Effect In Wild-type Mice Research Articles

Favorable effects of omega-3 polyunsaturated fatty acids in attentional control and conversion rate to psychosis in 22q11.2 deletion syndrome

Omega-3-polyunsaturated-fatty-acids were suggested against cognitive dysfunctions and conversion to psychosis. However, a recent multicenter trial found no effect in reducing conversion rates in individuals at risk of developing schizophrenia. Patients’ genetic heterogeneity and the timing of treatment might influence omega-3 efficacy. Here, we addressed the impact of omega-3 early treatment in both mice and human subjects with a 22q11.2 genetic hemi-deletion (22q11DS), characterized by cognitive dysfunctions and high penetrance of schizophrenia. We first tested the behavioural and cognitive consequences of adolescent exposure to normal or omega-3-enriched diets in wild-type and 22q11DS (LgDel/+) mice. We then contrasted mouse data with those gathered from sixty-two patients with 22q11DS exposed to a normal diet or supplemented with omega-3 during pre-adolescence/adolescence. Adolescent omega-3 exposure had no effects in wild-type mice. However, this treatment ameliorated distractibility deficits revealed in LgDel/+ mice by the Five Choice Serial Reaction Time Task (5CSRTT). The omega-3 improvement in LgDel/+ mice was selective, as no other generalized cognitive and non-cognitive effects were evident. Similarly, omega-3-exposed 22q11DS patients showed long-lasting improvements on distractibility as revealed by the continuous performance test (CPT). Moreover, omega-3-exposed 22q11DS patients showed less risk of developing an Ultra High Risk status and lower conversion rate to psychosis. Our convergent mouse-human findings represent a first analysis on the effects of omega-3 early treatment in 22q11DS. The beneficial effects in attentional control and transition to psychosis could support the early use of omega-3 supplementation in the 22q11DS population.

μ-Opioid receptors in primary sensory neurons are involved in supraspinal opioid analgesia

Both inhibiting ascending nociceptive transmission and activating descending inhibition are involved in the opioid analgesic effect. The spinal dorsal horn is a critical site for modulating nociceptive transmission by descending pathways elicited by opioids in the brain. μ-Opioid receptors (MORs, encoded by Oprm1) are highly expressed in primary sensory neurons and their central terminals in the spinal cord. In the present study, we tested the hypothesis that MORs expressed in primary sensory neurons contribute to the descending inhibition and supraspinal analgesic effect induced by centrally administered opioids. We generated Oprm1 conditional knockout (Oprm1-cKO) mice by crossing AdvillinCre/+ mice with Oprm1flox/flox mice. Immunocytochemical labeling in Oprm1-cKO mice showed that MORs are completely ablated from primary sensory neurons and are profoundly reduced in the superficial spinal dorsal horn. Intracerebroventricular injection of morphine or fentanyl produced a potent analgesic effect in wild-type mice, but such an effect was significantly attenuated in Oprm1-cKO mice. Furthermore, the analgesic effect produced by morphine or fentanyl microinjected into the periaqueductal gray was significantly greater in wild-type mice than in Oprm1-cKO mice. Blocking MORs at the spinal cord level diminished the analgesic effect of morphine and fentanyl microinjected into the periaqueductal gray in both groups of mice. Our findings indicate that MORs expressed at primary afferent terminals in the spinal cord contribute to the supraspinal opioid analgesic effect. These presynaptic MORs in the spinal cord may serve as an interface between ascending inhibition and descending modulation that are involved in opioid analgesia.

Long-Term Clearance of Senescent Cells Prevents the Hematopoietic Stem Cell Aging in Naturally Aged Mice

Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma

BackgroundMelanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results.MethodsIn this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9−/−)).ResultspIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice.ConclusionsThe combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.

