Molina Mhatre

Antioxidants in Central Nervous System Diseases: Preclinical Promise and Translational Challenges

Oxidative damage is strongly implicated in the pathogenesis of neurodegenerative diseases including Alzheimers disease, amyotrophic lateral sclerosis, Huntingtons disease, Parkinsons disease and stroke (brain ischemia/reperfusion injury). The availability of transgenic and toxin-inducible models of these conditions has facilitated the preclinical evaluation of putative antioxidant agents ranging from prototypic natural antioxidants such as vitamin E (alpha-tocopherol) to sophisticated synthetic free radical traps and catalytic oxidants. Literature review shows that antioxidant therapies have enjoyed general success in preclinical studies across disparate animal models, but little benefit in human intervention studies or clinical trials. Recent high-profile failures of vitamin E trials in Parkinsons disease, and nitrone therapies in stroke, have diminished enthusiasm to pursue antioxidant neuroprotectants in the clinic. The translational disappointment of antioxidants likely arises from a combination of factors including failure to understand the drug candidates mechanism of action in relationship to human disease, and failure to conduct preclinical studies using concentration and time parameters relevant to the clinical setting. This review discusses the rationale for using antioxidants in the prophylaxis or mitigation of human neurodiseases, with a critical discussion regarding ways in which future preclinical studies may be adjusted to offer more predictive value in selecting agents for translation into human trials.

Oxidative stress and neuroinflammation in Alzheimer's disease and amyotrophic lateral sclerosis: Common links and potential therapeutic targets

Many neurological diseases, including Alzheimers disease (AD) and amyotrophic lateral sclerosis (ALS), are now recognized to share atypical inflammatory reactions as a major pathological feature. Neuroinflammation can both be a cause, and a consequence, of chronic oxidative stress. Cytokine-stimulated microglia generate copious amounts of reactive oxygen and reactive nitrogen species, creating a stress upon ambient neurons. Conversely, oxidants can stimulate pro-inflammatory gene transcription in glia, leading to various inflammatory reactions. This review compares literature regarding neuroinflammation in AD and ALS, with special emphasis on roles played by tumor necrosis factor alpha (TNFalpha) and aberrant arachidonic acid metabolism in the genesis of chronic oxidative conditions. Based on our observations made in the G93A-SOD1 mouse model of ALS, and a body of Alzheimers disease findings, we hypothesize a prominent pathological role for the TNFalpha-signaling axis and neuroinflammation in the pathogenesis of both diseases. A discussion is made regarding the relevance of neuroinflammation to potential therapeutic implications for both ALS and AD.

Thrombin, a mediator of neurotoxicity and memory impairment

Thrombin has been found in neuritic plaques in Alzheimers disease (AD). Also, traumatic brain injury, where neurons are exposed to high thrombin levels, is associated with an increased incidence of AD. Our objective was to determine the effects of thrombin administered in vivo on cognitive function and neuropathology. Rats were trained using a radial eight-arm maze and then thrombin (25 or 100 nM, 0.25 microl/h, 28 days) or vehicle was delivered via intracerebroventricular infusion. Animals that received 100 nM thrombin demonstrated cognitive impairments including deficits in reference memory and an increase in task latency. Also, significant neuropathology was detected in these animals such as enlargement of cerebral ventricles, an increased number of TUNEL-positive cells, astrogliosis, and an increase in the immunoreactivity for phosphorylated neurofilament, and apolipoprotein-E fragments. Thrombin-induced changes in cognitive function and ventricular enlargement were inhibited by hirudin. These findings demonstrate that thrombin is a mediator of neurotoxicity and cognitive deficits and suggest that inhibition of thrombin may be a treatment strategy for AD- or head trauma-associated cognitive deficits.

Diazepam during prior ethanol withdrawals does not alter seizure susceptibility during a subsequent withdrawal.

