Madeliene Stump

Acid-sensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity

Acid-sensing ion channel 1A (ASIC1A) is abundant in the nucleus accumbens (NAc), a region known for its role in addiction. Because ASIC1A has been suggested to promote associative learning, we hypothesized that disrupting ASIC1A in the NAc would reduce drug-associated learning and memory. However, contrary to this hypothesis, we found that disrupting ASIC1A in the mouse NAc increased cocaine-conditioned place preference, suggesting an unexpected role for ASIC1A in addiction-related behavior. Moreover, overexpressing ASIC1A in rat NAc reduced cocaine self-administration. Investigating the underlying mechanisms, we identified a previously unknown postsynaptic current during neurotransmission that was mediated by ASIC1A and ASIC2 and thus well positioned to regulate synapse structure and function. Consistent with this possibility, disrupting ASIC1A altered dendritic spine density and glutamate receptor function, and increased cocaine-evoked plasticity, which resemble changes previously associated with cocaine-induced behavior. Together, these data suggest that ASIC1A inhibits the plasticity underlying addiction-related behavior and raise the possibility of developing therapies for drug addiction by targeting ASIC-dependent neurotransmission.

PPARγ Regulation in Hypertension and Metabolic Syndrome

Dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) activity leads to significant alterations in cardiovascular and metabolic regulation. This is most keenly observed by the metabolic syndrome-like phenotypes exhibited by patients carrying mutations in PPARγ. We will summarize recent findings regarding mechanisms of PPARγ regulation in the cardiovascular and nervous systems focusing largely on PPARγ in the smooth muscle, endothelium, and brain. Canonically, PPARγ exerts its effects by regulating the expression of target genes in these cells, and we will discuss mechanisms by which PPARγ targets in the vasculature regulate cardiovascular function. We will also discuss emerging evidence that PPARγ in the brain is a mediator of appetite and obesity. Finally, we will briefly review how novel PPARγ activators control posttranslational modifications of PPARγ and their prospects to offer new therapeutic options for treatment of metabolic diseases without the adverse side effects of thiazolidinediones which strongly activate transcriptional activity of PPARγ.

Endothelial PPAR-γ provides vascular protection from IL-1β-induced oxidative stress

Loss of peroxisome proliferator-activated receptor (PPAR)-γ function in the vascular endothelium enhances atherosclerosis and NF-κB target gene expression in high-fat diet-fed apolipoprotein E-deficient mice. The mechanisms by which endothelial PPAR-γ regulates inflammatory responses and protects against atherosclerosis remain unclear. To assess functional interactions between PPAR-γ and inflammation, we used a model of IL-1β-induced aortic dysfunction in transgenic mice with endothelium-specific overexpression of either wild-type (E-WT) or dominant negative PPAR-γ (E-V290M). IL-1β dose dependently decreased IκB-α, increased phospho-p65, and increased luciferase activity in the aorta of NF-κB-LUC transgenic mice. IL-1β also dose dependently reduced endothelial-dependent relaxation by ACh. The loss of ACh responsiveness was partially improved by pretreatment of the vessels with the PPAR-γ agonist rosiglitazone or in E-WT. Conversely, IL-1β-induced endothelial dysfunction was worsened in the aorta from E-V290M mice. Although IL-1β increased the expression of NF-κB target genes, NF-κB p65 inhibitor did not alleviate endothelial dysfunction induced by IL-1β. Tempol, a SOD mimetic, partially restored ACh responsiveness in the IL-1β-treated aorta. Notably, tempol only modestly improved protection in the E-WT aorta but had an increased protective effect in the E-V290M aorta compared with the aorta from nontransgenic mice, suggesting that PPAR-γ-mediated protection involves antioxidant effects. IL-1β increased ROS and decreased the phospho-endothelial nitric oxide synthase (Ser(1177))-to-endothelial nitric oxide synthase ratio in the nontransgenic aorta. These effects were completely abolished in the aorta with endothelial overexpression of WT PPAR-γ but were worsened in the aorta with E-V290M even in the absence of IL-1β. We conclude that PPAR-γ protects against IL-1β-mediated endothelial dysfunction through a reduction of oxidative stress responses but not by blunting IL-1β-mediated NF-κB activity.

