Wild-type Controls Research Articles

Use and Reuse of Animal Behavioral, Molecular, and Biochemical Data in Alzheimer's Disease Research: Focus on 3Rs and Saving People's Tax Dollars.

Several decades of research on cell and animal models contributed tremendously to understanding human diseases. Particularly, research on rodents and non-human primates revealed that animal research is a major and important component in biomedical research in learning complex pathophysiological processes. Further, animal research helped us to understand human diseases, such as Alzheimer's disease. In addition, animal research has also helped us to test hundreds of drugs and develop treatments for human use. Researchers can gain a better understanding of key biological and physiological processes in humans by comparing them to laboratory animals. Based on their relevance and resemblance to people, or even usual living conditions, scientists rationalize the use of particular animal models in their studies. It is suggested that in the National Institutes of Health and other agencies-funded research, animal models should be carefully selected to study the biology and pathophysiology of human health and diseases such as Alzheimer's disease and other dementias. However, it is critical to use a minimum number of animals for human research. Further, it is also noted that the use and reuse of behavioral, molecular, and biochemical data from wild-type (WT) control mice with mutant lines of disease models, as long as the genetic background is the same in both WT and disease mice. On the other hand, anonymous readers have challenged the use and reuse of WT mice data for comparison. In the current article, we discuss the minimum utility of animals, covering the 3Rs, Replacement, Reduction, and Refinement, and also discuss the use and reuse of behavioral, molecular, and biochemical data.

Cerebellar activity in PINK1 knockout rats during volitional gait.

Preclinical models of Parkinson's disease are imperative to gain insight into the neural circuits that contribute to gait dysfunction in advanced stages of the disease. A PTEN-induced putative kinase 1 knockout early-onset model of Parkinson's disease may be a useful rodent model to study the effects of neurotransmitter degeneration caused by a loss of PTEN-induced putative kinase 1 function on brain activity during volitional gait. The goal of this study was to measure changes in neural activity at the cerebellar vermis at 8 months of age. It was found that gait deficits, except run speed, were not significantly different from age-matched wild-type controls, as previously reported. PTEN-induced putative kinase 1 knockout (n = 4) and wild-type (n = 4) rats were implanted with a micro-electrocorticographic array placed over cerebellar vermis Lobules VI (a-c) and VII. Local field potential recordings were obtained during volitional gait across a runway. Power spectral analysis and coherence analysis were used to quantify network oscillatory activity in frequency bands of interest. Cerebellar vermis power was hypoactive in the beta (VIb, VIc and VII) and alpha (VII) bands at cerebellar vermis Lobules VIb, VIc and VII in PTEN-induced putative kinase 1 knockout rats compared with wild-type controls during gait (P < 0.05). These results suggest that gait improvement in PTEN-induced putative kinase 1 knockout rats at 8 months may be a compensatory mechanism attributed to movement corrections caused by a decreased inhibition of the alpha band of cerebellar vermis Lobule VII and beta band of Lobules VIb, VIc and VII. The PTEN-induced putative kinase 1 knockout model may be a valuable tool for understanding the circuit mechanisms underlying gait dysfunction in patients with early-onset Parkinson's disease with a functional loss of PTEN-induced putative kinase 1. Future studies investigating the cerebellar vermis as a potential biomarker and therapeutic target for the treatment of gait dysfunction in Parkinson's disease are warranted.

Investigating recovery after a spontaneous intracerebral haemorrhage in zebrafish larvae.

