Phenotype Of Mice Research Articles

Reduction of orexin-expressing neurons and a unique sleep phenotype in the Tg-SwDI mouse model of Alzheimer’s disease

Sleep disturbances are common in Alzheimer’s disease (AD) and AD-related dementia (ADRD). We performed a sleep study on Tg-SwDI mice, a cerebral amyloid angiopathy (CAA) model, and age-matched wild-type (WT) control mice. The results showed that at 12 months of age, the hemizygous Tg-SwDI mice spent significantly more time in non-rapid eye movement (NREM) sleep (44.6 ± 2.4% in Tg-SwDI versus 35.9 ± 2.5% in WT) and had a much shorter average length of wake bout during the dark (active) phase (148.5 ± 8.7 s in the Tg-SwDI versus 203.6 ± 13.0 s in WT). Histological analysis revealed stark decreases of orexin immunoreactive (orexin-IR) neuron number and soma size in these Tg-SwDI mice (cell number: 2187 ± 97.1 in Tg-SwDI versus 3318 ± 137.9 in WT. soma size: 109.1 ± 8.1 μm2 in Tg-SwDI versus 160.4 ± 6.6 μm2 in WT), while the number and size of melanin-concentrating hormone (MCH) immunoreactive (MCH-IR) neurons remained unchanged (cell number: 4256 ± 273.3 in Tg-SwDI versus 4494 ± 326.8 in WT. soma size: 220.1 ± 13.6 μm2 in Tg-SwDI versus 202.0 ± 7.8 μm2 in WT). The apoptotic cell death marker cleaved caspase-3 immunoreactive (Caspase-3-IR) percentage in orexin-IR neurons was significantly higher in Tg-SwDI mice than in WT controls. This selective loss of orexin-IR neurons could be associated with the abnormal sleep phenotype in these Tg-SwDI mice. Further studies are needed to determine the cause of the selective death of orexin-IR cells and relevant effects on cognition impairments in this mouse model of microvascular amyloidosis.

Assessment of Long-Term Safety and Efficacy of Purple Sweet Potato Color (PSPC) and Myo-Inositol (MI) Treatment for Motor Related and Behavioral Phenotypes in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a rare inherited metabolic disease caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase in galactose metabolism. The condition develops as a potentially fatal illness during the newborn period, but its acute clinical manifestations can be alleviated through a galactose restricted diet. Nonetheless, such dietary intervention is inadequate in preventing significant long-term consequences, including neurological impairments, growth restriction, cognitive delays, and, for most females, primary ovarian insufficiency. At present, no effective therapy exists to stop the progression of these complications, highlighting the urgent need for new treatment approaches to be developed. Supplements have been used in the treatment of other inborn errors of metabolism; however, they are not typically included in the clinical therapeutic regimen for CG. Recently, our research team has demonstrated that two generally recognized as safe supplements (purple weet potato color, PSPC and myo-inositol, MI) have been effective in partially restoring functions in the ovaries of our GalT-KO mouse model. However, the toxicological profile of both PSPC and MI has not been determined. In this study, we investigated the acute (30 days) and chronic (180 days) oral toxicities of PSPC and MI both in WT control and GalT-KO mice. Furthermore, our study aims to evaluate the effectiveness of oral feeding of PSPC and MI in correcting motor-related and behavioral phenotypes in GalT-KO mice. The long-term treatment of MI at a lower dose demonstrated promising improvements in motor deficit and anxiety driven hyperactivity in the mutant mice.

Comparison of naturalization mouse model setups uncover distinct effects on intestinal mucosa depending on microbial experience

