Control Mice Research Articles

Short Term Acid Sphingomyelinase Deficiency Exerts Proinflammatory and Antiapoptotic Effects During LPS-induced Lung Injury in Mice.

Lysosomal acid sphingomyelinase (ASM; SMPD1) deficiency causes Niemann-Pick Disease (NPD) that, in Type B, manifests with interstitial lung disease and susceptibility to infections. Constitutional Smpd1 (Smpd1-/- mice) deletion causes lung inflammation with foamy dysfunctional macrophages, but is protective against acute lung injury. It is unknown whether these manifestations are from progressive accumulation of sphingomyelin, decreased ceramide, or compensatory alterations in sphingolipid metabolism. We developed a conditional knockout mouse, Smpd1fl/flxCAGG-CreERTM, induced by tamoxifen (5 weeks), with decreased Smpd1 expression (by 75%) and ASM activity (by up to 40%). We investigated how brief post-developmental ASM insufficiency affects lung sphingolipids and pathology including following lipopolysaccharide (LPS)-induced injury. Compared to controls, Smpd1fl/fl mice exhibited modest sphingomyelin elevation with lower palmitoyl/lignoceroyl ceramide (C16/C24) ratios, increased de novo sphingolipid synthesis and sphingosine-1 phosphate levels. At 3 days following LPS instillation (20μg) control mice had increased lung (neutrophilic and monocytic) inflammation and apoptosis; Smpd1fl/fl mice showed more exuberant inflammation with reduced apoptosis, particularly in endothelial cells. During repair (6-9 days), Smpd1fl/fl lungs had increased cell proliferation with reduced autophagosome-tagging p62/SQSTM1. These results indicate that prior to significant lysosomal lipid storage, ASM insufficiency inhibits stress-induced lung apoptosis and promotes compensatory sphingolipid changes that favor exuberant inflammatory responses to LPS. Overall, ASM inhibition limits lung vascular injury and stimulates repair following inflammatory insults. These results provide novel insights into the function of ASM in the lung, which are relevant to understanding the pathogenesis and complications of NPD and the role of distinct sphingolipid metabolites in lung injury and repair.

Lrtm1 - A Novel Sensor of Insulin Signaling and Regulator of Metabolism and Activity.

Insulin regulates glucose uptake and metabolism in muscle via the insulin receptor. Here we show that Lrtm1 (Leucine Rich Repeats and Transmembrane Domains 1), a protein of unknown function enriched in insulin-responsive metabolic tissues, senses changes in insulin signaling in muscle and serves as a regulator of metabolic response. Thus, whole-body Lrtm1 deficient mice exhibit a reduced the percentage of fat mass, increased percentage of lean mass, and enhanced glucose tolerance and insulin sensitivity compared to control mice, under both chow and high fat diet conditions. Lrtm1 whole-body deficiency also affects dopamine signaling in the brain leading to hyperactivity. The improvements in glucose and insulin tolerance, but not the behavioral or body composition changes, are also observed in skeletal muscle-specific Lrtm1 knockout mice. These effects occur with no change in classical insulin receptor-Akt signaling Thus, Lrtm1 senses changes in insulin receptor signaling and serves as a novel post-receptor regulator of metabolic and behavioral activity.

Submandibular gland removal decreases avoidance of bitter taste in mice.

Saliva plays a crucial role in digestion, including taste perception, food breakdown, chewing, swallowing, and bolus formation. Saliva is mainly produced by three pairs of major glands: parotid, submandibular, and sublingual glands. To evaluate the effect of each salivary gland on taste preference, we conducted a 48 h two-bottle preference test using mouse models in which the parotid glands (PG), submandibular glands (SMG), or sublingual glands (SLG) were surgically removed. The taste preferences for the five basic tastes of the PG- and SLG-removed mice were similar to those of the control mice. However, in SMG-removed mice, the avoidance of bitter compounds was significantly decreased. These findings indicate that the SMG plays an important role in bitter taste perception among the three major salivary glands. To investigate the reasons for this preference change, we examined the impact of salivary gland removal on the expression of taste-related molecules in the taste buds. No apparent changes were observed in the expression of these molecules after salivary gland removal. When comparing the protein concentration and composition of saliva between the control and salivary gland removal groups, we found that, although the protein concentration did not change significantly, its composition was substantially altered by SMG removal. These results suggest that changes in protein composition in saliva may be one of the factors responsible for the altered bitter preferences observed in the SMG-removed mice.

Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

Sustained β-adrenergic activation induces cardiac fibrosis characterized by excessive deposition of extracellular matrix (ECM). Prostaglandin E2 (PGE2) receptor EP4 is essential for cardiovascular homeostasis. This study aims to investigate the roles of cardiomyocyte (CM) and cardiac fibroblast (CF) EP4 in isoproterenol (ISO)-induced cardiac fibrosis. By crossing the EP4f/f mice with α-MyHC-Cre or S100A4-Cre mice, this work obtains the CM-EP4 knockout (EP4f/f-α-MyHCCre+) or CF-EP4 knockout (EP4f/f-S100A4Cre+) mice. The mice of both genders are subcutaneously injected with ISO (5 mgkg-1day-1) for 7 days. Compared to the control mice, both EP4f/f-α-MyHCCre+ and EP4f/f-S100A4Cre+ mice show a significant improvement in cardiac diastolic function and fibrosis as assessed by echocardiography and histological staining, respectively. In the CMs, inhibition of EP4 suppresses ISO-induced TGF-β1 expression via blocking the cAMP/PKA pathway. In the CFs, inhibition of EP4 reversed TGF-β1-triggers production of ECM via preventing the formation of the TGF-β1/TGF-β receptor complex and blocks CF proliferation via suppressing the ERK1/2 pathway. Furthermore, double knockout of the CM- and CF-EP4 or administration of EP4 antagonist, grapiprant, markedly improves ISO-induced cardiac diastolic dysfunction and fibrosis. Collectively, this study demonstrates that both CM-EP4 and CF-EP4 contribute to β-adrenergic activation-induced cardiac fibrosis. Targeting EP4 may offer a novel therapeutic approach for cardiac fibrosis.

Radio-protective effects of ultra-fine bubble hydrogen water and serum protein responses in whole-body radiation-exposed mice

Many studies have demonstrated hydrogen’s therapeutic and preventive effects on various diseases. Its selective antioxidant properties against hydroxyl radicals, which are responsible for the indirect effects of ionizing radiation, may make it worthy of attention as a new radio-protector. We recently developed new hydrogen water that is more stable and has higher antioxidant activity by using ultra-fine bubbles. In this study, female C57BL/6J mice given ad libitum access to ultra-fine bubble hydrogen water (UBHW) were subjected to whole-body irradiation (WBI) with X-rays, and the radio-protective effect of UBHW was evaluated. WBI with 6.0 Gy (sub-lethal dose) resulted in a 30-day survival rate of 100% in UBHW-fed mice, compared with 37% in control mice. In the case of WBI with 6.5 Gy (lethal dose), while the control mice died out in about 3 weeks, the 30-day survival rate improved to 40% by UBHW due to the high scavenging activity of hydroxy radicals. Twenty-six serum proteins involved in inflammatory and immune responses were significantly identified in UBHW-fed mice by proteomics, and UBHW may enhance and regulate these functions, resulting in reduced damage in mice exposed to WBI. We conclude that UBHW has good potential in radio-protection, with evidence that warrants further research efforts in this field.

MCU expression in hippocampal CA2 neurons modulates dendritic mitochondrial morphology and synaptic plasticity

Neuronal mitochondria are diverse across cell types and subcellular compartments in order to meet unique energy demands. While mitochondria are essential for synaptic transmission and synaptic plasticity, the mechanisms regulating mitochondria to support normal synapse function are incompletely understood. The mitochondrial calcium uniporter (MCU) is proposed to couple neuronal activity to mitochondrial ATP production, which would allow neurons to rapidly adapt to changing energy demands. MCU is uniquely enriched in hippocampal CA2 distal dendrites compared to proximal dendrites, however, the functional significance of this layer-specific enrichment is not clear. Synapses onto CA2 distal dendrites readily express plasticity, unlike the plasticity-resistant synapses onto CA2 proximal dendrites, but the mechanisms underlying these different plasticity profiles are unknown. Using a CA2-specific MCU knockout (cKO) mouse, we found that MCU deletion impairs plasticity at distal dendrite synapses. However, mitochondria were more fragmented and spine head area was diminished throughout the dendritic layers of MCU cKO mice versus control mice. Fragmented mitochondria might have functional changes, such as altered ATP production, that could explain the structural and functional deficits at cKO synapses. Differences in MCU expression across cell types and circuits might be a general mechanism to tune mitochondrial function to meet distinct synaptic demands.

