Male Knockout Mice Research Articles

The loss of the hormonal function of bilirubin induces adiposity in male and female mice

The prevalence of obesity and its related comorbidities, including diabetes and metabolic dysfunction associated fatty liver disease (MAFLD), are on the rise globally, indicating the importance of new therapeutics for these diseases. Interestingly, serum bilirubin levels are negatively correlated with obesity, diabetes, and MAFLD. We have previously generated pegylated bilirubin nanoparticles (PEGBR) and demonstrated their therapeutic potential in the treatment of obesity and MAFLD. However, it’s unknown whether their beneficial effects are due to bilirubin’s antioxidant properties or its hormonal function by binding to and activating the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa). To determine this, we used female and male hepatocyte-specific PPARa knockout mice ( PparaHepKO) and floxed ( Pparaflox) control mice on a high-fat diet for 30 weeks to induce adiposity and MAFLD. The mice were treated with vehicle or PEGBR (30 mg/kg) for six weeks. In female Pparaflox mice, PEGBR significantly lowered body weight, but not in the female PparaHepKO mice. A similar trend was seen in the male Pparaflox mice but was not statistically significant. PEGBR significantly reduced hepatic fat mass and triglycerides in the female and male Pparaflox mice but not in the PparaHepKO mice. Using lipidomics, we determined the lipid composition of the liver and how PEGBR changed it. In the male Pparaflox mice, PEGBR significantly reduced 109 lipid species, whereas, in the male PparaHepKO mice, PEGBR only reduced 7 species (93.6% reduction). In the female PparaHepKO mice, there was a 2.7% reduction in the number of lipid species significantly decreased by PEGBR compared to Pparaflox controls. Male Pparaflox mice had an increase in plasma high-density lipoprotein cholesterol and a decrease in very-low-density particle number with PEGBR, but this was not seen in the male PparaHepKO or female mice. In addition to protecting against the development of MAFLD, PEGBR significantly reduced fasting blood glucose and insulin levels only in the Pparaflox mice. Overall, the data indicates that the protective effects of bilirubin on obesity, MAFLD, and glucose tolerance are due to its hormonal function through PPARa. This study also confirms the therapeutic potential of bilirubin nanoparticles for obesity and its associated comorbidities. This work was supported by the National Institutes of Health F31HL170972 (Z.A.K.) and R01DK121797 (T.D.H.J.) and R01DA058933 (T.D.H.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Closed-loop recruitment of striatal interneurons prevents compulsive-like grooming behaviors

Compulsive behaviors have been associated with striatal hyperactivity. Parvalbumin-positive striatal interneurons (PVIs) in the striatum play a crucial role in regulating striatal activity and suppressing prepotent inappropriate actions. To investigate the potential role of striatal PVIs in regulating compulsive behaviors, we assessed excessive self-grooming—a behavioral metric of compulsive-like behavior—in male Sapap3 knockout mice (Sapap3-KO). Continuous optogenetic activation of PVIs in striatal areas receiving input from the lateral orbitofrontal cortex reduced self-grooming events in Sapap3-KO mice to wild-type levels. Aiming to shorten the critical time window for PVI recruitment, we then provided real-time closed-loop optogenetic stimulation of striatal PVIs, using a transient power increase in the 1–4 Hz frequency band in the orbitofrontal cortex as a predictive biomarker of grooming onsets. Targeted closed-loop stimulation at grooming onsets was as effective as continuous stimulation in reducing grooming events but required 87% less stimulation time, paving the way for adaptive stimulation therapeutic protocols.

Loss of AC10 Impairs Neutrophil Recruitment and Alters Bacterial Colonization in the Lung During Bacterial Pneumonia

