Dysfunction In Female Mice Research Articles

Mitochondrial Membranes of Human SH-SY5Y Neuroblastoma Cells Express Serotonin 5-HT7 Receptor.

Mitochondria in neurons contribute to energy supply, the regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. In recent years, several studies have highlighted that the neurotransmitter serotonin (5-HT) plays an important role in mitochondrial biogenesis in cortical neurons, and regulates mitochondrial activity and cellular function in cardiomyocytes. 5-HT exerts its diverse actions by binding to cell surface receptors that are classified into seven distinct families (5-HT1 to 5-HT7). Recently, it was shown that 5-HT3 and 5-HT4 receptors are located on the mitochondrial membrane and participate in the regulation of mitochondrial function. Furthermore, it was observed that activation of brain 5-HT7 receptors rescued mitochondrial dysfunction in female mice from two models of Rett syndrome, a rare neurodevelopmental disorder characterized by severe behavioral and physiological symptoms. Our Western blot analyses performed on cell-lysate and purified mitochondria isolated from neuronal cell line SH-SY5Y showed that 5-HT7 receptors are also expressed into mitochondria. Maximal binding capacity (Bmax) obtained by Scatchard analysis on purified mitochondrial membranes was 0.081 pmol/mg of 5-HT7 receptor protein. Lastly, we evaluated the effect of selective 5-HT7 receptor agonist LP-211 and antagonist (inverse agonist) SB-269970 on mitochondrial respiratory chain (MRC) cytochrome c oxidase activity on mitochondria from SH-SY5Y cells. Our findings provide the first evidence that 5-HT7 receptor is also expressed in mitochondria.

Combined HIV-1 Tat and oxycodone activate the hypothalamic-pituitary-adrenal and -gonadal axes and promote psychomotor, affective, and cognitive dysfunction in female mice

The majority of HIV+ patients present with neuroendocrine dysfunction and ~50% experience co-morbid neurological symptoms including motor, affective, and cognitive dysfunction, collectively termed neuroHIV. In preclinical models, the neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat), promotes neuroHIV pathology that can be exacerbated by opioids. We and others find gonadal steroids, estradiol (E2) or progesterone (P4), to rescue Tat-mediated pathology. However, the combined effects of Tat and opioids on neuroendocrine function and the subsequent ameliorative capacity of gonadal steroids are unknown. We found that conditional HIV-1 Tat expression in naturally-cycling transgenic mice dose-dependently potentiated oxycodone-mediated psychomotor behavior. Tat increased depression-like behavior in a tail-suspension test among proestrous mice, but decreased it among diestrous mice (who already demonstrated greater depression-like behavior); oxycodone reversed these effects. Combined Tat and oxycodone produced apparent behavioral disinhibition of anxiety-like responding which was greater on diestrus than on proestrus. These mice made more central entries in an open field, but spent less time there and demonstrated greater circulating corticosterone. Tat increased the E2:P4 ratio of circulating steroids on diestrus and acute oxycodone attenuated this effect, but repeated oxycodone exacerbated it. Corticotropin-releasing factor was increased by Tat expression, acute oxycodone exposure, and was greater on diestrus compared to proestrus. In human neuroblastoma cells, Tat exerted neurotoxicity that was ameliorated by E2 (1 or 10 nM) or P4 (100, but not 10 nM) independent of oxycodone. Oxycodone decreased gene expression of estrogen and κ-opioid receptors. Thus, neuroendocrine function may be an important target for HIV-1 Tat/opioid interactions.

Mineralocorticoid Receptor and Endothelial Dysfunction in Hypertension.

To review the latest reports of the contributions of the endothelial mineralocorticoid receptor to endothelial dysfunction and hypertension to begin to determine the clinical potential for this pathway for hypertension treatment. Endothelial mineralocorticoid receptor expression is sex-specifically increased in female mice and humans compared with males. Moreover, the expression of endothelial mineralocorticoid receptors is increased by endothelial progesterone receptor activation and naturally occurring fluctuations in progesterone levels (estrous, pregnancy) predict endothelial mineralocorticoid receptor expression levels in female mice. These data follow many previous reports that have indicated that endothelial mineralocorticoid receptor deletion is protective in the development of obesity- and diabetes-associated endothelial dysfunction in female mouse models. These studies have more recently been followed up by reports indicating that both intact endothelial mineralocorticoid receptor and progesterone receptor expression are required for obesity-associated, leptin-mediated endothelial dysfunction in female mice. In addition, the intra-endothelial signaling pathway for endothelial mineralocorticoid receptors to induce dysfunction requires the intact expression of α-epithelial sodium channels (αENaC) in endothelial cells in females. Endothelial mineralocorticoid receptors are sex-specifically upregulated in the vasculature of females, a sex difference which is driven by endothelial progesterone receptor activation, and increased activity of these endothelial mineralocorticoid receptors is a crucial mediator of endothelial dysfunction, and potentially hypertension, in obese female experimental models.