Collagen Hybridizing Peptide-Based Radiotracers for Molecular Imaging of Collagen Turnover in Pulmonary Fibrosis.

Pulmonary fibrosis is a characteristic feature of interstitial lung disease. Current clinical diagnostic methods provide a snapshot of the lung structure without information on disease activity. Collagen hybridizing peptides offer the opportunity to detect collagen remodeling through their hybridization with denatured collagen. Here, we sought to develop a 99mTc-labeled collagen hybridizing tracer to track denatured collagen invivo and validate it in a murine model of pulmonary fibrosis. Methods: Imaging agents consisting of a polyhistidine or a poly-histidine-glutamic acid [(HE)3] peptide connected to an N-terminal targeting moiety with 9 glycine-proline-hydroxyproline repeats [(GPO)9] through a 3-glycine linker were synthesized. After radiolabeling with 99mTc-tricarbonyl, the labeled products' purity and stability were evaluated by high-performance liquid chromatography and γ-well counting, and their biodistributions were compared in mice. To induce pulmonary fibrosis, the lungs of 8- to 10-wk-old mice were exposed to bleomycin (or saline as control). At 3 wk after induction, SPECT/CT imaging with 99mTc-(HE)3-(GPO)9 was performed 1 h after injection and was followed by tissue collection to assess 99mTc-(HE)3-(GPO)9 biodistribution by γ-well counting and to evaluate lung histology. The specificity of the tracer uptake was assessed using a scrambled homolog. A group of animals underwent serial imaging 3 and 8-10 wk after induction. Results: The specific activity of the final radiolabeled product was 70.3 ± 14.8 GBq/µmol. Radiolabeled tracers were stable in blood for at least 2 h and showed rapid blood clearance. 99mTc-(HE)3-(GPO)9 showed lower liver uptake in biodistribution studies and was selected for invivo imaging studies. SPECT/CT imaging of bleomycin-treated mice 3 wk after induction showed higher specific 99mTc-(HE)3-(GPO)9 lung uptake than that of control mice (P < 0.01) and that of bleomycin-treated mice 8-10 wk after induction, when fibrosis was resolved (P < 0.05). There was a significant correlation between lung uptake quantified by SPECT/CT and γ-well counting (Pearson R = 0.83, P < 0.001) and significant correlations between tracer uptake and indices of tissue fibrosis. Conclusion: 99mTc-(HE)3-(GPO)9 enables SPECT imaging of collagen turnover in pulmonary fibrosis. This approach expands the scope of existing diagnostic tools in fibrosis and can lead to better patient management by monitoring the effect of antifibrotic therapies.

Analysis of a mouse germ cell tumor model establishes pluripotency-associated miRNAs as conserved serum biomarkers for germ cell cancer detection

Malignant testicular germ cells tumors (TGCTs) are the most common solid cancers in young men. Current TGCT diagnostics include conventional serum protein markers, but these lack the sensitivity and specificity to serve as accurate markers across all TGCT subtypes. MicroRNAs (miRNAs) are small non-coding regulatory RNAs and informative biomarkers for several diseases. In humans, miRNAs of the miR-371-373 cluster are detectable in the serum of patients with malignant TGCTs and outperform existing serum protein markers for both initial diagnosis and subsequent disease monitoring. We previously developed a genetically engineered mouse model featuring malignant mixed TGCTs consisting of pluripotent embryonal carcinoma (EC) and differentiated teratoma that, like the corresponding human malignancies, originate in utero and are highly chemosensitive. Here, we report that miRNAs in the mouse miR-290-295 cluster, homologs of the human miR-371-373 cluster, were detectable in serum from mice with malignant TGCTs but not from tumor-free control mice or mice with benign teratomas. miR-291-293 were expressed and secreted specifically by pluripotent EC cells, and expression was lost following differentiation induced by the drug thioridazine. Notably, miR-291-293 levels were significantly higher in the serum of pregnant dams carrying tumor-bearing fetuses compared to that of control dams. These findings reveal that expression of the miR-290-295 and miR-371-373 clusters in mice and humans, respectively, is a conserved feature of malignant TGCTs, further validating the mouse model as representative of the human disease. These data also highlight the potential of serum miR-371-373 assays to improve patient outcomes through early TGCT detection, possibly even prenatally.

