibited increased sponse (SI Appendix, Fig. S1B). levels of cholesterol esters, triglycerides, and phosphatidylcholine As GARP is an important mediator of protein sorting in the (Fig. 2A). endosomal system (7, 19, 20), we next tested whether the Previously, we showed that inhibition of sphingolipid synthesis Vps54 wobbler mutation affects protein homeostasis. For this, we by treatment with myriocin, a specific and potent inhibitor of first compared the abundance of proteins in wild-type and serine palmitoyl transferase (21), reduced sphingolipid accumu- wobbler MEFs by mass spectrometry of stable isotope labeling lation in yeast or HeLa cells depleted of GARP (14). Consistent with amino acids in cell culture (SILAC)-labeled cells (SI Ap- with this result, the sphingolipid abnormalities in wobbler MEFs pendix, Fig. S1C and Dataset S1). Proteins were reproducibly were suppressed when wobbler MEFs were cultured for 16 h in measured in both genotypes (SI Appendix, Fig. S1D). MEFs from the presence of myriocin (Fig. 2 A and B, Bottom). In particular, wobbler mice had reduced levels of numerous proteins of the the levels of sphingosine, sphinganine, ceramides, sphingomye- retromer complex (SI Appendix, Fig. S1C). The levels of proteins lin, and were all reduced with myriocin of the Wiskott-Aldrich syndrome protein and scar homolog treatment. In contrast, myriocin treatment had no effect on the (WASH) and GARP complexes were mostly unchanged. elevated levels of glycerolipids or neutral lipids in wobbler MEFs We next examined the subcellular proteomes of Golgi-enriched (Fig. 2A). Thus, MEFs from wobbler mice have increased levels fractions from wild-type and wobbler MEFs (lysosome-enriched of multiple sphingolipids, similar to what was found with GARP fractions could not be purified reliably from wobbler MEFs). mutant yeast or HeLa cells, and these abnormalities could be The enrichment scheme is shown in SI Appendix, Fig. S1E.We lessened by blocking sphingolipid synthesis. immunoblotted for Golgi proteins and found levels of GNPTG Inasmuch as wobbler mice develop progressive loss of large and B3GALNT were comparable between wild-type and wobbler motor neurons in the spinal cord (12), we also analyzed the lipid MEFs (Fig. 1F). However, we found differences in processing of content of the spinal cords of 4-mo-old wild-type and wobbler B3GALNT in the postnuclear supernatant. After tandem mass tag mice (Fig. 2C). Similar to the findings in MEF cells, the levels of (TMT) labeling of peptides and proteomic analyses by mass sphingosine were nearly twofold higher in spinal cord samples spectrometry, we found that wobbler Golgi-enriched fractions from wobbler mice. The levels of , which is syn- contained normal levels of Golgi markers (e.g., GOLGA1, thesized from ceramide, were reduced in the spinal cord. In GNPTG, and GOLGA2) but had a striking enrichment of nu- contrast, ceramide and sphinganine levels appeared to be un- merous lysosomal proteins (Fig. 1B and Dataset S2). Of 254 changed in spinal cords of wobbler mice (Fig. 2C).

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Fig. 2. The wobbler mutation of the GARP complex leads to accumulation of sphingolipid intermediate species in wobbler MEFs and spinal cords. (A) Lipidomics analysis of different classes of lipids shows a strong accumulation of sphingolipid species in wobbler MEF, that is rescued by myriocin treatment (n = 4 biological replicates). ***P < 0.001 and ****P < 0.0001 by one-way ANOVA with Bonferroni post hoc test. ChE: cholesterol esters, PE: phosphatidylethanolamine, PC: phosphatidylcholine. (B) Schematic representations of sphingolipid metabolism pathway, illustrating the effect of the GARP mutation, enzymes up-regulated and/or mislocalized from lysosome to Golgi and the changes in lipids upon myriocin treatment. Sphingolipid enzymes in the biosynthesis and degradation pathway but not identified by mass spectrometry in those experiments are shown. (C) Lipidomics analysis of different classes of lipids shows a strong accumulation in sphingolipid species in the spinal cord of wobbler mice (n = 4 to 6 biological replicates). *P < 0.05 and ****P < 0.0001 by unpaired t test. So: sphingosine, Sa: sphinganine, SM: sphingomyelin, CL: cardiolipin. No changes in phospholipids, PS and PE, ceramide (Cer) triglyceride (TG) or diacylglycerol (DG).

