Phenotype Of Knockout Mice Research Articles

Speos: an ensemble graph representation learning framework to predict core gene candidates for complex diseases

Understanding phenotype-to-genotype relationships is a grand challenge of 21st century biology with translational implications. The recently proposed “omnigenic” model postulates that effects of genetic variation on traits are mediated by core-genes and -proteins whose activities mechanistically influence the phenotype, whereas peripheral genes encode a regulatory network that indirectly affects phenotypes via core gene products. Here, we develop a positive-unlabeled graph representation-learning ensemble-approach based on a nested cross-validation to predict core-like genes for diverse diseases using Mendelian disorder genes for training. Employing mouse knockout phenotypes for external validations, we demonstrate that core-like genes display several key properties of core genes: Mouse knockouts of genes corresponding to our most confident predictions give rise to relevant mouse phenotypes at rates on par with the Mendelian disorder genes, and all candidates exhibit core gene properties like transcriptional deregulation in disease and loss-of-function intolerance. Moreover, as predicted for core genes, our candidates are enriched for drug targets and druggable proteins. In contrast to Mendelian disorder genes the new core-like genes are enriched for druggable yet untargeted gene products, which are therefore attractive targets for drug development. Interpretation of the underlying deep learning model suggests plausible explanations for our core gene predictions in form of molecular mechanisms and physical interactions. Our results demonstrate the potential of graph representation learning for the interpretation of biological complexity and pave the way for studying core gene properties and future drug development.

The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo

ObjectiveIn vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. MethodsWe investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. ResultsTrib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. ConclusionsOur findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

Defective function of α-ketoglutarate dehydrogenase exacerbates mitochondrial ATP deficits during complex I deficiency

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the affected regions of the NDUFS4 KO brain. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. The defective production of KGDHC derived succinyl-CoA resulted in decreased mitochondrial substrate level phosphorylation (SLP), further aggravating the existing oxidative phosphorylation (OXPHOS) ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Modeling succination of a cysteine in the spatial vicinity of the DLST active site or introduction of succinomimetic mutations recapitulates these metabolic deficits. Our data demonstrate that the biochemical deficit extends beyond impaired Complex I assembly and OXPHOS deficiency, functionally impairing select components of the TCA cycle to drive metabolic perturbations in affected neurons.

OR19-03 SIK2 Regulates Granulosa Cell Response to Exogenous Gonadotropins

Abstract Disclosure: E. Hayes: None. O. Lakomy: None. M. Hassan: None. R. Filzen: None. M. Rodriguez Esquivel: None. M. Foretz: None. C.O. Stocco: Grant Recipient; Self; NIH R01HD097202. Forty percent of cases of infertility are attributed to ovulatory defects. Successful ovulation requires the coordinated development of the ovarian follicle, the functional unit of the ovary that is composed of the oocyte surrounded by steroidogenic granulosa cells. However, the mechanisms that govern follicle development remain incompletely understood. Previously, our group showed that loss of salt-inducible kinase 2 (SIK2) in a murine knockout model resulted in a significantly increased number of ovulated oocytes in response to an ovarian hyperstimulation protocol. Here, we aimed to further characterize the reproductive phenotype of SIK2 knockout mice, as well as to understand the role of SIK2 in granulosa cells by creating and characterizing a granulosa cell-specific knockdown of SIK2 using the Cre-Lox system. Loss of SIK2 resulted in no changes in the number of litters or litter size as well as a normal estrus cycle. Interestingly, granulosa cell-specific knockdown of SIK2 resulted in a significantly increased number of ovulated oocytes in response to ovarian hyperstimulation. However, SIK2 knockout mice do not ovulate more oocytes under physiologic conditions. Furthermore, knockdown of SIK2 in granulosa cells resulted in significantly increased Cyp19a1 gene expression and serum estradiol concentration. The data reveal that SIK2 plays an important role in the regulation of the response of granulosa cells to gonadotropins. These data support the potential of SIK2 as a pharmacologic target for improving oocyte yield for women receiving gonadotropins during in vitro fertilization as a treatment to trigger follicular maturation and ovulation. SIK2-regulated mechanisms could reveal new targets for the development of innovative methods of contraception. Presentation Date: Saturday, June 17, 2023

Defective branched-chain amino acid catabolism in dorsal root ganglia contributes to mechanical pain.

