Ron J. Bouchard

Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis.

Glycogen synthase kinase-3β (GSK-3β) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3β that ultimately induce neuronal death are unknown. Here, we show that GSK-3β phosphorylates and regulates the activity of Bax, a pro-apoptotic Bcl-2 family member that stimulates the intrinsic (mitochondrial) death pathway by eliciting cytochrome c release from mitochondria. In cerebellar granule neurons undergoing apoptosis, inhibition of GSK-3β suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Baxα fusion protein and the conformational activation of endogenous Bax. GSK-3β directly phosphorylated Baxα on Ser163, a residue found within a species-conserved, putative GSK-3β phosphorylation motif. Coexpression of GFP-Baxα with a constitutively active mutant of GSK-3β, GSK-3β(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Baxα, but not a Ser163Ala mutant of Baxα, in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3β promoted the localization of Baxα to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Baxα nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Baxσ) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3β. In a similar manner, either mutation or deletion of the identified GSK-3β phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3β exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

Glutathione Binding to the Bcl-2 Homology-3 Domain Groove: A MOLECULAR BASIS FOR BCL-2 ANTIOXIDANT FUNCTION AT MITOCHONDRIA*

Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4). Given the opposing effects of Bcl-2 and Bax/BH3-only proteins on the redox state of mitochondria, we hypothesized that the antioxidant function of Bcl-2 is antagonized by its interaction with the BH3 domains of pro-apoptotic family members. Here, we show that BH3 mimetics that bind to a hydrophobic surface (the BH3 groove) of Bcl-2 induce GSH-sensitive mitochondrial dysfunction and apoptosis in cerebellar granule neurons. BH3 mimetics displace a discrete mitochondrial GSH pool in neurons and suppress GSH transport into isolated rat brain mitochondria. Moreover, BH3 mimetics and the BH3-only protein, Bim, inhibit a novel interaction between Bcl-2 and GSH in vitro. These results suggest that Bcl-2 regulates an essential pool of mitochondrial GSH and that this regulation may depend upon Bcl-2 directly interacting with GSH via the BH3 groove. We conclude that this novel GSH binding property of Bcl-2 likely plays a central role in its antioxidant function at mitochondria.

Inactivation of the Myocyte Enhancer Factor-2 Repressor Histone Deacetylase-5 by Endogenous Ca2//Calmodulin-dependent Kinase II Promotes Depolarization-mediated Cerebellar Granule Neuron Survival

Cerebellar granule neuron (CGN) survival depends on activity of the myocyte enhancer factor-2 (MEF2) transcription factors. Neuronal MEF2 activity is regulated by depolarization via a mechanism that is presently unclear. Here, we show that depolarization-mediated MEF2 activity and CGN survival are compromised by overexpression of the MEF2 repressor histone deacetylase-5 (HDAC5). Furthermore, removal of depolarization induced rapid cytoplasm-to-nuclear translocation of endogenous HDAC5. This effect was mimicked by addition of the calcium/calmodulin-dependent kinase (CaMK) inhibitor KN93 to depolarizing medium. Removal of depolarization or KN93 addition resulted in dephosphorylation of HDAC5 and its co-precipitation with MEF2D. HDAC5 nuclear translocation triggered by KN93 induced a marked loss of MEF2 activity and subsequent apoptosis. To selectively decrease CaMKII, CGNs were incubated with an antisense oligonucleotide to CaMKIIα. This antisense decreased CaMKIIα expression and induced nuclear shuttling of HDAC5 in CGNs maintained in depolarizing medium. Selectivity of the CaMKIIα antisense was demonstrated by its lack of effect on CaMKIV-mediated CREB phosphorylation. Finally, antisense to CaMKIIα induced caspase-3 activation and apoptosis, whereas a missense control oligonucleotide had no effect on CGN survival. These results indicate that depolarization-mediated calcium influx acts through CaMKII to inhibit HDAC5, thereby sustaining high MEF2 activity in CGNs maintained under depolarizing conditions.