Rationale: Pseudomonas aeruginosa is a gram-negative bacterium and a common cause of nosocomial pneumonia that can progress to acute respiratory distress syndrome (ARDS), characterized by hypoxia, pulmonary edema, elevated bicarbonate, and widespread inflammation. Neutrophil recruitment is a critical line of defense against P. aeruginosa; however, excessive, or dysregulated neutrophil infiltration can exacerbate tissue damage and contribute to the severity of ARDS. Recently, AC10 has been implicated in neutrophil transendothelial migration via CD99. Despite the growing recognition and significance of AC10 in various physiological processes, its role in neutrophil recruitment during ARDS remains poorly understood. Thus, herein we hypothesize that loss of AC10 attenuates neutrophil recruitment during pneumonia and increase bacterial colonization in the lung. Methods: Wild-type and AC10 knockout mice were intratracheally inoculated with increasing doses of P. aeruginosa. Twenty fours after infection, bronchoalveolar lavage fluid (BALF) was collected and the percent of PMNs in the BALF determined. In separate experiments, mice were intratracheally inoculated with P. aeruginosa for 6 or 24 hours and organs (lungs, kidney, liver, spleen and brain) harvested, homogenized and the homogenate cultured overnight on Pseudomonas isolation agar for quantification of colony forming units (CFUs). Results: Our data confirmed CD99 expression in rat pulmonary microvascular endothelial cells and whole lung of both wild type and AC10 knockout mice. While there was no difference in the percentage of PMNs in the BALF between male wild type and knockout animals at any of the bacterial doses tested, in the female AC10 knockout mice, there was a decrease in the percentage of cells PMNs in the BALF compared to female wild type litter mates at several doses tested (1x103 up to 1x105, p<0.05). At a higher dose of P. aeruginosa (2.5x106), there was no difference in bacterial clearance between male WT and AC10 knockout mice, but a trend toward an increase in the CFU harvested from lung tissue 6 hours post infection in female KO mice. Conclusion: Thus, CD99 is expressed in the pulmonary endothelium. Further, in female mice, AC10 plays a role in neutrophil recruitment into the airspace. Further studies are required to resolve whether AC10 plays a role in bacterial clearance in female mice in a model of P. aeruginosa pneumonia. HL121513. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiac hypertrophy that affects hyperthyroidism occurs independently of the NLRP3 inflammasome.

Cardiac hypertrophy (CH) is an adaptive response to maintain cardiac function; however, persistent stress responses lead to contractile dysfunction and heart failure. Although inflammation is involved in these processes, the mechanisms that control cardiac inflammation and hypertrophy still need to be clarified. The NLRP3 inflammasome is a cytosolic multiprotein complex that mediates IL-1β production. The priming step of NLRP3 is essential for increasing the expression of its components and occurs following NF-κB activation. Hyperthyroidism triggers CH, which can progress to maladaptive CH and even heart failure. We have shown in a previous study that thyroid hormone (TH)-induced CH is linked to the upregulation of S100A8, leading to NF-κB activation. Therefore, we aimed to investigate whether the NLRP3 inflammasome is involved in TH-induced CH and its potential role in CH pathophysiology. Hyperthyroidism was induced in NLRP3 knockout (NLRP3-KO), Caspase-1-KO and Wild Type (WT) male mice of the C57Bl/6J strain, aged 8-12 weeks, by triiodothyronine (7μg/100g BW, i.p.) administered daily for 14 days. Morphological and cardiac functional analysis besides molecular assays showed, for the first time, that TH-induced CH is accompanied by reduced NLRP3 expression in the heart and that it occurs independently of the NLRP3 inflammasome and caspase 1-related pathways. However, NLRP3 is important for the maintenance of basal cardiac function since NLRP3-KO mice had impaired diastolic function and reduced heart rate, ejection fraction, and fractional shortening compared with WT mice.

HSD17B1 Compensates for HSD17B3 Deficiency in Fetal Mouse Testis but Not in Adults.

Hydroxysteroid (17β) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.

Naoxintong capsule remodels gut microbiota and ameliorates early-stage atherosclerosis in apolipoprotein E-deficient mice

BackgroundNaoxintong capsule (NXT) is a compound traditional Chinese medicine prescription with demonstrated effect for the treatment of cardiovascular and cerebrovascular diseases including atherosclerosis (AS). However, the pharmacological mechanisms of NXT in ameliorating early-stage AS are still unclear, especially regarding the role of gut microbiota. PurposeThis study is aiming to evaluate the therapeutic effect of NXT against early-stage AS, and further illustrate the potential correlations among AS, gut microbiota, and NXT. MethodsThirty-two male ApoE knockout mice (C57BL/6 background) were fed with a high cholesterol diet (HCD) for 4 weeks to establish an early-stage AS model. NXT in two different dosages and simvastatin (Simv) were than administrated for another 8 weeks. Lipid metabolism indicators and inflammation levels were measured with corresponding assay kits. Changes in blood vessels, liver lesions, and intestinal barrier proteins were evaluated with different staining methods. Furthermore, the gut microbiota structure was analyzed using 16S rRNA sequencing technology, while GC–MS was utilized to determine the fecal contents of short-chain fatty acids (SCFAs). ResultsAdministration of NXT significantly ameliorated obesity, hyperlipidemia, systemic inflammation, vasculopathy, liver injury, and intestinal barrier disorder in AS mice. Administration of NXT also significantly regulated the gut microbiota disturbance and increased the total contents of fecal SCFAs in AS mice. Furthermore, acetic acid content and the relative abundance of Faecalibacterium in feces were proposed as potential therapeutic biomarkers of NXT for AS treatment as indicated via the correlation analysis. ConclusionThis study demonstrated that NXT could effectively treat early-stage AS induced by HCD in mice. NXT regulated the gut microbiota and metabolites, maintained intestinal homeostasis, and improved the systemic inflammatory response. These findings may provide robust experimental support for the clinical use of NXT for AS treatment.