Targeted inhibition of menin promotes β-catenin-mediated GLP-1 expression and improves glucose tolerance in high-fat diet-induced obese mice.

Glucagon-like peptide-1 (GLP-1), derived from enteroendocrine cells, is a pivotal hormone crucial for blood glucose regulation. Menin, encoded by the MEN1 gene and known for its tumor suppressor role, is abundantly expressed in the intestine. Previous research has demonstrated that acute Men1 excision reverses preexisting glucose intolerance in high-fat diet-fed mice. However, its impact on GLP-1 expression in enteroendocrine cells has not been investigated. In the present study, both the knockdown of Men1 and the administration of the MI-463 menin inhibitor increased GLP-1 expression in glucose-stimulated STC-1 cells. Additionally, administering MI-463 to obese mice significantly elevated GLP-1 levels in both ileal epithelial cells and serum. Mechanistically, menin inhibition enhanced the nuclear accumulation of β-catenin, allowing it to bind TCF7L2, thereby increasing glucagon gene (Gcg) transcription. Furthermore, compared with control mice, mice with intestinal epithelial cell-specific Men1 knockdown exhibited significant improvements in glucose tolerance under fat challenge, which was correlated with elevated GLP-1 levels. These findings suggest that menin-mediated regulation of GLP-1 expression may be an important mechanism through which menin inhibiton alleviates type 2 diabetes.

Antimicrobial peptide AP2 ameliorates Salmonella Typhimurium infection by modulating gut microbiota

BackgroundEndogenous antimicrobial peptides and proteins are essential for shaping and maintaining a healthy gut microbiota, contributing to anti-inflammatory responses and resistance to pathogen colonization. Salmonella enterica subsp. enterica serovar Typhimurium (ST) infection is one of the most frequently reported bacterial diseases worldwide. Manipulation of the gut microbiota through exogenous antimicrobial peptides may protect against ST colonization and improve clinical outcomes.ResultsThis study demonstrated that oral administration of the antimicrobial peptide AP2 (2 µg /mouse), an optimized version of native apidaecin IB (AP IB), provided protective effects against ST infection in mice. These effects were evidenced by reduced ST-induced body weight loss and lower levels of serum inflammatory cytokines. A 16 S rRNA-based analysis of the cecal microbiota revealed that AP2 significantly modulated the gut microbiota, increasing the relative abundance of Bifidobacterium while decreasing that of Akkermansia at the genus level. Furthermore, the transplantation of fecal microbiota from AP2-treated donor mice, rather than from Control mice, significantly reduced cecal damage caused by ST and decreased the concentration of ST by one order of magnitude after infection.ConclusionsThese findings reveal a novel mechanism by which exogenous antimicrobial peptides mitigate Salmonella Typhimurium infection through the modulation of gut microbiota.

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.

Dysbiotic oral microbiota-derived kynurenine, induced by chronic restraint stress, promotes head and neck squamous cell carcinoma by enhancing CD8+ T cell exhaustion

BackgroundChronic restraint stress (CRS) is a tumour-promoting factor. However, the underlying mechanism is unknown.ObjectiveWe aimed to investigate whether CRS promotes head and neck squamous cell carcinoma (HNSCC) by altering the...

KLF12 Aggravates Angiotensin II-Induced Cardiac Remodeling in Male Mice by Transcriptionally Inhibiting SMAD7.