Complement C3 activation regulates the production of tRNA-derived fragments Gly-tRFs and promotes alcohol-induced liver injury and steatosis

Complement is known to play a role in alcoholic fatty liver disease (AFLD), but the underlying mechanisms are poorly understood, thereby constraining the development of a rational approach for therapeutic intervention in the complement system. C3 deficiency has been shown to impart protective effects against ethanol-induced hepatic steatosis and inflammation. Here we demonstrate a protection effect in wild-type mice by treatment with CR2-Crry, a specific inhibitor of C3 activation. The expression of glycine transfer (t) RNA-derived fragments (Gly-tRFs) is upregulated in ethanol-fed mice and inhibition of Gly-tRFs in vivo decreases chronic ethanol feeding-induced hepatosteatosis without affecting inflammation. The expression of Gly-tRF was downregulated in C3-deficient or CR2-Crry-treated mice, but not in C5-deficient mice; Gly-tRF expression was restored by the C3 activation products C3a or Asp (C3a-des-Arg) via the regulation of CYP2E1. Transcriptome profiling of hepatic tissues showed that Gly-tRF inhibitors upregulate the expression of sirtuin1 (Sirt1) and subsequently affect downstream lipogenesis and β-oxidation pathways. Mechanistically, Gly-tRF interacts with AGO3 to downregulate Sirt1 expression via sequence complementarity in the 3′ UTR. Notably, the expression levels of C3d, CYP2E1 and Gly-tRF are upregulated, whereas Sirt1 is decreased in AFLD patients compared to healthy controls. Collectively, our findings suggest that C3 activation products contribute to hepatosteatosis by regulating the expression of Gly-tRF. Complement inhibition at the C3 activation step and treatment with Gly-tRF inhibitors may be potential and precise therapeutic approaches for AFLD.

Inhibition of RPTPβ/ζ blocks ethanol-induced conditioned place preference in pleiotrophin knockout mice

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ. Interestingly, pharmacological inhibition of RPTPβ/ζ reduces ethanol consumption and blocks ethanol-induced conditioned place preference (CPP) in wild type mice. Since PTN-knockout (Ptn−/−) mice are more sensitive to the conditioning effects of alcohol, we aimed to test the effects of MY10, a small-molecule inhibitor of RPTPβ/ζ, on ethanol-induced CPP in Ptn−/− mice. The data presented here demonstrate for the first time that a regular dose of MY10, known to block ethanol consumption and reward in wild type mice, also blocks the rewarding effects of ethanol in the more vulnerable individuals lacking PTN, the endogenous inhibitor of RPTPβ/ζ. In addition, since MY10 readily penetrates the blood brain barrier (BBB), we tested its effects in a series of behavioural tests in Ptn+/+ and Ptn−/− mice. The data indicate that MY10 does not cause gross behavioural effects in wild type mice. However, MY10 tended to induce anxiolytic effects in Ptn−/− mice in the elevated plus maze paradigm. Overall, the data indicate that MY10 rescues Ptn−/− mice from their increased susceptibility to the conditioning effects of ethanol and may induce anxiolytic effects in individuals with reduced or absent PTN functions. Further studies are needed to confirm the potential of pharmacological inhibition of RPTPβ/ζ as a new therapeutic strategy in the treatment of anxiety-related disorders.

Investigating the Role of Serotonin Transporter, Plasma Membrane Monoamine Transporter, and Organic Cation Transporter 3 in the Antidepressant‐like Effects of Ketamine

