The role of poly(ADP-ribose) polymerase-1 in CNS disease.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that contributes to both neuronal death and survival under stress conditions. PARP-1 is the most abundant of several PARP family members, accounting for more than 85% of nuclear PARP activity, and is present in all nucleated cells of multicellular animals. When activated by DNA damage, PARP-1 consumes nicotinamide adenine dinucleotide (NAD+) to form branched polymers of ADP-ribose on target proteins. This process can have at least three important consequences in the CNS, depending on the cell type and the extent of DNA damage: 1) Poly(ADP-ribose) formation on histones and on enzymes involved in DNA repair can prevent sister chromatid exchange and facilitate base-excision repair; 2) poly(ADP-ribose) formation can influence the action of transcription factors, notably nuclear factor kappaB, and thereby promote inflammation; and 3) extensive PARP-1 activation can promote neuronal death through mechanisms involving NAD+ depletion and release of apoptosis inducing factor from the mitochondria. PARP-1 activation is thereby a key mediator of neuronal death during excitotoxicity, ischemia, and oxidative stress, and PARP-1 gene deletion or pharmacological inhibition can markedly improve neuronal survival in these settings. PARP-1 activation has also been identified in Alzheimer's disease and in experimental allergic encephalitis, but the role of PARP-1 in these disorders remains to be established.

A ligand-receptor pair that triggers a non-apoptotic form of programmed cell death.

Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair-the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK(1)R)-that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)R-transfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.

Glial glycogen stores affect neuronal survival during glucose deprivation in vitro.

Glia perform several energy-dependent functions that may aid neuronal survival under pathological conditions. Glycogen is the major energy reserve in brain, and it is localized almost exclusively to astrocytes. Using murine cortical cell cultures containing both glia and neurons, we examined the effect of altered glial glycogen stores on neuronal survival following glucose deprivation. As previously reported, cultures exposed for several hours to media lacking glucose developed widespread neuronal degeneration without glial degeneration. If glial astrocyte glycogen content was increased to 2-3 times control levels by a 24-h pretreatment with 1 microM insulin or 0.5 mM methionine sulfoximine (MSO), glucose deprivation-induced neuronal degeneration was attenuated. These protective effects were blocked if glycogen levels were reduced back to control levels by a 30-min exposure to 1 mM dibutyryl cyclic AMP or 20 microM norepinephrine prior to glucose deprivation. Astrocyte glycogen stores may be an important factor influencing neuronal survival under conditions of energy substrate limitation.

Acidosis causes failure of astrocyte glutamate uptake during hypoxia.

Failure of glutamate uptake during ischemia can lead to neurotoxic accumulations of glutamate in brain extracellular space. Hypoxia and acidosis are metabolic consequences of ischemia that may individually or in combination impair glutamate uptake. We used primary rat astrocyte cultures to study the effects of acidosis, chemical hypoxia, and the combination of acidosis plus chemical hypoxia on glutamate uptake. Chemical hypoxia alone reduced uptake by 35-45%. Reduction in pH from 7.4 to 5.8 also caused a significant but incomplete inhibition of glutamate uptake, and this effect was more pronounced in medium buffered with CO2/bicarbonate. However, the combination of chemical hypoxia plus acidosis reduced glutamate uptake to below 10% of controls. Astrocyte ATP levels, like glutamate uptake, were significantly reduced by chemical hypoxia and further reduced by the combination of hypoxia plus acidosis. Acidosis under normoxic conditions had no significant effect on astrocyte ATP levels. These results suggest two mechanisms by which acidosis may contribute to failure of astrocyte glutamate uptake during ischemia: Acidosis may act in concert with hypoxia to cause ATP depletion, and acidosis may also have direct effects on glutamate transporters unrelated to effects on cellular ATP levels. pH effects on glutamate uptake may be an important factor affecting neuronal survival during incomplete ischemia.

Glucose can fuel glutamate uptake in ischemic brain.

