Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment

Abstract

Abstract:Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain-sensitive cytoskeletal proteins microtubule-associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24-h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I-specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca 2~-dependentprotease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown. Key Words: Microtubule-associated protein 2-Spectrin -Cytoskeleton -Glutamate-Calcium -Calpain l-RiluzoIe-CNS injury.

Calpain 1 Knockdown Improves Tissue Sparing and Functional Outcomes after Spinal Cord Injury in Rats

“…19,26,27 This suggests that of the several calpain isoforms present in the CNS, 28 excessive activation of calpain 1 is a major contributor to the neurodegeneration and resultant loss of motor function. Calpain 1 activation is relatively rapid after SCI, 13 and the pre-injury knockdown would be optimal for neuroprotection. This may account for the improvement in BBB scores observed as early as 3 days after injury.…”

“…Calpain 1 levels were reduced using lentiviral CAPN1 short hairpin ribonucleic acid (shRNA) injected into the rat spinal cord. Because of the relatively rapid calpain activation that occurs after SCI compared with the several days needed for knockdown of calpain protein levels using RNA interference or antisense approaches, 12,13 it was necessary to administer the lentiviral CAPN1 siRNA 1 week pre-injury. Locomotor function, lesion volume, and tissue sparing were evaluated post-injury.…”

Dendritic and Synaptic Pathology in Experimental Autoimmune Encephalomyelitis

“…MAP2 loss may also be induced by excitotoxicity, because treatment with either a glutamate release inhibitor rilozole or calpain inhibitors reduced MAP2 loss in spinal cord trauma models. 37 However, it remains to be determined whether exposure to other inflammatory factors, such as free radicals, inflammatory cytokines, or proteases may also result in dendritic beading or MAP2 loss.…”

Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

“…6F). Loss of MAP2 has been considered as an early marker for neuronal damage following cerebral ischemia (25), spinal cord injury (26), and traumatic brain injury (27); therefore, potential toxicities of FUS G156E and FUS P525L to neurons would be reflected by reduced intensity of MAP2 immunostaining in primary neurons. In addition, primary neurons expressing HA-FUS G156E and HA-FUS P525L often exhibited abnormal cell shapes (e.g.…”