Impaired branched-chain amino acid (BCAA) catabolism has recently been implicated in the development of mechanical pain, but the underlying molecular mechanisms are unclear. Here, we report that defective BCAA catabolism in dorsal root ganglion (DRG) neurons sensitizes mice to mechanical pain by increasing lactate production and expression of the mechanotransduction channel Piezo2. In high-fat diet-fed obese mice, we observed the downregulation of PP2Cm, a key regulator of the BCAA catabolic pathway, in DRG neurons. Mice with conditional knockout of PP2Cm in DRG neurons exhibit mechanical allodynia under normal or SNI-induced neuropathic injury conditions. Furthermore, the VAS scores in the plasma of patients with peripheral neuropathic pain are positively correlated with BCAA contents. Mechanistically, defective BCAA catabolism in DRG neurons promotes lactate production through glycolysis, which increases H3K18la modification and drives Piezo2 expression. Inhibition of lactate production or Piezo2 silencing attenuates the pain phenotype of knockout mice in response to mechanical stimuli. Therefore, our study demonstrates a causal role of defective BCAA catabolism in mechanical pain by enhancing metabolite-mediated epigenetic regulation.

Abstract P432: CTRP1 Contributes To Angiotensin II-Induced Hypertension Via Activation Of Intrarenal Renin

C1q/TNF-related protein 1(CTRP1) is generally regarded as an adipokine and its circulating level is elevated in patients with hypertension. However, the functional role and mechanism of CTRP1 in blood pressure (BP) regulation largely remains elusive. In the present study, we investigated the BP phenotype of global CTRP1 knockout mice (termed CTRP1 KO) during angiotensin II (AngII) infusion and further explored the underlying mechanism involving the intrarenal renin. At baseline, 4-mo-old CTRP1 KO mice exhibited no developmental abnormalities or had normal BPs as compared with wild-type controls (WT). AngII infusion (300 ng/min/kg) induced a 3-fold increase in plasma and urinary CTRP1 levels as assessed by ELISA, but it was undetectable in CTRP1 KO mice. By radiotelemetry, AngII-infused CTRP1 KO mice exhibited reduced MAP as compared with AngII-infused WT mice (MAP [mmHg] in Day 7: 124.1 ± 2.4 in CTRP1 KO mice vs. 135 ± 3.1 in WT mice, n = 4 for each group, p < 0.05 by Student’s t test). This reduction was accompanied by a diminished response in intrarenal renin expression, as evidenced by urinary total prorenin/renin excretion ([ng/24h]: 0.51 ± 0.08 in WT/Vehicle vs. 1.22 ± 0.12 in WT/AngII; 0.32 ± 0.11 in CTRP1 KO/Vehicle vs. 0.54 ± 0.09 in CTRP1 KO/AngII; p < 0.05 by Two-way ANOVA analysis) and renal medullary mRNA levels (qRT-PCR: 1.00 ± 0.18 in WT/Vehicle vs. 3.73 ± 0.59 in WT/AngII; 0.52 ± 0.21 in CTRP1 KO/Vehicle vs. 1.31 ± 0.37 in CTRP1 KO/AngII, p < 0.05 by Two-way ANOVA analysis). In contrast, renal cortical renin mRNA was suppressed by AngII infusion more significantly in WT than in CTRP1 KO mice. In cultured mouse collecting duct-derived M-1 cells, treatment with recombinant human CTRP1-His protein (hCTRP1-His) at 20 nM for 24 hours consistently upregulated renin expression at both protein (Densitometry: 1.00 ± 0.23 in Vehicle group vs. 2.58 ± 0.42 in hCTRP1-His group, p < 0.05) and mRNA levels (qRT-PCR: 1.00 ± 0.12 in Vehicle group vs. 11.60 ± 1.21 in hCTRP1-His group, p < 0.05) levels. Collectively, these findings provide evidence favoring CTRP1 as a regulator of intrarenal renin mediating AngII-induced hypertension and further suggest CTRP1 as a potential mediator of adipose-renal crosstalk.

Neuropilin 2 in osteoblasts regulates trabecular bone mass in male mice.

Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts. We first examined the in vitro effect of the calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on Nrp2 transcription in osteoblasts. We next generated mice with a conditional deletion of Nrp2 in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional Nrp2 knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D3 analog WY 1048 or with 1,25(OH)2D3. We show that Nrp2 expression is induced by 1,25(OH)2D3 in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of Nrp2 in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global Nrp2 knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic Nrp2 expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D3 analog WY 1048 (0.4 μg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting Nrp2 from the osteoclast lineage did not result in a bone phenotype, even though in vitro osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH)2D3 (0.5 μg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic Nrp2 expression is suggested to regulate bone homeostasis in a sex-specific manner.

Anabolic phenotype in cartilage-specific mitogen-inducible gene-6 knockout mice is independent of transforming growth factor-α

Background/objectiveOsteoarthritis (OA) is a whole joint disorder with no disease modifying treatment currently available. The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays an important role in cartilage/bone development and its ligand transforming growth factor-α (TGFα) is upregulated in OA. In contrast, Mitogen-inducible gene 6 (Mig6) is a negative regulator of EGFR, and cartilage-specific Mig-6 deletion results in anabolic effects on cartilage and formation of chondro-osseus nodules (CON). We aimed to attenuate EGFR signaling by inhibiting TGFα production in cartilage-specific Mig6 deficient mice, to test whether this would prevent the formation of CONs. MethodsWe generated double knockout mice by crossing cartilage-specific Mig-6fl/flCol2a1-Cre+/− and whole-body Tgfa± mice to generate experimental and control wild-type mice. Knee and elbow sections were used to examine articular cartilage thickness, cell density, and osteoclast presence. Additionally, immunohistochemistry was completed to analyze phospho-EGFR and SOX9. ResultsMig-6 deficient mice display cartilage thickening and CONs at 12 weeks in both the elbow and knee joints, which is independent of TGFα ligand presence. Similarly, articular cartilage cell density is increased in Mig6-cKO/Tgfa-KO and Mig6-cKOmice, but not Tgfa-KO mice, and displays increased SOX9 and phospho-EGFR staining. ConclusionThe articular cartilage displays increased thickness/cell density and CON formation independent of the presence of TGFα, suggesting the anabolic phenotype in the Mig6-deficient mice is independent of TGFα/EGFR binding. The anabolic phenotype may be due to an alternative EGFR ligand activation, or other non-EGFR specific mechanism. More research is required to elucidate the exact pathway responsible for the anabolic effects.

Molecular dissection and testing of PRSS37 function through LC–MS/MS and the generation of a PRSS37 humanized mouse model

The quest for a non-hormonal male contraceptive pill for men still exists. Serine protease 37 (PRSS37) is a sperm-specific protein that when ablated in mice renders them sterile. In this study we sought to examine the molecular sequelae of PRSS37 loss to better understand its molecular function, and to determine whether human PRSS37 could rescue the sterility phenotype of knockout (KO) mice, allowing for a more appropriate model for drug molecule testing. To this end, we used CRISPR-EZ to create mice lacking the entire coding region of Prss37, used pronuclear injection to create transgenic mice expressing human PRSS37, intercrossed these lines to generate humanized mice, and performed LC–MS/MS of KO and control tissues to identify proteomic perturbances that could attribute a molecular function to PRSS37. We found that our newly generated Prss37 KO mouse line is sterile, our human transgene rescues the sterility phenotype of KO mice, and our proteomics data not only yields novel insight into the proteome as it evolves along the male reproductive tract, but also demonstrates the proteins significantly influenced by PRSS37 loss. In summary, we report vast biological insight including insight into PRSS37 function and the generation of a novel tool for contraceptive evaluation.

Rapamycin improves social and stereotypic behavior abnormalities induced by pre-mitotic neuronal subset specific Pten deletion.

