Takahiro Miyawaki

Dysregulation of mTOR signaling in fragile X syndrome.

Fragile X syndrome, the most common form of inherited mental retardation and leading genetic cause of autism, is caused by transcriptional silencing of the Fmr1 gene. The fragile X mental retardation protein (FMRP), the gene product of Fmr1, is an RNA binding protein that negatively regulates translation in neurons. The Fmr1 knock-out mouse, a model of fragile X syndrome, exhibits cognitive deficits and exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression at CA1 synapses. However, the molecular mechanisms that link loss of function of FMRP to aberrant synaptic plasticity remain unclear. The mammalian target of rapamycin (mTOR) signaling cascade controls initiation of cap-dependent translation and is under control of mGluRs. Here we show that mTOR phosphorylation and activity are elevated in hippocampus of juvenile Fmr1 knock-out mice by four functional readouts: (1) association of mTOR with regulatory associated protein of mTOR; (2) mTOR kinase activity; (3) phosphorylation of mTOR downstream targets S6 kinase and 4E-binding protein; and (4) formation of eukaryotic initiation factor complex 4F, a critical first step in cap-dependent translation. Consistent with this, mGluR long-term depression at CA1 synapses of FMRP-deficient mice is exaggerated and rapamycin insensitive. We further show that the p110 subunit of the upstream kinase phosphatidylinositol 3-kinase (PI3K) and its upstream activator PI3K enhancer PIKE, predicted targets of FMRP, are upregulated in knock-out mice. Elevated mTOR signaling may provide a functional link between overactivation of group I mGluRs and aberrant synaptic plasticity in the fragile X mouse, mechanisms relevant to impaired cognition in fragile X syndrome.

Ischemic insults promote epigenetic reprogramming of μ opioid receptor expression in hippocampal neurons

Transient global ischemia is a neuronal insult that induces delayed, selective death of hippocampal CA1 pyramidal neurons. A mechanism underlying ischemia-induced cell death is activation of the gene silencing transcription factor REST (repressor element-1 silencing transcription factor)/NRSF (neuron-restrictive silencing factor) and REST-dependent suppression of the AMPA receptor subunit GluR2 in CA1 neurons destined to die. Here we show that REST regulates an additional gene target, OPRM1 (μ opioid receptor 1 or MOR-1). MORs are abundantly expressed by basket cells and other inhibitory interneurons of CA1. Global ischemia induces a marked decrease in MOR-1 mRNA and protein expression that is specific to the selectively vulnerable area CA1, as assessed by quantitative real-time RT-PCR, Western blotting, and ChIP. We further show that OPRM1 gene silencing is REST-dependent and occurs via epigenetic modifications. Ischemia promotes deacetylation of core histone proteins H3 and H4 and dimethylation of histone H3 at lysine-9 (H3-K9) over the MOR-1 promoter, an signature of epigenetic gene silencing. Acute knockdown of MOR-1 gene expression by administration of antisense oligodeoxynucleotides to hippocampal slices in vitro or injection of the MOR antagonist naloxone to rats in vivo affords protection against ischemia-induced death of CA1 pyramidal neurons. These findings implicate MORs in ischemia-induced death of CA1 pyramidal neurons and document epigenetic remodeling of expression of OPRM1 in CA1 inhibitory interneurons.

Repressor element-1 silencing transcription factor (REST)-dependent epigenetic remodeling is critical to ischemia-induced neuronal death

Dysregulation of the transcriptional repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor is important in a broad range of diseases, including cancer, diabetes, and heart disease. The role of REST-dependent epigenetic modifications in neurodegeneration is less clear. Here, we show that neuronal insults trigger activation of REST and CoREST in a clinically relevant model of ischemic stroke and that REST binds a subset of “transcriptionally responsive” genes (gria2, grin1, chrnb2, nefh, nfκb2, trpv1, chrm4, and syt6), of which the AMPA receptor subunit GluA2 is a top hit. Genes with enriched REST exhibited decreased mRNA and protein. We further show that REST assembles with CoREST, mSin3A, histone deacetylases 1 and 2, histone methyl-transferase G9a, and methyl CpG binding protein 2 at the promoters of target genes, where it orchestrates epigenetic remodeling and gene silencing. RNAi-mediated depletion of REST or administration of dominant-negative REST delivered directly into the hippocampus in vivo prevents epigenetic modifications, restores gene expression, and rescues hippocampal neurons. These findings document a causal role for REST-dependent epigenetic remodeling in the neurodegeneration associated with ischemic stroke and identify unique therapeutic targets for the amelioration of hippocampal injury and cognitive deficits.

Zinc-Dependent Multi-Conductance Channel Activity in Mitochondria Isolated from Ischemic Brain

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear. Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, ΔN-BCL-xL. The findings implicate ΔN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant ΔN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate ΔN-BCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria.

