Atherosclerosis (AS) is a chronic inflammatory disorder underlying most cardiovascular events. Sialic acid (SIA), a terminal metabolite of glycolipid catabolism, modulates vascular injury, but its role in endothelial dysfunction remains unclear. To investigate whether N-acetylneuraminic acid (Neu5Ac) accelerates AS development. ApoE⁻/⁻ mice were fed a high-fat diet to induce AS. Lesion burden was assessed by Oil Red O staining, plaque morphology by H&E staining, reactive oxygen species and macrophage polarization by flow cytometry, and signaling alterations by Western blotting. Neu5Ac markedly amplified systemic inflammation, enhanced atherosclerotic plaque formation, and disrupted lipid homeostasis. Neu5Ac exacerbates AS through pro-inflammatory, pro-lipid, and chemotactic/angiogenic mechanisms, highlighting potential therapeutic targets.

Xanthinuria type II is a rare hereditary disorder caused by mutations in the MOCOS gene, leading to dual deficiency of xanthine dehydrogenase and aldehyde oxidase. To establish a robust animal model for this condition, we generated Mocos knock-in (KI) rats carrying the Arg419Ter nonsense mutation identified in Japanese patients. Homozygous KI rats exhibited severe growth retardation, anemia, and reduced survival, with all individuals dying by 14 weeks of age. Biochemical analyses revealed elevated levels of hypoxanthine and xanthine, along with decreased uric acid in both serum and urine, confirming xanthinuria. Homozygous KI rats also showed increased blood creatinine (CRE) and urea nitrogen (UN), and decreased urinary CRE and UN, indicating renal dysfunction. Histopathological examination showed obstructive nephropathy characterized by tubular atrophy, crystal deposition, and inflammation. Compared to existing mouse models, Mocos KI rats demonstrated extended lifespan, enabling more detailed investigation of disease mechanisms. This rat model provides a valuable tool for studying the pathogenesis of xanthinuria type II and exploring potential therapeutic strategies.

Macrophages can develop into pro-inflammatory M1-like macrophages and anti-inflammatory M2-like macrophages when stimulated by distinct internal environment. Dynamic changes of the two kinds of macrophages play key roles in atherosclerosis progression. The study aims to explore the role of RNF10 in regulating macrophage polarization during atherosclerosis. Mice with macrophage-specific depletion of RNF10 (RNF10/ApoE) and control mice (RNF10/ ApoE) mice were fed with high-fat diet to generate atherosclerotic lesion, from which peritoneal macrophages were isolated and transfected with RNF10-overexpressing vector. Murine macrophages, RAW264.7, were transfected with RNF10-overexpressing vector or RNF10 siRNA and stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The RNF10/ApoE mice showed greater atherosclerotic lesions, more resident macrophages, higher expression of iNOS (M1-like macrophage marker), and lower expression of Arginase-1 (M2-like macrophage marker) than the RNF10/ApoE mice. RNF10 overexpression could reduce expressions of IL-1β, IL-6, and iNOS (M1 marker genes), increase expressions of IL-10 and Arg-1 (M2 marker genes) in the peritoneal macrophages isolated from RNF10/ApoE mice. RNF10 overexpression reduced lipid accumulation in ox-LDL-induced foam cells, whereas RNF10 silencing yielded opposite results. Our data suggest that RNF10 is associated with M1-like macrophage suppression and M2-like increase, indicating RNF10 in macrophages has an anti-atherosclerotic role.