Treatment of wobbler Mice with an Inhibitor of Sphingolipid diet containing 2.2 mg/kg of myriocin, a concentration previously Synthesis Markedly Improves Outcomes and Survival. We next used to effectively inhibit sphingolipid synthesis in vivo (22). At tested the hypothesis that accumulation of toxic sphingolipid the administered dose, we found no evidence of hepatotoxicity intermediates leads to neurodegeneration in wobbler mice by due to myriocin, as assessed by measuring plasma levels of the treating these mice with the sphingolipid synthesis inhibitor liver enzymes aspartate aminotransferase (AST) and alanine myriocin. After weaning, we fed wild-type and wobbler mice a aminotransferase (ALT) after 10 or 50 wk of myriocin treatment

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Fig. 3. Inhibiting sphingolipid synthesis improves locomotor activity and grip strength in wobbler mouse. (A) Locomotion activity was measured in metabolic cages equipped with laser beams. Total activity over 6 h shown as area under the curve (AUC). ****P < 0.0001 by one-way ANOVA and Tukey’s multiple comparison (n = 4 to 8 per group) (B, C, and D) Boxplots representing the weekly monitoring of weight, wellness score, and grip strength for the untreated and myriocin-treated wobbler mice (n = 14 to 15 per genotype). Blue regions highlight the interquartile range for untreated wobbler controls at week 10. Scores for wild type marked by gray line (C and D). (E) After 10 wk of myriocin treatment, wobbler mice exhibit improved wellness index and grip strength. **P = 0.002 and P = 0.006 by Mann–Whitney test, for wellness index and grip strength, respectively. (n = 8 and 12 for untreated and myriocin-treated wobbler, respectively.)

(SI Appendix, Fig. S2A), although ALT levels trended higher at mice, and myriocin treatment restored activity levels to normal 50 wk. (Fig. 3A). Corroborating this result, video recordings showed In an initial study, we treated wobbler mice with myriocin for greater mobility for myriocin-treated wobbler mice than un- 10 wk to study disease-related outcomes and neuropathology. treated wobbler controls (Movies S1–S4). The motor neuron loss in wobbler mice results in forelimb pa- We also assessed neurodegeneration-related outcomes, in- ralysis and impaired mobility and grip strength (23). We assessed cluding body weight, a wellness index, and grip strength, in mobility in untreated and myriocin-treated mice by monitoring treated wobbler mice over 10 wk. Myriocin-treated wild-type home-cage locomotor activity for 6 h (the study time was re- mice showed a modest reduction in weight gain (∼4 g) over stricted from 24 to 6 h owing to the inability of wobbler mice to the study period (SI Appendix, Fig. S2B), suggesting some effect feed and drink in the metabolic cages). The locomotor activity of of the diet in wild-type mice. The wobbler mice gained weight wild-type mice was unaffected by myriocin (Fig. 3A). Locomotor much more slowly than wild-type mice, but untreated- and activity was reduced by ∼50% in wobbler mice versus wild-type myriocin-treated wobbler mice gained weight at similar rates

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Fig. 4. Inhibiting sphingolipid synthesis improves neuropathology outcomes and increases the survival rate of wobbler mouse. (A) Hematoxylin and eosin staining of the ventral horn in the gray matter from lower cervical spinal cord shows the presence of different stages of dying motor neurons in wobbler mice, compared to wild-type mice, as illustrated in the Insets.(B) Reduced astrocyte gliosis in wobbler mice as revealed by GFAP staining in the ventral horn of the lower cervical spinal cord (black arrowheads show astrogliosis). (Scale bar, 50 μm.) (C) Quantification of the average number of astrocytes (GFAP-positive cells) in the ventral horn of the lower cervical spinal cord (one-way ANOVA P = 0.003, Mann–Whitney between untreated and treated wobbler **P = 0.0047). (D) Myriocin treatment improved the survival rate of wobbler mice during a 1-y treatment (log-rank test: P = 0.003) (n = 17 to 18 mice per group).