Abstract Introduction Concerns regarding the translational value of preclinical mouse models have been addressed by introducing various approaches of “naturalizing” research mice, which provide them with more diverse microbiomes and physiological immune responses. We have previously shown that “feralized” mice, i.e., inbred laboratory mice raised in a farmyard-like, microbe-rich environment exhibit a shifted gut microbiota, matured immunophenotype, and reduced severity of colorectal cancer. Similar studies occasionally involve co-housing with wild or pet-store-raised mice as microbial donors integrating species-specific commensals and pathogens. To what extent these different practices of microbial exposure are crucial for the resulting mouse phenotype remains unclear. Methods Here, we present the first side-by-side comparison of different methods to naturalize laboratory mice: co-housing with wild-caught house mice, feralization in a farmyard-like habitat only, or a combination of the two, with conventional clean laboratory mice as a reference. Results Independent of the method, the naturalized colon-mucosa microbiota was colonized by several Helicobacter species, and the colonic intestinal epithelial cells of naturalized mice displayed elevated expression of genes encoding antimicrobial peptides, mucus components, and reactive-oxygen-species-producing enzymes. They further showed significantly increased resident memory T cells in the colonic lamina propria and effector memory T cells in the mesenteric lymph nodes. The most pronounced changes of these parameters occurred in mice co-housed with wild-caught mice, while feralized mice displayed phenotypes that were intermediate between laboratory and co-housed mice. Conclusion These findings enhance our understanding of naturalization model setups and effects on the gut barrier and immune system, thereby aiding future decisions on the utilization of naturalized mouse models.

Nuclear farnesoid X receptor protects against bone loss by driving osteoblast differentiation through stabilizing RUNX2

The delicate balance between bone formation by osteoblasts and bone resorption by osteoclasts maintains bone homeostasis. Nuclear receptors (NRs) are now understood to be crucial in bone physiology and pathology. However, the function of the Farnesoid X receptor (FXR), a member of the NR family, in regulating bone homeostasis remains incompletely understood. In this study, in vitro and in vivo models revealed delayed bone development and an osteoporosis phenotype in mice lacking FXR in bone marrow mesenchymal stem cells (BMSCs) and osteoblasts due to impaired osteoblast differentiation. Mechanistically, FXR could stabilize RUNX2 by inhibiting Thoc6-mediated ubiquitination, thereby promoting osteogenic activity in BMSCs. Moreover, activated FXR could directly bind to the Thoc6 promoter, suppressing its expression. The interaction between RUNX2 and Thoc6 was mediated by the Runt domain of RUNX2 and the WD repeat of Thoc6. Additionally, Obeticholic acid (OCA), an orally available FXR agonist, could ameliorate bone loss in an ovariectomy (OVX)-induced osteoporotic mouse model. Taken together, our findings suggest that FXR plays pivotal roles in osteoblast differentiation by regulating RUNX2 stability and that targeting FXR may be a promising therapeutic approach for osteoporosis.

Epstein-Barr Virus-Induced 3 Attributes to TLR7-Mediated Splenomegaly.

Epstein-Barr virus-induced 3 (EBI3) functions as a component of the heterodimer cytokine IL-27, which regulates innate and acquired immune responses. The expression of EBI3 gene is induced by Toll-like receptors (TLRs). Repeated treatment with imiquimod (IMQ), a TLR7 agonist, induces splenomegaly and cytopaenia due to increased splenic function. Although immune cell activation is speculated to play a role in chronic infection-mediated splenomegaly, the detailed mechanisms remain unknown. This study shows that IMQ treatment induces marked splenomegaly and severe bicytopaenia (anaemia and thrombocytopaenia) in wild-type mice. In IMQ-treated mice, myeloid cell populations in the spleen increased, and extramedullary haematopoiesis was observed. RNA-seq analysis revealed the upregulation of type I interferon (IFN)-related genes in the spleens of IMQ-treated mice. IMQ-induced pathological changes were partially mitigated by EBI3 deficiency. To investigate the mechanism of the improved phenotypes in the Ebi3 KO mice, we examined the involvement of IL-27, a heterodimer of EBI3 and IL-27p28. The expression of Il27a, which encodes IL-27p28, was increased in the spleen and peripheral blood by IMQ treatment. Furthermore, IL-27 stimulation upregulated type I IFN-related genes in bone marrow-derived macrophage cultures without type I IFN. These findings suggest that EBI3 deficiency mitigated IMQ-mediated pathological changes, presumably via a lack of IL-27 formation. Our study thus provides insights into the molecular mechanisms underlying chronic infection-mediated splenomegaly.