Intracerebral haemorrhage is a debilitating stroke sub-type with high morbidity and mortality rates. For survivors, rehabilitation is a long process, and with no available therapeutics to limit the immediate pathophysiology of the haemorrhage, recovery is dependent on individual neuroplasticity. We have previously shown that zebrafish larvae can be used to model spontaneous brain haemorrhage. Zebrafish exhibit innate recovery mechanisms and are often used as a model system for investigation into regeneration after injury, including injury to the nervous system. Here, we investigate the spontaneous and immediate recovery in zebrafish larvae following an intracerebral haemorrhage at 2 days post-fertilisation, during pre-protected stages and over the first 3 weeks of life. We have shown that following the onset of bleed at ∼2 days post-fertilisation zebrafish are capable of clearing the haematoma through the ventricles. Brain cell damage associated with intracerebral haemorrhage is resolved within 48 h, and this recovery is associated with survival rates equal to wildtype and non-haemorrhaged sibling control animals. Larvae express more nestin-positive neural progenitor cells 24 h after injury when the most damage is observed, and through mass spectrometry analysis, we have determined that these cells are highly proliferative and may specially differentiate into oligodendrocytes. This study provides an insight into the haematoma resolution processes in a live, intact organism, and may suggest potential therapeutic approaches to support the recovery of intracerebral haemorrhage patients.

Akt1 deficiency does not affect fiber type composition or mitochondrial protein expression in skeletal muscle of male mice.

Insulin-like growth factor-1-induced activation of ATP citrate lyase (ACLY) improves muscle mitochondrial function through an Akt-dependent mechanism. In this study, we examined whether Akt1 deficiency alters skeletal muscle fiber type and mitochondrial function by regulating ACLY-dependent signaling in male Akt1 knockout (KO) mice (12-16 weeks old). Akt1 KO mice exhibited decreased body weight and muscle wet weight, with reduced cross-sectional areas of slow- and fast-type muscle fibers. Loss of Akt1 did not affect the phosphorylation status of ACLY in skeletal muscle. The skeletal muscle fiber type and expression of mitochondrial oxidative phosphorylation complex proteins were unchanged in Akt1 KO mice compared with the wild-type control. These observations indicate that Akt1 is important for the regulation of skeletal muscle fiber size, whereas the regulation of muscle fiber type and muscle mitochondrial content occurs independently of Akt1 activity.

Abstract 10: Adrenal Zona Glomerulosa Long Form Leptin Receptor (LepRb) Protects from Leptin-Mediated Vascular Disorders in Female Mice

Introduction: Previous work from our group demonstrated that leptin is a major contributor to obesity-associated cardiovascular disease, in females, by acting on the adrenal gland to promote aldosterone production. Hypothesis: This leptin-mediated aldosterone secretion is dependent on the presence of the long form leptin receptor (LepRb) in adrenal aldosterone synthase (AS) expressing cells. Methods: We generated AS LepRb deficient mice, ASKO and WT control (ASWT), by crossing LepRb flox/flox mice with AS-Cre mice to investigate the role of LepRb in AS expressing cells in leptin-mediated cardiovascular alteration in females and also generated db/AS + mice that have LepRb only in AS expressing cells by crossing LepR TBflox/flox (db/db) with AS-Cre mice to compare their phenotype. Results: Under baseline conditions, ASKO female mice exhibited no alteration in adrenal AS, plasma aldosterone, systolic blood pressure, and aortic endothelium-dependent relaxation. To mimic obese conditions, we submitted ASWT and ASKO to chronic leptin infusion for a week. Leptin increased adrenal AS expression, aldosterone levels, and blood pressure and impaired endothelial function, which were further elevated in ASKO compared to ASWT, suggesting protective effects of AS LeRb. Both mineralocorticoid receptor (MR) antagonist. eplerenone, or AS antagonist, fadrazole, rescued endothelial function in ASWT and ASKO mice treated with leptin, indicating that leptin-mediated endothelial dysfunction is still aldosterone-dependent in ASKO mice. Restoration of LepRb in AS expressing cells in db/db decreased adrenal AS and plasma aldosterone and improved endothelial function further indicating that AS LepRb blunts leptin-mediated aldosterone production. On a molecular level, leptin treatment elevated aorta NOX1 expression in ASWT, which was further increased in ASKO. Aldosterone treatment increased NOX1 expression in cultured microvascular endothelial cells. Incubation of aortic rings with a selective NOX1 inhibitor restored endothelial function in both ASWT and ASKO treated with leptin, suggesting leptin-mediated aldosterone increased NOX1 expression in the aorta, which causes endothelial dysfunction. Conclusion: These data indicate that AS LepRb protects from leptin-mediated aldosterone production and vascular dysfunction by mitigating the effects of leptin on aldosterone production and suggests that other isoforms of LepR are involved in stimulating aldosterone production.