Adverse left ventricular remodeling and subsequent heart failure remain a major cause of patient morbidity and mortality worldwide. The KLF family of transcription factors plays crucial roles in heart injury. KLF12 (Krüppel-like factor 12) is a transcription factor that regulates multiple disease processes, although the specific role of KLF12 in cardiac remodeling remains unclear. In our study, we observed a significant upregulation of KLF12 expression in remodeling hearts. The increased expression of KLF12 primarily originated from cardiac fibroblasts during the fibrotic response induced by angiotensin II. To investigate the effects of KLF12, we performed RNA-seq and found that KLF12 overexpression significantly upregulated the cardiac remodeling associated pathway. Hence, we generated adult mice with cardiac fibroblast-specific overexpression of KLF12 using lentivirus or miRNA (miR-1/133TS) technology. Compared with control mice, KLF12-miR1/133TS transfected mice exhibited exacerbated cardiac remodeling and function. Mechanistically, we discovered that KLF12 directly binds to the promoter of Smad7, leading to the activation of the TGF-β (transforming growth factor beta)-Smad3 pathway. In conclusion, KLF12 promoted the development of angiotensin II-induced cardiac remodeling in male mice. Targeting KLF12 may be a promising therapeutic approach to treat cardiac remodeling.

Blood exosome connexins and small RNAs related to demyelinating disease activity.

To assess blood exosome (Ex)-connexin (Cx)43 (encoded by GJA1) and its truncated isoforms in multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD), which show distinct alterations in astroglial Cx43. Serum Exs from 48 patients with MS (34 relapsing-remitting, 14 secondary-progressive), 35 with NMOSD, 20 with other inflammatory neurologic diseases (OIND), and 17 healthy controls (HC) were subjected to quantitative Western blotting for Cx43, single-molecule array for neurofilament-L, and quantitative polymerase chain reaction for non-coding RNAs detected by RNA sequencing. Sera from control and astroglia-specific Cx43 inducible conditional knockout (Cx43-icKO) mice with experimental autoimmune encephalomyelitis (EAE) were also tested. Ex-GJA1-29k was markedly higher in MS than in NMOSD, OIND, and HC; it successively increased at relapse, remission, and secondary progression, and positively correlated with disability scores. Ex-hsa-miR-133b and other hsa-miRs that bind to full-length Cx43 were significantly lower in secondary-progressive MS than in HC, and Ex-hsa-miR-133b was negatively correlated with disability scores. Ex-GJA1-11k expression was lower in NMOSD at relapse than in HC and OIND, and was negatively correlated with disability score worsening and Ex-neurofilament-L levels. NMOSD at relapse had significantly higher expression of small nucleolar RNA (SNORD37, SNORD95, and SNORD97) than HC, and SNORD37 and SNORD95 showed strong negative correlations with disability scores. Control mice showed increased Ex-GJA1-43k and -29k during EAE; this effect was markedly reduced in Cx43-icKO mice with attenuated EAE. Blood Ex-Cx43-truncated isoforms and small non-coding RNAs, which partially come from brain astroglia, are distinctly dysregulated in MS and NMSOD.

Discovering the anti-parasitic activity of melatonin loaded lecithin/chitosan nanoparticles against giardiasis and cryptosporidiosis in Balb/c infected mice

BackgroundGiardia duodenalis and Cryptosporidium parvum are the primary causes of diarrhea with global attention due to the severe pathophysiological changes leading to mortality. During this study, we explored the biological protozoal contaminants (Giardia and Cryptosporidium spp.) in some areas of the Nile River. Then, we evaluated effectiveness of melatonin (Mel) and melatonin loaded lecithin/chitosan nanoparticles (Mel-LCNPs) against giardiasis and cryptosporidiosis in mice models using parasitological and inflammatory response examination.ResultsThe number of positive samples for Cryptosporidium was higher than that for Giardia with percentage of 46.67% and 40.0%, respectively. Prior to treatment, the physical characterization (hydrodynamic size and zeta potential) and in vitro characterization of Mel-LCNPs were carried. Mel-LCNPs revealed a hydrodynamic size of 78.8 nm and a zeta potential of − 27.2 mV. Furthermore, they have powerful antioxidant and anti-inflammatory properties, while displaying minimal anticoagulant and cytotoxic effects during in vivo evaluation. Mel was consistently discharged from nanoparticles in a regulated and enduring manner for 36 h. Moreover, Mel in NPs has an entrapment efficiency (EE) of 33.6% and a drug loading capacity (DLC) of 7.2% and significant reduction (100% and 99.4%, respectively) in the shedding of Giardia cysts and Cryptosporidium oocysts. This reduction was higher than that observed with Mel alone or LCNPs alone on the 14th day post-infection. Moreover, mice of group V, which received Mel-LCNP treatment, exhibited significantly normal levels of interleukin-4 (IL-4) and interferon-gamma (IFN-γ) as well as healthy control mice group (group I).ConclusionMel-LCNPs were highly effective preparations against giardiasis and cryptosporidiosis in Balb/c mice experimentally infected with proved antioxidant, anti-inflammatory, and immunological modulatory characteristics.