Abstract B008: Understanding myeloid and lymphoid cell heterogeneity following radiotherapy and its impact on metastasis development in breast cancer

Abstract Breast cancer (BC) is the most frequent malignancy in women worldwide and the gold standard for its treatment currently entails resection of the primary tumor, followed by radiotherapy (RT), chemotherapy and/or immunotherapy. Specifically, adjuvant RT directed at the breast has been successfully employed to improve the risk of local recurrences, but the main obstacle to full remission of most BC patients remains the development of metastasis. RT directed at the primary tumor (pRT) acts via a combination of direct cytotoxicity and immunomodulatory effects on the local tumor microenvironment (TME), whereas its impact at distal sites and on the metastatic TME is still poorly understood. Thus, we hypothesized that pRT-driven systemic effects could change the nature and function of immune cell populations at distal sites, affecting survival and growth of disseminated tumor cells and ultimately influencing the development of metastasis. Analysis of scRNAseq data from murine lungs at the pre-metastatic stage showed that pRT towards a tumor-bearing breast induces alterations within the lung immune landscape, particularly suggesting a dual role of neutrophils and alterations in their interactions with the adaptive immune system. Moreover, we demonstrated that in a mouse model of metastasis, experimentally induced via intravenous (IV) injection of 4T1 cells, pRT directed to a tumor-bearing breast induces a significant increase in lung metastasis, accompanied by numerical and functional differences in lymphoid and myeloid cells which collectively suggest the establishment of a local immunosuppressive microenvironment. Interestingly, the metastatic increase observed in irradiated mice resembled the phenotype of mice IV injected with 4T1 cells in absence of a primary tumor, thus when there is no pre-activation of the immune system prior to the IV injection. This result suggested that pRT could alter systemic anti-tumor immunity in our model, resulting in metastatic growth. Therefore, current experiments are focusing on elucidating the specific immune populations involved in this effect by selectively depleting cell types of interest, such as neutrophils and Treg cells. Moreover, we are further investigating the mechanisms influencing DTCs’ outgrowth via scRNAseq of lungs bearing niche-labelling metastasis, which allow to identify changes occurring specifically in the metastatic niche. Finally, we are confirming the effects observed in our experimentally-induced metastatic model also in a clinically relevant mouse model, comprising both the spontaneous metastatic spread of 4T1 cells and the resection of the primary tumor prior to RT, resulting in a therapeutic protocol more similar to current clinical practice. Our data show a pro-metastatic effect of RT, possibly driven by alterations in the activation of the immune system. Results from this project could better inform on RT’s influence on immune cell behavior in the metastatic TME, allowing to identify relevant targets for novel immunotherapy approaches against metastatic disease. Citation Format: Alessandra Perini, Marti Brucoli, Sophie Acton, Luigi Ombrato. Understanding myeloid and lymphoid cell heterogeneity following radiotherapy and its impact on metastasis development in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B008.

Reduced autoimmunity associated with deletion of host CD73.

CD73 is ubiquitously expressed and regulates critical functions across multiple organ systems. The sequential actions of CD39 and CD73 accomplish the conversion of adenosine triphosphate to adenosine and shift the adenosine triphosphate-driven proinflammatory immune cell milieu toward an anti-inflammatory state. This immunological switch is a major mechanism by which regulatory T (Treg) cells control inflammation. Foxp3 engages in Treg development and function. Foxp3 mutations result in the scurfy (SF) mouse phenotype and a rapidly lethal lymphoproliferative syndrome. We generated double knockout (KO) mouse (CD73KOSF) by breeding heterozygous Foxp3sf/J females to CD73KO male mice to remove host CD73. We initially aimed to use these mice to identify a specific probiotic-CD73 effect, previously shown for Limosilactobacillus reuteri DSM 17938. We expected CD73 deletion to enhance the severity of autoimmunity in SF mice. However, we unexpectedly observed that KO of host CD73 in SF mice clinically reduced the severity of autoimmunity including reduced ear thickness, increased ear size, and less deformed ears, along with less dry and brittle skin. KO of CD73 in SF mice significantly reduced the numbers of CD4+ and CD8+T cells in spleen and blood. We identified that KO of CD73 in SF mice reduced the numbers of T cells in the thymus compared with those in SF mice, indicating that the milder clinical phenotype may be due to reduced central and peripheral lymphoproliferation. These new findings suggest targeting CD73 could improve T cell-mediated dermatitis, one of the most common symptoms in Treg deficiency-associated primary immune deficiencies.

Single-cell decoding of human islet cell type-specific alterations in type 2 diabetes reveals converging genetic- and state-driven β-cell gene expression defects.

