Wild-type Control Mice Research Articles

Multiple tissue-specific epigenetic alterations regulate persistent gene expression changes following developmental DES exposure in mouse reproductive tissues

ABSTRACT Clinically, developmental exposure to the endocrine disrupting chemical, diethylstilboestrol (DES), results in long-term male and female infertility. Experimentally, developmental exposure to DES results in abnormal reproductive tract phenotypes in male and female mice. Previously, we reported that neonatal DES exposure causes ERα-mediated aberrations in the transcriptome and in DNA methylation in seminal vesicles (SVs) of adult mice. However, only a subset of DES-altered genes could be explained by changes in DNA methylation. We hypothesized that alterations in histone modification may also contribute to the altered transcriptome during SV development. To test this idea, we performed a series of genome-wide analyses of mouse SVs at pubertal and adult developmental stages in control and DES-exposed wild-type and ERα knockout mice. Neonatal DES exposure altered ERα-mediated mRNA and lncRNA expression in adult SV, including genes encoding chromatin-modifying proteins that can impact histone H3K27ac modification. H3K27ac patterns, particularly at enhancers, and DNA methylation were reprogrammed over time during normal SV development and after DES exposure. Some of these reprogramming changes were ERα-dependent, but others were ERα-independent. A substantial number of DES-altered genes had differential H3K27ac peaks at nearby enhancers. Comparison of gene expression changes, H3K27ac marks and DNA methylation marks between adult SV and adult uterine tissue from ovariectomized mice neonatally exposed to DES revealed that most of the epigenetic changes and altered genes were distinct in the two tissues. These findings indicate that the effects of developmental DES exposure cause reprogramming of reproductive tract tissue differentiation through multiple epigenetic mechanisms.

RNF186/EPHB2 Axis Is Essential in Regulating TNF Signaling for Colorectal Tumorigenesis in Colorectal Epithelial Cells.

The receptor tyrosine kinase EPHB2 (EPH receptor B2) is highly expressed in many human cancer types, especially in gastrointestinal cancers, such as colorectal cancer. Several coding mutations of the EPHB2 gene have been identified in many cancer types, suggesting that EPHB2 plays a critical role in carcinogenesis. However, the exact functional mechanism of EPHB2 in carcinogenesis remains unknown. In this study, we find that EPHB2 is required for TNF-induced signaling activation and proinflammatory cytokine production in colorectal epithelial cells. Mechanistically, after TNF stimulation, EPHB2 is ubiquitinated by its E3 ligase RNF186. Then, ubiquitinated EPHB2 recruits and further phosphorylates TAB2 at nine tyrosine sites, which is a critical step for the binding between TAB2 and TAK1. Due to defects in TNF signaling in RNF186-knockout colorectal epithelial cells, the phenotype of colitis-propelled colorectal cancer model in RNF186-knockout mice is significantly reduced compared with that in wild-type control mice. Moreover, we find that a genetic mutation in EPHB2 identified in a family with colorectal cancer is a gain-of-function mutation that promoted TNF signaling activation compared with wild-type EPHB2. We provide evidence that the EPHB2-RNF186-TAB2-TAK1 signaling cascade plays an essential role in TNF-mediated signal transduction in colorectal epithelial cells and the carcinogenesis of colorectal cancer, which may provide potential targets for the treatment of colorectal cancer.

RF01 | PMON44 Absence of Kisspeptin in KNDy Neurons of Mice Causes Sexually Dimorphic Metabolic Dysfunction on a High-Fat Diet

