Renal ischemia-reperfusion (I/R) remains a leading cause of acute renal failure in both native and transplanted kidneys. Hypoxia-inducible factor (HIF)-1α is a protective factor against renal I/R injury (rIRI). However, the downstream mechanisms through which HIF-1α exerts its protective effects in rIRI remain to be fully elucidated. rIRI was induced in heterozygous HIF-1α knockout (hKO) mice. To establish an in vitro model of rIRI, a human tubular cell line (HK2) was subjected to hypoxia-reoxygenation (H/R). rIRI-induced hKO mice exhibited elevated serum creatinine levels compared with rIRI-induced wild-type (WT) mice. Furthermore, tubular cell death was observed earlier in WT mice during the initial phase of I/R, whereas it was reduced in hKO mice. Phagocytosis of damaged tubular cells by macrophages was diminished in hKO mice, suggesting that the clearance of cellular debris plays a critical role in renal tissue repair and regeneration. Furthermore, glutathione-specific γ-glutamyl cyclotransferase 1 (CHAC1), a known cell death inducer, was upregulated in the tubular cells of WT mice but not hKO mice following I/R. The overexpression of CHAC1 in HK2 cells induced cell death, whereas siRNA-mediated CHAC1 knockdown attenuated cell death in HK2 cells subjected to H/R. These findings collectively suggest that CHAC1 plays a pivotal role in regulating tubular cell death during rIRI. Our findings indicate that controlled cell death induction is essential for rIRI recovery. CHAC1, a key factor in this process, is a potential therapeutic target for rIRI.NEW & NOTEWORTHY Here, we reported that HIF-1α upregulates glutathione-specific γ-glutamyl cyclotransferase 1 (CHAC1), a regulator of cell death and oxidative stress, in rIRI. Our results suggested that CHAC1 plays a pivotal role in regulating tubular cell death during rIRI, and the controlled tubular cell death induced by CHAC1 is essential for rIRI recovery. This proposes novel mechanisms underlying rIRI recovery.

Lrp10 insufficiency upregulates mRNA and protein of neurotoxic α-synuclein and causes degeneration of substantia nigra dopaminergic neurons in heterozygous or homozygous Lrp10 knockout mice.

Increased concentrations of neuroblastoma suppressor of tumorigenicity 1 (NBL1) in the blood have been associated with disease progression in diabetic kidney disease (DKD) and IgA nephropathy. However, it is unclear whether NBL1 is a causal factor for kidney disease and what is driving these increased concentrations in the blood. To test this, we evaluated Nbl1 heterozygous knockout (Nbl1+/-) mice in two models of kidney injury, X-linked Alport syndrome (XLAS) and chronic low-dose cisplatin treatment, and compared them with wild-type (WT) controls. In parallel, we assessed serum NBL1, kidney function, and damage, and performed a genetic analysis for the drivers of NBL1 concentrations in two independent cohorts of genetically diverse Diversity Outbred mice with XLAS (DO-XLAS), analyzing each cohort separately. Serum NBL1 was consistently associated with reduced glomerular filtration rate (GFR) across both DO-XLAS cohorts, whereas correlations with albumin-to-creatinine ratio (ACR) were variable between cohorts, and not consistently replicated. In both XLAS and cisplatin models, partial reduction of NBL1 (∼50%) in Nbl1+/- mice did not alter GFR, ACR, or histological injury relative to WT controls. Genetic analysis of NBL1 concentrations in our DO-XLAS cohorts identified associations with loci on Chromosomes 4 and 17. Together, these findings indicate that elevated serum NBL1 reflects kidney injury and, under partial reduction, does not alter disease severity, consistent with NBL1 functioning as a biomarker rather than a causal driver of kidney disease.NEW & NOTEWORTHY Elevated NBL1 in blood correlates with end-stage kidney disease in humans with diabetic kidney disease. NBL1 also correlates with renal phenotypes in a cohort of genetically diverse mice with X-linked Alport syndrome. Studies in two different mouse models of kidney disease reveal that elevated NBL1 is not causal to kidney injury, positioning NBL1 as a biomarker with potential applicability across etiologies and clarifying its role as a consequence of renal pathology.