The p75 Neurotrophin Receptor Can Induce Autophagy and Death of Cerebellar Purkinje Neurons

The cellular mechanisms underlying Purkinje neuron death in various neurodegenerative disorders of the cerebellum are poorly understood. Here we investigate an in vitro model of cerebellar neuronal death. We report that cerebellar Purkinje neurons, deprived of trophic factors, die by a form of programmed cell death distinct from the apoptotic death of neighboring granule neurons. Purkinje neuron death was characterized by excessive autophagic-lysosomal vacuolation. Autophagy and death of Purkinje neurons were inhibited by nerve growth factor (NGF) and were activated by NGF-neutralizing antibodies. Although treatment with antisense oligonucleotides to the p75 neurotrophin receptor (p75ntr) decreased basal survival of cultured cerebellar neurons, p75ntr-antisense decreased autophagy and completely inhibited death of Purkinje neurons induced by trophic factor withdrawal. Moreover, adenoviral expression of a p75ntr mutant lacking the ligand-binding domain induced vacuolation and death of Purkinje neurons. These results suggest that p75ntr is required for Purkinje neuron survival in the presence of trophic support; however, during trophic factor withdrawal, p75ntr contributes to Purkinje neuron autophagy and death. The autophagic morphology resembles that found in neurodegenerative disorders, suggesting a potential role for this pathway in neurological disease.

The pesticide rotenone induces caspase‐3‐mediated apoptosis in ventral mesencephalic dopaminergic neurons

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian‐like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone‐induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase‐3 in TH+ neurons. The caspase‐3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone‐induced death. Furthermore, this protective effect lasted for at least 32 h post‐rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase‐3‐mediated apoptosis. Understanding the mechanism of rotenone‐induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinsons disease.

Caspase Inhibition Protects against Reovirus-Induced Myocardial Injury In Vitro and In Vivo

ABSTRACT Viral myocarditis is a disease with a high morbidity and mortality. The pathogenesis of this disease remains poorly characterized, with components of both direct virus-mediated and secondary inflammatory and immune responses contributing to disease. Apoptosis has increasingly been viewed as an important mechanism of myocardial injury in noninfectious models of cardiac disease, including ischemia and failure. Using a reovirus murine model of viral myocarditis, we characterized and targeted apoptosis as a key mechanism of virus-associated myocardial injury in vitro and in vivo. We demonstrated caspase-3 activation, in conjunction with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and annexin binding, in cardiac myocytes after myocarditic viral infection in vitro. We also demonstrated a tight temporal and geographical correlation between caspase-3 activation, histologic injury, and viral load in cardiac tissue after myocarditic viral infection in vivo. Two pharmacologic agents that broadly inhibit caspase activity, Q-VD-OPH and Z-VAD(OMe)-FMK, effectively inhibited virus-induced cellular death in vitro. The inhibition of caspase activity in vivo by the use of pharmacologic agents as well as genetic manipulation reduced virus-induced myocardial injury by 40 to 60% and dramatically improved survival in infected caspase-3-deficient animals. This study indicates that apoptosis plays a critical role in mediating cardiac injury in the setting of viral myocarditis and is the first demonstration that caspase inhibition may serve as a novel therapeutic strategy for this devastating disease.

Endoplasmic reticulum stress and trophic factor withdrawal activate distinct signaling cascades that induce glycogen synthase kinase-3β and a caspase-9-dependent apoptosis in cerebellar granule neurons

Abstract Loss of trophic or activity-dependent survival signals is commonly recognized as a stimulus for neuronal apoptosis and may play a significant role in neurodegeneration. Recent data have also implicated endoplasmic reticulum (ER) stress as an important factor in some neurodegenerative conditions. However, whether shared or unique apoptotic cascades are activated by trophic factor withdrawal (TFW) versus ER stress in primary neurons has not previously been investigated. In primary cultures of rat cerebellar granule neurons (CGNs), the ER stressor brefeldin A activated a discrete pathway involving the following: (1) stimulation of the ER resident kinase PERK, (2) enhanced phosphorylation of the translation initiation factor eIF2α, and (3) increased expression and nuclear localization of the transcription factor Gadd153/CHOP. ER stress-induced CGN apoptosis was blocked by an antagonist of IP3 receptor-mediated Ca2+ release, 2-aminoethoxydiphenyl borate (2-APB), and by expression of ER-targeted Bcl-2. In contrast, CGN apoptosis elicited by TFW (i.e., removal of serum and depolarizing extracellular potassium) did not display any ER stress component nor was it blocked by either 2-APB or ER-Bcl-2. Despite these apparent differences, both brefeldin A and TFW induced dephosphorylation (activation) of glycogen synthase kinase-3β (GSK-3β). Moreover, inhibitors of GSK-3β (IGF-I, lithium) and caspase-9 (LEHD-fmk) significantly protected CGNs from apoptosis induced by either ER stress or TFW. These data indicate that ER stress and TFW elicit distinct signals that activate GSK-3β and intrinsic apoptosis in neurons.