Pancreatic islets maintain glucose homeostasis through coordinated action of their constituent endocrine and affiliate cell types and are central to type 2 diabetes (T2D) genetics and pathophysiology. Our understanding of robust human islet cell type-specific alterations in T2D remains limited. Here, we report comprehensive single cell transcriptome profiling of 245,878 human islet cells from a 48-donor cohort spanning non-diabetic (ND), pre-diabetic (PD), and T2D states, identifying 14 distinct cell types detected in every donor from each glycemic state. Cohort analysis reveals ∼25-30% loss of functional beta cell mass in T2D vs. ND or PD donors resulting from (1) reduced total beta cell numbers/proportions and (2) reciprocal loss of 'high function' and gain of senescent β-cell subpopulations. We identify in T2D β-cells 511 differentially expressed genes (DEGs), including new (66.5%) and validated genes (e.g., FXYD2, SLC2A2, SYT1 ), and significant neuronal transmission and vitamin A metabolism pathway alterations. Importantly, we demonstrate newly identified DEG roles in human β-cell viability and/or insulin secretion and link 47 DEGs to diabetes-relevant phenotypes in knockout mice, implicating them as potential causal islet dysfunction genes. Additionally, we nominate as candidate T2D causal genes and therapeutic targets 27 DEGs for which T2D genetic risk variants (GWAS SNPs) and pathophysiology (T2D vs. ND) exert concordant expression effects. We provide this freely accessible atlas for data exploration, analysis, and hypothesis testing. Together, this study provides new genomic resources for and insights into T2D pathophysiology and human islet dysfunction.

IL-1R1 and IL-18 Signals Regulate Mesenchymal Stromal Cells in an Aged Murine Model of Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are age-related diseases characterized by bone marrow (BM) dysfunction and an increased risk of developing acute leukemia. While there is growing evidence highlighting the crucial role of the BM microenvironment (BMME) in MDS, the specific influence of inflammation on BMME changes, as well as the potential benefits of targeting cytokines therapeutically, remain to be elucidated. We previously found interleukin-1 (IL-1) to be a driver of aging phenotypes of BMME and hematopoietic stem and progenitor cells (HSPCs). In the current study, BM samples from patients with MDS demonstrated upregulated levels of IL-1 family cytokines including IL-18. Utilizing highly purified primary BM-derived mesenchymal stromal cells (MSCs), both interleukin-1b (IL-1b) and IL-18 were found to exert direct effects on MSCs, thus influencing their ability to support HSPCs as well as erythroid progenitors. This confirms the significant involvement of both these IL-1 family cytokines in regulating the BM niche. Furthermore, targeting IL-1 receptor type I (IL-1R1) mitigated these aging phenotypes in elderly mice. We subsequently employed an age-appropriate murine model of MDS by transplanting NUP98-HOXD13 transgenic mice (NHD13Tg) cells into aged wild-type mice. Treatment with inhibitors targeting interleukin-1 receptor-associated kinase 4 (IRAK4) and NLR family pyrin domain containing 3 (NLRP3) reversed the proliferation of dysfunctional MSCs and enhanced their functionality. Additionally, IRAK4 inhibition selectively suppressed MDS clonal cells while sparing non-MDS cells in the BM. These findings suggest that targeting IL-1 signaling holds promise for MDS treatment by addressing the underlying myeloid malignancy and restoring the altered BMME via BM-MSCs.

Impact of dapagliflozin on metabolic phenotype, hormone levels, and fertility in female mice after prolonged high-fat diet.

A long-term high-fat diet (HFD) cause obesity and infertility through hypothalamic inflammation and insulin resistance, leading to metabolic abnormalities and ovulation dysfunction. The sodium-glucose cotransporter 2 inhibitors (SGLT2i) have emerged as a treatment for type 2 diabetic patients, regulating adipose tissue metabolism, hypothalamic inflammation, and ovulation in women with polycystic ovary syndrome (PCOS). The study aimed to investigate the pharmacological effects of dapagliflozin on improving insulin resistance, energy metabolism, sex hormones, and fertility in female mice following prolonged consumption of HFD. At 6 weeks of age, female mice were fed a HFD and treated with dapagliflozin. Serum hormone concentrations and inflammatory factors in mice aged 28 weeks or 38 weeks were quantified using ultrasensitive enzyme-linked immunosorbent assays (ELISAs). Metabolic parameters were also assessed and documented at different stages of the experiment. At 34 weeks of age, half of the experimental mice in each of the four groups fed with standard chow were mated with male mice. Pregnancy rate, abortion rate, pregnancy-related deaths, and perinatal outcomes were systematically recorded. After 16 weeks of HFD feeding, dapagliflozin significantly attenuated visceral fat deposition, weight gain, glucose intolerance, and insulin resistance induced by the diet. However, these effects diminished after 32 weeks. Unexpectedly, neither HFD nor dapagliflozin treatment elicited any significant changes in serum IL-6 and TNFα levels. Throughout the experiment period, dapagliflozin exhibited favorable effects on reproductive function along with insulin sensitivity and luteinizing hormone (LH) release from the pituitary gland. In conclusion, this study demonstrates that dapagliflozin alleviated HFD-induced reproductive dysfunction independently of obesity, peripheral tissue insulin resistance, and systemic inflammation, suggesting its potential as a promising treatment for diet-related ovulation disorders.