Abstract P338: Impaired Endothelial-Dependent Vasorelaxation and Myogenic Response in Renal Afferent Arterioles of Diabetic mice: Role of Endothelial NOX1, and New Gene Pathways.

Diabetes increases the risk of cardiovascular disease and hypertension, in part, by decreasing arterial vasodilation through decreased NO bioavailability and increased superoxide. In the kidney, type 1 diabetes induces a decrease in renal filtration, proteinuria, inflammation, and fibrosis, increasing the incidence of kidney disease. In diabetic rats and rabbits, prone to renal disease, diabetes blunts acetylcholine (Ach)-induced vasorelaxation of afferent arterioles, in part, by enhancing endothelial superoxide. However, it is unclear whether diabetes affects afferent arteriole vasodilation and myogenic response in mice. We hypothesize that diabetes increases endothelial NOX1 mediated superoxide production in mouse afferent arterioles, decreasing Ach-induced vasodilation. We dissected and perfused afferent arterioles from male diabetic Akita mice 12-14 weeks of age and measured the effect Ach (10 -7 -10 -5 M) on luminal diameter after pre-constriction with norepinephrine. Diabetic Akita mice had a largely reduced Ach-induced dilation at all doses of Ach (80±6% average inhibition in dilation, p&lt;0.025, n=6). To study the role of NOX1, we used the inhibitor ML171 (5µM). When added to the endothelial lumen, NOX1 inhibition completely restored Ach-induced dilation of diabetic Akita mice, which was not different from WT controls. In Control C57 mice, ML171 did not change Ach-induced dilation in Aff-Art (n=6). We then examined the myogenic response in diabetic mice by measuring lumen diameter after step increases in perfusion pressure (60-140 mmHg). Afferent arterioles of diabetic Akita mice did not constrict when pressure was increased, even at 140 mmHg, whereas WT mice showed normal myogenic response (delta constriction 60-140 mmHg WT: 2.15±0.41 vs Akita: 0.37±0.32 µm, WT p&lt;0.01 from baseline, Akita n.s from baseline). To understand the mechanisms behind these effects we performed RNAseq in manually micro-dissected afferent arterioles from 12-week-old Akita and WT mice. Bioinformatics analysis showed 530 DEGs (FDR pAdj&lt;0.05, n=6). Most of the DEGs are involved in endothelial cell function/metabolism, smooth muscle cell contractility, activated inflammatory pathways, and free radical/NO production. We conclude that early diabetes has a large effect on renal afferent arteriole reactivity by affecting the expression of hundreds of genes. These effects are likely involved in hypertension and kidney damage progression in type 1 diabetes.

Abstract P377: Short-Chain Fatty Acids Improve Cardiac Function in a Hypertensive Rat Model of Heart Failure

Hypertension-induced ventricular remodeling contributes significantly to the progression of heart failure (HF). The global increase in HF prevalence results in heightened cardiovascular morbidity and mortality, yet treatment options remain limited. The gut microbiota ferments indigestible dietary fibers, producing essential metabolites such as short-chain fatty acids (SCFAs), amino acids metabolites. We hypothesize that direct oral administration of SCFAs modulates mucosal immune cells, thereby ameliorating the heart failure with preserved ejection fraction (HFpEF) phenotype in the double-transgenic rat (dTGR) model. Four-week-old male rats, double-transgenic for human renin and angiotensinogen (dTGRs), were utilized. These dTGR rats exhibit classical symptoms of HFpEF, including hypertension, inflammation, and fibrosis in the heart and kidneys. The rats were randomized into five experimental groups: healthy non-transgenic vehicle-treated controls (wild type), vehicle-treated dTGR, and dTGR treated with either acetate, propionate, or butyrate. Following a 3-week treatment period, comprehensive phenotyping was conducted, including echocardiography, Millar-tip catheterization, gene expression analysis, and histological examination. SCFA administration improves diastolic function, as evidenced by alterations in the end-diastolic pressure-volume relationship measured via Millar-tip catheter. Notably, butyrate treatment had the most pronounced effect on alleviating diastolic dysfunction and reducing blood pressure. Additionally, gene expressions such as atrial natriuretic peptide, connective tissue growth factor, and myosin heavy chain alpha and beta showed improvement in SCFA-treated dTGR HFpEF rats compared to vehicle-treated counterparts. Furthermore, SCFA treatment ameliorated cardiac inflammation. Our data suggest that SCFAs, particularly butyric acid, enhance hypertensive diastolic dysfunction and cardiac gene expression in dTGR rats, underscoring their therapeutic potential in HF.