Twenty percent of adults are diagnosed with major depressive disorder, which is typically treated with selective serotonin reuptake inhibitors (SSRIs). However, SSRIs take approximately six weeks to produce therapeutic effects. Recently, low doses of ketamine, a noncompetitive N‐methyl‐ D‐aspartate (NMDA) receptor antagonist, have been shown to produce rapid and long‐lasting antidepressant effects. Studies that have begun to examine the mechanisms underlying these effects have focused on intracellular changes mediated by NMDA and/or AMPA receptors. However, the traditional view of the etiology of depression involves the need for an increase in extracellular serotonin to regulate mood. Surprisingly, there appears to be little research into the effects of ketamine on serotonin. One study found an increase in extracellular serotonin following ketamine, consistent with results from another study showing that ketamine inhibited serotonin uptake. Together, these findings suggest a role for serotonin in the antidepressant‐like effects of ketamine, and putatively one involving transporters capable of serotonin uptake. The serotonin transporter (SERT) is an obvious candidate, but recent evidence points to the plasma membrane monoamine transporter (PMAT) and organic cation transporter (OCT3) as important players in serotonin uptake as well. In order to investigate a role for these transporters in the action of ketamine to inhibit serotonin uptake, we used in vivo chronoamperometry to measure serotonin clearance from extracellular fluid of CA3 region of the hippocampus. In wildtype mice, ketamine robustly inhibited serotonin clearance. Ongoing chronoamperometry studies are using knockout lines of SERT, PMAT and OCT3 mice to parse apart their roles in the action of ketamine to inhibit serotonin clearance. Parallel studies using these knockout (KO) lines in the forced swim test support the involvement of all three transporters in the antidepressant‐like effect of ketamine. As expected, ketamine (32 mg/kg) produced robust antidepressant‐like effects in wildtype mice, but in marked contrast, was without effect in SERT KO, PMAT KO, and OCT3 KO mice. Ketamine did not influence locomotor activity in SERT KO and wildtype mice, ruling out differences in the action of ketamine on general activity accounting for the contrasting effect of ketamine in the FST between these genotypes. Experiments assessing the locomotor actions of ketamine in PMAT KO and OCT3 KO mice are ongoing. Together, these data suggest that blockade of SERT, PMAT, and/or OCT3 may be important for the antidepressant‐like effects of ketamine.Support or Funding InformationR01 MH093320R01 MH106978T32 NS082145This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long-Term Feeding of Soy Protein Attenuates Choline Deficient-Induced Adverse Effects in Wild Type Mice and Prohibitin 1 Deficient Mice Response More Sensitively.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, however the exact cause of NAFLD remains unknown. Methionine, an essential amino acid, is the first limiting amino acid of soy protein, and its deficiency is suggested to cause hepatocyte damage and NAFLD. The objective of this study is to examine the changes in NAFLD susceptibility with soy protein consumption and deterioration due to prohibitin 1 (PHB1) deficiency, an important protein in hepatic mitochondrial function. In this study, liver-specific phb1 +/− mice and wild-type mice were fed a normal diet, choline-deficient diet (CDD), or soy protein diet without choline (SPD) for 16 weeks. Using hematoxylin and eosin staining, we showed that SPD attenuates symptoms of hepatocyte damage and lipid accumulation induced by CDD in mouse liver. The liver damage in mice fed the SPD was alleviated by decreasing lipogenic markers and by increasing anti-inflammatory markers. Furthermore, mRNA expression of genes involved in hepatic methionine metabolism was significantly lower in liver-specific phb1 +/− mice fed with a SPD compared with wild-type mice fed with a SPD. These data suggest a CDD can cause non-alcohol related liver damage, which can be attenuated by a SPD in wild-type mice. These phenomena were not observed in liver-specific phb1 +/− mice. It may therefore be concluded that SPD attenuates CDD-induced liver damage in wild-type mice, and that PHB1 deficiency blocks the beneficial effects of SPD against CDD-induced liver damage.

Dietary ascorbic acid restriction in GNL/SMP30-knockout mice unveils the role of ascorbic acid in regulation of somatic and visceral pain sensitivity

Cav3.2 T-type Ca2+ channels are expressed in the primary afferents and play a pronociceptive role. The activity of Cav3.2 is enhanced by H2S, a gasotransmitter, and suppressed by ascorbic acid (vitamin C) through metal-catalyzed oxidation of the Zn2+-binding His191 in Cav3.2. Since rodents, but not humans, are capable of synthesizing ascorbic acid, the present study examined the role of ascorbic acid in nociceptive processing, using the mice lacking GNL/SMP30, an enzyme essential for ascorbic acid biosynthesis. Intraplantar and intracolonic administration of NaHS, an H2S donor, caused somatic allodynia and referred hyperalgesia, respectively, and repeated treatment with paclitaxel produced neuropathic allodynia in wild-type mice, all of which were suppressed by ascorbic acid or T-type Ca2+ channel blockers. Dietary ascorbic acid restriction caused dramatic decreases in plasma and tissue ascorbic acid levels in GNL/SMP30-knockout, but not wild-type, mice. The ascorbic acid restriction enhanced the somatic and visceral hypersensitivity following intraplantar and intracolonic NaHS, respectively, and paclitaxel-induced neuropathy in GNL/SMP30-knockout mice, while it had no such effect in wild-type mice. Together, our data unveil the critical role of ascorbic acid in regulating somatic and visceral pain sensitivity and support accumulating clinical evidence for the usefulness of ascorbic acid in pain management.

