Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase methods
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Glccer and GCS expression are up-regulated in muscle of SOD1(G86R) mice
Following the analysis described earlier, we also took advantage of our previously published transcriptome of muscle in SOD1(G86R) mice ( ). We performed a joint enrichment analysis of transcriptomics and lipidomics data using the IMPaLA web interface ( ). 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 ( ). 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 (, ). HPLC analysis revealed a comparable ag...
GCS expression and GlcCer are up-regulated in response to surgically induced muscle denervation
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. A). Expression of the acetylcholine receptor...
GCS inhibition blocks the metabolic changes following muscle denervation and delays the recovery of motor function
In ALS, as well as in response to denervation, muscle atrophy is associated with changes in the metabolic phenotype of the fibers and modifications in their contractility and fatigability. These fibers undergo a shift from a fast-twitch to a slow-twitch type with more sustained activity and increased resistance to fatigue, characteristics that are typical of muscles with a high degree of oxidative, fatty acid metabolism ( - ). To determine the effects of inhibiting GCS enzymatic activity during muscle denervation, we treated WT mice, submitted to sciatic nerve crush, with daily intraperitoneal injections of the specific GCS inhibitor N -(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) ( ). AMP-DNM decreased muscle GlcCer content by 33% (Fig. A). Although this treatment did not influence the extent of muscle fiber atrophy 10 days following crush injury (Fig. B), it b...
GCS inhibition blocks the metabolic changes following muscle denervation and delays the recovery of motor function
Effect of inhibition of GCS enzymatic activity on motor function recovery after nerve lesion in WT mice. ( A ) Relative mRNA levels of AChR-α (left panel) and AChR-ε (right panel) in muscle of WT mice submitted to sciatic nerve crush in the absence (Vehicle) or presence of AMP-DNM. Ipsilateral and contralateral muscles were processed 10 days after lesion. * P < 0.05 versus corresponding contralateral muscle, n = 4. ( B and C ) Proportion of properly innervated neuromuscular junctions after 10 days of sciatic nerve crush, as determined by co-labeling with anti-synaptophysin antibody (green) and rhodamine-conjugated α-bungarotoxin (red). A total of 100-150 neuromuscular junctions per animal were analyzed. Examples of labeled neuromuscular junctions are shown in C. Scale bar, 500 µm. * P < 0.05 versus vehicle, n = 5-6. ( D ) Representative electromyographi...
GCS inhibition alters neuromuscular junction integrity and modifies the expression of metabolic genes in muscle of SO...
Considering the detrimental effect of the inhibition of GCS on motor axis regeneration, we hypothesized that loss of enzymatic activity would predispose SOD1(G86R) mice to develop the disease phenotype earlier. To test this hypothesis, we treated SOD1(G86R) mice with AMP-DNM for 10 days, starting at the pre-symptomatic stage. We did not observe behavioral signs of disease onset, such as impaired locomotion or abnormal hind limb extension reflexes. However, we found that SOD1(G86R) mice treated with AMP-DNM showed reduced hind limb grip strength (Fig. A). After treatment, the number of innervated synapses in treated and untreated mice was unchanged (Fig. B). In contrast, a more detailed analysis of the integrity of the postsynaptic apparatus revealed important morphological modifications (Fig. C). Specifically, the degree of fragmentation of the motor endplates was increased in treated...
Materials and Methods
FVB/N male mice, overexpressing SOD1(G86R), were maintained in our animal facility at 23°C with a 12 h light/dark cycle. Genotyping was performed according to Ripps and coworkers ( ). Mice had water and regular A04 rodent chow ad libitum and were fasted overnight before sacrifice. At 75 days of age (referred as to the pre-symptomatic group), mice were without signs of motor impairment, as determined by the absence of electromyographic abnormalities, as well as muscle expression of AChR-α and number of spinal cord motor neurons comparable to WT mice. At ∼100 days of age (referred as to the symptomatic group), mice showed apparent signs of paresis, or partial paralysis, in at least one limb, coinciding with the alteration of the above-mentioned parameters ( ). WT male littermates served as controls. To induce peripheral nerve injury, mice were anesthetized with ketamine...
Lipid extraction
Tissue samples (25 mg for lumbar spinal cord, and 25-50 mg for soleus muscle) were gently thawed on crushed ice and homogenized with 335 µl precooled methanol. Lipids were extracted by orbital agitation using a Precellys system (Bertin Technologies, Saint-Quentin-en-Yvelines, France) in a propylene tube containing ceramic beads. Two homogenization steps at 5000 rpm for 30 s each were performed at room temperature. Homogenates were transferred into glass conic tubes. To recover the maximum volume of homogenate, Precellys tubes, beads and tips were washed with additional 335 µl precooled methanol. Then, homogenates were mixed with 1340 µl chloroform and centrifuged at 2000 rpm for 5 min at 4°C. The organic phase was transferred into a conical glass tube and washed with 400 µl precooled 0.9% NaCl. After centrifugation at 2000 rpm for 5 min at 4°C, the...
