Ren-Wu Chen

Postinsult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat ischemia/reperfusion model

Lithium has long been a primary drug used to treat bipolar mood disorder, even though the drugs therapeutic mechanisms remain obscure. Recent studies demonstrate that lithium has neuroprotective effects against glutamate-induced excitotoxicity in cultured neurons and in vivo. The present study was undertaken to examine whether postinsult treatment with lithium reduces brain damage induced by cerebral ischemia. We found that s.c. injection of lithium dose dependently (0.5–3 mEq/kg) reduced infarct volume in the rat model of middle cerebral artery occlusion/reperfusion. Infarct volume was reduced at a therapeutic dose of 1 mEq/kg even when administered up to 3 h after the onset of ischemia. Neurological deficits induced by ischemia were also reduced by daily administration of lithium over 1 week. Moreover, lithium treatment decreased the number of neurons showing DNA damage in the ischemic brain. These neuroprotective effects were associated with an up-regulation of cytoprotective heat shock protein 70 (HSP70) in the ischemic brain hemisphere as determined by immunohistochemistry and Western blotting analysis. Lithium-induced HSP70 up-regulation in the ischemic hemisphere was preceded by an increase in the DNA binding activity of heat shock factor 1, which regulates the transcription of HSP70. Physical variables and cerebral blood flow were unchanged by lithium treatment. Our results suggest that postinsult lithium treatment reduces both ischemia-induced brain damage and associated neurological deficits. Moreover, the heat shock response is likely to be involved in lithiums neuroprotective actions. Additionally, our studies indicate that lithium may have clinical utility for the treatment of patients with acute stroke.

Regulation of c-Jun N-terminal kinase, p38 kinase and AP-1 DNA binding in cultured brain neurons: roles in glutamate excitotoxicity and lithium neuroprotection.

In rat cerebellar granule cells, glutamate induced rapid activation of c‐Jun N‐terminal kinase (JNK) and p38 kinase to phosphorylate c‐Jun (at Ser63) and p53 (at Ser15), respectively, and a subsequent marked increase in activator protein‐1 (AP‐1) binding that preceded apoptotic death. These glutamate‐induced effects and apoptosis could largely be prevented by long‐term (7 days) pretreatment with 0.5–2 mm lithium, an antibipolar drug. Glutamates actions could also be prevented by known blockers of this pathway, MK‐801 (an NMDA receptor blocker), SB 203580 (a p38 kinase inhibitor) and curcumin (an AP‐1 binding inhibitor). The concentration‐ and time‐dependent suppression of glutamates effects by lithium and curcumin correlated well with their neuroprotective effects. These results suggest a prominent role of JNK and p38, as well as their downstream AP‐1 binding activation and p53 phosphorylation in mediating glutamate excitotoxicity. Moreover, the neuroprotective effects of lithium are mediated, at least in part, by suppressing NMDA receptor‐mediated activation of the mitogen‐activated protein kinase pathway.

Nuclear Translocation of Glyceraldehyde-3-Phosphate Dehydrogenase Isoforms During Neuronal Apoptosis

Abstract : Treatment with cytosine β‐d‐arabinoside (AraC ; 300 μM) induced a time‐dependent accumulation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase‐3. Moreover, Asp‐Glu‐Val‐Asp‐fluoromethyl ketone (DEVD‐fmk), a CPP32‐selective antagonist, dosedependently suppressed AraC‐induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil‐DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC‐treated cells. The more alkaline isoforms, 1‐3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4‐6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h ; a 4‐h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside‐induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC‐induced neuronal death.

β-Amyloid peptide-induced death of PC 12 cells and cerebellar granule cell neurons is inhibited by long-term lithium treatment

Treatment of rat pheochromocytoma cells (PC 12) cells with beta-amyloid peptide-(1-42) for 24 h induced a concentration-dependent decrease in cellular redox activity in the dose range of 1 to 20 microM. These effects were markedly attenuated by pretreatment with 2 mM LiCl for 7 days, whereas 1-day pretreatment was ineffective. Measurements of live and dead cells by double-staining with fluorescein diacetate and propidium iodide, respectively revealed that protracted lithium pretreatment attenuated PC 12 cell death induced by beta-amyloid-(1-42) and cerebellar granule cell death induced by beta-amyloid-(25-35). Preceding PC 12 cell death, beta-amyloid peptide elicited a slight decrease in protein levels of Bcl-2. Conversely, 7-day pretreatment with lithium resulted in an approximate doubling of Bcl-2 protein levels in cells treated with or without beta-amyloid peptide-(1-42). Lithium-induced Bcl-2 upregulation was temporally associated with the cytoprotective effects of this drug. Thus, lithium protection against beta-amyloid peptide neurotoxicity might involve Bcl-2 overexpression, and lithium treatment for Alzheimers disease should be reexamined.