The mechanistic target of rapamycin (mTOR) pathway is a signaling system integral to neural growth and migration. In both patients and rodent models, mutations to the phosphatase and tensin homolog gene (PTEN) on chromosome 10 results in hyperactivation of the mTOR pathway, as well as seizures, intellectual disabilities and autistic behaviors. Rapamycin, an inhibitor of mTOR, can reverse the epileptic phenotype of neural subset specific Pten knockout (NS-Pten KO) mice, but its impact on behavior is not known. To determine the behavioral effects of rapamycin, male and female NS-Pten KO and wildtype (WT) mice were assigned as controls or administered 10 mg/kg of rapamycin for 2 weeks followed by behavioral testing. Rapamycin improved social behavior in both genotypes and stereotypic behaviors in NS-Pten KO mice. Rapamycin treatment resulted in a reduction of several measures of activity in the open field test in both genotypes. Rapamycin did not reverse the reduced anxiety behavior in KO mice. These data show the potential clinical use of mTOR inhibitors by showing its administration can reduce the production of autistic-like behaviors in NS-Pten KO mice.

252-LB: Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release from Macrophages and Impairs Glucose-Stimulated Insulin Secretion

Pancreatic islet inflammation plays an important role in the pathogenesis of diabetes mellitus. Islet macrophages, which have emerged as a key player in islet inflammation, express Dectin-2 (D2), one of the C-type lectin receptors recognizing α-mannans, while endocrine cells such as α-cells and β-cells lack the expression of D2. To elucidate the role of D2 in glucose metabolism and β-cell function, we analyzed the phenotype of D2 knockout (D2KO) mice. D2KO mice exhibited significantly impaired glucose tolerance compared to wild-type (WT) mice, although there were no significant differences in insulin sensitivity. Glucose-stimulated insulin secretion (GSIS) was significantly reduced in isolated islets from D2KO mice compared to WT mice. Comprehensive analysis of RNA-seq in isolated islets from D2KO and WT mice revealed that D2 deficiency induced immune cell infiltration and inflammation in pancreatic islets. Furthermore, RNA-seq analysis in peritoneal macrophages from D2KO and WT mice showed that macrophages from D2KO mice acquired an inflammatory phenotype and exhibited enhanced NF-κB signaling compared to those from WT mice. Next, to determine whether humoral factors secreted from macrophages affect GSIS, we examined GSIS using the culture supernatants of macrophages from D2KO and WT mice. The culture supernatants of macrophages from D2KO mice suppressed GSIS compared to WT mice. Furthermore, the multiplex cytokine assay revealed that IL-1α and IL-6 were significantly increased in the culture supernatants from macrophages of D2KO mice. Finally, we found that the stimulation of IL-1α and IL-6 reduced GSIS in β-cells. These findings suggest that macrophages from D2KO mice suppress GSIS by increasing IL-1α and IL-6 production. This study provides new insights into the mechanism by which D2 is involved in the pathogenesis of pancreatic islet inflammation. Disclosure M. Fujita: None. T. Miyazawa: None. K. Uchida: None. N. Uchida: None. T. Hatayama: None. S. Nakamura: None. Y. Takeichi: None. Y. Miyachi: None. Y. Ogawa: None.

Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP.

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase-activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Hippocampal versus cortical deletion of cholinergic receptor muscarinic 1 in mice differentially affects post-translational modifications and supramolecular assembly of respiratory chain-associated proteins, mitochondrial ultrastructure, and respiration: implications in Alzheimer's disease.

