Yong Ho Che

Tumor Necrosis Factor Induces Bcl-2 and Bcl-x Expression through NFκB Activation in Primary Hippocampal Neurons

Emerging data indicate that tumor necrosis factor (TNF) exerts a neuroprotective effect in response to brain injury. Here we examined the mechanism of TNF in preventing neuronal death in primary hippocampal neurons. TNF protected neurons against hypoxia- or nitric oxide-induced injury, with an increase in the anti-apoptotic proteins Bcl-2 and Bcl-x as determined by Western blot and reverse transcriptase-polymerase chain reaction analysis. Treatment of neurons with an antisense oligonucleotide to bcl-2 mRNA or that to bcl-x mRNA blocked the up-regulation of Bcl-2 or Bcl-x expression, respectively, and partially inhibited the neuroprotective effect induced by TNF. Moreover, adenovirus-mediated overexpression of Bcl-2 significantly inhibited hypoxia- or nitric oxide-induced neuronal death. To examine the possible involvement of a transcription factor, NFκB, in the regulation of Bcl-2 and Bcl-x expression in TNF-treated neurons, an adenoviral vector capable of expressing a mutated form of IκB was used to infect neurons prior to TNF treatment. Expression of the mutant NFκB completely inhibited NFκB DNA binding activity and inhibited both TNF-induced up-regulation of Bcl-2 and Bcl-x expression and neuroprotective effect. These findings indicate that induction of Bcl-2 and Bcl-x expression through NFκB activation is involved in the neuroprotective action of TNF against hypoxia- or nitric oxide-induced injury.

ORP150 protects against hypoxia/ischemia-induced neuronal death

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum–associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3–like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.

Changes in mRNA for post-synaptic density-95 (PSD-95) and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection.

When the axon of motoneurons is transected, the number of synaptic boutons contacting the cell body is decreased, and the recovery of synapses depends on muscle reinnervation. Post-synaptic density-95 (PSD-95) is a protein which is located at the post-synaptic density (PSD) and it plays a pivotal role in regulating synaptic plasticity and synaptogenesis. In addition, PSD-95 binds with neuronal nitric oxide synthase (nNOS), which is competitively inhibited by carboxy-terminal PDZ ligand of nNOS (CAPON) and, thereby, nNOS activity is thought to be regulated by PSD-95 and CAPON. We investigated the changes in mRNA for PSD-95, CAPON and nNOS in the facial motor nucleus of adult rats following axotomy, by in situ hybridization, in combination with the time course of muscle reinnervation, by retrograde tracing and nNOS protein expression, by examining nicotinamide adenine nucleotide phosphate diaphorase (NADPH-d) activity. Signals of mRNA for PSD-95 and CAPON were initially expressed in the facial motoneurons, transiently decreased following axotomy and gradually recovered to the control level. When reinnervation of the axotomized nerve into muscle was observed, mRNA expression of PSD-95 and CAPON started to recover in the facial motoneurons. It was also found that mRNA and protein expression of nNOS started to increase in the axotomized facial motoneurons just prior to the recovery of mRNA expression of PSD-95 and CAPON. These results suggest that PSD-95 and CAPON are involved in synaptogenesis and/or recovery of synaptic function in motoneurons after axotomy.

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re‐establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up‐regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over‐expression of P311 induces p21WAF1/Cip1 expression, leading PC12 cells to differentiate and to have neuron‐like morphologies. Adenovirus‐mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.

Changes in mRNA for choline transporter-like protein following facial nerve transection.

When the axon of a motoneuron is transected, axonal regrowth occurs to reconnect it to the correct target. During the regeneration period, a large amount of new membrane synthesis is required for the axons to extend. Choline is an important metabolite in all cells because of the major contribution of phosphatidylcholine and sphingomyelin to the production of membranes. Therefore, choline uptake is necessary for axonal elongation. We cloned rat choline transporter-like protein 1 (rCTL1) as an upregulated gene in the axotomized facial motor nucleus by differential display polymerase chain reaction using adult rat facial nerve axotomy model. rCTL1 belongs to the choline transporter-like protein family, which takes up choline. We investigated the changes in rCTL1 mRNA levels in the facial motor nucleus of adult rats following axotomy by in situ hybridization. In the facial motoneurons signals of rCTL1 mRNA were rarely expressed, were transiently increased following axotomy and gradually returned to the control level. These results suggest that rCTL1 is involved in activated choline uptake for membrane synthesis in motoneurons following nerve transection.

Changes in mRNA of protein inhibitor of neuronal nitric oxide synthase following facial nerve transection

Protein inhibitor of neuronal nitric oxide synthase (PIN) is reported as the protein inhibiting neuronal nitric oxide synthase (nNOS) activity by preventing dimerization of nNOS. It was also reported that PIN inhibits the activity of all nitric oxide synthase (NOS) isozymes. We examined the effects of facial nerve transection on PIN mRNA and NOS expression by in situ hybridization for PIN mRNA and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining. PIN mRNA was initially expressed and transiently increased from 3 to 5 days and returned to the basal level at 7 days after axotomy in the motoneurons of the facial nucleus. NADPH-d-positive motoneurons were found from 7 days post-operation in the facial nucleus. These results suggest that PIN may interact with NOS from 7 days post-operation.

Changes in mRNA for VAMPs following facial nerve transection

Vesicle-associated membrane proteins (VAMPs) are involved in synaptic vesicle recycling and exocytosis in neurons. Here we report the changes in mRNA expression for VAMPs (VAMP1, -2 and -3) in the facial motor nucleus of adult rats following axotomy by in situ hybridization. Signals for VAMP2 and -3 mRNAs in the facial nucleus were increased from 3 to 28 days after axotomy. On the contrary, VAMP1 mRNA, which was abundantly expressed in the control facial nucleus, was transiently decreased from 3 to 21 days after axotomy. Differential regulation of VAMPs may reflect distinct roles in nerve regeneration.

Involvement of Bcl-2 family and caspase-like protease in NO-mediated neuronal apoptosis

To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1beta with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1beta was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N(G)-monomethyl-L-arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of Bcl-2 and up-regulation of Bax protein levels.

Expression of ORP150 protects neurons from hypoxia-ischemic damage

Department of Anatomy and Neuroscience, Osaka University Medical School lmmunohistochemical analysis which indicate the expression of hemeoxygase-I (HO-l ) m glial cells surrouding the TUNEL positive neurons suggests the role of this stress protein in the regulation of neuronal cell death. Exposure of cultured astrocytes to hypoxia (~02 about IO torr) induced the expression of a 33 kDa stress protein. vvhich was identified as HOI (ECC I. 14.99.3) by Western blot, Northern bot. and parallel induction of this stress protein by, chemical stresses. In consistence with the induction of HO-l in astrocytes by hypoxia, broassay using platelet showed the production of carbon monoxide from cultured astrocytes after reoxygenation. The presence of CO in the medium decelerated the hypoxia-mediated apoptotic type of cell death in cultured cerebellar neurons via lowering the activity of CPP32/Yama, a key enzyme regulating apoptotic type of cell death. Further exposure to CO of hypoxic neurons caused the increase of cellular cGMP level. Consistently, addition of cGMP analogue, 8-bromo cGMP, to the hvpoxic culture. resulted in the maintenance of neuronal cellular viability as well as the suppression of CPP32:Yama activitv. These data describe another pathway through vvhich astrocytes exert neurotrophic function in the setting of ischemic cerebrovascular drseases.