Abstract Kisspeptin neurons in the arcuate nucleus (ARC) of the hypothalamus are considered as the gonadotropin-releasing hormone (GnRH) pulse generator to control the cascade of hormone secretions that constitute the reproductive axis. These neurons typically co-express Neurokinin B and Dynorphin and are thus dubbed KNDy neurons. In addition, KNDy neuron kisspeptin is hypothesized to be a major sensor and regulator of metabolic homeostasis while relaying energy status to the hypothalamic-pituitary-gonad (HPG) axis. However, the direct metabolic impact of KNDy neuron kisspeptin has not been well-characterized. To explore this role, we examined the metabolic profile of our previously established KNDy neuron-specific kisspeptin knock-out mouse model (Pdyn-Cre/Kissfl/fl, or KO). To do so, we performed glucose tolerance tests, EchoMRI body composition analysis, and measured body weight in wild type (WT) control or KO mice fed with either regular chow or a high-fat diet (HFD, 60% kcal fat, Research Diets, New Brunswick, NJ) for 12 weeks post-weaning. At 4 weeks on the HFD, KO females weighed significantly more than HFD WT females, which continued through the remaining 8 weeks. Additionally, we found significantly decreased glucose tolerance and increased fat mass in HFD KO females compared to HFD WT females. However, KO males exhibited no significant differences in body weight, body composition, or glucose tolerance between the genotypes on either diet. This data suggests that KNDy neuron kisspeptin is critical for metabolic homeostasis and preventing metabolic dysfunction when challenged with a high-fat diet. Our findings further suggest a sexual dimorphism whereby KNDy neuron kisspeptin performs this action predominantly in females. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Blocking Autophagy in M1 Macrophages Enhances Virus Replication and Eye Disease in Ocularly Infected Transgenic Mice.

Macrophages are one of the first innate immune infiltrates in the cornea of mice following ocular infection with herpes simplex virus 1 (HSV-1). Using gamma interferon (IFN-γ) and interleukin-4 (IL-4) injections to polarize macrophages into M1 and M2, respectively, and in M1 and M2 conditional knockout mice, we have shown that M1 macrophages play an important role in suppressing both virus replication in the eye and eye disease in HSV-1-infected mice. Autophagy is also important in controlling HSV infection and integrity of infected cells. To determine if blocking autophagy in M1 and M2 macrophages affects HSV-1 infectivity and eye disease, we generated two transgenic mouse strains expressing the HSV-1 γ34.5 autophagy gene under the M1 promoter (M1-γ34.5) or the M2 promoter (M2-γ34.5). We found that blocking autophagy in M1 macrophages increased both virus replication in the eyes and eye disease in comparison to blocking autophagy in M2 macrophages or wild-type (WT) control mice, but blocked autophagy did not affect latency-reactivation. However, blocking autophagy affected fertility in both M1 and M2 transgenic mice. Analysis of 62 autophagy genes and 32 cytokines/chemokines from infected bone marrow-derived macrophages from M1-γ34.5, M2-γ34.5, and WT mice suggested that upregulation of autophagy-blocking genes (i.e., Hif1a, Mtmr14, mTOR, Mtmr3, Stk11, and ULK2) and the inflammatory tumor necrosis factor alpha (TNF-α) gene in M1-γ34.5 transgenic mice correlated with increased pathogenicity, while upregulation of proautophagy genes (Nrbf2 and Rb1cc1) in M2-γ34.5 macrophages correlated with reduced pathogenicity. The in vivo and in vitro responses of M1-γ34.5 and M2-γ34.5 transgenic mice to HSV-1 infection were independent of the presence of the γ34.5 gene in wild-type HSV-1. Our results suggest that M1 macrophages, but not M2 macrophages, play an important role in autophagy relative to primary virus replication in the eye and eye disease in infected mice. IMPORTANCE Autophagy plays a critical role in clearing, disassembling, and recycling damaged cells, thus limiting inflammation. The HSV-1 γ34.5 gene is involved in neurovirulence and immune evasion by blocking autophagy in infected cells. We found that blocking autophagy in M1 macrophages enhances HSV-1 virus replication in the eye and eye disease in ocularly infected transgenic mice. Our results also show the suppressive effects of γ34.5 on immune responses to infection, suggesting the importance of intact autophagy in M1 but not M2 macrophages in controlling primary infection and eye disease.

Hepatic CYP2B10 is highly induced by binge ethanol and contributes to acute-on-chronic alcohol-induced liver injury.

The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.

Moringa Oleifera Alleviates Aβ Burden and Improves Synaptic Plasticity and Cognitive Impairments in APP/PS1 Mice.

