The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell lay er of the cerebellum. Concurrently, it has been shown that intracerebral in jection of pituitary adenylate cyclase-activating polypeptide (PACAP) induc es a transient increase of the thickness of the cerebellar cortex, In the p resent study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubatio n of granule neurons with PACAP for 24 h promoted cell survival and prevent ed DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptot ic cell death, Time-course studies revealed that PACAP causes a prolonged i nhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibit ion of caspase-3 activity. Incubation of granule cells with both dibutyryl- cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibi tory effect of PACAP on caspase-3, Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor cheleryt hrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule ne urons from apoptotic cell death through a protein kinase A- and protein kin ase C-dependent inhibition of caspase-3 activity.