Combinatorial Preconditioning of Rat Brain Cultures with Subprotective Ethanol and Resveratrol Concentrations Promotes Synergistic Neuroprotection.

Preconditioning brain cultures with moderate concentrations of ethanol (EtOH) or trans-resveratrol (RES), key red wine constituents, can prevent amyloid-ß (Aß) neurotoxicity. Past studies have indicated that moderate EtOH activates synaptic N-methyl-D-aspartate receptors (NMDAR) that, in part, signal via protein kinase C (PKC) to increase protective antioxidant proteins such as peroxiredoxin-2 (Prx2). RES preconditioning also is reported to involve NMDAR and PKC. However, although moderate, the EtOH and RES concentrations used have been noticeably above circulating levels from two glasses of wine, a daily intake linked to reduced risk of cognitive decline among older social drinkers. Given their mechanistic parallels, we speculated that subprotective EtOH and RES concentrations in a combinatorial preconditioning paradigm might elicit synergistic neuroprotection. To examine this notion, rat cerebellar cultures were pretreated with 10 mM EtOH (circulating concentration after ~ 2 drinks), 5 µM RES, EtOH + RES combinatorially, or media alone (controls). After 3 days, media were removed, and fresh media aliquots containing Aß25-35 (25 µM) were added. Assessing apoptosis 24 h later with Hoescht 33342, neurodegeneration did not differ from controls in cultures separately preconditioned with 10 mM EtOH or 5 µM RES. However, apoptosis was prevented in combinatorially preconditioned cultures. Also, immunoblotting revealed elevated Prx2 levels due to combinatorial pretreatment that correlated with subsequent neuroprotection, whereas Prx2 was unchanged in separately pretreated cultures. Although the protective mechanisms require clarification, synergistically upregulated NMDAR-PKC-Prx2 (and other antioxidant proteins) is a reasonable component. These findings imply that EtOH + RES antioxidant synergy could be involved in neurobenefits attributed to low-moderate wine consumption.

Human immunodeficiency virus type 1 gp120 and ethanol coexposure in rat organotypic brain slice cultures: Curtailment of gp120-induced neurotoxicity and neurotoxic mediators by moderate but not high ethanol concentrations.

Human immunodeficiency virus type 1 (HIV-1) envelope protein gp120, implicated with other retroviral proteins in acquired immunodeficiency syndrome (AIDS)-related dementia, causes neuronal degeneration by inciting cascades of neurotoxic mediators from glia. It also may facilitate neuronal glutamate (N-methyl-D-aspartate, NMDA) receptor-mediated excitotoxicity by interacting at the glycine coagonist site. The authors reported that preconditioning rat organotypic hippocampal-cortical slice cultures subchronically with ethanol at concentrations occurring during moderate drinking (20 to 30 mM) prevented gp120's induction of neurotoxic mediators and intracellular calcium, as well as neuronal death. The authors now find that the acute copresence of ethanol in moderate as opposed to high concentrations similarly blocks the retroviral protein's neurotoxic effects in brain slice cultures, assessed with lactate dehydrogenase (LDH) release and propidium iodide (PI) labeling. As with ethanol preconditioning, neuroprotection against gp120 by moderate ethanol coexposure appears secondary to abrogation of the retroviral protein's early induction of arachidonic acid (AA), glutamate, and superoxide (but not nitric oxide) elevations/release. Additionally, experiments indicate that 30 mM ethanol is sufficient to inhibit the NMDA receptor, particularly in the presence of added glycine, thus hindering potential direct neuronal stimulation by gp120. However, in contrast to moderate ethanol, 100 mM ethanol, a concentration tolerated only in chronic alcoholics, potentiates gp120-dependent neurotoxicity (PI labeling) in the hippocampal CA1 region, augments LDH release, and fails to curtail gp120's actions on AA, glutamate, and superoxide-but does suppress nitric oxide induction. The results indicate dominant roles for AA, superoxide, and glutamate-mediated oxidative stress in gp120's neurotoxic mechanism, but perhaps a less important role for NMDA receptor stimulation, which would be constrained at both ethanol concentrations employed. We suggest that ethanol's concentration-dependent, two-edged sword behavior could alter the development of dementia in HIV-1-infected individuals during social consumption or abuse. Further studies are needed to elucidate the differing apparently glial effects of the two concentrations of ethanol.