Calcium/calmodulin-dependent protein kinase II (CAMKII) is a critical regulator of cardiac electrophysiology. However, the role of the four bases deletion polymorphism in Camk2d which codes delta subunit of CAMKII, particularly those involving intron sequences, remains poorly understood. This study aimed to investigate the impact of Camk2d c.1044+125_128delGTTT missing polymorphism on cardiac morphology and arrhythmogenesis in normal adult Sprague-Dawley (SD) rats. A total of 85 SD rats were genotyped by Sanger sequencing, revealing a distribution of 25.9% wild-type (WT), 48.2% heterozygous, and 25.9% homozygous variants. Echocardiography, Hematoxylin-Eosin staining, Masson's trichrome staining and transmission electron microscopy indicated no significant differences in cardiac structure or baseline function among the three groups. In freely moving rats, premature atrial arrhythmias were detected in 2 of 9 WT rats, 1 of 9 heterozygous rats, and 1 of 9 homozygous rats. Premature ventricular contractions (PVCs) were observed in none of 9 WT or homozygous rats, 3 of 9 heterozygous rats, with one heterozygous rat exhibiting frequent PVCs. Electrical programmed stimulation revealed a higher incidence of inducible atrial fibrillation in homozygous rats compared to WT rats and a higher incidence of inducible ventricular tachycardia in heterozygous rats compared to WT rats. These findings suggest that deletion polymorphism in the intron sequences of Camk2d are unexpectedly common in normal SD rat populations and that such polymorphism predispose to ventricular arrhythmias without overt structural heart disease. Our study highlights the potential arrhythmogenic risk associated with non-coding DNA sequence alterations in Camk2d and underscores the importance of genetic screening in experimental animal models.

Chemotherapy-induced alopecia (CIA) is one of the most apparent symptoms of side effects in a cancer patient undergoing chemotherapy using anti-cancer drugs, resulting in distress and a lower quality of life. Hence, this study investigated the protective and regenerative effects of Philippine stingless bee propolis on CIA in a murine model. Female C57BL/6N mice were subjected to hair cycle synchronization through depilation, followed by cyclophosphamide (CYP) administration to induce hair loss and graying. Daily topical application of 99.5% ethanol extracted propolis diluted twice with water was performed for 30 days. Results revealed that propolis-treated mice exhibited increased folliculogenesis and epidermal thickness, but not hair length, and improved melanogenesis compared to controls. Immunohistochemical and gene expression analyses revealed increased Ki67 proliferative cells and reduced apoptosis (TUNEL cells) at the early 48 hours of topical treatment. Moreover, propolis upregulated expressions of Lef1 and melanogenic genes (Tyr, Tyrp1, Dct) at 30 days of treatment. These findings suggest that Philippine stingless bee propolis promotes hair follicle regeneration and melanocyte function, offering a potential natural therapeutic approach for CIA.

At the National Center of Geriatric and Gerontology (NCGG), aged mice and rats are used in research on aging and the treatment and prevention of gerontological diseases. Some of the most commonly used mouse strains in our center and general research were the C57BL/6J (B6J) and C57BL/6N (B6N). In this study, hematological and biochemical changes related to age, strain, and sex, from 3 months (mo) to 24 mo, were characterized every 3 mo in the B6J and B6N strains. Hematological results showed that in B6J males at 24 mo, the levels of WBC, especially lymphocytes, were higher than in the B6N strain. In males B6J, the number of CD4+ T cells did not decrease significantly between 6 and 24 mo, but in females and strain B6N, the number of CD4+ T cells decreased significantly. The levels of red blood cells (RBC) and hemoglobin (HGB) were reduced with age in all strains, while the number of platelets (PLT) increased. Biochemical parameters, Blood urea nitrogen (BUN) and Creatinine (CRE) in B6J males were significantly higher than in the other groups at 24 mo. Glutamate oxalacetate transaminase/aspartate aminotransferase (GOT/AST) and glutamate pyruvate transaminase/alanine aminotransferase (GPT/ALT) levels were higher in the B6N strain than the B6J strain at 24 mo. The present results revealed significant variations in hematological and biochemical parameters between the two strains and between sexes as a result of genetic and hormonal differences in laboratory mice.