Introduction: In a previous retrospective study using postmortem human brain tissues, we demonstrated that loss of Cholinergic Receptor Muscarinic 1 (CHRM1) in the temporal cortex of a subset of Alzheimer's patients was associated with poor survival, whereas similar loss in the hippocampus showed no such association. Mitochondrial dysfunction underlies Alzheimer's pathogenesis. Therefore, to investigate the mechanistic basis of our findings, we evaluated cortical mitochondrial phenotypes in Chrm1 knockout (Chrm1-/-) mice. Cortical Chrm1 loss resulted in reduced respiration, reduced supramolecular assembly of respiratory protein complexes, and caused mitochondrial ultrastructural abnormalities. These mouse-based findings mechanistically linked cortical CHRM1 loss with poor survival of Alzheimer's patients. However, evaluation of the effect of Chrm1 loss on mouse hippocampal mitochondrial characteristics is necessary to fully understand our retrospective human tissue-based observations. This is the objective of this study. Methods: Enriched hippocampal and cortical mitochondrial fractions (EHMFs/ECMFs, respectively) derived from wild-type and Chrm1-/- mice were used to measure respiration by quantifying real-time oxygen consumption, supramolecular assembly of oxidative phosphorylation (OXPHOS)-associated proteins by blue native polyacrylamide gel electrophoresis, post-translational modifications (PTMs) by isoelectric focusing (IEF), and mitochondrial ultrastructure by electron microscopy. Results: In contrast to our previous observations in Chrm1-/- ECMFs, EHMFs of Chrm1-/- mice significantly increased respiration with a concomitant increase in the supramolecular assembly of OXPHOS-associated proteins, specifically Atp5a and Uqcrc2, with no mitochondrial ultrastructural alterations. IEF of ECMFs and EHMFs from Chrm1-/- mice showed a decrease and an increase, respectively in a negatively charged (pH∼3) fraction of Atp5a relative to the wild-type mice, with a corresponding decrease or increase in the supramolecular assembly of Atp5a and respiration indicating a tissue-specific signaling effect. Discussion: Our findings indicate that loss of Chrm1 in the cortex causes structural, and physiological alterations to mitochondria that compromise neuronal function, whereas Chrm1 loss in the hippocampus may benefit neuronal function by enhancing mitochondrial function. This brain region-specific differential effect of Chrm1 deletion on mitochondrial function supports our human brain region-based findings and Chrm1-/- mouse behavioral phenotypes. Furthermore, our study indicates that Chrm1-mediated brain region-specific differential PTMs of Atp5a may alter complex-V supramolecular assembly which in turn regulates mitochondrial structure-function.

Kidney-Related Function of Mitochondrial Protein Mitoregulin.

A small protein, Mitoregulin (Mtln), localizes in mitochondria and contributes to oxidative phosphorylation and fatty acid metabolism. Mtln knockout mice develop obesity on a high-fat diet, demonstrating elevated cardiolipin damage and suboptimal creatine kinase oligomerization in muscle tissue. Kidneys heavily depend on the oxidative phosphorylation in mitochondria. Here we report kidney-related phenotypes in aged Mtln knockout mice. Similar to Mtln knockout mice muscle mitochondria, those of the kidney demonstrate a decreased respiratory complex I activity and excessive cardiolipin damage. Aged male mice carrying Mtln knockout demonstrated an increased frequency of renal proximal tubules' degeneration. At the same time, a decreased glomerular filtration rate has been more frequently detected in aged female mice devoid of Mtln. An amount of Mtln partner protein, Cyb5r3, is drastically decreased in the kidneys of Mtln knockout mice.

Novel genes associated with a placental phenotype in knockout mice also respond to cellular stressors in primary human trophoblasts

IntroductionPlacental insufficiency is a leading cause of intrauterine growth restriction, contributing to perinatal morbidity and mortality. The molecular regulation of placental development and what causes placental insufficiency is poorly understood. Recently, a panel of genes were found to cause significant placental dysmorphologies in mice with severely growth restricted off-spring. We aimed to assess whether these genes were also implicated in human intrauterine growth restriction. MethodsWe explored the expression of nine genes in primary cytotrophoblast cells in hypoxic (n = 6) and glucose starvation (n = 5) conditions in vitro. We also explored whether the genes were dysregulated in intrauterine growth restricted human placental samples (n = 11), with (n = 20) or without preeclampsia compared to gestationally matched controls (<34 weeks gestation) (n = 17). ResultsHypoxic stress significantly upregulated the expressions of BRD2 (p = 0.0313), SMG9 (p = 0.0313) genes. In contrast, glucose starvation significantly suppressed Kif1bp (p = 0.0089) in primary cytotrophoblasts. The FRYL, NEK9, CHTOP, PSPH, ATP11A, HM13 genes did not change under hypoxia or glucose starvation conditions. The expression of these genes was not altered in placenta from patients with intrauterine growth restriction, compared to gestationally matched controls. DiscussionWe demonstrate that some of the genes that cause a placental phenotype in mice, respond to hypoxic and glucose mediated stress in human cytotrophoblast isolations. Despite this, they are unchanged in placenta from patients with intrauterine growth restriction. Therefore, dysregulation of these genes is less likely to contribute to preterm intrauterine growth restriction in humans.

MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin.