(Fig. 3B). At each week of the study period, a wellness index [a (glial fibrillary acidic protein). As reported (27), wobbler mice combined score of activity, status of fur, eyes, tremor, and pos- showed increased astrogliosis in the cervical region of their spi- ture (ref. 24 and SI Appendix, Fig. S2C)] was assessed for each nal cords (Fig. 4 B and C). Importantly, we found that GFAP mouse in the different groups. Remarkably, myriocin treatment staining in the spinal cord of mice treated with myriocin was improved the wellness index of wobbler mice, and these out- markedly decreased (Fig. 4 B and C), consistent with reduced comes were easily visible by inspection of the mice (SI Appendix, degenerative processes in their neurons. Levels of choline Fig. S2D). As previously reported (25), wobbler mice had greatly acyltransferase-positive neurons were similar among the study reduced grip strength in their forelimbs starting at 3 wk of age. groups (SI Appendix, Fig. S3A). Of interest, the amounts of Therefore we measured the time mice were able to remain on a IBA1-positive cells were increased in myriocin-treated spinal vertical grid (Fig. 3D). Grip strength of wobbler mice was cords of either genotype (SI Appendix, Fig. S3B), suggesting that markedly improved with myriocin treatment (Fig. 3D and Movies inhibition of serine palmitoyl transferase increases microglia S5–S7). density independently of genotype, for unclear reasons. In par- Given that wobbler phenotypes result from degeneration of allel, we also attempted to analyze the fate of neuromuscular motor neurons (12, 26), we analyzed spinal cords of 3-mo-old junctions in the forelimbs of wobbler mice. As expected with the untreated or myriocin-treated wobbler and wild-type mice for death of motor neurons and the muscular atrophy, staining of the histological signs of neurodegeneration (Fig. 4). In the gray nicotinic acetylcholine receptor (using α-bungarotoxin) revealed matter of the spinal cord of wobbler mice but not in the wild-type damage to the neuromuscular junctions in wobbler mice, which mice, we found numerous dying motor neurons, as shown by appeared to be improved with myriocin treatment (SI Appendix, their swollen or vacuolated appearance (Fig. 4A). Dying motor Fig. S3A and C). neurons were identified in both untreated and myriocin-treated Mice harboring the wobbler mutation exhibit marked reduc- wobbler mice. Astrocytes have been implicated as key cell types tions in lifespan (13, 28). Indeed, in our study, we found that in modulating neurodegeneration due to lipid abnormalities 50% of wobbler mice did not survive to 100 d, whereas all of the (26), possibly by buffering the accumulation of specific lipids. To wild-type mice survived for over a year (Fig. 4D). To test whether assess the role of astrocytes in neurodegeneration of wobbler sphingolipids were causally involved in mediating wobbler mor- mice, we determined the amount of associated astrogliosis by tality, we treated a separate cohort of wobbler mice with and immunohistochemical staining of the astrocyte marker GFAP without myriocin in their diets and tested whether myriocin

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1913956117 Petit et al. Downloaded by guest on September 30, 2021 treatment improved survival. Remarkably, myriocin treatment mice. The wobbler mutant was identified in 1956 and exhibits markedly improved the survival rate at 100 d from 50% to about features of ALS, including progressive degenerative loss of mo- 90% and at 1 y, half the cohort were still alive. Myriocin treat- tor neurons and markedly diminished survival (9). We now show ment extended maximal lifespan of wobbler mice to well over that these mice have alterations in sphingolipid metabolism and, a year (Fig. 4D, P = 0.003 versus untreated wobbler controls). importantly, that the neurological disease phenotypes and com- promised survival of these mice can be significantly ameliorated Discussion by inhibition of sphingolipid synthesis. We note that our study In this study of the wobbler murine model, we show that defects may underestimate the treatment effect of myriocin, since the in retrograde trafficking due to a Vps54 mutation of the GARP weakness in wobbler mice leads to poor feeding, thereby likely complex result in sphingolipid abnormalities that are associated with reducing the amount of drug that was administered. Although missorting of lysosomal sphingolipid degradation enzymes. More- myriocin is not clinically utilized, other inhibitors of the first step over, we show