Search Results (1,174)

Sprouty-related proteins with enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain (SPREDs) are negative regulators of the Ras/MAPK signaling pathway and are known to modulate developmental and endocrine processes. While the roles of SPRED1 and SPRED2 are increasingly understood, the physiological relevance of SPRED3 remains elusive. To elucidate its function, we generated SPRED3 knockout (KO) mice and performed phenotypic, molecular, and hormonal analyses. SPRED3-deficient mice exhibited growth retardation and a non-Mendelian genotype distribution. X-Gal staining revealed Spred3 promoter activity in the thyroid, adrenal gland, pituitary, cerebral cortex, and kidney. Hormonal profiling identified elevated thyroid-stimulating hormone (TSH) and reduced thyroxine (T₄) levels, indicating primary hypothyroidism. Thyroidal extracellular signal-regulated kinase (ERK) signaling was mildly reduced in SPRED3 KO mice, and immunoblotting revealed altered expression of autophagy regulators, including reduced sequestosome 1 (p62), increased autophagy-related gene 5 (ATG5), as well as an elevated microtubule-associated protein 1 light chain 3 (LC3) II/I ratio and a decreased pBeclin/Beclin ratio in SPRED3 KO mice. Our findings indicate that SPRED3 is involved in thyroidal homeostasis and plays a regulatory role in autophagy processes within the thyroid gland. Full article

Background: Pulmonary fibrosis (PF), the end-stage manifestation of interstitial lung disease, is defined by excessive extracellular matrix deposition and alveolar destruction. Activated fibroblasts, the primary matrix producers, rely heavily on dysregulated glucose metabolism for their activation. While Salt Inducible Kinase 2 (SIK2) regulates glycolytic pathways in oncogenesis, its specific contributions to fibroblast activation and therapeutic potential in PF pathogenesis remain undefined. This study elucidates the functional role of SIK2 in PF and assesses its viability as a therapeutic target. Methods: SIK2 expression/localization in fibrosis was assessed by Western blot and immunofluorescence. Fibroblast-specific Sik2 KO mice evaluated effects on bleomycin-induced fibrosis. SIK2’s role in fibroblast activation and glucose metabolism impact (enzyme expression, metabolism assays, metabolites) were tested. SIK2 inhibitors were screened and evaluated therapeutically in fibrosis models. Results: It demonstrated significant SIK2 upregulation, specifically within activated fibroblasts of fibrotic lungs from both PF patients and murine models. Functional assays demonstrated that SIK2 is crucial for fibroblast activation, proliferation, and migration. Mechanistically, SIK2 enhances fibroblast glucose metabolism by increasing the expression of glycolysis-related enzymes. Additionally, this study demonstrated that the SIK2 inhibitor YKL06-061 effectively inhibited PF in both bleomycin and FITC-induced PF mouse models with the preliminary safety profile. Furthermore, we identified a novel therapeutic application for the clinically approved drug fostamatinib, demonstrating it inhibits fibroblast activation via SIK2 targeting and alleviates PF in mice. Conclusions: Our findings highlight SIK2 as a promising therapeutic target and provide compelling preclinical evidence for two distinct anti-fibrotic strategies with significant potential for future PF treatment. Full article

Neuroinflammation is the major contributor to the pathology of neonatal hypoxic–ischemic (HI) brain injury. Our previous studies have demonstrated that microRNA210 (miR210) inhibition with antisense locked nucleic acid (LNA) inhibitor mitigates neuroinflammation and provides neuroprotection after neonatal HI insult. However, the underlying mechanisms remain elusive. In the present study, using miR210 knockout (KO) mice and microglial cultures, we tested the hypothesis that miR210 promotes microglial activation and neuroinflammation through suppressing mitochondrial function in microglia after HI. Neonatal HI brain injury was conducted on postnatal day 9 (P9) wild-type (WT) and miR210 knockout (KO) mouse pups. We found that miR210 KO significantly reduced brain infarct size at 48 h and improved long-term locomotor functions assessed by an open field test three weeks after HI. Moreover, miR210 KO mice exhibited reduced IL1β levels, microglia activation and immune cell infiltration after HI. In addition, in vitro studies of microglia exposed to oxygen–glucose deprivation (OGD) revealed that miR210 inhibition with LNA reduced OGD-induced expression of Il1b and rescued OGD-mediated downregulation of mitochondrial iron–sulfur cluster assembly enzyme (ISCU) and mitochondrial oxidative phosphorylation activity. To validate the link between miR210 and microglia activation, isolated primary murine microglia were transfected with miR210 mimic or negative control. The results showed that miR210 mimic downregulated the expression of mitochondrial ISCU protein abundance and induced the expression of proinflammatory cytokines similar to the effect observed with ISCU silencing RNA. In summary, our results suggest that miR210 is a key regulator of microglial proinflammatory activation through reprogramming mitochondrial function in neonatal HI brain injury. Full article

