Gene expression changes in C57BL/6J and DBA/2J mice following prenatal alcohol exposure.

BACKGROUND: Prenatal alcohol exposure can result in fetal alcohol spectrum disorders (FASD). Not all women who consume alcohol during pregnancy have children with FASD and studies have shown that genetic factors can play a role in ethanol teratogenesis. We examined gene expression in embryos and placentae from C57BL/6J (B6) and DBA/2J (D2) mice following prenatal alcohol exposure. B6 fetuses are susceptible to morphological malformations following prenatal alcohol exposure while D2 are relatively resistant. METHODS: Male and female B6 and D2 mice were mated for 2 hours in the morning, producing 4 embryonic genotypes: true-bred B6B6 and D2D2, and reciprocal B6D2 and D2B6. On gestational day 9, dams were intubated with 5.8 g/kg ethanol, an isocaloric amount of maltose dextrin, or nothing. Four hours later, dams were sacrificed and embryos and placentae were harvested. RNA was extracted, labeled and hybridized to Affymetrix Mouse Genome 430 v2 microarray chips. Data were normalized, subjected to analysis of variance and tested for enrichment of gene ontology molecular function and biological process using the Database for Annotation, Visualization and Integrated Discovery (DAVID). RESULTS: Several gene classes were differentially expressed in B6 and D2 regardless of treatment, including genes involved in polysaccharide binding and mitosis. Prenatal alcohol exposure altered expression of a subset of genes, including genes involved in methylation, chromatin remodeling, protein synthesis, and mRNA splicing. Very few genes were differentially expressed between maltose-exposed tissues and tissues that received nothing, so we combined these groups for comparisons with ethanol. While we observed many expression changes specific to B6 following prenatal alcohol exposure, none were specific for D2. Gene classes up- or down-regulated in B6 following prenatal alcohol exposure included genes involved in mRNA splicing, transcription, and translation. CONCLUSIONS: Our study identified several classes of genes with altered expression following prenatal alcohol exposure, including many specific for B6, a strain susceptible to ethanol teratogenesis. Lack of strain specific effects in D2 suggests there are few gene expression changes that confer resistance. Future studies will begin to analyze functional significance of the expression changes.

Insulin-like growth factor-I prevents the accumulation of autophagic vesicles and cell death in Purkinje neurons by increasing the rate of autophagosome-to-lysosome fusion and degradation.

Continuous macroautophagic activity is critical for the maintenance of neuronal homeostasis; however, unchecked or dysregulated autophagy can lead to cell death. Cultured Purkinje neurons die by an autophagy-associated cell death mechanism when deprived of trophic support. Here, we report that insulin-like growth factor-I (IGF-I) completely blocked the autophagy-associated cell death of Purkinje neurons. To examine the mechanism by which IGF-I influences autophagy, neurons were infected with adeno-RFP-LC3 and subjected to trophic factor withdrawal, and the size and number of autophagosomes were analyzed by live-cell fluorescence imaging. In control neurons, autophagy occurred at a constitutive low level with most autophagosomes measuring less than 0.75 microm. Trophic factor withdrawal increased the number and size of autophagosomes with most autophagosomes ranging between 0.75 and 1.5 microm and some reaching 1.5-2.25 microm. IGF-I added at the time of trophic factor withdrawal prevented the accumulation of the larger autophagosomes; however, it had no effect on the conversion of LC3, an indicator of autophagy induction. Instead, the rate of autophagosome-to-lysosome fusion measured by colocalization of RFP-LC3 and LysoSensor Green was accelerated by IGF-I. Treating the neurons with bafilomycin A(1) in the presence of IGF-I led to the accumulation of autophagosomes even larger than those induced by trophic factor withdrawal alone, indicating that IGF-I regulates autophagic vesicle turnover. Finally, the effect of IGF-I on autophagy was mediated by an Akt/mTOR-de pend ent and an ERK-independent pathway. These data suggest a novel role for IGF-I in protecting Purkinje neurons from autophagy-associated cell death by increasing autophagy efficiency downstream of autophagy induction.

Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.

Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human beta-amyloid peptide (Abeta) in order to examine the mechanism(s) of Abeta-toxicity. In this model, Abeta expression causes autophagosome accumulation, thereby mimicking a pathology found in brains of Alzheimer's disease patients. Furthermore, we demonstrate that decreased insulin-receptor signaling [using the daf-2(e1370) mutation] suppresses Abeta-induced paralysis by a mechanism that requires autophagy. Surprisingly, the daf-2 mutation also decreases Abeta-induced autophagosome accumulation. These observations can be explained by a model in which decreased insulin-receptor signaling promotes the maturation of autophagosomes into degradative autolysosomes, whereas Abeta impairs this process. Consistent with this model, we find that RNAi-mediated knock-down of lysosomal components results in enhanced Abeta-toxicity and autophagosome accumulation. Also, Abeta; daf-2(e1370) nematodes contain more lysosomes than either Abeta or control strains. Finally, we demonstrate that decreased insulin-receptor signaling promotes the autophagic degradation of Abeta.

