Prenatal alcohol exposure impairs autophagy in neonatal brain cortical microvessels

Abstract

Brain developmental lesions are a devastating consequence of prenatal alcohol exposure (PAE). We recently showed that PAE affects cortical vascular development with major effects on angiogenesis and endothelial cell survival. The underlying molecular mechanisms of these effects remain poorly understood. This study aimed at characterizing the ethanol exposure impact on the autophagic process in brain microvessels in human fetuses with fetal alcohol syndrome (FAS) and in a PAE mouse model. Our results indicate that PAE induces an increase of autophagic vacuole number in human fetal and neonatal mouse brain cortical microvessels. Subsequently, ex vivo studies using green fluorescent protein (GFP)-LC3 mouse microvessel preparations revealed that ethanol treatment alters autophagy in endothelial cells. Primary cultures of mouse brain microvascular endothelial cells were used to characterize the underlying molecular mechanisms. LC3 and p62 protein levels were significantly increased in endothelial cells treated with 50 mM ethanol. The increase of autophagic vacuole number may be due to excessive autophagosome formation associated with the partial inhibition of the mammalian target of rapamycin pathway upon ethanol exposure. In addition, the progression from autophagosomes to autolysosomes, which was monitored using autophagic flux inhibitors and mRFP–EGFP vector, showed a decrease in the autolysosome number. Besides, a decrease in the Rab7 protein level was observed that may underlie the impairment of autophagosome–lysosome fusion. In addition, our results showed that ethanol-induced cell death is likely to be mediated by decreased mitochondrial integrity and release of apoptosis-inducing factor. Interestingly, incubation of cultured cells with rapamycin prevented ethanol effects on autophagic flux, ethanol-induced cell death and vascular plasticity. Taken together, these results are consistent with autophagy dysregulation in cortical microvessels upon ethanol exposure, which could contribute to the defects in angiogenesis observed in patients with FAS. Moreover, our results suggest that rapamycin represents a potential therapeutic strategy to reduce PAE-related brain developmental disorders.

Proteomic Analysis of Baboon Cerebral Artery Reveals Potential Pathways of Damage by Prenatal Alcohol Exposure*

“…However, alcohol gastric administration does not allow answering the question whether ethanol itself or rather its metabolites serve as molecular triggers of proteome changes observed in our analysis. For example, impairment of cerebral microvessel endothelium autophagy by PAE in mouse fetuses was ablated by 4-methylpyrazole, which blocked formation of the ethanol metabolite acetaldehyde (66). The role of ethanol metabolites in the ultimate effects of PAE on the fetal cerebral artery proteome remains to be established.…”

Alcohol and the Developing Brain: Why Neurons Die and How Survivors Change

“…The cell death is not necessarily and exclusively caused by impairments of the neurotransmitter system. Indirect effects, such as those related to the disruption of brain or uterine vasculature, may play a role in alcohol-induced cell death [ 14 , 15 , 16 ].…”

Glutamate controls vessel-associated migration of GABA interneurons from the pial migratory route via NMDA receptors and endothelial protease activation

“…Because it has been demonstrated that during cortical development, interneurons migrate tangentially while closely interacting with the vasculature of the pial migratory route (PMR) [2,10,11], these data suggest that the endothelial NMDA receptor would contribute in the vessel-associated interneuron migration. Consistent with this hypothesis, several recent data showed that the migration of GABAergic neurons is regulated by excitatory neurons [12] and that exposure of mouse neonate cortices to molecules with NMDA-antagonist properties such as ketamine, alcohol, or MK801 resulted in endothelial cell autophagy followed by massive apoptotic death of migrating tangential GABA interneurons present in the superficial cortical layers [13][14][15][16][17].…”