Astrocytes in culture can maintain glutamate uptake during hypoxia if glucose is available. To determine whether this capacity is shared by brain in situ, extracellular glutamate levels were measured in ischemic brain under conditions of continued glucose delivery. Microdialysis probes were placed bilaterally in caudate nuclei of rats and perfused with artificial cerebrospinal fluid (CSF) containing either 30 or 0 mM glucose. Global cerebral ischemia was induced by cardiac arrest. Dialysate collected from probes not perfused with glucose showed a 50-fold increase in glutamate levels over the 60 min following cardiac arrest. Addition of glucose to the perfusate reduced the glutamate rise to < 20% of the levels attained in the glucose-free probes. The glucose effect was negated by the addition of 0.5 mM of the glutamate uptake blocker threo-beta-hydroxyaspartate to the artificial CSF. These results show that oxygen is not required to maintain efficient uptake of extracellular glutamate in brain and suggest that elevations in extracellular glutamate levels during ischemia result from metabolic perturbations other than hypoxia.

Secobarbital attenuates excitotoxicity but potentiates oxygen-glucose deprivation neuronal injury in cortical cell culture.

We examined the effects of secobarbital and other sedative-hypnotic barbiturates on the neuronal death induced by exposure to excitatory amino acids or deprivation of oxygen or glucose in mouse cortical cell cultures. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate, and kainate toxicities were attenuated in a concentration-dependent fashion by high concentrations of secobarbital or thiopental. Antagonism of NMDA toxicity was not overcome by increasing NMDA concentration and not mimicked by gamma-aminobutyrate. Despite these antiexcitotoxic actions, secobarbital exacerbated the neuronal death induced by deprivation of either glucose alone or oxygen and glucose together; death induced by oxygen deprivation alone was little affected. Thiopental and methohexital also increased oxygen-glucose deprivation injury. A possible explanation for this injury potentiation was provided by the observation that secobarbital enhanced the cellular ATP depletion induced by combined oxygen-glucose deprivation. Deleterious effects on ATP production may counterbalance the protective effects of barbiturates under some conditions.

Effect of dichloroacetate on recovery of brain lactate, phosphorus energy metabolites, and glutamate during reperfusion after complete cerebral ischemia in rats.

The effects of dichloroacetate (DCA) on brain lactate, intracellular pH (pHi), phosphocreatine (PCr), and ATP during 60 min of complete cerebral ischemia and 2 h of reperfusion were investigated in rats by in vivo 1H and 31P magnetic resonance spectroscopy; brain lactate, water content, cations, and amino acids were measured in vitro after reperfusion. DCA, 100 mg/kg, or saline was infused before or immediately after the ischemic period. Preischemic treatment with DCA did not affect brain lactate or pHi during ischemia, but reduced lactate and increased pHi after 30 min of reperfusion (p < 0.05 vs. controls) and facilitated the recovery of PCr and ATP during reperfusion. Postischemic DCA treatment also reduced brain lactate and increased pHi during reperfusion compared with controls (p < 0.05), but had little effect on PCr, ATP, or Pi during reperfusion. After 30 min of reperfusion, serum lactate was 67% lower in the postischemic DCA group than in controls (p < 0.05). The brain lactate level in vitro was 46% lower in the postischemic DCA group than in controls (p < 0.05). DCA did not affect water content or cation concentrations in either group, but it increased brain glutamate by 40% in the preischemic treatment group (p < 0.05). The potential therapeutic effects of DCA on brain injury after complete ischemia may be mediated by reduced excitotoxin release related to decreased lactic acidosis during reperfusion.

A semiautomated method for measuring brain infarct volume.

An accurate, reproducible method for determining the infarct volumes of gray matter structures is presented for use with presently available image analysis systems. Areas of stained sections with optical densities above that of a threshold value are automatically recognized and measured. This eliminates the potential error and bias inherent in manually delineating infarcted regions. Moreover, the volume of surviving normal gray matter is determined rather than that of the infarct. This approach minimizes the error that is introduced by edema, which distorts and enlarges the infarcted tissue and surrounding white matter.