In pharmaceutical development, weight loss is occasionally observed in monkeys during non-clinical toxicity studies and can be difficult to differentiate from drug effects. This study retrospectively analyzed data from control group monkeys without drug treatment to investigate the incidence of weight loss and its physiological and pathological characteristics. We also investigated potential improvements through enhanced animal welfare. In the 4- and 13-week toxicity studies conducted at the test facility from 2010 to 2022, 684 control group monkeys were investigated. Among them, 3 animals in the 4-week toxicity studies and 5 animals in the 13-week toxicity studies showed a weight change rate of less than -10%, resulting in an incidence rate of 1.2%. However, these animals had adequate food consumption. Animals in the 4-week toxicity studies showed signs of stress in histopathology. Additionally, 2/3 animals in the 4-week toxicity studies had decreased blood glucose levels and 1/5 animal in the 13-week toxicity study fell into a crouching posture, suggesting hypoglycemia that was alleviated with glucose administration, indicating stress-induced metabolic abnormalities. From 2015, an enrichment program was implemented to improve animal welfare. Prior to this program, 2.4% of animals showed a weight change rate of less than -10%, which dropped to 0.25% post-implementation, suggesting the program's effectiveness in reducing stress. These results clarify the characteristics of animals that lose weight during toxicity studies and suggest that improving animal welfare can reduce the incidence rate.

Allergic rhinitis (AR) is an inflammatory disorder driven primarily by aberrant T helper 2 (Th2) differentiation in CD4⁺ T cells. Although dual-specificity phosphatase 5 (DUSP5) has been implicated in inflammatory and autoimmune regulation, its role in AR remains unexplored. In this study, an AR mouse model was established via intraperitoneal sensitization and intranasal challenge with ovalbumin. We observed significant downregulation of DUSP5 expression in the nasal mucosa, particularly within CD4⁺ cells. To elucidate its function, a lentiviral vector overexpressing DUSP5 was constructed and used to transduce naive CD4⁺ T cells isolated from BALB/c mouse spleens. Overexpression of DUSP5 suppressed Th2-specific cytokine production and inhibited Th2 differentiation. Mechanistic investigations using a luciferase reporter assay revealed that Dusp5 is transcriptionally repressed by SRY-box transcription factor 11 (SOX11), a known transcription factor that promotes the progression of AR. Furthermore, DUSP5 overexpression counteracted the pro-Th2 effects mediated by SOX11. These results demonstrate that DUSP5, transcriptionally inhibited by SOX11, attenuates AR-associated inflammation by restraining Th2 differentiation. Our findings identify DUSP5 as a potential therapeutic target for AR.

Endothelial dysfunction (ED) plays a pivotal role in the pathogenesis of hypertension and its associated vascular complications. Qingda granule (QDG) exhibits significant antihypertensive properties and demonstrates therapeutic potential in ameliorating vascular dysfunction. This study aimed to explore QDG's role in alleviating endothelial injury in hypertension. An L-NAME (Nω-Nitro-L-arginine methyl ester)-induced hypertensive mouse model was used to evaluate the effects of QDG on blood pressure and endothelial function. Endothelial function was assessed through histological analysis, nitric oxide (NO) quantification, and vascular response measurements. To explore underlying mechanisms, network pharmacology was conducted using databases such as HERB, SwissTargetPrediction and STRING. Key pathways related to inflammation and cell adhesion were identified. Based on these findings, immunohistochemical staining was conducted to analyze the expression of phosphorylation of NF-kappaB p65 (p-NF-κB p65), NF-κB p65, intercellular adhesion molecule-1 (ICAM-1), and tumor necrosis factor-α (TNF-α) in vascular tissues. QDG treatment significantly reduced blood pressure, increased NO levels, and enhanced endothelial nitric oxide synthase (eNOS) expression in L-NAME-induced hypertensive mice, indicating its potential to restore endothelial function. Experimental validation further confirmed that QDG markedly suppressed the expression of p-NF-κB p65, TNF-α, and ICAM-1 in vascular tissues. These results suggest that QDG alleviates hypertension-induced ED primarily by inhibiting inflammation and endothelial adhesion via the NF-κB signaling pathway. Overall, QDG presents a promising therapeutic candidate for managing hypertension and its vascular complications.