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.

Female mice exhibit similar long-term plasticity and microglial properties between the dorsal and ventral hippocampal poles

The hippocampus is a heterogenous structure that exhibits functional segregation along its longitudinal axis. We recently showed that in male mice, microglia, the brain’s resident immune cells, differ between the dorsal (DH) and ventral (VH) hippocampus, impacting long-term potentiation (LTP) mainly through the CX3CL1-CX3CR1 signaling. Here, we assessed the specific features of the hippocampal poles in female mice, demonstrating a similar LTP amplitude in VH and DH in both control and Cx3cr1 knock-out mice. In addition, the expression levels of Cx3cr1 and Cx3cl1 mRNA do not differ between the two poles in control mice. These data support the critical role of the CX3CL1-CX3CR1 signaling in setting the physiological amount of plasticity, equally between poles in females. Although BDNF is higher in DH compared to VH, the expression levels of inflammatory markers involved in plasticity and of phagocytosis markers in microglia are comparable between the two poles. In accordance, microglia soma and arborization area/perimeter, and microglial ultrastructure are similar across regions, with the exception of microglial density, cells arborization solidity and circularity that are higher in DH. Understanding the molecular processes underlying microglial sex differences and their potential implications for plasticity in specific brain regions is of major importance in physiological and pathological conditions.

Multiomics Analysis Reveals Therapeutic Targets for Chronic Kidney Disease With Sarcopenia.

The presence of sarcopenia in patients with chronic kidney disease (CKD) is associated with poor prognosis. The mechanism underlying CKD-induced muscle wasting has not yet been fully explored. This study investigates the influence of renal secretions on muscles using multiomics sequencing. The kidney transcriptome analysis by RNA-seq and protein profiling by tandem mass tag (TMT), serum TMT and muscle TMT were performed in CKD established using 0.2% adenine and control mice. Spp1 recombinant protein was used to study its effect on myotube atrophy invitro. In animal experiments on CKD, pharmacological inhibition of Spp1 was used to explore the role of Spp1 in skeletal muscle wasting. Transcriptome analysis was performed to identify differentially expressed genes (DEGs) in the gastrocnemius muscle following Spp1 pharmacological inhibition. In the renal transcriptome and TMT, 503 and 377 proteins/genes respectively were co-upregulated and co-downregulated. In the serum TMT of CKD and normal control (NC) mice, 22 upregulated and 7 downregulated differentially expressed proteins (DEPs) showed the same expression patterns as those in the kidney transcriptome and TMT analysis. Based on bioinformatics analysis and reported studies, we selected Spp1 for further validation. Spp1 recombinant protein was added to C2C12 myotubes invitro, and the results indicated that Spp1 significantly increased the protein levels of the muscle atrophy marker (Murf-1) and promoted the smaller myotubes (all p < 0.05). Compared with NC mice, Spp1 mRNA and protein levels were significantly upregulated in the kidneys of CKD mice, and the serum concentration of Spp1 was also markedly increased (all p < 0.05). In animal experiments, pharmacological inhibition of Spp1 increased the weights of gastrocnemius and tibialis anterior muscles (p < 0.05) and improved muscle atrophy phenotype. Transcriptome analysis showed that DEGs in the gastrocnemius muscle following Spp1 pharmacological inhibition were enriched in protein digestion and absorption, glucagon signalling pathway, apelin signalling pathway and ECM-receptor interaction pathway. Our study is the first to establish a regulatory network of kidney-muscle crosstalk to explore the potential mechanism of CKD-related sarcopenia. Employing multiomics analysis, cellular assessment and animal experiments, we have identified that Spp1 could potentialy serve as a promising therapeutic target for CKD patients with sarcopenia.

The effect of modulation Piezo2 by IGF-1 on tactile hypersensitivity in BTBR model mice.