The kallikrein–kinin system and its B1 and B2 receptors are key regulators in metabolic disorders such as obesity, diabetes, and insulin resistance. Obesity, a chronic and multifactorial condition often associated with comorbidities like type 2 diabetes and dyslipidemia, remains poorly understood at the metabolic level. The kinin B2 receptor (B2R) is involved in blood pressure regulation and glucose metabolism, promoting glucose uptake in skeletal muscle via bradykinin. Studies in B2R-KO mice demonstrate that the absence of this receptor predisposes animals to glucose intolerance under a high-fat diet and impairs adaptive thermogenesis, indicating a protective role for B2R in metabolic homeostasis and insulin sensitivity. In contrast, the kinin B1 receptor (B1R) is inducible under pathological conditions and is activated by kinin metabolites. Mouse models lacking B1R exhibit improved metabolic profiles, including protection against high-fat diet-induced obesity and insulin resistance, enhanced energy expenditure, and increased leptin sensitivity. B1R inactivation in adipocytes enhances insulin responsiveness and glucose tolerance, supporting its role in the development of insulin resistance. Moreover, B1R deficiency improves energy metabolism and thermogenic responses to adrenergic and cold stimuli, promoting the activation of brown adipose tissue and the browning of white adipose tissue. Collectively, these findings suggest that B1R and B2R represent promising therapeutic targets for the treatment of metabolic disorders. Full article

Background/Objectives: The receptor for advanced glycation end products (RAGEs) has been implicated in obesity and metabolic dysfunction. However, its precise role in diet-induced obesity remains unclear. Methods: In this study, we investigated the metabolic consequences of RAGE knockout (RAGE KO) in mice subjected to a Western diet (WD). Results: Our findings demonstrate that RAGE KO mice remained significantly leaner than their wild-type (WT) counterparts when fed a WD, exhibiting reduced body weight gain and smaller adipocyte size. Indirect calorimetry revealed that RAGE KO mice had increased oxygen consumption and locomotor activity compared to WT mice, indicating enhanced energy expenditure. Mitochondrial respiration assays indicated significantly greater oxygen consumption in RAGE KO animals. Additionally, systemic inflammation markers, such as TNF-α, were significantly lower in RAGE KO mice when fed a WD, indicating a reduction in diet-induced inflammatory responses. Conclusions: These findings suggest that RAGE plays a key role in metabolic homeostasis, and its deletion confers resistance to obesity and metabolic disruption induced by a Western diet. Targeting RAGE may provide a novel therapeutic approach for combating obesity and related metabolic disorders. Full article

Background/Objectives: Ulcerative colitis (UC) is characterized by the interplay between immune responses and dysbiosis in disease development. Aiming to provide additional insights into disease development and potential treatment strategies, the present study investigates the local effect of oral treatment with polysaccharides obtained from Auricularia auricula’s submerged culture in an experimental model of DSS-induced colitis and its impact on lesion resolution. Methods: The structure and monosaccharide composition of Auricularia polysaccharides were characterized through Nuclear Magnetic Resonance (NMR). To evaluate the effect of this polysaccharide on the murine model, wild-type and Dectin-1 knockout mice were treated or not with the exopolysaccharide (EPS) while under DSS consumption. During the experimental period, feces samples were collected to evaluate microbial shifts during disease development, and, finally, the colonic tissue was analyzed to assess the inflammatory process and cytokine production. Results: The EPS composition showed a polymeric mixture of glucans and fucogalactomannans. The treatment of the wild-type DSS-induced colitis group improved the inflammatory response by increasing gut–homeostatic cytokines, such as interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α). The Dectin-1 KO mice group did not show the same enhancement after EPS treatment. The microbiome analysis revealed a difference in the genotype, and the treatment modified the DSS microbiome modulation, with nine and four ASVs in WT and Dectin-1 KO mice, respectively. Conclusions: The EPS treatment demonstrated therapeutic potential in treating inflammatory intestinal diseases by modulating cytokine secretion and microbiota composition, which is dependent on the Dectin-1 receptor’s carbohydrate recognition. Full article