OR25-1 Conditional Deletion of Gonadotropic Androgen Receptor Prevents Elevated Androgen Induced Reproductive Dysfunction in Female Mice

Androgen and its receptor (AR) play a critical role in reproductive function, under both normal physiological and clinically pathophysiological conditions. Many women with hyperandrogenemia suffer irregular menses, oligo-anovulation and infertility. Whether high androgen levels directly or secondarily affect reproduction is unknown. Further, most animal models of hyperandrogenemia induced infertility are associated with obesity which adds a confounding variable to analysis. Therefore, we created a lean elevated androgen model by implantation of a low dose dihydrotestosterone (DHT) that separates the effects of obesity from elevated androgen. Reproduction is tightly controlled by the hypothalamus-pituitary-gonadal axis, androgen could theoretically alter neuroendocrine function at the level of brain and/or pituitary. The goal of this study is to understand how high androgen signaling via the androgen receptor (AR) in pituitary gonadotropes affects reproductive function. Mice with disruption of the Ar gene specifically in pituitary gonadotropes (PitARKO) were produced to explore the role of elevated androgen on the development of reproductive dysfunction in female mice. PitARKO-DHT mice showed significantly improved estrous cyclicity and fertility compared to DHT treated control littermates with intact pituitary Ar (Con-DHT). During 90 days of mating, PitARKO-DHT mice had a significantly increased number of litters (2.3±0.6 vs 0.5±0.3) and number of pups (5.3±2.8 vs 17.0±4.5) compared to Con-DHT mice. The improved reproductive function in PitARKO-DHT mice is partially due to improved pituitary responsiveness to GnRH stimulation. Ex vivo pituitary primary culture confirmed that DHT significantly reduced both basal and GnRH stimulated LH secretion. In vivo, LH secretion is preserved in PitARKO-DHT mice after GnRH stimulation compared to CON-DHT mice. GnRH induces the release of gonadotropins via an increase in cytosolic Ca2+ concentration, and reduced cytoplasmic Ca2+ concentrations may interfere with exocytosis of granules of LH. GEM, a calcium binding protein, inhibits voltage-gated Ca2+ channel activity and reduces Ca2+ influx in gonadotropes, which can affect gonadotropin secretion. We observed that Gem mRNA levels were significantly increased in Con-DHT treated mice (6 fold higher) compared to PitARKO-DHT mice. Further, the intracellular calcium influx signals were dramatically attenuated in Con-DHT mice compared to Con-no DHT mice, and were preserved in PitARKO-DHT mice after GnRH stimulation. These findings highlight gonadotrope AR as an extra ovarian regulator playing an important role in reproductive physiology and that high androgens may impact function through AR regulated Gem expression.

OR05-4 Androgen Induced Metabolic Dysfunction in Female Mice is Prevented by Deletion of Liver Androgen Receptor

Women with elevated androgen often exhibit hyperinsulinemia and insulin resistance. Many models have been created to produce metabolic dysfunction by manipulating androgen levels. Previously we reported that hyperandrogenemia produced by implantation of a DHT pellet may induce whole body insulin resistance through hepatic androgen receptor (AR) in vitro. Therefore, we conditionally disrupted the hepatic AR both developmentally (by breeding the albumin-CRE mouse (alb-CRE; liver-specific Cre expression) with ARfl/fl mice (LivARKO)) and acutely (by tail vein injection in ARfl/fl mice of adeno-associated virus with albumin promoter driven Cre vector (adLivARKO)). Impaired glucose tolerance, pyruvate tolerance, and insulin tolerance were prevented in both LivARKO-DHT and adLivARKO-DHT mice compared to control mice treated with DHT (ARfl/fl; cre-; Con-DHT) mice. Further, the glucose infusion rate is significantly reduced in LivARKO-DHT mice compared to Con-DHT mice as measured during hyperinsulinemia-euglycemia clamp suggesting improved insulin-induced glucose disposal. We observed that primary hepatocytes treated with DHT from both women and female mice showed reduced PI3K-pAKT and pFOXO1 activation by insulin, and increased ex vivo glucose production. LivARKO primary hepatocytes were resistant to DHT induced attenuated insulin signaling and increased gluconeogenesis (ex vivo) and this was confirmed in LivARKO in vivo. These data support a paradigm in which in females, elevated androgen disrupts metabolic function via hepatic AR.