Slitrk4 is required for the development of inhibitory neurons in the fear memory circuit of the lateral amygdala.

The Slitrk family consists of six synaptic adhesion molecules, some of which are associated with neuropsychiatric disorders. In this study, we aimed to investigate the physiological role of Slitrk4 by analyzing Slitrk4 knockout (KO) mice. The Slitrk4 protein was widely detected in the brain and was abundant in the olfactory bulb and amygdala. In a systematic behavioral analysis, male Slitrk4 KO mice exhibited an enhanced fear memory acquisition in a cued test for classical fear conditioning, and social behavior deficits in reciprocal social interaction tests. In an electrophysiological analysis using amygdala slices, Slitrk4 KO mice showed enhanced long-term potentiation in the thalamo-amygdala afferents and reduced feedback inhibition. In the molecular marker analysis of Slitrk4 KO brains, the number of calretinin (CR)-positive interneurons was decreased in the anterior part of the lateral amygdala nuclei at the adult stage. In in vitro experiments for neuronal differentiation, Slitrk4-deficient embryonic stem cells were defective in inducing GABAergic interneurons with an altered response to sonic hedgehog signaling activation that was involved in the generation of GABAergic interneuron subsets. These results indicate that Slitrk4 function is related to the development of inhibitory neurons in the fear memory circuit and would contribute to a better understanding of osttraumatic stress disorder, in which an altered expression of Slitrk4 has been reported.

Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

Mutation of the Thap4 gene causes dwarfism and testicular anomalies in rats and mice.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7Mb and 101.2Mb on rat chromosome 9. Nucleotide sequence analysis identified 2bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

AZGP1 deficiency promotes angiogenesis in prostate cancer

BackgroundLoss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive.MethodAZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples.ResultNeither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1−/− mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues.ConclusionAZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

WHAMM functions in kidney reabsorption and polymerizes actin to promote autophagosomal membrane closure and cargo sequestration.

The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.

Mesenchymal-specific Alms1 knockout in mice recapitulates metabolic features of Alström syndrome

ObjectiveAlström Syndrome (AS), caused by biallelic ALMS1 mutations, includes obesity with disproportionately severe insulin resistant diabetes, dyslipidemia, and fatty liver. Prior studies suggest that hyperphagia is accounted for by loss of ALMS1 function in hypothalamic neurones, whereas disproportionate metabolic complications may be due to impaired adipose tissue expandability. We tested this by comparing the metabolic effects of global and mesenchymal stem cell (MSC)-specific Alms1 knockout. MethodsGlobal Alms1 knockout (KO) mice were generated by crossing floxed Alms1 and CAG-Cre mice. A Pdgfrα-Cre driver was used to abrogate Alms1 function selectively in MSCs and their descendants, including preadipocytes. We combined metabolic phenotyping of global and Pdgfrα+ Alms1-KO mice on a 45% fat diet with measurements of body composition and food intake, and histological analysis of metabolic tissues. ResultsAssessed on 45% fat diet to promote adipose expansion, global Alms1 KO caused hyperphagia, obesity, insulin resistance, dyslipidaemia, and fatty liver. Pdgfrα-cre driven KO of Alms1 (MSC KO) recapitulated insulin resistance, fatty liver, and dyslipidaemia in both sexes. Other phenotypes were sexually dimorphic: increased fat mass was only present in female Alms1 MSC KO mice. Hyperphagia was not evident in male Alms1 MSC KO mice, but was found in MSC KO females, despite no neuronal Pdgfrα expression. ConclusionsMesenchymal deletion of Alms1 recapitulates metabolic features of AS, including fatty liver. This confirms a key role for Alms1 in the adipose lineage, where its loss is sufficient to cause systemic metabolic effects and damage to remote organs. Hyperphagia in females may depend on Alms1 deficiency in oligodendrocyte precursor cells rather than neurones. AS should be regarded as a forme fruste of lipodystrophy.