Autism spectrum disorder (ASD) is classified as a neurodevelopmental disorder. Individuals with ASD exhibit a higher incidence of tactile hypersensitivity. However, the underlying mechanisms remain unclear. The dorsal root ganglion (DRG) plays a crucial role in influencing tactile processing. This study aims to integrate RNA sequencing (RNA-seq) and molecular biology experiments to identify key molecules involved in tactile hypersensitivity in ASD, further investigate related mechanisms, and develop effective intervention strategy. Using BTBR as the ASD model mouse and wild-type C57BL/6J as the control mouse, the differences in tactile sensitivity between them was compared. DRG were collected for RNA-seq analysis. Immunofluorescence and Enzyme-linked immunosorbent assay (ELISA) techniques were employed to validate the identified key molecules. And combined western blot to investigate the associated regulatory pathways. BTBR mice exhibit tactile hypersensitivity, which are associated with the upregulation of IGF-1 in the DRG. IGF-1 regulates the expression of Piezo2 ion channels. Inhibition of the IGF-1/Piezo2 pathway can significantly alleviate tactile hypersensitivity and social deficits in BTBR mice. Additionally, gentle touch intervention has been shown to reduce the overexpression of IGF-1/Piezo2 in the DRG, thereby ameliorating ASD symptoms. The upregulation of the IGF-1/Piezo2 pathway in DRG may serve as a potential mechanism for tactile hypersensitivity observed in BTBR mice. Restoring the normalization of the IGF-1/Piezo2 is crucial for alleviating tactile hypersensitivity and synergistically rescues social deficits. Gentle touch intervention has the potential to ameliorate these behaviors through regulating IGF-1/Piezo2, positioning it as a promising strategy for ASD.

Abstract 95: LILRB4 Inhibition Alleviates Ischemic Brain Injury by Reducing Microglial Necroptosis

Objective: Microglia, the central nervous system's immune cells, are crucial in neuroinflammation after ischemic brain injury. Previous single-cell and spatial transcriptomics showed that LILRB4 was upregulated in microglia after MCAO in C57BL/B6J mice, but its role is unclear. This study investigates LILRB4's function and mechanism in ischemic injury. Methods: In C57BL/B6J mice, LILRB4 expression was analyzed at various time points post-MCAO using Q-PCR, Western blot, and flow cytometry. A microglia-specific LILRB4 knockout mouse model (LILRB4-cKO) and control mice (LILRB4 fl/fl ) were established, and MCAO was induced. Infarct volume and behavioral changes were assessed by MAP2 staining, mNSS scoring, grip strength, gait, and rotarod tests. qPCR and Western blot were used to detect brain inflammatory factors and DAMPs, while microglial morphology, cell counts, and RIPK3 levels were analyzed by immunofluorescence and flow cytometry. In vitro, LILRB4 expression changes post-OGD were validated, and LILRB4 knockdown BV2 cells (sh-LILRB4 BV2) were constructed. Necroptosis was induced using RIPK3 inhibitor and OGD, and necroptosis markers were detected. The JASPAR database was used to identify ATF2 as a potential RIPK3 transcription factor, and its role in gene regulation was validated by transfecting ATF2 plasmid into sh-LILRB4 BV2 cells. Results: LILRB4 expression in microglia was upregulated after MCAO, peaking at day 3. LILRB4-cKO mice showed larger infarct volumes, greater neurological deficits, and higher pro-inflammatory cytokines (TNF, IL-6, IL-1β) than LILRB4 fl/fl mice. Microglia from LILRB4-cKO mice had larger cell bodies, shorter processes, reduced numbers, and more RIPK3 + necroptotic cells. In primary microglia and sh-LILRB4 BV2 cells, OGD increased LILRB4, RIPK3, MLKL, and cytokines, which were suppressed by GSK-872. The JASPAR database identified ATF-2 as a potential RIPK3 transcription factor, and its role in promoting RIPK3 transcription was confirmed. The JASPAR database identified ATF-2 as a potential RIPK3 transcription factor, and its role in promoting RIPK3 transcription was confirmed. Knockdown of ATF-2 reduced the elevated levels of inflammatory cytokines, DAMPs, and necroptosis markers induced by LILRB4 deletion. Conclusion: LILRB4 inhibits microglial necroptosis through the ATF2/RIPK3 pathway, reducing neuroinflammation and offering neuroprotection. Therefore, LILRB4 may serve as a potential therapeutic target for ischemic brain injury