Ethanol pre-exposure suppresses HIV-1 glycoprotein 120-induced neuronal degeneration by abrogating endogenous glutamate/Ca2+-mediated neurotoxicity.

The neurotoxic mechanism of HIV-1 envelope glycoprotein 120 (gp120) involves glutamatergic (NMDA) receptor/Ca2+-dependent excitotoxicity, mediated in part via glia. Pro-inflammatory cytokines also may have roles. We have reported that pre-exposure of brain cultures to 'physiological' ethanol concentrations (20-30 mM) protects against neuronal damage from HIV-1 gp120, but not from the direct receptor agonist, NMDA. Using lactate dehydrogenase assays and propidium iodide staining of rat organotypic hippocampal-entorhinal cortical slice cultures we determined that ethanol's suppression of gp120 neurotoxicity required at least 4 days of pretreatment. The gp120-induced neurotoxicity was accompanied by interleukin-6 elevations that were not affected by the pretreatment. However, gp120 induced substantial, early increases in extracellular glutamate levels that were blocked by ethanol pretreatment, conceivably abrogating excitotoxicity. Consistent with abrogation of excitotoxic pathways, fura-2 imaging showed selective deficits in gp120-dependent intracellular Ca2+ responses in ethanol-pretreated slices. Gp120 is believed to increase glutamate levels by both stimulating release and inhibiting (re)uptake. Results with a labeled glutamate analog, D-[3H]aspartate, revealed that gp120's inhibition of glutamate uptake, rather than its stimulation of release, was abolished after ethanol. Further studies indicated that two converging effects of ethanol pretreatment may underlie the abolishment of gp120-mediated glutamate uptake inhibition: (a) blockade of gp120-induced release (ostensibly from glia) of arachidonic acid, an inhibitor of astroglial glutamate reuptake, and (b) modest proliferation and activation of astroglia upon gp120 stimulation--which are likely to augment glutamate transporters. Thus, as with gp120 itself, glia and glutamate/arachidonic acid regulation appear to be important targets for ethanol. Since moderate ethanol consumption is as common among HIV-infected individuals as in the general population, this newly recognized neuroprotective (and apparently anti-excitotoxic) effect of ethanol withdrawal in vitro could be important, but it requires further study before its significance, if any, is understood.

Increased beta-carboline 9N-methyltransferase activity in the frontal cortex in Parkinson's disease.

Enzymatic beta-carboline N-methyltransferase activities generate N-methylated beta-carbolinium cations that are analogs of the parkinsonian-producing neurotoxin MPP+. We measured beta-carboline-2N-methyltransferase and beta-carboline-9N-methyltransferase activities in the supernatant and particulate fractions from postmortem human brains. These N-methyltransferase activities were assessed in the substantia nigra, putamen, and frontal cortex from control and Parkinson's disease cases. No significant differences were measured in any brain region in particulate and supernatant fraction beta-carboline 2N-methyltransferase activity or particulate fraction beta-carboline 9N-methyltransferase activity. Likewise, supernatant fraction beta-carboline 9N-methyltransferase activity was similar in the putamen and substantia nigra from Parkinson's disease and control cases. Unexpectedly, supernatant fraction beta-carboline 9N-methyltransferase activity was increased fourfold in Parkinson's disease frontal cortex (P < 0.05), suggesting that beta-carboline N-methylation may play a role in Parkinson's disease.

HIV-I gpI20 neurotoxicity in brain cultures is prevented by moderate ethanol pretreatment.

