Substance use disorders are common among women veterans, many of which are of childbearing age. Drinking during pregnancy may lead to Fetal Alcohol Spectrum Disorders (FASD), a leading cause of intellectual disability. Research on novel mechanisms involved in FASD, which may lead to innovative interventions, is therefore a topic highly relevant to the VA mission. Hippocampal alterations are associated with deficits in learning and memory in individuals with FASD. The extracellular matrix (ECM) plays a major role in brain development and astrocytes are major regulators of the brain ECM. Critical gaps in knowledge remain concerning the mechanisms by which ethanol alters neuronal development in the fetal hippocampus hampering the development of therapies for FASD. Indeed, there is no published literature on the effects of alcohol exposure during the third trimester of human gestation-equivalent on astrocyte gene expression in vivo. Furthermore, dysregulation of the brain ECM mediated by alterations in extracellular proteases is involved in many neuropathological conditions and in addiction. However, very little is known about the role of the ECM and extracellular proteases in FASD in vivo. Finally, the link between changes in astrocyte-released ECM modulators and dendritic development following neonatal alcohol exposure has not been investigated. Preliminary results suggest that developmental alcohol exposure alters the ECM through the modulation of extracellular protease systems in the hippocampus. Indeed, Adamts5 expression, encoding for a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a protease that degrades lecticans, and tissue plasminogen activator (tPA), which can activate ADAMTSs, are both upregulated in the hippocampus of animals neonatally exposed to ethanol. Furthermore, we observed decreased levels of lectican sulfated glycosaminoglycans (sGAGs) and increased proteolysis of the lectican brevican in these animals. We also observed down-regulation of Mmp14 and Mmp15 encoding for matrix metalloproteinases (MMP)14 and MMP15 and increased protein levels of one major target of these MMPs: laminin. All of these changes are consistent with an ethanol-induced increase in dendritic arborization in pyramidal hippocampal neurons, as lecticans are inhibitors and laminin is a strong inducer of dendritic arborization. The overall hypothesis of this proposal is that ethanol alters the expression and activity of astrocyte extracellular proteases leading to ECM remodeling and increased dendritic arborization in the hippocampus.
In aim 1, the hypothesis that developmental ethanol exposure increases the expression and activity of ADAMTSs that degrade lecticans in part via an increase in tPA expression leading to ADAMTS activation resulting in the degradation of lecticans and increased dendritic arborization in the neonatal brain will be explored.
In aim 2, the hypothesis that ethanol exposure decreases MMP14 and MMP15 leading to increased laminin protein levels and increased dendritic complexity will be explored.
Aims 1 and 2 will employ qRT-PCR, Western blot, confocal microscopy, Golgi-Cox staining followed by morphometric analysis with the software Neurolucida, and AAV6 viral construct injections into the hippocampus to overexpress ADAMTS5 and tPA and to silence MMP14 and MMP15.
In aim 3 the modulation of gene expression by ethanol in neonatal hippocampal astrocytes of Aldh1l1-EGFP-Rpl10a mice using the translating ribosome affinity purification (TRAP) methodology will be examined. The effects of neonatal alcohol exposure on the expression of target genes encoding for ECM proteins and proteins involved in the remodeling of the ECM as well as on astrocyte global gene expression will be analyzed. We will isolate astrocyte mRNA in the engineered Aldh1l1-EGFP-Rpl10a mouse model that expresses a modified ribosomal protein Rpl10a with an eGFP tag (EGFP-Rpl10a) in cells expressing Aldh1l1 (a highly specific astrocytic marker) using the TRAP method. This study will unveil novel astrocyte-mediated effects of ethanol on extracellular proteases leading to changes in ECM composition and neuronal development.
Women are playing a growing role in the U.S. military and women are the fastest growing group of new users of the VA health care system. Between 2005 and 2015 the number of women veterans enrolled in the VA health care system increased 84%. Alcohol abuse in reproductive-age women represents a particularly severe problem because in utero alcohol exposure can cause FASD, a leading cause of intellectual and behavioral disabilities. Approximately 50% of women veterans enrolled in the VA health care system are of childbearing age. Recent studies reported that 25% of women veterans have unhealthy alcohol use. This study will unveil novel astrocyte-mediated effects of ethanol on extracellular proteases leading to changes in ECM composition and neuronal development. A better understanding of these effects of alcohol will lead to therapeutic interventions that are not only specific for a given molecular target, but also for a given cellular target and, for this reason, safer. The proposed studies are an essential first step towards achieving this goal.