Translocator Protein Ligand Protects against Neurodegeneration in the MPTP Mouse Model of Parkinsonism.

Parkinson's disease is the second most common neurodegenerative disease, after Alzheimer's disease. Parkinson's disease is a movement disorder with characteristic motor features that arise due to the loss of dopaminergic neurons from the substantia nigra. Although symptomatic treatment by the dopamine precursor levodopa and dopamine agonists can improve motor symptoms, no disease-modifying therapy exists yet. Here, we show that Emapunil (AC-5216, XBD-173), a synthetic ligand of the translocator protein 18, ameliorates degeneration of dopaminergic neurons, preserves striatal dopamine metabolism, and prevents motor dysfunction in female mice treated with the MPTP, as a model of parkinsonism. We found that Emapunil modulates the inositol requiring kinase 1α (IRE α)/X-box binding protein 1 (XBP1) unfolded protein response pathway and induces a shift from pro-inflammatory toward anti-inflammatory microglia activation. Previously, Emapunil was shown to cross the blood-brain barrier and to be safe and well tolerated in a Phase II clinical trial. Therefore, our data suggest that Emapunil may be a promising approach in the treatment of Parkinson's disease.SIGNIFICANCE STATEMENT Our study reveals a beneficial effect of Emapunil on dopaminergic neuron survival, dopamine metabolism, and motor phenotype in the MPTP mouse model of parkinsonism. In addition, our work uncovers molecular networks which mediate neuroprotective effects of Emapunil, including microglial activation state and unfolded protein response pathways. These findings not only contribute to our understanding of biological mechanisms of translocator protein 18 (TSPO) function but also indicate that translocator protein 18 may be a promising therapeutic target. We thus propose to further validate Emapunil in other Parkinson's disease mouse models and subsequently in clinical trials to treat Parkinson's disease.

Leptin‐Induced Endothelial Dysfunction is Mediated by Endothelial Mineralocorticoid Receptor Epithelial Sodium Channel Activation in Female Mice

Rising rates of hypertension are significantly associated with rising rates of obesity. While the mechanisms of obesity‐associated hypertension have been heavily investigated, these studies have overwhelmingly favored men and male rodents as research subjects. Therefore, the sex‐specific mechanisms of obesity‐associated hypertension require investigation. Our lab has previously shown that the adipocyte‐derived hormone leptin, whose levels are drastically increased in obesity, is required for hypertension and endothelial dysfunction in obese female mouse models, the development of which is dependent on leptin‐induced aldosterone secretion and subsequent mineralocorticoid receptor (MR) activation. The epithelial sodium channel alpha (ENaC), whose transcription is directly upregulated by MR activation, has also been implicated in endothelial dysfunction in female mice. We therefore sought to test the hypothesis that endothelial MR activation mediates leptin‐induced endothelial dysfunction via increased activation of ENaC. To test that endothelial MR activation, specifically, is required for leptin‐induced endothelial dysfunction we infused female endothelial cell‐specific MR knockout mice (ECMR KO) with leptin (0.9 mg/kg/day) via subcutaneous osmotic minipump. Wild‐type mice (WT) developed endothelial dysfunction, as assessed by vascular (aortic rings) relaxation to acetylcholine (10−9 M–10−5 M, nonlinear fit curve), in response to leptin infusion (P<0.05, two‐way ANOVA, n=4–6). However, ECMR KO female mice infused with leptin were protected from endothelial dysfunction (n=6–7), indicating that endothelial MR activation mediates leptin‐induced endothelial dysfunction in female mice. In contrast, no effect of leptin nor endothelial MR deletion was observed on endothelial function in male WT and ECMR KO mice (n=2–5). To assess if MR‐mediated ENaC activation plays a role in leptin‐induced endothelial dysfunction, leptin‐infused female mice were treated with amiloride (ENaC antagonist, 100 ug/day in drinking water) which ablated endothelial dysfunction in female leptin‐infused mice (P<0.05, two‐way ANOVA, n=4), indicating that leptin‐induced endothelial dysfunction requires ENaC activation. In addition, we sought to assess the potential for leptin to upregulate endothelial cell ENaC expression in female mice. Isolated endothelial cells, which were obtained from the aorta via CD31‐specific magnetic bead sorting, were extracted from female Agouti hyperleptinemic obese mice and RT‐PCR analysis for mRNA expression of ENaC demonstrated elevated ENaC expression in hyperleptinemic mice (10.8±0.5‐fold change from lean control female mice, n=2–3). These elevations in endothelial ENaC mRNA expression are associated with elevated plasma aldosterone in hyperleptinemic female mice, indicating that leptin‐induced aldosterone induces endothelial dysfunction via increased MR activation, which ultimately leads to elevated ENaC expression and activation. Therefore, MR‐stimulated ENaC upregulation may play a crucial role in the development of leptin‐mediated cardiovascular diseases in obese females.Support or Funding InformationThis work was supported by the following: NIH 1R01HL130301‐01, AHA 16IRG27770047, NIH 5F32HL136191‐0This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective mechanisms of triptorelin against tripterygium glycoside-induced ovarian dysfunction