Uncontrolled activation of autophagy following ischemia/reperfusion (I/R) injury leads to cell death. The superfamily of ankyrin repeat proteins (N-Ank protein) was reported to be involved in autophagy regulation and cardiac protection. Bioinformatics analysis was performed (GSE61592 and GSE160516) and ten N-Ank proteins were differentially expressed in I/R models. Retinoic acid-induced protein 14 (RAI14), a member of N-Ank protein family, was upregulated in I/R-injured cardiac tissue and was first selected for research. A mouse I/R model was established by ligating the left anterior descending coronary artery to induce 90 min of ischemia, followed by 72 h of reperfusion. RAI14 was found upregulated in ischemic penumbra. RAI14 overexpression in cardiac tissue by injecting adeno-associated virus-9-RAI14 plasmid system via tail vein improved cardiac function and reduced infarct and apoptosis. Furthermore, the activated autophagy in ischemic penumbra of I/R mice was reversed by RAI14 overexpression along with decreased microtubule-associated protein 1 light chain 3 beta (LC3) II and increased autophagy receptor p62 expressions. RAI14 silence showed an opposite effect. A cell model was established by using mouse cardiomyocytes HL-1 underwent hypoxia/reoxygenation (H/R) treatment. Similarly, H/R also enhanced RAI14 expression and RAI14 overexpression inhibited H/R-induced apoptosis and autophagy in HL-1 cells. Mechanistically, autophagy inhibitor, the AKT/mTOR pathway, was found to be suppressed in mouse and cell models whereas RAI14 overexpression activated this pathway. Collectively, we demonstrated that compensatory increase of RAI14 inhibited I/R-induced myocardial injury by preventing excessive autophagy through activating the AKT/mTOR pathway, which providing an idea to explore strategies for preventing I/R injury.

Euthanasia agents should induce a rapid and painless loss of consciousness, followed by cardiopulmonary arrest and subsequent brain death. Injectable drugs such as pentobarbital sodium are commonly used for laboratory rodents due to their quick and smooth action. However, the discontinuation of pharmaceutical-grade pentobarbital sodium and secobarbital sodium in Japan, along with a global shortage of pentobarbital in late 2020, has increased the demand for new injectable euthanasia drugs. In Japan, the combination of medetomidine, midazolam, and butorphanol (MMB), as well as a newer formulation in which midazolam is replaced with alfaxalone (MAB), have been widely used as balanced anesthesia for rodents. To evaluate their potential as alternative euthanasia agents in mice, we compared mortality rates and the time intervals to the loss of the righting reflex, respiratory arrest, and cardiac arrest following anesthetic administration. An intraperitoneal injection of MAB at five times the anesthetic dose induced death within 10 min with the loss of the righting reflex, respiratory arrest, and cardiac arrest occurring at 1.5 min, 4 min, and 9 min respectively, in all mice, which was comparable to those observed with 300 mg/kg of secobarbital. In contrast, none of the mice administered MMB at five times the anesthetic dose experienced cardiopulmonary arrest within 30 min. Intraperitoneal overdose of MAB induces rapid and irreversible death, supporting its potential use as an effective euthanasia agent in mice.

Archiving and sharing cryopreserved rat sperm can improve animal experiments' reliability, reproducibility, and sustainability in the scientific community. When sharing cryopreserved sperm from genetically engineered rats, a shipment system is required. Generally, a dry shipper, which can maintain at below -150°C, is the most widely used for sperm transport. However, using it for shipping cryopreserved sperm faces some difficulties, such as the risk of transporting hazardous materials (liquid nitrogen), its high cost, and the round-trip fee. Recently, the shipment of cryopreserved mouse sperm with dry ice at -79°C has been alternatively accepted in the scientific community. However, its outcome in terms of the fertilization and developmental abilities of the cryopreserved rat sperm was not examined. Therefore, this study aimed to examine the fertilization and developmental abilities of cryopreserved rat sperm after being stored in a deep freezer (-80°C) and dry ice (-79°C). We also demonstrated the transport of cryopreserved rat sperm in a Styrofoam box with dry ice. The fertilization rate of cryopreserved sperm stored in a deep freezer or dry ice was comparable to that in liquid nitrogen. In the transport experiment, the rat sperm transported between Kumamoto and Hokkaido maintained a high fertilization rate, and live pups were obtained from the embryos derived from the transported sperm. Fertilization and developmental abilities of cryopreserved rat sperm were maintained after shipment using a Styrofoam box with dry ice for storage.

