TREATMENT AND PREVENTION OF AGING RELATED-DISEASE AND/OR AGING BY THE INHIBITION OF SPHINGOLIPIDS

Abstract

The present invention relates to an inhibitor of sphingolipids for use in the treatment or prevention of an aging related-disease and/or aging.

Claims

1. An inhibitor of sphingolipid adapted for use in treatment or prevention of a muscle disease.

2. The inhibitor of sphingolipid for use of claim 1, wherein the muscle disease comprises frailty comprising sarcopenia and/or muscle atrophy, and/or is sarcopenia and optionally is frailty comprising sarcopenia and/or muscle atrophy.

3. The inhibitor of sphingolipid for use of claim 2, wherein the frailty comprises (i) sarcopenia and/or muscle atrophy, and (ii) cognitive impairment.

4. The inhibitor of sphingolipid for use of claim 3, wherein the cognitive impairment is senile dementia.

5. The inhibitor of sphingolipid for use of claim 1, wherein the sphingolipid comprises sphinganines, sphingosines, ceramides, dihydroceramides, sphingomyelins, deoxysphingolipids optionally 1-deoxysphinganine) or any combination thereof.

6. The inhibitor of sphingolipid for use of claim 1, wherein the inhibitor is an inhibitor of one or more enzymes involved in biosynthesis of sphingolipid.

7. The inhibitor of sphingolipid for use of claim 6, wherein the one or more enzymes is or are selected from SPTLC1, SPTLC2, SPTLC3, KDSR, CERS1, CERS2, CERS3, CERS4, CERS5, CERS6, SGMS1, SGMS2, SMPD1, SMPD2, SMPD3, SMPD4, ASAH1, ASAH2, ASAH2B, ASAH2C, ACER1, ACER2, ACER3, SPHK1 and SGPP1.

8. The inhibitor of sphingolipid for use of claim 6, wherein the inhibitor inhibits (i) expression of a nucleic acid molecule encoding one or more enzymes being involved in biosynthesis of sphingolipid, or (ii) enzymatic activity of an enzyme being involved in biosynthesis of sphingolipid.

9. The inhibitor of sphingolipid for use of claim 8, wherein (I) the inhibitor of (i) is selected from a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease, and/or (II) the inhibitor of (ii) is selected from a small molecule, an antibody or antibody mimetic, and an aptamer.

10. The inhibitor of sphingolipid for use of claim 9, wherein the antibody mimetic is selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.

11. The inhibitor of sphingolipid for use of claim 9, wherein the small molecule of (ii) is myriocin or a small molecule inhibitor selected from Table A, or a salt or ester thereof.

12. The inhibitor of sphingolipid for use of claim 6, wherein the inhibitor edits a genome at a location encoding an enzyme being involved in biosynthesis of sphingolipid.

13. The inhibitor of sphingolipid for use of claim 1, wherein the muscle disease is inclusion body myositis or a muscular dystrophy, optionally Duchenne muscular dystrophy.

14. A method for treating and/or preventing muscle disease comprising administering an inhibitor of sphingolipid to a subject in need thereof.

Description

[0117] The figures show.

[0118] FIG. 1. Sphingolipid de novo synthesis is activated upon aging in skeletal muscle. (a) Scheme of the sphingolipid de novo synthesis pathway. (b) Transcript abundance of enzymes of sphingolipid de novo synthesis pathway in human skeletal muscle from individuals in the Gene-Tissue Expression (GTEx) project (n=491). (c) Total ceramide levels in liver, brain, skeletal muscle, and plasma of young (8-week old, n=10) and aged (24-month-old, n=10) C57BL/6JRj mice. (d) Concentrations of individual ceramide species in mouse quadriceps muscle. (e) Transcript abundance of enzymes of sphingolipid de novo synthesis pathway in skeletal muscle of young and aged individuals. (f) Correlation of transcripts of sphingolipid de novo synthesis pathway in human skeletal muscle (GTEx, n=491). (g) Correlation of transcripts of sphingolipid de novo synthesis pathway in skeletal muscle of 42 genetically diverse BXD strains. (h) A factor loading plot (biplot) showing the effects of the enzymes of sphingolipid de novo synthesis pathway on two first principal components (SphPC1 and SphPC2) in human skeletal muscle (GTEx). (i) Proportion of variance explained by each principal component of the sphingolipid de novo synthesis pathway. (j) Contribution of each transcript to the SphPC1 in human skeletal muscle (GTEx). (k) Correlation of SphPC1 in skeletal muscle with measurements of muscle mass and function in BXD mice. (l) Correlation of Sptlc1 in skeletal muscle with measurements of muscle mass and function in BXD mice. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0119] FIG. 2. Inactivation of sphingolipid de novo synthesis increases muscle mass and improves muscle function. Aged (18-month-old) C57BL/6JRj mice were treated with intraperitoneal injections of myriocin (MYR) for 5 months @ 0.4 mg/kg/3 times per week. (a) Total ceramide levels in liver, brain, skeletal muscle, and plasma. (b) Concentrations of individual ceramide species in mouse quadriceps muscle. (c) Lean body mass measured before and after treatment, and its change. (d) Gastrocnemius and tibialis anterior (TA) mass. (e) Hematoxylin and eosin (H&E) staining of TA muscle. Scale bar, 50 μm. (f) Proportion of fibers with centralized nuclei. Distribution (g), mean of fiber minimal Feret diameter (h), and cross-sectional area (CSA) (i) in TA muscle. Comparison of maximal running distance and duration (j), aerobic capacity (k), grip strength (l), latency and maximal speed of rotarod test (m), and latency of beam crossing (n) between aged DMSO and myriocin treated mice. For all experiments, n=6-12 per group. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0120] FIG. 3. Inhibition sphingolipid de novo synthesis increases MuSC proliferation and tissue count. RNA-seq transcriptome analysis from quadriceps muscle of aged mice treated with DMSO or myriocin. Volcano plot (a) of the genesets (GO categories) with nomalized enrichment score (NES) and adjusted p-value for each geneset given. Enrichment plot of the ‘Striated muscle contraction’ (b), the most upregulated GO category by myriocin. Comparison of transcripts of Myf5, Myod1, and Myog (c), and the MuSC marker Pax7 (d). (e) Correlation of SphPC1, i.e. the first principal component of the sphingolipid de novo synthesis pathway, with PAX7 in human skeletal muscle (GTEx, n=491). (f) Correlation of SphPC1 with Pax7 in mouse skeletal muscle (BXD, n=42). Number of freshly isolated MuSCs from total hindlimbs musculature (g), normalized to muscle weight (h) from young, aged, and aged mice treated with myriocin. (i) FACS contour plot of a7 integrinCD34Sca-1-CD45-CD31-CD11b-cells which correspond to MuSCs isolated from aged and aged mice treated with myriocin. Representative images (j) and quantification (k) of PAX7-immunostained cells in TA muscle. DAPI, 4′6-diamino-2-phenylindole. Scale bar, 50 μm. Immunocytochemistry (l) and quantification (m) of Ki67 positive cells from freshly isolated MuSCs from aged C57BL/6JRj mice after 72 h incubation in DMSO or myriocin containing medium. Scale bar, 50 μm. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0121] FIG. 4. Sphingolipid depletion improves MuSC function and regenerative capacity in vivo. (a) Aged C57BL/6JRj mice were treated with myriocin, and upon sacrifice, freshly isolated MuSCs were transplanted into the TA of either cartdiotoxin damaged aged C57BL/6JRj or mdx recipient mice. Representative images and quantification of PAX7 (b-c) and dystrophin (d-e) immunostained TA muscle from mdx recipients 7 days after transplantation. Scale bar, 50 μm. Representative images and quantification of PAX7 (f-g) and embryonic myosin heavy chain (eMyHC) (h-i) immunostained TA muscle from aged C57BL/6JRj mice at 7 days after transplantation. Scale bar, 50 μm in PAX7 staining. Scale bar, 250 μm in eMyHC staining. Representative images and quantification of H&E stained TA from aged C57BL/6JRj recipients at 7 days (j-k) and 14 days (I-m) after transplantation. Scale bar, 50 μm. Maximal running distance (n), grip strength (o), and latency on a rotarod (p) before and 7 days after transplantation. The change relative to baseline is reported. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0122] FIG. 5. Sphingolipid depletion cell-autonomously activates muscle differentiation program in MuSCs and myoblasts. (a) Immunocytochemistry and quantification of MYOD from freshly isolated MuSCs from aged C57BL/6JRj mice after 72 h incubation in DMSO or myriocin containing medium. (b) Immunocytochemistry and quantification of MYOG from freshly isolated MuSCs from aged C57BL/6JRj mice. Immunocytochemistry (c), fusion index (d), myotube area (e), and expression of myogenesis markers (f) from C2C12 myoblasts grown in DMSO and myriocin containing medium. Immunocytochemistry (g), fusion index (h), myotube area (i), and gene expression of myogenesis markers (j) from C2C12 myoblasts silenced for Sptlc1 using CRISPR-Cas9. Immunocytochemistry (k), fusion index (l), myotube area (m), and gene expression of myogenesis markers (n) from C2C12 myoblasts silenced for Cers2 using CRISPR-Cas9. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test. Scale bar, 50 μm.