Identification of Cellular Sources of IL-2 Needed for Regulatory T Cell Development and Homeostasis.

The cytokine IL-2 is critical for promoting the development, homeostasis, and function of regulatory T (Treg) cells. The cellular sources of IL-2 that promote these processes remain unclear. T cells, B cells, and dendritic cells (DCs) are known to make IL-2 in peripheral tissues. We found that T cells and DCs in the thymus also make IL-2. To identify cellular sources of IL-2 in Treg cell development and homeostasis, we used Il2FL/FL mice to selectively delete Il2 in T cells, B cells, and DCs. Because IL-15 can partially substitute for IL-2 in Treg cell development, we carried out the majority of these studies on an Il15-/- background. Deletion of Il2 in B cells, DCs, or both these subsets had no effect on Treg cell development, either in wild-type (WT) or Il15-/- mice. Deletion of Il2 in T cells had minimal effects in WT mice but virtually eliminated developing Treg cells in Il15-/- mice. In the spleen and most peripheral lymphoid organs, deletion of Il2 in B cells, DCs, or both subsets had no effect on Treg cell homeostasis. In contrast, deletion of Il2 in T cells led to a significant decrease in Treg cells in either WT or Il15-/- mice. The one exception was the mesenteric lymph nodes where significantly fewer Treg cells were observed when Il2 was deleted in both T cells and DCs. Thus, T cells are the sole source of IL-2 needed for Treg cell development, but DCs can contribute to Treg cell homeostasis in select organs.

Enhanced extinction of aversive memories in mice lacking SPARC-related protein containing immunoglobulin domains 1 (SPIG1/FSTL4).

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity related to learning and memory. We previously reported that SPARC-related protein containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4, FSTL4) binds to pro-BDNF and negatively regulates BDNF maturation; however, its neurological functions, particularly in learning and memory, have not yet been elucidated. We herein examined the electrophysiological and behavioral phenotypes of Spig1-knockout (Spig1-KO) mice. Adult Spig1-KO mice exhibited greater excitability and facilitated long-term potentiation (LTP) in the CA1 region of hippocampal slices than age- and sex-matched wild-type (WT) mice. Facilitated LTP was reduced to the level of WT by the bath application of an anti-BDNF antibody to hippocampal slices. A step-through inhibitory avoidance learning paradigm revealed that the extinction of aversive memories was significantly enhanced in adult Spig1-KO mice, while they showed the normal acquisition of aversive memories; besides, spatial reference memory formation was also normal in the standard Morris water maze task. An intracerebroventricular (icv) injection of anti-BDNF in the process of extinction learning transiently induced the recurrence of aversive memories in Spig1-KO mice, but exerted no effects in WT mice. These results indicate a critical role for SPIG1 in BDNF-mediated synaptic plasticity in extinction of inhibitory avoidance memory.

DJ‐1 protects against renal mitochondrial oxidative stress and T cell infiltration