FOXP1 is differentially active during development of murine vasopressin and oxytocin magnocellular neurons.

Neurofibromatosis type 1 (NF1) is a genetic condition presenting with variable symptomatology, however most individuals will demonstrate cognitive and behavioural difficulties, including autism. Using a heterozygous germline knockout mouse model of NF1 (Nf1 +/-), we performed in-depth behavioural evaluations encompassing learning and memory, stereotypy, social interaction, anxiety- and depression-like behaviour. Anatomical and functional studies of the brain and gastrointestinal tract were followed by the first investigation of gut microbiota composition (via full-length 16S rRNA sequencing) in a Nf1 +/- mouse model. The cognitive and autism-like behavioural phenotype seen in Nf1 +/- mice was accompanied by a striking increase in relative brain size which is highly relevant to clinical NF1. Furthermore, brain size was correlated with behaviour, supporting a potential mechanistic link. Nf1 +/- mice showed significant alterations in gut microbiota composition vs. Nf1 +/+ wild-type controls, with males additionally showing significant changes to species abundance of the Clostridium and Blautia genera, and the Lachnospiraceae family, findings which partially overlap with those in preclinical and clinical autism. Composition of associated functional pathways was not globally altered, however +/- mice showed significant changes in a pyrimidine deoxynucleotide biosynthesis pathway. In male Nf1 +/- mice, we also identified a genotype-specific host-microbial signature, pointing towards a mechanistic link between gut microbiome composition and brain size. These findings significantly expand our understanding of brain and behavioural abnormalities in this preclinical model of NF1 and, importantly, have uncovered the gut microbiome as a highly promising new area of research and a potential therapeutic target for these symptom clusters.

Microexons of 3 to 27 nucleotides selectively regulated in the vertebrate nervous system have attracted attention as new elements for modifying the function of neuronal proteins. Protein tyrosine phosphatase δ (PTPRD) is one of presynaptic hubs for neuronal synaptic organization. Alternative splicing (AS) of three microexons, meA3, meA6, and meB, encoding 3, 6, and 4 amino acid-peptides, respectively, imparts structural diversity to PTPRD, due to which the resulting eight splice variants exhibit distinct synaptogenic properties. However, the regulatory mechanisms of AS and physiological significance of the AS code for Ptprd gene remain largely unknown. Here, we report the AS of the three microexons is genetically regulated to generate spatiotemporally distinct expression pattern of eight Ptprd splice variants across brain regions and is modulated by neuronal activity. We identified both the intronic splicing enhancer region (ISE) for meB contributing to the spatiotemporal patterning of the AS and the neuronal activity-dependent intronic splicing silencer region for meB (ISS). Heterozygous deletion of the ISE in mice led to a decreased meB selection rate by ~25% with unaltered total amount of PTPRD protein and caused severe sensory, motor, social, and emotional behavioral abnormalities but no obvious changes in learning and memory, while heterozygous Ptprd knockout mice with unaltered meB selection rate and ~50% decrease in total PTPRD protein showed much fewer behavioral abnormalities. Interestingly, deletion of the ISS for meB caused selective impairments in motor learning and fear memory. Our findings demonstrate the spatiotemporal and activity-dependent AS code for Ptprd meB plays a crucial role in proper behavioral development.

BackgroundThe gene DPP6 has been associated with behavioral phenotypes of alcohol use disorder (AUD) in recent human genome wide association studies. DPP6 encodes an auxiliary subunit that modulates A-type voltage-gated potassium channels, particularly Kv4.2.MethodsTo further assess the role of this gene in ethanol-related traits, we tested Dpp6 knockout (KO) and wild-type mice for ethanol (EtOH) conditioned place preference (CPP), locomotor activity, and ethanol-induced anxiolysis.ResultsMale homozygous KO mice (HOM) showed greater preference for the ethanol-paired (2 g/kg) context compared to wild type littermates (WT) and heterozygous KO mice (HET), while female mice showed no genotypic difference. HOM of both sexes exhibited greater novelty-induced hyperactivity in the CPP apparatus than HET and WT mice in the first two minutes. In a separate experiment, HOM mice showed enhanced locomotor activity following a 1.5 g/kg EtOH injection; however, they also displayed greater locomotor activity during habituation, suggesting basal locomotor differences. Following 1.5 and 2 g/kg injections, HOM mice exhibited EtOH-induced anxiolysis in the first 5 minutes, while the HET and WT mice did not. Lastly, HOM mice displayed a significant sedative response compared to WT animals following a 2 g/kg injection of ethanol.ConclusionsUltimately, these findings validate a role for Dpp6 in modulating ethanol’s rewarding, anxiolytic, and sedative effects in a sex-dependent manner.

