Plasminogen activator system homeostasis and its dysregulation by ethanol in astrocyte cultures and the developing brain.

In utero alcohol exposure can cause fetal alcohol spectrum disorders (FASD), characterized by structural brain abnormalities and long-lasting behavioral and cognitive dysfunction. Neuronal plasticity is affected by in utero alcohol exposure and can be modulated by extracellular proteolysis. Plasmin is a major extracellular serine-protease whose activation is tightly regulated by the plasminogen activator (PA) system. In the present study we explored the effect of ethanol on the expression of the main components of the brain PA system in sex-specific cortical astrocyte primary cultures in vitro and in the cortex and hippocampus of post-natal day (PD) 9 male and female rats. We find that ethanol alters the PA system in astrocytes and in the developing brain. In particular, the expression of tissue-type PA (tPA), encoded by the gene Plat, is consistently upregulated by ethanol in astrocytes in vitro and in the cortex and hippocampus in vivo. Astrocytes exhibit endogenous plasmin activity that is increased by ethanol and recombinant tPA and inhibited by tPA silencing. We also find that tPA is expressed by astrocytes of the developing cortex and hippocampus in vivo. All components of the PA system investigated, with the exception of Neuroserpin/Serpini1, are expressed at higher levels in astrocyte cultures than in the developing brain, suggesting that astrocytes are major producers of these proteins in the brain. In conclusion, astrocyte PA system may play a major role in the modulation of neuronal plasticity; ethanol-induced upregulation of tPA levels and plasmin activity may be responsible for altered neuronal plasticity in FASD.

Astrocyte Dysfunction Induced by Alcohol in Females but Not Males.

Chronic alcohol abuse is associated with brain damage in a sex-specific fashion, but the mechanisms involved are poorly described and remain controversial. Previous results have suggested that astrocyte gene expression is influenced by ethanol intoxication and during abstinence in vivo. Here, bioinformatic analysis of astrocyte-enriched ethanol-regulated genes in vivo revealed ubiquitin pathways as an ethanol target, but with sexually dimorphic cytokine signaling and changes associated with brain aging in females and not males. Consistent with this result, astrocyte activation was observed after exposure in female but not male animals, with reduced S100ß levels in the anterior cingulate cortex and increased GFAP(+) cells in the hippocampus. In primary culture, the direct effects of chronic ethanol exposure followed by recovery on sex-specific astrocyte function were examined. Male astrocyte responses were consistent with astrocyte deactivation with reduced GFAP expression during ethanol exposure. In contrast, female astrocytes exhibited increased expression of Tnf, reduced expression of the neuroprotective cytokine Tgfb1, disrupted bioenergetics and reduced excitatory amino acid uptake following exposure or recovery. These results indicate widespread astrocyte dysfunction in ethanol-exposed females and suggest a mechanism that may underlie increased vulnerability to ethanol-induced neurotoxicity in females.

Females uniquely vulnerable to alcohol-induced neurotoxicity show altered glucocorticoid signaling.

Women are more sensitive to the harmful effects of alcohol (EtOH) abuse than men, yet the underlying mechanisms remain poorly understood. Previous gene expression analysis of the medial prefrontal cortex (mPFC) following a chronic intoxication paradigm using continuous 72 h vapor inhalation found that females, but not males, exhibit an inflammatory response at peak withdrawal that is associated with cell damage. Given that glucocorticoids can function as anti-inflammatories, are known to increase with EtOH exposure, and influence neurotoxicity, we hypothesized that males and females may exhibit an altered corticosterone (CORT) response following chronic intoxication. Analysis of serum CORT levels revealed the expected increase during withdrawal with no difference between males and females, while control males but not females exhibited higher CORT concentrations than naive animals. Glucocorticoid signaling characterized using focused qPCR arrays identified a sexually dimorphic response in the mPFC during withdrawal, particularly among astrocyte-enriched genes. These genes include aquaporin-1 (Aqp1), sphingosine kinase 1 (Sphk1) and connective tissue growth factor (Ctgf); genes associated with inflammatory signaling, and tissue damage and repair. Bioinformatic analysis also revealed activation of inflammatory signaling and cell death pathways in females. Confirmation studies showed that female mice exhibited significant neuronal degeneration within the anterior cingulate cortex (ACC). By contrast, EtOH exposure lead to a significant reduction in cell death in males. Thus, distinct glucocorticoid signaling pathways are associated with sexually dimorphic neurotoxicity, suggesting one mechanism by which EtOH-exposed females are particularly vulnerable to the damaging effects of alcohol in the CNS.

Elevated testosterone in females reveals a robust sex difference in altered androgen levels during chronic alcohol withdrawal.

The endocrine disruption associated with alcohol (ethanol) abuse in both males and females is widely recognized. Ethanol intoxication and withdrawal in males results in significant reductions in androgen levels. Less is known about female alcoholics, and because the changes in testosterone concentrations remain controversial, we systematically characterized changes in sex steroids after chronic ethanol exposure and withdrawal in both sexes. Testosterone and 17ß-estradiol concentrations were determined during chronic high intoxication, over a withdrawal time course, and following a period of abstinence using a genetic model of withdrawal vulnerability, the Withdrawal Seizure-Resistant (WSR) and -Prone (WSP) selected lines. In males, testosterone concentrations were significantly lower in intoxicated WSP mice after chronic ethanol exposure, and were dramatically and transiently reduced during the withdrawal period in both WSR and WSP lines. In contrast, testosterone levels were increased in intoxicated WSP females and in both WSR and WSP mice during withdrawal. Chronic ethanol exposure disrupted normal estrous cycling in WSP mice, associated with hyperandrogenemia while intoxicated. In abstinence, elevated testosterone was observed in both sexes but only in WSR mice. Estrogen levels were modestly reduced during withdrawal in both WSR and WSP lines, predominantly in males. These findings identify a mechanism based on altered androgen signaling that likely contributes to sex-specific responses during withdrawal. However, only WSR mice showed similar elevations in androgen long after withdrawal in both sexes, suggesting that genotype is an important determinant of steroid responses after abstinence. Increased androgen signaling in females as a consequence of chronic ethanol exposure may play an important and relatively uncharacterized role in sexually dimorphic responses to alcohol abuse.