Epigallocatechin-3-gallate (EGCG) is a major flavonoid component of green tea that displays antiapoptotic effects in numerous models of neurotoxicity. Although the intrinsic free radical scavenging activity of EGCG likely contributes to its antiapoptotic effect, other modes of action have also been suggested. We systematically analyzed the antiapoptotic action of EGCG in primary cultures of rat cerebellar granule neurons (CGNs). The dose-dependent protective effects of EGCG were determined after coincubation with eight different stimuli that each induced neuronal apoptosis by distinct mechanisms. Under these conditions, EGCG provided significant neuroprotection only from insults that induce apoptosis by causing mitochondrial oxidative stress. Despite this selective antiapoptotic effect, EGCG did not significantly alter the endogenous activities or expression of Mn(2+)- superoxide dismutase, glutathione peroxidase, Nrf2, or Bcl-2. Subfractionation of CGNs after incubation with (3)H-EGCG revealed that a striking 90-95% of the polyphenol accumulated in the mitochondrial fraction. These data demonstrate that EGCG selectively protects neurons from apoptosis induced by mitochondrial oxidative stress. This effect is likely due to accumulation of EGCG in the mitochondria, where it acts locally as a free radical scavenger. These properties of EGCG make it an interesting therapeutic candidate for neurodegenerative diseases involving neuronal apoptosis triggered by mitochondrial oxidative stress.

Rho family GTPases (eg., RhoA, Rac1 and Cdc42) are monomeric G-proteins that act as key transducers of extracellular signals to the actin cytoskeleton. In the nervous system, Rho family GTPases are essential regulators of neuronal growth cone motility, axonal migration, and dendritic spine morphogenesis. Given these vital functions, it is perhaps not surprising that mutations in several proteins involved in Rho GTPase signaling are causative in some forms of mental retardation. In addition, numerous recent studies have identified Rho family GTPases as central players in the molecular pathways that determine neuronal survival and death. Interestingly, individual Rho family members have been shown to play either a pro-death or pro-survival role in the nervous system depending on both the type of neuron and the particular neurodegenerative insult involved. This review summarizes current work demonstrating a critical role for Rho family GTPases and their effectors in the regulation of neuronal development, survival, and death. These findings may be particularly relevant in the context of specific neurodegenerative disorders in which Rho family GTPase function is altered, such as loss-of-function of the Rac1 guanine nucleotide exchange factor, alsin, in juvenile-onset amyotrophic lateral sclerosis.

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 (RGDdependent) 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.

The adolescent transition from juvenile to adult is marked by anatomical and functional remodeling of brain networks. Currently, the cellular and synaptic level changes underlying the adolescent transition are only coarsely understood. Here, we use two-photon imaging to make time-lapse observations of long-range axons that innervate the frontal cortex in the living brain. We labeled cells in the orbitofrontal cortex (OFC) and basolateral amygdala (BLA) and imaged their axonal afferents to the dorsomedial prefrontal cortex (dmPFC). We also imaged the apical dendrites of dmPFC pyramidal neurons. Images were taken daily in separate cohorts of juvenile (P24–P28) and young adult mice (P64–P68), ages where we have previously discovered differences in dmPFC dependent decision-making. Dendritic spines were pruned across this peri-adolescent period, while BLA and OFC afferents followed alternate developmental trajectories. OFC boutons showed no decrease in density, but did show a decrease in daily bouton gain and loss with age. BLA axons showed an increase in both bouton density and daily bouton gain at the later age, suggesting a delayed window of enhanced plasticity. Our findings reveal projection specific maturation of synaptic structures within a single frontal region and suggest that stabilization is a more general characteristic of maturation than pruning.

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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 proapoptotic 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.

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 GSHsensitive 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.

The critical processes of mitochondrial fission and fusion are regulated by members of the dynamin family of GTPases. Imbalances in mitochondrial fission and fusion contribute to neuronal cell death. For example, increased fission mediated by the dynamin-related GTPase, Drp1, or decreased fusion resulting from inactivating mutations in the OPA1 GTPase, cause neuronal apoptosis and/or neurodegeneration. Recent studies indicate that post-translational processing regulates OPA1 function in non-neuronal cells and moreover, aberrant processing of OPA1 is induced during apoptosis. To date, the post-translational processing of OPA1 during neuronal apoptosis has not been examined. Here, we show that cerebellar granule neurons (CGNs) or neuroblastoma cells exposed to pro-apoptotic stressors display a novel N-terminal cleavage of OPA1 which is blocked by either pan-caspase or caspase-8 selective inhibitors. OPA1 cleavage occurs concurrently with mitochondrial fragmentation and cytochrome c release in CGNs deprived of depolarizing potassium (5K condition). Although a caspase-8 selective inhibitor prevents both 5K-induced OPA1 cleavage and mitochondrial fragmentation, recombinant caspase-8 fails to cleave OPA1 in vitro. In marked contrast, either caspase-8 or caspase-3 stimulates OPA1 cleavage in digitonin-permeabilized rat brain mitochondria, suggesting that OPA1 is cleaved by an intermembrane space protease which is regulated by active caspases. Finally, the N-terminal truncation of OPA1 induced during neuronal apoptosis removes an essential residue (K301) within the GTPase domain. These data are the first to demonstrate OPA1 cleavage during neuronal apoptosis and they implicate caspases as indirect regulators of OPA1 processing in degenerating neurons.