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

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    "name": "Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase methods",
    "description": "Evidence-backed execution summary for Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase methods from Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase.",
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        "name": "Glccer and GCS expression are up-regulated in muscle of SOD1(G86R) mice",
        "text": "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..."
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        "position": 2,
        "name": "GCS expression and GlcCer are up-regulated in response to surgically induced muscle denervation",
        "text": "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..."
      },
      {
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        "position": 3,
        "name": "GCS inhibition blocks the metabolic changes following muscle denervation and delays the recovery of motor function",
        "text": "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..."
      },
      {
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        "position": 4,
        "name": "GCS inhibition blocks the metabolic changes following muscle denervation and delays the recovery of motor function",
        "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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        "position": 5,
        "name": "GCS inhibition alters neuromuscular junction integrity and modifies the expression of metabolic genes in muscle of SO...",
        "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..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Materials and Methods",
        "text": "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..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Lipid extraction",
        "text": "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..."
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        "name": "UPLC/TOF-MS",
        "text": "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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        "@type": "HowToTool",
        "name": "Glccer and GCS expression are up-regulated in muscle of SOD1(G86R) mice"
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        "@type": "HowToTool",
        "name": "GCS expression is up-regulated in atrophic TDP-43-positive myofibers in ALS patients"
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      {
        "@type": "HowToTool",
        "name": "GCS inhibition alters neuromuscular junction integrity and modifies the expression of metabolic genes in muscle of SO..."
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      {
        "@type": "HowToTool",
        "name": "Lipid extraction"
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        "name": "UPLC/TOF-MS"
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        "name": "HPLC"
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        "name": "Immunoperoxidase staining"
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        "@type": "HowToTool",
        "name": "Muscle histochemistry"
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        "@type": "HowToSupply",
        "name": "Glccer and GCS expression are up-regulated in muscle of SOD1(G86R) mice"
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        "name": "GCS inhibition blocks the metabolic changes following muscle denervation and delays the recovery of motor function"
      },
      {
        "@type": "HowToSupply",
        "name": "GCS inhibition blocks the metabolic changes following muscle denervation and delays the recovery of motor function"
      },
      {
        "@type": "HowToSupply",
        "name": "GCS inhibition alters neuromuscular junction integrity and modifies the expression of metabolic genes in muscle of SO..."
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        "name": "UPLC/TOF-MS"
      },
      {
        "@type": "HowToSupply",
        "name": "Quantitative RT-PCR"
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        "@type": "HowToSupply",
        "name": "Western blot"
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        "name": "Immunoperoxidase staining"
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      "@type": "ScholarlyArticle",
      "headline": "Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase",
      "datePublished": "2015",
      "author": [
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          "@type": "Person",
          "name": "Alexandre Henriques"
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          "@type": "Person",
          "name": "Vincent Croixmarie"
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          "name": "David A. Priestman"
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        {
          "@type": "Person",
          "name": "Angela Rosenbohm"
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        {
          "@type": "Person",
          "name": "Sylvie Dirrig-Grosch"
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        {
          "@type": "Person",
          "name": "Eleonora D'Ambra"
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          "@type": "Person",
          "name": "Mylene Huebecker"
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          "@type": "Person",
          "name": "Ghulam Hussain"
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        {
          "@type": "Person",
          "name": "Claire Boursier-Neyret"
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        {
          "@type": "Person",
          "name": "Andoni Echaniz-Laguna"
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          "@type": "Person",
          "name": "Albert C. Ludolph"
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          "name": "Frances M. Platt"
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          "name": "Bernard Walther"
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          "name": "Michael Spedding"
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          "name": "Jean-Philippe Loeffler"
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        {
          "@type": "Person",
          "name": "Jose-Luis Gonzalez De Aguilar"
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      "identifier": "10.1093/hmg/ddv439"
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