Maria Teresa Fernández-Sánchez

Neuronal Sensitization and Its Behavioral Correlates in a Rat Model of Neuropathy Are Prevented by a Cyclic Analog of Orphenadrine

N-methyl-D-aspartic acid (NMDA) is an agonist at the homonymous receptor implicated in the development of neuronal sensitization and its behavioral correlates. An effective modulation of the NMDA effects, achieved also by uncompetitive antagonists, could contribute to controlling pain symptoms in several neuropathic syndromes. Because nefopam is a known analgesic derivative of orphenadrine and of its congener diphenhydramine, both uncompetitive NMDA receptor antagonists, we tested the effect of nefopam on the developing pain and neuronal anomalies in an animal model of chronic pain with NMDA receptor involvement. A single intraperitoneal injection of nefopam was administered twenty minutes prior to the chronic constriction injury of the sciatic nerve (CCI rats). In the first 10 days, nefopam (30 mg/kg) significantly decreased behavioral signs of neuropathic pain and the stimulus-evoked electrophysiological anomalies in recordings at 14 days, with only slight manifestation afterwards. The dose of 20 mg/kg was ineffective. Nefopam injected after constriction was ineffective. In normal non-operated rats, Nefopam had no effect on the electrophysiological and behavioral parameters. Iontophoretic nefopam (1 mM, 50-80 nA, positive current) in normal rats did not change the spontaneous neuronal activity, but reduced the mean response to noxious stimuli and the concurrent iontophoretic NMDA evoked activity. In CCI rats, iontophoretic nefopam did not significantly modify the spontaneous hyperactivity but reduced significantly both the frequency of the responses to noxious stimuli, and the duration of the afterdischarge. We propose that nefopam exerts a preventive analgesic effect, with a possible role in modulating NMDA receptor-mediated effects in central sensitization.

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity.

Summary. Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 μM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol.

Terfenadine induces toxicity in cultured cerebellar neurons: a role for glutamate receptors.

SummaryExposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2h exposure, 20µM terfenadine reduced the number of surviving neurons by 75%, and as low as 10nM terfenadine induced significant neurotoxicity after 5 days of exposure. Neuronal sensitivity to terfenadine changed with age in culture, and at 25 days in culture neurons appeared to be much less sensitive than at 5 or 9–17 days in culture. Neurotoxicity by terfenadine could not be prevented by high concentrations of histamine (5 mM), but it was significantly delayed by blocking NMDA or non-NMDA glutamate receptors with MK-801 or CNQX respectively, suggesting the involvement of excitatory transmission mediated by glutamate in the neurotoxicity induced by terfenadine in these neurons. We also found that the presence of terfenadine (5,µM) unveiled the potential excitotoxicity of the non-NMDA receptor agonist AMPA (100µM), and reduced the concentration of glutamate necessary to induce excitotoxicity, compared to untreated cultures. These results suggest a role for terfenadine in the modulation of the excitotoxic response mediated in cerebellar neurons through ionotropic glutamate receptors.

Glutamate and Neurodegeneration

Psychiatric diseases have stimulated human interest since ancient times, evoking mixed feelings of fear, compassion, appreciation of originality, and medical impotence. The search for their biological causes and therapeutical remedies has been predominantly inspired either by fantasy, as retracted in the famous masterpiece of Fig. 1, or by religious convictions, similarly to other diseases involving consciousness, such as epilepsy. Following the development of neuroscience, the problems of the mind started to receive attention according to the scientific method and to biological hypothesis, and the discovery of useful psychoactive drugs revolutionized our understanding of the nature of psychiatric diseases. Curiously, at the same time as antipsychotics began to be known as such, another molecule, glutamate, began to be known for both its neurotoxic properties (1), and its potential for being an excitatory neurotransmitter in the central nervous system (CNS) (2). However, in the early 1960s only few molecules, such as acetylcholine, norepinephrine, dopamine, and γ-aminobutyric acid could be definitively considered to play a role as neurotransmitters. Thus, it should not be surprising that glutamate, a widespread amino acid, struggled to establish its role as the most important excitatory neurotransmitter in the brain. Furthermore, after the initial observation of Lucas and Newhouse (1), most of the evidence assigning a neurodegenerative role to glutamate came from studies where glutamate and its analogs were either administered peripherally in high doses or injected directly in the CNS, two experimental conditions that did not suggest a neurodegenerative role for endogenous glutamate.