Abstract P353: Sexual dimorphism of blood pressure responses to angiotensin II in rats subjected to early life circadian stress

Various forms of early life stress (ELS) lead to a greater risk of hypertension, but no studies have examined stress produced by circadian disruption. We hypothesize that circadian stress during adolescence will increase sensitivity to a hypertensive stimulus in adulthood recapitulating other models of early life stress (ELS). We exposed male and female rats 1-2 weeks post-weaning to 6-hour phase advances (shifts) in the light/dark cycle each week for 4 weeks. To assess whether the molecular clock contributes to the response to ELS, we performed these studies in both Bmal1 knockout (KO) rats and littermate wildtype (WT) controls, Bmal1 is a core circadian clock transcription factor. At 10-12 weeks of age, baseline mean arterial pressure (MAP) was measured (telemetry) followed by a 2-wk angiotensin II (AngII) infusion (250 ng/kg/min, s.c.). At baseline, non-ELS KO rats of both sexes had lower MAP compared to WT littermates, but this genotype effect was lost in ELS rats. Thus, exposure to circadian stress early in life has only a modest effect on baseline blood pressure in rats lacking Bmal1. AngII increased MAP in all groups but was significantly attenuated in male ELS rats compared to non-ELS controls of both genotypes (p stress = 0.039, p genotype =0.979, p SxG =0.917, 2-way ANOVA), but this attenuation was not evident in female rats (p stress = 0.497, p genotype =0.041, p SxG =0.772). We also observed a significant phase advance in peak MAP in male non-ELS rats of both genotypes that was absent in ELS males (p Genotype =0.030, p AngII &lt;0.001, p AngIIxGT =0.025). In contrast, there were no significant differences in peak MAP time in any female rats. The response to AngII was attenuated in ELS male, but not female rats. These data do not support our original hypothesis that exposure to circadian stress during adolescence induces hypersensitization to Ang II in rats but does reveal an impact on the timing of peak MAP in male rats. We propose that the attenuated MAP response to AngII seen in male rats following exposure to specific types of circadian stress during the post-weaning period may pre-condition rats against a subsequent hypertensive stimulus.

MiR-10a regulates cell death and inflammation in adipose tissue of male mice with diet-induced obesity

ObjectiveAdipose tissue remodeling plays a critical role in obesity-induced metabolic dysfunction, but the underlying molecular mechanisms remain incompletely understood. This study investigates the role of miR-10a-5p in adipose tissue inflammation and metabolic dysfunction induced by a high fat diet (HFD). MethodsMale miR-10a knockout (KO) mice were fed a HFD to induce obesity for up to 16 weeks. RNA sequencing (RNA-seq) analysis was performed to profile mRNA expression and assess the effects of miR-10a-5p KO in gonadal white adipose tissue (gWAT). Additional analyses included immunoblotting, qPCR, histological examination, and validation of the miR-10a-5p target sequence using a dual-luciferase reporter assay. ResultsmiR-10a-5p was highly expressed in gWAT but decreased after 8 weeks of HFD feeding. Over the 16-week HFD period, miR-10a KO mice exhibited greater weight gain and reduced energy expenditure compared to wild-type (WT) controls. gWAT of miR-10a KO mice on a HFD showed an increased population of proinflammatory macrophages, elevated inflammation, and increased cell death, characterized by upregulated apoptosis and necrosis markers. This was also associated with increased triglyceride accumulation in the liver. Mechanistically, the proapoptotic gene Bcl2l11 was identified as a direct target of miR-10a-5p. Loss of miR-10a-5p led to BIM-mediated adipocyte death and inflammation, contributing to mitochondrial metabolic dysregulation, increased fibrosis marker expression, and the onset of inflammation in adipose tissue. ConclusionsThis study demonstrates the significant role of miR-10a-5p and its downstream target BIM in regulating adipocyte death during diet-induced obesity. This signaling pathway presents a potential therapeutic target for modulating obesity-induced inflammation and cell death in adipose tissue.