Alternative educational tools, such as training videos and simulators, are recommended in the education of laboratory animal science. However, evidence supporting their educational utility in the training of rodent experimental techniques remains limited. In this study, we assessed the utility of alternative educational tools in the practice of laboratory animal science for novice veterinary students. 149 students participated in a stepwise program beginning with lectures, followed by preparatory learning sessions using training videos and two types of mouse simulators (a silicone-based model and fabric toy mouse), and then hands-on training with live mice. The program covered basic techniques: habituation, restraint, and vaginal smear sampling for estrous cycle determination. A survey-based evaluation was conducted to assess the educational utility of alternative educational tools. The contribution of each preparatory resource (videos, lectures, simulators, printed materials, and notes) to skill acquisition was evaluated, showing that videos, lectures, and printed materials highly contributed. The training videos were rated as more necessary than the simulators for skill acquisition. Psychological evaluation showed that 84% of students experienced anxiety before practice. A positive correlation was found between anxiety levels and frequency of use for all three tools, and students reported that all tools were effective in reducing anxiety during practice. All techniques showed high proficiency rates. Our findings suggest that integrating alternative tools with live-animal training promotes technical skill acquisition, enhances psychological readiness, and supports 3Rs-based laboratory animal practice.

Chronic kidney disease (CKD) is a complicated systemic disease displaying various pathophysiological symptoms including mineral bone disorder (CKD-MBD). Ideally, early intervention for CKD-MBD would be desirable, however, there is not enough evidence regarding treatment of CKD-MBD, especially in its early stages, due to its multifactorial pathophysiology and the difficulty in generating adequate animal models. In this study, we evaluated the efficacy of a tissue nonspecific alkaline phosphatase (TNAP) inhibitor, SBI-425 in a CKD-MBD animal model, produced by a combination of nephrectomy and high inorganic phosphate (P) diet. This combination induced renal damage, and significantly elevated blood urea nitrogen (BUN). Plasma levels of fibroblast growing factor 23 (FGF-23), parathyroid hormone (PTH) and phosphate were also elevated, leading to ectopic calcification in the kidneys, particularly in the renal tubules. We orally administered SBI-425 twice daily for 12 weeks at doses of 1 and 10 mg/kg, and this treatment significantly inhibited the progression of calcium deposition in the renal tubules. Furthermore, SBI-425 effectively prevented the deterioration of plasma parameters, BUN, FGF-23, PTH, and phosphate. In conclusion, our findings suggest that TNAP inhibition can effectively slow the progression of CKD-MBD by inhibiting the calcification in the renal tubules. These results may have implications for better clinical care of patients with CKD.

In 2016, an outbreak of Rattus norvegicus polyomavirus 2 (RatPyV2) infection was reported in a colony of X-linked severe combined immunodeficiency (XSCID) rats in the United States. While RatPyV2 infection persists asymptomatically in immunocompetent rats, immunodeficient XSCID rats develop variable respiratory symptoms, emaciation, impaired breeding performance, and systemic deteriorating condition. RatPyV2 is an epitheliotropic virus targeting epithelial cells of the salivary glands, Harderian glands, extraorbital lacrimal glands, respiratory system, and reproductive or accessory reproductive organs. Histopathologically, the formation of large basophilic nuclear inclusion bodies in the infected epithelial cells is a characteristic feature, along with hyperplasia or dysplasia. Glandular atrophy and loss, accompanied by fibrosis and mononuclear cell infiltration, are also observed in the salivary glands, Harderian glands, and extraocular lacrimal glands. In particular, the parotid salivary glands are prone to be severely and extensively affected with relatively severe and diffuse lesions even at one month of age. Severely affected animals also develop interstitial pneumonia. Among target tissues, the parotid salivary glands appear to be higher susceptible to RatPyV2, therefore pathological examination and PCR examination of the salivary glands, including the parotid salivary glands, are essential for the diagnosis of RatPyV2 infection. This review paper provides a comprehensive summary of the features (clinical signs, pathological findings, and transmission), diagnostic methods, and prevalence of RatPyV2 infection, based on our research and reports from research groups in the United States.