Alzheimer’s disease is a global public health problem and the most common form of dementia. Due to the failure of many single therapies targeting the two hallmarks, Aβ and Tau, and the multifactorial etiology of AD, there is now more and more interest in nutraceutical agents with multiple effects such as Moringa oleifera (MO) that have strong anti-oxidative, anti-inflammatory, anticholinesterase, and neuroprotective virtues. In this study, we treated APP/PS1 mice with a methanolic extract of MO for four months and evaluated its effect on AD-related pathology in these mice using a multitude of behavioral, biochemical, and histochemical tests. Our data revealed that MO improved behavioral deficits such as anxiety-like behavior and hyperactivity and cognitive, learning, and memory impairments. MO treatment abrogated the Aβ burden to wild-type control mice levels via decreasing BACE1 and AEP and upregulating IDE, NEP, and LRP1 protein levels. Moreover, MO improved synaptic plasticity by improving the decreased GluN2B phosphorylation, the synapse-related proteins PSD95 and synapsin1 levels, the quantity and quality of dendritic spines, and neurodegeneration in the treated mice. MO is a nutraceutical agent with promising therapeutic potential that can be used in the management of AD and other neurodegenerative diseases.

Cathepsin L was involved in vascular aging by mediating phenotypic transformation of vascular cells

Vascular media and adventitia-induced remodeling plays an important role in vascular aging. However, the mechanism remains unclear. This study aims to investigate the mechanisms underlying vascular aging. Transcriptome analysis revealed that the expression of cathepsin L (CTSL) significantly decreased in arteries of old mice (24 months old) compared with that in arteries of young mice (4 months old), which was confirmed by immunohistochemistry and Western blot. The expression of CTSL in adventitia fibroblasts (AFs) and vascular smooth muscle cells (VSMCs) of aged mice was lower than that of young mice. Compared with wild-type control mice, CTSL knockout (CTSL - /−) mice had increased collagen deposition (fibrosis) and decreased telomerase activity and LC3Ⅱ/ LC3Ⅰratio. The expression of mammalian target of rapamycin (mTOR) and osteopontin (OPN) increased in aortas of CTSL−/−mice compared with that in aortas of wild-type control mice. In vitro, lentivirus-mediated CTSL knockdown induced VSMCs senescence and AFs transformed into myofibroblasts (MFs). Rapamycin, a mTOR inhibitor, inhibited CTSL deficiency induced VSMCs senescence, osteopontin (OPN) secretion and AFs migration. In conclusion, the decreased level of CTSL with age may participate in vascular aging by promoting the phenotypic transformation of vascular cells.

Overexpression of miR-210 ameliorates endothelial dysfunction in type 2 diabetic mice

Abstract Background MicroRNA (miR)-210 plays a protective role in many cardiometabolic diseases, and its levels are reduced in whole blood, erythrocytes, and plasma in type 2 diabetes mellitus (T2DM). Our recent study demonstrated that miR-210 is downregulated in carotid artery plaques from patients with T2DM compared to non-diabetic patients. However, the functional role of miR-210 in endothelial dysfunction is not fully understood. Purpose This study aimed to investigate the potential therapeutic value of miR-210 overexpression against T2DM-associated endothelial dysfunction. Methods miR-210 transgenic mice (TG) at age of 8 weeks were subjected to Western Diet (WD) for 12 weeks. During the last 10 days of the diet regime, doxycycline or vehicle dissolved in drinking water was given to switch on miR-210 (miR-210/on) expression or keep it off (miR-210/off), respectively. Age-matched wild-type (WT) control mice received normal chow through the same period. Additionally, WT and T2DM db/db mice at age of 15–20 weeks received tail vein injections of miR-210 mimic or miR-210 scramble oligonucleotides. All animals were euthanized at the end of the treatment or 72h after the i.v. injections for determination of endothelial function by acetylcholine-induced endothelium-dependent relaxation (EDR) of isolated aortic segments from all groups of mice using wire myographs. The expression of protein tyrosine phosphatase 1B (PTP1B; a miR-210 target protein) and the levels of 4-hydroxynonenal (4-HNE; an oxidative stress marker) were measured in aortic segments by immunohistochemistry. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). Results EDR in aortic segments from miR-210/off TG mice fed WD and T2DM db/db mice injected with miR-210 scramble was significantly impaired compared to vessels from WT controls fed with regular chow (Fig. 1A, B). Of note, EDR was markedly improved or even restored in aortae from miR-210/on TG mice treated with WD and T2DM db/db mice injected with miR-210 mimic (Fig. 1A, B). Furthermore, the expression of PTP1B and the levels of 4-HNE, which is formed by lipid peroxidation, were significantly elevated in the aortae from miR-210/off TG mice treated with WD when compared to WT controls (Fig. 2A–D). The expression was attenuated in aortae of miR-210/on TG mice treated with WD compared to miR-210/off TG mice (Fig. 2A–D). There was a significant increase in the expression of PTP1B and a trend to increased 4-HNE levels in aortae of db/db mice injected with miR-210 scramble vs. control mice (Fig. 2E–H). A significant reduction in PTP1B but not 4-HNE was observed in db/db mice injected with miR-210 mimic (Fig. 2E–H). Conclusion Genetic and pharmacological overexpression of miR-210 ameliorates endothelial dysfunction in mice fed with WD and db/db mice. Increasing miR-210 levels may become a potential treatment strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation

Saprophytic bacteria and fungi colonize stearoyl coenzyme-A desaturase-1 knockout skin.

Lipids synthesized on the skin are critical to the antimicrobial barrier. Skin lipids also facilitate survival of lipophilic skin commensals in an otherwise dry and acidic ecological landscape. Thus, skin-specific stearoyl-coenzyme A desaturase 1 knockout mice (Scd1ΔK14 ) with sebocyte atrophy and decreased synthesis of monounsaturated fatty acids, triglycerides and wax diesters have dry, inflamed skin. Here, we used 16S rRNA (V1-V2 and V1-V9) and internal transcribed spacer 1 (ITS1) amplicon sequencing to compare bacterial and fungal skin microbiomes between Scd1ΔK14 mice and wildtype control mice (Scd1fl/fl ) in a barrier facility. Saprophytic bacteria including Sporosarcina spp. and Staphylococcus lentus and saprophytic fungi including Alternaria infectoria were found in higher relative abundance in the Scd1ΔK14 group (ANCOM). Analysis of community diversity (Shannon index) revealed greater fungal alpha diversity in the Scd1ΔK14 group (p=0.009, Kruskal-Wallis). Principal coordinates analysis (Bray-Curtis dissimilarity) showed that both bacterial (p=0.002, PERMANOVA) and fungal communities (p=0.006, PERMANOVA) of the Scd1ΔK14 group were unique from the wildtype group. Altogether, these results suggest that sebaceous gland-derived lipids normally restrict the skin microbiome, and in the absence of these lipids, a greater diversity of opportunistic organisms are able to colonize the surface of skin.

Ablation of the Presynaptic Protein Mover Impairs Learning Performance and Decreases Anxiety Behavior in Mice.

The presynaptic protein Mover/TPRGL/SVAP30 is absent in Drosophila and C. elegans and differentially expressed in synapses in the rodent brain, suggesting that it confers specific functions to subtypes of presynaptic terminals. In order to investigate how the absence of this protein affects behavior and learning, Mover knockout mice (KO) were subjected to a series of established learning tests. To determine possible behavioral and cognitive alterations, male and female 8-week-old KO and C57Bl/6J wildtype (WT) control mice were tested in a battery of memory and anxiety tests. Testing included the cross maze, novel object recognition test (NOR), the Morris water maze (MWM), the elevated plus maze (EPM), and the open field test (OF). Mover KO mice showed impaired recognition memory in the NOR test, and decreased anxiety behavior in the OF and the EPM. Mover KO did not lead to changes in working memory in the cross maze or spatial reference memory in the MWM. However, a detailed analysis of the swimming strategies demonstrated allocentric-specific memory deficits in male KO mice. Our data indicate that Mover appears to control synaptic properties associated with specific forms of memory formation and behavior, suggesting that it has a modulatory role in synaptic transmission.

Long-term gamma transcranial alternating current stimulation improves the memory function of mice with Alzheimer's disease.