[0123] FIG. 6. Genetic evidence points to beneficial effects of sphingolipid depletion in humans. Immunocytochemistry (a), myotube diameter (b), and myotube area (c) of human primary myoblasts treated with DMSO or myriocin. All data are shown mean±SEM. *P<0.05 with Student's two-tailed T test. Scale bar, 50 μm. Haploblock structure of human SPTLC1 (d) and SPTLC2 (e) loci. (f) Associations of SPTLC1 and SPTLC2 cis-eQTLs rs10820917 and rs8013312 with mRNA levels of SPTLC1 and SPTL2 in GTEx, respectively, and senior fitness test (SFT) score, and its component traits in Helsinki Birth Cohort Study. The forest plot represents 3 with 95% confidence interval (CI). (g) Violin plots of skeletal muscle mRNA, SFT score, and distance in a 6-min walking test as a function of SPTLC1 and SPTLC2 cis-eQTLs rs10820917 and rs8013312, respectively.

[0124] FIG. 7. Levels of Sphinganine (SA) (a) and sphingosine (SO) (b) in quadriceps of young mice, aged mice, and aged mice treated with myriocin. See FIG. 10 for details.

[0125] FIG. 8. Sphingolipid inhibition counteracts Duchenne muscular dystrophy. (a) Transcripts of the enzymes of sphingolipid biosynthetic pathway are upregulated in skeletal muscle of patients with muscular dystrophies. Running distance (b), running time (c), grip strength (d), latency on a rotarod (e), and plasma creatine kinase (f) after down-hill running in mdx mice treated with myriocin. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.$

[0126] FIG. 9. Sphingolipid depletion protects against frailty phenotypes. (a) Distance moved in 10 mins. (b) Average velocity during 10 minutes of observation. (c) Glucose tolerance test. (d) Time exploring familiar and novel objects. (e) Recognition index. Aged (18-month-old) C57BL/6JRj mice were treated with intraperitoneal injections of myriocin (MYR) for 5 months @ 0.4 mg/kg/3 times per week.

[0127] FIG. 10. Sphingomyelin and deoxysphingolipid levels in skeletal muscle in aging and upon myriocin treatment. Concentrations of total sphingomyelin (SM) in liver, brain, skeletal muscle, and plasma of young (8-week old, n=10) and aged (24-month-old, n=10) C57BL/6JRj mice (a), and concentrations of individual sphingomyelin species in quadriceps of young and aged (b) C57BL/6JRj mice. Concentrations of 1-deoxysphinganine (c) in quadriceps muscle of young, aged, and aged mice treated with myriocin. (d) A factor loading plot (biplot) showing the effects of the enzymes of sphingolipid de novo synthesis pathway on two first principal components (SphPC1 and SphPC2) in BXD mouse skeletal muscle. (e) Proportion of variance explained by each principal component of the sphingolipid de novo synthesis pathway. (f) Contribution of each transcript to the SphPC1 in mouse skeletal muscle (BXD). Concentrations of total sphingomyelin in liver, brain, muscle, and plasma (g), and individual sphingomyelin species (h) in quadriceps muscle of aged mice and aged mice treated with myriocin. All data are shown mean±SEM. n=6-10 per group. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0128] FIG. 11. Inhibition sphingolipid de novo synthesis increases MuSC proliferation and tissue count. Upregulated (a) and downregulated (b) GO categories in quadriceps muscle of aged C57BL/6JRj mice with myriocin started at 18 months of age in RNA sequencing. (c) Volcano plot of individual genes displaying log of nominal p-value (vertical axis) and log.sub.2 fold change (horizontal axis) in quadriceps of myriocin treated mice. (d) Quantification of PAX7 positive cells per section in quadriceps muscle. All data are shown mean±SEM. ***P<0.001 with Student's two-tailed T test.

[0129] FIG. 12. Sphingolipid depletion improves MuSC function and muscle regeneration in vivo. Aged 18-month-old C57BL/6JRj mice were treated with myriocin, and upon sacrifice, freshly isolated MuSCs were transplanted to either aged C57BL/6JRj or mdx mice. Representative images (a) and quantification of inflammatory area (b) of H&E stained TA from mdx recipients at 7 days and 14 days after transplantation. Scale bar, 50 μm. (c) Quantification of PAX 7 positive cells per section in aged C57BL/6JRj and mdx recipient mice. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0130] FIG. 13. Single clone CRISPR mediated Sptlc1 knockout promotes muscle cell differentiation in C2C12 myoblasts in a dose-dependent manner. (a) Location guide RNA (gRNA) binding sites in Sptlc1 for two different gRNAs, gRNA1 and gRNA2. (b) Changes in DNA induced by gRNA1 and gRNA2 in C2C12 myoblasts. gRNA1 transfection induced deletions in two homologous chromosomes resulting homozygous Sptlc1 knockout (Sptlc1.sup.−/−). gRNA2 induced a long insertion in one homologous chromosome leaving the other chromosome intact, resulting in heterozygous knockout (Sptlc1.sup.+/−). (c) Verification of Sptlc1.sup.+/− and Sptlc1.sup.−/− knockout C2C12 cell lines using Western blot. Fusion index (d), myotube diameter (e), transcript expression of myogenesis markers (f), and immunocytochemistry (g) from C2C12 myoblasts with heterozygous and homozygous loss-of-function of Sptlc1 using single clone CRISPR-Cas9. Scale bar, 50 μm. (h) Polyclonal silencing of Sptlc1 and Cers2. Verification of knockouts using Western blot. All data are shown mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 with Student's two-tailed T test.

[0131] FIG. 14. Linkage disequilibrium (LD) structure of cis-eQTL of human SPTLC1 and SPTLC2. (a) LD structure of SPTLC1 (a) and SPTLC2 (b) cis-eQTLs denoted by r.sup.2 measurements rom Phase 3 (Version 5) of the 1000 Genomes Project.

[0132] FIG. 15. Inhibition of ceramide synthase improves grip strength upon aging. Aged C57BL/6JRj male mice (20 months of age) were treated with P053, an inhibitor of ceramide synthase suitable for oral administration. P053 recovered the loss in grip strength seen in aged mice.

[0133] FIG. 16. Quantitative Proteostat signal normalized over the number of cells in human primary myoblasts from IBM donors (above) and APPSwe-expressing C2C12 myoblasts (below) decreases after treatment with 30 μM MYR for 24 h. Values are expressed as mean±s.e.m. ***P:0.001; ****P:0.0001. Differences between two groups were assessed using Student's two-tailed t-tests (error bars: 95% confidence intervals).

[0134] FIG. 17. Myriocin improves proteostasis in aged mice. Values are expressed as mean±s.e.m. *P≤0.05. Differences between two groups were assessed using Student's two-tailed t-tests (error bars: 95% confidence intervals).

[0135] The examples illustrate the invention.

EXAMPLE 1—CERAMIDES ACCUMULATE IN SKELETAL MUSCLE UPON AGING

[0136] Sphingolipid de novo synthesis pathway produces ceramides and other sphingolipids by using fatty acids and amino acids as substrates (FIG. 1a). SPT converts L-serine and palmitoyl-CoA to 3-ketosphinganine, which is rapidly converted to sphinganine. Coupling of sphinganine to long-chain fatty acid is accomplished by one of 6 distinct mammalian ceramide synthases of which CERS2 is the most abundant in skeletal muscle (FIG. 1b). To study how aging affects the activity of sphingolipid de novo synthesis pathway in skeletal muscle in vivo, total ceramide content of different organs in young (2-month-old) and aged (2-year-old) mice was compared. We observed an increase in skeletal muscle ceramides was observed (FIG. 1c). Here the focus was on skeletal muscle sphingolipids and potential benefits upon their reduction.

[0137] The trend of age-dependent increase in skeletal muscle ceramides was global, comprising ceramide species with different acyl lengths (FIG. 1d). The most pronounced increase upon aging was observed for Cer(d18:1/24:1), and the most abundant ceramide in muscle, Cer(d18:1/18:0), displayed a 20% increase upon aging (FIG. 1d). Differences in skeletal muscle sphingomyelin (SM), a sphingolipid consisting of phosphocholine and ceramide, were less consistent between young and aged mice (FIG. 10a-b). Deoxysphingolipids are synthesized by SPT through conjugation of L-alanine rather than L-serine with fatty acid, and have been implicated in disease conditions, such as diabetes. Similar to canonical sphingolipids, there was a trend of deoxysphinganine (doxSA) accumulation in skeletal muscle upon aging (FIG. 10c).

