Male Knockout Research Articles

A sex-specific effect of M4 muscarinic cholinergic autoreceptor deletion on locomotor stimulation by cocaine and scopolamine

ObjectiveAcetylcholine modulates the activity of the direct and indirect pathways within the striatum through interaction with muscarinic M4 and M1 receptors. M4 receptors are uniquely positioned to regulate plasticity within the direct pathway and play a substantial role in reward and addiction-related behaviors. However, the role of M4 receptors on cholinergic neurons has been less explored. This study aims to fill this gap by addressing the role of M4 receptors on cholinergic neurons in these behaviors.MethodsTo investigate the significance of M4-dependent inhibitory signaling in cholinergic neurons we created mutant mice that lack M4 receptors on cholinergic neurons. Cholinergic neuron-specific depletion was confirmed using in situ hybridization. We aimed to untangle the possible contribution of M4 autoreceptors to the effects of the global M4 knockout by examining aspects of basal locomotion and dose-dependent reactivity to the psychostimulant and rewarding properties of cocaine, haloperidol-induced catalepsy, and examined both the anti-cataleptic and locomotion-inducing effects of the non-selective anticholinergic drug scopolamine.ResultsBasal phenotype assessment revealed no developmental deficits in knockout mice. Cocaine stimulated locomotion in both genotypes, with no differences observed at lower doses. However, at the highest cocaine dose tested, male knockout mice displayed significantly less activity compared to wild type littermates (p = 0.0084). Behavioral sensitization to cocaine was similar between knockout and wild type mice. Conditioned place preference tests indicated no differences in the rewarding effects of cocaine between genotypes. In food-reinforced operant tasks knockout and wild type mice successfully acquired the tasks with comparable performance results. M4 receptor depletion did not affect haloperidol-induced catalepsy and scopolamine reversal of catalepsy but attenuated scopolamine-induced locomotion in females (p = 0.04). Our results show that M4 receptor depletion attenuated the locomotor response to high doses of cocaine in males and scopolamine in females, suggesting sex-specific regulation of cholinergic activity.ConclusionDepletion of M4 receptors on cholinergic neurons does not significantly impact basal behavior or cocaine-induced hyperactivity but may modulate the response to high doses of cocaine in male mice and the response to scopolamine in female mice. Overall, our findings suggest that M4-dependent autoregulation plays a minor but delicate role in modulating specific behavioral responses to pharmacological challenges, possibly in a sex-dependent manner.

DEP-1 is a brain insulin receptor phosphatase that prevents the simultaneous activation of counteracting metabolic pathways

A healthy metabolism relies on precise regulation of anabolic and catabolic pathways. While insulin deficiency impairs anabolism, insulin resistance in obesity causes metabolic dysfunction, especially via altered brain insulin receptor (IR) activity. Density-enhanced phosphatase 1 (DEP-1) negatively modulates the IR in peripheral tissues. Our study shows that DEP-1 is an insulin-regulated gene, dysregulated in obesity, and uncovers its role in brain insulin signaling, impacting both anabolic and catabolic pathways. Neuro-2a cells lacking DEP-1 demonstrated heightened IR phosphorylation upon acute insulin stimulation. This coincided with simultaneous AMP-activated protein kinase (AMPK) activation, which governs catabolic pathways, due to increased phospholipase C-gamma 1 signaling. These opposing pathways in male DEP-1 forebrain-specific knockout mice resulted in elevated lipolysis in white adipose tissue and fat oxidation in brown adipose tissue, with enhanced sympathetic activation and β-adrenergic receptor expression. In conclusion, DEP-1 deficiency causes the simultaneous activation of IR and AMPK signaling in the brain, with enhanced sympathetic activity in adipose tissues.

Sex differences in the role of AKAP12 in behavioral function of middle-aged mice

A-kinase anchoring protein 12 (AKAP12) is a key scaffolding protein that regulates cellular signaling by anchoring protein kinase A (PKA) and other signaling molecules. While recent studies suggest an important role for AKAP12 in the brain, including cognitive functions, its role in middle-aged mice and potential sex differences are not fully understood. Therefore, this study investigated the effects of AKAP12 on cognitive and exploratory behavior in middle-aged mice, focusing on sex differences. Cognitive function was assessed using the spontaneous Y-maze test and the novel object recognition test (NORT). No significant sex differences in cognitive function were found in middle-aged C57BL/6J mice; however, female mice showed greater exploratory behavior during the NORT. In addition, both middle-aged male and female Akap12 knockout (KO) mice performed similarly to wild-type (WT) mice in the Y-maze test, but had lower discrimination indices in the NORT, suggesting a potential role for AKAP12 in short-term memory. Notably, exploratory behavior was suppressed in female Akap12 KO mice compared to WT mice, whereas male Akap12 KO mice did not show this effect. There were no significant differences in movement distance and velocity during the Y-maze test and NORT between WT and KO mice of either sex. These results indicate that AKAP12 affects cognitive function and exploratory behavior in middle-aged mice and that these effects differ between sexes.