that treatment of wobbler mice with myriocin, an in sphingolipid synthesis catalyzed by serine palmitoyl trans- inhibitor of the first step of sphingolipid synthesis, markedly im- ferase have been developed for other diseases (for examples see proved many outcomes for wobbler-associated neurodegeneration, refs. 34, 35). Thus, a path toward development of therapeutically including grip strength, neuropathology, and survival. These findings useful agents seems feasible. provide support for the hypothesis that neurodegeneration from Our data are consistent with accumulating evidence that defects in retrograde trafficking are due, at least in part, to abnor- implicates sphingolipid abnormalities as a cause of neuro- malities in sphingolipid metabolism that result from protein sorting degeneration. For instance, in ALS and AD substantial changes defects. of sphingolipids, among other lipid species, have been reported Consistent with our previous findings for GARP mutations in and suggested to cause neurodegeneration (29, 30, 36). Fur- yeast and HeLa cells (14), we found here that the sphingoid long- thermore, genetic defects of lysosomal lipid degradation en- chain bases, primarily sphinganine and sphingosine, accumulated zymes have long been recognized as causing inborn errors of in MEF cells derived from wobbler mice, and sphingosine accu- metabolism that result in severe neurological diseases (e.g., mulated in spinal cords. The accumulation of sphingoid bases to Gaucher’s, Sandhoff’s, and Tay-Sach’s diseases), and excess levels in cells has long been known to be cytotoxic (14, 16, causing Parkinson’s disease include sphingolipid catabolic en- 29, 30). The mechanisms of this cytotoxicity remain unclear, but zymes, such as Gba1 and Gba2 (37 –39). In addition, other Par- might involve permeabilization of cellular organelles or in- kinsonism genes might affect retrograde trafficking to alter terference with normal lipid metabolism (14, 31, 32). Accumu- sphingolipid metabolism, as recently shown for PLA2G6 (18). lation of long-chain bases in lysosomes is associated with Mutations in (dihydro)ceramide synthase 1 (CERS1) in mice NEUROSCIENCE lysosome dysfunction, including changes in calcium signaling or lead to early-onset cerebellar ataxia and Purkinje cell degenera- lysosomal leakage (31, 33). tion, and similar to our findings, long-chain sphingoid bases have The reasons for accumulation of long-chain bases (and other been implicated as the cause for the neuropathology (16, 40). sphingolipids) in GARP deficiency is unclear. Increased sphin- These findings suggest that the accumulation of intermediates of ganine levels reflect an increase of a sphingolipid synthesis in- sphingolipid metabolism can lead to neurodegeneration. Impor- termediate, and increased sphingosine reflects a degradation tantly, the current study shows that inhibiting the accumulation of pathway intermediate. This suggests that the abnormalities of toxic sphingolipid species may provide a therapeutic avenue for sphingolipid metabolism are complex and likely due to de- treating such neurodegenerative diseases. rangement of multiple steps in sphingolipid metabolism. Con- sistent with this, we found mislocalization of both sphingolipid Methods biosynthetic and degradative enzymes to the Golgi-enriched Cell Culture. MEFs were generated from wobbler mice as described (41) using fraction in wobbler MEFs. One possibility to explain these re- a heterozygous breeding pair with point mutation L697Q at exon 23. MEFs sults is that there is increased flux through sphingolipid bio- were collected at embryonic day 13 and genotyped for L597Q mutation wobbler using primer sequences 5′-TTTTTACACTGGAAATCTTCAAGCCTTAAAAGGCCT synthesis in the ER and Golgi in MEFs, combined with ′ ′ mislocalized activity of sphingolipid degradation enzymes to the TAAAAATCTGGATC-3 and 5 -GATGAACGACCTGGGTCTCCAGTCTGTCATCAC CTCTTCTGTTCCCAGATTTCGGCCATA-3′ and restriction digestion using BstYI Golgi apparatus. Although these enzymes are likely not opti- (NEB) as described by the Mutant Mouse Resource and Research Center mally active in the less acidic pH of the Golgi versus the lyso- (MMRRC). To immortalize MEFs, cultures were passaged at least 20 times at some, they may still possess some activities. In agreement with a 1:3 dilution every 3 d or until cells were 80 to 90% confluent. Wild-type our observations, mutations impairing retromer function also and wobbler MEFs were grown in Dulbecco’s modified Eagle medium lead to the cellular accumulation of the sphingolipid ceramide (DMEM) supplemented with glutamine, 10% fetal bovine serum (FBS), and and the long-chain bases sphinganine and, particularly, sphin- 1% penicillin/streptomycin supplement (all from Invitrogen) and maintained gosine (18). Possibly, the increased levels of the neutral ceram- at 37 °C in a 5% CO2 incubator. MEFs were grown on a 15-cm culture dish idases Asah1 and Asah2 that we found in wobbler cells could treated with myriocin or dimethyl sulfoxide (DMSO) until cells reached ∼ aggravate this metabolic dysregulation. 