The role of the cohesin complex depends on the cohesin loader proteins NIPBL and MAU2. While NIPBL variants are a major cause of Cornelia de Lange Syndrome (CdLS), the role of MAU2 in disease is unclear. We describe 18 individuals carrying 15 heterozygous MAU2 variants and demonstrate pathogenicity through functional analyses. In-frame MAU2 variants predominantly impair NIPBL–MAU2 interaction, whereas truncating variants cause MAU2 haploinsufficiency and lead to NIPBL reduction. Most individuals exhibit a DNA methylation profile compatible with the CdLS episignature. We also describe two MAU2-specific episignatures that reflect variant-dependent molecular consequences. Affected individuals display a wide range of phenotypes, from classic CdLS to milder presentations, with short stature and microcephaly as major features. A heterozygous Mau2 knockout mouse model recapitulates these traits, confirming the causal role of MAU2 disruption in vivo. Our study establishes MAU2 as a CdLS-associated gene and delineates a MAU2-related chromatinopathy with variable expressivity.

δ-catenin haploinsufficiency is sufficient to alter behaviors and glutamatergic synapses in mice.

Dravet syndrome (DS) is a prototypical developmental and epileptic encephalopathy caused by mutations in the SCN1A gene, leading to loss of function of the voltage-gated sodium channel Naᵥ1.1. The latter causes early onset drug-resistant seizures and enduring cognitive and behavioral deficits. In this pathological context, the implication of astrocytes remains insufficiently explored. Using a heterozygous Scn1a knockout (Scn1a+/-) mouse model that recapitulates the DS-human phenotype, we examined astrocyte remodeling at landmark disease stages, as defined by video-electroencephalography and behavioral readouts. From initial disease aggravation (postnatal day [PN] 20-35) to long-term stabilization (up to PN90), Scn1a+/- mice showed increased hippocampal and cortical glial fibrillary acidic protein (GFAP) transcript and protein levels compared to age-matched control littermates and to an earlier presymptomatic (<PN20) time point. During the aggravation phase in Scn1a+/- mice, astrocyte branching, revealed by GFAP histological analysis and by intracellular delivery of Alexa Fluor 488 in hippocampal slices, was increased but not sustained long-term. Importantly, these disease-stage-dependent astrocyte modifications were not associated with macroscopic hippocampal sclerosis or cortical atrophy. To further study astrocyte remodeling, we used biocytin diffusion following single-astrocyte loading to reveal an expanded astrocyte-astrocyte network in Scn1a+/- mice long-term, along with increased connexin (Cx30 and Cx43) protein levels. An ethidium bromide uptake assay indicated impaired astrocytic hemichannel function in Scn1a+/- mice. Regionally, these long-term cellular and network astrocyte modifications coincided with augmented posttetanic synaptic potentiation. In DS, astrocytes undergo long-term remodeling independent of tissue damage. We discuss the association between astrocyte network changes and seizures, as well as synaptic and cognitive deficits.