CREB Downregulation in Vascular Disease. A Common Response to Cardiovascular Risk

Objective—To examine the impact of low-density lipoprotein (LDL), an established mediator of atherosclerosis, on the transcription factor cAMP-response element-binding protein (CREB), which is a regulator of vascular smooth muscle cell (VSMC) quiescence. Methods and Results—VSMC CREB content is diminished in rodent models of diabetes and pulmonary hypertension. We examined aortic CREB content in rodent models of aging, hypertension, and insulin resistance, and we determined nuclear CREB protein in the medial VSMC of high-fat-fed LDL receptor-null mice. There was significant loss of CREB protein in all models. In vitro, primary culture rat aortic VSMC exposed to LDL and oxidized LDL exhibited a rapid, transient increase in CREB phosphorylation and transient phosphorylation/activation of Akt, ERK, JNK, ans p38 MAPK. Exposure to oxidized LDL, but not to LDL, for 24 to 48 hours decreased CREB protein in a dose-dependent fashion and led to nuclear exclusion of CREB. Pharmacological reactive oxygen species scavengers and inhibition of ERK activation blocked oxidized LDL-mediated CREB downregulation. Conclusion—These data support a model wherein loss of VSMC CREB protein, which renders these cells more susceptible to activation and apoptosis, is a common pathological response to vascular injury and potentially contributes to plaque progression.

Inhibition of Rac GTPase triggers a c‐Jun‐ and Bim‐dependent mitochondrial apoptotic cascade in cerebellar granule neurons

Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin‐mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro‐survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP‐loading of Rac1 was maintained in CGNs by integrin‐mediated (RGD‐dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase‐mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant–negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase‐9 and ‐3, whereas dominant–negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c‐Jun activation, and induction of the BH3‐only protein Bim. Similarly, c‐Jun activation and Bim induction were observed with N17Rac1. A c‐jun N‐terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK‐specific inhibitor, SP600125, significantly decreased ToxB‐induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c‐Jun/Bim‐mediated mitochondrial apoptosis.

A myocyte enhancer factor 2D (MEF2D) kinase activated during neuronal apoptosis is a novel target inhibited by lithium

Depolarization promotes the survival of cerebellar granule neurons via activation of the transcription factor myocyte enhancer factor 2D (MEF2D). Removal of depolarization induces hyperphosphorylation of MEF2D on serine/threonine residues, resulting in its decreased DNA binding and susceptibility to caspases. The subsequent loss of MEF2-dependent gene transcription contributes to the apoptosis of granule neurons. The kinase(s) that phosphorylates MEF2D during apoptosis is currently unknown. The serine/threonine kinase, glycogen synthase kinase-3 beta (GSK-3 beta), plays a pro-apoptotic role in granule neurons. To investigate a potential role for GSK-3 beta in MEF2D phosphorylation, we examined the effects of lithium, a non-competitive inhibitor of GSK-3 beta, on MEF2D activity in cultured cerebellar granule neurons. Lithium inhibited caspase-3 activation and chromatin condensation in granule neurons induced to undergo apoptosis by removal of depolarizing potassium and serum. Concurrently, lithium suppressed the hyperphosphorylation and caspase-mediated degradation of MEF2D. Moreover, lithium sustained MEF2 DNA binding and transcriptional activity in the absence of depolarization. Lithium also attenuated MEF2D hyperphosphorylation and apoptosis induced by calcineurin inhibition under depolarizing conditions, a GSK-3 beta-independent model of neuronal death. In contrast to lithium, MEF2D hyperphosphorylation was not inhibited by forskolin, insulin-like growth factor-I, or valproate, three mechanistically distinct inhibitors of GSK-3 beta. These results demonstrate that the kinase that phosphorylates and inhibits the pro-survival function of MEF2D in cerebellar granule neurons is a novel lithium target distinct from GSK-3 beta.