A double knockout for zinc transporter 8 and somatostatin in mice reveals their distinct roles in regulation of insulin secretion and obesity

BackgroundBoth zinc transporter 8 (ZnT8) and somatostatin (Sst) play crucial roles in the regulation of insulin and glucagon secretion. However, the interaction between them in controlling glucose metabolism was not well understood. The aim of this study was to explore the interactive effects of a double knockout of Znt8 and Sst on insulin and glucose metabolism in mice.MethodsCo-expression of ZnT8 with hormones secreted from gastrointestinal endocrine cells of mice was determined using immunofluorescence. Male Znt8 knockout (Znt8KO), Sst knockout (SstKO), double knockout for Sst and Znt8 (DKO), and the wild-type (WT) mice were fed a regular chow diet (CD) or a high-fat diet (HFD) at 3 weeks old for 15 weeks. Weights and fasting or fed glucose levels were determined. Glucose and insulin tolerance tests were performed; metabolic-relevant hormone levels including insulin, glucagon, glucagon-like peptide 1, Pyy, and leptin were determined.ResultsZnT8 is co-expressed with Sst in a subpopulation of endocrine D cells in the gastrointestinal tract. The absence of ZnT8 expression resulted in an increased density of the dense cores in the secretory granules of the D cell. DKO mice had reduced weight compared to WT when maintained on the CD. Compared to Znt8KO and SstKO, DKO mice did not show significant differences in fed or fasting blood glucose level regardless of dietary conditions. However, the CD-fed DKO mice had impaired insulin secretion without alterations in islet morphology or numbers. Moreover, DKO mice displayed diet-induced insulin resistance and disrupted secretion of metabolic-related hormones.ConclusionsSomatostatin as well as a normal insulin sensitivity are required for normalizing glucose metabolism in Znt8KO mice. ZnT8 may play a role in regulating fat mass and leptin secretion. These findings shed light on the multifaceted nature of Znt8 and Sst interactions, opening new avenues to understand their roles in controlling glucose metabolism and fat mass.

Compensatory mechanisms that maintain androgen production in mice lacking key androgen biosynthetic enzymes

AbstractTestosterone and dihydrotestosterone (DHT) are essential for male development and fertility. In the canonical androgen production pathway, testosterone is produced in the testis by HSD17B3; however, adult male Hsd17b3 knockout (KO) mice continue to produce androgens and are fertile, indicating compensatory mechanisms exist. A second, alternate pathway produces DHT from precursors other than testosterone via 5α‐reductase (SRD5A) activity. We hypothesized that the alternate pathway contributes to androgen bioactivity in Hsd17b3 KO mice. To investigate contributions arising from and interactions between the canonical and alternate pathways, we pharmacologically inhibited SRD5A and ablated Srd5a1 (the predominant SRD5A in the testis) on the background of Hsd17b3 KO mice. Mice with perturbation of either the canonical or both pathways exhibited increased LH, testicular steroidogenic enzyme expression, and normal reproductive tracts and fertility. In the circulation, alternate pathway steroids were increased in the absence of HSD17B3 but were reduced by co‐inhibition of SRD5A1. Mice with perturbations of both pathways produced normal basal levels of intratesticular testosterone, suggesting the action of other unidentified hydroxysteroid dehydrogenase(s). Strikingly, testicular expression of another SRD5A enzyme, Srd5a2, was markedly increased in the absence of Hsd17b3, suggesting a compensatory increase in SRD5A2 to maintain androgen bioactivity during HSD17B3 deficiency. Finally, we observed elevated circulating concentrations of the 11‐keto‐derivative of DHT, suggesting compensatory extra‐gonadal induction of bioactive 11‐keto androgen production. Taken together, we conclude that, in the absence of the canonical pathway of androgen production, multiple intra‐ and extra‐gonadal mechanisms cooperate to maintain testosterone and DHT production, supporting male development and fertility.