The HIV-1 coat protein gp 20, a potent neurotoxin that may underlie AIDS dementia, activates glia to cause neurotoxicity via the NMDA receptor and perhaps other routes. We find that pretreating cultures of rat organotypic cortical/hippocampal slices or cerebellar granule cells subchronically with ethanol in physiological concentrations (20-30 mM; 6 days) largely or even completely inhibits neurodegeneration due to gp120. However, NMDA-induced neurotoxicity appears unaffected by moderate ethanol pretreatment, indicating that ethanol's neuroprotection against gp120 is upstream of the NMDA receptor, possibly at a glial activation stage. The results could lead to a better understanding of relationships between ethanol, glia and neurodegeneration, particularly in AIDS.

Brain neuronal degeneration caused by episodic alcohol intoxication in rats: effects of nimodipine, 6,7-dinitro-quinoxaline-2,3-dione, and MK-801.

Rats repeatedly intoxicated with alcohol (ethanol, three times daily) over a 4-day period display neuronal degeneration in the dentate gyrus; entorhinal, piriform, insular, orbital, and perirhinal cortices; and in the olfactory nerve fibers and terminals in the olfactory bulb. Postulating a role for excitotoxicity, we have attempted to prevent the degeneration using antagonists that are neuroprotective in this type of brain damage. In an initial study, continuous subcutaneous infusion of a high dose of the glutamate/NMDA receptor antagonist MK-801 (2 mg/kg/day) by itself caused extensive neuronal degeneration in several brain regions and severe behavioral intoxication that precluded survival if combined with high blood alcohol levels (approximately 300 mg/dl). Moreover, the lower, nonneurotoxic blood alcohol levels (approximately 150 mg/dl) that were compatible with survival worsened the MK-801-induced brain damage. In a subsequent experiment, daily intraperitoneal injections of a lower dose of MK-801 (1 mg/kg/day) resulted in no MK-801 toxicity and, when combined with neurotoxic levels of alcohol, no reduction in alcohol-induced neurotoxicity. Nimodipine, a voltage-gated Ca2+ channel blocker, reduced the neuronal damage in the dentate gyrus, but greatly increased it in the piriform cortex when administered intragastrically at 600 mg/kg/day; it provided no protection from alcohol-dependent degeneration when given intragastrically at 100 mg/kg/day. Continuous intracerebroventricular delivery of 0.24 to 0.29 mg/day of 6,7-dinitro-quinoxaline-2,3-dione, a glutamate/alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptor antagonist, failed to diminish alcohol-dependent neuronal damage in any brain region. We conclude that brain damage from episodic "binge" alcohol intoxication is not primarily mediated by excitotoxic mechanisms, implying that other, nonexcitotoxic pathophysiological mechanisms, are involved. Furthermore, MK-801, far from protecting from the alcohol-induced damage, at high doses causes widespread neuropathology that is significantly potentiated by alcohol.

Brain damage due to episodic alcohol exposure in vivo and in vitro: furosemide neuroprotection implicates edema-based mechanism.