Alzheimer's disease (AD) patients frequently experience seizures, sleep disturbances, and other forms of neural network dysfunction that accelerate cognitive decline. Although tau-lowering therapies may alleviate these features, they also risk disrupting essential physiological functions of tau, leading to motor impairments. We hypothesized that targeted mutations within tau's proline-rich domain-regions critical for binding SH3-containing proteins implicated in seizures and excitotoxicity-could selectively disrupt pathological interactions while preserving normal cognition and behaviour. To test this, we generated two tau knockin mouse lines: AxxA6, carrying proline-to-alanine mutations in the sixth PxxP motif, and R221A, containing an arginine-to-alanine substitution at residue 221. Tau-protein interactions were evaluated using proximity ligation assays in cultured hippocampal neurons and co-immunoprecipitation-mass spectrometry of cortical lysates. To model epilepsy, Kv1.1 heterozygous knockout mice were crossed with tau knockin mice. Mice underwent 24-hour cortical EEG recordings. Seizure susceptibility was assessed following intraperitoneal kainic acid (25 mg/kg). Hippocampal slice electrophysiology was used to measure epileptic bursting after picrotoxin/4-AP application. Comprehensive motor and cognitive testing were performed in AxxA6 and R221A lines at young and older ages. Both variants reduced tau's binding to the SH3-containing proteins BIN1, PLCγ1, and p85⍺/PI3K, with AxxA6 specifically decreasing Fyn interaction (P < 0.0001). Coimmunoprecipitation-mass spectrometry revealed variant-specific alterations in tau interactomes, including increased synaptotagmin-5 binding in both lines (P < 0.05). AxxA6 knockin mice displayed unique resistance to kainic-acid-induced seizures. AxxA6 knockin also reduced epileptic spike rates in Kv1.1-/- mice (P = 0.02), along with improved beta power during REM (P < 0.05), and rescued sleep disruptions (P < 0.002). Both AxxA6 and R221A prevented the increase in epileptiform bursting in Kv1.1-/- hippocampal slices after picrotoxin/4-AP (P < 0.05) and improved survival in Kv1.1-/- mice. Motor function, cognition, and body weight were preserved in both lines across ageing (3-7 and 14-18 months), in contrast to age-related weight gain and motor deficits in tau knockout mice. These findings demonstrate that precision targeting of tau's sixth PxxP motif can selectively disrupt pathological protein interactions while preserving physiological function, offering a promising therapeutic strategy to mitigate tau-driven neuronal and network dysfunction without compromising cognitive or motor health.

Neurodegenerative disorders such as frontotemporal dementia (FTD) have strong hereditary links, yet these genes do not have full penetrance and environmental influences determine the lifetime risk of disease development. Better understanding of the environmental risk factors that determine age of onset, progression, and severity is needed. How these risk factors interact with genetic predisposition for these disorders will allow clinicians to provide better lifestyle recommendations for people with a familial history and deliver more personalized medicine. Here we examine the dose-dependent effects of the gene encoding progranulin (Grn), one of the most common mutations associated with familial FTD. We utilize both homozygous loss and heterozygous knockdown of Grn with the objective of assessing how a western diet consisting of high-fat and high-carbohydrate intake modulates the inflammatory and metabolic hallmarks in middle-aged mice. We found that while full Grn loss leads to heighted antigen presentation machinery and immune cell infiltration in the brain after obesogenic diet, a heterozygous gene primarily affects the periphery. Yet, further examination by RNA sequencing reveals that heterozygous mice have a disruption of MAPK signaling in the brain highlighting early disruption in the neuronal landscape. Our findings are consistent with reports that in individuals with genetic predisposition for FTD due to a GRN mutation, a western-style diet exacerbates the cellular stress in the peripheral immune system and affects the function of the prefrontal cortex. These data further support the use of heterozygous Grn knockout mice as a model for prodromal FTD in addition to the more common Grn full knockout which may not as accurately reflect disease onset biology.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary microvascular loss and obliterative remodeling driven by endothelial dysfunction. Low penetrance of only BMPR2 mutations causing metabolic shifts in pulmonary microvascular endothelial cells (PMVECs), suggests role of additional genetic modifiers. Genetic screening of PAH PMVECs identified carboxylesterase1 (CES1)-an endoplasmic reticulum (ER) enzyme involved in lipid metabolism and detoxification-as a candidate regulator of endothelial metabolism and angiogenesis. We hypothesize that CES1 loss promotes endothelial dysfunction via metabolic reprogramming, lipotoxicity, and oxidative stress. PAH/healthy PMVECs and lung tissues were obtained from transplant donors and commercial sources. CES1 knockdown and CES1 overexpression was performed in PMVECs for functional assays. Animal studies were done on CES1 heterozygous knockout (HET KO) and endothelial-specific knockout (ECKO) mice exposed to normoxia or hypoxia. CES1 expression was significantly reduced in PAH PMVECs and vascular lesions. CES1-deficient PMVECs exhibited increased apoptosis, reactive oxygen species (ROS) production, mitochondrial fragmentation, ER stress, and impaired angiogenesis. CES1 loss caused lipid droplet accumulation, reduced fatty acid oxidation, and glycolytic shift-phenotypes reversed by CES1 restoration. CES1 transcription was induced by BMPR2 via NRF2 activation, a key regulator of redox and metabolic homeostasis. CES1-deficient mice developed severe pulmonary hypertension (PH) under hypoxia, with extensive vascular remodeling, right ventricular dysfunction, and dysregulated angiogenesis and lipid metabolism pathways. CES1 is essential for pulmonary endothelial homeostasis and modifier of BMPR2 signaling. Restoring CES1 expression may serve as potential therapeutic strategy in reversing endothelial dysfunction and small-vessel loss.

