Source Paper
Alexandre Henriques, Vincent Croixmarie, David A. Priestman, Angela Rosenbohm, Sylvie Dirrig-Grosch et al.
Human Molecular Genetics • 2015
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. 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. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets.
Objective: To induce peripheral nerve injury via sciatic nerve ablation and study motor function recovery and nerve degeneration in SOD1(G86R) transgenic mice
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FVB/N male mice overexpressing SOD1(G86R) were maintained in animal facility at 23°C with 12 hour light/dark cycle. Mice had water and regular A04 rodent chow provided ad libitum.
Note: 12 hour light/dark cycle maintained throughout
“FVB/N male mice, overexpressing SOD1(G86R), were maintained in our animal facility at 23°C with a 12 h light/dark cycle. Mice had water and regular A04 rodent chow ad libitum”
Mice were fasted overnight before sacrifice
“were fasted overnight before sacrifice”
Mice were anesthetized with ketamine chlorohydrate at 100 mg/kg and xylazine at 5 mg/kg
Note: Intraperitoneal or intramuscular injection route not specified
“mice were anesthetized with ketamine chlorohydrate (100 mg/kg) and xylazine (5 mg/kg)”
The sciatic nerve was exposed at mid-thigh level through surgical incision
Note: Surgical site location: mid-thigh level
“The sciatic nerve was exposed at mid-thigh level”
The sciatic nerve was crushed with fine forceps for 30 seconds
Note: This is one of two injury methods used; crush injury was used for mice receiving AMP-DNM treatment
“crushed with fine forceps for 30 s”
A 3-mm section of the sciatic nerve was removed with microscissors
Note: This is the primary ablation method described in the experiment title
“a 3-mm section removed with microscissors”
The skin incision was sutured closed
“The skin incision was sutured, and mice were allowed to recover”
Mice were allowed to recover from anesthesia following surgery
Note: Recovery period duration not specified
“mice were allowed to recover”
Mice subjected to sciatic nerve crush were treated with daily intraperitoneal injections of AMP-DNM at 25 mg/kg in 0.9% NaCl containing 5% DMSO for 10 days
Note: Treatment duration corresponds to typical time frame required for motor function recovery under normal conditions
“mice subjected to sciatic nerve crush were treated with daily intraperitoneal injections of N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM, Cayman Chemical Company, Ann Arbor, MI) for 10 days”
Mice were killed by decapitation after deep anesthesia with 120 mg/kg sodium pentobarbital
“Mice were killed by decapitation after deep anesthesia with 120 mg/kg sodium pentobarbital”
Lumbar spinal cord and muscle were rapidly dissected, frozen in liquid nitrogen and stored at −80°C
Note: Rapid freezing in liquid nitrogen performed immediately after dissection
“Lumbar spinal cord and muscle were rapidly dissected, frozen in liquid nitrogen and stored at −80°C”
For immunolabeling experiments, dissected muscle samples were fixed with 4% paraformaldehyde and stored in PBS at 4°C
Note: Alternative to frozen storage for immunolabeling studies
“For immunolabeling experiments, dissected muscle samples were fixed with 4% paraformaldehyde and stored in PBS at 4°C”
WT male littermates served as controls. At 75 days, mice were without signs of motor impairment. At ~100 days, mice showed apparent signs of paresis or partial paralysis in at least one limb.
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