80% confluency and collected for subsequent lipid or protein analysis. It is unclear why lysosomal enzymes are mistargeted to the Lipidomics. Lipids were extracted from cells or tissue according to Folch’s Golgi apparatus if the primary defect in GARP mutations is in method (42). The organic phase of each cell culture sample was normalized retrograde trafficking from the endosome to the Golgi. A pos- by total protein (using bicinchoninic acid [BCA] assay), whereas tissue sam- sible explanation is that mistrafficking is a secondary conse- ples were normalized according to phosphatidylcholine due to variations in quence of failure to recycle trafficking receptors, such as the the protein measurements. Samples were routinely subjected to two rounds mannose-6-phosphate receptor, sortilins, or other proteins that of extraction. The high-performance liquid chromatography-mass specrom- are required for bringing lysosomal proteins from the secretory etry (HPLC-MS) method was adopted from refs. 43, 44. Briefly, HPLC analysis pathway to the lysosome (19, 20). Indeed, we found doubled employed a C30 reverse-phase column (Thermo Acclaim C30, 2.1 × 250 mm, amounts of both cation-dependent and -independent mannose- 3 μm, operated at 55 °C; Thermo Fisher Scientific) connected to a Dionex 6-phosphate receptors in the Golgi fraction of wobbler MEFs, UltiMate 3000 HPLC system and a QExactive orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a heated electrospray ionization similar to what was recently reported for VPS51 mutations as (HESI) probe and performed with run conditions previously described (45). well as a fivefold increase of the sortilin-related receptor (Fig. 2 – Samples were injected in positive and negative ionization modes and two and Datasets S2 S4). technical replicates, respectively. Processing of raw data was performed Remarkably, treatment of wobbler mice with myriocin was using LipidSearch software (Thermo Fisher Scientific/Mitsui Knowledge In- sufficient to ameliorate many of the phenotypes found in these dustries) (46, 47). The assembled results were exported to R-Studio where all

Petit et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 30, 2021 identified lipids were included for subsequent analyses if they fulfilled the water. Mice were weaned at 3 to 4 wk of age and put on either a myriocin- following LipidSearch-based criteria: 1) reject equal to zero, 2) main grade A supplemented diet (2.2 mg/kg of diet) or the same diet without myriocin or main grade B and APValue < 0.01 for at least half of the samples, and 3) (ResearchDiet, New Brunswick, NJ) ad libitum for 10 wk. wobbler and lit- no missing values across all samples. Based on these filters, 666 out of 2,211 termate wild types were weighed regularly and observed. During observa- lipids were present in all samples and displayed high accuracy of identifi- tion, the mice were scored for parameters of wellness, as depicted in cation (Dataset S5). Further quality controls used pairwise correlations be- supplementary data. This score consists of parameters adapted from a tween replicates and principal component analysis (PCA) (FactomineR published wellness score (24). Grip strength was recorded weekly by placing package) (48), comparing sample groups (SI Appendix, Fig. S4). Main areas of mice on a vertical grid for 30 s. For locomotion studies, mice were placed in the included lipids were summarized according to their class, and the total cages equipped with laser beams for a total of 6 h (3 h with light and 3 h in class abundance was compared between sample groups using one-way the dark). Analysis between these groups showed no differences due to ANOVA, followed by a Bonferroni post hoc test. gender. At the end of the 10-wk study, mice were anesthetized with iso- flurane, blood was collected, and then the mice were perfused with PBS. Immunofluorescence and Organelle Analysis in Cells. For standard immuno- Brain, liver, and spinal cord were collected for further analysis. AST and ALT fluorescence