ATP13A2 is a lysosomal polyamine transporter with loss of function mutations linked to multiple neurodegenerative disorders including Parkinson’s disease (PD). Knockout of ATP13A2 in mice leads to age-related sensorimotor impairments and in the brain lipofuscinosis, gliosis, and modest alpha-synuclein (αSyn) pathology. However, few studies have included ATP13A2 heterozygous mice as a comparison. In the present study, the effect of reduced or complete loss of ATP13A2 function on behavior, αSyn, gliosis, dopamine, and polyamines were determined in mice. Male and female ATP13A2 wildtype (WT), heterozygous (Het), and knockout (KO) mice were assessed behaviorally at 3, 12, and 18 months of age. In the brain, αSyn, phosphorylated αSyn, and GFAP were measured in the prefrontal cortex, striatum, ventral midbrain, and cerebellum. Polyamine and neurotransmitter analyses were performed in the same brain regions. Similar to previous studies, KO mice developed motor impairments and widespread gliosis in the brain. In addition, polyamine content was altered in Het and KO mice. In contrast, Het mice showed impairments in cognitive function and an age-related increase in αSyn in the brain. These results indicate potentially different pathological mechanisms when ATP13A2 is reduced compared to when it is knocked out and may have important implications for disease modification in synucleinopathies including PD. Full article

The Carbohydrate-Responsive Element-Binding Protein (ChREBP) is a glucose-sensitive transcription factor that regulates the carbohydrate and lipid metabolism. We investigated its cell-type-specific role in hepatocarcinogenesis using a chemically induced mouse model. Additionally, we examined the functions of its isoforms, ChREBPα and ChREBPβ. After the diethylnitrosamine (DEN) administration, we analyzed hepatocellular adenomas and carcinomas in systemic ChREBP-knockout (KO), hepatocyte-specific ChREBP-KO (L-KO), and wildtype (WT) mice at 4, 12, and 36 weeks using histology, morphometry, proliferation measurements, immunohistochemistry, a Western blot, and a quantitative PCR. Tumors developed 36 weeks after the DEN administration in 27% of WT mice but less frequently in KO (18%) and L-KO (9%) mice. However, preneoplastic foci were less common in KO mice but not in L-KO mice (39% vs. 9%; p < 0.05). L-KO hepatocytes exhibited lower proliferation, while KO tumors showed the downregulation of AKT/mTOR signaling, glycolysis, and lipogenesis compared to WT tumors. Our results showed that the liver-specific loss of ChREBPα, while ChREBPβ remained active, significantly reduced the tumor progression, suggesting an oncogenic role for ChREBPα. In contrast, the systemic knockout of both ChREBPα and ChREBPβ reduced the tumor initiation but did slightly prevent tumor progression, indicating that ChREBPβ may exert tumor-suppressive functions. Full article

Despite the abundant expression of the microRNA-degrading Translin (TN)/Trax (TX) complex in midbrain dopaminergic (DA) neurons and its implication in neuropsychiatric disorders, its cell-autonomous roles in metabolic and behavioral responses remain unclear. To address this, we generated DA neuron-specific conditional knockout (cKO) mice for Tsn (TN) or Tsnax (TX) using DAT-Cre. Immunostaining confirmed efficient TX loss in Tsnax cKO DA neurons without affecting TN, while Tsn deletion abolished TX expression, revealing asymmetric protein dependency. Body composition analysis showed no alterations in adiposity in either cKO model. Locomotor responses to acute or repeated administration of cocaine (20 mg/kg) or amphetamine (2.5 mg/kg) were unchanged in Tsn or Tsnax cKO mice. Furthermore, amphetamine-induced conditioned place preference (1 mg/kg) was unaffected. These results demonstrate that the TN/TX complex within DA neurons is dispensable for regulating adiposity, psychostimulant-induced locomotion (both acute and sensitized), or amphetamine reward-related behavior, suggesting its critical functions may lie outside these specific domains. Full article

