Investigation of the role of neuroserpin in neurite outgrowth and learning and memory
Neuroserpin is a serine protease inhibitor, or serpin, that is expressed in the nervous system and inhibits the protease tissue plasminogen activator (tPA). Neuroserpin has been shown to play a role in neurite outgrowth but con icting results have been reported. Neuroserpin has also been proposed to play a role in learning and memory but direct evidence for such a role is lacking. e overall goals of this thesis were to further investigate a role for neuroserpin in neurite outgrowth and to study a possible role of neuroserpin in learning and memory. To achieve these goals, recombinant adeno-associated viral (rAAV) vectors were prepared to mediate neuroserpin overexpression both in vitro and in vivo. e effect of neuroserpin overexpression on neurite outgrowth was investigated in rat dissociated dorsal root ganglion cultures. Overexpression of neuroserpin was successfully achieved in this model system using rAAV vectors, however, the timescale of expression was beyond the ideal time for the study of neurite outgrowth. Direct application of recombinant neuroserpin to these cultures revealed no effects of neuroserpin treatment on neurite outgrowth. e effect of neuroserpin knockdown on neurite outgrowth was studied in NGF-treated PC12 cells. Neuroserpin knockdown was achieved by the use of short hairpin RNA (shRNA) which led to changes in the neurite extension and cell morphology of the PC12 cells. When quantitative measures of cell and neurite morphology were related to the level of neuroserpin knockdown achieved by different shRNA sequences, a complex relationship between cell phenotype and neuroserpin level emerged. is suggested that neuroserpin may play a role in neurite outgrowth via multiple mechanisms and that an unknown cofactor may mediate the function of neuroserpin. To investigate the role of neuroserpin in learning and memory in vivo, localized overexpression of neuroserpin in the rat hippocampus was achieved through the targeted injection of rAAV vectors. Behavioural studies on rats overexpressing neuroserpin provided no evidence for a role of neuroserpin in learning and memory. Interestingly, no changes in tPA proteolytic activity were detected in the brains of these rats despite a large increase in the levels of neuroserpin expression. Moreover, there was a signi cant decrease in the levels of postsynaptic density protein 95 (PSD-95), a major postsynaptic scaffolding protein, in rats overexpressing neuroserpin, indicating some involvement of neuroserpin in synaptic plasticity. Taken together, the results from this thesis support a role of neuroserpin in neurite outgrowth and synaptic plasticity. In addition, these results, together with previous studies, raise doubts about the physiological role of neuroserpin as an inhibitor of tPA, and suggest the involvement of an unknown cofactor in regulating neuroserpin function.
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