The number of cycles of alcohol detoxification is suggested to be an important variable in the predisposition to severe withdrawal seizures in alcohol-dependent individuals. Several clinical studies have suggested that exposure to repeated alcohol withdrawals may lead to increased severity of subsequent withdrawal episodes. Consistent with these observations, exposure to multiple cycles of ethanol withdrawal in our previous study significantly increased sensitivity to the convulsive effects of the GABA(A) receptor inverse agonist, Ro15-4513, in comparison to continuous ethanol exposure with no intermittent withdrawals. There was also a selective increase in the occurrence of spontaneous spike and sharp wave (SSW) activity in the EEG recorded from hippocampal area CA(3) in proportion to the number of withdrawal episodes experienced. It is hypothesized that during such repeated episodes of ethanol intoxication and withdrawal, changes in neuronal excitation during prior withdrawals could serve as initially subconvulsive kindling stimuli that might eventually result in the increased severity of the withdrawal syndrome. There is some evidence of the successful suppression of such neuronal excitation during acute ethanol withdrawal by positive modulators of the GABA(A) receptor. In the present study, the benzodiazepine agonist, diazepam, at a dose (4.0 mg/kg) that suppresses acute withdrawal symptoms, when administered during intermittent withdrawals, did not alter seizure sensitivity during a subsequent nonmedicated withdrawal. Diazepam treatment during prior withdrawals also did not have any effect on the multiple withdrawal-associated increase in SSW activity in hippocampal area CA(3) during an untreated withdrawal. This finding suggests that suppression of acute withdrawal symptoms by diazepam does not prevent long-lasting changes in CNS function resulting from repeated exposures to ethanol withdrawal.

Increased Ro15-4513–Induced Seizures Following Multiple Ethanol Withdrawals

Clinical research into the etiology of ethanol withdrawal seizures has shown an increase in the number and severity of seizures with increasing numbers of withdrawal episodes. The aim of the present study was to determine the effects of multiple ethanol withdrawals on the seizure sensitivity to the GABA(A) receptor inverse agonist Ro15-4513. In this study, three groups of laboratory rats received varying amounts of either continuous or intermittent ethanol exposure. A fourth group (Naive) received no ethanol exposure. Eight hours following the last withdrawal from chronic ethanol exposure, animals were tested for sensitivity to Ro15-4513-induced motor convulsions. Seizure sensitivity was significantly increased in all ethanol-treated groups compared to ethanol-naive controls, which did not exhibit any convulsive responses to this dose of Ro15-4513. Furthermore, rats exposed to multiple ethanol withdrawals exhibited significantly higher sensitivity to drug-induced seizures than did animals experiencing only a single ethanol withdrawal. Although the specific mechanism of this enhanced convulsant effect of Ro15-4513 following multiple ethanol withdrawals remains to be determined, these results suggest an involvement of GABA(A)-benzodiazepine receptors in this multiple withdrawal phenomenon.

Opiate delta-2-receptor antagonist naltriben does not alter discriminative stimulus effects of ethanol

The ability of a selective 2-opiate receptor antagonist, naltriben, to modulate ethanol discrimination was investigated in a rat model using a drug discrimination procedure. Rats were trained to discriminate ethanol (1.25 g/kg, IP) from saline on a fixed-ratio schedule, FR10. Once rats had acquired the ethanol-saline discrimination, ethanol dose-response tests were conducted with 15-min pretest injections. Following the characterization of the ethanol dose-response curve, the effect of naltriben on ethanols discriminative stimulus was assessed by administering naltriben (0. 032-5.6 mg/kg, IP) 15 min before the ethanol administration. In the present study, naltriben did not have any modulatory effect on ethanol discrimination, suggesting that either Delta(2)-opiate receptors are not involved in the formation of ethanols discriminative stimulus or the antagonism of Delta(2)-opiate receptors is not sufficient to alter ethanols compound discriminative stimulus.

Caloric restriction retards the aging associated changes in γ-aminobutyric acidA receptor gene expression in rat cerebellum

It is widely accepted that calorie restriction is an effective way of delaying the aging process. Also, there is an indication that the beneficial effects exerted by dietary manipulation may be due to a direct effect at the molecular level like gene expression. The studies were conducted to determine whether calorie restriction prevents any age-related changes in the structural and molecular aspects of the GABAA-BZ receptor. In aged (24-month old diet ad libitum) rats, the binding of [35S]t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced in the cerebellum. In contrast, [35S]TBPS binding remained unchanged in the cerebellum of calorie restricted old rats. In order to evaluate the molecular basis of these changes, the alpha sub-unit mRNA levels were measured. The GABAA receptor alpha1 sub-unit mRNA level remained unchanged in both the old groups of rats. The alpha2 subunit mRNA level was significantly decreased in the cerebellum of aged rats (24-month old ad libitum), whereas it remained unchanged in the cerebellum of calorie restricted old animals. These findings indicate a selective age and diet related modulation in the stoichiometry of the GABAA receptor in aging.

