Adenovirus-mediated gene transfer of inhibitors of apoptosis proteins delays apoptosis in cerebellar granule neurons

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans . Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity, Cerebellar granule neuron s undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxi c cell death in response to glutamate. We examined the endogenous expressio n of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IA P1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by sem iquantitative reverse PCR and immunoblot analysis in cultured cerebellar gr anule neurons. Cerebellar granule neurons express significant levels of RIA P2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detect ed. RIAP2 mRNA content and protein levels did not change when cells were sw itched from 25 to 5 mit? potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamat e exposure, we used recombinant adenoviral vectors to target XIAP, human IA P1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-in duced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fr agmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In con trast, treatment with 100 mu M or 1 mM glutamate did not induce caspase act ivity and adenoviral-mediated expression of IAPs had no influence on subseq uent excitotoxic cell death.