Isoxazole-based molecules restore NK cell immune surveillance in hepatocarcinogenesis by targeting TM4SF5 and SLAMF7 linkage

Dynamic communication between hepatocytes and the environment is critical in hepatocellular carcinoma (HCC) development. Clinical immunotherapy against HCC is currently unsatisfactory and needs more systemic considerations, including the identification of new biomarkers and immune checkpoints. Transmembrane 4 L six family member 5 (TM4SF5) is known to promote HCC, but it remains unclear how cancerous hepatocytes avoid immune surveillance and whether avoidance can be blocked. We investigated how TM4SF5-mediated hepatic tumorigenesis avoids surveillance by natural killer (NK) cells, which are prevalent in the liver, and whether the avoidance can be blocked by anti-TM4SF5 agents. We used comprehensive structure activity relationship analysis to identify TM4SF5-specific isoxazole (TSI)-based small molecules that inhibit TM4SF5-mediated effects. TM4SF5 expressed by hepatocytes reduced NK cell cytotoxicity by downregulating stimulatory ligands/receptors, including signaling lymphocytic activation molecule family member 7 (SLAMF7). TM4SF5 bound SLAMF7 depending on N-glycosylation and caused intracellular trafficking of SLAMF7 from the plasma membrane to lysosomes for degradation. TSI treatments in cell lines and animal models of HCC blocked this binding, intracellular trafficking, and downregulation, resulting in higher levels of stimulatory NK cell ligands. In mouse xenograft models, TSI treatment abrogated HCC development by increasing the abundance and dispersion of Slamf7-positive cells in liver tissues, recapitulating the phenotype of Tm4sf5-knockout mice and indicating TSI-mediated restoration of NK cell surveillance. These findings suggest that TSIs can inhibit TM4SF5-mediated liver carcinogenesis by increasing NK cell surveillance.

L1CAM+ extracellular vesicles derived from the serum of adolescents with major depressive disorder induce depression-like phenotypes in adolescent mice.

It has been reported that L1 cell adhesion molecule (L1CAM) antibody can capture neuron-derived extracellular vesicles (NDEVs) derived from peripheral blood. This antibody is significantly associated with occurrence of adult psychiatric disorders. However, the role and mechanism of L1CAM+ EVs (L1+ EVs) in adolescent with major depressive disorder (AMDD) is not well understood. This research aimed to explore the function and potential mechanism of L1+ EVs and miRNAs genes in AMDD. L1+ EVs derived from the serum of AMDD and healthy controls (HC) were transplanted into adolescent mice via tail vein. Their effects were explored using behavioral tests, hippocampal Nissl staining, and whole genome mRNA sequencing. MiRNAs expression in L1+ EVs was evaluated by whole-genome sequencing and qRT-PCR. Bioinformatics analysis was employed to explore the possible pathogenic molecular mechanisms of these miRNAs in AMDD. Transplantation of L1+ EVs from AMDD induced depression-like behavior and hippocampal neuronal damage in adolescent mice and aberrant expression of 298 mRNA genes. The molecular signals related to MDD were enriched in the top pathways of the differentially expressed genes. Compared with HC, miR-375-3p and miR-200a-3p were upregulated in L1+ EVs from AMDD, miR-375-3p was also increased in the hippocampus of AMDD serum L1+ EVs-recipient mice. Bioinformatics analysis revealed that miR-375-3p might modulate the network of molecules associated with the MAPK pathway via protein interaction involving hippocampal differential genes Cadm2, Cacna2d1, and Casz1. MiR-375-3p might contribute to L1+ EVs-induced AMDD. L1+ EVs miR-375-3p and miR-200a-3p could potentially serve as potential biomarkers for AMDD.