Comparison of Extracellular and Intracellular Blood Compartments Highlights Redox Alterations in Alzheimer's and Mild Cognitive Impairment Patients

BACKGROUND Many studies suggest oxidative stress as an early feature of Alzheimers Disease (AD). However, evidence of established oxidative stress in AD peripheral cells is still inconclusive, possibly due to both, differences in the type of samples and the heterogeneity of oxidative markers used in different studies. OBJECTIVE The aim of this study was to evaluate blood-based redox alterations in Alzheimers Disease in order to identify a peculiar disease profile. METHOD To that purpose, we measured the activity of Superoxide Dismutase, Catalase and Glutathione Peroxidase both in the extracellular and the intracellular blood compartments of AD, MCI and control subjects. The amount of an open isoform of p53 protein (unfolded p53), resulting from oxidative modifications was also determined. RESULTS Decreased SOD, increased GPx activity and higher p53 open isoform were found in both AD and MCI plasma compared to controls. In blood peripheral mononuclear cells, SOD activity was also decreased in both AD and MCI, and unfolded p53 increased exquisitely in younger AD males compared to controls. CONCLUSION Overall, these data highlight the importance of considering both extracellular and intracellular compartments, in the determination of antioxidant enzyme activities as well as specific oxidation end-products, in order to identify peculiar blood-based redox alterations in AD pathology.

The amnesic shellfish poison domoic acid enhances neurotoxicity by excitatory amino acids in cultured neurons

SummaryA recent episode of human intoxication by cultured mussels containing a rare excitatory amino acid named domoic acid, received particular attention for its neurological implications. The intoxication produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. We now report that in neuronal cultures the neurotoxicity of a domoic acid-containing mussel extract is the result of domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high amounts in mussel tissue. Moreover, we show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity. We present evidence suggesting that the neurotoxic synergism may be due to a reduction of Mg+ + block at the NMDA receptor-associated channel, following activation of NON-NMDA receptors by domoic acid.

Cellular and Molecular Responses of Cultured Neurons to Stressful Stimuli

Synaptic function is critical for the brain to process experiences dictated by the environment requiring change over the lifetime of the organism. Experience-driven adaptation requires that receptors, signal transduction pathways, transcription and translational mechanisms within neurons respond rapidly over its lifetime. Adaptive responses communicated through the rapid firing of neurons are dependent upon the integrity and function of synapses. These rapid responses via adaptation underlie the organisms ability to perceive, learn, remember, calculate and plan. Glutamate, the endogenous neurotransmitter required for physiological excitation in the brain, is critically involved in neuronal adaptive responses and in the pathophysiology of neurodegenerative disorders. Using neuronal experimental systems, we will discuss how compounds with low dose effects mediated via glutamate receptors can result either in a neuroprotective or neurotoxic response. Because the brain has evolved to respond rapidly to environmental cues, exposure of neurons to stressful stimuli can result in a pivotal response toward either synaptic adaptation or dysfunction and neuronal cell death. Understanding how neurons adapt to stressful stimuli will provide important clues toward the development of strategies to protect the brain against neurodegeneration.

