Doug Lobner

Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?

Neuronal apoptosis induced in cortical cultures by exposure to serum deprivation, staurosporine, nifedipine, or C2-ceramide was assayed by lactate dehydrogenase (LDH) release or inhibition of 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction. The protective effects of neurotrophin-4, Z-Val-Ala-Asp-fluoromethylketone (ZVAD), and cycloheximide against each insult were also assayed. The level of injury for each insult was similar whether determined by LDH release or inhibition of MTT reduction, but effects of anti-apoptotic agents were assay dependent. ZVAD and cycloheximide protected neurons from nifedipine-induced death, when assayed by LDH release, but not MTT reduction. In contrast, only cycloheximide attenuated C2-ceramide-induced LDH release, while ZVAD and cycloheximide actually enhanced the C2-ceramide induced inhibition of MTT reduction. Counting of trypan blue positive cells provided results consistent with values obtained using the LDH assay. These results indicate that both LDH release and MTT reduction accurately determine apoptotic death of neurons. However, the MTT assay does not always correctly quantify neuroprotective effects, this likely reflects differences in the point of the death pathway that the neuroprotective agents act. Therefore, while the MTT assay is of limited value in assessing the efficacy of neuroprotective strategies, it may provide information regarding whether specific anti-apoptotic agents act up or downstream of mitochondrial dysfunction.

Repeated N-Acetylcysteine Administration Alters Plasticity-Dependent Effects of Cocaine

Cocaine produces a persistent reduction in cystine–glutamate exchange via system xc− in the nucleus accumbens that may contribute to pathological glutamate signaling linked to addiction. System xc− influences glutamate neurotransmission by maintaining basal, extracellular glutamate in the nucleus accumbens, which, in turn, shapes synaptic activity by stimulating group II metabotropic glutamate autoreceptors. In the present study, we tested the hypothesis that a long-term reduction in system xc− activity is part of the plasticity produced by repeated cocaine that results in the establishment of compulsive drug seeking. To test this, the cysteine prodrug N-acetylcysteine was administered before daily cocaine to determine the impact of increased cystine–glutamate exchange on the development of plasticity-dependent cocaine seeking. Although N-acetylcysteine administered before cocaine did not alter the acute effects of cocaine on self-administration or locomotor activity, it prevented behaviors produced by repeated cocaine including escalation of drug intake, behavioral sensitization, and cocaine-primed reinstatement. Because sensitization or reinstatement was not evident even 2–3 weeks after the last injection of N-acetylcysteine, we examined whether N-acetylcysteine administered before daily cocaine also prevented the persistent reduction in system xc− activity produced by repeated cocaine. Interestingly, N-acetylcysteine pretreatment prevented cocaine-induced changes in [35S]cystine transport via system xc−, basal glutamate, and cocaine-evoked glutamate in the nucleus accumbens when assessed at least 3 weeks after the last N-acetylcysteine pretreatment. These findings indicate that N-acetylcysteine selectively alters plasticity-dependent behaviors and that normal system xc− activity prevents pathological changes in extracellular glutamate that may be necessary for compulsive drug seeking.

β-N-methylamino-l-alanine induces oxidative stress and glutamate release through action on system Xc−

beta-N-methylamino-l-alanine (BMAA) is a non-protein amino acid implicated in the neurodegenerative disease amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC) on Guam. BMAA has recently been discovered in the brains of Alzheimers patients in Canada and is produced by various species of cyanobacteria around the world. These findings suggest the possibility that BMAA may be of concern not only for specific groups of Pacific Islanders, but for a much larger population. Previous studies have indicated that BMAA can act as an excitotoxin by acting on the NMDA receptor. We have shown that the mechanism of neurotoxicity is actually three-fold; it involves not only direct action on the NMDA receptor, but also activation of metabotropic glutamate receptor 5 (mGluR5) and induction of oxidative stress. We now explore the mechanism by which BMAA activates the mGluR5 receptor and induces oxidative stress. We found that BMAA inhibits the cystine/glutamate antiporter (system Xc(-)) mediated cystine uptake, which in turn leads to glutathione depletion and increased oxidative stress. BMAA also appears to drive glutamate release via system Xc(-) and this glutamate induces toxicity through activation of the mGluR5 receptor. Therefore, the oxidative stress and mGluR5 activation induced by BMAA are both mediated through action at system Xc(-). The multiple mechanisms of BMAA toxicity, particularly the depletion of glutathione and enhanced oxidative stress, may account for its ability to induce complex neurodegenerative diseases.