Protective Role for Tissue Inhibitor of Metalloproteinase-4, a Novel Peroxisome Proliferator–Activated Receptor-γ Target Gene, in Smooth Muscle in Deoxycorticosterone Acetate–Salt Hypertension

Loss of peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) function causes hypertension, whereas its activation lowers blood pressure. Evidence suggests that these effects may be attributable to PPAR&ggr; activity in the vasculature. However, the specific transcriptional targets of PPAR&ggr; in vessels remain largely unidentified. In this study, we examined the role of smooth muscle PPAR&ggr; during salt-sensitive hypertension and investigated its transcriptional targets and functional effect. Transgenic mice expressing dominant-negative PPAR&ggr; (S-P467L) in smooth muscle cells were more prone to deoxycorticosterone acetate–salt–induced hypertension and mesenteric arterial dysfunction compared with nontransgenic controls. Despite similar morphometry at baseline, vascular remodeling in conduit and small arteries was enhanced in S-P467L after deoxycorticosterone acetate–salt treatment. Gene expression profiling in aorta and mesenteric arteries revealed significantly decreased expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in S-P467L. Expression of TIMP-4 was increased by deoxycorticosterone acetate–salt treatment, but this increase was ablated in S-P467L. Interference with PPAR&ggr; activity either by treatment with a PPAR&ggr; inhibitor, GW9662, or by expressing P467L PPAR&ggr; markedly suppressed TIMP-4 in primary smooth muscle cells. PPAR&ggr; binds to a PPAR response element (PPRE) in chromatin close to the TIMP-4 gene in smooth muscle cells, suggesting that TIMP-4 is a novel target of PPAR&ggr;. The interference with PPAR&ggr; and decrease in TIMP-4 were accompanied by an increase in total matrix metalloproteinase activity. PPAR&ggr;-mediated loss of TIMP-4 increased, whereas overexpression of TIMP-4 decreased smooth muscle cell migration in a scratch assay. Our findings highlight a protective mechanism induced by PPAR&ggr; in deoxycorticosterone acetate–salt treatment, establishing a novel mechanistic link between PPAR&ggr; and TIMP-4.

Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance

Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions.

Hypertension-Causing Mutation in Peroxisome Proliferator–Activated Receptor γ Impairs Nuclear Export of Nuclear Factor-κB p65 in Vascular Smooth Muscle

Selective expression of dominant negative (DN) peroxisome proliferator–activated receptor &ggr; (PPAR&ggr;) in vascular smooth muscle cells (SMC) results in hypertension, atherosclerosis, and increased nuclear factor-&kgr;B (NF-&kgr;B) target gene expression. Mesenteric SMC were cultured from mice designed to conditionally express wild-type (WT) or DN-PPAR&ggr; in response to Cre recombinase to determine how SMC PPAR&ggr; regulates expression of NF-&kgr;B target inflammatory genes. SMC-specific overexpression of WT-PPAR&ggr; or agonist-induced activation of endogenous PPAR&ggr; blunted tumor necrosis factor &agr; (TNF-&agr;)–induced NF-&kgr;B target gene expression and activity of an NF-&kgr;B–responsive promoter. TNF-&agr;–induced gene expression responses were enhanced by DN-PPAR&ggr; in SMC. Although expression of NF-&kgr;B p65 was unchanged, nuclear export of p65 was accelerated by WT-PPAR&ggr; and prevented by DN-PPAR&ggr; in SMC. Leptomycin B, a nuclear export inhibitor, blocked p65 nuclear export and inhibited the anti-inflammatory action of PPAR&ggr;. Consistent with a role in facilitating p65 nuclear export, WT-PPAR&ggr; coimmunoprecipitated with p65, and WT-PPAR&ggr; was also exported from the nucleus after TNF-&agr; treatment. Conversely, DN-PPAR&ggr; does not bind to p65 and was retained in the nucleus after TNF-&agr; treatment. Transgenic mice expressing WT-PPAR&ggr; or DN-PPAR&ggr; specifically in SMC (S-WT or S-DN) were bred with mice expressing luciferase controlled by an NF-&kgr;B–responsive promoter to assess effects on NF-&kgr;B activity in whole tissue. TNF-&agr;–induced NF-&kgr;B activity was decreased in aorta and carotid artery from S-WT but was increased in vessels from S-DN mice. We conclude that SMC PPAR&ggr; blunts expression of proinflammatory genes by inhibition of NF-&kgr;B activity through a mechanism promoting nuclear export of p65, which is abolished by DN mutation in PPAR&ggr;.