Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis James C. Dodgea,1, Christopher M. Treleavena, Joshua Pachecoa, Samantha Coopera,ChannaBaoa, Marissa Abrahama, Mandy Cromwella, S. Pablo Sardia, Wei-Lien Chuanga, Richard L. Sidmanb,1,2,SengH.Chenga, and Lamya S. Shihabuddina aRare Diseases Science, Genzyme, a Sanofi Company, Framingham, MA 01701; and bBeth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 Contributed by Richard L. Sidman, May 7, 2015 (sent for review January 12, 2015; reviewed by David V. Schaffer) Recent genetic evidence suggests that aberrant glycosphingolipid mediates the hydrolysis of Cer, are linked to forms of spinal metabolism plays an important role in several neuromuscular muscular atrophy that are not caused by the more frequent mu- diseases including hereditary spastic paraplegia, hereditary sen- tations in the survival motor neuron 1 gene (non-5q SMA) (15). sory neuropathy type 1, and non-5q spinal muscular atrophy. Here, Moreover, hereditary spastic paraplegia (HSP), a disease with cor- we investigated whether altered glycosphingolipid metabolism is ticospinal tract and in some cases, spinal cord MN degeneration, a modulator of disease course in amyotrophic lateral sclerosis (ALS). is attributed to mutations in GM2 synthase (16) and the non- Levels of ceramide, glucosylceramide, galactocerebroside, lactosyl- lysosomal glucosylceramidase, GBA2 (17). Last, patients with adult- ceramide, globotriaosylceramide, and the gangliosides GM3 and onset Tay-Sachs disease (GM2 gangliosidosis), a disease triggered GM1 were significantly elevated in spinal cords of ALS patients. by a deficiency in hexosaminidase (HEX), a lysosomal enzyme Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosi- that hydrolyzes GM2 to GM3, have been reported in some in- dase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and stances to display a disease phenotype that closely mimics ALS β-galactosidase) mediating glycosphingolipid hydrolysis were also (18–20). Interestingly, HEX mRNA is up-regulated within MNs elevated up to threefold. Increased ceramide, glucosylceramide, of transgenic mice expressing the mutant human SOD1 protein G93A GM3, and hexosaminidase activity were also found in SOD1G93A (i.e., SOD1 mice), a familial model of ALS (21, 22). Collec- mice, a familial model of ALS. Inhibition of glucosylceramide syn- tively, these findings suggest that titration of GSLs is important to thesis accelerated disease course in SOD1G93A mice, whereas infu- maintaining neuromuscular system homeostasis. Similar to other sion of exogenous GM3 significantly slowed the onset of paralysis metabolic aspects thought to influence ALS disease progression and increased survival. Our results suggest that glycosphingolipids (23), GSL bioactivity in the neuromuscular system is likely exerted – SI Ap- are likely important participants in pathogenesis of ALS and merit through an inverted U-shaped dose response curve ( pendix further analysis as potential drug targets. ,Fig.S1). Healthy (or eustatic) GSL levels are achieved in the central, optimal range of the curve, whereas GSL levels that motor neuron disease | SALS | FALS | mouse models neurological disease promote features of disease (or cacostasis) are associated with either end of the curve (i.e., instances of GSL insufficiency or excess). Here, we investigated whether disrupted GSL homeostasis myotrophic lateral sclerosis (ALS) is a progressive neuro- contributes to neurodegeneration in ALS. We show that altered Adegenerative disorder characterized by selective loss of motor GSL metabolism is a manifestation of sporadic and familial ALS neurons (MNs) within the CNS. Although our understanding of the genetic basis of ALS has advanced greatly in recent years (1), Significance the adverse biological processes that converge on the neuromus- cular axis to drive both MN death and neuropathological features in additional cell types remain largely unknown. Glycosphingolipids Glycosphingolipids are a heterogeneous group of membrane (GSLs) are a heterogeneous group of membrane lipids formed lipids formed through the covalent linkage of a glycan moiety to ceramide. Genetic evidence suggests that aberrant glyco- through the covalent linkage of a glycan moiety to ceramide sphingolipid metabolism plays an important role in several neu- (Cer; see SI Appendix,Fig.S1for an overview of GSL metabolism). romuscular diseases. Here, we investigated whether alterations in Glucosylceramide (GlcCer) and galactosylceramide (GalCer) are glycosphingolipids contribute to neurodegeneration in amyo- GSLs with a single sugar residue: glucose and galactose respec- trophic lateral sclerosis (ALS). We show that ALS patients and tively. The