Protein nucleic acid deglycase (DJ‐1) exerts a protective role against oxidative stress. DJ‐1−/− mice have increased systolic BP (30±3%) relative to wild‐type (WT) littermates. This study aimed to determine the mechanisms involved in the oxidative stress‐mediated hypertension due to DJ‐1 germline deletion. At baseline, no differences were found in sodium excretion, renal expression of renin, NADPH activity or serum creatinine levels between DJ‐1−/− and WT mice (n=5–12). Histological assessment by H&E and Picro Sirius demonstrated no kidney morphological abnormalities associated to DJ‐1 deletion. However, renal expression of nitrotyrosine was elevated in DJ‐1−/− mice (+176.8±31% vs. WT mice, n=5), and urinary excretion of the renal damage marker KIM‐1 was increased in DJ‐1−/− mice (148±22% vs WT, n=4), indicating the presence of oxidative stress and kidney damage in DJ‐1−/− mice. In addition, mRNA expression of mitochondrial heat shock protein mtHSP60, but not mtHSP40, was increased in kidneys from DJ‐1−/− mice (2.9±0.1 fold changes of WT, n=4), evidencing mitochondrial oxidative stress in these mice. Conversely, levels of renal endoplasmic reticulum (ER) stress and renal inflammation were similar between the genotypes, indicating that inflammation was not altered in this animal model, despite the high mitochondrial ROS production. Two week treatment with the membrane‐permeable radical scavenger Tempol normalized BP (vehicle vs. tempol: 118±2 vs. 100±1%, n=4, p<0.05), renal malondialdehyde production (160±23% vs 109±15% vs WT, n=4, p<0.05) and KIM‐1 excretion (‐58±3% vs WT, n=4, p<0.05) in DJ‐1−/− mice, but had no effect in WT mice. Serum nitrite/nitrate levels in DJ‐1−/− mice treated with tempol were significantly elevated compared to vehicle‐treated DJ‐1−/− mice (172±30% DJ‐1−/− tempol vs. vehicle, n=4, p<0.05), suggesting a role of nitric oxide on the high blood pressure observed in this model. To examine if lack of DJ‐1 leads to exaggerated renal inflammation and renal damage after an insult, unilateral ureter obstruction (UUO) was performed in DJ‐1−/− and WT mice. After 2 weeks of UUO, increased cortical T‐cell infiltration was observed in WT mice (225±101%; sham vs. UUO, n=3–4; p<0.05) and it was dramatically exaggerated in the DJ‐1−/− mice (67±14%; WT UUO vs. DJ‐1−/− UUO, n=3; p<0.05). In conclusion, deletion of DJ‐1 leads to mitochondrial oxidative stress‐induced hypertension possibly, due to decreased nitric oxide bioavailability, without altering sodium excretion, renal NAPDH activity, inflammation, morphology or ER stress levels. Our results suggest a protective role of DJ‐1 against renal infiltration of T‐cells in the UUO model of kidney disease and may have important implications in the prevention of renal diseases.Support or Funding InformationFunded by NIH T32 DK007545 to CDM and NIH/NHLBI 5P01 HL074940‐10) and NIH/NHLBI 5P01 HL068686‐11 to PAJThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury

γδ T cells located near the epithelial barrier are integral components of local inflammatory and innate immune responses. We have previously reported the presence of choroidal γδ T cells in a model of chronic degeneration of the retinal pigment epithelium (RPE). The goals of the current study were to further define the functions of choroidal γδ T cells and to explore the underlying mechanisms of their action. Our data demonstrate that choroidal γδ T cells are activated by RPE injury in response to NaIO3 treatment, and that they express genes that encode immunosuppressive cytokines, such as IL-4 and IL-10. γδ-T-cell-deficient mice developed profound RPE and retinal damage at doses that caused minimal effects in wild-type mice, and adoptive transfer of γδ T cells prevented sensitization. Intravitreal injection of IL-4 and IL-10 ameliorated RPE toxicity that was induced by NaIO3Ex vivo coculture of γδ T cells with RPE explants activated the production of anti-inflammatory cytokines via an aryl hydrocarbon receptor (AhR)-dependent mechanism. AhR deficiency abolished the protective effects of γδ T cells after adoptive transfer. Collectively, these findings define important roles for choroid γδ T cells in maintaining tissue homeostasis in the outer retina.-Zhao, Z., Liang, Y., Liu, Y., Xu, P., Flamme-Wiese, M. J., Sun, D., Sun, J., Mullins, R. F., Chen, Y., Cai, J. Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

Metabolic and Blood Pressure Effects of Walnut Supplementation in a Mouse Model of the Metabolic Syndrome.