The circadian clock is a self-sustaining oscillator with a period of approximately 24 h, enabling organisms to anticipate daily recurring events, such as sunrise and sunset. Since the circadian period is not exactly 24 h and the environmental day length varies throughout the year, the clock must be periodically reset to align an organism’s physiology with the natural light/dark cycle. This synchronization, known as entrainment, is primarily regulated by nocturnal light, which can be replicated in laboratory settings using a 15 min light pulse (LP) and by assessing locomotor activity. An LP during the early part of the dark phase delays the onset of locomotor activity, resulting in a phase delay, whereas an LP in the late dark phase advances activity onset, causing a phase advance. The clock gene Period 2 (Per2) plays a key role in this process. To investigate its contributions, we examined the effects of Per2 deletion in neurons versus astrocytes using glia-specific GPer2 (Per2/GfapCre) knockout (KO) and neuronal-specific NPer2KO (Per2/NesCre) mice. All groups were subjected to Aschoff type II protocol, where an LP was applied at ZT14 or ZT22 and the animals were released into constant darkness. As control, no LP was applied. Phase shift, period, amplitude, total activity count, and rhythm instability were assessed. Our findings revealed that mice lacking Per2 in neurons (NPer2) exhibited smaller phase delays and larger phase advances compared to control animals. In contrast, mice with Per2 deletion specifically in glial cells including astrocytes (GPer2) displayed normal clock resetting. Interestingly, the absence of Per2 in either of the cell types resulted in a shorter circadian period compared to control animals. These results suggest that astrocytic Per2 is important for maintaining the circadian period but is not required for phase adaptation to light stimuli. Full article

In placental mammals, the co-option of vertebrate orthologous ATP1B4 genes has profoundly altered the properties of the encoded BetaM proteins, which function as bona fide β-subunits of Na,K-ATPases in lower vertebrates. Eutherian BetaM acquired an extended Glu-rich N-terminal domain resulting in the complete loss of its ancestral function and became a skeletal and cardiac muscle-specific component of the inner nuclear membrane. BetaM is expressed at the highest level during perinatal development and is implicated in gene regulation. Here we report the long-term consequences of Atp1b4 ablation on metabolic parameters in adult mice. Male BetaM-deficient (Atp1b4−/Y) mice have remarkably lower body weight and adiposity than their wild-type littermates, despite higher food intake. Indirect calorimetry shows higher energy expenditure (heat production and oxygen consumption) with a greater spontaneous locomotor activity in Atp1b4−/Y males. Their lower respiratory exchange ratio suggests a greater reliance on fat metabolism compared to their wild-type counterparts. Consistently, Atp1b4−/Y KO mice exhibit enhanced β-oxidation in skeletal muscle, along with improved glucose and insulin tolerance. These robust metabolic changes induced by Atp1b4 disruption demonstrate that eutherian BetaM plays an important role in regulating adult mouse metabolism. This demonstrates that bypassing the co-option of Atp1b4 potentially reduces susceptibility to obesity. Thus, Atp1b4 ablation leading to the loss of evolutionarily acquired BetaM functions serves as a model for a potential alternative pathway in mammalian evolution. Full article

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted social communication and repetitive behaviors. Prenatal stress is critical in neurodevelopment and increases risk for ASD, particularly in those with greater genetic susceptibility to stress. Docosahexaenoic acid (DHA) is one of the most abundant ω-3 fatty acids in the membrane phospholipids of the mammalian brain, and dietary DHA plays an important role in brain development and maintenance of brain structure. In this study, we investigated whether peri-natal supplementation of DHA can alleviate autistic-like behaviors in a genetic risk/stress mouse model and how it alters lipid peroxidation activity and GABAergic system gene expression in the forebrain. Pregnant heterozygous serotonin transporter knockout (SERT-KO) and wild-type (WT) dams were placed in either non-stressed control conditions or chronic variable stress (CVS) conditions and fed either a control diet or a DHA-rich (1% by weight) diet. Offspring of each group were assessed for anxiety and autism-associated behavior at post-natal day 60 using an open field test, elevated plus maze test, repetitive behavior, and the 3-chamber social approach test. A liquid chromatography-mass spectrometry (LC-MS)-based method was used to follow changes in levels of lipid peroxidation products in the cerebral cortex. Male offspring of prenatally stressed SERT-het KO dams exhibited decreased social preference behaviors and increased repetitive grooming behaviors compared to WT control offspring. Moreover, DHA supplementation in male SERT-het mice decreased frequency of grooming behaviors albeit showing no associated effects on social behaviors. Regardless of stress conditions, supplementation of DHA to the WT mice did not result in alterations in grooming nor social interaction in the offspring. Furthermore, no apparent changes were observed in the lipid peroxidation products comparing the stressed and non-stressed brains. Gad2 was downregulated in the cortex of female offspring of prenatally stressed SERT-KO dams, and this change appeared to be rescued by DHA supplementation in offspring. Gad2 was upregulated in the striatum of male offspring of prenatally stressed SERT-KO dams, but DHA did not significantly alter the expression compared to the control diet condition. Full article