Chronic Thrombin Exposure Results in an Increase in Apolipoprotein-E Levels

Studies have shown that individuals with both a history of traumatic brain injury and inheritance of apolipoprotein E‐4 (ApoE4) allele are associated with a poor neurologic outcome and an increased risk for Alzheimers disease. We assessed the hypothesis that thrombin released during brain injury causes an increase in apolipoprotein‐E levels and such increase in the levels of apolipoprotein‐E4 isoform may have amyloidogenic effects. Rats received either thrombin (100 nm, 0.25 μl/hr, 28 days) or vehicle via intracerebroventricular (i.c.v.) infusion. Thrombin treatment increased apolipoprotein‐E levels in hippocampus as compared to vehicle treatment (P < 0.001). Infusion of human apolipoprotein‐E4 (0.6 ng/hr, i.c.v., 56 days) into rats resulted in β‐amyloid deposition and increased the number of GFAP‐positive astrocytes. ApoE4 infusion also resulted in significant spatial memory deficits. These findings suggest that thrombin released during brain injury may contribute to an increase in apolipoprotein‐E levels. Such increase in Apolipoprotein‐E4 isoform facilitates β‐amyloid deposition and cognitive deficits.

5-HT3 receptor antagonist ICS 205-930 alters the discriminative effects of ethanol

The ability of a selective 5-hydroxytryptamine (5-HT(3)) receptor antagonist, ICS 205-930 (3-tropanyl-indole-1-carboxylate, tropisetron), to block the discriminative stimulus effects of ethanol was investigated in rats that were trained to discriminate ethanol (1.25 g/kg ip) from saline with food as the reinforcement. Prior administration of ICS 205-930, at the dose of 0.01 mg/kg, significantly decreased ethanols discriminative stimulus effect at ED(75) dose of ethanol, while higher doses of ICS 205-930 (10 and 17 mg/kg) showed enhancement of ethanols discriminative effects at ED(0), ED(25), and ED(50) doses of ethanol. Under conditions where ICS 205-930 (10, 17 mg/kg) was tested alone, rats responded exclusively on the saline-appropriate lever. These effects occurred without significantly altering response rates or blood ethanol concentrations. The results suggest that the 5-HT(3) antagonist ICS 205-930 at lower concentration decreases, and at higher concentration enhances the discriminative stimulus effects associated with a lower to moderate dose of ethanol.

Chapter 14 – Oxidative Stress and Neuroinflammation in Alzheimer's Disease and Amyotrophic Lateral Sclerosis: From Biology to Therapeutic Strategies

Oxidative stress (OS) and neuroinflammation appear to be common pathogenetic mechanisms in neurodegenerative disorders. Several studies show markers of oxidative damage and neuroinflammation—such as glial activation, up-regulation of cytokines, lipid peroxidation, and protein and DNA oxidation—in the affected brain regions. Neuroinflammation and oxidative stress can be a cause or a consequence of disease pathology. Cytokine-stimulated microglia generates significant amounts of reactive oxygen and nitrogen species, thereby leading to various signal transduction pathways and resulting in neurotoxicity. Conversely, oxidants can stimulate proinflammatory gene transcription in glia, thereby leading to various inflammatory reactions. Findings by Tamagno et al . support the concept that OS and β-amyloid (Aβ) production are strictly interrelated events. An age-dependent increase in reactive oxygen species (ROS) may be directly responsible for a switch of amyloid precursor protein (APP) cleavage to Aβ production resulting in sporadic AD. The presence of a vicious feedback loop in AD pathogenesis is proposed in the chapter, where disease pathology causes microglial activation, up-regulation of proinflammatory cytokines, and free-radical generation, which subsequently stimulate Aβ generation and Aβ, subsequently, further stimulates various inflammatory pathways.