Preeclampsia is a severe hypertensive disorder of pregnancy associated with low SIRT1 (sirtuin 1) levels in trophoblasts. Single-cell sequencing showed abnormal activation of trophoblast Rarres2 (retinoic acid receptor responder 2) and macrophage Cmklr1 (chemokine-like receptor 1) at the maternal-fetal interface in systemic Sirt1 heterozygous knockout mice. This study investigated how low SIRT1 in trophoblasts increases RARRES2 expression, affecting macrophage polarization and preeclampsia pathogenesis. We conducted coculture experiments to analyze trophoblast RARRES2 and macrophage CMKLR1 interactions, performed luciferase and chromatin immunoprecipitation assays to validate transcription factors for RARRES2 in trophoblasts, and utilized mass spectrometry and immunoprecipitation to identify transcriptional coregulators. cKO (trophoblast-specific Sirt1 knockout) mice were generated and treated with Rarres2 knockout or progesterone supplementation to validate the role of the SIRT1/RARRES2 axis in preeclampsia pathogenesis and prevention by progesterone. Finally, we measured RARRES2 and SIRT1 levels in the plasma of patients with preeclampsia. Low-SIRT1 expression in trophoblasts promoted M1-type macrophage polarization and inhibited trophoblast invasion, mediated by the RARRES2-CMKLR1 interaction. SIRT1 regulated RARRES2 expression in trophoblasts by recruiting NCOR2 (nuclear receptor corepressor 2). cKO mice showed preeclampsia-like symptoms and RARRES2-CMKLR1 activation at the maternal-fetal interface, which were reversed by Rarres2 knockout or progesterone supplementation. Notably, RARRES2 levels were higher and were a risk factor, whereas SIRT1 levels were lower and were a protective factor for preeclampsia in early pregnancy. This study highlights SIRT1's potential role in regulating abnormal trophoblast-macrophage interactions at the maternal-fetal interface in preeclampsia and offers a new strategy for its early prediction and prevention.

Deficiency of NR2C2 accelerates senescence of testicular Leydig cells and infertility in male mice.

Phosphatidylinositol-binding clathrin assembly protein (PICALM) is a clathrin adaptor essential for clathrin-mediated endocytosis. Genome-wide association studies (GWAS) have consistently identified PICALM as one of the most significant genetic susceptibility loci for late-onset sporadic Alzheimer’s disease (AD). However, the functional impact of the most validated AD-associated variant, rs3851179, remains unclear. Here, we examined PICALM mRNA and protein expression in post-mortem AD brains with reference to rs3851179 genotype. We found that PICALM mRNA levels were significantly increased in AD brains compared with controls, and that the protective rs3851179T allele was associated with reduced PICALM mRNA levels relative to the non-protective rs3851179C allele. In contrast, PICALM levels were significantly reduced in AD brain lysates compared with controls. PICALM expression did not significantly differ between carriers of the protective and non-protective alleles. Analysis of the mRNA-to-protein ratio revealed a significant dissociation between transcript and protein levels, suggesting relatively reduced protein expression efficiency in cases carrying the non-protective CC genotype. To assess whether reduced PICALM levels influence tau pathology, we used Picalm heterozygous knockout (Picalm+/−) mice, which express approximately 50% of the wild-type Picalm protein. Following stereotaxic injection of pathological tau extracted from AD brains, both wild-type and Picalm+/− mice developed tau pathology; however, the extent of tau accumulation did not significantly differ between genotypes. Together, these findings indicate that although PICALM protein level is reduced in AD, this reduction does not appear to affect tau propagation in this model. Therefore, the AD susceptibility associated with PICALM variant likely arises from mechanisms other than tau spread, possibly involving other aspects of autophagy, endocytic or vascular function.