BackgroundThe main manifestation of Alzheimer’s disease (AD) in patients and animal models is impaired memory function, characterized by amyloid-beta (Aβ) deposition and impairment of gamma oscillations that play an important role in perception and cognitive function. The therapeutic effect of gamma band stimulation in AD mouse models has been reported recently. Transcranial alternating current stimulation (tACS) is an emerging non-invasive intervention method, but at present, researchers have not completely understood the intervention effect of tACS. Thus, the intervention mechanism of tACS has not been fully elucidated, and the course of treatment in clinical selection also lacks theoretical support. Based on this issue, we investigated the effect of gamma frequency (40 Hz) tACS at different durations in a mouse model of AD.Materials and methodsWe placed stimulating electrodes on the skull surface of APP/PS1 and wild-type control mice (n = 30 and n = 5, respectively). Among them, 20 APP/PS1 mice were divided into 4 groups to receive 20 min 40 Hz tACS every day for 1–4 weeks. The other 10 APP/PS1 mice were equally divided into two groups to receive sham treatment and no treatment. No intervention was performed in the wild-type control mice. The short-term memory function of the mice was examined by the Y maze. Aβ levels and microglia in the hippocampus were measured by immunofluorescence. Spontaneous electroencephalogram gamma power was calculated by the average period method, and brain connectivity was examined by cross-frequency coupling.ResultsWe found that the long-term treatment groups (21 and 28 days) had decreased hippocampal Aβ levels, increased electroencephalogram spontaneous gamma power, and ultimately improved short-term memory function. The treatment effect of the short-term treatment group (7 days) was not significant. Moreover, the treatment effect of the 14-day treatment group was weaker than that of the 21-day treatment group.ConclusionThese results suggest that long-term gamma-frequency tACS is more effective in treating AD by reducing Aβ load and improving gamma oscillation than short-term gamma-frequency tACS.

Activation of uroepithelial 5-HT4R inhibits mechanosensory activity of murine bladder afferent nerves.

Serotonin (5-HT) is known to act via multiple 5-HT receptors at spinal and supraspinal levels to regulate micturition. However, the contribution of peripheral 5-HT and its receptors in bladder physiology and pathology is not very well understood, despite evidence showing expression of multiple 5-HT receptors in the bladder wall and 5-HT may activate bladder afferent nerves. The current study was designed to investigate the possible role of 5-HT4R in modulation of the sensitivity of bladder afferents to bladder filling. Immunofluorescent staining showed abundant 5-HT4R immunoreactivity largely confined to the uroepithelium in wild type (WT) but not 5-HT4R−/− mice. In the ex vivo bladder-pelvic nerve preparation, intravesical application of the 5-HT4R agonist RS67333 (1–30 μm) caused concentration-dependent decreases of the pelvic nerve response to bladder filling. Such effect was not observed in the presence of 5-HT4R antagonist GR125487 or in 5-HT4R−/− preparations. A cohort of 5-HT4R−/− and WT control mice were treated with intraperitoneal injections of cyclophosphamide (CYP) (75 mg/kg, three times at 2 days interval) to induce chronic cystitis. Void spot analysis showed that CYP-treated 5-HT4R−/− mice urinated more frequently than their CYP-treated WT counterparts. Concomitantly, bladder afferents of CYP-treated 5-HT4R−/− mice displayed exaggerated sensitivity to bladder filling in comparison with the CYP-treated WT controls. These data suggest that 5-HT4R expressed on uroepithelial cells plays an inhibitory role in mechanosensory transduction in the bladder. Loss of 5-HT4R-mediated inhibition may enhance bladder afferent sensitivity and exacerbate bladder overactivity in pathological conditions. We propose that 5-HT4R agonists might be exploited for the treatment of overactive and painful bladder symptoms.

Deficiency of Jamc Leads to Congenital Nuclear Cataract and Activates the Unfolded Protein Response in Mouse Lenses.

PurposeThe malfunction of junctional adhesion molecule C (JAM-C) has been reported to induce congenital cataract in humans and mice; however, specific characters and the mechanism of this cataract are still unclear. This study aimed to characterize abnormal lens development in Jamc knockout mice and clarify the underlying mechanism.Methods Jamc knockout mice backcrossed onto the C57BL/6 genetic background were used for this research. Slit-lamp and darkfield images showed the cataract phenotype of Jamc−/− mice. Hematoxylin and eosin staining was performed to visualize the morphological and histological features. RNA sequencing was applied to detect differentially expressed genes. Quantitative RT-PCR, western blot, and immunofluorescence were used to determine the level of unfolded protein response (UPR)-related genes. TUNEL staining was utilized to label cell death.Results Jamc knockout mice exhibited nuclear cataract with abnormal lens morphology and defective degradation of nuclei and organelles in lens fiber cells. Compared with wild-type control mice, the expression level of BiP, CHOP, TRIB3, and CHAC1, genes involved in endoplasmic reticulum stress and the UPR, were highly upregulated in Jamc−/− lenses, suggesting that abnormal lens development was accompanied by UPR activation. Moreover, increased cell death was also found in Jamc−/− lenses.ConclusionsCongenital nuclear cataract caused by Jamc deficiency is accompanied by defective degradation of nuclei and organelles in lens fiber cells, lens structure disorder, and UPR activation, suggesting that JAM-C is required for maintaining normal lens development and that UPR activation is involved in cataract formation in Jamc-deficient lenses.