System xc− Activity and Astrocytes Are Necessary for Interleukin-1β-Mediated Hypoxic Neuronal Injury

The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1β (IL-1β) contributes to the pathogenesis of hypoxic–ischemic brain damage. In vivo, IL-1 receptor type I (IL-1RI)-deficient mice showed smaller infarcts and less neurological deficits than wild-type animals after a 90 min reversible middle cerebral artery occlusion. In vitro, IL-1β mediated an enhancement of hypoxic neuronal injury in murine cortical cultures that was lacking in cultures derived from IL-1RI null mutant animals and was blocked by the IL-1 receptor antagonist or an IL-1RI blocking antibody. This IL-1β-mediated potentiation of hypoxic neuronal injury was associated with an increase in both cellular cystine uptake ([cystine]i) and extracellular glutamate levels ([glutamate]e) and was prevented by either ionotropic glutamate receptor antagonism or removal of l-cystine, suggesting a role for the cystine/glutamate antiporter (System xc−). Indeed, dual System xc−/metabotropic glutamate receptor subunit 1 (mGluR1) antagonism but not selective mGluR1 antagonism prevented neuronal injury. Additionally, cultures derived from mGluR1-deficient mice exhibited the same potentiation in injury after treatment with IL-1β as wild-type cultures, an effect prevented by System xc−/mGluR1 antagonism. Finally, assessment of System xc− function and kinetics in IL-1β-treated cultures revealed an increase in velocity of cystine transport (Vmax), in the absence of a change in affinity (Km). Neither the enhancement in [cystine]i, [glutamate]e, or neuronal injury were observed in chimeric cultures consisting of IL-1RI+/+ neurons plated on top of IL-1RI−/− astrocytes, highlighting the importance of astrocyte-mediated alterations in System xc− as a novel contributor to the development and progression of hypoxic neuronal injury.

BDNF or IGF-I potentiates free radical-mediated injury in cortical cell cultures

Free radical-mediated damage to cultured cortical neurons was induced by a 24 h exposure to Fe2+ (30 µM) or an inhibitor of γ-glutamylcysteine synthetase, l-buthionine-[S,R]-sulfoximine (BSO, 1 mM). As expected, neuronal death was blocked by inclusion of the free radical scavenger trolox during the Fe2+ or BSO exposure. However, unexpectedly, pretreatment of the cultures with BDNF or IGF-I markedly potentiated neuronal death. This growth factor-potentiated death was still blocked by trolox, but was insensitive to glutamate antagonists. Concurrent addition of cycloheximide with the growth factors prevented injury potentiation. Present findings suggest that growth factors may increase free radical-induced neuronal death by mechanisms dependent upon protein synthesis.

In Vitro Neurotoxic Evaluation of Root-end–filling Materials

Root-end-filling materials have been tested for toxicity on several cell types, but their toxicity has not been tested on neurons. In this study we evaluated the neurotoxicity in murine cerebral cortical cell cultures of four commonly used root-end-filling materials: mineral trioxide aggregate, amalgam, Super EBA, and Diaket. Standardized amounts of each material were placed on culture-well inserts, allowing the material to be exposed to the culture bathing media without causing physical disruption of the cells. Cell death was quantified by assaying release of the cytosolic enzyme lactate dehydrogenase. Exposure of cortical cultures to freshly mixed or 7-day-old MTA did not cause significant neuronal death, whereas exposure to freshly mixed or 7-day-old amalgam, Super EBA, and Diaket resulted in significant neuronal death (p < .05). Thus, each material, except for mineral trioxide aggregate, can induce neurotoxicity, even when allowed to set thoroughly.

Synergistic toxicity of the environmental neurotoxins methylmercury and β-N-methylamino-L-alanine.