Sex differences in cognition, anxiety-phenotype and therapeutic effect of metformin in the aged apoE-TR mice

BackgroundApolipoprotein E4 (ApoE4) is associated with an increased risk of Alzheimer’s disease (AD), depression, and anxiety, which were reported to improve after the administration of metformin. However, sex influence on the effect of ApoE4 and metformin on cognition and mental health is poorly understood.MethodsApoE3-TR and apoE4-TR mice of both sexes were randomly assigned to the normal saline and metformin groups from 13 months to 18 months of age. Behavior tests (MWM, EPM, OFT, TST, FST) were conducted to assess cognition, anxiety, and depression-like behaviors. The mice’s blood glucose was also recorded.ResultsMale aged apoE4-TR mice are more vulnerable to cognitive decline than females. Metformin improves the spatial memory of female, but not male apoE3-TR mice and female apoE4-TR mice while aggravating the cognitive impairment of male apoE4-TR mice. The anxiety-like phenotypes in male apoE4-TR mice are more severe than in male apoE3-TR mice, while metformin ameliorates the anxiety-like behaviors in the male apoE4-TR mice but not in male apoE3-TR mice. In addition, metformin alleviates depression-like behaviors in male and female apoE4-TR mice. The hypoglycemic effect of metformin is insignificant in both male and female apoE4-TR mice.ConclusionsMale sex exacerbates APOE4-related cognitive impairment and anxiety in aged mice and is insensitive to the cognition improvement effect of metformin in the aged apoE3 mice. Male sex with APOE4 may experience more severe cognitive impairment after treatment with metformin while sensitive to the anti-anxiety effects of metformin. These findings identify sex-specific effects on ApoE4-based dementia, anxiety prevention, and therapy, emphasizing the importance of further sex dimension analyses in vivo and clinical studies.

Systematic ocular phenotyping of 8,707 knockout mouse lines identifies genes associated with abnormal corneal phenotypes

PurposeCorneal dysmorphologies (CDs) are typically classified as either regressive degenerative corneal dystrophies (CDtrs) or defective growth and differentiation-driven corneal dysplasias (CDyps). Both eye disorders have multifactorial etiologies. While previous work has elucidated many aspects of CDs, such as presenting symptoms, epidemiology, and pathophysiology, the genetic mechanisms remain incompletely understood. The purpose of this study was to analyze phenotype data from 8,707 knockout mouse lines to identify new genes associated with the development of CDs in humans.Methods8,707 knockout mouse lines phenotyped by the International Mouse Phenotyping Consortium were queried for genes associated with statistically significant (P < 0.0001) abnormal cornea morphology to identify candidate CD genes. Corneal abnormalities were investigated by histopathology. A literature search was used to determine the proportion of candidate genes previously associated with CDs in mice and humans. Phenotypes of human orthologues of mouse candidate genes were compared with known human CD genes to identify protein-protein interactions and molecular pathways using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Protein Analysis Through Evolutionary Relationships (PANTHER), and Kyoto Encyclopedia of Genes and Genomes.ResultsAnalysis of data from 8,707 knockout mouse lines identified 213 candidate CD genes. Of these, 37 (17%) genes were previously known to be associated with CD, including 14 in the mouse, 16 in humans, and 7 in both. The remaining 176 (83%) genes have not been previously implicated in CD. We also searched publicly available RNAseq data and found that 131 of the total 213 (61.5%) were expressed in adult human corneal tissue. STRING analysis showed several interactions within and between candidate and established CD proteins. All cellular pathways of the established genes were found in the PANTHER analysis of the candidate genes. Several of the candidate genes were implicated in corneal disease, such as TGF-ß signaling. We also identified other possible underappreciated mechanisms relevant to the human cornea.ConclusionsWe identified 213 mouse genes that resulted in statistically significant abnormal corneal phenotypes in knockout mice, many of which have not previously been implicated in corneal pathology. Bioinformatic analyses implicated candidate genes in several signaling pathways which are potential therapeutic targets.

