Michael E. Charness

Ethanol and the Nervous System

ALCOHOLISM is a major social, economic, and public health problem throughout much of the world. In the United States alone, the annual cost of lost productivity and health expenses related to alcoh...

Differential effects of ethanol antagonism and neuroprotection in peptide fragment NAPVSIPQ prevention of ethanol-induced developmental toxicity

NAPVSIPQ (NAP), an active fragment of the glial-derived activity-dependent neuroprotective protein, is protective at femtomolar concentrations against a wide array of neural insults and prevents ethanol-induced fetal wastage and growth retardation in mice. NAP also antagonizes ethanol inhibition of L1-mediated cell adhesion (ethanol antagonism). We performed an Ala scanning substitution of NAP to determine the role of ethanol antagonism and neuroprotection in NAP prevention of ethanol embryotoxicity. The Ser-Ile-Pro region of NAP was crucial for both ethanol antagonism and protection of cortical neurons from tetrodotoxin toxicity (neuroprotection). Ala replacement of either Ser-5 or Pro-7 (P7A-NAP) abolished NAP neuroprotection but minimally changed the efficacy of NAP ethanol antagonism. In contrast, Ala replacement of Ile-6 (I6A-NAP) caused a decrease in potency (>2 logarithmic orders) with only a small reduction (<10%) in the efficacy of NAP neuroprotection but markedly reduced the efficacy (50%) and the potency (5 logarithmic orders) of NAP ethanol antagonism. Ethanol significantly reduced the number of paired somites in mouse whole-embryo culture; this effect was prevented significantly by 100 pM NAP or by 100 pM P7A-NAP, but not by 100 pM I6A-NAP. The structure–activity relation for NAP prevention of ethanol embryotoxicity was similar to that for NAP ethanol antagonism and different from that for NAP neuroprotection. These findings support the hypothesis that NAP antagonism of ethanol inhibition of L1 adhesion plays a central role in NAP prevention of ethanol embryotoxicity and highlight the potential importance of ethanol effects on L1 in the pathophysiology of fetal alcohol syndrome.

Ethanol differentially regulates G proteins in neural cells

Long-term incubation of clonal neural cell lines with ethanol differentially reduces the stimulation of cAMP accumulation by hormones and cholera toxin. In the NG108-15 neuroblastoma chi glioma hybrid cell line, this heterologous desensitization was associated with a 42% reduction in the expression of Gs alpha and no significant change in Gi alpha. By contrast, ethanol treatment of the parental neuroblastoma cell line N18TG2 caused little loss of response to hormones or cholera toxin and no significant change in Gs alpha or Gi alpha. Ethanol induced heterologous desensitization in N1E-115 neuroblastoma cells; however, this cell line showed a dose-dependent increase in Gi alpha and a later decrease in Gs alpha. Thus, ethanol causes heterologous desensitization of hormone-stimulated cAMP accumulation by different mechanisms in related neural cell lines.

Neurotransmitter receptor binding: regional distribution in human brain.

INVESTIGATIONS of neurotransmitter levels and enzyme activities in human postmortem brains have helped elucidate the pathophysiology of conditions such as Parkinsons disease (HORNYKIEWICZ, 1972) and Huntingtons chorea (SHOULSON & CHASE, 1975; BIRD & IVERSEN, 1976). Recently it has become possible to measure specific synaptic receptor binding for a variety of neurotransmitters in mammalian brain (SNYDER & BENNETT, 1976). Initial studies on human postmortem caudate nucleus and cerebral cortex have indicated that receptor binding for y-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT). the b-adrenergic receptor, the opiate receptor and the muscarinic cholinergic receptor can be readily detected with levels of receptor binding comparable to those reported for rat and monkey brain (ENNA et al., 1976; KUHAR et al., 1973; HILEY 8z BIRD, 1974). These studies led to the discovery that 5-HT and muscarinic cholinergic receptor binding are markedly reduced in the caudate nucleus of brains from Huntingtons chorea patients (ENNA et al., 1976; HILEY & BIRD, 1974). To apply these techniques in disease states, it is desirable first to ascertain the detailed regional distribution of neurotransmitter receptor binding in human postmortem brain. In this study we report regional variations in binding to the GABA, 5-HT, 8-adrenergic, dopamine, opiate and muscarinic cholinergic receptors as well as enzyme activities for glutamic acid decarboxylase (EC 4.1.1.15) and choline acetyltransferase (EC 2.3.1.6). Human brains were obtained from patients who had been hospitalized at the time of death. No brains were used from individuals who had a disorder with any type of CNS involvement. In all cases, less than 6 h had elapsed between death and refrigeration, and less than 24 h had elapsed between death and autopsy. At autopsy, the entire brain was placed on ice and immediately dissected. After dissection, the various brain regions were frozen at 20°C until analysis. For analysis, each brain was homogenized in 10 uol of H 2 0 using a Brinkmann Polytron PT-10 (setting 6 ) for 1 min. Portions of t h s homogenate were assayed for muscarinic cholinergic receptors using as a ligand [3H]quinuclidinyl benzilate ([HIQNB), a potent and specific muscarinic antagonist (YAMAMURA & SNYDER, 1974; YAMAMURA & KUHAR, 1975; YAMAMURA et al., 1974), ChAc (MCCAMAN & HUNT, 1965), and GAD activities (ALBERS & BRADY, 1959). The remaining homogenate was centri-

Frequent neurologic toxicity associated with amiodarone therapy

Fifty-four consecutive patients were treated with amiodarone for symptomatic ventricular tachycardia or ventricular fibrillation refractory to treatment with conventional antiarrhythmic drugs. A reversible neurologic syndrome of tremor, ataxia, and occasionally peripheral neuropathy without nystagmus, dizziness, encephalopathy, or long-tract signs developed in 54% of the patients and was the most common reason for altering or discontinuing drug therapy. Neurologic side effects improved or resolved within 2 days to 4 weeks of decreasing or discontinuing amiodarone. Frequent neurologic toxicity is a hitherto undescribed complication of amiodarone therapy. Wider recognition of this syndrome will avoid unnecessary and costly diagnostic evaluation.