The aim of this study was to investigate the protective effects of Triptorelin (TRI) on Tripterygium Glycoside-induced Ovarian Dysfunction (TG-OD). Thirty mice with normal estrous cycles were randomly divided into three groups (n=10): the control group (group A) was orally administered 0.35 ml of saline daily; the TH group (group B) was orally administered 0.35 ml of TG solution from the 8th d for 10 w continuously; the TG+TRI group (group C) was subcutaneously injected with 0.1 mg/kg TRI daily, as well as orally administered 0.35 ml of TG solution from the 8th d for 10 w continuously. All mice were sacrificed during the 11th week to detect blood anti-Mullerian hormone, follicle-stimulating hormone, luteinizing hormone, and estradiol; calculate the ovarian index; and observe the morphologies of ovarian tissues. Mice in group C were more flexible, and had increased weight, reduced folliclestimulating hormone and luteinizing hormone, and increased anti-Mullerian hormone and estradiol; additionally, ovarian follicles and mature follicles increased. TG caused gonad and ovarian dysfunction in female mice; TRI exhibited protective effects against TG-induced gonad and ovarian dysfunction in female mice.

Discovery of GnIH and Its Role in Hypothyroidism-Induced Delayed Puberty

It is known that hypothyroidism delays puberty in mammals. Interaction between the hypothalamo-pituitary-thyroid (HPT) and hypothalamo-pituitary-gonadal (HPG) axes may be important processes in delayed puberty. Gonadotropin-inhibitory hormone (GnIH) is a newly discovered hypothalamic neuropeptide that inhibits gonadotropin synthesis and release in quail. It now appears that GnIH is conserved across various mammals and primates, including humans, and inhibits reproduction. We have further demonstrated that GnIH is involved in pubertal delay induced by thyroid dysfunction in female mice. Hypothyroidism delays pubertal onset with the increase in hypothalamic GnIH expression and the decrease in circulating gonadotropin and estradiol levels. Thyroid status regulates GnIH expression by epigenetic modification of the GnIH promoter region. Furthermore, knockout of GnIH gene abolishes the effect of hypothyroidism on delayed pubertal onset. Accordingly, it is considered that GnIH is a mediator of pubertal disorder induced by thyroid dysfunction. This is a novel function of GnIH that interacts between the HPT-HPG axes in pubertal onset delay. This mini-review summarizes the structure, expression, and function of GnIH and highlights the action of GnIH in pubertal disorder induced by thyroid dysfunction.

Stimulation of the brain serotonin receptor 7 rescues mitochondrial dysfunction in female mice from two models of Rett syndrome

Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioral and physiological symptoms. Mutations in the methyl CpG binding protein 2 gene (MECP2) cause more than 95% of classic cases, and currently there is no cure for this devastating disorder. Recently we have demonstrated that neurobehavioral and brain molecular alterations can be rescued in a RTT mouse model, by pharmacological stimulation of the brain serotonin receptor 7 (5-HT7R). This member of the serotonin receptor family, crucially involved in the regulation of brain structural plasticity and cognitive processes, can be stimulated by systemic repeated treatment with LP-211, a brain-penetrant selective agonist. The present study extends previous findings by demonstrating that LP-211 treatment (0.25 mg/kg, once per day for 7 days) rescues mitochondrial respiratory chain impairment, oxidative phosphorylation deficiency and the reduced energy status in the brain of heterozygous female mice from two highly validated mouse models of RTT (MeCP2-308 and MeCP2-Bird mice). Moreover, LP-211 treatment completely restored the radical species overproduction by brain mitochondria in the MeCP2-308 model and partially recovered the oxidative imbalance in the more severely affected MeCP2-Bird model. These results provide the first evidence that RTT brain mitochondrial dysfunction can be rescued targeting the brain 5-HT7R and add compelling preclinical evidence of the potential therapeutic value of LP-211 as a pharmacological approach for this devastating neurodevelopmental disorder.