[0138] Transcript abundance of the enzymes of sphingolipid de novo biosynthesis pathway between young and aged human individuals in a publicly available dataset (GSE25941) was next compared. Consistent with increased muscle ceramides, many transcripts of these enzymes were upregulated upon aging, including SPTLC1, KDSR, CERS2, and CERS5 (FIG. 1e). The only down-regulated enzyme was CERS1 (FIG. 1e) whose expression is relatively low in skeletal muscle in human Genotype-Tissue Expression (GTEx) database (FIG. 1B). In general, there was a strong positive correlation between different transcripts of the sphingolipid de novo biosynthesis pathway in post-mortem skeletal muscle biopsies in human GTEx dataset (n=491) (FIG. 1f). Only CERS1 transcript displayed a negative correlation with the other enzymes. Similarly, in the mouse BXD strains, a recombinant inbred mouse population with substantial genetic heterogeneity, the transcripts were directly correlated with each other (FIG. 1g). These results suggest that sphingolipid de novo biosynthesis pathway is under tightly coordinated transcriptional control.

[0139] Following the strong correlations between transcripts of the pathway, principal component analysis of the sphingolipid de novo biosynthesis pathway in humans (FIG. 1h) and mice (FIG. 10d-f) was conducted. The first principal component of the expression of different enzymes of the sphingolipid de novo synthesis pathway (SphPC1) explained 30.6% of the variance in human skeletal muscle expression (FIG. 1i). Of the transcripts of the pathway, Sptlc1 contributed the most, accounting for 21% of SphPC1 variability (FIG. 1j). In BXD mouse reference population correlations were examined between SphPC1 and different parameters of muscular fitness, including measures of muscle mass and function, components of sarcopenia. Negative correlation was observed between SphPC1 and both gastrocnemius and soleus muscle masses, as well as performance on a treadmill and maximal aerobic capacity (FIG. 1k). Sptlc1 skeletal muscle expression showed similar correlations with these phenotypes (FIG. 1l). These findings demonstrate that the expression of sphingolipid de novo biosynthesis is inversely correlated with muscle mass and function, and suggest its involvement in sarcopenia.

EXAMPLE 2—INHIBITION OF SPHINGOLIPID DE NOVO SYNTHESIS PREVENTS LOSS OF MUSCLE MASS AND FUNCTION IN AGING

[0140] To examine whether a causal relationship could underlie the correlation between SphPC1 and muscle mass and function, it was tested whether inhibition of sphingolipid de novo biosynthesis pathway could protect against age-related muscle dysfunction. Aged (18-month-old) C57Bl/6JRj mice were treated for 5 months on chow diet with myriocin, a specific inhibitor of SPT, the first and rate-limiting enzyme of the sphingolipid de novo synthesis pathway, and whose expression in skeletal muscle was negatively correlated with muscular fitness in BXDs. Myriocin treatment reduced total ceramide contents of skeletal muscle (FIG. 2a) as well as individual ceramide species in a global fashion (FIG. 2b), confirming the efficacy of the compound in skeletal muscle. In addition to ceramides, myriocin treatment reduced skeletal muscle deoxysphingolipid contents, yet with smaller effect than L-serine derived canonical sphingolipids (FIG. 10c).

[0141] Importantly, myriocin treatment improved body composition of aged mice. Myriocin delayed age-dependent decline in lean body mass (FIG. 2c), and increased muscle mass. Myriocin treated mice displayed greater gastrocnemius and TA mass than DMSO treated controls (FIG. 2d). Improved muscle morphology was evident in histological analysis of tibialis anterior (TA) muscles of myriocin treated mice (FIG. 2e), manifested by reduced number of centralized nuclei (FIG. 2f), a hallmark of muscle aging, and larger cross-sectional area of muscle fibers (FIG. 2g-i). Myriocin treatment also counteracted age-related muscle dysfunction. Aged mice treated with myriocin demonstrated improved exercise performance and muscle strength, evidenced by the increased running distance and time on a treadmill (FIG. 2j), improved aerobic capacity (FIG. 2k), and grip strength (FIG. 2l). Myriocin treated mice also displayed better muscle coordination, as shown by their improved performance in the rotarod test (FIG. 2m), and faster crossing of a beam (FIG. 2n). Overall, myriocin treatment improved muscle morphology and counteracted age-related loss of muscle mass, strength, endurance, and coordination, indicating protection against age-related sarcopenia.

EXAMPLE 3—SPHINGOLIPID DEPLETION ENHANCES PROLIFERATIVE CAPACITY OF MUSCLE STEM CELLS

[0142] To identify biological pathways whose modulation could explain improved muscle function upon myriocin treatment, transcriptomes of quadriceps muscle of aged myriocin and DMSO treated mice using RNA sequencing were compared. Gene set enrichment analysis (GSEA) ranking transcripts based on their fold change were performed. The most downregulated pathways by myriocin treatment were related to the extensively studied role of sphingolipids in lipid metabolism and inflammation, while upregulated pathways were related to muscle contraction and differentiation (FIG. 3a-b, 11a-c), suggesting improved regeneration. Indeed, in a targeted analysis of classical transcription factors regulating myogenesis, Myog and Myf5 were upregulated in skeletal muscle upon myriocin treatment (FIG. 3c).

[0143] Muscle stem cells (MuSCs) are capable of giving rise to mature muscle fibers, and their regenerative capacity has been reported to be impaired upon aging. The expression of Pax7, a specific marker of MuSCs, was upregulated in the quadriceps muscle of myriocin treated mice (FIG. 3d). Consistent with this, PAX7 expression correlated negatively with the first PC of the ceramide synthetic pathway in both human (FIG. 3e) and mouse (FIG. 3f) skeletal muscle. To study whether increased Pax7 transcript abundance in skeletal muscle reflects elevated MuSC count, a7 integrin/CD34 double positive cells were isolated from the hind-limbs using fluorescence-activated cell sorting (FACS). Consistent with previous reports the number of MuSCs was decreased in hindlimbs of aged mice as compared to young mice, and myriocin treatment restored the MuSC pool of aged mice close to that of young mice (FIG. 3g-i). Tissue sections of TA from myriocin treated mice revealed elevated number of PAX7.sup.+ muscle cells (FIG. 3j-k, FIG. 11d), suggesting increased MuSC proliferation, and MuSCs isolated from untreated aged mice and cultured ex vivo in myriocin containing medium resulted in higher Ki67.sup.+ MuSC count (FIG. 3l-m), indicating that myriocin treatment enhances MuSC proliferation. Upon aging, there is a decline in self-renewal and proliferative potential of MuSCs, and our findings suggest that sphingolipid depletion could counteract these defects.

EXAMPLE 4—MYRIOCIN PRIMES MUSCS FOR ACCELERATED REGENERATION

[0144] Decline in tissue regenerative potential is a major hallmark of mammalian aging, including skeletal muscle. It was next asked the question whether myriocin treatment induces functional improvement of MuSCs, boosting their regenerative capacity. MuSCs from aged mice treated or untreated with myriocin for 5 months were isolated, and transplanted into tibialis anterior (TA) muscles of recipient mice. The recipient mice were either aged (20-month-old) C57Bl/6JRj or aged (1-year-old) mdx mice, a mouse model of Duchenne muscular dystrophy lacking dystrophin protein (FIG. 4a). Before MuSC transplantation, injury was induced by cardiotoxin injection to TA of all recipient mice. After 7 days of transplantation, mdx mice displayed elevated count of PAX7.sup.+ cells, indicating more efficient MuSC proliferation (FIGS. 4b-c and 12c). MuSCs isolated from myriocin treated mice stimulated myogenesis of dystrophin-positive fibers, verifying the functionality of newly transplanted MuSCs (FIG. 4d-e). At both 7 and 14 days after transplantation the inflammatory area was also smaller (FIG. 12a-b) in mdx recipients of MuSCs isolated from myriocin treated mice. Aged C57Bl/6JRj recipients of MuSCs isolated from myriocin treated mice exhibited similar morphologic changes in muscle to mdx recipients, manifested by increased count of PAX7.sup.+ cells per field (FIG. 4f-g) and per section (FIG. 12c), higher embryonic myosin heavy chain (eMyHC) positive area (FIG. 4h-i), indicating increased muscle regeneration, and smaller inflammatory area at both 7 and 14 days after injury (FIG. 4j-m). Thus, transplantation of MuSCs derived from myriocin treated donors induce major morphological improvements following cardiotoxin-induced muscle injury.

