Yoav Littner

Rescue of neuronal migration deficits in a mouse model of fetal Minamata disease by increasing neuronal Ca2+ spike frequency

In the brains of patients with fetal Minamata disease (FMD), which is caused by exposure to methylmercury (MeHg) during development, many neurons are hypoplastic, ectopic, and disoriented, indicating disrupted migration, maturation, and growth. MeHg affects a myriad of signaling molecules, but little is known about which signals are primary targets for MeHg-induced deficits in neuronal development. In this study, using a mouse model of FMD, we examined how MeHg affects the migration of cerebellar granule cells during early postnatal development. The cerebellum is one of the most susceptible brain regions to MeHg exposure, and profound loss of cerebellar granule cells is detected in the brains of patients with FMD. We show that MeHg inhibits granule cell migration by reducing the frequency of somal Ca2+ spikes through alterations in Ca2+, cAMP, and insulin-like growth factor 1 (IGF1) signaling. First, MeHg slows the speed of granule cell migration in a dose-dependent manner, independent of the mode of migration. Second, MeHg reduces the frequency of spontaneous Ca2+ spikes in granule cell somata in a dose-dependent manner. Third, a unique in vivo live-imaging system for cell migration reveals that reducing the inhibitory effects of MeHg on somal Ca2+ spike frequency by stimulating internal Ca2+ release and Ca2+ influxes, inhibiting cAMP activity, or activating IGF1 receptors ameliorates the inhibitory effects of MeHg on granule cell migration. These results suggest that alteration of Ca2+ spike frequency and Ca2+, cAMP, and IGF1 signaling could be potential therapeutic targets for infants with MeHg intoxication.

ETHANOL CAUSES THE REDISTRIBUTION OF L1 CELL ADHESION MOLECULE IN LIPID RAFTS

J. Neurochem. (2011) 119, 859–867.

L1 Cell Adhesion Molecule Signaling Is Inhibited by Ethanol In Vivo

BACKGROUND Fetal alcohol spectrum disorder is an immense public health problem. In vitro studies support the hypothesis that L1 cell adhesion molecule (L1) is a target for ethanol (EtOH) developmental neurotoxicity. L1 is critical for the development of the central nervous system. It functions through signal transduction leading to phosphorylation and dephosphorylation of tyrosines on its cytoplasmic domain. The function of L1 is also dependent on trafficking through lipid rafts (LRs). Our hypothesis is that L1 is a target for EtOH neurotoxicity in vivo. Our objective is to demonstrate changes in L1 phosphorylation/dephosphorylation and LR association in vivo. METHODS Rat pups on postnatal day 6 are administered 4.5, 5.25, and 6 g/kg of EtOH divided into 2 doses 2 hours apart, then killed. Cerebella are rapidly frozen for assay. Blood is analyzed for blood EtOH concentration. L1 tyrosine phosphorylation is determined by immunoprecipitation and dephosphorylation of tyrosine 1176 determined by immunoblot. LRs are isolated by sucrose density gradient, and the distribution of L1 in LRs is determined. RESULTS EtOH at all doses reduced the relative amount of Y1176 dephosphorylation as well as the relative amount of L1 phosphorylated on other tyrosines. The proportion of L1 present in LRs is significantly increased in pups who received 6 g/kg EtOH compared to intubated controls. CONCLUSIONS L1 is a target for EtOH developmental neurotoxicity in vivo.

Inhibition of Cerebellar Granule Cell Turning by Alcohol

Ectopic neurons are often found in the brains of fetal alcohol spectrum disorders (FASD) and fetal alcohol syndrome (FAS) patients, suggesting that alcohol exposure impairs neuronal cell migration. Although it has been reported that alcohol decreases the speed of neuronal cell migration, little is known about whether alcohol also affects the turning of neurons. Here we show that ethanol exposure inhibits the turning of cerebellar granule cells in vivo and in vitro. First, in vivo studies using P10 mice demonstrated that a single intraperitoneal injection of ethanol not only reduces the number of turning granule cells but also alters the mode of turning at the EGL-ML border of the cerebellum. Second, in vitro analysis using microexplant cultures of P0-P3 mouse cerebella revealed that ethanol directly reduces the frequency of spontaneous granule cell turning in a dose-dependent manner. Third, the action of ethanol on the frequency of granule cell turning was significantly ameliorated by stimulating Ca(2+) and cGMP signaling or by inhibiting cAMP signaling. Taken together, these results indicate that ethanol affects the frequency and mode of cerebellar granule cell turning through alteration of the Ca(2+) and cyclic nucleotide signaling pathways, suggesting that the abnormal allocation of neurons found in the brains of FASD and FSA patients results, at least in part, from impaired turning of immature neurons by alcohol.

Elevated fatty acid ethyl esters in meconium of sheep fetuses exposed in utero to ethanol--a new animal model.

Specific fatty acid ethyl esters (FAEE) in meconium of newborns have been shown to correlate with maternal ethanol exposure. An animal model is needed to assess the validity of this biomarker. We hypothesized that the pregnant/fetal sheep is a feasible animal model for validating FAEE as a biomarker of prenatal ethanol exposure. Nine pregnant ewes were treated during the third trimester with different i.v. ethanol doses. The control group consisted of 14 pregnant ewes exposed to similar volumes of saline. On gestational d 133, the fetuses were delivered and meconium samples removed. FAEEs were quantified by gas chromatography-flame ionization detection. FAEEs were found in both control and ethanol exposed fetuses. Ethyl oleate, ethyl linoleate, and ethyl arachidonate levels were significantly higher in the ethanol-exposed sheep. Ethyl oleate was the FAEE that correlated most strongly with alcohol ingestion during pregnancy and had the greatest area under the curve (0.94). Using a cut-off value of 131 ng/g ethyl oleate dry weight, sensitivity was 89% and specificity was 100%. In conclusion, pregnant ewes are a feasible model for validating biomarkers of prenatal ethanol exposure. Ethyl oleate, ethyl linoleate, and ethyl arachidonate may be useful biomarkers of prenatal alcohol exposure.

Neuronal Cell Migration in Fetal Alcohol Syndrome

Maternal alcohol consumption during pregnancy can cause serious birth defects, of which fetal alcohol syndrome (FAS) is the most devastating. Recognized by characteristic craniofacial abnormalities and growth deficiency, this condition produces severe alcohol-induced damage in the developing brain. FAS children experience ataxia, deficits in intellectual functioning, difficulties in learning, memory, problem-solving, and attention. Multiple aspects of central nervous system development can be affected by alcohol exposure, but the most striking abnormalities are in neuronal cell migration. Although the cellular mechanisms by which alcohol affects the migration of immature neurons are not fully understood, recent studies reveal that Ca2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. An acute administration of ethanol reduces the frequency of transient Ca2+ elevations in migrating neurons and cGMP levels, and increases cAMP levels. Experimental manipulations of these second messenger pathways, through stimulating Ca2+ and cGMP signaling or inhibiting cAMP signaling, completely reverses the action of ethanol on neuronal cell migration in vitro as well as in vivo. Each second-messenger has multiple but distinct downstream targets, including CaMKII, calcineurin, PP1, Rho GTPase, MAPK, and PI3K. Therefore, the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. In this chapter, using cerebellar granule cell migration as a model system, we first describe how alcohol exposure impairs the migration of immature neurons, and then discuss the signaling mechanisms involved.