experiments, cells were grown on glass-bottom 24-well plates measurements were performed by the Analytical Core at the University of and fixed with 4% formaldehyde in phosphate-buffered saline (PBS). Cells Massachusetts Mouse Metabolic Phenotyping Center using plasma samples were permeabilized in 0.1% saponin and 0.1% bovine serum albumin (BSA). from 3- or 12-mo-old male and female mice. Blocking was performed with 3% BSA and 0.1% Triton X-100 or 0.1% sa- ponin and 5% normal goat serum. Primary and secondary antibody dilutions SILAC Proteomics of Total Cell Proteins. For SILAC experiments, cells were were performed in the same solution used for blocking. Alexa 488-, Alexa cultured with heavy or light lysine and arginine (Cambridge Isotope) and 555-, and Alexa 647-conjugated secondary antibodies were obtained from dialyzed FBS (Thermo Fisher Scientific). The cells were incubated in medium Invitrogen. Nuclei were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole supplemented with 1 μM myriocin (MilliporeSigma) for the indicated times (DAPI) (Invitrogen) during one of the postsecondary antibody washes. Mi- (or DMSO as a negative control). tochondria and nuclei were identified in live-cell imaging experiments with SILAC-labeled cell lysates were reduced using 5 mM dithiothreitol (DTT) MitoTracker (Thermo Fisher Scientific) and Hoechst 33342 (1 μg/mL for (MilliporeSigma) at 37 °C for 1 h, followed by alkylation of cysteine residues 10 min; Cell Signaling Technology) staining, respectively. Postacquisition using 15 mM iodoacetamide (MilliporeSigma) in the dark at room temper- image analysis was performed with ImageJ (NIH), ImageJ plug in, DiAna (49), ature for 1 h. Excessive iodoacetamide was quenched with 10 mM DTT. and Cell Profiler software (50) to measure the average LAMP1, GGOLGA2, or Proteins were precipitated by the addition of nine volumes of ice-cold ace- MitoTracker staining per cell. Using the ImageJ plugin DiAna, LAMP1 par- tone and one volume of methanol and incubated at −80 °C for 2 h. Pre- ticles were segmented using spot thresholding at default settings and a cipitated proteins were centrifuged for 1 h at 4500 × g and 4 °C. After Gaussian thresholding method, using a 2-pixel radius for Golgi particles. washing with methanol, proteins were resolubilized in 100 mM NaOH aided Distances were measured from the center of LAMP1 particles to the edge of by sonication at 4 °C, and the solution was brought to pH 7.5 with 200 mM the Golga2 signal in cells costained for lysosomes and the Golgi. The distance Hepes-free acid. Protein concentrations were determined using a bicincho- from the center of two LAMP1 particles was normalized to total cell area. ninic acid assay kit (Pierce), followed by equal mixing of proteins at 1:1 ratio Lysosomal area was analyzed using auto local threshold Phansalkar method (light:heavy labels). Proteins were trypsinized using sequencing grade tryp- radius 15 and the analyze particle function on ImageJ. Hematoxylin and sin (Promega) at 37 °C for 16 h. Digested peptides were subsequently eosin (H&E) and GFAP staining were performed on the spinal cords by the desalted using self-packed C18 STAGE tips (3M Empore) (51), and analyzed Histopathology Core and the Rodent Histopathology Core of Dana-Farber/ on an Orbitrap Q-Exactive HF (Thermo Scientific) mass spectrometer coupled Harvard Cancer Center in Boston, MA. GFAP-positive sections were quanti- to an Easy nanoLC 1000 (Thermo Scientific) with a flow rate of 300 nl/min. fied by blinded investigators. Stationary phase buffer was 0.5% formic acid, and mobile phase buffer was 0.5% (vol/vol) formic acid in acetonitrile. A gradient of increasing organic Antibodies and Immunoblotting. The following primary antibodies were used proportion was used for peptide separation (5 to 40% [vol/vol] acetonitrile in our experiments: tubulin (Sigma-Aldrich), LAMP1 (1D4B) (Developmental over 265 min). Peptides were separated on self-packed analytical column Studies Hybridoma Bank), Golga2/Gm130 (BD Transduction Laboratories), Hexa using PicoTip emitter (New Objective) using Reprosil Gold 120 C18 (Novus Biologicals), CtsD (Novus Biologicals), Psap (Proteintech), Npc1 (Pro- (Dr. Maisch) 1.9-μm particle size resin. The mass spectrometer operated in teintech), Actin (Cell Signaling), α-bungarotoxin (Abcam), and Neurofilament-H data-dependent acquisition mode with a top 10 method at a mass range of (Cell Signaling). 