Alfred Stracher

Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition

Inhibition of muscle degeneration by the tripeptide calpain inhibitor, leupeptin, was tested in vivo in a dystrophin‐deficient mdx murine model. In a short‐term control study, intramuscular administration of leupeptin for 30 days inhibited muscle degeneration as assessed by histologic analysis. Calpain inhibition could be correlated with retention of myofiber size and our results suggest that this may be a promising treatment modality in human Duchenne muscular dystrophy.

Calpain Inhibitors as Therapeutic Agents in Nerve and Muscle Degeneration

ABSTRACT: It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists that, when deciphered, could lead to our understanding of a variety of diseases that result in tissue necrosis, as well as offer potential for therapeutic intervention. In recent years progress toward elucidating this common pathway has been accelerated through the studies of a number of laboratories, including our own, on the role of the protease calpain in this process. Thus, in a variety of disorders, such as stroke, spinal cord injury, traumatic nerve injury, Parkinsons disease, amyotrophic lateral sclerosis (ALS), Alzheimers disease, muscular dystrophy, cataract formation, unregulated calpain proteolysis, initiated via dysregulation of calcium ion homeostasis, participates in the pathogenesis and is a potentially unifying mechanistic event.

Phosphorylation of platelet actin binding protein protects against proteolysis by calcium dependent sulfhydryl protease.

When Actin Binding Protein (ABP) isolated from human blood platelets is phosphorylated in vitro with a cyclic AMP dependent kinase it becomes resistant to proteolysis by the Calcium Dependent Sulfhydryl Protease (CDSP). This protection against proteolytic cleavage is specific for CDSP since phosphorylation of ABP does not protect against proteolysis by trypsin, papain and thermolysin. Thus, there appears to be a distinct phosphorylation site on the ABP molecule which is essential for regulating the initial proteolytic degradation of ABP by CDSP.

Localization and inhibition of calcium-activated neutral protease (CANP) in primate skeletal muscle and peripheral nerve

Localization of calcium-activated neutral protease in normal monkey (Cebus apella) skeletal muscle and peripheral nerve was studied by application of the indirect immunofluorescent and peroxidase-antiperoxidase techniques for light and electron microscopy. In muscle, the protease was demonstrated in association with endomysial collagen fibrils, basal lamina, sarcolemma, and Z-bands and also at neuromuscular junctions. In nerve, the enzyme was demonstrated in association with endoneurial collagen fibrils, basal lamina, axolemma, and neurofilaments of both myelinated and unmyelinated axons. Intramuscular injections of the thiol protease inhibitor, leupeptin, abolished Ca-activated neutral protease immunoreactivity in both muscle and peripheral nerve. These data suggest that the protease is localized both intracellularly and extracellularly in normal primate muscle and peripheral nerve. Inhibition of basal lamina associated neutral protease by leupeptin after denervation may hold significance for protecting this structure against degradation and preserving it as a neurotrophic lattice for axon regeneration.

Inhibition of neural and muscle degeneration after epineural neurorrhaphy

Investigations were undertaken on the regeneration of transected rat sciatic nerves. The ability of the protease inhibitor leupeptin to inhibit wallerian degeneration and muscle atrophy was evaluated. After transection of a sciatic nerve and immediate neurorrhaphy, animals were treated with leupeptin for a period of 1 week to 6 months. Our results indicate a significant increase in the numbers of myelinated and unmyelinated neurofibers in the distal segment of treated nerves. Peroxidase tracer, injected intramuscularly, was transported by retrograde axonal flow and was observed to label increased numbers of treated axons both distal and proximal to the repair site. This finding suggests that treated neurofibers are functionally viable. Evaluation of muscle showed that secondary muscular atrophy is also significantly inhibited by leupeptin.

Calcium-induced degeneration of the cytoskeleton in monkey and human peripheral nerves

Biopsy specimens of human and monkey peripheral nerves, when incubated in calcium containing media, showed a loss of neurofilaments and microtubules with replacement by granular debris. Cytoskeletal structures remained intact when incubated in calcium-free media. Disruption of neurofilaments and microtubules in calcium containing media was inhibited by the thiol protease inhibitor, leupeptin. Similar incubations of excised Pacinian corpuscles revealed evidence of early terminal axon degeneration in the presence of calcium. These data substantiate the hypothesis that neural cytoskeletal degradation in primates and in man is calcium-mediated.