One potential factor contributing to the susceptibility of these cells to premature death arises from the cytotoxic effects of amyloid-␤ (A␤) peptide deposition at or near sites of neuronal degeneration. Cultured human Franc ¸ois G.

Neuronal death, which follows ischemic injury or is triggered by excitotoxins, can occur by both apoptosis and necrosis. Caspases, which are not directly required for necrotic cell death, are central mediators of the apoptotic program. Here we demonstrate that caspases cleave and inactivate the plasma membrane Ca 2+ pump (PMCA) in neurons and non-neuronal cells undergoing apoptosis. PMCA cleavage impairs intracellular Ca 2+ handling, which results in Ca 2+ overload. Expression of non-cleavable PMCA mutants prevents the disturbance in Ca 2+ handling, slows down the kinetics of apoptosis, and markedly delays secondary cell lysis (necrosis). These findings suggest that caspase-mediated cleavage and inactivation of PMCAs can lead to necrosis, an event that is reduced by caspase inhibitors in brain ischemia.

Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal prostaglandin E 2 (PGE 2 ) synthase in the cyclooxygenase pathway. Inhibitors of mPGES-1 may block PGE 2 production and relieve inflammatory symptoms. To test the hypothesis, we evaluated the antipyretic and analgesic properties of a novel and selective mPGES-1 inhibitor, MF63 [2-(6-chloro-1H-phenanthro-[9,10-d]imidazol-2-yl)isophthalonitrile], in animal models of inflammation. MF63 potently inhibited the human mPGES-1 enzyme (IC 50 ϭ 1.3 nM), with a high degree (Ͼ1000-fold) of selectivity over other prostanoid synthases. In rodent species, MF63 strongly inhibited guinea pig mPGES-1 (IC 50 ϭ 0.9 nM) but not the mouse or rat enzyme. When tested in the guinea pig and a knock-in (KI) mouse expressing human mPGES-1, the compound selectively suppressed the synthesis of PGE 2 , but not other prostaglandins inhibitable by nonsteroidal anti-inflammatory drugs (NSAIDs), yet retained NSAID-like efficacy at inhibiting lipopolysaccharide-induced pyresis, hyperalgesia, and iodoacetate-induced osteoarthritic pain. In addition, MF63 did not cause NSAID-like gastrointestinal toxic effects, such as mucosal erosions or leakage in the KI mice or nonhuman primates, although it markedly inhibited PGE 2 synthesis in the KI mouse stomach. Our data demonstrate that mPGES-1 inhibition leads to effective relief of both pyresis and inflammatory pain in preclinical models of inflammation and may be a useful approach for treating inflammatory diseases.

Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model. Hypoxic-ischemic (H-I)1 encephalopathy in the prenatal and perinatal period is a major cause of morbidity and mortality and often results in cognitive impairment, seizures, and motor impairment leading to cerebral palsy (1, 2). Many studies of neonatal H-I brain injury have utilized the well characterized Levine model in which unilateral carotid ligation is followed by exposure to hypoxia in postnatal day (P) 7 rats (3-5). This model of H-I results in a reproducible pattern of hemispheric injury ipsilateral, but not contralateral, to the carotid ligation (5-7). There are prominent features of both apoptosis and necrosis when this model is performed in neonatal rats and mice (1, 8 -11). Inhibition of caspases utilizing a pan-caspase inhibitor partially protects against brain injury after neonatal H-I injury in this model (12), and similar inhibitors have been shown to partially protect against ischemic injury in adult models (13-16). Previously utilized peptide-based caspase inhibitors (e.g. Boc-D-fmk, z-VAD-fmk, z-DEVD-fmk) required relatively large doses in vivo for their protective effects, and at high concentrations, their effects are more likely to be less selective. Thus, although these studies suggest a role for caspases, the specific caspases and other proteases, which contribute to brain injury after neonatal H-I, have not been clarified.Caspases are a family of cysteine asp...