From the Cover: Selective Enhancement of Domoic Acid Toxicity in Primary Cultures of Cerebellar Granule Cells by Lowering Extracellular Na+ Concentration

Domoic acid (DOM) is an excitatory amino acid analog of kainic acid (KA) that acts through glutamic acid (GLU) receptors, inducing a fast and potent neurotoxic response. Here, we present evidence for an enhancement of excitotoxicity following exposure of cultured cerebellar granule cells to DOM in the presence of lower than physiological Na+ concentrations. The concentration of DOM that reduced by 50% neuronal survival was approximately 3 µM in Na+-free conditions and 16 µM in presence of a physiological concentration of extracellular Na+. The enhanced neurotoxic effect of DOM was fully prevented by AMPA/KA receptor antagonist, while N-methyl-D-aspartate-receptor-mediated neurotoxicity did not seem to be involved, as the absence of extracellular Na+ failed to potentiate GLU excitotoxicity under the same experimental conditions. Lowering of extracellular Na+ concentration to 60 mM eliminated extracellular recording of spontaneous electrophysiological activity from cultured neurons grown on a multi electrode array and prevented DOM stimulation of the electrical activity. Although changes in the extracellular Na+ concentration did not alter the magnitude of the rapid increase in intracellular Ca2+ levels associated to DOM exposure, they did change significantly the contribution of voltage-sensitive calcium channels (VScaCs) and the recovery time to baseline. The prevention of Ca2+ influx via VSCaCs by nifedipine failed to prevent DOM toxicity at any extracellular Na+ concentration, while the reduction of extracellular Ca2+ concentration ameliorated DOM toxicity only in the absence of extracellular Na+, enhancing it in physiological conditions. Our data suggest a crucial role for extracellular Na+ concentration in determining excitotoxicity by DOM.

A NEW PUTATIVE EARLY BIOMARKER FOR A BLOOD-BASED TIMELY DIAGNOSIS FOR ALZHEIMER’S DISEASE

2-Aminoadipic acid Amines TG(51:3) Lipids: Triglycerides 3-Hydroxyisovaleric acid Organic acids Tyrosine Amines TG(54:6) Lipids: Triglycerides TG(50:4) Lipids: Triglycerides S-3-Hydroxyisobutyric acid Organic acids TG(56:8) Lipids: Triglycerides Methyldopa Amines 8-iso-PGF2a (15-F2t-IsoP) Oxidative stress: Isoprostane TG(48:3) Lipids: Triglycerides O-Acetylserine Amines TG(48:2) Lipids: Triglycerides Methylmalonic acid Organic acids TG(46:2) Lipids: Triglycerides Valine Amines TG(50:3) Lipids: Triglycerides TG(52:4) Lipids: Triglycerides TG(52:5) Lipids: Triglycerides TG(56:7) Lipids: Triglycerides TG(48:0) Lipids: Triglycerides Ornithine Amines SM(d18:1/23:0) Lipids: Sphingomyelins SM(d18:1/20:1) Lipids: Sphingomyelins TG(48:1) Lipids: Triglycerides TG(58:10) Lipids: Triglycerides Poster Presentations: Tuesday, July 18, 2017 P1025

AN OPEN ISOFORM OF P53 AS AN EARLY BIOMARKER OF BLOOD REDOX ALTERATIONS IN ALZHEIMER’S DISEASE: DEVELOPMENT OF AN EASY AND REPRODUCIBLE ASSAY

plasma of 5-, 7-, 9and 12-month-old female transgenic mice using sandwich-ELISA to define a relationship between the plasma Ab and AD progression and, also, to confirm the diagnostic potential of the plasma Ab prior to the plaque formation. Results:We found that plasma Ab(1–42) concentration increases with age, while the concentration of Ab(1–42) in the cerebrospinal fluid (CSF) decreases in 3xTg-AD mice, if measurements were made before formation of ThS-positive plaques in the brain.Conclusions:There is an inverse correlations between the plasma and CSF Ab(1–42) levels until plaques form in transgenic mice’s brains and that the plasma Ab concentration possesses the diagnostic potential as a biomarker for diagnosis of early AD stages.