IL-10 is not required for the alleviation of collagen-induced arthritis by non-lethal malarial infection in mice

We previously reported that Plasmodium yoelii 17XNL (Py), a non-lethal rodent malarial parasite, could suppress collagen-induced arthritis (CIA) and increase the production of T cell-derived interleukin (IL)-10. However, it remained unclear whether IL-10 is essential for the Py-induced suppression of CIA. Male IL-10 knockout (KO) DBA/1 J mice were immunized with bovine type II collagen (CII) and subsequently infected with Py at one week post-immunization. The development of arthritis was evaluated by an arthritis score up to 6 weeks post-immunization. At 3 weeks post-immunization, cytokine production from splenocytes and serum anti-CII IgG/IgG1/IgG2a levels were compared between non-infected control mice and Py-infected mice. Py infection inhibited the development of CIA in IL-10KO mice until 4 weeks post-immunization, after which the arthritis score reached levels comparable with the control mice. Both pro-arthritic (IL-17 and TNF-α) and anti-arthritic (IFN-γ and IL-4) cytokines were down-regulated during the periods of parasitemia, while no significant differences were observed in levels of anti-CII IgG antibodies. Our findings indicate that Py alleviates CIA via IL-10-independent mechanisms.

Humanin-G Ameliorates Hemorrhage-Induced Acute Lung Injury in Mice Through AMPKα1-Dependent and -Independent Mechanisms.

Background/Objectives: The severity of acute lung injury is significantly impacted by age and sex in patients with hemorrhagic shock. AMP-activated protein kinase (AMPK) is a crucial regulator of energy metabolism but its activity declines with aging. Humanin is a mitochondrial peptide that exerts cytoprotective effects in response to oxidative stressors and is associated with longevity. Using a mouse model of hemorrhagic shock that mimics the clinical condition of adult patients, we investigated whether treatment with a humanin analog, humanin-G, mitigates lung injury and whether its mechanisms of action are dependent on the catalytic AMPKα1 subunit activation. Methods: Male and female AMPKα1 wild-type (WT) and knock-out (KO) mice (8-13 months old) were subjected to hemorrhagic shock by blood withdrawal, followed by resuscitation with shed blood and lactated Ringer's solution. The mice were treated with PEGylated humanin-G or vehicle and euthanized 3 h post-resuscitation. Results: Sex- and genotype-related differences were observed after hemorrhagic shock as lung neutrophil infiltration was more pronounced in the male AMPKα1 WT mice than the female WT mice; also, the male AMPKα1 KO mice experienced a significant decline in mean arterial blood pressure when compared to the male WT mice after resuscitation. The scores of histological lung injury were similarly elevated in all the male and female AMPKα1 WT and KO mice when compared to the control mice. At molecular analysis, acute lung injury was associated with the downregulation of AMPKα1/α2 catalytic subunits in the WT mice, whereas an increased activation of the signal transducer and activator of transcription-3 (STAT3) was observed in all the vehicle-treated groups. The in vivo administration of humanin-G ameliorated histological lung damage in all the groups of animals and ameliorated mean arterial blood pressure in the male AMPKα1 KO mice. The in vivo administration of humanin-G lowered lung neutrophil infiltration in the male and female AMPKα1 WT mice only but not in the KO mice. The beneficial results of humanin-G correlated with the lung cytosolic and nuclear activation of AMPKα in the male and female AMPKα1 WT groups, whereas STAT3 activation was not modified. Conclusions: In adult age, hemorrhage-induced acute lung injury manifests with sex-dependent characteristics. Humanin-G has therapeutic potential and the AMPKα1subunit is an important requisite for its inhibitory effects on lung leucosequestration, but not for the amelioration of lung alveolar structure or the hemodynamic effects of the peptide.

KDM5C is a sex-biased brake against germline gene expression programs in somatic lineages.

