The Role of Caspase-3 Activation in Phencyclidine-Induced Neuronal Death in Postnatal Rats

Abstract

This study determined the role of caspase-3 in phencyclidine (PCP)-induced neurodegeneration in postnatal rats. PCP administration to postnatal day 7 rats induced a dose-dependent increase in caspase-3 enzymatic activity in frontal cortex, striatum, and hippocampus. Enzymatic activation was present at 4 h, peaked between 6 and 12 h, and disappeared by 24 h. Further, cleaved caspase-3-immunoreactive neurons were detected as early as 2 h in the cortex, and were found throughout the brain, including, in addition, the thalamus and striatum. Within the cingulate, frontal, parietal, and retrosplenial cortices, immunoreactivity was specific for layers II-IV (especially layer II). Neurons positive for both silver staining and terminal deoxynucleotidyl transferase biotin-d-UTP nick-end labeling (TUNEL) were found in the same brain regions and subregions. Double labeling experiments confirmed that cleaved caspase-3 and TUNEL were coexpressed in many neurons in all brain regions and subregions studied. Temporal studies revealed that procaspase-3 cleavage preceded TUNEL staining by about 3 h, with many neurons being positive for both caspase-3 and TUNEL 9 h after PCP treatment. In organotypic corticostriatal slices, PCP caused a concentration-and time-dependent cleavage of procaspase-3 that was also colocalized with TUNEL staining in layers II-IV of the parietal cortex. Caspase-3 activation again preceded PCP-induced DNA damage assessed by TUNEL. PCP-induced neuronal death in vitro as measured by TUNEL staining was blocked 85% by Ac-AAVALLPAVLLALLAPDEVD-CHO, a cell-permeable selective caspase-3 inhibitor. These data demonstrate that caspase-3 activation plays a necessary role in the regionally selective neuronal death induced by PCP in the developing rat brain.

The Role of Akt-GSK-3β Signaling and Synaptic Strength in Phencyclidine-Induced Neurodegeneration

“…The metabolic half-life of PCP in adults is about 4.5 h (Shelnutt et al, 1999), but these data are unavailable for pups at this stage of maturity. This profile of pAkt Ser473 parallels the change in caspase-3 activation in rat pups treated with PCP observed previously by this laboratory (Wang and Johnson, 2007). The reduction in pAkt Ser473 was also observed in dissociated neuronal cultures (Figure 2d and e).…”

“…PCP-induced toxicity was concentrationdependent with a half maximal effect observed at 6574.3 nM, which correlates well with its binding affinity of 50-100 nM for NMDA receptors (Johnson and Jones, 1990). As previously reported in corticostriatal slices (Wang and Johnson, 2007), PCP (1 mM) caused activation of caspase-3 within 3 h after PCP treatment, with the peak effect observed at 12 h before decaying at 48 h ( Figure 1d). It is possible that the toxic effect of PCP seen in vivo could partially result from increased extracellular dopamine (DA) secondary to inhibition of the DA transporter by PCP (Rothman et al, 1989;Alagarsamy et al, 1997).…”

“…Second, cultured neurons were exposed to PCP and it was also found to robustly reduce pGSK-3b Ser9 (Figure 5d). It has been reported that caspase-3 activation plays a major role in PCP-induced neurotoxicity (Wang and Johnson, 2007). Caspase-3 is downstream of, and activated by, GSK-3b (Song et al, 2002).…”

Lithium Protection of Phencyclidine-Induced Neurotoxicity in Developing Brain: The Role of Phosphatidylinositol-3 Kinase/Akt and Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways

“…Slice samples used for caspase-3 activity assay were always collected 12 h after PCP treatment. Caspase-3 activity was measured as described previously (Wang and Johnson, 2007). In brief, slices were sonicated in ice-cold lysis buffer containing 25 mM HEPES, pH 7.4, 5 mM MgCl 2 , 1.5 mM EDTA, 1.0 mM EGTA, 1 mM dithiothreitol, 0.1% Triton X-100, and 1% protease inhibitor cocktail.…”

“…This study used cultures of organotypic brain slices, an in vitro model that conserves the biologically relevant structural and functional features of in vivo tissues (Vickers and Fisher, 2004), while also allowing manipulation of drugs that can not easily gain access to the brain in vivo. Corticostriatal slice cultures were prepared as prescribed previously (Wang and Johnson, 2007). In brief, 2-day-old rat pups were sacrificed by decapitation.…”