Adult rats intubated with a single dose of ethanol (alcohol; approximately 5 g/kg) for 5 to 10 successive days incur neurodegeneration in the entorhinal cortex, dentate gyrus, and olfactory bulbs accompanied by cerebrocortical edema and electrolyte (Na+, K+) accumulation. The brain damage is not lessened by cotreatment with the NMDA receptor antagonist MK-801; also, as reported elsewhere, MK-801 as well as non-NMDA receptor and Ca2+ channel antagonists are not neuroprotective in a similar, but more compressed, intoxication protocol. However, cotreatment with the electrolyte transport inhibitor/diuretic furosemide reduces alcohol-dependent cerebrocortical damage by 75-85% while preventing brain hydration and electrolyte elevations; olfactory bulb neurodegeneration is not attenuated. In parallel in vitro studies, rat organotypic entorhinal/hippocampal slice cultures exposed to alcohol (50-200 mM) 15 h/day for 6 days, mirroring episodic intoxication in vivo, demonstrate concentration-related release of the cytotoxic indicator, lactate dehydrogenase. Analogous to the in vivo findings, furosemide blocks this alcohol-induced in vitro cytotoxicity. Our results showing neuroprotection by furosemide indicate that brain edema and swelling are essential events in the brain damage induced by episodic alcohol exposure. Furosemide and related agents might be useful as neuroprotective agents in alcohol abuse. We suggest that the neurodegeneration is elicited in part by edema-dependent oxidative stress, but the regional selectivity of the damage may be best explained by physical (mechanical) compression of the limbic cortex against the adjacent tympanic bulla and subsequent neuronal cytoskeletal collapse. A scheme for these apparently nonexcitotoxic metabolic and mechanical pathways initiated by repeated alcohol exposure is proposed.

Characterization of brain beta-carboline-2-N-methyltransferase, an enzyme that may play a role in idiopathic Parkinson's disease.

The activity of beta-carboline-2-N-methyltransferase results in the formation of neurotoxic N-methylated beta-carbolinium compounds. We have hypothesized that these N-methylated beta-carbolinium cations may contribute to the development of idiopathic Parkinson's disease. This report describes experiments undertaken to optimize assay conditions for bovine brain beta-carboline-2-N-methyltransferase activity. The activity of beta-carboline-2-N-methyltransferase is primarily localized in the cytosol, has a pH optimum of 8.5-9, and obeys Michaelis-Menten kinetics with respect to its substrates, 9-methylnorharman (9-MeNH) and S-adenosyl-L-methionine (SAM). Kinetic constants, KM and Vmax, with respect to 9-MeNH, are 75 microM and 48 pmol/h/mg protein, respectively. The KM for SAM is 81 microM and the Vmax is 53 pmol/h/mg protein. In addition, enzyme activity is inhibited by S-adenosyl-L-homocysteine (SAH) or zinc, and is increased 2-fold in the presence of iron or manganese. Enzyme characterization is a prerequisite to the purification of this N-methyltransferase from bovine brain as well as comparison of its activity in human brain from control and Parkinson's disease individuals.

Binge ethanol-induced brain damage in rats: effect of inhibitors of nitric oxide synthase.

Testing the possible role of endogenous nitric oxide (NO) in the neurotoxicity of ethanol, we examined how two different NO synthase (NOS) inhibitors affected the extent cerebrocortical and olfactory neuronal damage in a modified "binge intoxication" rat model (Collins et al., Alcohol Clin. Exp. Res. 20:284-292, 1996). Male rats intragastrically fed ethanol (6.5 to 12 g/kg/day) in nutrient solution three times daily for 4 days also received NG-nitro-L-arginine methyl ester by chronic intracerebroventricular infusion or 7-nitro-indazole by daily intraperitoneal injection; control rats were given nutrient solution only and/or vehicles. Blood ethanol levels did not differ among the ethanol-treated groups. The amount of ethanol-dependent neuronal degeneration in the entorihinal cortex, dentate gyrus, and olfactory bulb glomeruli--visualized with the de Olmos cupric silver stain and quantitatively assessed in the binge-intoxicated rats--was either unchanged or significantly increased by the NOS inhibitors. Although the efficacies of the inhibitors cannot be directly compared because of various NOS forms were probably inhibited to differing extents, the results do not support the idea that endogenous NO is a neurotoxic mediator of ethanol's effects. Rather NO may have a modest neuroprotectant role in this model of early brain damage induced by ethanol. In addition, the NOS that is localized histochemically as NADPH diaphorase was present primarily in regions and/or cells not damaged by binge ethanol treatment. Assuming that NADPH diaphorase represents most of the NO forming enzyme(s) this suggests a transcellular mechanism for NO. A further observation was that hippocampal CA pyramidal neuron degeneration was extensive in rats infused centrally with NG-nitro-L-arginine methyl ester.