[0145] To test whether the functional improvement of MuSCs upon myriocin treatment also translates into better muscle function of recipient mice, tests of muscle function were performed before and 7 days after MuSC transplantation. After 7 days of MuSC transplantation, aged C57Bl/6JRj mice, which had received MuSCs from myriocin treated mice, displayed improved exercise capacity (FIG. 4n), grip strength (FIG. 4o), and increased latency in rotarod test (FIG. 4p). These findings demonstrate that myriocin-induced improvement in MuSC function also translates into better muscle function.

EXAMPLE 5—SPHINGOLIPID DEPLETION ACTIVATES MYOGENIC DIFFERENTIATION IN MUSCLE PROGENITOR CELLS

[0146] Inhibition of sphingolipid de novo synthesis pathway has previously been linked to metabolic benefits, including improved insulin sensitivity which might affect muscle function. To study whether sphingolipid depletion has a cell-autonomous effect on myogenesis and muscle regeneration, freshly isolated MuSCs in 30 μM myriocin containing culture medium were incubated. Myriocin elevated both MYOD and MYOG protein levels (FIG. 5a-b), indicating that sphingolipid depletion primes MuSCs for myogenic differentiation. Then the effects of myriocin were examined in myoblasts, a later stage myogenic progenitor cell using mouse C2C12 myoblasts. Myriocin accelerated myoblast differentiation (FIG. 5c), as shown by greater fusion index (FIG. 5d) and myotube area (FIG. 5e), and induced a myogenic transcript signature featuring upregulation of myogenic transcription factors, such as Myog, as well markers of mature myotubes, including myosin heavy chain subunits Myh4 and Myh1 (FIG. 5f).

[0147] To corroborate the effects of the sphingolipid de novo synthesis pathway on myogenesis, members of the pathway were silenced using polyclonal CRISPR-Cas9 genome editing. Knockout of Sptlc1 induced myoblast differentiation (FIG. 5g), as determined by quantification of fusion index (FIG. 5h), myotube area (FIG. 5i), and myogenic transcript signature similar to myriocin treated cells (FIG. 5j). Also Cers2 was silenced, the most abundant ceramide synthase in skeletal muscle, downstream of SPT. Inactivation of Cers2 led to accelerated myogenesis (FIG. 5k-m), featuring similar gene expression signature to that observed after myriocin treatment (FIG. 5n). Thus, enzymes downstream of SPT are involved in myogenic programming in a cell-autonomous manner.

[0148] To further validate our findings, homozygous (Sptlc1.sup.−/−) and heterozygous (Sptlc1.sup.+/−) single clone knockouts of Sptlc1 were generated using CRISPR-Cas9 in C2C12 myoblasts (FIG. 13a-c). The single clone knockouts promoted myogenesis in allele dose-dependent manner, with Sptlc1.sup.−/− myoblasts displaying greater myotube area than Sptlc1.sup.+/− or EV cells (FIG. 13d-g). While the polyclonal Sptlc1 and Cers2 knockouts had milder effects on gene expression signature than myriocin (FIG. 5 j,n), the Sptlc1.sup.−/− single clone knockouts induced magnitude similar to myriocin (FIG. 13f). Thus, SPT abundance dose-dependently influences muscle differentiation.

EXAMPLE 6—GENETIC VARIANTS REDUCING SPT EXPRESSION ARE ASSOCIATED WITH IMPROVED FITNESS IN AGED HUMANS

[0149] To determine whether sphingolipid depletion could stimulate muscle differentiation in human cell lines, human primary myoblasts were treated with myriocin. Consistent with mouse myoblasts, myriocin accelerated human myoblast differentiation, displaying larger myotube area (FIG. 6a-c). Thus, SPT inhibition could enhance muscle maintenance in human muscles.

[0150] It was finally investigated whether the sphingolipid de novo synthesis pathway is involved in age-related muscle dysfunction in humans by gathering evidence from human genetic studies. The objective was to first examine the region near SPTLC1 and SPTLC2 to identify loci that associate with the expression of these genes in skeletal muscle (cis-expression quantitative trait loci (cis-eQTL)), and then test whether these cis-eQTLs are associated with muscular fitness of aged individuals. The regions near SPTLC1 and SPTLC2 were both spanned by 4 haploblocks (r.sup.2>0.2) (FIG. 6d). Using skeletal muscle gene expression data from the GTEx project, cis-eQTLs for SPTLC1 and SPTLC2 (Tables 1-2) were identified in tight linkage disequilibrium within the gene (FIG. 14a-b).

TABLE-US-00002 TABLE 1 Cis-eQTLs of SPTLC1 in skeletal muscle rs10820917 rs10820919 rs7869504 rs7038823 SPTLC1 β P β P β P β P SPTLC1 mRNA −0.088 0.0019 −0.088 0.0019 −0.084 0.0012 −0.084 0.0015 SFT 2.99 0.04 2.99 0.04 0.50 0.69 1.74 0.21 6-min walk 0.99 0.03 0.99 0.03 0.74 0.06 1.00 0.02 Back scratch −0.12 0.81 −0.12 0.81 −0.52 0.24 −0.40 0.41 Chair stand 0.58 0.076 0.58 0.077 0.21 0.44 0.31 0.31 Sit and reach 0.61 0.23 0.61 0.24 0.06 0.89 0.25 0.60 Arm curl 0.99 0.01 0.99 0.01 0.01 0.98 0.62 0.098 Grip strength 0.99 0.0047 0.99 0.0046 0.33 0.29 0.84 0.0079

TABLE-US-00003 TABLE 2 Cis-eQTLs of SPTLC2 in skeletal muscle rs8013312 rs10145519 rs12588277 SPTLC2 β P β P β P SPTLC2 −0.13 0.0014 −0.13 0.002 −0.12 0.002 mRNA SFT 4.64 0.0036 4.80 0.0053 4.35 0.006 6-min 1.51 0.0027 1.36 0.012 1.40 0.005 walk Back 1.38 0.013 1.38 0.021 1.37 0.01 scratch Chair 0.43 0.22 0.24 0.54 0.36 0.31 stand Sit and 1.27 0.02 1.71 0.004 1.21 0.029 reach Arm 0.03 0.94 0.13 0.78 −0.013 0.98 curl Grip 0.38 0.33 0.63 0.13 0.48 0.22 strength

[0151] The cis-eQTL with the largest effect of SPTLC1, rs10820917, was located within a haploblock spanning 250 kb region between SPTLC1 and its neighboring ROR2 (Haploblock 1) while the most significant cis-eQTL for SPTLC2 was located 680 kb upstream of the gene (FIG. 6d). The major alleles (C) of rs10820917 and rs8013312 were associated with reduced transcript abundance of SPTLC1 and SPTLC2, respectively, in a dose-dependent manner (FIG. 6e-f). They were exclusively associated with SPT transcript levels, and not with any of the neighboring genes (Table 3-4).

TABLE-US-00004 TABLE 3 Association of skeletal muscle transcripts of neighboring genes with SPTLC1 cis-eQTL rs10820917. SPTLC1 rs10820917 NES P ASPN −0.013 0.7 AUH −0.014 0.67 BICD2 0.023 0.32 CENPP 0.0043 0.93 ECM2 −0.018 0.53 FGD3 −0.015 0.76 IARS −0.027 0.37 IPKK 0.039 0.24 NFIL3 −0.013 0.64 NOL8 0.0025 0.93 ROR2 0.089 0.099 SPTLC1 0.088 0.0019 SUSD3 −0.0098 0.87 ZNF484 −0.00012 1

TABLE-US-00005 TABLE 4 Association of skeletal muscle transcripts of neighboring genes with SPTLC2 cis-eQTL rs8013312. SPTLC2 rs8013312 NES P ADCK1 −0.026 0.44 AHSA1 0.056 0.1 ANGEL1 0.0037 0.92 GSTZ1 0.034 0.28 IRF2BPL −0.0038 0.92 NRXN3 0.004 0.94 SLIRP −0.016 0.45 SNW1 −0.0019 0.92 SPTLC2 0.13 0.0014 TMED8 0.0013 0.97 TMEM63C −0.053 0.3 VASH1 −0.024 0.48 VIPAS39 0.011 0.71 ZDHHC22 −0.065 0.22

[0152] The SPT protein complex consists of both SPTLC1 and SPTLC2 encoded subunits, and to model the genetic effects of the entire protein complex on its gene expression, a two-locus genetic score was constructed indicating the total number of C alleles within SPTLC1 rs10820917 and SPTLC2 rs8013312 loci. The average gene expression of SPTLC1 and SPTLC2 was dose-dependently associated with the C allele score, individuals homozygous for C allele in both SPTLC1 rs10820917 and SPTLC2 rs8013312 loci displaying the lowest SPTLC1-SPTLC2 expression (FIG. 6e-f).

