Disturbances in cellular ion gradients by excitotoxicity promote apoptosis through activation of the Bcl-2 family member Bim.

Excitotoxicity resulting from excessive Ca 2ϩ influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca 2ϩ levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16 -E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca 2ϩ homeostasis, a persistent depolarization of mitochondrial membrane potential (⌬ m ), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca 2ϩ increases, sensitivity to bax gene deletion, and delayed ⌬ m depolarization and Ca 2ϩ deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/ glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

The effect of highly active antiretroviral therapy (HAART) on the natural history of anal squamous intraepithelial lesions (ASIL)-the likely anal cancer precursor-and anal human papillomavirus (HPV) infection is unknown. ASIL severity and level of anal HPV DNA were evaluated among HIV-positive men who have sex with men (MSM) for at least 6 months before initiation of HAART. The results were compared with those from a 6-month period after initiation of HAART. Anal swabs for cytology and HPV studies were obtained, followed by high-resolution anoscopy and biopsy. Among men whose most severe pre-HAART diagnosis was atypical squamous cells of undetermined significance or low-grade ASIL, 18% (confidence interval [CI], 6-31%, 7 of 38) progressed and 21% (CI, 8-34%, 8 of 38) regressed 6 months after starting HAART. Seventeen percent (CI, 0-38%, 2 of 12) of study subjects who began with a normal diagnosis developed ASIL. Only 4% (CI, 0-10%, 1 of 28) of study subjects with high-grade ASIL regressed to normal. There was no reduction in the proportion of study subjects who tested positive for HPV DNA or HPV DNA levels after HAART initiation. The ASIL and HPV data were similar to those of the pre-HAART comparison period. These results indicate that HAART has little effect on either ASIL or HPV in the first 6 months after HAART initiation.

Prolonged seizures (status epilepticus) are associated with brain region-specific regulation of apoptosis-associated signaling pathways. Bcl-2 homology domain 3-only (BH3) members of the Bcl-2 gene family are of interest as possible initiators of mitochondrial dysfunction and release of apoptogenic molecules after seizures. Previously, we showed expression of the BH3-only protein Bim increased in the rat hippocampus but not neocortex following focal-onset status epilepticus. Here, we examined Bim expression in mice and compared seizure-damage between wild-type and Bim-deficient animals. Status epilepticus induced by intra-amygdala kainic acid caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and post-translational activation of Bim, including CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Up-regulation of bim mRNA was evident after 2 h and Bim protein was increased from 4-24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared to wild-type animals following seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death following kainic acid treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus and neocortex damage in Bim-deficient mice was comparable to wild-type animals. These results demonstrate region-specific differential contributions of Bim to seizure-induced neuronal death. KeywordsApoptosis; Bax; Bcl-2; FKHR; Hippocampus; JNK; Neuroprotection; Temporal lobe epilepsy Signaling pathways associated with the orchestration of apoptosis may contribute to neuronal death after experimental seizures.1 Both caspase-dependent and -independent apoptosis pathways have been implicated. 1 The Bcl-2 gene family are critical upstream regulators of the mitochondrial (intrinsic) apoptotic pathways. 2 Prolonged seizures (status epilepticus), and glutamate excitotoxicity in certain models, cause mitochondrial dysfunction and release of NIH Public Access Author ManuscriptCell Death Differ. Author manuscript; available in PMC 2010 October 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript apoptogenic molecules, including cytochrome c ,3 and apoptosis inducing factor (AIF). 4 Support for involvement of Bcl-2 family members comes from several observations. Seizures cause multi-domain pro-apoptotic Bax to cluster around mitochondria at the time of cytochrome c release, 5 and Bax-deficient neurons have been reported to be abnormally resistant to excitotoxicity. 6 Moreover, over-expression of anti-apoptotic Bcl-xl reduces cell death after seizure-like insults in vivo ,7 and, conversely, mice deficient in anti-apoptotic Bcl-w are abnormally vulnerable to hippocampal damage after seizures. 3 However, a functional role for Bax in excitotoxicity has been excluded by some studies. 4 Also, calcium exposure of mitochondria alone, 8 and calpain activity, 9 can trigger release...

We used intracellular microelectrodes to record the membrane potential (Vm) of intact murine colonic smooth muscle. Electrical activity consisted of spike complexes separated by quiescent periods (Vm≈−60 mV). The spike complexes consisted of about a dozen action potentials of approximately 30 mV amplitude. Tetraethylammonium (TEA, 1–10 mM) had little effect on the quiescent periods but increased the amplitude of the action potential spikes. 4‐Aminopyridine (4‐AP, ? 5 mM) caused continuous spiking. Voltage clamp of isolated myocytes identified delayed rectifier K+ currents that activated rapidly (time to half‐maximum current, 11.5 ms at 0 mV) and inactivated in two phases (τf= 96 ms, τs= 1.5 s at 0 mV). The half‐activation voltage of the permeability was −27 mV, with significant activation at −50 mV. TEA (10 mM) reduced the outward current at potentials positive to 0 mV. 4‐AP (5 mM) reduced the early current but increased outward current at later times (100–500 ms) consistent with block of resting channels relieved by depolarization. 4‐AP inhibited outward current at potentials negative to −20 mV, potentials where TEA had no effect. Qualitative PCR amplification of mRNA identified transcripts encoding delayed rectifier K+ channel subunits Kv1.6, Kv4.1, Kv4.2, Kv4.3 and the Kvβ1.1 subunit in murine colon myocytes. mRNA encoding Kv 1.4 was not detected. We find that TEA‐sensitive delayed rectifier currents are important determinants of action potential amplitude but not rhythmicity. Delayed rectifier currents sensitive to 4‐AP are important determinants of rhythmicity but not action potential amplitude.