Abstract 122: Effect Of Tie2-cre Endothelial Caveolin-1 Deficiency Mice On Murine Cardiovascular And Metabolic Phenotypes

In humans, caveolin-1 (cav-1) gene polymorphisms confer a metabolic syndrome (MetS) phenotype and higher aldosterone (Aldo) levels. Further, global cav-1 knock out (KO) mice recapitulate the MetS phenotype, with both cardiovascular (CV) and metabolic dysfunction. We have shown that vascular dysfunction in the cav-1 KO is dissociated from the metabolic phenotype and is mediated by an endothelial crosstalk between cav-1 and the Aldo-mineralocorticoid receptor (MR) signaling pathway. Hence, we hypothesized endothelial cav-1 deficiency in mice is a major contributor to CV (but not metabolic) dysfunction.To assess this hypothesis, we generated Tie-2 mediated endothelium-specific cav-1 KO (ECKO) and appropriate genetic wild type (WT) control mice and studied their metabolic and CV traits in response to 1) changes in dietary salt intake (0.03% (LS) vs. 1.6% Na + (HS)) and 2) 3-weeks of Aldo infusion (200μg/kg/day) during sodium loading. Our results showed no difference in glucose, lipids and HOMA-IR in ECKO vs. WT mice, irrespective of the dietary intervention. However, on a HS (but not a LS) diet, ECKO vs. WT mice displayed similar CV dysfunction as previously shown in the full cav-1 KO: increased Aldo levels (73.7±11.1 vs 47.8±4.7ng/dl, p<0.05) and systolic BP (116.4±1.5 vs111±1.4 mmHg, p<0.015). Further, ECKO vs. WT mice had increased cardiac MR, eNOS, PAI-1 and CD68 expression (p<0.05 for each) as well as left ventricular hypertrophy (echocardiogram, p<0.05). In addition, 3-week HS diet led to higher albuminuria (p<0.05) and kidney/body weight ratios in ECKO vs. WT (7.2±0.2 vs 6.3±0.1). Surprisingly, as compared to placebo, Aldo infusion induced significant CV and renal damage only in the WT, but not in the ECKO. However, as compared to WT, Aldo infused ECKO had increased cardiac hypertrophy (5.9±0.2 vs 5.2±0.1) and molecular markers noted above (p<0.05 for each).In conclusion, lack of endothelial cav-1 in the mouse segregated the metabolic and CV phenotypes, and the associated CV phenotype resembles that described for the global cav-1 KO. Further, the CV and renal damage in the ECKO at baseline cannot be further enhanced by Aldo infusion. These data confirm our hypothesis that endothelial cav-1 is a major player in CV but not metabolic homeostasis.

Aldehyde dehydrogenase 2 and arrhythmogenesis.

Cardiac arrhythmia is a common cardiovascular disease that leads to considerable economic burdens and significant global public health challenges. Despite the remarkable progress made in recent decades, antiarrhythmic therapy remains suboptimal. Aldehyde dehydrogenase 2 (ALDH2), a critical detoxifying enzyme, catalyzes toxic aldehydes and protects individuals from damage caused by oxidative stress. Accumulating evidence has demonstrated that ALDH2 activation has potential antiarrhythmic benefits. The correlation between ALDH2 deficiency and arrhythmogenesis has been widely recognized. In this review, we summarize recent research on the potential role of ALDH2 activation and antiarrhythmic protection, as well as the role played by the ALDH2∗2 polymorphism (rs671) in promoting arrhythmic risk. Additionally, we discuss important new findings illustrating the use of ALDH2 activators, which may prove to be promising antiarrhythmic therapy agents.