Differing effects of copper,zinc superoxide dismutase overexpression on neurotoxicity elicited by nitric oxide, reactive oxygen species, and excitotoxins.

Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons (p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2'7'-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Overexpression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.

Alcohol exacerbates behavioral and neurochemical effects of rat spinal cord trauma.

Acute alcohol intoxication may exacerbate the consequences of central nervous system trauma, although the mechanism is uncertain. Effects of acute ethanol administration on behavioral and neurochemical changes were examined in rats after traumatic spinal cord injury. Survival rates were reduced and posttraumatic neurologic function worsened in ethanol-treated as compared with saline-treated controls. Ethanol-treated rats had significantly lower tissue levels of excitatory amino acids and higher levels of free fatty acids, thromboxane, and lactic acid than did controls. Tissue magnesium concentration was significantly reduced by trauma and recovered more slowly in ethanol-treated rats. Enhanced phospholipid hydrolysis with free fatty acid and thromboxane accumulation, increased release of excitatory amino acids, and decreased tissue magnesium levels may each serve to worsen secondary tissue damage and diminish neurologic recovery after spinal cord injury associated with acute alcohol intoxication.

Intravenous heparin for acute stroke: what can we learn from the megatrials?

Recently published large clinical trials of heparin and aspirin in acute stroke-the International Stroke Trial, Chinese Acute Stroke Trial, and Trial of ORG 10172 in Acute Stroke Treatment--fail to show a net benefit from heparin. None of these trials used i.v., dose-adjusted, unfractionated heparin as generally employed in the United States. However, the control groups in these trials provide data on acute stroke recurrence in large numbers of patients, and these stroke recurrence rates can be used to establish an upper limit for the potential efficacy of antithrombotic therapy. The rates of recurrent ischemic stroke in the control groups of these trials were low, ranging from 0.6 to 2.2% per week. The low rates of recurrent stroke observed in these groups, coupled with the morbidity and mortality associated with i.v. heparin in this patient population, argue against routine use of i.v. heparin in the acute stroke period.

A microangiopathic syndrome of encephalopathy, hearing loss, and retinal arteriolar occlusions.

A syndrome consisting of a subacute encephalopathy, sensorineural hearing loss, and retinal arteriolar occlusions is described in two women. Laboratory investigations did not reveal any systemic vasculitis. CT and cerebral angiography showed no abnormalities, but magnetic resonance imaging revealed small, discrete lesions in the white matter. Biopsy of cortical brain from one patient showed disseminated microinfarcts in the gray matter as well as sclerosis of small vessels. This syndrome is characterized as an occlusive vasculopathy rather than vasculitis, and should be considered in evaluations of young women presenting with encephalopathy and hearing loss.

NADPH-diaphorase (nitric oxide synthase)-reactive amacrine cells of rabbit retina: putative target cells and stimulation by light.

In the mammalian retina there are two populations of nitric oxide synthase-containing amacrine cells that stain with the nicotinamide adenine dinucleotide phosphate-diaphorase reaction. To determine the response of these neurons to light, immunoreactivity to Fos proteins was used as a marker of synaptic activation. Fos immunoreactivity is absent in dark-adapted retinas, but 70% of large, Type I nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells and 5-10% of the smaller but more numerous Type II nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells contain Fos proteins after light stimulation. To localize putative cellular targets of nitric oxide in the retina, retinas were stained immunocytochemically for cyclic GMP after the local administration of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. Both compounds induce strong cyclic GMP immunoreactivity in ON cone bipolar cells. The data suggest that the light-induced inward current in ON cone bipolar cells is enhanced by a nitric oxide-cyclic GMP pathway and that the major source of nitric oxide is the nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells in the rabbit retina.

Sensory stimulation induces local cerebral glycogenolysis: demonstration by autoradiography.