Metabolic dysfunction in female mice with disruption of 5α-reductase 1.

5α-Reductases irreversibly catalyse A-ring reduction of pregnene steroids, including glucocorticoids and androgens. Genetic disruption of 5α-reductase 1 in male mice impairs glucocorticoid clearance and predisposes to glucose intolerance and hepatic steatosis upon metabolic challenge. However, it is unclear whether this is driven by changes in androgen and/or glucocorticoid action. Female mice with transgenic disruption of 5α-reductase 1 (5αR1-KO) were studied, representing a ‘low androgen’ state. Glucocorticoid clearance and stress responses were studied in mice aged 6 months. Metabolism was assessed in mice on normal chow (aged 6 and 12 m) and also in a separate cohort following 1-month high-fat diet (aged 3 m). Female 5αR1-KO mice had adrenal suppression (44% lower AUC corticosterone after stress), and upon corticosterone infusion, accumulated hepatic glucocorticoids (~27% increased corticosterone). Female 5αR1-KO mice aged 6 m fed normal chow demonstrated insulin resistance (~35% increased area under curve (AUC) for insulin upon glucose tolerance testing) and hepatic steatosis (~33% increased hepatic triglycerides) compared with controls. This progressed to obesity (~12% increased body weight) and sustained insulin resistance (~38% increased AUC insulin) by age 12 m. Hepatic transcript profiles supported impaired lipid β-oxidation and increased triglyceride storage. Female 5αR1-KO mice were also predisposed to develop high-fat diet-induced insulin resistance. Exaggerated predisposition to metabolic disorders in female mice, compared with that seen in male mice, after disruption of 5αR1 suggests phenotypic changes may be underpinned by altered metabolism of glucocorticoids rather than androgens.

Daily exercise prevents diastolic dysfunction and oxidative stress in a female mouse model of western diet induced obesity by maintaining cardiac heme oxygenase-1 levels

ObjectiveObesity is a global epidemic with profound cardiovascular disease (CVD) complications. Obese women are particularly vulnerable to CVD, suffering higher rates of CVD compared to non-obese females. Diastolic dysfunction is the earliest manifestation of CVD in obese women but remains poorly understood with no evidence-based therapies. We have shown early diastolic dysfunction in obesity is associated with oxidative stress and myocardial fibrosis. Recent evidence suggests exercise may increase levels of the antioxidant heme oxygenase-1 (HO-1). Accordingly, we hypothesized that diastolic dysfunction in female mice consuming a western diet (WD) could be prevented by daily volitional exercise with reductions in oxidative stress, myocardial fibrosis and maintenance of myocardial HO-1 levels. Materials/MethodsFour-week-old female C57BL/6J mice were fed a high-fat/high-fructose WD for 16weeks (N=8) alongside control diet fed mice (N=8). A separate cohort of WD fed females was allowed a running wheel for the entire study (N=7). Cardiac function was assessed at 20weeks by high-resolution cardiac magnetic resonance imaging (MRI). Functional assessment was followed by immunohistochemistry, transmission electron microscopy (TEM) and Western blotting to identify pathologic mechanisms and assess HO-1 protein levels. ResultsThere was no significant body weight decrease in exercising mice, normalized body weight 14.3g/mm, compared to sedentary mice, normalized body weight 13.6g/mm (p=0.38). Total body fat was also unchanged in exercising, fat mass of 6.6g, compared to sedentary mice, fat mass 7.4g (p=0.55). Exercise prevented diastolic dysfunction with a significant reduction in left ventricular relaxation time to 23.8ms for exercising group compared to 33.0ms in sedentary group (p<0.01). Exercise markedly reduced oxidative stress and myocardial fibrosis with improved mitochondrial architecture. HO-1 protein levels were increased in the hearts of exercising mice compared to sedentary WD fed females. ConclusionsThis study provides seminal evidence that exercise can prevent diastolic dysfunction in WD-induced obesity in females even without changes in body weight. Furthermore, the reduction in myocardial oxidative stress and fibrosis and improved HO-1 levels in exercising mice suggests a novel mechanism for the antioxidant effect of exercise.