There is extensive evidence that walnut consumption is protective against cardiovascular disease and diabetes in the healthy population, but the beneficial effects of walnut consumption in individuals with the metabolic syndrome (MetS) remain uncertain. We compared a range of cardio-metabolic traits and related tissue gene expression associated with 21 weeks of dietary walnut supplementation in a mouse model of MetS (MetS-Tg) and wild-type (WT) mice (n = 10 per genotype per diet, equal males and females). Compared to standard diet, walnuts did not significantly alter food consumption or body weight trajectory of either MetS-Tg or WT mice. In MetS-Tg mice, walnuts were associated with reductions in oral glucose area under the curve (gAUC, standard diet 1455 ± 54, walnut 1146 ± 91, p = 0.006) and mean arterial blood pressure (MAP, standard diet 100.6 ± 1.9, walnut 73.2 ± 1.8 mmHg, p < 0.001), with neutral effects on gAUC and MAP in WT mice. However, in MetS-Tg mice, walnuts were also associated with trends for higher plasma cholesterol (standard diet 4.73 ± 0.18, walnut 7.03 ± 1.99 mmol/L, p = 0.140) and triglyceride levels (standard diet 2.4 ± 0.5, walnut 5.4 ± 1.6 mmol/L, p = 0.061), despite lowering cholesterol and having no effect on triglycerides in WT mice. Moreover, in both MetS-Tg and WT mice, walnuts were associated with significantly increased liver expression of genes associated with metabolism (Fabp1, Insr), cell stress (Atf6, Ddit3, Eif2ak3), fibrosis (Hgf, Sp1, Timp1) and inflammation (Tnf, Ptpn22, Pparg). In conclusion, dietary walnuts were associated with modest favourable effects in WT mice, but a combination of beneficial and adverse effects in MetS-Tg mice, and up-regulation of hepatic pro-fibrotic and pro-inflammatory genes in both mouse strains.

Prodepressant- and anxiogenic-like effects of serotonin-selective, but not noradrenaline-selective, antidepressant agents in mice lacking α2-containing GABAA receptors.

Deficits in neuronal inhibition via gamma-aminobutyric acid (GABA) type A receptors (GABAA-Rs) are implicated in the pathophysiology of major depressive disorder and the therapeutic effects of current antidepressant treatments, however, the relevant GABAA-R subtype as defined by its alpha subunit is still unknown. We previously reported anxiety- and depressive-like behavior in alpha2+/- and alpha2-/- mice, respectively (Vollenweider, 2011). We sought to determine whether this phenotype could be reversed by chronic antidepressant treatment. Adult male mice received 4 or 8mg/kg fluoxetine or 53mg/kg desipramine in their drinking water for four weeks before undergoing behavioral testing. In the novelty suppressed feeding test, desipramine had anxiolytic-like effects reducing the latencies to bite and to eat the pellet in both wild-type and alpha2+/- mice. Surprisingly, 4mg/kg fluoxetine had anxiogenic-like effects in alpha2+/- mice increasing latency to bite and to eat while 8mg/kg fluoxetine increased the latency to eat in both wild-type and alpha2+/- mice. In the forced swim and tail suspension tests, chronic desipramine treatment increased latency to immobility in wild-type and alpha2-/- mice. In contrast, chronic fluoxetine treatment increased immobility in alpha2-/- mice in both tasks while generally having no effect in wild-type mice. These findings suggest that in preclinical paradigms of anxiety and behavioral despair the antidepressant-like effects of desipramine are independent of alpha2-containing GABAA-Rs, while a reduction in alpha2 expression leads to an increased sensitivity to anxiogenic- and prodepressant-like effects with chronic fluoxetine treatment, pointing to a potential role of alpha2-containing GABAA-Rs in the response to serotonin-selective antidepressants.

Abstract 616: Potentiation of Endothelium-Dependent Vasodilation by Adenylate Cyclase Type 9 Inactivation is Associated With Increased Endothelial Cell Signaling in Mouse Femoral Arteries