As aging affects the appearance of the skin, anti-aging research has intensified in dermatology, skincare, and aesthetic medicine. Because natural aging takes a very long time, one essential anti-aging approach is to pharmacologically mimic aging, such as with D-galactose treatment. Hairless mice (HR-1) have been extensively used in skin research because of their lack of body hair and ease of animal care. In the present study, HR-1 mice were treated with D-galactose to determine whether detrimental effects were induced in the skin. After 3 months of D-galactose treatment, AGEs in the skin significantly increased. On the other hand, no signs of skin disorders (dermal thickness, type I collagen content, expression of various genes, collagen synthesis, and degradation signals) were observed. Even when the concentration of D-galactose increased, no apparent changes in dermal thickness were observed. These findings suggest that D-galactose treatment induces AGEs accumulation but no further detrimental effects in the HR-1 skin.

In vivo imaging enables real-time detection of excitation and emission signals and is useful for the noninvasive evaluation of temporal changes in biological tissues. The near-infrared fluorescent protein iRFP can be used for deep-tissue imaging because it emits light at wavelengths that are less attenuated by biological tissues. However, autofluorescence originating from diet, tissues, and the imaging environment can interfere with fluorescence detection; therefore, appropriate animal pretreatment and optimization of imaging conditions are essential. We generated two mouse strains: Alb reporter mice, in which enhanced green fluorescent protein (eGFP) and iRFP713 genes were tandemly inserted downstream of the Albumin gene, and hairless mice (Hr), carrying a mutation in the hairless gene. Their offspring were used in in vivo imaging experiments to investigate: (i) the localization of eGFP and iRFP713 fluorescence, (ii) the influence of hair on fluorescence detection, and (iii) suitable filter combinations for fluorescence detection. In the resulting mice, liver-specific expression of both eGFP and iRFP713 was observed at the same anatomical location. Although autofluorescence was more prominent in hairless mice than in furred mice, signal detection was improved either by using longer-wavelength excitation/emission filters or by applying spectral unmixing to separate the target signal. These findings provide practical guidance for optimizing in vivo fluorescence imaging conditions using standard IVIS platforms.

Uncovering protein interaction networks in vivo is essential for understanding physiological and pathological processes. Here, we report the generation of a novel knock-in mouse model expressing miniTurbo, a highly active biotin ligase, fused to the endogenous Usp46 gene. This model enables proximity-dependent biotinylation (BioID) of USP46-associated proteins in the brain. In adult mice, biotinylation was induced by feeding a 0.1% biotin diet. We further evaluated whether the combination of miniTurbo and dietary biotin supplementation is effective for BioID in the developing brain. Biotinylation was successfully induced in embryonic and neonatal brains via maternal biotin intake, demonstrating the transfer of biotin to the offspring through the placenta during pregnancy and through milk during lactation. This strategy enables proximity labeling under physiological conditions without invasive procedures, such as repetitive subcutaneous injections, during developmental stages. Using mass spectrometry, we identified USP46-proximal proteins, including known cofactors WDR48 and WDR20, in the adult brain. Gene Ontology analysis revealed enrichment in postsynaptic pathways, consistent with known localization of USP46. Among the identified proteins, PLPP3, a phospholipid phosphatase, was significantly downregulated in the hippocampus of Usp46-knockout mice. These findings establish the USP46-miniTurbo knock-in mouse as a powerful tool for in vivo interactome analysis and provide new insights into the molecular functions of USP46 in the brain.