Abstract MP33: A20 in the Kidney Epithelium Attenuates Angiotensin II-induced Hypertension by Constraining Renal Tubular NHE3 Expression

Whereas the ubiquitin editor A20 limits NF-κB-mediated signaling in myeloid leukocytes, the functions of A20 in renal epithelial cells during hypertension have not been described. We recently found that A20 in the kidney tubule ameliorates hypertension and cardiac hypertrophy induced by chronic angiotensin (Ang) II infusion (500ng/kg/min) in mice. Here we report that following 3 weeks of hypertension, the kidneys of mice with tubular deletion of A20 in the kidney epithelium (“A20 iKKO” = A20 flox/flox Pax8-rtTA TetO-Cre + ) have augmented protein expression of the proximal tubular sodium transporter NHE3 (1.5±0.1 vs. 1.0±0.1; p &lt;0.01) but similar levels of ENaC subunit expression in the collecting tubule ( p =NS for all 3 subunits) vs wild-type (WT) controls. Moreover, kidneys from A20 iKKO mice have upregulated mRNA levels for the T cell chemokine CCL5 (2.4±0.3 vs. 1.0±0.3; p &lt;0.01) and higher proportions of effector memory CD8 + T cells (51.2±2.3 vs. 39.3±2.9 % of CD8 + ; p =0.013) compared to WTs despite similar numbers of effector memory T cells in the spleen ( p =NS). To examine whether A20 in the kidney attenuates hypertension by constraining tubular NHE3 expression, we bred A20 iKKO mice with an NHE3 flox/flox line, thereby generating mice with double A20 and NHE3 deletion in the kidney tubule (“Dual KKO”) mice. At baseline, kidneys from Dual KKO mice showed robust reductions in mRNA for A20 (0.5±0.1 vs. 1.0±0.1; p =0.022) and NHE3 (0.2±0.05 vs. 1.0±0.15; p =0.002) compared to WT littermates. During the first ten days of Ang II infusion, mean arterial pressures (MAPs) in the Dual KKO mice were lower than the MAPs previously recorded in our A20 iKKO cohort (129±3 vs. 152±4 mmHg; p &lt;0.01). Moreover, during 3 weeks of Ang II infusion, Dual KKO and WT mice maintained similar MAPs (145±7 vs. 149±13 mmHg; p =0.78), and heart weight to body weight ratios (5.9±0.3 vs. 5.5±0.2 mg/g; p =0.37) in concomitant measurements. Thus, A20 in the kidney tubule blunts the chronic hypertensive response by curtailing NHE3 expression. Our data further suggest that A20 in the nephron can blunt renal inflammation without altering systemic T cell immunity. Stimulating the A20 signaling pathway in the kidney may thus attenuate NHE3-dependent hypertension without conferring attendant risks of global immunosuppression.

Abstract P424: Podocyte Cell-Specific Guanylyl Cyclase/Natriuretic Peptide Receptor-A is Required for Blood Pressure and Renal Hemodynamics in Sex-Specific Manner

The cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) that increases intracellular production of cGMP and stimulates natriuresis, diuresis, and vasorelaxant activities, thereby, reduces blood pressure (BP) and fluid volume. The role of ANP/NPRA signaling in podocytes, highly specialized epithelial cells covering the outer surfaces of renal glomerular capillaries, remains unclear. The objective of this study was to determine the effect of conditional deletion of podocyte (PD)-specific Npr1 (encoding GC-A/NPRA) in gene-knockout (KO) male and female mice in a sex-dependent manner. Tamoxifen-treated wild-type control (PD Npr1 f/f; WT), heterozygous (PD-Cre- Npr1 f/+; HT), and gene-knockout (PD-Cre- Npr1 f/-; KO) mice were fed a normal-, low-, or high-salt diet for 4 weeks. Podocytes isolated from HT and KO male and female mice showed complete absence of Npr1 mRNA and NPRA protein compared to WT mice. On a normal-salt diet the mean arterial pressure (MAP) and heart rate (HT) were significantly greater in both male and female PD- Npr1 KO mice compared with WT mice as measured by radiotelemetry method. On a high-salt diet, the systolic blood pressure (SBP) was significantly higher (p&lt;0.01; p&lt;0.001) in the PD- Npr1 HT mice (male:108.8 ± 1.10; female: 97 ± 0.80) and KO mice (male:114.60 ± 1; female: 100.10 ± 1.10) compared with WT mice (male: 100.60 ± 0.80; female: 93.30 ± 0.80) at 4 weeks. On normal-, low-, and high-salt diets, plasma creatinine, and urinary protein were significantly increased (p&lt;0.05; p&lt;0.01; p&lt;0.001), while plasma total protein and albumin were significantly reduced (p&lt;0.05; p&lt;0.01; p&lt;0.001) in the Npr1 HT and KO male and female mice compared to WT mice. These changes were significantly greater in males than females. On a normal-salt diet, the glomerular filtration rate (GFR) was significantly (p&lt;0.05; p&lt;0.01; p&lt;0.001) decreased in PD Npr1 HT mice (male: 0.67 ± 0.06; female: 0.81 ± 0.09) and KO mice (male:0.46 ± 0.04; female: 0.69 ± 0.05) compared with WT mice (male:1.1 ± 0.07; female: 1.2 ± 0.1). These observations suggest that in podocyte, ANP/NPRA/cGMP signaling may be crucial in the maintenance and regulation of GFR and hypertension and point as a biomarker of BP and renal hemodynamics in a sex-dependent manner.

Mechanisms of GTN-induced migraine: Role of NOS isoforms, sGC and peroxynitrite in a migraine relevant mouse model.

Migraine research has highlighted the pivotal role of nitric oxide (NO) in migraine pathophysiology. Nitric oxide donors such as glyceryl trinitrate (GTN) induce migraine attacks in humans, whereas spontaneous migraine attacks can be aborted by inhibiting NO production. The present study aimed to investigate how GTN triggers migraine through its three nitric oxide synthase (NOS) isoforms (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) via a suspected feed-forward phenomenon. Migraine-relevant hypersensitivity was induced by repeated injection of GTN in an in vivo mouse model. Cutaneous tactile sensitivity was assessed using von Frey filaments. Signaling pathways involved in this model were dissected using non-selective and selective NOS inhibitors, knockout mice lacking eNOS or nNOS and their wild-type control mice. Also, we tested a soluble guanylate cyclase inhibitor and a peroxynitrite decomposition catalyst (Ntotal = 312). Non-selective NOS inhibition blocked GTN-induced hypersensitivity. This response was partially associated with iNOS, and potentially nNOS and eNOS conjointly. Furthermore, we found that the GTN response was largely dependent on the generation of peroxynitrite and partly soluble guanylate cyclase. Migraine-relevant hypersensitivity induced by GTN is mediated by a possible feed-forward phenomenon of NO driven mainly by iNOS but with contributions from other isoforms. The involvement of peroxynitrite adds to the notion that oxidative stress reactions are also involved.

The Sperm-Associated Antigen 11A (Spag11a) Knockout Mice Display Sub-Fertility and Perturbations in the Sperm Proteome.

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm-associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild-type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function.