300 to 2,000 Da. Mass spectrometry files were analyzed by MaxQuant (52) version 1.5.2.8 qRT-PCR Analysis. Total RNA was extracted from cells using Qiazol and RNeasy with the Uniprot mouse database downloaded on November 2016, counting Mini kit (Qiagen). From total RNA, cDNA was reverse transcribed using iScript 52,026 protein entries. MaxQuant analysis included an initial search with a cDNA synthesis kit (Bio-Rad). Power SYBR Green Master Mix (Thermo) with precursor mass tolerance of 20 ppm for mass recalibration. In the main the following primers was used in qRT-PCR analysis: mTfeb: 5′-CCACCCCAG Andromeda search, precursor mass and fragment mass were searched with CCATCAACAC-3′ (forward primer), 5′-CAGACAGATACTCCCGAACCTT-3′ (re- initial mass tolerance of 4.5 ppm. The search included variable modifications verse primer); mHexa: 5′-TGGCCCCAGTACATCCAAAC-3′ (forward primer), of methionine oxidation and N-terminal acetylation and fixed modification 5′-GGTTACGGTAGCGTCGAAAGG-3′ (reverse primer); mGba: 5′-GCCAGGCTC of carbamidomethyl cysteine. Minimum peptide length was set to six amino ATCGGATTCTTC-3′ (forward primer), 5′-CACGGGGTCAAGAGAGTCAC-3′ (re- acids, and up to two missed cleavages were allowed. The false discovery rate verse primer); mLAMP1: 5′-CAGCACTCTTTGAGGTGAAAAAC-3′ (forward was set to 0.05 for peptide and protein identifications; however, proteins primer), 5′-ACGATCTGAGAACCATTCGCA-3′ (reverse primer); mCtsd: 5′-GCT that were independently identified in at least three replicates (n ≥ 3) were TCCGGTCTTTGACAACCT-3′ (forward primer), 5′-CACCAAGCATTAGTTCTC considered in the final analysis. For comparisons between samples, we used CTCC-3′ (reverse primer); mCyclophilin: 5′-TGGAAGAGCACCAAGACAACA-3′ a labeling scheme based on multiplicity 2: Arg0/Lys0 (light label) and Lys8/ (forward primer), 5′-TGCCGGAGTCGACAATGAT-3′ (reverse primer); mXbp1s: Arg10 (heavy label), respectively. A minimum of two ratio counts was used 5′-GGTCTGCTGAGTCCGCAGCAGG-3′ (forward primer), 5′-AGGCTTGGTGTA to determine the normalized protein intensity. Protein table was filtered to TACATGG -3′ (reverse primer); mXbp1t: 5′-TTGTCACCTCCCCAGAACATC-3′ eliminate the identifications from the reverse database and common (forward primer), 5′-TCCAGAATGCCCAAAAGGAT-3′ (reverse primer); mBip: contaminants. 5′-GCCAGGCTCATCGGATTCTTC-3′ (forward primer), 5′-CACGGGGTCAAG AGAGTCAC-3′ (reverse primer); and mChop: 5′-CAGCACTCTTTGAGGTGA Partial Purification of Golgi Membranes. All steps were carried out at 4 °C. For AAAAC-3′ (forward primer), 5′-ACGATCTGAGAACCATTCGCA-3′ (reverse each genotype, cells grown to 80 to 90% confluency in 15 cm × 2.5 cm (D × primer). Expression was normalized to the average of cyclophilin levels. H) plates (10 each × 3 replicates = 30 plates) were washed twice with PBS (Corning). The cells were carefully detached in PBS containing protease in- Mouse Experiments. All animal studies followed guidelines issued by Harvard hibitors (MilliporeSigma) (2 to 3 mL/plate) using a cell lifter and transferred University’s institutional animal care and use committees. The mouse strain to 50-mL tubes. Plastic Pasteur pipettes were used to handle cells and cell (NFR/N genetic background) used for this research project, NFR.B-Vps54wr/ fractions. The cells were centrifuged at 500 × g for 5 min, supernatants were Mmmh, RRID:MMRRC_030340-MU, was obtained from the MMRRC at the discarded, and the cells were washed in homogenization buffer (HB) (0.5 M

University of Missouri, a NIH-funded strain repository, and was donated to sucrose, 5 mM MgCl2, 0.1 M KH2PO4/K2HPO4 buffer, pH 6.7, protease in- the MMRRC by Beth A. Bauer, DVM, University of Missouri. Mice were hibitors). The cells were centrifuged at 500 × g for 5 min, supernatants were housed at a 12-h light/12-h dark cycle with ad libitum access to food and discarded, and cells were resuspended in 4 to 5 mL of HB. The cells were

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1913956117 Petit et al. Downloaded by guest on September 30, 2021 homogenized with 15 to 20 strokes in a Dounce-type homogenizer/Teflon Peptides were dried via vacuum centrifugation and desalted by C18 stage tip pestle (Thomas Scientific). The homogenate was centrifuged at 1,500 × g for (3M Empore). Peptides were resuspended in 200 mM EPPS (pH 8.5), quantified 10 min, and the resulting postnuclear supernatant (PNS) collected. The PNS using micro BCA (Thermo Scientific), and labeled with TMT 6-plex reagents samples were filtered using 100-μm cell strainers (Corning), normalized (Thermo Scientific) for 1 h at room temperature with samples from wild-type based on their concentration (Bradford assay), and each PNS was loaded in