The division of labor among cellular lineages is a pivotal step in the evolution of multicellularity. In mammals, the soma-germline boundary is formed during early embryogenesis, when genes that drive germline identity are repressed in somatic lineages through DNA and histone modifications at promoter CpG islands (CGIs). Somatic misexpression of germline genes is a signature of cancer and observed in select neurodevelopmental disorders. However, it is currently unclear if all germline genes use the same repressive mechanisms and if factors like development and sex influence their dysregulation. Here, we examine how cellular context influences the formation of somatic tissue identity in mice lacking lysine demethylase 5c (KDM5C), an X chromosome eraser of histone 3 lysine 4 di and tri-methylation (H3K4me2/3). We found male Kdm5c knockout (-KO) mice aberrantly express many tissue-specific genes within the brain, the majority of which are unique to the germline. By developing a comprehensive list of mouse germline-enriched genes, we observed Kdm5c -KO cells aberrantly express key drivers of germline fate during early embryogenesis but late-stage spermatogenesis genes within the mature brain. KDM5C binds CGIs within germline gene promoters to facilitate DNA CpG methylation as embryonic stem cells differentiate into epiblast-like cells (EpiLCs). However, the majority of late-stage spermatogenesis genes expressed within the Kdm5c -KO brain did not harbor promoter CGIs. These CGI-free germline genes were not bound by KDM5C and instead expressed through ectopic activation by RFX transcription factors. Furthermore, germline gene repression is sexually dimorphic, as female EpiLCs require a higher dose of KDM5C to maintain germline silencing. Altogether, these data revealed distinct regulatory classes of germline genes and sex-biased silencing mechanisms in somatic cells.

Psilocybin reduces grooming in the SAPAP3 knockout mouse model of compulsive behaviour

Psilocybin is a serotonergic psychedelic compound which shows promise for treating compulsive behaviours. This is particularly pertinent as compulsive disorders require research into new pharmacological treatment options as the current frontline treatments such as selective serotonin reuptake inhibitors, require chronic administration, have significant side effects, and leave almost half of the clinical population refractory to treatment.In this study, we investigated psilocybin administration in male and female SAPAP3 knockout (KO) mice, a well-validated mouse model of obsessive compulsive and related disorders. We assessed the effects of acute psilocybin (1 mg/kg, intraperitoneal) administration on head twitch and locomotor behaviour as well as anxiety- and compulsive-like behaviours at multiple time-points (1, 3 and 8 days post-injection).While psilocybin did not have any effect on anxiety-like behaviours, we revealed that acute psilocybin administration led to enduring reductions in compulsive behaviour in male SAPAP3 KO mice and reduced grooming behaviour in female wild-type (WT) and SAPAP3 KO mice. We also found that psilocybin increased locomotion in WT littermates but not in SAPAP3 KO mice, suggesting in vivo serotonergic dysfunctions in KO animals. On the other hand, the typical head-twitch response following acute psilocybin (confirming its hallucinogenic-like effect at this dose) was observed in both genotypes.Our novel findings suggest that acute psilocybin may have potential to reduce compulsive-like behaviours (up to 1 week after a single injection). Our study can inform future research directions as well as supporting the utility of psilocybin as a novel treatment option for compulsive disorders.

Dopamine D3 receptor mediates natural and methamphetamine rewards via regulating the expression of miR-29c in the nucleus accumbens of mice

The dopamine D3 receptor (D3R), principally confined to the nucleus accumbens (NAc), is involved in regulating natural and drug rewards; however, the molecular mechanisms underlying the associated process remain unclear. Earlier research has reported the concurrent influence of D3R and miR-29c expressed in the NAc on methamphetamine (METH)-induced reward behaviors and microglial activation, hinting at regulatory roles in reward processing. Herein, we performed viral manipulation-mediating D3R/miR-29c overexpression and inhibition in the whole NAc in male D3R knockout and wild-type mice to investigate this potential relationship. Behavioral responses to the rewarding stimuli were assessed using sucrose preference score, METH-induced locomotor sensitization, and METH-induced conditioned place preference tests. Overall, we observed a notable decrease in the behavioral response to sucrose and METH in D3R-deficient mice, accompanied by the downregulation of miR-29c expression in the NAc. Diminished responses to those rewarding stimuli in D3R-deficient mice primarily stemmed from the reduction of GSK3β activity and subsequent down-regulation of miR-29c in the NAc. Microglial activation in the NAc mediates the effect of D3R-miR-29c deficiency on the reward effects of sucrose and METH. Pharmacological suppression of microglial activity rescued the reduced response in mice lacking D3R-miR-29c in the NAc. Overall, this study revealed the mechanism by which D3R regulates both natural and drug rewards via miR-29c in the murine NAc, highlighting the role of the NAc D3R-miR-29c pathway as a critical regulator of rewards, and providing new insights into the role of NAc D3R-miR-29c in encoding rewarding experiences.

Ninjurin1 deficiency differentially mitigates colorectal cancer induced by azoxymethane and dextran sulfate sodium in male and female mice.