Abstract 6832: Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC

Abstract Background: Lemur tail kinase 3 (LMTK3) is a serine/threonine protein kinase with roles in multiple cellular pathways, including Wnt signaling, KIT modulation, and the estrogen receptor pathway; identifying it as a potential target in various cancers. LMTK3 is highly expressed within the central nervous system (CNS) and has been demonstrated to influence neuronal signaling. This study aimed to investigate the potential connection between LMTK3 and neuronal signaling pathways in colorectal cancer (CRC). Methods: Using the Cre/loxP system, we generated a C57BL/6 mouse strain that lacks the expression of LMTK3. 1 × 106 MC38 cells were subcutaneously implanted into 8 weeks old male and female wildtype (WT) and knockout (KO) mice. Tumor growth was measured until endpoint was reached. Tumors were then processed for RNA sequencing. Differentially expressed genes (DEGs) for each comparison group: WT Female vs WT Male, WT Female vs KO Female, WT Male vs KO Male, and KO Female vs KO Male were analyzed. Ingenuity pathway analysis (IPA) was done for identification of upstream regulators involved and affected cellular pathways. Expression levels of DEGs for glutamate receptor subunits were validated using qRT-PCR. Results: Both male and female LMTK3 KO mice showed highly significant (p<0.01) reduced tumor size when compared to WT mice of the same sex. Additionally, the KO female tumors were significantly (p<0.05) smaller when compared to the KO males. RNA sequencing revealed 761 DEGs in KO and WT female and 133 DEGs in KO female and male cohort comparisons (q<0.05). IPA results showed significant (p<0.01, Z-score←2) alterations in pathways including glutaminergic receptor signaling, neurovascular coupling, CREB signaling in neurons, and synaptic long-term depression in the WT vs KO female tumor cohort comparison. Within these pathways DEGs: CACNA1E, CACNA2DA, CACNG2, GRIA1, GRIA2, GRIN1, and GRIN2B, were downregulated in KO females. Upstream regulator analysis identified transcription regulators such as OTX2 and NEUROD1 as well as beta-estradiol to be significantly inhibited (p<0.01, Z-score←2) in KO females. qRT-PCR analysis showed significant (p<0.05) decreased expression in GRIA1, GRIN1, and GRIN2B as well as GRIN2A and GRIN2C in KO females when compared to WT females or KO males, validating RNA seq results. Conclusions: These results showed the involvement of LMTK3 in the gut-brain axis and its effect on CRC tumor growth. Downregulation of multiple neuronal pathways and genes suggest that LMTK3 may influence neuron signaling and neurotransmitter release to produce stimulatory or inhibitory effects within the tumor environment. The concurrent inhibition of beta-estradiol in LMTK3 KO tumors allude to possible sex differences the kinase’s effect on the observed tumor growth. Further studies are needed to confirm the mechanism of interaction of LMTK3 within these pathways. Citation Format: Lesly Torres-Gonzalez, Shivani Soni, Yan Yang, Goar Smbatyan, Jae Ho Lo, Pooja Mittal, Francesca Battaglin, Indrakant K. Singh, Priya Jayachandran, Sandra Algaze, Alexandra Wong, Karam Ashouri, Wu Zhang, Georgios Giamas, Joshua Millstein, Heinz-Josef Lenz. Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6832.

An evolutionarily conserved olfactory receptor is required for sex differences in blood pressure.

Sex differences in blood pressure are well-established, with premenopausal women having lower blood pressure than men by ~10 millimeters of mercury; however, the underlying mechanisms are not fully understood. We report here that sex differences in blood pressure are absent in olfactory receptor 558 knockout (KO) mice. Olfr558 localizes to renin-positive cells in the kidney and to vascular smooth muscle cells. Female KOs exhibit increased blood pressure and increased pulse wave velocity. In contrast, male KO mice have decreased renin expression and activity, altered vascular reactivity, and decreased diastolic pressure. A rare OR51E1 (human ortholog) missense variant has a statistically significant sex interaction effect with diastolic blood pressure, increasing diastolic blood pressure in women but decreasing it in men. In summary, our findings demonstrate an evolutionarily conserved role for OLFR558/OR51E1 to mediate sex differences in blood pressure.