Abstract 128: Loss Of Gstm1 Augments Kidney Neutrophil Population In Angii-induced Hypertension

Background: Glutathione-S-transferase Mu1( Gstm1 ) gene, a member of Glutathione-S-transferases (GST) superfamily, encodes an enzyme that functions in the detoxification of electrophilic compounds. The common GSTM1 deletion variant in humans is associated with increased risks of chronic kidney disease (CKD) progression and incident end stage kidney failure. We reported that deletion of Gstm1 increases oxidative stress, kidney inflammation and injury in angiotensin II induced hypertension (Ang II-HTN) in mice. At 4 weeks of Ang II-HTN, neutrophils, CD4+T cells, and F4/80+ cells were significantly increased in Gstm1 knockout (KO) kidney. While a link between oxidative stress and immune activation in hypertension has been well documented, the molecular mechanism by which Gstm1 regulates immune cell activation in hypertension is unknown. Methods: To further characterize the inflammatory response in GSTM1 deficiency, Gstm1 KO and wild-type (WT) male mice at ~ 12 weeks of age were subjected to Angiotensin II at 1000 ng/kg per minute for 4 days via a mini osmotic pump. The kidney cells and bone marrow cells were isolated for flow cytometry analysis. Results: At baseline, there were no differences in inflammatory cell populations including neutrophils between WT and Gstm1 KO mice. At Day 4 of Ang II-HTN, Gstm1 KO kidneys exhibited about a 2.3 fold increase in neutrophil population (CD45 + Ly6G + CD11b + ), compared to WT control mice (10 WT and 8 Gstm1 KO mice, p=0.0075). Interestingly, CD45+ bone marrow cells isolated from both Gstm1 KO and WT mice have similar percentile of neutrophil populations ; however, neutrophils in Gstm1 KO bone marrow express relatively higher levels of CXCR2. Unlike at 4 weeks, there was no difference in other inflammatory cells at Day 4. Conclusion: Deletion of Gstm1 increases renal neutrophil population early in Ang II-HTN and CXCR2 expression in bone marrow neutrophils. Gstm1 may play a role in regulating neutrophil migration and recruitment. Further studies are needed to delineate whether GSTM1 deficiency drives kidney injury via early neutrophil migration, which could shed light on potential therapeutic target for CKD progression in those genetically susceptible.

Coenzyme Q10 Supplementation Increases Removal of the ATXN3 Polyglutamine Repeat, Reducing Cerebellar Degeneration and Improving Motor Dysfunction in Murine Spinocerebellar Ataxia Type 3.

Coenzyme Q10 (CoQ10), a well-known antioxidant, has been explored as a treatment in several neurodegenerative diseases, but its utility in spinocerebellar ataxia type 3 (SCA3) has not been explored. Herein, the protective effect of CoQ10 was examined using a transgenic mouse model of SCA3 onset. These results demonstrated that a diet supplemented with CoQ10 significantly improved murine locomotion, revealed by rotarod and open-field tests, compared with untreated controls. Additionally, a histological analysis showed the stratification of cerebellar layers indistinguishable from that of wild-type littermates. The increased survival of Purkinje cells was reflected by the reduced abundance of TUNEL-positive nuclei and apoptosis markers of activated p53, as well as lower levels of cleaved caspase 3 and cleaved poly-ADP-ribose polymerase. CoQ10 effects were related to the facilitation of the autophagy-mediated clearance of mutant ataxin-3 protein, as evidenced by the increased expression of heat shock protein 27 and autophagic markers p62, Beclin-1 and LC3II. The expression of antioxidant enzymes heme oxygenase 1 (HO-1), glutathione peroxidase 1 (GPx1) and superoxide dismutase 1 (SOD1) and 2 (SOD2), but not of glutathione peroxidase 2 (GPx2), were restored in 84Q SCA3 mice treated with CoQ10 to levels even higher than those measured in wild-type control mice. Furthermore, CoQ10 treatment also prevented skeletal muscle weight loss and muscle atrophy in diseased mice, revealed by significantly increased muscle fiber area and upregulated muscle protein synthesis pathways. In summary, our results demonstrated biochemical and pharmacological bases for the possible use of CoQ10 in SCA3 therapy.

Altered membrane properties but unchanged intrinsic excitability and spontaneous postsynaptic currents in an aged APP swe /PS1dE9 model of Alzheimer's disease.