This study investigated the role of Ninjurin1 (Ninj1), encoding a small transmembrane protein, in colitis-associated colon tumorigenesis in relation to sex hormones. Male and female wild-type (WT) and Ninj1 knockout (KO) mice were treated with azoxymethane (AOM) and dextran sulfate sodium (DSS), with or without testosterone propionate (TP). At week 2 (acute colitis stage), Ninj1 KO exhibited an alleviation in the colitis symptoms in both male and female mice. The M2 macrophage population increased and CD8+ T cell population decreased only in the female Ninj1 KO than in the female WT AOM/DSS group. In the female AOM/DSS group, TP treatment exacerbated colon shortening in the Ninj1 KO than in the WT. At week 13 (tumorigenesis stage), male Ninj1 KO mice had fewer tumors, but females showed similar tumors. In the WT AOM/DSS group, females had more M2 macrophages and fewer M1 macrophages than males, but this difference was absent in Ninj1 KO mice. In the Ninj1 KO versus WT group, the expression of pro-inflammatory mediators and Ho-1 and CD8+ T cell populations decreased in both female and male Ninj1 KO mice. In the WT group, M2 macrophage populations were increased by AOM/DSS treatment and decreased by TP treatment. However, neither treatment changed the cell populations in the Ninj1 KO group. These results suggest that Ninj1 is involved in colorectal cancer development in a testosterone-dependent manner, which was different in male and female. This highlights the importance of considering sex disparities in understanding Ninj1's role in cancer pathogenesis.

An in vitro and in vivo efficacy evaluation of gene therapy candidate SBT101 in mouse models of adrenomyeloneuropathy and in NHPs

Adrenomyeloneuropathy is a progressive neurodegenerative disease caused by pathogenic variants in the ABCD1 gene, resulting in very-long-chain fatty acid (VLCFA) accumulation that leads to dying-back axonopathy. Our candidate gene therapy, SBT101 (AAV9-human ABCD1 [hABCD1]), aims to ameliorate pathology by delivering functional copies of hABCD1 to the spinal cord. Transduced cells produce functional ABCD1 protein, thereby repairing the underlying biochemical defect. Invitro and invivo mouse studies were conducted to assess the biochemical and functional efficacy of SBT101 and show effective delivery to target tissues involved in the disease pathology: spinal cord and dorsal root ganglia. Administration of SBT101 to mixed glial cell cultures from Abcd1-Null mice, and to male Abcd1 knockout (Abcd1 -/y ) and double-knockout (Abcd1 -/y /Abcd2 -/- ) mice led to increased hABCD1 production and reduced VLCFA. Double-knockout mice also exhibited improved grip strength. Furthermore, we conducted biodistribution and safety assessments in nonhuman primates. Six-hour intrathecal lumbar infusions demonstrated effective transduction throughout target tissues,supporting the clinical feasibility of the procedure. SBT101 was well tolerated, with no observed SBT101-relatedmortality or clinical signs. These findings not only providepreclinical efficacy data for SBT101 but also inform clinically relevant SBT101 dose selection for patients with adrenomyeloneuropathy.

8030 Sexually Dimorphic Metabolic Consequences Of CCN5/WISP2 Gene Deletion Revealed A Male-Specific Pattern Of Gene Expression

Abstract Disclosure: M. Yang: None. V. Xega: None. M. Zakikhani: None. J. Liu: None. Matricellular protein CCN5/WISP2 is a novel target of IGF-1 and WNT signaling within the pancreatic islets. Our previous research suggests CCN5 promotes β-cell proliferation and survival through Akt/PKB and focal adhesion kinase signaling pathways. More recently, we have reported sexual dimorphic features of CCN5 gene deletion and overexpression. Specifically, male knockout mice demonstrated improved insulin sensitivity and glucose tolerance upon high-fat diet (HFD)-induced obesity, while females were unaffected (ENDO 2022). On the other hand, male aP2-CCN5 transgenic mice that overexpress CCN5 in adipose tissues were partially protected from HFD-streptozotocin induced diabetes, with improved glucose tolerance; females exhibited impaired glucose tolerance and exacerbated diabetes (ENDO 2023). Our findings indicate that an increased level of CCN5 protects β-cells, but only in male mice. This protection cannot be solely attributed to the pro-islet effect and suggests the involvement of extra-pancreatic and sex-specific roles. This study indirectly screened normal CCN5 gene expression by detecting β-galactosidase activity, measured in X-gal-stained tissues before fixation and sectioning. In order to create the gene deletion through homologous recombination, LacZ was inserted between exons 2 and 5 of the CCN5 gene. As a result, male knockout mice exhibited a distinct blue staining in the pseudostratified columnar epithelium with stereocilia of the epididymis and vas deferens. This type of epithelium comprises a single layer of cells but appears to have multiple layers due to the nuclei being at different levels. The epithelium possesses long projections made of actin filaments and plays a crucial role in absorbing, transporting, and maturing sperm cells. The staining was also clear in Leydig cells of the testis, which produce testosterone, which was confirmed using immunohistochemistry and authentic CCN5 antibody. In female mice, however, only a few cells in the ovary and scattered staining was observed in the endometrial glands of the uterus. Since androgens promote energy expenditure, lipolysis, and insulin sensitivity, knockout or overexpression of CCN5 may potentially affect androgen and/or sperm production, leading to metabolic consequences in males. Our results from in vitro, knockout, and overexpression models support the notion that CCN5 promotes β-cell growth and survival against diabetes and further suggest that it possesses sex-specific, metabolic effects. Presentation: 6/2/2024

