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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

GCS expression in denervated muscle of WT mice. Relative mRNA levels of AChR-α (A), AChR-ε (B), GCS (C) and RUNX1 (D) in muscle of WT mice submitted to sciatic nerve crush or axotomy (Axo). Muscles were processed at the indicated days post-lesion. Ipsilateral muscles (red bars) were compared with contralateral muscles (white bars) in the same animal. *P < 0.05 versus corresponding contralateral muscle, #P < 0.05 versus preceding time point following nerve crush, n = 5–7. (E) GCS protein levels, as determined by western blot (a representative blot is shown in the left panel), in muscle of WT mice submitted to nerve crush as in A. Right panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05, n = 8. (F) Representative photomicrographs showing GCS immunostaining on cross-sections of contralateral and ipsilateral muscle as in A. Scale bar, 50 µm. (G) HPLC quantification of GlcCer (left panel) and several GSLs (right panel) in muscle of WT mice submitted to sciatic nerve crush as in A. Only detected main peaks of gangliosides are shown. *P < 0.05 versus corresponding contralateral muscle, n = 7–8.
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DDV439F5: GCS expression in denervated muscle of WT mice. Relative mRNA levels of AChR-α (A), AChR-ε (B), GCS (C) and RUNX1 (D) in muscle of WT mice submitted to sciatic nerve crush or axotomy (Axo). Muscles were processed at the indicated days post-lesion. Ipsilateral muscles (red bars) were compared with contralateral muscles (white bars) in the same animal. *P < 0.05 versus corresponding contralateral muscle, #P < 0.05 versus preceding time point following nerve crush, n = 5–7. (E) GCS protein levels, as determined by western blot (a representative blot is shown in the left panel), in muscle of WT mice submitted to nerve crush as in A. Right panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05, n = 8. (F) Representative photomicrographs showing GCS immunostaining on cross-sections of contralateral and ipsilateral muscle as in A. Scale bar, 50 µm. (G) HPLC quantification of GlcCer (left panel) and several GSLs (right panel) in muscle of WT mice submitted to sciatic nerve crush as in A. Only detected main peaks of gangliosides are shown. *P < 0.05 versus corresponding contralateral muscle, n = 7–8.

Mentions: To understand GCS up-regulation in muscle during ALS, we investigated the response of GCS to peripheral nerve injury in WT mice, as a way of mimicking ALS-like muscle denervation. Crushing the sciatic nerve for a few seconds is used as a model of hind limb denervation. This is followed by subsequent recovery owing to reinnervation in about 10 days. In contrast, axotomy, cutting and removing several millimeters of the sciatic nerve, causes denervation that persists for several weeks in WT mice. Muscle expression of the acetylcholine receptor alpha subunit (AChR-α) was strongly stimulated 3 days after crush injury in WT mice, at levels comparable to those found after 15 days following axotomy. This stimulatory effect was reduced by half, 15 days following the crush injury, when mice already exhibit signs of motor function recovery (Fig. 5A). Expression of the acetylcholine receptor epsilon subunit (AChR-ε) was significantly down-regulated 3 days following the crush injury and 15 days following axotomy, but expression was fully restored 15 days following crush (Fig. 5B). The decreased expression of AChR-α, together with the increased expression of AChR-ε, is interpreted as a sign of muscle re-innervation. Under these conditions, the pattern of expression of GCS (Fig. 5C), as well as that of RUNT-related transcription factor-1 (RUNX1) (Fig. 5D), a transcriptional activator of GCS (40), was similar to that of AChR-α. Western blot (Fig. 5E) and histological analysis (Fig. 5F) confirmed increased amounts of GCS in denervated muscle 3 days following crush injury. Furthermore, HPLC analysis revealed a significant increase in GlcCer, as well as GM3 and GM2 gangliosides, 10 days following crush injury (Fig. 5G). These findings reflect the observation in SOD1(G86R) mice and provide compelling evidence of the stimulatory effect of denervation on GlcCer and GSL accumulation.Figure 5.