Adenylate cyclase (AC) activity in vessels is associated with vasodilation. The AC family includes 10 members of which AC9 is the least studied. We recently demonstrated that Adcy9 inactivation in mice protects from atherosclerosis in the aorta and potentiates endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) in femoral arteries (FA). Our objectives were 1) to determine Adcy9 expression in FA; 2) investigate endothelial signalling pathways associated with Adcy9 inactivation-induced potentiation of ACh-induced EDV; and 3) to assess the effect of Adcy9 inactivation on EDV during flow-mediated dilation (FMD). FA were isolated from 8 to 13 week-old Adcy9- inactivated ( Adcy9 Gt ) and wild-type (WT) littermate mice. Adcy9 expression was studied in WT FA tissue section by in situ hybridization (ISH) using Adcy9 and negative control probes. Diameter of pressurized (80 mmHg) FA (internal diameter 288±3 μm) was studied in response to ACh (n=7-14) and shear stress (n=12) up to 20 dynes/cm 2 after preconstriction with phenylephrine. To study the role of endothelial signalling pathways in response to ACh-induced EDV, FA were incubated in absence or presence of inhibitors for nitric oxide (NO) synthase (L-NNA 0.1 μM), cyclooxygenase (meclofenamate 1 μM) and endothelial hyperpolarization (TRAM-34 10 μM plus apamin 0.1 μM). ISH with Adcy9 probe resulted in numerous dots in the vascular wall of the FA. Meclofenamate and the combination of TRAM-34 plus apamin decreased maximal (E max ) EDV to ACh, respectively, by 40 and 17% in Adcy9 Gt (P&lt;0.01) but had no effect in WT mice. L-NNA reduced E max to ACh by 26% in WT (P&lt;0.01) and 50% in Adcy9 Gt (P&lt;0.01). Adcy9 inactivation tended to increase FMD to shear stress ranging from 6 to 20 dynes/cm 2 : At 10 dynes/cm 2 , FMD was increased from 25.4±4.9% in WT to 37.4±4.9% in Adcy9 Gt mice (P=0.083, n=12 per group). In conclusion, our data show that FA express Adcy9 and that Adcy9 inactivation increases the effects of endothelial NO, hyperpolarization and cyclooxygenase pathways in EDV. A numerical increase in FMD in Adcy9 Gt compared to WT confirms the potentiating effect of Adcy9 inactivation on EDV. The potentiation of endothelial dilatory pathways appears to be associated with the anti-atherosclerotic effects of Adcy9 inactivation.

Effect of aging and Alzheimer's disease-like pathology on brain monoamines in mice

Aging is the greatest single risk factor of the neurodegenerative disorder Alzheimer's disease (AD). The monoaminergic system, including serotonin (5-HT), dopamine (DA) and noradrenaline (NA) modulates cognition, which is affected in AD. Changes in monoamine levels have been observed in AD, but these can both be age- and/or disease-related. We examined whether brain monoamine levels change as part of physiological aging and/or AD-like disease in APPSWE/PS1ΔE9 (APP/PS1) transgenic mice. The neocortex, hippocampus, striatum, brainstem and cerebellum of 6-, 12-, 18- and 24-month-old B6C3 wild-type (WT) mice and of 18-month old APP/PS1 and WT mice were analysed for 5-HT, DA and NA contents by high pressure liquid chromatography (HPLC), along with neocortex from 14-month-old APP/PS1 and WT mice. While, we observed no aging effect in WT mice, we detected region-specific changes in the levels of all monoamines in 18-month-old transgenic compared with WT mice. This included reductions in 5-HT (−30%), DA (−47%) and NA (−32%) levels in the neocortex and increases of 5-HT in the brainstem (+18%). No changes were observed in any of the monoamines in the neocortex from 14-month-old APP/PS1 mice. In combination, these findings indicate that aging alone is not sufficient to affect brain monoamine levels, unlike the APPSWE/PS1ΔE9 genotype.

AHCODA-DB: a data repository with web-based mining tools for the analysis of automated high-content mouse phenomics data

BackgroundSystematic, standardized and in-depth phenotyping and data analyses of rodent behaviour empowers gene-function studies, drug testing and therapy design. However, no data repositories are currently available for standardized quality control, data analysis and mining at the resolution of individual mice.DescriptionHere, we present AHCODA-DB, a public data repository with standardized quality control and exclusion criteria aimed to enhance robustness of data, enabled with web-based mining tools for the analysis of individually and group-wise collected mouse phenotypic data. AHCODA-DB allows monitoring in vivo effects of compounds collected from conventional behavioural tests and from automated home-cage experiments assessing spontaneous behaviour, anxiety and cognition without human interference. AHCODA-DB includes such data from mutant mice (transgenics, knock-out, knock-in), (recombinant) inbred strains, and compound effects in wildtype mice and disease models. AHCODA-DB provides real time statistical analyses with single mouse resolution and versatile suite of data presentation tools. On March 9th, 2017 AHCODA-DB contained 650 k data points on 2419 parameters from 1563 mice.ConclusionAHCODA-DB provides users with tools to systematically explore mouse behavioural data, both with positive and negative outcome, published and unpublished, across time and experiments with single mouse resolution. The standardized (automated) experimental settings and the large current dataset (1563 mice) in AHCODA-DB provide a unique framework for the interpretation of behavioural data and drug effects. The use of common ontologies allows data export to other databases such as the Mouse Phenome Database. Unbiased presentation of positive and negative data obtained under the highly standardized screening conditions increase cost efficiency of publicly funded mouse screening projects and help to reach consensus conclusions on drug responses and mouse behavioural phenotypes. The website is publicly accessible through https://public.sylics.com and can be viewed in every recent version of all commonly used browsers.