Ischemic preconditioning blocks BAD translocation, Bcl-xL cleavage, and large channel activity in mitochondria of postischemic hippocampal neurons

Transient forebrain or global ischemia induces delayed neuronal death in vulnerable CA1 pyramidal cells with many features of apoptosis. A brief period of ischemia, i.e., ischemic preconditioning, affords robust protection of CA1 neurons against a subsequent more prolonged ischemic challenge. Here we show that preconditioning acts via PI3K/Akt signaling to block the ischemia-induced cascade involving mitochondrial translocation of Bad, assembly of Bad with Bcl-xL, cleavage of Bcl-xL to form its prodeath fragment, ΔN-Bcl-xL, activation of large-conductance channels in the mitochondrial outer membrane, mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), caspase activation, and neuronal death. These findings show how preconditioning acts to prevent the release of cytochrome c and Smac/DIABLO from mitochondria and to preserve the integrity of the mitochondrial membrane. The specific PI3K inhibitor LY294002 administered in vivo 1 h before or immediately after ischemia or up to 120 h later significantly reverses preconditioning-induced protection, indicating a requirement for sustained PI3K signaling in ischemic tolerance. These findings implicate PI3K/Akt signaling in maintenance of the integrity of the mitochondrial outer membrane.

Acute estradiol protects CA1 neurons from ischemia-induced apoptotic cell death via the PI3K/Akt pathway.

Global ischemia arising during cardiac arrest or cardiac surgery causes highly selective, delayed death of hippocampal CA1 neurons. Exogenous estradiol ameliorates global ischemia-induced neuronal death and cognitive impairment in male and female rodents. However, the molecular mechanisms by which a single acute injection of estradiol administered after the ischemic event intervenes in global ischemia-induced apoptotic cell death are unclear. Here we show that acute estradiol acts via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling cascade to protect CA1 neurons in ovariectomized female rats. We demonstrate that global ischemia promotes early activation of glycogen synthase kinase-3beta (GSK3beta) and forkhead transcription factor of the O class (FOXO)3A, known Akt targets that are related to cell survival, and activation of caspase-3. Estradiol prevents ischemia-induced dephosphorylation and activation of GSK3beta and FOXO3A, and the caspase death cascade. These findings support a model whereby estradiol acts by activation of PI3K/Akt signaling to promote neuronal survival in the face of global ischemia.

N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, whether these events have a causal role in ischemia-induced neuronal death is unclear. We found that the Bcl-2 and Bcl-xL inhibitor ABT-737, which enhances death of tumor cells, protected rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-xL is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment, ΔN-Bcl-xL. We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-xL–induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-xL, we generated knock-in mice expressing a caspase-resistant form of Bcl-xL. The knock-in mice exhibited markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings suggest that truncated Bcl-xL could be a potentially important therapeutic target in ischemic brain injury.

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (β-pompilidotoxin) derived from wasp venom

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom, on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 microM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 microM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the presynaptic axons, causing summation of EPSPs. In the presence of 10 microM CNQX and 50 microM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors.

Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils.

Developing animals are known to be resistant to cerebral ischemia. To investigate the mechanisms by which developing animals exhibit ischemic resistance, we examined the changes in intracellular calcium ([Ca2+]i) after oxygen-glucose deprivation (OGD) using hippocampal slices from gerbils. We found that increases of [Ca2+]i in hippocampal CA1 neurons is significantly less after OGD in developing gerbils than in adults. Western blot analysis of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors (AMPARs) showed that GluR2 expression, but not that of the other AMPARs is significantly higher in developing gerbils than in adults. Expression of the anti-apoptotic proteins such as HSP70, Bcl-XL, and plasma membrane Ca2+-ATPase type1 (PMCA1) are not higher in the developing gerbils than in adults. These results suggest that the higher expression of GluR2 is important for the smaller increases in [Ca2+]i and enhanced resistance to ischemia-induced neuronal damage in developing animals.

Differential effects of novel wasp toxin on rat hippocampal interneurons

We studied the effects of a wasp toxin beta-pompilidotoxin (beta-PMTX) on rat hippocampal CA1 interneurons by the current-clamp technique. The firing patterns of pyramidal neurons and pyramidale interneurons were not affected by beta-PMTX, but in oriens and radiatum interneurons, beta-PMTX converted the action potentials to prolonged depolarizing potentials by slowing the inactivation of Na(+) channels. In lacunosum moleculare interneurons, beta-PMTX induced initial bursting spikes followed by block of succeeding spikes. Comparison of beta-PMTX with a sea anemone toxin, ATX II, revealed that ATX II altered the firing properties of pyramidal neurons and pyramidale interneurons that were unchanged by beta-PMTX. Our results suggest that beta-PMTX modulates Na(+) currents in CA1 interneurons differently in various CA1 neurons and the toxin is useful to classify Na(+) channel subtypes.