The ε-containing GABA (A) receptors (GABAARs), a lesser-studied subtype within the GABAAR family, have garnered attention due to their distinct pharmacological properties and potential involvement in brain injury. Zolpidem (ZPM), a widely used Z-drug, is known to induce paradoxical effects in patients with brain injury, although the underlying molecular mechanisms remain unclear. In this study, a chronic cerebral hypoperfusion (CCH) rat model was established using Permanent Bilateral Occlusion of the Common Carotid Arteries (PBOCCA), followed by administration of ZPM at doses of 1.0, 2.0, and 4.0 mg/kg. Behavioral assessments demonstrated that the 1.0 mg/kg dose of ZPM significantly improved spatial learning and memory acquisition (P<0.01) and enhanced memory retention (P<0.001), whereas higher doses resulted in sedation and cognitive impairment. Immunohistochemical analysis revealed an upregulation of the ε subunit expression in the hippocampal CA1 and CA3 regions of CCH rats (P<0.05), suggesting alterations in receptor composition in response to cerebral hypoperfusion. Further investigation of ZPM's interaction with ε-containing GABAARs (specifically the α1β2ε subtype) was conducted using in silico techniques. Molecular docking identified the α1+/ε- binding interface as a favorable ZPM binding site, with key residues being either conserved or suitably replaced. Molecular dynamics simulations demonstrated that ZPM stabilizes the receptor while permitting conformational flexibility, consistent with its role as a positive allosteric modulator. These findings provide evidence that ZPM interacts with ε-containing GABAARs, potentially explaining its paradoxical effects observed in brain injury models.

Emerging evidence indicates that oxidative stress in skeletal muscle is a prerequisite for sarcopenia in diabetic patients. In this study, we show that ubiquitin-specific protease (USP) 2 mitigates the accumulation of reactive oxygen species (ROS) in mature muscle cells. Treatment with ML364, a canonical USP2 inhibitor, robustly increased mitochondrial ROS in mouse C2C12 myotubes and caused an accompanying increase in the glutathione disulfide (GSSG)/glutathione (GSH) ratio. ML364 also caused mitochondrial damage in C2C12 myotubes, resulting in a reduction in intracellular adenosine triphosphate levels. Correspondingly, under diabetic condition, the muscle-specific Usp2-knockout (msUsp2KO) C57BL/6N mice exhibited a significantly higher lipid peroxide level and GSSG/GSH ratio in skeletal muscle than the control mice. The msUsp2KO mice also exhibited augmented insulin resistance and glucose intolerance, but showed no obvious deterioration in muscle weight or histology relative to the control mice. However, damaged mitochondria in the soleus muscle were more frequently observed in msUsp2KO mice than in the control mice. Together, these data suggest that USP2 mitigates ROS accumulation and subsequent mitochondrial damage in muscle cells in mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that causes the selective loss of motor neurons. A histopathological hallmark of ALS is the cytoplasmic aggregation of TDP-43, a ubiquitously expressed RNA-binding protein involved in transcription and splicing regulation. To prevent abnormal accumulation, TDP-43 controls its expression levels through an autoregulatory feedback loop. While most ALS studies have focused on pathogenic variants that impair the protein function of TDP-43, the mechanisms underlying endogenous TDP-43 dysregulation mediated by non-coding elements, including the 3´ untranslated region (3´UTR), remain incompletely understood. In this study, we generated a mouse model carrying a targeted deletion of the Tardbp 3´UTR that encompasses the TDP-binding region, polyadenylation signals, and alternative intronic sequences. Our findings demonstrate that the Tardbp 3´UTR is essential for normal mouse development. Loss of this region led to decreased Tardbp mRNA expression and embryonic lethality after gastrulation. Young heterozygous mice were phenotypically normal with no overt disruption in TDP-43 autoregulation. However, aged heterozygous mice displayed mild locomotor dysfunction accompanied by a modest increase in spinal cord TDP-43 protein levels and a reduction in motor neuron numbers. These findings indicate that regulatory elements within the Tardbp 3´UTR play a pivotal role in normal development and contribute to TDP-43 pathology relevant to ALS.