Neuronal degeneration in rat cerebrocortical and olfactory regions during subchronic "binge" intoxication with ethanol: possible explanation for olfactory deficits in alcoholics.

Severe, repetitive ("binge") ethanol intoxication in adult rats (intragastric delivery 3 times daily for 4 days in a modification of the Majchrowicz method) precipitates neuronal degeneration in selected cerebral cortical regions involved in memory and olfaction, confirming the results of Switzer and colleagues (Anat. Rec. 202: 186a, 1982). Neuronal damage was visualized with the de Olmos cupric silver technique for degenerating neurons and processes (argyrophilia), and was quantitated by total counts and densities of argyrophilic cells/fields. The specificity of the degeneration provides a neuropathological basis for the olfactory memory deficits in chronic alcoholics. In highly intoxicated rats, argyrophilia was most extensive among hippocampal dentate gyrus granule cells, pyramidal neurons in layer 3 of the entorhinal cortex, and olfactory nerve terminals in the olfactory bulb. Degenerating pyramidal neurons were also consistently seen in the insular cortex and olfactory cortical regions, such as the piriform and perirhinal cortices. There were few argyrophilic neurons in the CA regions of the hippocampus and none in the cerebellum--regions generally shown to have cell loss in long-term ethanol feeding models--but degenerating mossy fibers in the CA2 region were observed. Degeneration was maximal before the peak period of abstinence symptoms in this model, because argyrophilic densities were no greater 36 hr, compared with 8 hr after the last ethanol dose. High blood ethanol levels were required, because argyrophilia, absent from isocaloric controls, also was only evident in ethanol-intoxicated rats with mean blood ethanol levels for days 2 to 4 above 300 mg/dl; however, it increased substantially between 350 and 550 mg/dl. The resemblance of the argyrophilic distribution to the regional neuropathology that occurs in experimental seizures indicates that the ethanol-induced degeneration may have an excitotoxic basis. Progressive reductions in the seizure threshold (e.g., kindling phenomena that have been documented during binge ethanol intoxication) might be associated with excitotoxic hyperactivity during the repetitive nadirs between high blood and brain ethanol peaks. However, direct toxic actions of ethanol or its metabolites could also be involved. Overall, the model should be useful for studying mechanisms of ethanol-induced selective cortical and olfactory brain damage.

Methyl-beta-carbolinium analogs of MPP+ cause nigrostriatal toxicity after substantia nigra injections in rats.

Eleven beta-carbolinium compounds (beta C+s) and MPP+ were stereotaxically injected (40-200 nmol in 5 microliter of vehicle) unilaterally into the substantia nigra of anesthetized adult male Sprague-Dawley rats. The rats were sacrificed after three weeks. The ipsilateral striatum was analyzed for dopamine and DOPAC levels with HPLC. The brainstem injection site was fixed and cut coronally. The largest lesion area in each animal was measured using NIH IMAGE. Three beta C+s produced lesions whose mean areas were nearly as large as that produced by MPP+ (defined as 100%): 2,9-Me2-harman (94%), 2-Me-harmol (74%), and 2,9-Me2-norharman (57%). Three other compounds produced somewhat smaller lesions: 2-Me-harmaline (34%), 6-MeO-2-Me-harman (29%), and 2-Me-harmine (25%). The remaining compounds were ineffective (< or = 12%): norharman, 2-Me-norharman, 2-Me-harman, harmine, and 2-Me-6-MeO-harmalan. A 40 nmol dose of MPP+ reduced ipsilateral striatal dopamine to 0.6% of control. None of the beta C+s approached this, although several did significantly reduce striatal dopamine at doses of either 40 nmol (2,9-Me2-harman (37%), 2,9-Me2-norharman (42%), and 2-Me-harman (63%)) or 200 nmol (2-Me-harmaline (23%), norharman (63%), and 2-Me-norharman (64%)). There was a moderate negative correlation between lesion size and dopamine level (r = -0.65). There were also moderately strong correlation between lesion size and dopamine level (r = -0.65). There were also moderately strong correlations (r = 0.39-0.78) between the beta C+ nigral lesion area or striatal dopamine level potencies and their previously described IC50 values for inhibiting mitochondrial respiration or their toxicity to PC12 cells in culture. Interestingly, our correlation analysis revealed a remarkably strong correlation between beta C+ Ki MAO-A values and their toxicity to PC12 LDH release (r = -0.84) or PC12 protein loss (r = 0.79). Although beta C+s appear to be less specific toxins than MPP+, their levels in human substantia nigra are 8-20-fold higher than in cortex, making their role as relatively selective nigral toxins in Parkinson's disease plausible.