successive addition of galactose and sialic acid moie- model mice display disease-related changes in spinal cord glyco- ties to GlcCer results in the synthesis of gangliosides (e.g., GM3, sphingolipids levels and in the enzymes that regulate their GM2, and GM1) (2). GSLs are especially abundant in the CNS metabolism. Importantly, we demonstrate that inhibition of and have bioactive roles in metabolism, growth factor signaling, glycosphingolipid synthesis in ALS model mice exacerbated dis- oligodendrocyte differentiation, neuroinflammation, angiogene- ease progression, whereas administration of GM3, a subtype of – — sis, and pathways of cell death (2 9) all of which are thought to glycosphingolipids, slowed it, thus implicating glycosphingolipids participate in ALS disease pathogenesis. as potentially important participants in ALS pathogenesis and Several lines of evidence suggest that aberrant changes in GSL potential targets for future drug development. homeostasis may contribute to disease pathogenesis in ALS. Ev- idence includes the detection of unique gangliosides (10), high Author contributions: J.C.D. designed research; J.C.D., C.M.T., J.P., S.C., C.B., M.A., S.P.S., titer serum auto-antibodies to GM2 and GM1 (11, 12), and ele- and W.-L.C. performed research; M.C. contributed new reagents/analytic tools; J.C.D., vated GM2 levels within the motor cortex of ALS patients (13). R.L.S., S.H.C., and L.S.S. analyzed data; and J.C.D., R.L.S., S.H.C., and L.S.S. wrote the paper. Furthermore, a number of neuromuscular diseases are associated Reviewers included: D.V.S., University of California, Berkeley. with mutations in genes that regulate the metabolism of Cer and The authors declare no conflict of interest. GSLs. For example, hereditary sensory neuropathy type I (HSNT1), Freely available online through the PNAS open access option. a disease that features dorsal root ganglion cell and MN degen- 1To whom correspondence may be addressed. Email: [email protected] or eration, is attributed to mutations in serine palmitoyltransferase [email protected]. long chain base subunit-1 (SPTLC1), the rate-limiting enzyme in 2Present address: Schepens Eye Research Institute, Department of Ophthalmology, Cer synthesis. Notably, certain mutations in SPTLC1 associated Harvard Medical School, Boston, MA 02114. with HSNT1 result in elevated levels of Cer and GlcCer (14). In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. addition, mutations in acid ceramidase (ASHA1), an enzyme that 1073/pnas.1508767112/-/DCSupplemental. 8100–8105 | PNAS | June 30, 2015 | vol. 112 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1508767112 Downloaded by guest on September 30, 2021 and that modulation of GSL levels in SOD1G93A mice significantly in MB SOD1G93A mice, which is very similar to what was seen affects disease course. human ALS tissue. Interestingly, similar fluctuations (i.e., an initial significant decline in SYMP and ES mice followed by a resurgence Results in MB mice relative to SYMP mice) were observed for almost all GSL Levels Are Increased in the Spinal Cords of ALS Patients. To de- GSLs downstream of GlcCer including LacCer, GL3 and GM1, termine whether GSL levels are modified in the spinal cords of with the only exception being GM3. Total levels of GM3 were ALS patients, we examined cervical spinal tissue samples collected significantly elevated at all phases of disease (SI Appendix,Fig.S3) at autopsy from six male ALS patients and six age-matched male and specific isoforms displayed progressive accumulation (Fig. 2 controls. In contrast to earlier studies that examined total GSL and SI Appendix,TableS2). Disease-related fluctuations in GalCer levels in the whole spinal cord (10), we analyzed gray matter (GM) and SPM, additional lipids derived from Cer (Fig. 1), were similar and ventral white matter (VWM) samples separately, because to several of the GSLs analyzed with levels (for major isoforms) regional variation in total ganglioside levels within segments of being significantly (P < 0.0001) depressed in SYMP and ES mice spinal cord have previously been reported (24). We also measured followed by a rebound in MB mice (Fig. 2). individual molecular isoforms of GSLs because of their distinct biological functions (25). Lipids analyzed include ceramide (Cer), Enzymes Involved in Modulating GSL Levels Are Altered in ALS Patients sphingomyelin (SPM), glucosylceramide (GlcCer), galactosylcer- and SOD1G93A Mice. Next, we asked whether GSL accumulation amide (GalCer), lactosylceramide (LacCer), globotriaosylcer- within the spinal cords of ALS patients was due to impaired en- amide(GL3),andthegangliosidesGM3andGM1(SI Appendix, zymatic action either within lysosomes or other