Updated clinical guidelines for diagnosing fetal alcohol spectrum disorders

The adverse effects of prenatal alcohol exposure constitute a continuum of disabilities (fetal alcohol spectrum disorders [FASD]). In 1996, the Institute of Medicine established diagnostic categories delineating the spectrum but not specifying clinical criteria by which diagnoses could be assigned. In 2005, the authors published practical guidelines operationalizing the Institute of Medicine categories, allowing for standardization of FASD diagnoses in clinical settings. The purpose of the current report is to present updated diagnostic guidelines based on a thorough review of the literature and the authors’ combined expertise based on the evaluation of >10u2009000 children for potential FASD in clinical settings and in epidemiologic studies in conjunction with National Institute on Alcohol Abuse and Alcoholism–funded studies, the Collaborative Initiative on Fetal Alcohol Spectrum Disorders, and the Collaboration on FASD Prevalence. The guidelines were formulated through conference calls and meetings held at National Institute on Alcohol Abuse and Alcoholism offices in Rockville, MD. Specific areas addressed include the following: precise definition of documented prenatal alcohol exposure; neurobehavioral criteria for diagnosis of fetal alcohol syndrome, partial fetal alcohol syndrome, and alcohol-related neurodevelopmental disorder; revised diagnostic criteria for alcohol-related birth defects; an updated comprehensive research dysmorphology scoring system; and a new lip/philtrum guide for the white population, incorporating a 45-degree view. The guidelines reflect consensus among a large and experienced cadre of FASD investigators in the fields of dysmorphology, epidemiology, neurology, psychology, developmental/behavioral pediatrics, and educational diagnostics. Their improved clarity and specificity will guide clinicians in accurate diagnosis of infants and children prenatally exposed to alcohol.

Octanol antagonism of ethanol teratogenesis

Alcohol may cause birth defects in part by disrupting the developmentally critical L1 cell adhesion molecule. Because 1‐octanol antagonizes ethanol inhibition of L1‐mediated cell adhesion, we asked whether 1‐octanol also prevents ethanol teratogenicity. L1 was detected in control mouse embryos as early as gestational day 8 (GD8), an early stage of vulnerability to ethanol teratogenesis. Culture of GD8 embryos with ethanol increased apoptotic cell death and decreased numbers of somite pairs. 1‐Octanol markedly reduced both ethanol effects. The paradoxical inhibition of ethanol teratogenicity by a longer chain 1‐alcohol suggests a strategy for developing compounds that prevent alcohol‐related birth defects.

Neuroprotective effect of human osteogenic protein-1 in a rat model of cerebral hypoxia/ischemia

Possible neuroprotective actions of osteogenic protein-1 (OP-1) were evaluated in a rat model of cerebral hypoxia/ischemia. Intraperitoneal injection of 50 micrograms of OP-1 prior to bilateral carotid ligation and transient hypoxia in 12-day-old rats reduced cerebral infarct area from 44.8 +/- 3.3% in vehicle-injected controls to 29 +/- 4.9%. Treatment of 14-day-old rats with 20 micrograms of OP-1 1 h after hypoxia reduced mortality from 45% to 13%. OP-1 may represent a novel class of neuroprotective agents.

Peptide-mediated protection from ethanol-induced neural tube defects.

Ethanol inhibition of L1-mediated cell adhesion may contribute to the spectrum of neurological, behavioral and morphological abnormalities associated with prenatal ethanol exposure. We showed previously that the neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) antagonize ethanol inhibition of L1 adhesion and prevent ethanol-induced growth retardation in mouse whole embryo culture. Here we ask whether NAP and SAL also prevent ethanol-induced major malformations of the nervous system. Gestational day 8.0 (3–5 somites) C57BL/6J mouse embryos were grown for 6 h in control medium, 100 mM ethanol and 10–10M peptides and then maintained for an additional 20 h in control medium. At the end of the culture period, only embryos having 18–19 somite pairs were examined and compared for the degree of neural tube closure. Ethanol exposure resulted in neural tube defects (NTDs) consistent with total dysraphia and anencephaly. Co-incubation with ethanol and L-NAP (all L-amino acids), D-NAP (all D-amino acids) or SAL significantly increased the percentage of embryos that had begun to close their neural folds at the level of the forebrain/midbrain junction or that had progressed beyond this stage of closure. P7A-NAP (NAPVSIAQ), which lacks neuroprotective activity, but retains activity as an antagonist of ethanol inhibition of L1 adhesion, was effective in preventing ethanol-induced NTDs. In contrast, I6A-NAP (NAPVSAPQ), which shows reduced efficacy as an ethanol antagonist but retains its neuroprotective efficacy, did not significantly diminish the induction of NTDs by ethanol. These findings demonstrate the ability of NAP and SAL to prevent ethanol-induced NTDs and support the hypothesis that ethanol teratogenesis is caused in part by ethanol inhibition of L1-mediated cell adhesion.

Ethanol and opioid receptor signalling

Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate μ-opioid receptor binding by increasing receptor Bmax, and, in some cases, chronic ethanol exposure decreases the density or affinity of the μ-opioid receptors. By contrast, high concentrations of ethanol acutely decrease λ-opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the λ-opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the λ-opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the λ-opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of Gαs and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the λ-opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in Gαs or Gαi. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the λ-opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in Gαs and increases in Gαi. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.