Abstract P293: Endothelial Sodium Channel Activation Promotes Vascular Stiffness in Obese Female Mice

Obesity-associated arterial stiffening is an independent predictor of cardiovascular disease (CVD) events. Although premenopausal non-obese women are protected against CVD, aortic stiffening in obese women is more common than in men. This disproportionate increase in vascular stiffness in obese females may partly explain their loss of sex-related CVD protection. Recent studies have suggested a role for endothelial sodium channel (ENaC) activation in promotion of endothelial stiffness and suppression of flow-(nitric oxide) mediated vasodilation. Increased mineralocorticoid receptor (MR) activation mediated endothelial stiffness is promoted, in part, by ENaC activation. In this regard, we have recently reported increased aortic stiffness, MR and ENaC expression and endothelial dysfunction in female mice fed a high fat and high fructose diet (western diet [WD]). This increase in aortic stiffness was prevented by very low dose MR antagonism. Accordingly, we hypothesized that inhibition of MR-mediated ENaC activation by using a very low dose of the ENaC inhibitor, amiloride, would prevent arterial stiffening and vascular dysfunction in WD-fed female mice. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1mg/kg/day) for 16 weeks. Amiloride significantly attenuated WD-induced increases in aortic stiffness in vivo as measured by pulse wave velocity as well as in vitro endothelial stiffness as measured by atomic force microscopy. Moreover, incubation of aortic explants with very low dose of amiloride (1 μM) inhibited WD-induced aortic stiffness in aorta explants from WD-fed female mice. Amiloride also prevented WD-induced impairment in acetylcholine-induced aortic vasodilatation and flow-mediated dilation in mesenteric arteries. Taken together, these observations support a role for ENaC activation in diet-induced vascular stiffening in obese females.

Urinary bladder hypersensitivity and dysfunction in female mice following early life and adult stress

Early adverse events have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. Despite high clinical relevance and reports of stress-related symptom exacerbation, animal models investigating the contribution of early life stress to female urological pain are lacking. We examined the impact of neonatal maternal separation (NMS) on bladder sensitivity and visceral neuroimmune status both prior-to, and following, water avoidance stress (WAS) in adult female mice. The visceromotor response to urinary bladder distension was increased at baseline and 8d post-WAS in NMS mice, while colorectal sensitivity was transiently increased 1d post-WAS only in naïve mice. Bladder micturition rate and output, but not fecal output, were also significantly increased following WAS in NMS mice. Changes in gene expression involved in regulating the stress response system were observed at baseline and following WAS in NMS mice, and WAS reduced serum corticosterone levels. Cytokine and growth factor mRNA levels in the bladder, and to a lesser extent in the colon, were significantly impacted by NMS and WAS. Peripheral mRNA levels of stress-responsive receptors were differentially influenced by early life and adult stress in bladder, but not colon, of naïve and NMS mice. Histological evidence of mast cell degranulation was increased in NMS bladder, while protein levels of protease activated receptor 2 (PAR2) and transient receptor potential ankyrin 1 (TRPA1) were increased by WAS. Together, this study provides new insight into mechanisms contributing to stress associated symptom onset or exacerbation in patients exposed to early life stress.

Early and gender-specific differences in spinal cord mitochondrial function and oxidative stress markers in a mouse model of ALS.

IntroductionAmyotrophic lateral sclerosis (ALS) is a motor neuron disease with a gender bias towards major prevalence in male individuals. Several data suggest the involvement of oxidative stress and mitochondrial dysfunction in its pathogenesis, though differences between genders have not been evaluated. For this reason, we analysed features of mitochondrial oxidative metabolism, as well as mitochondrial chain complex enzyme activities and protein expression, lipid profile, and protein oxidative stress markers, in the Cu,Zn superoxide dismutase with the G93A mutation (hSOD1-G93A)- transgenic mice and Neuro2A(N2A) cells overexpressing hSOD1-G93A.Results and ConclusionsOur results show that overexpression of hSOD1-G93A in transgenic mice decreased efficiency of mitochondrial oxidative phosphorylation, located at complex I, revealing a temporal delay in females with respect to males associated with a parallel increase in selected markers of protein oxidative damage. Further, females exhibit a fatty acid profile with higher levels of docosahexaenoic acid at 30 days. Mechanistic studies showed that hSOD1-G93A overexpression in N2A cells reduced complex I function, a defect prevented by 17β-estradiol pretreatment. In conclusion, ALS-associated SOD1 mutation leads to delayed mitochondrial dysfunction in female mice in comparison with males, in part attributable to the higher oestrogen levels of the former. This study is important in the effort to further understanding of whether different degrees of spinal cord mitochondrial dysfunction could be disease modifiers in ALS.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0271-6) contains supplementary material, which is available to authorized users.