Age-related hearing loss (ARHL) is the most common sensory degenerative disease and can significantly impact the quality of life in elderly people. A previous study using GeneChip miRNA microarray assays showed that the expression of miR-29a changes with age, however, its role in hearing loss is still unclear. In this study, we characterized the cochlear phenotype of miR-29a knockout (miR-29a-/-) mice and found that miR-29a-deficient mice had a rapid progressive elevation of the hearing threshold from 2 to 5 months of age compared with littermate controls as measured by the auditory brainstem response. Stereocilia degeneration, hair cell loss and abnormal stria vascularis (SV) were observed in miR-29a-/- mice at 4 months of age. Transcriptome sequencing results showed elevated extracellular matrix (ECM) gene expression in miR-29a-/- mice. Both Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the key differences were closely related to ECM. Further examination with a transmission electron microscope showed thickening of the basilar membrane in the cochlea of miR-29a-/- mice. Five Col4a genes (Col4a1-a5) and two laminin genes (Lamb2 and Lamc1) were validated as miR-29a direct targets by dual luciferase assays and miR-29a inhibition assays with a miR-29a inhibitor. Consistent with the target gene validation results, the expression of these genes was significantly increased in the cochlea of miR-29a-/- mice, as shown by RT-PCR and Western blot. These findings suggest that miR-29a plays an important role in maintaining cochlear structure and function by regulating the expression of collagen and laminin and that the disturbance of its expression could be a cause of progressive hearing loss.

Single-cell RNA-seq uncovers dynamic processes orchestrated by RNA-binding protein DDX43 in chromatin remodeling during spermiogenesis

Mammalian spermatogenesis shows prominent chromatin and transcriptomic switches in germ cells, but it is unclear how such dynamics are controlled. Here we identify RNA helicase DDX43 as an essential regulator of the chromatin remodeling process during spermiogenesis. Testis-specific Ddx43 knockout mice show male infertility with defective histone-to-protamine replacement and post-meiotic chromatin condensation defects. The loss of its ATP hydrolysis activity by a missense mutation replicates the infertility phenotype in global Ddx43 knockout mice. Single-cell RNA sequencing analyses of germ cells depleted of Ddx43 or expressing the Ddx43 ATPase-dead mutant reveals that DDX43 regulates dynamic RNA regulatory processes that underlie spermatid chromatin remodeling and differentiation. Transcriptomic profiling focusing on early-stage spermatids combined with enhanced crosslinking immunoprecipitation and sequencing further identifies Elfn2 as DDX43-targeted hub gene. These findings illustrate an essential role for DDX43 in spermiogenesis and highlight the single-cell-based strategy to dissect cell-state-specific regulation of male germline development.

Gut microbiota from sigma-1 receptor knockout mice induces depression-like behaviors and modulates the cAMP/CREB/BDNF signaling pathway.

Depression is a common mental disorder that affects approximately 350 million people worldwide. Much remains unknown about the molecular mechanisms underlying this complex disorder. Sigma-1 receptor (Sig-1R) is expressed at high levels in the central nervous system. Increasing evidence has demonstrated a close association between the Sig-1R and depression. Recently, research has suggested that the gut microbiota may play a crucial role in the development of depression. Male Sig-1R knockout (Sig-1R KO) and wild-type (WT) mice were used for this study. All transgenic mice were of a pure C57BL/6J background. Mice received a daily gavage of vancomycin (100 mg/kg), neomycin sulfate (200 mg/kg), metronidazole (200 mg/kg), and ampicillin (200 mg/kg) for one week to deplete gut microbiota. Fecal microbiota transplantation (FMT) was conducted to assess the effects of gut microbiota. Depression-like behaviors was evaluated by tail suspension test (TST), forced swimming test (FST) and sucrose preference test (SPT). Gut microbiota was analyzed by 16s rRNA and hippocampal transcriptome changes were assessed by RNA-seq. We found that Sig-1R knockout induced depression-like behaviors in mice, including a significant reduction in immobility time and an increase in latency to immobility in the FST and TST, which was reversed upon clearance of gut microbiota with antibiotic treatment. Sig-1R knockout significantly altered the composition of the gut microbiota. At the genus level, the abundance of Alistipes, Alloprevotella, and Lleibacterium decreased significantly. Gut microbiota dysfunction and depression-like phenotypes in Sig-1R knockout mice could be reproduced through FMT experiments. Additionally, hippocampal RNA sequencing identified multiple KEGG pathways that are associated with depression. We also discovered that the cAMP/CREB/BDNF signaling pathway is inhibited in the Sig-1R KO group along with lower expression of neurotrophic factors including CTNF, TGF-α and NGF. Fecal bacteria transplantation from Sig-1R KO mice also inhibited cAMP/CREB/BDNF signaling pathway. In our study, we found that the gut-brain axis may be a potential mechanism through which Sig-1R regulates depression-like behaviors. Our study provides new insights into the mechanisms by which Sig-1R regulates depression and further supports the concept of the gut-brain axis.