Pannexin 3 deletion in mice results in knee osteoarthritis and intervertebral disc degeneration after forced treadmill running.

Pannexin 3 (Panx3) is a glycoprotein that forms mechanosensitive channels expressed in chondrocytes and annulus fibrosus cells of the intervertebral disc (IVD). Evidence suggests Panx3 plays contrasting roles in traumatic versus aging osteoarthritis (OA) and intervertebral disc degeneration (IDD). However, whether its deletion influences the response of joint tissue to forced useis unknown. The purpose of this study was to determine if Panx3 deletion in mice causes increased knee joint OA and IDD after forced treadmill running. Male and female wildtype (WT) and Panx3 knockout (KO) mice were randomized to either a no-exercise group (sedentary; SED) or daily forced treadmill running (forced exercise; FEX) from 24 to 30 weeks of age. Knee cartilage and IVD histopathology were evaluated by histology, while tibial secondary ossification centers were analyzed using microcomputed tomography (µCT). Both male and female Panx3 KO mice developed larger superficial defects of the tibial cartilage after forced treadmill running compared with SED WT mice. Additionally, Panx3 KO mice developed reduced bone volume, and female PANX3 KO mice had lengthening of the lateral tubercle at the intercondylar eminence. In the lower lumbar spine, both male and female Panx3 KO mice developed histopathological features of IDD after running compared to SED WT mice. These findings suggest that the combination of deleting Panx3 and forced treadmill running induces OA and causes histopathological changes associated with the degeneration of the IVDs in mice.

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse.

From a cohort of 167 infertile patients suffering from multiple morphological abnormalities of the flagellum (MMAF), pathogenic bi-allelic mutations were identified in the CCDC146 gene. In somatic cells, CCDC146 is located at the centrosome and at multiple microtubule-related organelles during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To decipher the molecular pathogenesis of infertility associated with CCDC146 mutations, a Ccdc146 knock-out (KO) mouse line was created. KO male mice were infertile, and sperm exhibited a phenotype identical to CCDC146 mutated patients. CCDC146 expression starts during late spermiogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets suggests that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 impacted all microtubule-based organelles such as the manchette, the head-tail coupling apparatus (HTCA), and the axoneme. Through this study, a new genetic cause of infertility and a new factor in the formation and/or structure of the sperm axoneme were characterized.

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

From a cohort of 167 infertile patients suffering from multiple morphological abnormalities of the flagellum (MMAF), pathogenic bi-allelic mutations were identified in the CCDC146 gene. In somatic cells, CCDC146 is located at the centrosome and at multiple microtubule-related organelles during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To decipher the molecular pathogenesis of infertility associated with CCDC146 mutations, a Ccdc146 knock-out (KO) mouse line was created. KO male mice were infertile, and sperm exhibited a phenotype identical to CCDC146 mutated patients. CCDC146 expression starts during late spermiogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets suggests that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 impacted all microtubule-based organelles such as the manchette, the head–tail coupling apparatus (HTCA), and the axoneme. Through this study, a new genetic cause of infertility and a new factor in the formation and/or structure of the sperm axoneme were characterized.

Abstract B049: Uncovering sex-biased immunometabolic roles for NIK in the GBM tumor microenvironment