The effect of medial frontal cortex lesions on cardiovascular conditioned emotional responses in the rat.

The effect of ventral medial frontal cortex (MFC) lesions on heart rate and blood pressure during conditioned emotional responses (CER) was investigated. Male Sprague-Dawley rats were divided into two groups: MFC-lesioned rats (n = 11) sustained bilateral lesions of the infralimbic and ventral prelimbic regions of the MFC via microinjection of the neurotoxin N-methyl-D-aspartate; Controls (n = 13) received sterile saline. Following a 2-week recovery period, all animals were trained; one of two tones served as the conditioned stimulus (CS) and a 2 mA footshock served as the unconditioned stimulus (US). The CS+ tone was consistently paired with the US, while the CS- tone was randomly paired with the US. Heart rate and blood pressure were recorded during CS+ and CS- presentations before and after administration of the following pharmacological agents: atropine, atenolol, and atropine + atenolol. All animals responded to the CS+ with increased BP compared to baseline; the increase was not significantly different between groups. Controls responded to the CS+ with increased HR, while MFC-lesioned animals displayed a bimodal HR response which was not significantly different from baseline, but was significantly different from Controls. Pharmacological blockade of the HR response revealed coactivation of the sympathetic and parasympathetic nervous systems during the CS+, with a significant decrease (52%) in the sympathetic tachycardia component of the CS+ HR response in MFC-lesioned rats as compared to Controls; the parasympathetic bradycardia component was not altered by MFC lesions. In all cases, CS- responses were smaller than the CS+ responses. Pharmacological analysis revealed that the CS- HR response was mediated by the sympathetic component only, which was also significantly reduced in MFC-lesioned animals as compared to Controls. This significant reduction in the sympathetically mediated HR component of both the reinforced CER (CS+) and the unreinforced CER (CS-) following ventral MFC lesions implies that the MFC is necessary for complete sympathetic activation of cardiovascular responses to both severely and mildly stressful stimuli. The role of the MFC in emotion is also discussed.

Differential cytotoxicities of N-methyl-beta-carbolinium analogues of MPP+ in PC12 cells: insights into potential neurotoxicants in Parkinson's disease.

N-Methylated beta-carbolinium cations that can form in vivo from environmental or endogenous beta-carbolines are putative neurotoxic factors in Parkinson's disease. The cytotoxicities of 11 N-methylated beta-carbolinium cations and N-methyl-4-phenylpyridinium cation (MPP+), the experimental parkinsonian neurotoxicant which the carbolinium cations structurally resemble, were examined using rat pheochromocytoma (PC12) cells cultured in "low energy" N-5 medium; cell death was estimated by released lactate dehydrogenase activity and viable cell protein. Of the eight N2-monomethylated beta-carbolinium cations utilized, only 2-methyl-harmalinium (harmaline-2-methiodide) was as cytotoxic as MPP+. Also, three N2(beta), N9(indole)-dimethylated beta-carbolinium cations displayed cytotoxic effects, with the simplest, 2,9-dimethylnorharmanium, approaching the effectiveness of MPP+ in PC12 cells cultured in N-5 medium. However, when PC12 cells grown in higher energy Dulbecco's modified Eagle's medium were utilized with selected effective cations, it was observed that the cultures were relatively resistant to MPP+ and 2,9-dimethylnorharmanium, but remained vulnerable to 2-methylharmalinium. The results are interpreted to mean that different cytotoxic mechanisms exist for the two most potent beta-carbolinium cations--namely, a mechanism for the 2,9-dimethyl-beta-carbolinium species that, as with MPP+, is conditional on mitochondrial ATP depletion, but a different (or additional) mechanism for 2-methylharmalinium that is independent of mitochondrial inhibition. The possible accumulation of these cytotoxic cations in Parkinson's disease is discussed in the context of these findings.

