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Published on: December 9, 2014
Cloning of m-calpain 80 kD subunit from the axonal degeneration-resistant WLD(S) mouse mutant
1Department of Neurology, Emory University, Atlanta, Georgia 30322, USA. jglas03@emory.edu
Abstract:
Calpains are calcium-activated cysteine proteases that are involved in cellular degradation in models of neurodegeneration. Calpains are the effectors of cytoskeletal disruption during axonal degeneration, a pathological feature of many neurological disorders. The WLD(S) mouse mutant is resistant to axonal degeneration and demonstrates prolonged survival of the cytoskeleton after nerve injury. To investigate the possibility that a mutation in calpain or abnormalities in calpain protein expression is responsible for the resistance to axonal degeneration seen in the WLD(S) mouse mutant, we 1) cloned and sequenced the large subunit of the high calcium-requiring form of calpain (m-calpain) from nervous system tissues of WLD(S) and from wild-type C57BL/6 mice, and 2) generated polyclonal m-calpain antibodies for comparison of relative protein levels by Western blot. We found our sequence for mouse m-calpain to be almost identical to another recently published mouse sequence, and the wild-type and WLD(S) sequences to be identical. Our fusion protein and peptide polyclonal antibodies were specific for the 80 kD subunit and recognized appropriate protein bands from pure m-calpain, fusion protein, and in tissue. There was no apparent difference in m-calpain expression in nerve or spinal cord in noninjured adult animals. These data suggest that a defect in m-calpain 80 kD subunit does not likely underlie the WLD(S) phenotype, but raise questions about other subunits of calpain and possibly other proteases.
Insights
The WLD(S) mouse mutant resists axonal degeneration. Researchers found no differences in the m-calpain gene or protein, suggesting this specific calpain subunit is not responsible for the protective effect.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Calpains are calcium-activated proteases crucial in cellular degradation and cytoskeletal disruption during neurodegeneration.
- Axonal degeneration is a key pathological feature in many neurological disorders.
- The WLD(S) mouse mutant exhibits resistance to axonal degeneration and prolonged cytoskeletal survival post-nerve injury.
Purpose of the Study:
- To investigate if mutations or expression abnormalities in calpain cause the WLD(S) mouse mutant's resistance to axonal degeneration.
- To analyze the large subunit of the high calcium-requiring calpain (m-calpain) in WLD(S) mice.
Main Methods:
- Cloning and sequencing of the m-calpain large subunit from WLD(S) and wild-type mouse nervous system tissues.
- Generation of polyclonal m-calpain antibodies for Western blot analysis of protein levels.
- Comparison of m-calpain sequences and protein expression between WLD(S) and wild-type mice.
Main Results:
- The m-calpain sequences from WLD(S) and wild-type mice were identical to each other and nearly identical to a published mouse sequence.
- Generated antibodies were specific for the 80 kD m-calpain subunit and detected appropriate protein bands.
- No significant differences in m-calpain expression were observed in nerve or spinal cord tissues of non-injured adult animals.
Conclusions:
- A defect in the m-calpain 80 kD subunit is unlikely to be the cause of the WLD(S) phenotype.
- Further investigation into other calpain subunits or proteases may be warranted to explain the WLD(S) resistance to axonal degeneration.