and wobbler cells in triplicate. Labeling was quenched by addition of 5% hy- the middle of a discontinuous sucrose step gradient. The gradients were droxylamine to each reaction and incubating for 15 min at room temperature. assembled in ultra-clear centrifuge tubes (14 × 95 mm/Beckman Coulter) As a ratio check for normalization, 5% of the labeled peptides was combined. with decreasing sucrose buffer (containing 5 mM MgCl2, 0.1 M KH2PO4/ Equal amounts of labeled peptides were combined, vacuum dried, and subjected K2HPO4 buffer, pH 6.7, protease inhibitors) densities from bottom to top: to high pH reverse-phase fractionation (Thermo Fisher Scientific) according to 1.3 M sucrose (∼2.5 mL) →0.86 M sucrose (∼3mL)→0.5 M PNS (∼4to5mL) manufacturer instructions. The six fractions were vacuum dried and desalted by →0.25 M sucrose (2 to 3 mL). The gradients were centrifuged using a SW40 Ti C18 stage tip. Peptides were resuspended in 5% acetonitrile/5% formic acid and rotor/swinging buckets (Beckman Coulter) at 100,000 × g for 1 h (slow ac- analyzed on an Orbitrap Fusion (Thermo Scientific) using synchronous precursor celeration/slow deceleration). The Golgi fraction at the 0.5 M/0.86 M sucrose selection (SPS)-MS (53). Peptides were identified using SEQUEST and TMT re- interface was collected, and the molarity of the fraction was adjusted to porter ion intensities used to determine relative peptide abundance as 1.15 M using a 2.0-M sucrose buffer solution. This fraction was layered at the described (53). bottom of a second sucrose step gradient and overlaid with equal volumes of decreasing sucrose buffer densities from bottom to top: 1.15 M Golgi Statistical Analyses. Data were analyzed using Prism (GraphPad Software), → → → fraction 1.0 M sucrose 0.86 M sucrose 0.25 M sucrose. The gradients and the tests are specified in the figure legends. Error bars represent the SD or × were centrifuged at 76,000 g for 3 h. The enriched Golgi fraction was SEM as indicated in the figure legend. collected at the 0.25 M/0.86 M sucrose interface, diluted in HB, and pelleted for 1 h at 150,000 × g. The Golgi pellet was resuspended in lysis buffer Data Availability. The mass spectrometry proteomics data have been de- (50 mM Tris, pH 7.5, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM posited to the ProteomeXchange Consortium via the PRIDE partner re- ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid [EGTA], pository with the dataset identifier PXD007981. 0.27 M sucrose, 150 mM NaCl, 0.5% Nonidet P-40, protease inhibitors, phosphatase inhibitors; Roche), sonicated, and clarified by centrifugation to ACKNOWLEDGMENTS. We thank the members of the J.W.H. and F&W (R.V.F. yield protein lysate. and T.C.W.) laboratories for comments on the manuscript; Dr. Alexander Bartelt for help with metabolic cage analyses; the Dana-Farber/Harvard TMT Quantitative Proteomics of Golgi-Enriched Proteins. Cells were incubated with Cancer Center in Boston, MA, for the use of the specialized Histopathology 25 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (MilliporeSigma) for Core and the Rodent Histopathology Core, which provided H&E staining 30 min at room temperature and alkylated with 15 mM chloroacetamide and GFAP staining of the spinal cords; and Gary Howard for editorial (MilliporeSigma) for 30 min at room temperature. The alkylation reaction was assistance. Dana-Farber/Harvard Cancer Center is supported in part by a

quenched with 10 mM DTT (MilliporeSigma) for 10 min at room temperature, National Cancer Institute Cancer Center Support Grant NIH 5 P30 CA06516. NEUROSCIENCE This work was supported by the Consortium for Frontotemporal Dementia and proteins were precipitated using chloroform/methanol. Protein pellets were Research (to C.S.P., T.C.W., and R.V.F.), the Mathers Foundation (to resuspended in 200 mM 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic T.C.W.), NIH R37NS083524 (to J.W.H.), NIH R01NS110395 (to J.W.H.), the acid (EPPS) (pH 8.5) and digested with Lys-C (Wako) for 2 h at 37 °C. Trypsin Harvard Brain Initiative ALS seed grant program (to J.W.H.), a generous (Promega)wasaddedtoeachsampleand incubated for an additional 6 h at gift from Ned Goodnow (to J.W.H.), the Canadian Institutes for Health 37 °C. Lys-C and trypsin were used at a protease:substrate ratio of 1:100 (wt/wt). Research (to S.S.), and the Howard Hughes Medical Institute (where Digestion reactions were stopped by addition of 5% formic acid for 30 min. T.C.W. is an investigator).

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