Endothelial Mineralocorticoid Receptor Deletion Prevents Diet-Induced Cardiac Diastolic Dysfunction in Females.

Overnutrition and insulin resistance are especially prominent risk factors for the development of cardiac diastolic dysfunction in females. We recently reported that consumption of a Western diet (WD) containing excess fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) for 16 weeks resulted in cardiac diastolic dysfunction and aortic stiffening in young female mice and that these abnormalities were prevented by mineralocorticoid receptor blockade. Herein, we extend those studies by testing whether WD-induced diastolic dysfunction and factors contributing to diastolic impairment, such as cardiac fibrosis, hypertrophy, inflammation, and impaired insulin signaling, are modulated by excess endothelial cell mineralocorticoid receptor signaling. Four-week-old female endothelial cell mineralocorticoid receptor knockout and wild-type mice were fed mouse chow or WD for 4 months. WD feeding resulted in prolonged relaxation time, impaired diastolic septal wall motion, and increased left ventricular filling pressure indicative of diastolic dysfunction. This occurred in concert with myocardial interstitial fibrosis and cardiomyocyte hypertrophy that were associated with enhanced profibrotic (transforming growth factor β1/Smad) and progrowth (S6 kinase-1) signaling, as well as myocardial oxidative stress and a proinflammatory immune response. WD also induced cardiomyocyte stiffening, assessed ex vivo using atomic force microscopy. Conversely, endothelial cell mineralocorticoid receptor deficiency prevented WD-induced diastolic dysfunction, profibrotic, and progrowth signaling, in conjunction with reductions in macrophage proinflammatory polarization and improvements in insulin metabolic signaling. Therefore, our findings indicate that increased endothelial cell mineralocorticoid receptor signaling associated with consumption of a WD plays a key role in the activation of cardiac profibrotic, inflammatory, and growth pathways that lead to diastolic dysfunction in female mice.

Chronic Alcohol Exposure is Associated with Decreased Neurogenesis, Aberrant Integration of Newborn Neurons, and Cognitive Dysfunction in Female Mice.

Neurological deficits of alcohol use disorder (AUD) have been attributed to dysfunctions of specific brain structures. Studies of alcoholic patients and chronic alcohol exposure animal models consistently identify reduced hippocampal mass and cogntive dysfunctions as a key alcohol-induced brain adaptation. However, the precise substrate of chronic alcohol exposure that leads to structural and functional impairments of the hippocampus is largely unknown. Using a calorie-matched alcohol feeding method, we tested whether chronic alcohol exposure targets neural stem cells and neurogenesis in the adult hippocampus. The effect of alcohol on proliferation of neural stem cells as well as cell fate determination and survival of newborn cells was evaluated via bromodeoxyuridine pulse and chase methods. A retrovirus-mediated single-cell labeling method was used to determine the effect of alcohol on the morphological development and circuitry incorporation of individual hippocampal newborn neurons. Finally, novel object recognition (NOR) and Y-maze tests were performed to examine whether disrupted neurogenesis is associated with hippocampus-dependent functional deficits in alcohol-fed mice. Chronic alcohol exposure reduced proliferation of neural stem cells and survival rate of newborn neurons; however, the fate determination of newborn cells remained unaltered. Moreover, the dendritic spine density of newborn neurons significantly decreased in alcohol-fed mice. Impaired spine formation indicates that alcohol interfered the synaptic connectivity of newborn neurons with excitatory neurons originating from various areas of the brain. In the NOR test, alcohol-fed mice displayed deficits in the ability to discriminate the novel object. Our study revealed that chronic alcohol exposure disrupted multiple steps of neurogenesis, including the production and development of newborn neurons. In addition, chronic alcohol exposure altered connectivity of newborn neurons with other input neurons. Decreased neurogenesis and aberrant integration of newborn neurons into hippocampal networks are closely associated with deficits in hippocampus-dependent cognitive functions of alcohol-fed mice.