Tissue nonspecific alkaline phosphatase deficiency impairs Purkinje cell development and survival in a mouse model of infantile hypophosphatasia

Loss-of-function mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene can result in hypophosphatasia (HPP), an inherited multi-systemic metabolic disorder that is well-known for skeletal and dental hypomineralization. However, emerging evidence shows that both adult and pediatric patients with HPP suffer from cognitive deficits, higher measures of depression and anxiety, and impaired sensorimotor skills. The cerebellum plays an important role in sensorimotor coordination, cognition, and emotion. To date, the impact of TNAP mutation on the cerebellar circuitry development and function remains poorly understood. The main objective of this study was to investigate the roles of TNAP in cerebellar development and function, with a particular focus on Purkinje cells, in a mouse model of infantile HPP. Male and female wild type (WT) and TNAP knockout (KO) mice underwent behavioral testing on postnatal day 13–14 and were euthanized after completion of behavioral tests. Cerebellar tissues were harvested for gene expression and immunohistochemistry analyses. We found that TNAP mutation resulted in significantly reduced body weight, shorter body length, and impaired sensorimotor functions in both male and female KO mice. These developmental and behavioral deficits were accompanied by abnormal Purkinje cell morphology and dysregulation of genes that regulates synaptic transmission, cellular growth, proliferation, and death. In conclusion, inactivation of TNAP via gene deletion causes developmental delays, sensorimotor impairment, and Purkinje cell maldevelopment. These results shed light on a new perspective of cerebellar dysfunction in HPP.

Myeloid deficiency of heparan sulfate 6-O-endosulfatases impairs bone marrow hematopoiesis

The heparan sulfate (HS) 6-O-endosulfatases or the Sulfs (Sulf1 and Sulf2) are the only known enzymes that can modify HS sulfation status extracellularly and have been shown to regulate diverse biological processes. The role of the Sulfs in bone marrow (BM) hematopoiesis is not known. In this study, we generated a novel mouse line with myeloid-specific deletion of the Sulfs by crossing Sulf1/2 double floxed mice with the LysM-cre line. The LysM-Sulf knockout (KO) male mice exhibited age-dependent expansion of hematopoietic stem cells and the granulocyte-monocyte lineages in the BM, whereas common lymphoid progenitors and B lymphocyte populations were significantly reduced. Although megakaryocytic and erythroid progenitors were not reduced in the BM, the LysM-Sulf KO males suffered age-dependent reduction of red blood cells (RBCs) and platelets in the peripheral blood, suggesting that the production of RBCs and platelets was arrested at later stages. In addition, LysM-Sulf KO males displayed progressive splenomegaly with extramedullary hematopoiesis. Compared to males, LysM-Sulf KO females exhibited a much-reduced phenotype, and ovariectomy had little effect. Mechanistically, reduced TGF-β/Smad2 but enhanced p53/p21 signaling were observed in male but not female LysM-Sulf KO mice. Finally, HS disaccharide analysis via LC-MS/MS revealed increased HS 6-O-sulfation in the BM from both male and female LysM-Sulf KO mice, however, the distribution of 6-O-sulfated motifs were different between the sexes with compensatory increase in Sulf1 expression observed only in LysM-Sulf KO females. In conclusion, our study reveals that myeloid deficiency of the Sulfs leads to multilineage abnormalities in BM hematopoiesis in an age- and sex-dependent manner.

Hepatic bile acid accretion correlates with cholestatic liver injury and therapeutic response in Cyp2c70 knockout mice with a humanized bile acid composition.