Reduced voluntary wheel running behaviour in Kiss1r knockout mice

Kisspeptin and its receptor, Kiss1r, are novel players in the central balance of energy intake and expenditure. Recent evidence also indicates that kisspeptin signalling is important in thermoregulation and generation of the circadian rhythm. We used global Kiss1r knockout mice (Kiss1r KO), which are hypogonadal and develop obesity, to determine the impact of kisspeptin on circadian related behaviour. Voluntary wheel running was examined in Kiss1r KO and wild-type (WT) mice, using gonad intact and gonadectomised (GDX) mice to account for the effects of kisspeptin on gonadal sex steroids. Intact male and female Kiss1r KO mice covered only 10% and 30% of the distance travelled each day by their respective WT controls. In all mice, most of the running activity occurred during the dark phase. GDX WT mice ran significantly less during dark periods than the intact WT. GDX Kiss1r KO male mice ran significantly less than the GDX WT male mice, but the decrease was attenuated compared to intact mice. There was no difference between the female GDX Kiss1r KO and GDX WT. In contrast to the obese phenotype that develops in Kiss1r KO mice, body mass at the end of the study was significantly lower in the GDX Kiss1r KO than it was in the GDX WT mice. The difference in wheel running activity was not associated with any histological change in WAT, BAT, or muscle diameter. No difference in immunohistochemistry expression was seen in lateral hypothalamic orexin neurons or dopamine neurons in the ventral tegmental area / substantia nigra. We observed increased Iba1 expression (activation of microglia) in the arcuate nucleus of male Kiss1r KO mice. Overall, the circadian locomotor activity in male Kiss1r KO mice appears dependent on kisspeptin signalling and the obese phenotype does not develop in Kiss1r KO mice when they engage in voluntary activity.

Age-related effects of optineurin deficiency in the mouse eye

Optineurin (OPTN) is a gene associated with familial normal tension glaucoma (NTG). While NTG involves intraocular pressure (IOP)-independent neurodegeneration of the visual pathway that progresses with age, how OPTN dysfunction leads to NTG remains unclear. Here, we generated an OPTN knockout mouse (Optn−/−) model to test the hypothesis that a loss-of-function mechanism induces structural and functional eye deterioration with aging. Eye anatomy, visual function, IOP, retinal histology, and retinal ganglion cell survival were compared to littermate wild-type (WT) control mice. Consistent with OPTN’s role in NTG, loss of OPTN did not increase IOP or alter gross eye anatomy in young (2–3 months) or aged (12 months) mice. When retinal layers were quantitated, young Optn−/− mice had thinner retina in the peripheral regions than young WT mice, primarily due to thinner ganglion cell-inner plexiform layers. Despite this, visual function in Optn−/− mice was not severely impaired, even with aging. We also assessed relative abundance of retinal cell subtypes, including amacrine cells, bipolar cells, cone photoreceptors, microglia, and astrocytes. While many of these cellular subtypes were unaffected by Optn deletion, more dopaminergic amacrine cells were observed in aged Optn−/− mice. Taken together, our findings showed that complete loss of Optn resulted in mild retinal changes and less visual function impairment, supporting the possibility that OPTN-associated glaucoma does not result from a loss-of-function disease mechanism. Further research using these Optn mice will elucidate detailed molecular pathways involved in NTG and identify clinical or environmental risk factors that can be targeted for glaucoma treatment.

The deletion of nuclear progesterone receptors from kisspeptin cells does not impair negative feedback in female mice.

Reproductive function in mammals depends on the ability of progesterone to suppress pulsatile gonadotrophin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Previous research identified that cells upstream from GnRH neurons expressing the nuclear progesterone receptor (PGR) are required for progesterone-negative feedback. However, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion is unknown. We aimed to address the hypothesis that PGR expressed by a neural population in the arcuate nucleus recently identified as the GnRH pulse generator, cells expressing Kisspeptin, Neurokinin B, and Dynorphin (KNDy cells), mediate progesterone negative feedback. To achieve this, we utilized female mice with the PGR gene conditionally deleted from kisspeptin cells (KPRKO mice) and observed a substantial decrease in the percentage of KNDy neurons co-expressing PGR mRNA (11% in KPRKO mice versus 86% in wildtype mice). However, KPRKO mice did not display changes in the frequency or amplitude of LH pulses in diestrus or estrus, nor in the ability of exogenous progesterone to blunt a post-castration rise in LH. Further, mRNA expression of arcuate kisspeptin and dynorphin, which are excitatory and inhibitory to GnRH secretion, respectively, remained unaltered in KPRKO mice compared to wildtype controls. Together, these findings show that the near-complete loss of PGR signaling from KNDy cells does not impact negative feedback regulation of GnRH pulse generation in mice, suggesting that feedback through this receptor can occur via a small number of KNDy cells or a yet unidentified cell population.