Abstract 131: Endothelial Cell Mineralocorticoid Receptor: A Critical Player in Aortic Stiffness and Cardiac Diastolic Dysfunction in Female Mice

Enhanced activation of mineralocorticoid receptors (MRs) impairs insulin metabolic signaling, increases oxidative stress, and induces maladaptive immune responses with associated CV abnormalities. Emerging information suggests that obesity and insulin resistance predict CV stiffness in females. However, the specific role of endothelial cell MR (ECMR) has not been explored. Accordingly, we hypothesized that ECMR signaling modulated by a western diet (WD) impairs insulin signaling and increases inflammation, fibrosis and CV stiffness in females. Four week-old ECMR knockout (ECMR -/- ) and wild-type female mice were fed a mouse chow or WD containing fat (46%), sucrose (17.5%), and high fructose (17.5%) for 16 weeks. WD prompted MR to bind the hormone response element (nGnACAnnnTGTnCn) on the site of ENaC promoter and induced an increase in ENaC expression that was associated with increased aortic and EC stiffness as determined by in vivo pulse wave velocity and ex vivo atomic force microscopy techniques, respectively The elevated aortic stiffness was accompanied by increased expression of cytokines IL-17, MCP-1 and M1 markers CD 86, and CD11c. ENaC expression was reduced in the ECMR -/- vasculature with a decrease in WD-increase in aortic and EC stiffness.. ECMR -/- also improved aortic vasorelaxation to Ach, SNP (10 -9 -10 -4 mol/L), and insulin (0.1- 300 ng/ml), which were impaired by WD. Additionally, ECMR -/- restored WD-induced cardiac diastolic dysfunction assessed by cardiac MRI and echocardiography. Diastolic dysfunction was related to cardiomyocyte hypertrophy, oxidative stress, and fibrosis and occurred with enhanced activation of S6 kinase-1, Erk 1/2, serine phosphorylation of IRS-1, inactivation of PI3K-AKT-eNOS signaling pathways and the pro-fibrotic TGF-β1/ Smad signaling pathway and increased macrophage pro-inflammatory polarization. ECMR -/- markedly attenuated the cardiac functional and changes signaling induced by WD. These findings suggest that increased ECMR signaling and associated ENaC activation plays a key role in WD induced insulin metabolic sinaling impairment, adaptive pro-inflammatory responses, macrophage polarization and associated aortic stiffness and cardiac diastolic dysfunction in females.

Involvement of HDL receptor SR-BI-mediated vitamin e uptake in murine neural tube closure

Involvement of HDL receptor SR-BI-mediated vitamin e uptake in murine neural tube closure

Mineralocorticoid receptor blockade prevents Western diet-induced diastolic dysfunction in female mice.

Overnutrition/obesity predisposes individuals, particularly women, to diastolic dysfunction (DD), an independent predictor of future cardiovascular disease. We examined whether low-dose spironolactone (Sp) prevents DD associated with consumption of a Western Diet (WD) high in fat, fructose, and sucrose. Female C57BL6J mice were fed a WD with or without Sp (1 mg·kg(-1)·day(-1)). After 4 mo on the WD, mice exhibited increased body weight and visceral fat, but similar blood pressures, compared with control diet-fed mice. Sp prevented the development of WD-induced DD, as indicated by decreased isovolumic relaxation time and an improvement in myocardial performance (<Tei index) and septal annular velocity (<E'-to-A' ratio), as assessed by echocardiography, as well as decreased diastolic relaxation time/increased diastolic initial filling rate, as assessed by MRI. The relationship between passive sarcomere length of cardiac myocytes and ventricular pressure was monitored using di-8-ANEPPS staining of the t-tubule network in hearts ex vivo. Sp administration led to longer sarcomere lengths at each pressure indicative of improved ventricular compliance in WD-fed mice. Sp also prevented left ventricular hypertrophy, interstitial fibrosis, and oxidative stress. Sp prevented the WD-induced increased expression of myocardial proinflammatory M1 macrophage markers monocyte chemoattractant protein-1 and CD11c and increased the expression of the anti-inflammatory M2 macrophage marker CD206. These findings demonstrate that WD-induced DD is associated with increased oxidant stress, fibrosis, and immune dysregulation. Mineralocorticoid receptor antagonism enhanced M2 macrophage polarization and ameliorated oxidant stress and fibrosis. This work supports a novel blood pressure-independent effect of MR antagonism as a strategy to prevent diet-induced DD in women. Mineralocorticoid antagonism; low-dose spironolactone; aldosterone;high-fat diet; high-fructose diet; oxidative stress; inflammation; cardiac hypertrophy; myocardial compliance.