Cyp2c70 knockout (KO) mice lack the liver enzyme responsible for synthesis of 6-hydroxylated muricholate bile acid species and possess a more hydrophobic human-like bile acid composition. Cyp2c70 KO mice develop cholestatic liver injury that can be prevented by the administration of an ileal bile acid transporter (IBAT) inhibitor. In this study, we investigated the potential of an ileal bile acid transporter (IBAT) inhibitor (SC-435) and steroidal farnesoid X receptor (FXR) agonist (cilofexor) to modulate established hepatobiliary injury and the consequent relationship of intrahepatic bile acid content and hydrophobicity to the cholestatic liver injury phenotype. Oral administration of SC-435, cilofexor, or combined treatment for 2 wk markedly reduced serum markers of liver injury and improved histological and gene expression markers of fibrosis, liver inflammation, and ductular reaction in male and female Cyp2c70 KO mice, with the greatest benefit in the combination treatment group. The IBAT inhibitor and FXR agonist significantly reduced intrahepatic bile acid content but not hepatic bile acid pool hydrophobicity, and markers of liver injury were strongly correlated with intrahepatic total bile acid and taurochenodeoxycholic acid accretion. Biomarkers of liver injury increased linearly with similar hepatic thresholds for pathological accretion of hydrophobic bile acids in male and female Cyp2c70 KO mice. These findings further support targeting intrahepatic bile acid retention as a component of treatments for cholestatic liver disease.NEW & NOTEWORTHY Bile acids are implicated as a common contributor to the pathogenesis and progression of cholestatic liver disease. Using a mouse model with a humanized bile acid composition, we demonstrated that mono and combination therapy using an IBAT inhibitor and FXR nonsteroidal agonist were effective at reducing hepatic bile acid accretion and reversing liver injury, without reducing hepatic bile acid hydrophobicity. The findings support the concept of a therapeutically tractable threshold for bile acid-induced liver injury.

Knockout of Sirtuin 3 in endothelial cells impairs endothelial-dependent relaxation and myogenic response in mice.

Sirtuin 3 has been shown to regulate endothelial function and coronary flow reserve in mice. Knockout of SIRT3 reduced endothelial nitric oxide synthase expression in the mouse hearts. In this study, we investigate whether endothelial SIRT3 regulates vascular function and myogenic responses in distal intramural branches of the left anterior descending coronary artery (CA) and middle cerebral artery (MCA) of mice. Both male and female endothelial SIRT3 knockout (SIRT3ECKO) mice and control SIRT3LoxP mice were used and CA and MCA were dissected and mounted in a myograph system. The myogenic response was evaluated by measuring changes in inner diameter in response to 20 mmHg stepwise increases in intraluminal pressure in PSS (active diameter) and Ca2+-free PSS (passive diameter). Acetylcholine (Ach)-induced endothelial-dependent relaxation (EDR) and sodium nitroprusside (SNP)-induced endothelial-independent relaxation (EIR) were examined. Our results showed that the myogenic responses were significantly impaired in both the CA and MCA of SIRT3ECKO mice. Furthermore, female mice had worsened myogenic response in MCA. In CA, EDR was abolished in both male and female SIRT3ECKO mice. Intriguingly, EIR was only reduced in the female mice. In MCA, EDR was reduced in male SIRT3ECKO mice, whereas EIR was decreased in both male and female mice. Female SIRT3ECKO mice had profound dysfunction in CA, whereas male mice exhibited more dysfunction in MCA. These data revealed a sex and organ-specific role of endothelial SIRT3 in vascular function and myogenic responses. Our study suggests that endothelial SIRT3 is necessary for maintaining vascular function and blood flow autoregulation.

Microglial TREM2 promotes phagocytic clearance of damaged neurons after status epilepticus

In the central nervous system, triggering receptor expressed on myeloid cells 2 (TREM2) is exclusively expressed by microglia and is critical for microglial proliferation, migration, and phagocytosis. Microglial TREM2 plays an important role in neurodegenerative diseases, such as Alzheimer’s disease and amyotrophic lateral sclerosis. However, little is known about how TREM2 affects microglial function within epileptogenesis. To investigate this, we utilized male TREM2 knockout (KO) mice within the intra-amygdala kainic acid seizure model. Electroencephalographic analysis, immunocytochemistry, and RNA sequencing revealed that TREM2 deficiency significantly promoted seizure-induced pathology. We found that TREM2 KO increased both the severity of acute status epilepticus and the number of spontaneous recurrent seizures characteristic of chronic focal epilepsy. Phagocytic clearance of damaged neurons by microglia was also impaired by TREM2 KO and reduced phagocytic activity correlated with increased spontaneous seizures. Analysis of human tissue from patients who underwent surgical resection for drug resistant temporal lobe epilepsy also showed a negative correlation between expression of the microglial phagocytic marker CD68 and focal to bilateral tonic-clonic generalized seizure history. These results indicate that microglial TREM2 and phagocytic activity are important to epileptogenic pathology.