Potential bioactivated neurotoxicants, N-methylated beta-carbolinium ions, are present in human brain.

Potential bioactivated neurotoxicants, 2-N-methyl-beta-carbolinium and 2,9-N,N'-dimethyl-beta-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norharmanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me2NH) were detected in almost all samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[beta] and 9[indole] nitrogens toward beta-carbolines was measured both in the cortex and in the nigra. 2[beta]-N-Methylation activity was significantly higher than 9[indole]-N-methylation activity in both regions. Recent studies show that beta-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP+, with respect to structure and neurotoxic activity. Such 'bioactivated' carbolinium ions could be endogenous causative factors in Parkinson's disease.

Mono-N-methylation of 1,2,3,4-tetrahydro-beta-carbolines in brain cytosol: absence of indole methylation.

In an accompanying report we demonstrated enzyme activity in guinea pig brain cell nuclei that catalyzes S-adenosylmethionine (SAM)-dependent N-methylations of heteroaromatic beta-carbolines (BCs) on the 2[beta]-nitrogen and subsequently on the 9[indole]-nitrogen, ultimately yielding N2,N9-dimethylated BCs. Presented here are the results of a parallel study of the N-methylation of 1,2,3,4-tetrahydro-BCs (THBCs), which form endogenously via condensations of tryptophan and its derived indoles with carbonyl compounds or, like their BC oxidation products, are environmental constituents and plant alkaloids. THBCs were enzymatically methylated on the 2[beta]-nitrogen by [3H]-SAM in undialyzed homogenates of rat or guinea pig brain, but [3H]methyl transfer to the 9[indole]-nitrogen was not observed. The structure of the 2[beta]-methyl THBC product was verified with capillary gas chromatography-mass spectrometry. Furthermore, whereas BC N-methylation was largely particulate and displayed micromolar Km values for BC substrate, THBC 2[beta]-N-methylation activity was cytosolic and displayed a relatively high (millimolar) Km for THBC substrate. The N-methylation of THBCs may be due to cytosolic N-methyltransferases that others have studied using different azaheterocyclics. Our overall studies indicate that N2,N9-dimethylated BCs could be unique neurotoxic factors that are bioactivated within brain by sequential N-methylations of BCs. These results suggest the possibility of an additional route to the putative 2,9-dimethylated toxins involving, as a first step, 2[beta]-N-methylation of environmental or endogenously derived THBCs in the brain and perhaps other organs.

Novel S-adenosylmethionine-dependent indole-N-methylation of beta-carbolines in brain particulate fractions.