Hepatic gene therapy rescues high-fat diet responses in circadian Clock mutant mice

ObjectiveCircadian Clock gene mutant mice show dampened 24-h feeding rhythms and an increased sensitivity to high-fat diet (HFD) feeding. Restricting HFD access to the dark phase counteracts its obesogenic effect in wild-type mice. The extent to which altered feeding rhythms are causative for the obesogenic phenotype of Clock mutant mice, however, remains unknown. MethodsMetabolic parameters of wild-type (WT) and ClockΔ19 mutant mice (MT) were investigated under ad libitum and nighttime restricted HFD feeding. Liver circadian clock function was partially rescued by hydrodynamic tail vein delivery of WT-Clock DNA vectors in mutant mice and transcriptional, metabolic, endocrine and behavioral rhythms studied. ResultsNighttime-restricted feeding restored food intake, but not body weight regulation in MT mice under HFD, suggesting Clock-dependent metabolic dysregulation downstream of circadian appetite control. Liver-directed Clock gene therapy partially restored liver circadian oscillator function and transcriptome regulation without affecting centrally controlled circadian behaviors. Under HFD, MT mice with partially restored liver clock function (MT-LR) showed normalized body weight gain, rescued 24-h food intake rhythms, and WT-like energy expenditure. This was associated with decreased nighttime leptin and daytime ghrelin levels, reduced hepatic lipid accumulation, and improved glucose tolerance. Transcriptome analysis revealed that hepatic Clock rescue in MT mice affected a range of metabolic pathways. ConclusionLiver Clock gene therapy improves resistance against HFD-induced metabolic impairments in mice with circadian clock disruption. Restoring or stabilizing liver clock function might be a promising target for therapeutic interventions in obesity and metabolic disorders.

Role of the adipose PPAR\u03b3-adiponectin axis in susceptibility to stress and depression/anxiety-related behaviors

Adaptive responses to stressful stimuli involving behavioral, emotional and metabolic changes are orchestrated by the nervous and endocrine systems. Adipose tissue has been recognized as a highly active metabolic and endocrine organ, secreting adipokines that operate as hormones to mediate the crosstalk with other organs including the brain. The role of adipose tissue in sensing and responding to emotional stress and in behavioral regulation, however, remains largely unknown. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a key transcriptional factor controlling adipokine gene expression. Here we show that chronic social defeat stress decreases messenger RNA and protein levels of PPARγ in adipose tissue of susceptible but not resilient mice, which was correlated with social avoidance behavior. A corresponding reduction in adipose adiponectin production was observed in susceptible mice. Rosiglitazone, a blood–brain barrier-impermeant PPARγ-selective agonist, elicited antidepressant- and anxiolytic-like behavioral effects in wild-type mice, with a concurrent increase in plasma adiponectin levels. These effects of rosiglitazone were absent in mice lacking adiponectin but having normal PPARγ expression in adipose tissue and brain. Moreover, pretreatment with the PPARγ-selective antagonist GW9662 blocked rosiglitazone-induced adiponectin expression and antidepressant/anxiolytic-like effects. Together, these results suggest that the behavioral responses to rosiglitazone are mediated through PPARγ-dependent induction of adiponectin. Our findings support an important role for the adipose PPARγ-adiponectin axis in susceptibility to stress and negative emotion-related behaviors. Selectively targeting PPARγ in adipose tissue may offer novel strategies for combating depression and anxiety.