Analyzing the relationship between gene expression and phenotype in space-flown mice using a causal inference machine learning ensemble

Spaceflight has several detrimental effects on human and rodent health. For example, liver dysfunction is a common phenotype observed in space-flown rodents, and this dysfunction is partially reflected in transcriptomic changes. Studies linking transcriptomics with liver dysfunction rely on tools which exploit correlation, but these tools make no attempt to disambiguate true correlations from spurious ones. In this work, we use a machine learning ensemble of causal inference methods called the Causal Research and Inference Search Platform (CRISP) which was developed to predict causal features of a binary response variable from high-dimensional input. We used CRISP to identify genes robustly correlated with a lipid density phenotype using transcriptomic and histological data from the NASA Open Science Data Repository (OSDR). Our approach identified genes and molecular targets not predicted by previous traditional differential gene expression analyses. These genes are likely to play a pivotal role in the liver dysfunction observed in space-flown rodents, and this work opens the door to identifying novel countermeasures for space travel.

Fasting activates optineurin-mediated mitophagy in chondrocytes to protect against osteoarthritis

Mitochondrial homeostasis plays a crucial role in the pathogenesis of osteoarthritis (OA), a chronic musculoskeletal disorder characterized by articular cartilage degeneration and chondrocyte apoptosis. However, molecular mechanisms underlying the association between mitophagy and OA remain unclear. Here, we aimed to investigate the role of the autophagy receptor protein optineurin (OPTN) in OA, and explore the effects of dietary intervention on OA symptoms and its relationship with OPTN-mediated mitophagy. Our findings showed the downregulation of OPTN in patients with OA. Using an Optn-knockout mouse model, we demonstrated that OPTN deficiency leads to impaired mitophagy, resulting in the accumulation of damaged mitochondria, increased production of reactive oxygen species, and chondrocyte apoptosis. Furthermore, fasting prevented OA progression by activating OPTN-mediated mitophagy and maintaining mitochondrial homeostasis in mice. The present study revealed a novel mechanism by which OPTN-mediated mitophagy influences chondrocytes and the OA phenotype in Optn-knockout mice, suggesting that OPTN-mediated mitophagy plays a crucial role in OA development and progression. This study provides new insights into the pathogenesis of OA and offers a potential avenue for the development of novel drugs targeting OPTN to mitigate OA progression.

Independent genetic strategies define the scope and limits of CDKL5 deficiency disorder reversal.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a neurodevelopmental syndrome caused by mutations in the X-linked CDKL5 gene. The early onset of CDD suggests that CDKL5 is essential during development, but post-developmental re-expression rescues multiple CDD-related phenotypes in hemizygous male mice. Since most patients are heterozygous females, studies in clinically relevant female models are essential. Here, we systematically compare phenotype reversal across age and sex using two independent mouse models of CDD. We find that early re-activation of endogenous Cdkl5 in heterozygous females reverses most phenotypes, except working memory. Later re-expression improves several traits but has limited effects on cognitive function. Seizure prevention is more effective with early intervention in heterozygous females but becomes limited after seizure onset. These findings demonstrate the robust potential of CDKL5 re-expression to reverse CDD-related phenotypes in both sexes while underscoring the critical impact of age and disease stage in designing clinical trials.

Inhaled xenon modulates microglia and ameliorates disease in mouse models of amyloidosis and tauopathy.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. Antiamyloid antibody treatments modestly slow disease progression in mild dementia due to AD. Emerging evidence shows that homeostatic dysregulation of the brain immune system, especially that orchestrated by microglia, plays an important role in disease onset and progression. Thus, a major question is how to modulate the phenotype and function of microglia to treat AD. Xenon (Xe) gas is a noble gas used in human patients as an anesthetic and a neuroprotectant used for treating brain injuries. Xe penetrates the blood-brain barrier, which could make it an effective therapeutic. To assess the effect of Xe on microglia and AD pathology, we designed a custom Xe inhalation chamber and treated several mouse models of AD with Xe gas. Xe treatment induced mouse microglia to adopt an intermediate activation state that we have termed pre-neurodegenerative microglia (pre-MGnD). This microglial phenotypic transition was observed in mouse models of acute neurodegeneration and amyloidosis (APP/PS1 and 5xFAD mice) and tauopathy (P301S mice). This microglial state enhanced amyloid plaque compaction and reduced dystrophic neurites in the APP/PS1 and 5xFAD mouse models. Moreover, Xe inhalation reduced brain atrophy and neuroinflammation and improved nest-building behavior in P301S mice. Mechanistically, Xe inhalation induced homeostatic brain microglia toward a pre-MGnD state through IFN-γ signaling that maintained the microglial phagocytic response in APP/PS1 and 5xFAD mice while suppressing the microglial proinflammatory phenotype in P301S mice. These results support the translation of Xe inhalation as an approach for treating AD.