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset, intractable epilepsy, motor and cognitive impairment, and autistic-like features. Although constitutive Cdkl5 knockout (KO) models have established the importance of CDKL5 during early brain development, CDKL5’s role in the mature brain remains poorly defined. Here, we employed an inducible, conditional KO model in which Cdkl5 is selectively deleted from forebrain glutamatergic neurons in adult mice to investigate the postdevelopmental functions of CDKL5. Using a total of 48 adult male mice, including Cdkl5^flox/Y(Cre⁺) (n = 30) and Cdkl5^flox/Y(Cre⁻) littermate controls (n = 18), we found that tamoxifen-induced Cdkl5 deletion led to prominent behavioral impairments, including deficits in motor coordination, reduced sociability, and impaired hippocampus-dependent spatial memory, while behavioral features such as hyperactivity and stereotypic jumping, typically present in germline KOs, were absent. Sensory functions, including olfaction and pain perception, were also preserved. At the cellular level, the loss of Cdkl5 resulted in a marked reduction in excitatory synapse density in the cortex and hippocampus, accompanied by increased numbers of immature dendritic spines and decreased mature spines. Neuronal loss in the hippocampal CA1 region and selective microglial activation in the cortex were also observed. These alterations closely resemble those seen in constitutive KO models, underscoring the ongoing requirement for CDKL5 expression in excitatory neurons for maintaining synaptic integrity and neuronal homeostasis in the adult brain. This study underscores the importance of temporally controlled models for investigating the mechanisms underlying CDD pathophysiology in the adult brain. Full article

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by bile duct inflammation and fibrosis, leading to cirrhosis and liver failure. Current therapies are limited to symptom management, with no approved treatments targeting fibrosis. We have identified the MAP kinase-activated protein kinase 2 (MK2) pathway as a potential therapeutic target for treating PSC due to its role in promoting inflammatory cytokine production and activation of fibroblasts. Thus, MDR2 knockout mice were treated therapeutically with MK2 inhibitors, which led to significantly reduced hepatic inflammation and fibrosis. Liver enzymes, collagen 1A1, and fibronectin were decreased in serum with MK2 inhibitor treatment. Furthermore, the production of IL-6, TNFα, CXCL5, collagen 1A1, and fibronectin was decreased in liver tissues and liver stellate cells, whereas the production of IL-10, G-CSF, and IL-22 was increased. MDR2KO mice treated with IL-22 also showed improvements in inflammation and fibrosis, along with increased IL-10 and G-CSF production. Taken together, we identified both a direct mechanism of MK2 regulation of fibrotic factors and an indirect cytokine-mediated mechanism whereby the levels of IL-22, IL-10, and G-CSF were increased with MK2 inhibition and contributed to decreased levels of fibrotic factors. These data suggest that the MK2 pathway is a promising treatment target for PSC. Full article

We previously showed that selective suppression of CD44 in the corneal epithelium leads to structural abnormalities in the mouse cornea. Our comparative studies of young and aged ocular biopsies revealed that CD44 expression is downregulated in aged corneas, while leucine-rich repeats and immunoglobulin-like domain 1 (Lrig1+) stem cells remain preserved in the peripheral limbus. These findings suggest an age-related shift in the corneal stem cell compartmentalization, characterized by impaired CD44 expression in the central cornea and preservation of Lrig1+ stem cells in the limbus, which become the main stem cells in the senescent cornea. To investigate this further, we performed topical tamoxifen-inducible, diphtheria toxin-mediated ablation of Lrig1+ stem cells in mouse corneas. We then assessed both activated and non-activated beta-catenin expression in wild-type (WT) and CD44 knockout (CD44KO) mice, given that CD44 modulates the Wingless-related integration site (Wnt) pathway. Our results indicate that two distinct stem cell populations operate in the mouse cornea: Lrig1-derived stem cells and Wnt-activity/CD44-dependent stem cells. The Lrig1-derived cells act as a reservoir of quiescent stem cells that regenerate the cornea upon injury, whereas under homeostatic conditions, the Wnt-activity/CD44-dependent stem cells are primarily responsible for corneal renewal. In the aged cornea, the loss of CD44 expression leads to reduced Wnt signaling, making the tissue increasingly dependent on Lrig1+ stem cells for regeneration. In mice, Lrig1+ stem cells are capable of sustaining permanent corneal renewal, even in the absence of CD44. Full article