Abstract The interconnected relationship between the central nervous system (CNS) and the immune system is crucial for brain function. Astrocytes, microglia and infiltrating macrophages perform diverse functions for the maintenance of CNS homeostasis, including inflammatory responses to injury or infection, tissue repair and immune surveillance of cancers such as glioblastoma (GBM). Indeed, these cells comprise up to 50% of GBM tumor mass, yet the impact of their interactions in regulating neuroinflammation and immunosuppression in the GBM tumor microenvironment is not well understood. We have recently found that NF-κB-Inducing Kinase (NIK), an essential activator of the noncanonical NF-κB pathway, governs the mitochondrial metabolic reprogramming that is required for the acquisition of pro-repair bone-marrow-derived macrophage (BMDM) immune effector functions. Although NIK is highly expressed in brain immune cells, including resident macrophages/microglia and astrocytes, little is known regarding NIK-dependent regulatory pathways controlling their immune cell effector functions in brain health and systemic responses to disease. Our preliminary data demonstrate that similar to BMDMs, NIK knockout (KO) microglia and astrocytes have impaired mitochondrial oxidative metabolism and skew towards pro-inflammatory phenotypes. Moreover, NIK KO mice harboring orthotopic syngeneic brain tumors exhibit improved survival and reduced microglia and macrophage tumor infiltration, suggesting that NIK functions in the TME to support immune cell interactions that promote GBM growth. Notably these effects were sex-biased, with male NIK KO mice having a more pronounced survival benefit. Additionally, we observe whole-body metabolic dysfunction in myeloid- and astrocyte-specific conditional NIK knockout mice, implying a potential NIK-dependent feedback relationship between CNS immune/glial cell functions and control of systemic metabolism. Overall, these results are consistent with poorer survival of patients with elevated NIK expression and provides a possible rationale for the higher incidence of GBM in males. Coupled with our previous work demonstrating that NIK has several GBM cell-intrinsic roles to promote tumor invasion and adaptive growth through control of mitochondrial fission and cellular metabolism, these new findings highlight the importance of understanding NIK-dependent immunometabolic functions in the brain. Therapeutic strategies targeting NIK may not only attenuate GBM cell invasion and render them vulnerable to metabolic stress, but also modulate metabolic rewiring of immune cells in the tumor microenvironment to improve patient outcomes. Citation Format: Justin N. Keeney, Caren Stuebe, Kathryn M. Pflug, L. Gerard Toussaint, Raquel Sitcheran. Uncovering sex-biased immunometabolic roles for NIK in the GBM tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B049.

Tex46 knockout male mice are sterile secondary to sperm head malformations and failure to penetrate through the zona pellucida.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida (ZP) penetration but research has yet to elucidate why. We generated testis expressed 46 (Tex46) knockout mice to investigate the essential roles of TEX46 in mammalian reproduction. We used RT-PCR to demonstrate that Tex46 was expressed exclusively in the male reproductive tract in mice and humans. We created Tex46-/- mice using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and analyzed their fertility. Tex46 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis to elucidate relationships between TEX46 and other acrosome biogenesis-related proteins. Mouse and human TEX46 are testis-enriched and encode a transmembrane protein which is conserved from amphibians to mammals. Loss of the mouse TEX46 protein causes male sterility primarily due to abnormal sperm head formation and secondary effects on sperm motility. Tex46 null spermatozoa morphologically lack the typical hooked sperm head appearance and fail to penetrate through the ZP. Electron microscopy of the testicular germ cells reveals malformation of the acrosomal cap, with misshapen sperm head tips and the appearance of a gap between the acrosome head and the nucleus. TEX46 is essential for sperm head formation, sperm penetration through the ZP, and male fertility in mice, and is a putative contraceptive target in men.

Liver Androgen Receptor Knockout Improved High-fat Diet Induced Glucose Dysregulation in Female Mice But Not Male Mice.

Previous research has indicated that liver androgen receptors may play a role in modulating disease. This study aims to investigate the pathophysiology of high-fat diet (HFD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. We performed metabolic tests on LivARKO female and male mice fed a HFD or a control diet (from Research Diets Inc.) during months 1 or 2 after starting the diet. Additionally, we performed Western blot and quantitative real-time PCR analysis on the livers of the mice to examine intermediates in the insulin signaling pathway. LivARKO-HFD female mice displayed no difference in glucose tolerance compared to female LivARKO-Control (Con) mice, whereas in wild-type female mice, HFD impaired glucose tolerance (IGT). Our data suggests that starting at 1 month, LivARKO may be protecting female mice from HFD-induced metabolic dysfunction. LivARKO-HFD female mice displayed significantly worse insulin sensitivity at 15 minutes compared to LivARKO-Con female mice, but, strangely, LivARKO-HFD female mice had significantly better insulin sensitivity at 60 and 90 minutes compared to LivARKO-Con female mice. Despite protecting against IGT, LivARKO did not protect against HFD-induced hyperinsulinemia in female mice. In contrast to females, male LivARKO-HFD mice displayed impaired glucose tolerance compared to male LivARKO-Con mice. Thus, LivARKO is not protective against HFD-induced glucose metabolic dysfunction in male mice. Lastly, LivARKO-HFD female mice maintained hepatic insulin sensitivity whereas LivARKO-HFD male mice displayed hepatic insulin resistance. These findings suggest that LivARKO delayed the onset of HFD-induced dysglycemia in female mice.