[0153] To test the effects of the identified cis-eQTLs on muscular fitness upon aging, individuals of the Helsinki Birth Cohort Study (N=2,003) were examined, of whom approximately 700 between 70 to 80 years of age underwent both dense marker genotyping and an extensive battery of physical fitness measurements. The objectively measured fitness tests includes arm curl, number of chair stands, chair sit and reach, back scratch, and 6-min walk test, and collectively constitutes the senior fitness test (SFT) score. It was first tested the association of the SPTLC1-SPTLC2 two-locus genetic score with SFT score, and observed that increased C allele count was dose-dependently associated with improved SFT score (P=0.01) (FIG. 6e-f). Of the SFT component traits, 6-min walking distance and chair sit and reach were associated with the SPTLC C allele count (P=0.03 and P=0.04), as well as improved grip strength, a trait not included in SFT test battery (P=0.006) (FIG. 6e). It was then analyzed the most significant cis-eQTL of SPTLC1 and SPTLC2 individually. The reduced expression associated C alleles of rs10820917 and rs8013312 in SPTLC1 and SPTLC2, were associated with higher SFT (P=0.04 and P=0.004) (FIG. 6e-f). Of the component traits, the major C allele of SPTLC1 rs10820917 was associated with improved performance in 6-min walking test (P=0.03), arm curl (P=0.01), as well as grip strength (P=0.004) while the major C allele of SPTLC2 rs8013312 was associated with increased 6-min walking distance (P=0.0003), sit and reach (P=0.02), and back scratch (P=0.01) (FIG. 6e-f). These genetic data, demonstrating that SPT transcript reducing genetic variants improve age-related fitness, are consistent with outcomes from our pharmacological approach in the aged mice, implying that SPT inhibition in human aging could deliver considerable fitness benefits.

EXAMPLE 7—MYRIOCIN TREATMENT REDUCED SPHINGANINE AND SPHINGOSINE LEVELS IN SKELETAL MUSCLE

[0154] Also muscle sphinganine (SA) and sphingosine (SO) levels were measured after myriocin treatment. Aged myriocin treated mice displayed lower levels of SA and SO in skeletal muscle (FIG. 10a-b). These findings indicate that myriocin induces a reduction of every metabolite of the pathway. Thus, inhibiting sphinganine or sphingosine production could confer fitness benefits.

[0155] Identification of Dihydroceramides as Suppressors of Age-Related Muscle Dysfunction and Muscle Differentiation

[0156] Also Degs1 in C2C12 myoblasts were silenced using DEGS1 CRISPR-Cas9 genome editing. Unlike silencing of Sptlc1 and Cers2, Degs1 silencing led to reduced myogenesis, as demonstrated by reduced myotube fusion index, area, and muscle related gene expression (FIG. 5o-r).

[0157] In humans, in the Helsinki Birth Cohort Study, it was demonstrated that the allele of a SNP associated with reduced mRNA expression of DEGS1 in GTEx skeletal muscle expression database, was associated with impaired measures of senior fitness, including a lower distance walked in a 6-walking test, and worse flexibility, as measured by sit and reach. This findings are in contrast to observations with Sptlc1 and Cers2 silencing which lead to enhanced myogenesis.

[0158] These findings demonstrate that dihydroceramide accumulation through DEGS1 inhibition impairs muscle growth and age-related muscular fitness.

EXAMPLE 8—SPHINGOLIPID DEPLETION IN MDX MICE ALLEVIATES DUCHENNE MUSCULAR DYSTROPHY

[0159] Using muscle biopsies from humans with muscular dystrophies upregulation of transcripts of the sphingolipid de novo biosynthetic pathways in patients with muscular dystrophies was observed, including SPTLC1, SPTLC2, KDSR, CERS2, CERS5, CERS6, and DEGS1 (FIG. 8a). The most significant upregulation was observed for Duchenne muscular dystrophy.

[0160] Next the activity of sphingolipid synthesis was inhibited in a mouse model of Duchenne muscular dystrophy. Mdx, a dystrophin deficient mouse model, was treated with myriocin (0.4 mg/kg/3 times a week intraperitoneally) for 2 months. After myriocin treatment, these mice displayed improved performance in uphill running test (FIG. 8b-c), improved grip strength (FIG. 8d), and improved performance in rotarod test (FIG. 8e). Furthermore, after downhill running, plasma levels of creatine kinase (CK) were lower in myriocin treated mice compared to controls (FIG. 8f). These findings demonstrate that inhibition of sphingolipid synthesis protects against muscular dystrophy.

EXAMPLE 9—SPHINGOLIPID DEPLETION ALLEVIATES FRAILTY SYNDROME

[0161] Human frailty syndrome is characterized by weakness, decreased walking speed, exhaustion, weight loss, and low physical activity. Myriocin treated mice displayed reduced weakness, as demonstrated by increased grip strength (FIG. 2l), increased ad libitum walking speed (FIG. 9b), reduced exhaustion, as demonstrated by improved performance on a treadmill and increased maximal oxygen consumption (FIG. 2j-k), higher lean body as well as muscle mass (FIG. 2c-d), and higher physical activity, as demonstrated by longer distance travelled ad libitum during 10 min (FIG. 9a). Frailty predicts falls in older people. Myriocin treated mice reached a higher speed before they fell off the rotarod (FIG. 2m), as well as were faster in crossing a beam (FIG. 2n), demonstrating their improved coordination. In object recognition test, myriocin treated mice spent more exploring both the familiar object and novel object as compared to their DMSO treated counterparts (FIG. 9d). Although this could be partly attributable to the higher physical activity of myriocin treated mice, the treated mice were more interested in the novel than familiar object as compared to untreated mice. Thus, sphingolipid synthesis inhibition improves memory, demonstrated by the higher recognition index (FIG. 9e). Collectively, our findings demonstrate that myriocin treatment prevents both physical and cognitive frailty in aged mice.

[0162] Frailty is associated with multimorbidity, including diabetes. Aged myriocin treated mice demonstrated improved glucose tolerance in an oral glucose tolerance test (FIG. 9c). Importantly, the myriocin treated were chow-fed, demonstrating that rather than high-fat diet induced insulin resistance, myriocin protect against age-related glucose intolerance. Our findings demonstrate inhibition of sphingolipid synthesis protects against age-related chronic diseases, and targeting of sphingolipid synthesis pathway could protect against age-related multimorbidity and reduce polypharmacy.

EXAMPLE 10—INHIBITION OF CERAMIDE SYNTHASE 1 IMPROVES GRIP STRENGTH IN AGING

[0163] Inhibition of ceramide synthase 1 producing 18:0 sphingolipids by P053, an oral inhibitor of CERS1, has recently been shown to produce beneficial metabolic effects (Turner et. al., 2018). However, its role in age-related sarcopenia has not been explored.

[0164] Mice were treated with P053 for 1 month 3.6 mg/kg/day in food (chow diet). Then grip strength was measured. After 1 month of treatment, P053 treated mice displayed improved grip strength, suggesting that inhibition of CERS1 could attenuate frailty and sarcopenia (FIG. 15). These findings demonstrate that inhibition of other enzymes of the sphingolipid pathway could provide beneficial health outcomes, and the suitability of sphingolipid pathway for pharmacological approaches in general.

EXAMPLE 11—PROTEOSTASIS

[0165] Mitochondrial dysfunction and collapse of cellular protein homeostasis (proteostasis) are hallmarks of several neuromuscular disorders, such as aging, and dystrophies (Wattin et al. (2018), Int J Mol Sci.; 19(1):178). Inclusion body myositis (IBM) is a muscle disease featuring impaired proteostasis, in addition to inflammation (Weihl and Pestronk (2010), Curr Opin Neurol.; 23(5):482-488).

[0166] Cell lines from IBM patients featuring impaired proteostasis were obtained. This was validated using Proteostat staining which targets general disruption in proteostasis. Myriocin treatment improved the phenotype of IBM cell lines, thus clearing protein aggregates from these cells (FIG. 16).

[0167] Myriocin also promoted proteostasis in the skeletal muscle of aged mice (Figure FIG. 17). The A11 staining, indicating increased deposition of oligomers in the formation of Amyloid beta, shows that myriocin cleared amyloid beta aggregates in these mice.

[0168] Together with improved clearance of amyloids from IBM cells treated with myriocin, these data demonstrate that inhibition of sphingolipid pathway improves proteostasis and could be used as a therapeutic strategy for proteostatic diseases.