S100A8/A9 Promotes Dendritic Cell-Mediated Th17 Cell Response in Sjögren's Dry Eye Disease by Regulating the Acod1/STAT3 Pathway.

To investigate the role of S100A8/A9 in the pathogenesis of Sjögren's dry eye disease (SjDED) and explore its potential mechanism of action. S100A8/A9 expression was determined by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Tear secretion, corneal fluorescein staining, and hematoxylin and eosin staining were used to evaluate the effect of paquinimod, a S100A8/A9 inhibitor, on dry eye disease in nonobese diabetic (NOD) mice. Immune cell infiltration and percentage were assessed by immunofluorescence and flow cytometry. Proinflammatory cytokine levels were examined by qRT-PCR or ELISA. The mechanism of action was analyzed using western blot, immunofluorescence, and chromatin immunoprecipitation. S100A8/A9 was upregulated in peripheral blood mononuclear cells of patients with SjDED and lacrimal glands (LGs) of SjDED mice. The upregulation of S100A8/A9 was correlated with the dry eye severity and inflammatory infiltration levels in LGs. Administration of paquinimod ameliorated clinical and histopathological phenotypes of SjDED mice and reduced the proportion of Th17 cells in LGs, lymph nodes, and spleens. Further experiments revealed that S100A8/A9 did not directly affect Th17 generation and function but upregulated the expression of major histocompatibility complex Ⅱ (MHC Ⅱ) and Th17-polarizing cytokines in dendritic cells (DCs) to augment Th17 cell response. Mechanistically, S100A8/A9 induced the expression of Acod1 and thereby promoted the activation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3), resulting in increased Il23a transcription. STAT3 activator reversed the therapeutic effect of paquinimod on SjDED mice. S100A8/A9 activated the Acod1/STAT3 pathway to promote DC-driven Th17 cell responses in SjDED. The S100A8/A9/Acod1/STAT3 pathway may represent a promising therapeutic target for SjDED.

Inhibition of IFITM3 in cerebrovascular endothelium alleviates Alzheimer's-related phenotypes.

Interferon-induced transmembrane protein 3 (IFITM3) modulates γ-secretase in Alzheimer's Disease (AD). Although IFITM3 knockout reduces amyloid β protein (Aβ) production, its cell-specific effect on AD remains unclear. Single nucleus RNA sequencing (snRNA-seq) was used to assess IFITM3 expression. Adeno-associated virus-BI30 (AAV-BI30) was injected to reduce IFITM3 expression in the cerebrovascular endothelial cells (CVECs). The effects on AD phenotypes in cells and AD mice were examined through behavioral tests, two-photon imaging, flow cytometry, Western blot, immunohistochemistry, and quantitative polymerase chain reaction assay (qPCR). IFITM3 expression was increased in the CVECs of patients with AD. Overexpression of IFITM3 in primary endothelial cells enhanced Aβ generation through regulating beta-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Aβ further increased IFITM3 expression, creating a vicious cycle. Knockdown of IFITM3 in CVECs decreased Aβ accumulation within cerebrovascular walls, reduced Alzheimer's-related pathology, and improved cognitive performance in AD transgenic mice. Knockdown of IFITM3 in CVECs alleviates AD pathology and cognitive impairment. Targeting cerebrovascular endothelial IFITM3 holds promise for AD treatment. Interferon-induced transmembrane protein 3 (IFITM3) expression was increased in the cerebrovascular endothelial cells (CVECs) of patients with Alzheimer's Disease (AD). Cerebrovascular endothelial IFITM3 regulates amyloid β protein (Aβ) generation through regulating beta-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Knockdown of IFITM3 in CVECs reduces Aβ deposits and improves cognitive impairments in AD transgenic mice. Cerebrovascular endothelial IFITM3 could be a potential target for the treatment of AD.