Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain.

Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. In this study, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2-4 h after exposure, followed by caspase 3 activation that peaked around 8 h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase 3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.

Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain.

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.

Tau phosphorylation and cleavage in ethanol-induced neurodegeneration in the developing mouse brain.

Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3beta (GSK-3beta) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3beta and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3beta inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3beta and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.

Developmental profiles of lipogenic enzymes and their regulators in the neonatal mouse brain.

It has been shown that lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), are highly expressed in the rodent brain during the early neonatal period and decline thereafter. However, cellular localization of these enzymes is unknown. Presently, we examined developmental changes in the levels and cellular localization of FAS and ACC, and their putative regulators, sterol-regulatory element-binding protein (SREBP)-1 and AMP-activated protein kinase (AMPK) in the mouse brain. Levels of these proteins including phosphorylated forms of ACC and AMPK decreased between postnatal day 4 (P4) and P19. Immunohistochemical studies indicated that FAS, ACC, AMPK, and SREBP-1 were expressed in neurons at P7, while FAS was found mostly in cells of oligodendrocyte lineage at P19. These studies suggest that neurons in the early neonatal brain are involved in do novo fatty acid synthesis.

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain.

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3beta (GSK-3beta), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3beta, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3beta, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

Ethanol alters lipid profiles and phosphorylation status of AMP-activated protein kinase in the neonatal mouse brain.

Previously, we have shown that ethanol-induced apoptosis in cultured neurons is accompanied by changes in cellular lipid profiles. In the present study, the effects of ethanol on brain lipid metabolism were studied using 7-day-old C57BL/6ByJ mice, which display apoptotic neurodegeneration upon exposure to ethanol. The brain lipids were extracted 4-24 h after the ethanol or saline treatment, and analyzed by TLC. We found that the levels of triglyceride, cholesterol ester, ceramide, and N-acylphosphatidylethanolamine increased significantly in the brains of ethanol-treated mice compared to those of saline-treated mice. Concomitantly, ethanol reduced Thr172 phosphorylation of AMP-activated protein kinase (AMPK) alpha subunits. Ethanol also reduced phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK and a lipogenic enzyme known to be activated by dephosphorylation. In contrast, lipid profiles of 19-day-old mouse brains, which scarcely manifested neurodegeneration upon ethanol exposure, were not significantly affected by ethanol. Also, the basal levels of Thr172-phosphorylated AMPK alpha were lower in these brains than in 7-day-old mouse brains, and no detectable changes in the phosphorylation status were observed by ethanol treatment. Our findings indicate that the ethanol-induced apoptotic neurodegeneration observed in mice during restricted developmental periods is accompanied by alterations in both the lipid content and the activity of AMPK in the brain.

Effects of gangliosides on ethanol-induced neurodegeneration in the developing mouse brain.

BACKGROUND: Ethanol exposure induces apoptotic neurodegeneration in the developing rodent brain during synaptogenesis. This process has been studied as a model for fetal alcohol syndrome. Previously, we have shown that gangliosides and LIGA20 (a semisynthetic derivative of GM1 ganglioside) attenuate ethanol-induced apoptosis in cultured neurons. In the present study, the effects of GM1 and LIGA20 on ethanol-induced apoptotic neurodegeneration were examined using an in vivo neonatal mouse model. METHODS: Seven-day-old C57BL/6By (B6By) mice were pretreated twice with intraperitoneal administration of GM1 (30 mg/kg), LIGA20 (2.5 mg/kg), or saline, followed by subcutaneous injection of either saline or ethanol (2.5 g/kg) twice with a 2 hours interval. Then the brains were: (1) perfusion-fixed 24 hours after the first ethanol injection, and the extent of neurodegeneration was assessed by cupric silver staining of the brain sections, or (2) perfusion-fixed 8 hours after the first ethanol injection, and the sections were immunostained with anti-cleaved (activated) caspase-3 antibody to evaluate caspase-3 activation. RESULTS: The comparison of cupric silver stained coronal sections indicates that ethanol-induced widespread neurodegeneration in the forebrains of B6By mice was reduced overall by GM1 and LIGA20 pretreatments. The extent of neurodegeneration detected by silver impregnation and activated caspase-3 immunostaining was quantified in the cingulate and retrosplenial cortices, which were the regions most severely affected by ethanol. The results indicate that GM1 and LIGA20 pretreatments induced statistically significant reductions-approximately 50% of the ethanol-treated samples-in silver impregnation and activated caspase-3 immunostaining. No significant differences were observed between saline controls and samples treated with GM1 or LIGA20 alone. CONCLUSIONS: These results indicate that GM1 and LIGA20, which have been shown to be neuroprotective against insults caused by various agents, partially attenuate ethanol-induced apoptotic neurodegeneration in the developing mouse brain.