Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment.

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENT cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.

Ethanol-induced modulation of GPR55 expression in human monocyte-derived dendritic cells is accompanied by H4K12 acetylation.

Inflammation supports the progression of alcohol-related organ injury. Recent research findings have linked ethanol exposure to changes in histone acetylation and deacetylation in the brain and in peripheral tissues, leading to ethanol-dependence related damage. One of the mechanisms that has been shown to play a major role during inflammation is the cannabinoid system. Previous research has demonstrated that ethanol can modulate cannabinoid receptors' functions. Our lab has shown that the G protein-coupled receptor (GPR55), a novel cannabinoid receptor, is upregulated in binge drinkers and in cells treated acutely with ethanol. Additionally, our group has also uncovered that chronic ethanol exposure leads to an increase in histone modifications, such as acetylation. However, the regulatory mechanism of GPR55 within the immune system under the influence of ethanol is poorly understood. Since changes in histone modifications might lead to changes in gene expression, we hypothesize that the mechanism of ethanol-induced upregulation of GPR55 is linked to epigenetic changes on histone proteins. Taking into account previous findings from our lab, the goal of the present study was to determine whether there is any relevant association between histone hyperacetylation and the regulation of the novel cannabinoid receptor GPR55 in monocyte-derived dendritic cells (MDDCs) of human origin treated acutely with ethanol. Therefore, monocytes were isolated from buffy coats and allowed to differentiate into MDDCs. The cells were treated with ethanol for 24 h, harvested, fixed, and stained with antibodies against GPR55. As expected, based on previous findings, confocal microscopy showed that ethanol exposure increases GPR55 expression. In order to demonstrate the correlation between histone acetylation and GPR55 expression regulation, the cells were treated with ethanol, harvested, and then the chromatin was extracted and fractionated for chromatin immunoprecipitation (ChIP) assay, followed by real-time qPCR for the analysis of DNA fragments. The results showed an enrichment of the histone modification H4K12ac in the GPR55 gene of MDDCs treated with ethanol. Furthermore, siRNA against the histone acetyltransferase Tip60 (responsible for the acetylation of H4K12) resulted in a downregulation of GPR55. In conjunction, these results indicate that in the presence of ethanol, the upregulation of GPR55 expression is accompanied by H4K12 acetylation, which might have a significant effect in the ability of this innate immune system's cells to cope with cellular stress induced by ethanol. However, the causality of ethanol regulation of H4K12ac in GPR55 expression changes still lacks further elucidation; therefore, additional experimental approaches to confirm a significant causality between H4K12 acetylation and ethanol regulation of GPR55 are currently undergoing in our lab.

Trichostatin A Shows Transient Protection from Chronic Alcohol-Induced Reactive Oxygen Species (ROS) Production in Human Monocyte-Derived Dendritic Cells.

OBJECTIVE: The objective of this study was to understand whether histone deacetylase (HDACs) inhibitor Trichostatin A or TSA can block and/or reverse chronic alcohol exposure-induced ROS in human monocyte-derived dendritic cells (MDDCs). Additionally, since nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a known regulator of antioxidant responses, we studied the effects of alcohol and TSA on ROS production and modulation of Nrf2 by MDDCs. METHODS: Intra-cellular, extra-cellular, and total ROS levels were measured in MDDCs treated chronically with alcohol (0.1 and 0.2 % EtOH) using 2',7'-dichlorofluorescin diacetate (DCF-DA) followed by detection of ROS in microplate reader and imaging flow cytometer. Nrf2 expression was analyzed by qRT- PCR and western blot. In addition, NFE2L2 (Nrf2), class I HDAC genes HDAC1, HDAC2, and histone acetyltransferase genes KAT5 were analyzed in silico using the GeneMania prediction server. RESULTS: Our results confirmed alcohol's ability to increase intracellular ROS levels in MDDCs within minutes of treatment. Our findings have also demonstrated, for the first time, that TSA has a transient protective effect on MDDCs treated chronically with alcohol since the ability of TSA to reduce intracellular ROS levels is only detected up to 15 minutes post-chronic alcohol treatment with no significant protective effects by 10 hours. In addition, chronic alcohol treatment was able to increase the expression of the antioxidant regulator Nrf2 in a dose dependent manner, and the effect of the higher amount of alcohol (0.2%) on Nrf2 gene expression was significantly enhanced by TSA. CONCLUSION: This study demonstrates that TSA has a transient protective effect against ROS induced by chronic alcohol exposure of human MDDCs and chronic long-term exposure of MDDCs with alcohol and TSA induces cellular toxicity. It also highlights imaging flow cytometry as a novel tool to detect intracellular ROS levels. Overall, the effect of TSA might be mediated through Nrf2; however, further studies are needed to fully understand the molecular mechanisms.