EXAMPLE 12—MATERIAL AND METHODS

[0169] In Vivo Studies

[0170] Animals. Young (2-month-old) and aged (18-month-old) C57Bl/6JRj mice were purchased from Janvier Labs. EchoMRI measuring the fat and lean body mass, were measured before and after the treatment. The dose of myriocin was 0.4 mg/kg/3 times a week. Myriocin (Enzo Life Sciences, Farmingdale, N.Y.) was first dissolved in DMSO which was then mixed with PBS so that each mouse received 1.5 μL DMSO per injection. Animals were fed on a standard chow diet. All animals were housed in micro-isolator cages in a room illuminated from 7:00 AM to 7:00 PM with ad libitum access to diet and water. All the animal experiments are authorized by animal license 2890.1 and 3341 in Canton of Vaud, Switzerland.

[0171] Measurement of sphingolipids. Plasma (100 μL) and weighed tissue samples (20-60 mg) were transferred to 2 ml Safe-Lock PP-tubes, and extracted. In brief, samples were homogenized using two 6 mm steal beads on a Mixer Mill (Retsch, Haan, GER; 2×10 sec, frequency 30/s) in 700 μL MTBE/MeOH (3/1, v/v) containing 500 μmol butylated hydroxytoluene, 1% acetic acid, and 200 μmol of internal standards (IS, d18:1/17:0 ceramide, d18:1/17:0 sphingomyelin, Avanti Polar Lipids, Alabaster, Ala., USA) per sample. Total lipid extraction was performed under constant shaking for 30 min at RT. After addition of 140 μL dH2O and further incubation for 30 min on RT, samples were centrifuged at 1,000×g for 15 min to establish phase separation. 500 μL of the upper, organic phase were collected and dried under a stream of nitrogen. Lipids were resolved in 700 μL 2-propanol/methanol/water (7/2.5/1, v/v/v) for UPLC-MS analysis. Remaining tissues were dried, solubilized in NaOH (0.3 N) at 65° C. for 4 h and the protein content was determined using Pierce™ BCA reagent (Thermo Fisher Scientific, Waltham, Mass., USA) and BSA as standard.

[0172] Chromatographic separation was modified after Knittelfelder et al. using an ACQUITY-UPLC system (Waters Corporation), equipped with a Luna omega C18 column (2.1×50 mm, 1.6 μm; Phenomenex) starting a 20 min linear gradient with 80% solvent A (MeOH/H2O, 1/1, v/v; 10 mM ammonium acetate, 0.1% formic acid, 8 μM phosphoric acid). The column compartment was kept on 50° C. A EVOQ Elite™ triple quadrupole mass spectrometer (Bruker) equipped with an ESI source was used for detection of lipids in positive ionization mode. Lipid species were analyzed by selected reaction monitoring (ceramide, [M+H]+ to m/z 264.3, 22 eV, 60 ms; sphingomyelin, [M+H]+ to m/z 184.1, 20 eV, 40 ms; resolution 0.7 Q1/Q3). Data acquisition was done by MS Workstation (Bruker). Data were normalized for recovery, extraction-, and ionization efficacy by calculating analyte/IS ratios (AU) and expressed as AU/g tissue or AU/mL plasma.

[0173] Measurement of deoxysphingolipids. Plasma and muscle sphingolipids were processed using a method adapted from. Briefly, 10-15 mg of muscle sample was extracted with 500 μL of −20° C. methanol, 400 μL of ice-cold saline, 100 μL of ice-cold water and spiked with internal standard deoxysphinganine d3 (Avanti lipids). An aliquot (50 μL) of homogenate was dried under air and resuspended in RIPA buffer for protein quantification using BCA assay (BCA Protein Assay, Lambda, Biotech Inc., US). To the remaining homogenate, 1 mL of chloroform was added and the samples were vortexed for 5 min followed by centrifugation at 4° C. for 5 min at 15 000×G. The organic phase was collected and 2 μL of formic acid was added to the remaining polar phase, which was re-extracted with 1 mL of chloroform. Combined organic phases were dried and the pellet was resuspended in 500 μL of methanol and subsequent extraction steps were identical as described for plasma.

[0174] Fifty microliters of plasma was mixed with 500 μL of methanol and spiked with internal standard of deoxysphinganine d3 (Avanti lipids). The samples were placed on a mixer for 1 h at 37° C., centrifuged at 2800×G and the supernatant collected and acid hydrolyzed overnight at 65° C. with 75 μL of methanolic HCl (1N HCl, 10M H2O in methanol). Next, 100 μL of 10 M KOH was added to neutralize. 625 μL of chloroform, 100 μL of 2N NH4OH and 500 μL of alkaline water were added, the sample vortex-mixed and centrifuged for 5 min at 16 000 g. The lower organic phase was washed three times with alkaline water and dried under air. LCMS analysis was performed on an Agilent 6460 QQQ LC-MS/MS. Metabolite separation was achieved with a C18 column (Luna 100×2.1 mm, 3 μm, Phenomenex). Mobile phase A was composed of a 60:40 ratio of methanol:water and mobile phase B consisted of 100% methanol with 0.1% formic acid with 5 mM ammonium formate added to both mobile phases. The gradient elution program consisted of holding at 40% B for 0.5 min, linearly increasing to 100% B over 15 min, and maintaining it for 9 min, followed by re-equilibration to the initial condition for 10 min. The capillary voltage was set to 3.5 kV, the drying gas temperature was 350° C., the drying gas flow rate was 10 L/min, and the nebulizer pressure was 60 psi. Sphingoid bases were analyzed by SRM of the transition from precursor to product ions at associated optimized collision energies and fragmentor voltages (Table below). Sphingoid bases were then quantified from spiked internal standards of known concentration.

TABLE-US-00006 Sphingoid base Parent ion Daughter ion N m17:0 doxSA 272.4 254.4 13 m18:0 doxSA 286.3 268.4 13 m18:0 doxSA d3 289.3 271.5 13

[0175] Endurance running test. The exercise regimen on a treadmill commenced at a speed of 9 cm/s. As the mice were aged, an inclination of 0 degrees was used. Mice were considered to be exhausted, and removed from the treadmill if they received 7 or more shocks (0.2 mA) per minute for two consecutive minutes. The distance traveled and time before exhaustion were registered as maximal running distance and time.

[0176] Grip strength. Muscle strength was assessed by grip strength test. The grip strength of each mouse was measured on a pulldown grid assembly connected to a grip strength meter (Columbus Instruments). The mouse was drawn along a straight line parallel to the grip, providing peak force. The experiment was repeated three times, and the highest value was included in the analysis.

[0177] Rotarod test. The rotarod test measures muscle strength, coordination, and endurance. Mice were left undisturbed in the room for 30 min. The speed of the rotating cylinder (rotarod) increased from 0 to 40 rpm in 5 min. Each mouse had 3 trials per day for 3 consecutive days. The latency and speed the mouse reached it passive rotation or fall from the rotor was recorded, and the latency and speed of the best trial of the second day is presented.

[0178] Crossbar test. Crossbar test assesses active balance through the ability to balance while walking along an elevated beam to reach a dark end side where they are able hide. Since all the mice were easily able to cross a squared (3 cm) beam, and a circular beam of diameter 1.5 cm was too difficult to them, the latency was recorded to cross a circular beam of 3 cm. The mice were trained for one day before the actual trial was recorded, and the average of the latency of three trials per mouse is presented.

[0179] Stem cell isolation. Gastrocnemius, soleus, and quadriceps muscles from both hindlimbs were excised and transferred into PBS on ice. All muscles were trimmed, minced and digested with 2.5 U/ml of Dispase II (Roche) and 0.2% Collagenase B (Roche) in PBS for 30 mins at 37° C. Samples were then centrifuged at 50 g for 5 min followed by removing the supernatant and further digested for 20 mins at 37° C. twice. Muscle slurries were sequentially filtered through 100 μm and 40 μm cell strainers. The isolated cells were then washed in washing buffer (PBS+2.5% BSA) and resuspended in 800 μL of washing buffer. They were immediately stained with antibodies, including CD31 (1:800, eBioscience, eFluor450 conjugated); CD45 (1:200, eBioscience, eFluor450 conjugated); Sca-1 (1:1000, eBioscience, PE-Cy7 conjugated); CD11b (1:100, eBioscience, eFluor450) and CD34 (1:100, BD Pharmingen, FITC conjugated); alpha-7 integrin (1:50, RD system, eFluor700 conjugated) for 45 min at 4° C. Secondary staining was performed with propidium iodide (PI, Sigma) for 15 min at 4° C. in the dark. Stained cells were analyzed and sorted using the FACSAria II instrument (BD Biosciences). Debris and dead cells were excluded by forward scatter, side scatter and PI gating.