Brain glycogen stores are localized primarily to glia and undergo continuous utilization and resynthesis. To study the function of glycogen under normal conditions in brain, we developed an autoradiographic method of demonstrating local-glycogen utilization in the awake rat. The method employs labeling of brain glycogen with 14C(3,4)glucose, in situ microwave fixation of brain metabolism, and anhydrous tissue preparation. With this technique, tactile stimulation of the rat face and vibrissae was found to accelerate the utilization of labeled glycogen in brain regions known to receive sensory input from face and vibrissae: the contralateral somatosensory cortex and the ipsilateral trigeminal, sensory and motor nuclei. These findings demonstrate a link between neuronal activity and local glycogen utilization in mammalian brain and suggest that, like other tissues, brain may respond to sudden increases in energy demand in part by rapid glycolytic metabolism of glycogen. As cerebral glycogen is restricted primarily to glia, these observations also support a close coupling of glial energy metabolism with neuronal activity.

Phencyclidine induction of the hsp 70 stress gene in injured pyramidal neurons is mediated via multiple receptors and voltage gated calcium channels.

Non-competitive N-methyl-D-aspartate receptor antagonists, including phencyclidine, ketamine, and MK801, produce vacuoles and induce the hsp 70 stress gene in layer III pyramidal neurons of the rat cingulate cortex. This study shows that phencyclidine (50 mg/kg) induces hsp 70 messenger RNA and HSP70 stress protein primarily in pyramidal neurons in posterior cingulate and retrosplenial cortex, neocortex, insular cortex, piriform cortex, hippocampus, and in the basal nuclei of the amygdala. Several neurotransmitter receptor antagonists inhibited induction of HSP70 produced by phencyclidine (50 mg/kg): haloperidol (ED50 = 0.8 mg/kg), clozapine (ED50 = 1 mg/kg), valium (ED50 = 1 mg/kg), SCH 23390 (ED50 = 7 mg/kg) and muscimol (ED50 = 3 mg/kg). Baclofen had no effect. Nifedipine blocked the induction of HSP70 produced by phencyclidine in some regions (cingulate, neocortex, insular cortex) but only partially blocked HSP70 induction in other regions (piriform cortex, amygdala). These results suggest that phencyclidine injuries pyramidal neurons via dopamine D1, D2, D4, sigma and other receptors. Several factors appear to contribute to this unusual multi-receptor mediated injury. (1) Phencyclidine blocks N-methyl-D-aspartate receptors on GABAergic interneurons resulting in decreased inhibition of pyramidal neurons. This may help to explain why multiple excitatory receptors mediate the injury and why GABAA agonists decrease the injury produced by phencyclidine. (2) Phencyclidine blockade of an amine transporter helps explain why dopamine receptor antagonists ameliorate injury. (3) Phencyclidine depolarizes neurons and produces high, potentially damaging intracellular calcium levels probably by blocking K+ channels that may be linked to sigma receptors. Since nifedipine prevents injury in cingulate, insula, and neocortex, it appears that calcium entry through L-type voltage gated calcium channels plays a role in the pyramidal neuronal injury produced by phencyclidine in these regions. There are similarities between the cingulate neurons injured by phencyclidine and circuits recently hypothesized to explain receptor changes in cingulate gyrus of schizophrenic patients. The present and previous studies also provide approaches for decreasing the clinical side effects of N-methyl-D-aspartate receptor antagonists to facilitate their possible use in the treatment of ischemia and other disorders.

K(+)-induced reversal of astrocyte glutamate uptake is limited by compensatory changes in intracellular Na+.

Glutamate uptake is coupled to counter-transport of K+, and high external K+ concentrations can induce reversal of glutamate uptake in whole-cell patch-clamp and isolated membrane preparations. However, high external K+ causes little or no reversal of glutamate uptake in intact astrocytes, suggesting a regulatory mechanism not evident in membrane preparations. One mechanism by which intact cells could limit the effects of altered extracellular ion concentrations on glutamate transport is by compensatory changes in intracellular Na+ concentrations. This possibility was examined using astrocyte cultures treated in two ways to reduce the driving force for glutamate uptake: incubation in high K+ (with reciprocal reduction in Na+), and incubation with metabolic inhibitors to induce ATP depletion. ATP depletion produced a rise in intracellular Na+, a collapse of the membrane sodium gradient and a massive reversal of glutamate uptake. By contrast, incubation in high K+/low Na+ medium did not significantly alter the sodium gradient and did not induce glutamate uptake reversal. The sodium gradient was shown to be maintained under these conditions by compensatory reductions in intracellular Na+ that approximately matched the reductions in extracellular Na+. These findings suggest a mechanism by which astrocytes may limit reversal of glutamate uptake under high K+/low Na+ conditions, and further suggest a general mechanism by which Na(+)-dependent transport processes could be shielded from fluctuating extracellular ion concentrations.

