Calpain-Mediated Apoptosis in Motor Neurons of Adult Mouse Spinal Cord Slices

Motor neuron degeneration is a critical phenomenon during spinal cord injuries and some neurodegenerative diseases. However, the mechanisms by which cell death is induced in these neurons are poorly understood. One reason for this, is the lack of model systems in which the survival and death of adult motor neurons can be studied under controlled conditions. This thesis is an attempt to develop such a model system. Here, we tried to establish an in vitro model utilizing slices of adult mouse spinal cord. A respiratory assay (MTT) was used to assess cell viability in the slices, and cell death was evaluated by morphological features using fluorescent nuclear stains or biochemical feature of cell death using agarose gel electrophoresis of DNA. We could demonstrate that both slice thickness and the presence of serum in the culture medium affected slice survival. By using the optimal conditions, we showed that survival could be maintained in the slice cultures for a few days. In the cultured slices, both motor neurons and glial cells died by apoptosis. Interestingly, apoptosis was induced independent of caspase activation, excitotoxicity or free radical formation-classical inducers of apoptosis. Immunohistochemical studies revealed that two isoforms of the Ca2+ -dependent proteases, calpain I and calpain II, appeared in the nuclei of the motor neurons in response to injury and culturing. Calpain activation occurred both in the cytoplasm and the nuclei of the motor neurons as assessed by a fluorogenic calpain substrate. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved ?-fodrin fragment. Calpain inhibitors and chelation of Ca2+ by EGTA delayed apoptosis and prevented calpain activation in the motor neurons. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect on either apoptosis or calpain activation in the motor neurons. In conclusion, our results show that massive caspase-independent but calpain-dependent apoptosis is induced in motor neurons in response to injury and culturing and that inhibitors of calpain can delay apoptosis in the motor neurons of cultured slices