Guinea pig brain S-adenosylmethionine (SAM)-dependent N-methyltransferase activity toward physiologically relevant beta-carboline (BC) substrates was examined with reverse-phase HPLC and radiochemical detection. Representative BCs, norharman and harmine, were enzymatically methylated on the 2[beta]-nitrogen by [3H]CH3-SAM in undialyzed homogenates to yield 2[beta]-methylated BCs and subsequently on the 9[indole]-nitrogen to generate 2,9-dimethylated BC products. This may be the first account of mammalian indole N-methyl transfer. There was no HPLC evidence for 9-methyl BC or (from carbon methylation) 2,6-dimethyl BC products. Capillary gas chromatography-mass spectrometry analysis confirmed the structures of the 2,9-dimethyl and 2-methyl products of norharman. The 2[beta]- and 9[indole]-N-methylation activities were mainly in the nuclear fractions and were negligible in undialyzed cytosol. This differs from the cytosolic SAM-dependent N-methylations reported with other azaheterocyclics, including 1,2,3,4-tetrahydro-BCs. The involvement of a single enzyme was suggested because the two N-methyl transfers with BC substrate had similar subcellular activity patterns, regional brain distributions, and Km and Vmax values. Sequential N-methylation of various BCs that have been observed in vivo may be a unique route to centrally retained N2,N9-dimethylated beta-carbolinium ions. Because they resemble the synthetic parkinsonian toxicant, N-methyl-4-phenylpyridinium, with respect to structure and neurotoxic activity, such "bioactivated" carbolinium ions could be endogenous causative factors in Parkinson's disease.

Inhibition of mitochondrial succinate oxidation--similarities and differences between N-methylated beta-carbolines and MPP+.

N-Methylated beta-carbolinium compounds (N-Me-BCs), including 2-N-methyl and 2,9-N,N-dimethyl analogs, structural analogs of 1-methyl-4-phenylpyridinium (MPP+), may be endogenously bioactivated, MPP(+)-like toxins, capable of inducing parkinsonism. Both MPP+ and selected N-Me-BCs inhibit NADH-linked mitochondrial respiration (Complex I). We now show that both also inhibit succinate-supported (Complex II) respiration, the greatest inhibition (80%) being seen for 2,9-dimethylharmanium. Complex I inhibition occurs at MPP+ concentrations (IC50 = 0.17 mM) about one order of magnitude lower than Complex II inhibition (greater than 1.2 mM). In contrast, Complex I and Complex II inhibition by the N-Me-BCs tested occurred at similar concentrations (I, 0.1 mM; II, 0.25 mM) and concentrations similar to Complex I inhibition by MPP+. 2,9-N,N-Dimethyl-BCs, which are the permanently charged BC analogs of MPP+, show inhibitory characteristics similar to MPP+: slow onset of inhibition, potentiation by TPB, and reversal by DNP. The fact that succinate oxidation cannot bypass the Complex II inhibition by N-Me-BCs could enhance any chronic neurotoxicity of N-Me-BCs.

Indole-N-methylated beta-carbolinium ions as potential brain-bioactivated neurotoxins.

N-Methyl-4-phenylpyridinium ion (MPP+), a highly toxic metabolite produced in the brain from a street drug contaminant, is selectively taken up by nigrostriatal dopaminergic neurons and accumulated intraneuronally in mitochondria. There it inhibits respiration, causes neuronal death and, in primates, provokes a parkinsonian condition. It has been suggested that endogenously generated or activated agents resembling MPP+ may contribute to the development of Parkinson's disease. We report here that simple beta-carbolines derived from tryptophan or related open chain indoles, when specifically methyl-substituted on both (2[beta] and 9[indole]) available nitrogens, display mitochondrial inhibitory potencies and neurotoxic effects in vitro (PC12 cultures) and in vivo (striatal microdialysis) which approach or even surpass MPP+. These results take on physiological significance with our finding that brain enzyme activity catalyzes S-adenosylmethionine-dependent methylations of the beta- and indole-nitrogens in beta-carbolines that have been detected in vivo. The unusual 9[indole]-N-methyl transfer, previously unrecognized in animals, apparently requires prior methylation of the 2[beta]-nitrogen. Sequential di-N-methylation of endogenous or xenobiotic beta-carbolines to form unique, neurotoxic 2,9-N,N'-dimethyl-beta-carbolinium ions may serve as a brain bioactivation route in chronic neurodegenerative conditions such as Parkinson's disease.