UPLC/TOF-MS
Chromatography was performed on an Acquity UPLC system using an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm, Waters Corporation, Milford, MA) maintained at 50°C. Chromatographic flow rate was 0.5 ml/min, and run time was 18 min. Mobile phases were isopropanol (solvent A) and acetonitrile (solvent B). The starting condition was 100% solvent B, followed by a gradual increase (gradient type 6) of solvent A from 0 to 75% over the first 15 min. Then, 100% solvent B was reused from 15.1 to 17 min. The chromatographic system was coupled to a Micromass LCT Premier TOF/W mass spectrometer (Waters Corporation), equipped with an electrospray source operating in positive ion mode with a lockspray interface for accurate mass measurements. Source temperature was set at 150°C, with a cone gas flow of 50 l/h at 400°C and a nebulization gas flow of 750 l/h. Capillary volta...
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Following the analysis described earlier, we also took advantage of our previously published transcriptome of muscle in SOD1(G86R) mice ( ). We performed a joint enrichment analysis of transcriptomics and lipidomics data using the IMPaLA web interface ( ). 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 ( ). 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 (, ). 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. A). 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. B...
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. A). 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. B). The decreased expression of AChR-α, together with the increased expression of AChR-ε, is inter...
In ALS, as well as in response to denervation, muscle atrophy is associated with changes in the metabolic phenotype of the fibers and modifications in their contractility and fatigability. These fibers undergo a shift from a fast-twitch to a slow-twitch type with more sustained activity and increased resistance to fatigue, characteristics that are typical of muscles with a high degree of oxidative, fatty acid metabolism ( - ). To determine the effects of inhibiting GCS enzymatic activity during muscle denervation, we treated WT mice, submitted to sciatic nerve crush, with daily intraperitoneal injections of the specific GCS inhibitor N -(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) ( ). AMP-DNM decreased muscle GlcCer content by 33% (Fig. A). Although this treatment did not influence the extent of muscle fiber atrophy 10 days following crush injury (Fig. B), it blocked the expression of several genes critical for promoting oxidative metabolism following denervation (Fig. C). In particular, AMP-DNM significantly reduced the expression of the transcription factors PGC1α and PPARα, master activators of mitochondriogenesis and lipid catabolism, respec...
Effect of inhibition of GCS enzymatic activity on motor function recovery after nerve lesion in WT mice. ( A ) Relative mRNA levels of AChR-α (left panel) and AChR-ε (right panel) in muscle of WT mice submitted to sciatic nerve crush in the absence (Vehicle) or presence of AMP-DNM. Ipsilateral and contralateral muscles were processed 10 days after lesion. * P < 0.05 versus corresponding contralateral muscle, n = 4. ( B and C ) Proportion of properly innervated neuromuscular junctions after 10 days of sciatic nerve crush, as determined by co-labeling with anti-synaptophysin antibody (green) and rhodamine-conjugated α-bungarotoxin (red). A total of 100-150 neuromuscular junctions per animal were analyzed. Examples of labeled neuromuscular junctions are shown in C. Scale bar, 500 µm. * P < 0.05 versus vehicle, n = 5-6. ( D ) Representative electromyographic recordings in muscle of WT mice submitted to sciatic nerve crush in the absence (Crush, middle panel) or presence (Crush+AMP-DNM, lower panel) of GCS inhibitor. Upper panel shows non-denervated muscle. ( E ) Restoration of hind limb muscle grip strength in vehicle- or AMP-DNM-treated WT mice at th...
Considering the detrimental effect of the inhibition of GCS on motor axis regeneration, we hypothesized that loss of enzymatic activity would predispose SOD1(G86R) mice to develop the disease phenotype earlier. To test this hypothesis, we treated SOD1(G86R) mice with AMP-DNM for 10 days, starting at the pre-symptomatic stage. We did not observe behavioral signs of disease onset, such as impaired locomotion or abnormal hind limb extension reflexes. However, we found that SOD1(G86R) mice treated with AMP-DNM showed reduced hind limb grip strength (Fig. A). After treatment, the number of innervated synapses in treated and untreated mice was unchanged (Fig. B). In contrast, a more detailed analysis of the integrity of the postsynaptic apparatus revealed important morphological modifications (Fig. C). Specifically, the degree of fragmentation of the motor endplates was increased in treated SOD1(G86R) mice compared with untreated littermates (Fig. D). In addition, the number of gutter intersections per neuromuscular junction was decreased (Fig. E), which is interpreted as a loss of complexity. This morphological deterioration coincided with the presence of typical denervation markers,...