Metabolic mapping with cellular resolution: c-fos vs. 2-deoxyglucose.

2-Deoxyglucose (2DG) studies have been most useful in mapping activated regions of the nervous system. Cellular localization studies using 2DG have been less rewarding, but results are consistent with current views that increases of 2DG accumulation produced by synaptic activation represent increases in glycolytic glucose metabolism occurring mainly in presynaptic neuronal and possibly glial elements. Immediate early genes (IEGs), including the fos, jun, and NGFI-A families, are induced by a wide variety of intracellular signaling pathways. The nuclear localization of the protein products of these genes and their induction by a variety of stimuli make them useful in metabolic activation studies carried out at the cellular level. IEGs have been induced in neurons by osmotic, bacterial endotoxin, steroids, stress, and other hormonal stimuli; by light, auditory, painful, and other sensory stimuli; during stimulation of motor cortex and other motor behaviors; and by various drugs and toxins that act on a variety of neurotransmitter systems, including dopamine and glutamate. In addition, the localization of c-fos gene expression identifies cells that respond to growth factors in vivo. Retinal Muller cells, the major glial cell type of the retina, demonstrate nuclear Fos immunostaining after the intravitreal injection of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). This observation demonstrates that adult glia can respond to these growth factors in vivo. The investigation of early response gene expression may be particularly useful for elucidating the role of trophic factors in the cellular response to central nervous system injury.

HIV-1 envelope gp120 alters astrocytes in human brain cultures.

The majority of AIDS patients will experience some degree of dementia induced by human immunodeficiency virus (HIV-1). In this study, we report that treatment of human brain tissue with envelope gp120 of HIV-1 did not cause neuronal death but did cause astrocyte alterations and/or death. Human astrocyte cultures showed decreased expression of glial fibrillary acidic protein (GFAP), as well as the diminution of a major protein of 66 kDa. These findings are similar to the in vitro changes observed when astrocytes are exposed to ammonia and in vivo changes observed in experimental hepatic encephalopathy. We hypothesize that AIDS dementia may partially involve a perturbation of astrocyte function by gp120 that could indirectly impair neuronal function.

c-fos proto-oncogene expression in astrocytes associated with differentiation or proliferation but not depolarization.

Expression of the c-fos proto-oncogene in rat neocortical astrocytes in culture was examined using Northern blotting and immunocytochemistry. Marked induction of c-fos mRNA in astrocytes was observed after treatment with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), dibutyryl cyclic AMP (db-cAMP), and phorbol diester (TPA; 12-O-tetra-decanoylphorbol 13-acetate), which are known to induce the proliferation or differentiation of astrocytes. Increase of c-fos protein immunoreactivity (IR) was obtained after treatment with fetal calf serum, EGF, bFGF, db-cAMP and TPA. High concentrations of calcium ionophore A23187, which were lethal to cultured astrocytes, also increased c-fos protein-IR. Treatment with lower concentrations of calcium ionophore (which slightly increase Ca2+ uptake), high K+ and nerve growth factor had no detectable effect on c-fos expression. These results show that depolarization does not induce c-fos in astrocytes and suggest that c-fos may play a role in differentiation and proliferation of astrocytes.

Effects of transient global ischemia and kainate on poly(ADP-ribose) polymerase (PARP) gene expression and proteolytic cleavage in gerbil and rat brains.

Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg/kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.