Overexpression of native carbonic anhydrases increases carbon conversion efficiency in the methanotrophic biocatalyst Methylococcus capsulatus Bath.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4 and CO2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2 evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH4 into CO2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4 conversion bioprocesses.

Osteopontin Regulates Treg Cell Stability and Function with Implications for Anti-Tumor Immunity and Autoimmunity.

Foxp3-expressing regulatory T (Treg) cells represent the most highly immunosuppressive cell in the tumor microenvironment (TME) that halts effective anti-tumor immunity. Osteopontin (Opn), an extracellular matrix (ECM) glycophosphoprotein, plays key roles in many types of immune-related diseases and is associated with cancer aggressiveness when expressed by tumor cells. However, its role in Foxp3Treg heterogeneity, function, and stability in the TME is poorly defined. We generated mice with a Foxp3-specific deletion of Opn and assessed the ability of Opn-deficient Tregs to suppress inflammation. As these mice aged, they developed a scurfy-like syndrome characterized by aberrant and excessive activation of effector T cells. We evaluated and further confirmed the reduced suppressive capacity of Opn-deficient Tregs in an in vivo suppression assay of colitis. We also found that mice with Opn-deficient Foxp3+ Tregs have enhanced anti-tumor immunity and reduced tumor burden, associated with an unstable Treg phenotype, paralleled by reduced Foxp3 expression in tumor-infiltrating lymphocytes. Finally, we observed reduced Foxp3 and Helios expression in Opn-deficient Tregs compared to wild-type controls after in vitro activation. Our findings indicate that targeting Opn in Tregs reveals vigorous and effective ways of promoting Treg instability and dysfunction in the TME, facilitating anti-tumor immunity.

The Complement System Is Essential for Arteriogenesis by Enhancing Sterile Inflammation as a Relevant Step in Collateral Artery Growth.

Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 -/-) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 -/- mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 -/- mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 -/- mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68+) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68+/MRC1-) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 -/- mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

Inflammation and Tumor Progression: The Differential Impact of SAA in Breast Cancer Models.

Background: Previous research has shown that the Serum Amyloid A (SAA) protein family is intricately involved in inflammatory signaling and various disease pathologies. We have previously demonstrated that SAA is associated with increased colitis disease severity and the promotion of tumorigenesis. However, the specific role of SAA proteins in breast cancer pathology remains unclear. Therefore, we investigated the role of systemic SAA1 and SAA2 (SAA1/2) in a triple-negative breast cancer mouse model. Methods: Syngeneic breast tumors were established in wild-type mice, and mice lacking the SAA1/2 (SAADKO). Subsequently, tumor volume was monitored, species survival determined, the inflammatory profiles of mice assessed with a multiplex assay, and tumor molecular biology and histology characterized with Western blotting and H&E histological staining. Results: WT tumor-bearing mice had increased levels of plasma SAA compared to wild-type control mice, while SAADKO control and tumor-bearing mice presented with lower levels of SAA in their plasma. SAADKO tumor-bearing mice also displayed significantly lower concentrations of systemic inflammatory markers. Tumors from SAADKO mice overall had lower levels of SAA compared to tumors from wild-type mice, decreased apoptosis and inflammasome signaling, and little to no tumor necrosis. Conclusions: We demonstrated that systemic SAA1/2 stimulates the activation of the NLRP3 inflammasome in breast tumors, leading to the production of pro-inflammatory cytokines. This, in turn, promoted apoptosis and tumor necrosis but did not significantly impact tumor growth or histological grading.