Valproic acid inhibits histone deacetylase activity and suppresses excitotoxicity-induced GAPDH nuclear accumulation and apoptotic death in neurons.

ABSTRACTValproic acid (VPA), used to treat bipolar mood disorder and seizures, also inhibits histone deacetylase (HDAC). Here, we found that VPA and other HDAC inhibitors, butyrate and trichostatin A, robustly protected mature cerebellar granule cell cultures from excitotoxicity induced by SYM 2081 ((2S, 4R)-4-methylglutamate), an inhibitor of excitatory amino-acid transporters and an agonist of low-affinity kainate receptors. These neuroprotective effects required protracted treatment and were correlated with enhanced acetylated histone levels, indicating HDAC inhibition. SYM-induced excitotoxicity was blocked by MK-801 ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate), supporting that the toxicity was largely N-methyl-D-aspartate receptor dependent. SYM excitotoxicity had apoptotic characteristics and was prevented by a caspase inhibitor. SYM-induced apoptosis was associated with a rapid and robust nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene previously shown to be proapoptotic. VPA pretreatment suppressed SYM 2081-induced GAPDH nuclear accumulation, concurrent with its neuroprotective effects. Chromatin immunoprecipitation (ChIP) revealed that GAPDH is copresent with acetylated histone H3, including Lys9-acetylated histone, and that VPA treatment caused a time-dependent decrease in the levels of nuclear GAPDH with a concomitant increase in acetylated histones in the ChIP complex. Our results strongly suggest that VPA protects neurons from excitotoxicity through inhibition of HDAC activity and that this protective effect may involve suppression of excitotoxicity-induced accumulation of GAPDH protein in the nucleus.

Neuronal apoptosis induced by pharmacological concentrations of 3-hydroxykynurenine: characterization and protection by dantrolene and Bcl-2 overexpression.

Abstract : We have studied neurotoxicity induced by pharmacological concentrations of 3‐hydroxykynurenine (3‐HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT1‐7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose‐dependent manner by 3‐HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3‐HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3‐HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H2O2 was demonstrated in PC12 and GT1‐7 cells treated with 3‐HK, by measuring the increase in the fluorescent product, 2′,7′‐dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3‐HK failed to block 3‐HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3‐HK in these cell types is mediated primarily by H2O2, which is formed most likely by auto‐oxidation of 3‐HK in extracellular compartments. 3‐HK‐induced death of PC12 and GT1‐7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl‐2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl‐2 in GT1‐7 cells elicited by gene transfection suppressed 3‐HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up‐regulation of the level and function of Bcl‐2 protein.

Nuclear factor‐κB‐dependent cyclin D1 induction and DNA replication associated with N‐methyl‐D‐aspartate receptor‐mediated apoptosis in rat striatum

Cell cycle reentry has been found during apoptosis of postmitotic neurons under certain pathological conditions. To evaluate whether nuclear factor‐κB (NF‐κB) activation promotes cell cycle entry and neuronal apoptosis, we studied the relation among NF‐κB‐mediated cyclin induction, bromodeoxyuridine (BrdU) incorporation, and apoptosis initiation in rat striatal neurons following excitotoxic insult. Intrastriatally injected N‐methyl‐D‐aspartate receptor agonist quinolinic acid (QA, 60 nmol) elicited a rise in cyclin D1 mRNA and protein levels (P < 0.05). QA‐induced NF‐κB activation occurred in striatal neurons and nonneuronal cells and partially colocalized with elevated cyclin D1 immunoreactivity and TUNEL‐positive nuclei. QA triggered DNA replication as evidenced by BrdU incorporation; some striatal BrdU‐positive cells were identified as neurons by colocalization with NeuN. Blockade of NF‐κB nuclear translocation with the recombinant peptide NF‐κB SN50 attenuated the QA‐induced elevation in cyclin D1 and BrdU incorporation. QA‐induced internucleosomal DNA fragmentation was blunted by G1/S‐phase cell cycle inhibitors. These findings suggest that NF‐κB activation stimulates cyclin D1 expression and triggers DNA replication in striatal neurons. Excitotoxin‐induced neuronal apoptosis may thus result from, at least partially, a failed cell cycle attempt.