Rho family GTPase inhibition reveals opposing effects of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and Janus kinase/signal transducer and activator of transcription signaling cascades on neuronal survival.

Rho family GTPases promote the survival of certain neuronal populations. However, pro-survival and pro-death signaling pathways regulated downstream of Rho GTPases are largely unknown. Cerebellar granule neurons (CGNs) exposed to Clostridium difficile toxin B (ToxB), a monoglucosyltransferase that specifically inhibits Rho GTPases, die by a mitochondrial apoptotic cascade. Using a high-throughput immunoblotting screen (BD Powerblot), we found that ToxB markedly reduced the expression of Rac1 and c-Raf, upstream components of a Rac-dependent mitogen-activated protein (MAP) kinase pathway. Moreover, ToxB rapidly suppressed a p21-activated kinase/MAP kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling cascade that normally promotes degradation of the Bcl-2 homology-3 (BH3)-only protein Bim, a key initiator of mitochondrial apoptosis. In contrast to c-Raf down-regulation, ToxB enhanced expression of the transcription factor, signal transducer and activator of transcription-1 (STAT1). Both STAT1 up-regulation and apoptosis induced by ToxB were prevented by a pan-inhibitor of Janus kinases (JAKs), indicating that JAK/STAT signaling was pro-apoptotic in CGNs. Most significantly, direct inhibition of MEK was sufficient to trigger JAK-dependent STAT1 expression, suggesting that cross-talk between MEK/ERK and JAK/STAT pathways plays a key role in regulating neuronal survival. Finally, ERK dephosphorylation and STAT1 up-regulation induced by ToxB were mimicked by a dominant-negative (N17) mutant of Rac1. These data suggest that the MEK/ERK cascade functions downstream of Rac GTPase to actively repress pro-apoptotic JAK/STAT signaling in healthy CGNs.

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.

Distinct mechanisms of neuronal apoptosis are triggered by antagonism of Bcl-2/Bcl-x(L) versus induction of the BH3-only protein Bim.

Primary cerebellar granule neurons (CGNs) require depolarizing extracellular potassium for their survival. Removal of depolarizing potassium triggers CGN apoptosis that requires induction of Bim, a BH3-only Bcl-2 family member. Bim is classically thought to promote apoptosis by neutralizing pro-survival Bcl-2 proteins. To determine if this is the principal function of Bim in CGNs, we contrasted Bim-mediated apoptosis to neuronal death induced by HA14-1, a BH3-domain mimetic that antagonizes Bcl-2 and Bcl-x(L). HA14-1 elicited CGN apoptosis characterized by caspase 3 and 9 activation, cytochrome c release, conformational activation of Bax, and mitochondrial depolarization. HA14-1 provoked CGN apoptosis in the absence of Bim induction and negative regulators of Bim transcription did not prevent HA14-1-induced cell death. However, the antioxidant glutathione and its precursor, N-acetyl-l-cysteine, suppressed HA14-1-induced apoptosis. Similarly, apoptosis induced by either a structurally distinct Bcl-2/Bcl-x(L) inhibitor (compound 6) or Bcl-2 antisense oligonucleotides was diminished by glutathione. In contrast, antioxidants had no effect on CGN apoptosis provoked by either removal of depolarizing potassium or overexpression of a GFP-Bim fusion protein, two models of Bim-dependent death. These data show that antagonism of Bcl-2/Bcl-x(L) function elicits oxidative stress-dependent CGN apoptosis that is mechanistically distinct from Bim-mediated cell death. These results further indicate that, although Bcl-2/Bcl-x(L) antagonism is sufficient to induce neuronal apoptosis, Bim likely promotes neuronal death by interacting with additional proteins besides Bcl-2/Bcl-x(L).

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

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

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.

Inactivation of the myocyte enhancer factor-2 repressor histone deacetylase-5 by endogenous Ca(2+) //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 CaMKIIalpha. This antisense decreased CaMKIIalpha expression and induced nuclear shuttling of HDAC5 in CGNs maintained in depolarizing medium. Selectivity of the CaMKIIalpha antisense was demonstrated by its lack of effect on CaMKIV-mediated CREB phosphorylation. Finally, antisense to CaMKIIalpha 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.

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.

Insulin-like growth factor-I blocks Bcl-2 interacting mediator of cell death (Bim) induction and intrinsic death signaling in cerebellar granule neurons.

Cerebellar granule neurons depend on insulin-like growth factor-I (IGF-I) for their survival. However, the mechanism underlying the neuroprotective effects of IGF-I is presently unclear. Here we show that IGF-I protects granule neurons by suppressing key elements of the intrinsic (mitochondrial) death pathway. IGF-I blocked activation of the executioner caspase-3 and the intrinsic initiator caspase-9 in primary cerebellar granule neurons deprived of serum and depolarizing potassium. IGF-I inhibited cytochrome c release from mitochondria and prevented its redistribution to neuronal processes. The effects of IGF-I on cytochrome c release were not mediated by blockade of the mitochondrial permeability transition pore, because IGF-I failed to inhibit mitochondrial swelling or depolarization. In contrast, IGF-I blocked induction of the BH3-only Bcl-2 family member, Bim (Bcl-2 interacting mediator of cell death), a mediator of Bax-dependent cytochrome c release. The suppression of Bim expression by IGF-I did not involve inhibition of the c-Jun transcription factor. Instead, IGF-I prevented activation of the forkhead family member, FKHRL1, another transcriptional regulator of Bim. Finally, adenoviral-mediated expression of dominant-negative AKT activated FKHRL1 and induced expression of Bim. These data suggest that IGF-I signaling via AKT promotes survival of cerebellar granule neurons by blocking the FKHRL1-dependent transcription of Bim, a principal effector of the intrinsic death-signaling cascade.