[0180] Cardiotoxin-induced muscle damage and MuSC transplantation. MuSCs were transplanted from donor mice to recipient mice. The donor mice were aged male C57Bl/6JRj mice, whose treatment with myriocin was started at the age of 18 months. The recipient mice were either aged (18-month-old) male C57Bl/6JRj mice or 1-year-old male mdx mice. 50 μL of 20 μM Naje Mossambica mossambica cardiotoxin (Sigma) was injected intramuscularly into the tibialis anterior (TA) muscle of recipient mice before transplantation. 24 h after CTX injection, equal number (1500) of freshly isolated MuSCs from donor mice were injected intramuscularly into the TA muscle. Recipient mice were sacrificed at 7 and 14 days after transplantation.

[0181] Histology. TA muscles were harvested from anesthetized mice, and immediately embedded in Thermo Scientific™ Shandon™ Cryomatrix™ and frozen in isopentane, cooled in liquid nitrogen, for 1 min before being transferred to dry ice and stored at −80° C. 8 μm cryosections were incubated in 4% PFA for 15 min, washed three times for 10 min with PBS, incubated for antigen retrieval in pH 6.0 citrate buffer for 10 min at 95° C. (for PAX7 antibody), counterstained with DAPI, laminin (1:200, Sigma), PAX7 (1:200, DSHB, University of Iowa), dystrophin (1:100, Spring Bioscience) or eMyHC (1:50, DSHB, University of Iowa), coupled with Alexa-488 or Alexa-568 fluorochromes (Life Technology) and mounted with Dako Mounting Medium. Microscopy images of fluorescence from muscle fibers were analyzed using the ImageJ software. Centralized nuclei percent, minimal Feret diameter and cross-sectional area in TA muscles were determined using the ImageJ software quantification of laminin, dystrophin, and DAPI-stained muscle images from VS120-S6-W slides scanner (Olympus). A minimum of 2,000 fibers were used for each condition and measurement. The minimal Feret diameter is defined as the minimum distance between two parallel tangents at opposing borders of the muscle fiber. This measure has been found to be resistant to deviations away from the optimal cross-sectioning profile during the sectioning process. The mean cross-sectional area of muscle fibers was calculated as the average cross-sectional area of 2,000 fibers per from sample. 7-8 mouse samples per condition were used for histological quantification of donor mice. The eMyHC quantification is expressed as proportion of eMyHC positive signal over total TA cross sectional area. Following MuSC injection in CTX injected recipient mice, inflammation representing the regenerative stage of muscle was quantified by ImageJ software as proportion of inflammatory area over total area of the TA muscle cross section. The PAX7-positive cells were quantified as the average number of cells per field of more than 30 randomly chosen fields from a mouse TA. For each quantification, 3 or more mice were used.

[0182] Ex vivo analysis of MuSCs. MuSCs were isolated as described above, and seeded in 96- or 48-well plates. The cells were incubated in 30 μM myriocin containing medium (Ham's F-10 nutrient mixture, FBS 20%, basic fibroblast growth factor 2.5 ng/mL, penicillin 100 U/mL, streptomycin 100 μg/mL) for 72 hours. PFA 4% was applied for 15 min, the cells were washed three times for 10 min with PBS, and were blocked in 2% BSA in PBS. The cells were then incubated in primary antibodies MYOD (1:50, LabForce) and MYOG (1:50, Santa Cruz) overnight at 4° C. Secondary antibodies were coupled with Alexa-488 or Alexa-568 fluorochromes (Life Technology) and mounted with Dako Mounting Medium. Leica DMI 4000 microscope was used to image the cells. Quantification of the MYOD+ and MYOG+ cell number was based on more than 500 cells per condition.

[0183] Novel Object Recognition. The novel object recognition task is used to assess recognition memory of mice. It is based on the natural tendency of rodents to explore a novel object by comparison to a familiar one. A habituation phase is realized to familiarize animals to the arena context. 24 hours later, during the acquisition phase (learning phase), two identical objects are placed in the arena and presented to the mouse. During the retention phase (3 hours following the acquisition phase), one of the two familiar objects is replaced by a novel one. Measures of duration and number of exploration for each object are realized, during both acquisition and retention phases, and a Recognition Index is calculated.

[0184] c. Severity Grade

[0185] Grade 0

[0186] In Vitro Studies

[0187] Cell culture and cell transfection. The C2C12 mouse myoblast cell line was obtained from the American Type Culture Collection (CRL-1772™). C2C12 cells or clones were cultured in growth medium consisting of Dulbecco's modified Eagle's medium (Gibco, 41966-029), 20% Fetal Bovine Serum (Gibco, 10270-106) and 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, 15140-122). To induce differentiation, FBS was substituted with 2% horse serum (Gibco, 16050-122). Trypsin-EDTA 0.05% (GIBCO, 25300-062) was used to detach cells. Human skeletal muscle cells were obtained from Lonza (SkMC, #CC-2561) and cultured in growth medium consisting of DMEM/F12 (Gibco, 10565018), 20% Fetal Bovine Serum (Gibco, 10270-106) and 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, 15140-122). To induce differentiation, FBS was reduced to 2% and kept in culture. All cells were maintained at 37° C. with 5% CO2. Cell transfections were done using TransiT (Mirus) according to the manufacturer's protocol with a 3:1 ratio of transfection agent to DNA. C2C12 cells were grown confluent, and 30 μM myriocin or DMSO was added and cells were kept in growth medium for another 3 days. Sptlc1 clones were plated to reach confluency simultaneously and were kept in growth medium for 3 days before using them for immunocytochemistry or RNA isolation. The concentration of myriocin in medium for all experiments was 30 μM. A stock solution of 20 mM myriocin in DMSO was used dissolve myriocin, and a corresponding volume of DMSO without myriocin was used as control.

[0188] CRISPR guide RNA design and cloning. Two guide RNAs per gene were designed with the help of the online GPP web portal tool (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) using Streptococcus pyogenes PAM sequence (NGG). The guide RNAs with best predicted on- and off-target scores were selected. The guide RNA sequences are listed in Table 2. The oligonucleotides were synthesized (Microsynth, Switzerland) and cloned into the CRISPRv2 plasmid (addgene #52961) using the BsmBI restriction sides. The insertion by cloning was verified by Sanger sequencing (Microsynth, Switzerland). To test the efficiency of the guide RNAs, the cloned vectors were transiently transfected (TransIT, Mirus) in C2C12 cells, and 48 h after transfection RNA was isolated, reverse transcribed and gene expression was measured by RT-qPCR.

[0189] Creation of single clone Sptlc1 knockout in C2C12 myoblasts. C2C12 cells were transfected with the lentiCRISPR v2 plasmid containing the gRNAs targeting exon 1 of Sptlc1 or the empty vector lentiCRISPR v2 plasmid as a control. 36 h after transfection cells were selected with 2 μg/mL puromycin (Invivo gene; QLL3803A) for 3 d and single cell sorted. Five to ten different clones for each gRNA were grown without selection marker. DNA from these clones was isolated (Macherey-Nagel, 740952) and PCR amplified (see Table 2). The PCR product was gel purified (Machery-Nagel, 740609) and Sanger sequencing was performed to verify the clones with deletions or insertions. In one of the clones, Sptlc1 gRNA1 led to a homozygous knockout of Sptlc1 (Sptlc1−/−) whereas gRNA2 led to a heterozygous knockout of Sptlc1 (Sptlc+/−) in another clone.

[0190] Creation of polyclonal Sptlc1 or Cers2 knockouts in C2C12 myoblasts. Lentivirus were produced from lentiCRISPRv2 plasmids containing no gRNA (empty vector), Sptlc1 gRNA2 or Cers2 gRNA2 (Table 3) by co-transfection with the packaging of plasmids pMD2G and psPAX2 in HEK 293T cells using lipofectamine 2000. Viral supernatants were harvested 36 to 48 h post transfection. C2C12 were transduced with viral supernatant for 20 hours. 24 h later, cells were selected with 3 μg/mL puromycin for 3 days. Reduction in the target protein was confirmed by Western blotting.

[0191] Western blotting. C2C12 cells were lysed on ice in RIPA buffer composed of Tris HCl 50 mM, NaCl 5 M, EDTA 5 mM, SFS 0.1%, NAF 100 mM, sodium deoxycholate 5 mg/mL, and NP40 1% containing protease and phosphatase inhibitors (Roche). Protein concentrations were determined using Bradford method, and 23 μg of protein loaded on a 12% SDS-PAGE gel. After electrophoresis, proteins were separated by SDS-PAGE and transferred onto methanol activated polyvinylidene difluoride membranes. Blocking of the membranes was done in 5% milk-TBST for 1 h, and after wash, the membranes were incubated overnight with primary antibody anti-SPTLC1 (Proteintech) 5% BSA-TBST 1:1000 or anti-CERS2 (Sigma) in 3% BSA-TBST 1:1000. Incubation with secondary anti-rabbit polyclonal antibody was done in 5% BSA-TBST 1:2000. Antibody detection reactions were developed by enhanced chemiluminescence (Advansta), and imaged using the c300 imaging system (Azure Biosystems).

