Helena P. Bonner

Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

Excitotoxicity resulting from excessive Ca2+ influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca2+ 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 Ca2+ homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψm), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca2+ increases, sensitivity to bax gene deletion, and delayed Δψm depolarization and Ca2+ 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.

NMDA receptor-mediated excitotoxic neuronal apoptosis in vitro and in vivo occurs in an ER stress and PUMA independent manner.

Disruption of endoplasmic reticulum (ER) Ca2+ homeostasis and ER dysfunction have been suggested to contribute to excitotoxic and ischaemic neuronal injury. Previously, we have characterized the neural transcriptome following ER stress and identified the BH3‐only protein, p53 up‐regulated mediator of apoptosis (PUMA), as a central mediator of ER stress toxicity. In this study, we investigated the effects of excitotoxic injury on ER Ca2+ levels and induction of ER stress responses in models of glutamate‐ and NMDA‐induced excitotoxic apoptosis. While exposure to the ER stressor tunicamycin induced an ER stress response in cerebellar granule neurons, transcriptional activation of targets of the ER stress response, including PUMA, were absent following glutamate‐induced apoptosis. Confocal imaging revealed no long‐term changes in the ER Ca2+ level in response to glutamate. Murine cortical neurons and organotypic hippocampal slice cultures from PUMA+/+ and PUMA−/− animals provided no evidence of ER stress and did not differ in their sensitivity to NMDA. Finally, NMDA‐induced excitotoxic apoptosis in vivo was not associated with ER stress, nor did deficiency in PUMA alleviate the injury induced. Our data suggest that NMDA receptor‐mediated excitotoxic apoptosis occurs in vitro and in vivo in an ER stress and PUMA independent manner.

Caspase-3 cleavage and nuclear localization of caspase-activated dnase in human temporal lobe epilepsy

Programmed cell death (apoptosis) signaling pathways have been implicated in seizure-induced neuronal death and the pathogenesis of human temporal lobe epilepsy (TLE). End-stage DNA fragmentation during cell death may be mediated by nucleases including caspase-activated DNase (CAD), apoptosis-inducing factor (AIF) and endonuclease G. In the present study, we investigated the subcellular localization of these nucleases in resected hippocampus from TLE patients and autopsy controls. Subcellular fractionation determined levels of CAD were significantly higher in the nuclear fraction of TLE samples compared with controls, and semiquantitative immunohistochemistry revealed cleaved caspase-3 positive cells in TLE sections but not controls. While mitochondrial levels of AIF and endonuclease G were higher in TLE samples than controls, nuclear localization of AIF was limited and restricted to cells that were negative for cleaved caspase-3. Nuclear accumulation of endonuclease G was not found in TLE samples. These data support ongoing caspase-dependent apoptosis signaling in human TLE and suggest that interventions targeting such pathways may have potential as adjunctive neuroprotective therapy in epilepsy.

Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus

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 that expression of the BH3-only protein, Bcl-2 interacting mediator of cell death (Bim), increased in the rat hippocampus but not in the neocortex after focal-onset status epilepticus. In this study, 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 (KA) caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and posttranslational activation of Bim, such as CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Upregulation of bim mRNA was evident after 2 h and Bim protein increased between 4 and 24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared with wild-type animals after seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death after KA treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus, and neocortex damage in Bim-deficient mice was comparable with that in wild-type animals. These results show region-specific differential contributions of Bim to seizure-induced neuronal death.

Depletion of 14-3-3 zeta elicits endoplasmic reticulum stress and cell death, and increases vulnerability to kainate-induced injury in mouse hippocampal cultures.

14‐3‐3 proteins are ubiquitous signalling molecules that regulate development and survival pathways in brain. Altered expression and cellular localization of 14‐3‐3 proteins has been implicated in neurodegenerative diseases and in neuronal death after acute neurological insults, including seizures. Presently, we examined expression and function of 14‐3‐3 isoforms in vitro using mouse organotypic hippocampal cultures. Treatment of cultures with the endoplasmic reticulum (ER) stressor tunicamycin caused an increase in levels of 14‐3‐3 zeta within the ER‐containing microsomal fraction, along with up‐regulation of Lys‐Asp‐Glu‐Leu‐containing proteins and calnexin, and the selective death of dentate granule cells. Depletion of 14‐3‐3 zeta levels using small interfering RNA induced both ER stress proteins and death of granule cells. Treatment of hippocampal cultures with the excitotoxin kainic acid increased levels of Lys‐Asp‐Glu‐Leu‐containing proteins and microsomal 14‐3‐3 zeta levels and caused cell death within the CA1, CA3 and dentate gyrus of the hippocampus. Kainic acid‐induced damage was significantly increased in each hippocampal subfield of cultures treated with small interfering RNA targeting 14‐3‐3 zeta. The present data indicate a role for 14‐3‐3 zeta in survival responses following ER stress and possibly protection against seizure injury to the hippocampus.

Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the pro-apoptotic BH3-only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single-cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feed-forward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress.

Bax regulates neuronal Ca2+ homeostasis.

Excessive Ca2+ entry during glutamate receptor overactivation (“excitotoxicity”) induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca2+ and mitochondrial membrane potential (Δψm) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca2+ transients during the NMDA excitation period and did not exhibit the deregulation of Δψm that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca2+ dynamics during NMDA excitation, suggesting that Bax controlled Ca2+ signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca2+ levels using FRET-based sensors indicated that the effects of bax deficiency on Ca2+ handling were not due to enhanced cellular bioenergetics or increased Ca2+ uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca2+-induced permeability transition. However, when Ca2+ uptake into the sarco/endoplasmic reticulum was blocked with the Ca2+-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca2+ levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca2+ handling is critical for cell death signaling during periods of neuronal overexcitation.

Differential expression patterns of Puma and Hsp70 following proteasomal stress in the hippocampus are key determinants of neuronal vulnerability

J. Neurochem. (2010) 114, 606–616.

Bcl-2 homology domain 3-only proteins Puma and Bim mediate the vulnerability of CA1 hippocampal neurons to proteasome inhibition in vivo

Bcl‐2 homology domain 3 (BH3)‐only proteins are pro‐apoptotic Bcl‐2 family members that play important roles in upstream cell death signalling during apoptosis. Proteasomal stress has been shown to contribute to the pathology of cerebral ischaemia and many neurodegenerative disorders. Here we explored the contribution of BH3‐only proteins in mediating proteasome‐inhibition‐induced apoptosis in the murine brain in vivo. Stereotactic intrahippocampal microinjection of the selective proteasome inhibitor epoxomicin (2.5 nmol) induced a delayed apoptosis within only the CA1 hippocampal neurons and not neurons within the CA3 or dentate gyrus regions, a selective vulnerability similar to that seen during ischaemia. This injury developed over a time‐course of 3 days and was characterized by positive terminal deoxynucleotidyl transferase dUTP nick end labelling staining and nuclear condensation. Previous work from our laboratory has identified the BH3‐only protein p53‐upregulated mediator of apoptosis (Puma) as mediating proteasome‐inhibition‐induced apoptosis in cultured neural cells. Genetic deletion of puma reduced the number of terminal deoxynucleotidyl transferase dUTP nick end labelling‐positive cells within the CA1 following epoxomicin microinjection but it did not provide a complete protection. Subsequent studies identified the BH3‐only protein Bim as also being upregulated during proteasome inhibition in organotypic hippocampal slice cultures and after epoxomicin treatment in vivo. Interestingly, the genetic deletion of bim also afforded significant neuroprotection, although this protection was less pronounced. In summary, we demonstrate that the BH3‐only proteins Puma and Bim mediate the delayed apoptosis of CA1 hippocampal neurons induced by proteasome inhibition in vivo, and that either BH3‐only protein can only partly compensate for the deficiency of the other.

NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: An in vitro model for the study of neural precursor cell proliferation

We present a model for the study of injury-induced neurogenesis in the dentate gyrus (DG) in murine organotypic hippocampal slice cultures (OHCs). A brief exposure of 8-day-old hippocampal slice cultures to the glutamate receptor agonist N-methyl-d-aspartate (NMDA; 20-50µM for 30 min) caused a selective excitotoxic injury in the CA1 subfield of the hippocampus that matured over a period of 24h. The insult resulted in a prominent up-regulation of proliferating nuclei within the OHC dentate gyrus (DG), and a corresponding increase in Ki67/doublecortin double-positive cells in the SGZ of the dentate gyrus. 5-bromo-2-deoxyuridine (BrdU)-labelling of the OHCs for three days subsequent to the NMDA exposure revealed significantly increased BrdU incorporation within the DG (SGZ and GCL) of the hippocampus. Doublecortin immunofluorescence indicated a concurrent up-regulation of neuronal precursor cells specifically in the SGZ and GCL. Significantly increased BrdU incorporation could be detected up to 6-9 days after termination of the NMDA exposure. The model presented here enables easy manipulation and follow-up of injury-induced neuroblast proliferation in the DG that is amenable to the study of transgenic mice.