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Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase.

Henriques A, Croixmarie V, Priestman DA, Rosenbohm A, Dirrig-Grosch S, D'Ambra E, Huebecker M, Hussain G, Boursier-Neyret C, Echaniz-Laguna A, Ludolph AC, Platt FM, Walther B, Spedding M, Loeffler JP, Gonzalez De Aguilar JL - Hum. Mol. Genet. (2015)

Bottom Line: Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology.In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer).HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates.

View Article: PubMed Central - PubMed

Affiliation: Université de Strasbourg, UMR_S 1118, Strasbourg, France, INSERM, U1118, Mécanismes Centraux et Péripheriques de la Neurodégénérescence, Strasbourg, France.

No MeSH data available.


Related in: MedlinePlus

GlcCer/GSL metabolism in spinal cord and muscle of SOD1(G86R) mice. HPLC quantification of GlcCer and several GSLs in spinal cord (A and B) and muscle (D and E) of pre-symptomatic (PRE) and symptomatic (DIS) SOD1(G86R) mice compared with corresponding WT littermates. Only detected main peaks of gangliosides are shown in each tissue. *P < 0.05 versus corresponding WT, n = 6. Relative GCS mRNA levels in spinal cord (C) and muscle (F) of mice as in A. *P < 0.05 versus corresponding WT, n = 6–9. (G) GCS protein levels, as determined by western blot (upper panel), in muscle of mice as in A. Lower panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05 versus corresponding WT, n = 3. (H) Representative photomicrographs showing GCS immunostaining on cross-sections of WT and SOD1(G86R) muscle. Scale bar, 50 µm. (I) Representative photomicrographs showing mitochondrial SDH enzymatic activity and GCS immunostaining within the same myofibers on adjacent cross-sections. Scale bar, 50 µm.
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DDV439F3: GlcCer/GSL metabolism in spinal cord and muscle of SOD1(G86R) mice. HPLC quantification of GlcCer and several GSLs in spinal cord (A and B) and muscle (D and E) of pre-symptomatic (PRE) and symptomatic (DIS) SOD1(G86R) mice compared with corresponding WT littermates. Only detected main peaks of gangliosides are shown in each tissue. *P < 0.05 versus corresponding WT, n = 6. Relative GCS mRNA levels in spinal cord (C) and muscle (F) of mice as in A. *P < 0.05 versus corresponding WT, n = 6–9. (G) GCS protein levels, as determined by western blot (upper panel), in muscle of mice as in A. Lower panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05 versus corresponding WT, n = 3. (H) Representative photomicrographs showing GCS immunostaining on cross-sections of WT and SOD1(G86R) muscle. Scale bar, 50 µm. (I) Representative photomicrographs showing mitochondrial SDH enzymatic activity and GCS immunostaining within the same myofibers on adjacent cross-sections. Scale bar, 50 µm.

Mentions: Following the analysis described earlier, we also took advantage of our previously published transcriptome of muscle in SOD1(G86R) mice (32). We performed a joint enrichment analysis of transcriptomics and lipidomics data using the IMPaLA web interface (33). This analysis revealed that the metabolism of sphingolipids, including ceramide and GlcCer, was among the most significantly over-represented pathways linked to the differential expression of GCS (Supplementary Material, Table S3), which is the enzyme responsible for the conversion of ceramides into GlcCer (28). As there is no available transcriptome database of spinal cord in SOD1(G86R) mice, we could not perform a similar analysis in this tissue. Based on these findings, we focused on GlcCer, because it is the precursor for all the more complex GSLs with important biological functions (34,35). HPLC analysis revealed a comparable age-related decline in the total amount of GlcCer in spinal cord of both SOD1(G86R) and WT mice (Fig. 3A). In contrast, a significant up-regulation of GM1a, the major ganglioside in the central nervous system, was observed in SOD1(G86R) mice at the symptomatic stage compared with WT littermates (Fig. 3B). There was also a significant increase in GlcCer in muscle of symptomatic SOD1(G86R) mice (Fig. 3D). Concomitantly, levels of several gangliosides of the a-series, including GM3 and GM2, were significantly increased in this tissue (Fig. 3E). To investigate the reason for altered GlcCer levels, we measured GCS expression. GCS mRNA levels were significantly increased in muscle at both pre-symptomatic and symptomatic stages in SOD1(G86R) mice compared with WT littermates (Fig. 3F), but no differences were observed in spinal cord (Fig. 3C). Muscle GCS expression at 60 days of age, an earlier pre-symptomatic time point, was similar in SOD1(G86R) and WT mice (data not shown). Therefore, the up-regulation of GCS observed at 75 days of age can be considered as an early event preceding the onset of overt motor disease. To support these findings, we performed western blot analyses with actin or total protein content as an internal reference. In both cases, GCS protein amount was increased at the pre-symptomatic stage in SOD1(G86R) mice. This increase was similarly observed in symptomatic mice (Fig. 3G), although it was not noticeable after total protein normalization (data not shown). To gain further insight, muscle GCS expression was confirmed by immunohistological analysis of muscles from WT and SOD1(G86R) mice. We showed increased punctate GCS staining, frequently located at the border of myofibers (Fig. 3H), compatible with the reorganization of Golgi complex in denervated muscle (36). This pattern of staining was specifically observed in myofibers exhibiting less cross-sectional area and more metabolic oxidative capacity, as assessed by an increase in the presence of mitochondrial succinate dehydrogenase (SDH) (Fig. 3I).Figure 3.


Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase.

Henriques A, Croixmarie V, Priestman DA, Rosenbohm A, Dirrig-Grosch S, D'Ambra E, Huebecker M, Hussain G, Boursier-Neyret C, Echaniz-Laguna A, Ludolph AC, Platt FM, Walther B, Spedding M, Loeffler JP, Gonzalez De Aguilar JL - Hum. Mol. Genet. (2015)

GlcCer/GSL metabolism in spinal cord and muscle of SOD1(G86R) mice. HPLC quantification of GlcCer and several GSLs in spinal cord (A and B) and muscle (D and E) of pre-symptomatic (PRE) and symptomatic (DIS) SOD1(G86R) mice compared with corresponding WT littermates. Only detected main peaks of gangliosides are shown in each tissue. *P < 0.05 versus corresponding WT, n = 6. Relative GCS mRNA levels in spinal cord (C) and muscle (F) of mice as in A. *P < 0.05 versus corresponding WT, n = 6–9. (G) GCS protein levels, as determined by western blot (upper panel), in muscle of mice as in A. Lower panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05 versus corresponding WT, n = 3. (H) Representative photomicrographs showing GCS immunostaining on cross-sections of WT and SOD1(G86R) muscle. Scale bar, 50 µm. (I) Representative photomicrographs showing mitochondrial SDH enzymatic activity and GCS immunostaining within the same myofibers on adjacent cross-sections. Scale bar, 50 µm.
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DDV439F3: GlcCer/GSL metabolism in spinal cord and muscle of SOD1(G86R) mice. HPLC quantification of GlcCer and several GSLs in spinal cord (A and B) and muscle (D and E) of pre-symptomatic (PRE) and symptomatic (DIS) SOD1(G86R) mice compared with corresponding WT littermates. Only detected main peaks of gangliosides are shown in each tissue. *P < 0.05 versus corresponding WT, n = 6. Relative GCS mRNA levels in spinal cord (C) and muscle (F) of mice as in A. *P < 0.05 versus corresponding WT, n = 6–9. (G) GCS protein levels, as determined by western blot (upper panel), in muscle of mice as in A. Lower panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05 versus corresponding WT, n = 3. (H) Representative photomicrographs showing GCS immunostaining on cross-sections of WT and SOD1(G86R) muscle. Scale bar, 50 µm. (I) Representative photomicrographs showing mitochondrial SDH enzymatic activity and GCS immunostaining within the same myofibers on adjacent cross-sections. Scale bar, 50 µm.
Mentions: Following the analysis described earlier, we also took advantage of our previously published transcriptome of muscle in SOD1(G86R) mice (32). We performed a joint enrichment analysis of transcriptomics and lipidomics data using the IMPaLA web interface (33). This analysis revealed that the metabolism of sphingolipids, including ceramide and GlcCer, was among the most significantly over-represented pathways linked to the differential expression of GCS (Supplementary Material, Table S3), which is the enzyme responsible for the conversion of ceramides into GlcCer (28). As there is no available transcriptome database of spinal cord in SOD1(G86R) mice, we could not perform a similar analysis in this tissue. Based on these findings, we focused on GlcCer, because it is the precursor for all the more complex GSLs with important biological functions (34,35). HPLC analysis revealed a comparable age-related decline in the total amount of GlcCer in spinal cord of both SOD1(G86R) and WT mice (Fig. 3A). In contrast, a significant up-regulation of GM1a, the major ganglioside in the central nervous system, was observed in SOD1(G86R) mice at the symptomatic stage compared with WT littermates (Fig. 3B). There was also a significant increase in GlcCer in muscle of symptomatic SOD1(G86R) mice (Fig. 3D). Concomitantly, levels of several gangliosides of the a-series, including GM3 and GM2, were significantly increased in this tissue (Fig. 3E). To investigate the reason for altered GlcCer levels, we measured GCS expression. GCS mRNA levels were significantly increased in muscle at both pre-symptomatic and symptomatic stages in SOD1(G86R) mice compared with WT littermates (Fig. 3F), but no differences were observed in spinal cord (Fig. 3C). Muscle GCS expression at 60 days of age, an earlier pre-symptomatic time point, was similar in SOD1(G86R) and WT mice (data not shown). Therefore, the up-regulation of GCS observed at 75 days of age can be considered as an early event preceding the onset of overt motor disease. To support these findings, we performed western blot analyses with actin or total protein content as an internal reference. In both cases, GCS protein amount was increased at the pre-symptomatic stage in SOD1(G86R) mice. This increase was similarly observed in symptomatic mice (Fig. 3G), although it was not noticeable after total protein normalization (data not shown). To gain further insight, muscle GCS expression was confirmed by immunohistological analysis of muscles from WT and SOD1(G86R) mice. We showed increased punctate GCS staining, frequently located at the border of myofibers (Fig. 3H), compatible with the reorganization of Golgi complex in denervated muscle (36). This pattern of staining was specifically observed in myofibers exhibiting less cross-sectional area and more metabolic oxidative capacity, as assessed by an increase in the presence of mitochondrial succinate dehydrogenase (SDH) (Fig. 3I).Figure 3.

Bottom Line: Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology.In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer).HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates.

View Article: PubMed Central - PubMed

Affiliation: Université de Strasbourg, UMR_S 1118, Strasbourg, France, INSERM, U1118, Mécanismes Centraux et Péripheriques de la Neurodégénérescence, Strasbourg, France.

No MeSH data available.


Related in: MedlinePlus