Apoptosis as well necrosis plays a role in neuronal loss in various neurological diseases including hypoxic-ischemia, Alzheimer’s disease, and Parkinson’s disease. Up to now, caspase-mediated signaling pathways have been proposed as a common route of apoptosis. We applied proteomic approach to draw expression profiles of proteins specifically altered in the process of neuronal apoptosis. Cortical neurons deprived of serum underwent widespread apoptosis over the next 24 hr, which was sensitive to cycloheximide, a protein synthesis inhibitor, that was administered within 8 - 10 hr after serum deprivation. Protein samples were obtained from cortical neurons deprived of serum for 8 hr and subjected to two-dimensional gel electrophoresis (2-DE). The first dimension separation was performed on an immobilized pH gradient strip, which was then separated on SDS-PAGE for the second dimension. Thousands of protein spots were visualized by silver staining, analysed by image processing, and identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). We have identified 49 proteins that are mainly associated with metabolism, transcription, development, and synthetic pathways
Of them, tissue inhibitor of metalloproteinases-3 (TIMP-3 Tissue inhibitor of metalloproteinases-3 (TIMP-3), a pro-apoptotic protein in various cancer cells, was increased during serum deprivation-induced apoptosis (SDIA), but not during necrosis induced by excitotoxicity or oxidative stress. Levels of TIMP-3 were markedly increased in degenerating neurons in a transgenic model of familial amyotrophic lateral sclerosis and in brains of patients with Alzheimer’s disease. Upregulation of TIMP-3 following serum deprivation was accompanied by activation of the Fas death pathway. SDIA and activation of the Fas pathway were prevented by addition of an active MMP-3. Timp-3 deletion by RNA interference attenuated SDIA in N2a cells. These findings provide evidence that TIMP-3 is an upstream mediator of neuronal apoptosis and likely contributes to neuronal loss in neurodegenerative diseases such as amyotrophic lateral sclerosis or Alzheimer’s disease.