Neuronal hyperexcitability in Alzheimer’s disease (AD) models is thought to either contribute to the formation of amyloid beta plaques or result from their formation. Neuronal hyperexcitability has been shown in the cerebral cortex of the widely used young APPswe/PS1dE9 mice, which have accelerated plaque formation. However, it is currently unclear if hyperexcitability also occurs in CA1 hippocampal neurons of aged animals in this model. In the present work, we have compared intrinsic excitability and spontaneous synaptic inputs from CA1 pyramidal cells of 8-month-old APPswe/PS1dE9 and wildtype control mice. We find no change in intrinsic excitability or spontaneous postsynaptic currents (PSCs) between groups. We did, however, find a reduced input resistance and an increase in hyperpolarization-activated sag current. These results are consistent with findings from other aged AD model mice, including the widely used 5xFAD and 3xTg. Together these results suggest that neuronal hyperexcitability is not a consistent feature of all AD mouse models, particularly at advanced ages.

Behavioral defects and downregulation of hippocampal BDNF and nNOS expression in db/db mice did not improved by chronic TGF-β2 treatment.

Epidemiological evidence suggests that there is a link between diabetes and mood disorders, such as depression and anxiety. Although peripheral or central inflammation may explain this link, the molecular mechanisms are not fully understood and few effective treatments for diabetes or mood disorders are available. In the present study, we aimed to determine whether transforming growth factor (TGF)-β2, an anti-inflammatory substance, might represent a potential therapeutic agent for diabetes-related mood behaviors. TGF-β2 expression in the hippocampus is affected by anxiolytic drugs and stress exposure, it is able to cross the blood-brain barrier, and it is as an exercise-induced physiological adipokine that regulates glucose homeostasis. Therefore, we hypothesized that a chronic TGF-β2 infusion would ameliorate diabetes-related glucose intolerance and mood dysregulation. To determine the effects of the chronic administration of TGF-β2 on diabetes, we implanted osmotic pumps containing TGF-β2 into type 2 diabetic mice (db/db mice), and age-matched non-diabetic control wild type mice and db/db mice were infused with vehicle (PBS), for 12 consecutive days. To assess anxiety-like behaviors and glucose homeostasis, the mice underwent elevated plus maze testing and intraperitoneal glucose tolerance testing. Hippocampal and perigonadal visceral white adipose tissue perigonadal white adipose tissue samples were obtained 12 days later. Contrary to our hypothesis, TGF-β2 infusion had no effect on diabetes-related glucose intolerance or diabetes-related behavioral defects, such as inactivity. In db/db mice, the expression of inflammatory markers was high in pgWAT, but not in the hippocampus, and the former was ameliorated by TGF-β2 infusion. The expression of brain-derived neurotrophic factor and neuronal nitric oxide synthase, important regulators of anxiety-like behaviors, was low in db/db mice, but TGF-β2 infusion did not affect their expression. We conclude that although TGF-β2 reduces the expression of pro-inflammatory markers in the adipose tissue of diabetic mice, it does not ameliorate their obesity or mood dysregulation.

AT2 activation does not influence brain damage in the early phase after experimental traumatic brain injury in male mice

Antagonism of the angiotensin II type 1 receptor (AT1) improves neurological function and reduces brain damage after experimental traumatic brain injury (TBI), which may be partly a result of enhanced indirect angiotensin II type 2 receptor (AT2) stimulation. AT2 stimulation was demonstrated to be neuroprotective via anti-inflammatory, vasodilatory, and neuroregenerative mechanisms in experimental cerebral pathology models. We recently demonstrated an upregulation of AT2 after TBI suggesting a protective mechanism. The present study investigated the effect of post-traumatic (5 days after TBI) AT2 activation via high and low doses of a selective AT2 agonist, compound 21 (C21), compared to vehicle-treated controls. No differences in the extent of the TBI-induced lesions were found between both doses of C21 and the controls. We then tested AT2-knockdown animals for secondary brain damage after experimental TBI. Lesion volume and neurological outcomes in AT2-deficient mice were similar to those in wild-type control mice at both 24 h and 5 days post-trauma. Thus, in contrast to AT1 antagonism, AT2 modulation does not influence the initial pathophysiological mechanisms of TBI in the first 5 days after the insult, indicating that AT2 plays only a minor role in the early phase following trauma-induced brain damage.