FVB/N male mice, overexpressing SOD1(G86R), were maintained in our animal facility at 23°C with a 12 h light/dark cycle. Genotyping was performed according to Ripps and coworkers ( ). Mice had water and regular A04 rodent chow ad libitum and were fasted overnight before sacrifice. At 75 days of age (referred as to the pre-symptomatic group), mice were without signs of motor impairment, as determined by the absence of electromyographic abnormalities, as well as muscle expression of AChR-α and number of spinal cord motor neurons comparable to WT mice. At ∼100 days of age (referred as to the symptomatic group), mice showed apparent signs of paresis, or partial paralysis, in at least one limb, coinciding with the alteration of the above-mentioned parameters ( ). WT male littermates served as controls. To induce peripheral nerve injury, mice were anesthetized with ketamine chlorohydrate (100 mg/kg) and xylazine (5 mg/kg). The sciatic nerve was exposed at mid-thigh level and crushed with fine forceps for 30 s, or a 3-mm section removed with microscissors. The skin incision was sutured, and mice were allowed to recover. The hind limb, contralateral to the lesion, served...
Tissue samples (25 mg for lumbar spinal cord, and 25-50 mg for soleus muscle) were gently thawed on crushed ice and homogenized with 335 µl precooled methanol. Lipids were extracted by orbital agitation using a Precellys system (Bertin Technologies, Saint-Quentin-en-Yvelines, France) in a propylene tube containing ceramic beads. Two homogenization steps at 5000 rpm for 30 s each were performed at room temperature. Homogenates were transferred into glass conic tubes. To recover the maximum volume of homogenate, Precellys tubes, beads and tips were washed with additional 335 µl precooled methanol. Then, homogenates were mixed with 1340 µl chloroform and centrifuged at 2000 rpm for 5 min at 4°C. The organic phase was transferred into a conical glass tube and washed with 400 µl precooled 0.9% NaCl. After centrifugation at 2000 rpm for 5 min at 4°C, the organic phase was evaporated to dryness at 30°C under nitrogen. Residues were reconstituted with acetonitrile/isopropanol (1:1) (100 µl for lumbar spinal cord, and 60 µl for soleus muscle) and further diluted with solvent mixture before injection (1/40 for lumbar spinal cord, and 1/10...
Chromatography was performed on an Acquity UPLC system using an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm, Waters Corporation, Milford, MA) maintained at 50°C. Chromatographic flow rate was 0.5 ml/min, and run time was 18 min. Mobile phases were isopropanol (solvent A) and acetonitrile (solvent B). The starting condition was 100% solvent B, followed by a gradual increase (gradient type 6) of solvent A from 0 to 75% over the first 15 min. Then, 100% solvent B was reused from 15.1 to 17 min. The chromatographic system was coupled to a Micromass LCT Premier TOF/W mass spectrometer (Waters Corporation), equipped with an electrospray source operating in positive ion mode with a lockspray interface for accurate mass measurements. Source temperature was set at 150°C, with a cone gas flow of 50 l/h at 400°C and a nebulization gas flow of 750 l/h. Capillary voltage was set at 2.7 kV, with a cone voltage of 50 V. Scan acquisition time was 0.1 s, and interscan delay was 0.01 s, in dynamic range enhancement mode. Leucine enkephalin was used as lockmass compound, infused at a concentration of 50 pg/µl at an infusion flow rate of 50 µl/min. Isotopic [M...
Machine-readable layer
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"text": "Effect of inhibition of GCS enzymatic activity on motor function recovery after nerve lesion in WT mice. ( A ) Relative mRNA levels of AChR-α (left panel) and AChR-ε (right panel) in muscle of WT mice submitted to sciatic nerve crush in the absence (Vehicle) or presence of AMP-DNM. Ipsilateral and contralateral muscles were processed 10 days after lesion. * P < 0.05 versus corresponding contralateral muscle, n = 4. ( B and C ) Proportion of properly innervated neuromuscular junctions after 10 days of sciatic nerve crush, as determined by co-labeling with anti-synaptophysin antibody (green) and rhodamine-conjugated α-bungarotoxin (red). A total of 100-150 neuromuscular junctions per animal were analyzed. Examples of labeled neuromuscular junctions are shown in C. Scale bar, 500 µm. * P < 0.05 versus vehicle, n = 5-6. ( D ) Representative electromyographi..."
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"text": "Considering the detrimental effect of the inhibition of GCS on motor axis regeneration, we hypothesized that loss of enzymatic activity would predispose SOD1(G86R) mice to develop the disease phenotype earlier. To test this hypothesis, we treated SOD1(G86R) mice with AMP-DNM for 10 days, starting at the pre-symptomatic stage. We did not observe behavioral signs of disease onset, such as impaired locomotion or abnormal hind limb extension reflexes. However, we found that SOD1(G86R) mice treated with AMP-DNM showed reduced hind limb grip strength (Fig. A). After treatment, the number of innervated synapses in treated and untreated mice was unchanged (Fig. B). In contrast, a more detailed analysis of the integrity of the postsynaptic apparatus revealed important morphological modifications (Fig. C). Specifically, the degree of fragmentation of the motor endplates was increased in treated..."
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