SUCNR1 signaling in adipocytes controls energy metabolism by modulating circadian clock and leptin expression

Adipose tissue modulates energy homeostasis by secreting leptin, but little is known about the factors governing leptin production. We show that succinate, long perceived as a mediator of immune response and lipolysis, controls leptin expression via its receptor SUCNR1. Adipocyte-specific deletion of Sucnr1 influences metabolic health according to nutritional status. Adipocyte Sucnr1 deficiency impairs leptin response to feeding, whereas oral succinate mimics nutrient-related leptin dynamics via SUCNR1. SUCNR1 activation controls leptin expression via the circadian clock in an AMPK/JNK-C/EBPα-dependent manner. Although the anti-lipolytic role of SUCNR1 prevails in obesity, its function as a regulator of leptin signaling contributes to the metabolically favorable phenotype in adipocyte-specific Sucnr1 knockout mice under standard dietary conditions. Obesity-associated hyperleptinemia in humans is linked to SUCNR1 overexpression in adipocytes, which emerges as the major predictor of adipose tissue leptin expression. Our study establishes the succinate/SUCNR1 axis as a metabolite-sensing pathway mediating nutrient-related leptin dynamics to control whole-body homeostasis.

Behavioral phenotype, intestinal microbiome, and brain neuronal activity of male serotonin transporter knockout mice

The serotonin transporter (5-HTT) plays a critical role in the regulation of serotonin neurotransmission. Mice genetically deficient in 5-HTT expression have been used to study the physiological functions of 5-HTT in the brain and have been proposed as a potential animal model for neuropsychiatric and neurodevelopmental disorders. Recent studies have provided evidence for a link between the gut-brain axis and mood disorders. However, the effects of 5-HTT deficiency on gut microbiota, brain function, and behavior remain to be fully characterized. Here we investigated the effects of 5-HTT deficiency on different types of behavior, the gut microbiome, and brain c-Fos expression as a marker of neuronal activation in response to the forced swim test for assessing depression-related behavior in male 5-HTT knockout mice. Behavioral analysis using a battery of 16 different tests showed that 5-HTT−/− mice exhibited markedly reduced locomotor activity, decreased pain sensitivity, reduced motor function, increased anxiety-like and depression-related behavior, altered social behavior in novel and familiar environments, normal working memory, enhanced spatial reference memory, and impaired fear memory compared to 5-HTT+/+ mice. 5-HTT+/− mice showed slightly reduced locomotor activity and impaired social behavior compared to 5-HTT+/+ mice. Analysis of 16S rRNA gene amplicons showed that 5-HTT−/− mice had altered gut microbiota abundances, such as a decrease in Allobaculum, Bifidobacterium, Clostridium sensu stricto, and Turicibacter, compared to 5-HTT+/+ mice. This study also showed that after exposure to the forced swim test, the number of c-Fos-positive cells was higher in the paraventricular thalamus and lateral hypothalamus and was lower in the prefrontal cortical regions, nucleus accumbens shell, dorsolateral septal nucleus, hippocampal regions, and ventromedial hypothalamus in 5-HTT−/− mice than in 5-HTT+/+ mice. These phenotypes of 5-HTT−/− mice partially recapitulate clinical observations in humans with major depressive disorder. The present findings indicate that 5-HTT-deficient mice serve as a good and valid animal model to study anxiety and depression with altered gut microbial composition and abnormal neuronal activity in the brain, highlighting the importance of 5-HTT in brain function and the mechanisms underlying the regulation of anxiety and depression.