[0192] Myoblast proliferation assay in vitro. To measure cell proliferation in CRISPR mediated Sptlc1 knockout or myriocin-treated cells, 3000 cells per well were seeded in 96-well plates (Greiner bio-one, CELLSTAR, 655180), cultured in growth medium and either transfected (TransIT, Mirus) 24 h post seeding with 30 ng lentiCRISPR v2 plasmid (addgene #52961) containing a guide RNA (Table 2) or treated with 30 μM myriocin diluted in DMSO, or DMSO as control. Cell proliferation was measured according to the manufacturer's protocol (Cell proliferation ELISA BrdU, Roche). In brief, proliferating myoblasts were labelled with 10 μL/well BrdU for 2 h at 37° C., fixed with 200 μL/well for 30 min at room temperature and incubated with the supplied BrdU antibody for 90 min at room temperature. After washing three times with PBS, cells were incubated in 100 μL/well substrate solution for 10 min before measuring 370 nm absorbance using the ELISA plate reader (Perkin Elmer, Victor™ X4).

[0193] RNA isolation and real time qPCR. RNA was isolated using the RNeasy Mini kit (Qiagen, 74106) and reverse transcribed with the High-Capacity-RNA-to-cDNA kit (Thermo Fisher, 4387406). Gene expression was measured by qPCR using the Power SybrGreen Master mix (Thermo Fisher, 4367659). All quantitative polymerase chain reaction (PCR) results were calculated relative to the mean of the housekeeping gene Gapdh. The average of two technical replicates was used for each biological data point. Primer sets for quantitative reverse transcription PCR (q-RT-PCR) analyses are shown in Table 1.

[0194] Immunocytochemistry. C2C12 cells cultured on a sterilized cover slip in 6-well plates (Greiner bio-one, CELLSTAR, 657160) were fixed in Fixx solution (Thermo Scientific, 9990244) for 15 min and permeabilized in 0.1% Triton X-100 (Amresco, 0694) solution for 15 min at room temperature. Cells were blocked in 3% BSA for 1 h at room temperature to avoid unspecific antibody binding and then incubated with primary antibody over night at 4° C. with gentle shaking. MyHC was stained using the MF20 primary antibody (1:200, Invitrogen, 14-6503-82) for C2C12 cells and in Lonza muscle cells with a MYL2 antibody (1:140, Abcam, ab79935). The next day cells were incubated with secondary antibody (Thermo Fisher #A10037 for MF20 and #A-21206 for MYL2) for 1 h at room temperature and nuclei were labelled with DAPI. The immunofluorescence images were acquired using either fluorescence or confocal microscopy. The myofusion index was calculated as the ratio of nuclei within myotubes to total nuclei. The myotube diameter was measured for 8 myotubes per image using ImageJ. Myotube area was calculated as the total area covered by myotubes.

[0195] Gene expression and phenotype analysis in BXD mouse population. Quadriceps microarray data (Affymetrix Mouse Gene 1.0 ST) and phenotype data from BXD mouse genetic reference population were analyzed for Pearson correlations with R software. The first principal component of the ceramide biosynthetic pathway representing its expression in muscle was calculated by including the following genes: SPTLC1, SPTLC2, KDSR, CERS1, CERS2, CERS3, CERS4, CERS5, CERS6, DEGS1.

[0196] Quadriceps transcript profiling using RNA sequencing. Quadriceps muscles were collected and snap-frozen in liquid nitrogen from C57Bl/6JRj mice undergoing intraperitoneal myriocin treatment for 10 weeks on a chow diet starting at the age of 18 months. RNA was isolated using Direct-zol RNA kit (Zymo, Irvine, Calif.). RNA quality was assessed using Agilent 2100 BioAnalyzer (Agilent, Santa Clara, Calif.). Samples with RIN≥8, 28S/18S≥1.0, and c≥20 ng/μL were included in analyses. Using these criteria, 9 (4 DMSO and 5 myriocin) samples passed and 2 samples failed to pass quality control criteria. Sequencing libraries were prepared by BGI genomics using the DNBsea™ technology. Paired end sequencing with 100 cycles was performed using the BGISEQ-500 instrument. After removal of adaptor sequences and low quality reads, at least 56 million reads per sample was obtained. SOAPnuke was used to obtain clean reads (parameters -l 15, -q 0.2, -n 0.05). Reads were mapped using STAR aligner version 2.5.2b using the mouse GRCm38 genome assembly and the release 91 GTF annotation from Ensembl. Htseq-count version 0.6.0 was used to count the number of reads mapping to genes (mode=union, type=exon, idattr=gene_id). Transcript displaying higher expression than log 2(CPM+1)>0.5 in at least 3 samples were included in analyses. Differential gene expression analysis and expression normalization was performed using voom, ‘variance modeling at the observational level’, adjusting for sacrifice date. At individual gene level, no gene passed the multiple testing correction threshold. Benjamini-Hochberg correction for multiple testing was used. For gene set enrichment analysis using gene ontology (GO) categories, transcripts were ordered according to their log 2-transformed fold change, and 100,000 permutations were used. Adjusted p-value<0.05 was considered significant.

[0197] Human Studies

[0198] Young vs. old skeletal muscle microarrays: Gene expression analysis of young vs. old human muscle biopsies was obtained from publicly available dataset GSE25941. Briefly, a total of 36 subjects were included in the study. The young (n=15, 25±1 y) participants included 7 males and 8 females. The old (n=21, 78±1 y) participants included 10 males and 11 females. All subjects were healthy and had never been involved in any formal exercise. Skeletal muscle biopsies were obtained from the vastus lateralis in the basal state. Affymetrix Human Genome U133 Plus 2.0 Array platform was used to perform the microarray analysis.

[0199] Gene expression analysis from human skeletal muscle in Genotype-Tissue Expression (GTEx) project. For RNA gene expression analyses, 491 post-mortem skeletal muscle biopsies from the GTEx gene expression collected were employed. As measures of gene expression, residual expression levels of transcripts adjusting for the published GTEx v7 covariates was used. As for eQTL analyses, the GTEx v7 genotypes (dbGAP, approved request #10143-AgingX) was used. For the combined expression of SPTLC1 and SPTLC2, mean of the residual expression was used.

[0200] Helsinki Birth Cohort Study (HBCS): The HBCS includes 13,435 individuals born in Helsinki between 1934 and 1944. The senior fitness test (SFT) describing the physical performance of the participants was performed to 695 individuals. Here, a modified test battery was used, consisting of five components of the SFT: number of full arm stands in 30 seconds with arms folded across chest to assess lower body strength; number of biceps curls in 30 seconds while holding a hand weight (3 kg for men and 2 kg for women) to assess upper body strength; chair sit and reach to assess the lower body flexibility (from sitting position with leg extended at front of chair and hands reaching toward toes, number of cm (plus/minus) from extended fingers to tip of toe); number of meters walked in 6 minutes to measure aerobic endurance; and back scratch to assess upper body flexibility (with one hand reaching over shoulder and the other one up middle of back, distance (in cm) between extended middle fingers (plus/minus). The result of each test was expressed as age (for each 5-year group) and sex-standardized percentile scores. An overall test score was calculated by summarizing the normalized scores of the 5 SFT components. Isometric grip strength of the dominating hand was tested by a Newtest Grip Force dynamometer (Newtest Oy, Oulu, Finland). The maximum value of three squeezes was used in analyses.

[0201] DNA was extracted from blood samples and genotyping was performed with the modified Illumina 610k chip by the Wellcome Trust Sanger Institute, Cambridge, UK, according to standard protocols. Genomic coverage was extended by imputation using the 1000 Genomes Phase I integrated variant set (v3/April 2012; NCBI build 37/hg19) as the reference sample and IMPUTE2 software. Before imputation the following QC filters were applied: SNP clustering probability for each genotype >95%, Call rate >95% individuals and markers (99% for markers with MAF<5%), MAF>1%, HWE P>1×10-6. Moreover, heterozygosity, gender check and relatedness checks were performed and any discrepancies removed.

[0202] For identification of cis-eQTLs eQTLs±1 Mb were analyzed from the start and the end of SPTLC1 and SPTLC2. SNPs with minor allele frequency >10% were included. SNPs with r2>0.2 were incorporated in the same haploblock. As there were 4 haploblocks within both SPTLC1 and SPTLC2 region (300 kb), Bonferroni correction was used for 25 haploblocks (P<0.002) within the 2 Mb region studied for identification of cis-eQTLs. Linear regressions were performed with SNPtest assuming an additive genetic model. All models were adjusted for age, sex, highest education achieved (basic or less/upper secondary/lower tertiary/upper tertiary) and smoking (yes/no).