Fads2 knockout mice reveal that ALA prevention of hepatic steatosis is dependent on delta-6 desaturase activity

The production of the omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) relies on the delta-6 desaturase (D6D) enzyme encoded by the Fads2 gene. While EPA and DHA reduce hepatic triacylglycerol (TAG) storage and regulate lipogenesis, the independent impact of ALA is less understood. To address this gap in knowledge, hepatic fatty acid metabolism was investigated in male wild-type (WT) and Fads2 knockout (KO) mice fed diets (16% kcal from fat) containing either lard (no n-3 LCPUFA), flaxseed oil (ALA-rich), or menhaden oil (EPA/DHA rich) for 21 weeks. Fat content and composition, as well as markers of lipogenesis, glyceroneogenesis, and TAG synthesis, were analyzed using histology, gas chromatography, and reverse transcription quantitative PCR (RT-qPCR). Mice fed the menhaden diet had significantly lower hepatic TAG compared to both lard- and flax-fed mice, concomitant with changes in n-3 and n-6 LCPUFA in both TAG and phospholipid (PL) fractions (all P < 0.05). Flax-fed WT mice had lower liver TAG content compared to their KO counterparts. Menhaden-fed mice had significantly lower expression of key lipogenic (Scd1, Srebp-1c, Fasn, Fads1, and Fads2), glyceroneogenic (Pck1), and TAG synthesis (Agpat3) genes compared to lard, with flax-fed mice showing some intermediate effects. Gene expression effects were independent of D6D activity, since no differences were detected between WT and KO mice fed the same diet. This study demonstrates that EPA/DHA and not ALA itself is critical for the prevention of hepatic steatosis.

Abstract 09: Leucine Rich Repeat Containing 8A Anion Channels Modulate Vascular Reactivity in ApoE Knockout Mice

Volume-regulated anion channels (VRACs) are comprised of Leucine Rich Repeat Containing 8 (LRRC8) family proteins which include LRRC8A, B, C, D and E. We have proposed that these channels may allow influx of extracellular superoxide to drive inflammation and modulate cultured vascular smooth muscle cell (VSMC) inflammation. We previously demonstrated that LRRC8A knockdown or inhibition inhibits tumor necrosis factor alpha (TNFα)-induced inflammation. Mesenteric arteries from male smooth muscle-specific LRRC8A knockout (KO) mice displayed enhanced responsiveness to both endothelium-dependent (acetylcholine, ACh), and independent (sodium nitroprusside, SNP) vasodilators compared to vessels from WT mice. KO vessels were also protected from vascular dysfunction induced by TNFα in tissue culture (48hr exposure). We hypothesized that reduced VSMC inflammation in LRRC8A KO mice would protect vascular function in ApoE knockout (ApoE -/- ). We created ApoE -/- mice lacking LRRC8A only in VSMCs and assessed vascular reactivity of isolated mesenteric arteries by wire myography in female and male WT, Heterozygous (HET) and KO tissues after 14 weeks of high fat diet. WT vessels displayed enhanced in contraction compared to LRRC8A KO or HET (male PE max contraction of KCL (%), WT 177 ± 4.6 vs. KO 152 ± 3.3 or HET 153 ± 3.0, p &lt; 0.05, n=6-7). Relaxation responses to ACh were increased in female KO or HET compared to WT (ACh LogEC 50 , WT -7.2 ± 0.09 vs. KO -7.5 ± 0.09 or HET -7.4 ± 0.07, p &lt; 0.05, n=5-6). SNP-mediated relaxation were increased in male KO vessels compared to WT (SNP LogEC 50 , WT -8.1 ± 0.06 vs. KO -8.3 ± 0.06, p &lt; 0.05, n=7). These results demonstrate that loss (KO) or reduction (HET) of LRRC8A expression preserves vascular function in the face of the inflammatory response induced by hypercholesterolemia in ApoE -/- mice. These channels may represent a novel therapeutic target for preservation of vascular function and this may only require partial inhibition of current.