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)

GCS expression in denervated muscle of WT mice. Relative mRNA levels of AChR-α (A), AChR-ε (B), GCS (C) and RUNX1 (D) in muscle of WT mice submitted to sciatic nerve crush or axotomy (Axo). Muscles were processed at the indicated days post-lesion. Ipsilateral muscles (red bars) were compared with contralateral muscles (white bars) in the same animal. *P < 0.05 versus corresponding contralateral muscle, #P < 0.05 versus preceding time point following nerve crush, n = 5–7. (E) GCS protein levels, as determined by western blot (a representative blot is shown in the left panel), in muscle of WT mice submitted to nerve crush as in A. Right panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05, n = 8. (F) Representative photomicrographs showing GCS immunostaining on cross-sections of contralateral and ipsilateral muscle as in A. Scale bar, 50 µm. (G) HPLC quantification of GlcCer (left panel) and several GSLs (right panel) in muscle of WT mice submitted to sciatic nerve crush as in A. Only detected main peaks of gangliosides are shown. *P < 0.05 versus corresponding contralateral muscle, n = 7–8.
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DDV439F5: GCS expression in denervated muscle of WT mice. Relative mRNA levels of AChR-α (A), AChR-ε (B), GCS (C) and RUNX1 (D) in muscle of WT mice submitted to sciatic nerve crush or axotomy (Axo). Muscles were processed at the indicated days post-lesion. Ipsilateral muscles (red bars) were compared with contralateral muscles (white bars) in the same animal. *P < 0.05 versus corresponding contralateral muscle, #P < 0.05 versus preceding time point following nerve crush, n = 5–7. (E) GCS protein levels, as determined by western blot (a representative blot is shown in the left panel), in muscle of WT mice submitted to nerve crush as in A. Right panel shows quantification of immunoblots using actin protein levels as internal reference. *P < 0.05, n = 8. (F) Representative photomicrographs showing GCS immunostaining on cross-sections of contralateral and ipsilateral muscle as in A. Scale bar, 50 µm. (G) HPLC quantification of GlcCer (left panel) and several GSLs (right panel) in muscle of WT mice submitted to sciatic nerve crush as in A. Only detected main peaks of gangliosides are shown. *P < 0.05 versus corresponding contralateral muscle, n = 7–8.
Mentions: To understand GCS up-regulation in muscle during ALS, we investigated the response of GCS to peripheral nerve injury in WT mice, as a way of mimicking ALS-like muscle denervation. Crushing the sciatic nerve for a few seconds is used as a model of hind limb denervation. This is followed by subsequent recovery owing to reinnervation in about 10 days. In contrast, axotomy, cutting and removing several millimeters of the sciatic nerve, causes denervation that persists for several weeks in WT mice. Muscle expression of the acetylcholine receptor alpha subunit (AChR-α) was strongly stimulated 3 days after crush injury in WT mice, at levels comparable to those found after 15 days following axotomy. This stimulatory effect was reduced by half, 15 days following the crush injury, when mice already exhibit signs of motor function recovery (Fig. 5A). Expression of the acetylcholine receptor epsilon subunit (AChR-ε) was significantly down-regulated 3 days following the crush injury and 15 days following axotomy, but expression was fully restored 15 days following crush (Fig. 5B). The decreased expression of AChR-α, together with the increased expression of AChR-ε, is interpreted as a sign of muscle re-innervation. Under these conditions, the pattern of expression of GCS (Fig. 5C), as well as that of RUNT-related transcription factor-1 (RUNX1) (Fig. 5D), a transcriptional activator of GCS (40), was similar to that of AChR-α. Western blot (Fig. 5E) and histological analysis (Fig. 5F) confirmed increased amounts of GCS in denervated muscle 3 days following crush injury. Furthermore, HPLC analysis revealed a significant increase in GlcCer, as well as GM3 and GM2 gangliosides, 10 days following crush injury (Fig. 5G). These findings reflect the observation in SOD1(G86R) mice and provide compelling evidence of the stimulatory effect of denervation on GlcCer and GSL accumulation.Figure 5.

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