Determination of the environmental factors involved in neurodegenerative diseases has been elusive. Methylmercury and &bgr;-N-methylamino-L-alanine (BMAA) have both been implicated in this role. Exposure of primary cortical cultures to these compounds independently induced concentration-dependent neurotoxicity. Importantly, concentrations of BMAA (10–100 &mgr;M) that caused no toxicity alone potentiated methylmercury (3 &mgr;M) toxicity. In addition, concentrations of BMAA and methylmercury that had no effect by themselves on the main cellular antioxidant glutathione together decreased glutathione levels. Furthermore, the combined toxicity of methylmercury and BMAA was attenuated by the cell permeant form of glutathione, glutathione monoethyl ester. The results indicate a synergistic toxic effect of the environmental neurotoxins BMAA and methylmercury, and that the interaction is at the level of glutathione depletion.

Mechanisms of β-N-methylamino-L-alanine induced neurotoxicity

Abstract Since the initial discovery that the amino acid β-N-methylamino-L-alanine (BMAA) was a neurotoxin, a great deal has been learned about its mechanism of action. However, exactly how it causes death of motor neurons, and how its actions may interact with other neurotoxins or pathological conditions, is not well understood. The focus of study on the mechanism of BMAA toxicity has been on its action as a glutamate receptor agonist. There is evidence that BMAA has effects on all of the main types of glutamate receptors: NMDA, AMPA/kainate, and metabotropic receptors. However, recent results suggest that BMAA may also act through other mechanisms to induce neuronal death. One such action is on the cystine/glutamate antiporter (system xc-). Through its effect of system xc-, BMAA can induce oxidative stress and increase extracellular glutamate. This action of BMAA provides an attractive mechanism for the multiple neurological deficits that BMAA has been implicated in inducing.

Drug-Induced Plasticity Contributing to Heightened Relapse Susceptibility: Neurochemical Changes and Augmented Reinstatement in High-Intake Rats

A key in understanding the neurobiology of addiction and developing effective pharmacotherapies is revealing drug-induced plasticity that results in heightened relapse susceptibility. Previous studies have demonstrated that increased extracellular glutamate, but not dopamine, in the nucleus accumbens core (NAcc) is necessary for cocaine-induced reinstatement. In this report, we examined whether drug-induced adaptations that are necessary to generate cocaine-induced reinstatement also determine relapse vulnerability. To do this, rats were assigned to self-administer cocaine under conditions resulting in low (2 h/d; 0.5 mg/kg/infusion, i.v.) or high (6 h/d; 1.0 mg/kg/infusion, i.v.) levels of drug intake since these manipulations produce groups of rats exhibiting differences in the magnitude of cocaine-induced reinstatement. Approximately 19 d after the last session, cocaine-induced drug seeking and extracellular levels of glutamate and dopamine in the NAcc were measured. Contrary to our hypothesis, high-intake rats exhibited a more robust cocaine-induced increase in extracellular levels of dopamine but not glutamate. Further, increased reinstatement in high-intake rats was no longer observed when the D1 receptor antagonist SCH-23390 was infused into the NAcc. The sensitized dopamine response to cocaine in high-intake rats may involve blunted cystine–glutamate exchange by system xc−. Reduced 14C-cystine uptake through system xc− was evident in NAcc tissue slices obtained from high-intake rats, and the augmented dopamine response in these rats was no longer observed when subjects received the cysteine prodrug N-acetyl cysteine. These data reveal a role for drug-induced NAcc dopamine in heightened relapse vulnerability observed in rats with a history of high levels of drug intake.

Neurotrophic Factor Effects on Oxidative Stress-Induced Neuronal Death

Neurotrophic factors have been shown to potentiate necrotic neuronal death in cortical cultures. In this study we characterized the death induced by various oxidative insults and tested the effects of neurotrophic factors on that death. Treatment with fibroblast growth factor-2, neurotrophin-4, or insulin-like growth factor-1 potentiated neuronal cell death induced by iron-citrate (Fe) or buthionine sulfoximine (BSO), but not ethacrynic acid (EA). Neuronal death induced by each insult was blocked by the free radical scavenger, trolox. An analysis of the death indicated that Fe and BSO induced necrotic cell death, while EA induced apoptotic cell death. BSO and EA caused decreased cellular glutathione levels, whereas Fe had no effect on glutathione levels. Neurotrophic factors had no effect on the changes in glutathione. The results indicate that oxidative insults can induce either apoptotic or necrotic death and that the effects of neurotrophic factors are dependent on the type of cell death.