Distinct mechanisms of neuronal apoptosis are triggered by antagonism of Bcl-2/Bcl-x(L) versus induction of the BH3-only protein Bim.

Primary cerebellar granule neurons (CGNs) require depolarizing extracellular potassium for their survival. Removal of depolarizing potassium triggers CGN apoptosis that requires induction of Bim, a BH3-only Bcl-2 family member. Bim is classically thought to promote apoptosis by neutralizing pro-survival Bcl-2 proteins. To determine if this is the principal function of Bim in CGNs, we contrasted Bim-mediated apoptosis to neuronal death induced by HA14-1, a BH3-domain mimetic that antagonizes Bcl-2 and Bcl-x(L). HA14-1 elicited CGN apoptosis characterized by caspase 3 and 9 activation, cytochrome c release, conformational activation of Bax, and mitochondrial depolarization. HA14-1 provoked CGN apoptosis in the absence of Bim induction and negative regulators of Bim transcription did not prevent HA14-1-induced cell death. However, the antioxidant glutathione and its precursor, N-acetyl-l-cysteine, suppressed HA14-1-induced apoptosis. Similarly, apoptosis induced by either a structurally distinct Bcl-2/Bcl-x(L) inhibitor (compound 6) or Bcl-2 antisense oligonucleotides was diminished by glutathione. In contrast, antioxidants had no effect on CGN apoptosis provoked by either removal of depolarizing potassium or overexpression of a GFP-Bim fusion protein, two models of Bim-dependent death. These data show that antagonism of Bcl-2/Bcl-x(L) function elicits oxidative stress-dependent CGN apoptosis that is mechanistically distinct from Bim-mediated cell death. These results further indicate that, although Bcl-2/Bcl-x(L) antagonism is sufficient to induce neuronal apoptosis, Bim likely promotes neuronal death by interacting with additional proteins besides Bcl-2/Bcl-x(L).

Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis.

Glycogen synthase kinase-3beta (GSK-3beta) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3beta that ultimately induce neuronal death are unknown. Here, we show that GSK-3beta phosphorylates and regulates the activity of Bax, a pro-apoptotic Bcl-2 family member that stimulates the intrinsic (mitochondrial) death pathway by eliciting cytochrome c release from mitochondria. In cerebellar granule neurons undergoing apoptosis, inhibition of GSK-3beta suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax(alpha) fusion protein and the conformational activation of endogenous Bax. GSK-3beta directly phosphorylated Bax(alpha) on Ser163, a residue found within a species-conserved, putative GSK-3beta phosphorylation motif. Coexpression of GFP-Bax(alpha) with a constitutively active mutant of GSK-3beta, GSK-3beta(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax(alpha), but not a Ser163Ala mutant of Bax(alpha), in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3beta promoted the localization of Bax(alpha) to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax(alpha) nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax(sigma)) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3beta. In a similar manner, either mutation or deletion of the identified GSK-3beta phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3beta exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

The p75 neurotrophin receptor can induce autophagy and death of cerebellar Purkinje neurons.

The cellular mechanisms underlying Purkinje neuron death in various neurodegenerative disorders of the cerebellum are poorly understood. Here we investigate an in vitro model of cerebellar neuronal death. We report that cerebellar Purkinje neurons, deprived of trophic factors, die by a form of programmed cell death distinct from the apoptotic death of neighboring granule neurons. Purkinje neuron death was characterized by excessive autophagic-lysosomal vacuolation. Autophagy and death of Purkinje neurons were inhibited by nerve growth factor (NGF) and were activated by NGF-neutralizing antibodies. Although treatment with antisense oligonucleotides to the p75 neurotrophin receptor (p75ntr) decreased basal survival of cultured cerebellar neurons, p75ntr-antisense decreased autophagy and completely inhibited death of Purkinje neurons induced by trophic factor withdrawal. Moreover, adenoviral expression of a p75ntr mutant lacking the ligand-binding domain induced vacuolation and death of Purkinje neurons. These results suggest that p75ntr is required for Purkinje neuron survival in the presence of trophic support; however, during trophic factor withdrawal, p75ntr contributes to Purkinje neuron autophagy and death. The autophagic morphology resembles that found in neurodegenerative disorders, suggesting a potential role for this pathway in neurological disease.