Cisplatin (CP)-induced nephrotoxicity is a major clinical concern. Emerging evidence has revealed the critical role of PANoptosis, a coordinated cell death pathway, and neutrophil extracellular traps (NETs) in renal tubular damage. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) has been recognized as a potential modulator of inflammation and cell survival; however, its regulatory function and mechanism in acute kidney injury (AKI), especially CP-induced AKI, particularly concerning NETs and PANoptosis, remain poorly understood. This study investigates the central role of PPARγ and explores the therapeutic potential of its novel activator, O-alkyl and o-benzyl hesperetin derivative-1 L (HD-1L), in this context. Cultured renal tubular epithelial cells (mTECs) as well as a CP-induced AKI mouse model (20mg/kg, 72h) and renal ischemia-reperfusion injury (IRI) model​ were used. PPARγ heterozygous knockout mice, NET inhibitors (DNase I and GSK484), and pharmacological interventions (including the novel PPARγ agonist HD-1L and rosiglitazone [ROSI]) were used. The molecular mechanisms were assessed using western blotting, immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and cellular thermal shift assays (CETSA). PPARγ activity, NET markers (MPO, Cit-H3, and dsDNA), PANoptosis-related proteins (p-MLKL, GSDMD-N, and cleaved caspase-3), and reactive oxygen species (ROS) levels were quantified. CP triggered robust PANoptosis in the renal tissues, accompanied by elevated NETs and ROS-dependent NETosis. PPARγ activation significantly suppressed ROS production in neutrophils, thereby reducing NET formation. Mechanistically, NETs facilitate the release of cytoplasmic dsDNA, activate the AIM2 inflammasome, and promote PANoptosome assembly. Genetic PPARγ heterozygous knockout exacerbated renal injury and abolished protective effects, confirming the central role of PPARγ. HD-1L-induced activation of PPARγ reduced markers of PANoptosis and improved renal function in CP-AKI models. Furthermore, PPARγ agonism similarly protected against renal injury and suppressed the NETosis-PANoptosis axis in the IRI model. PPARγ is a pivotal checkpoint in CP-AKI by inhibiting ROS-NETosis-driven AIM2-mediated PANoptosis. This protective mechanism is also applicable to IRI-induced AKI, highlighting its broad relevance. HD-1L confers renoprotection through PPARγ activation, providing a novel therapeutic strategy against AKI.

Variants in the X-linked gene CASK are associated with neurodevelopmental defects. Animal model studies have demonstrated that conditions such as cerebellar hypoplasia, microcephaly, and optic nerve hypoplasia (ONH) are related to loss-of-function (LOF) in the CASK gene. CASK variants are associated with multiple ocular conditions spanning both anterior and posterior segments of the eye including retinopathies. Both Cask heterozygous knockout (+/−) mice and Cask knock-in (KI) mice with reduced Cask expression have been shown to display ONH. Cask (+/−) mice displayed no defects in retinal structure or function. Here, we have systematically examined the Cask (KI) mice. Our results demonstrate that the anterior segment of the eye in Cask (KI) mice does not display any obvious phenotype. Cask (KI) mice however show a reduced visual acuity in optomotor response. The retina of Cask (KI) mice does not exhibit any major changes in their structure, vasculature, or gene expression pattern. We, however, uncovered a specific dysfunction of the cone receptor in Cask (KI) mice using electroretinogram (ERG). Mechanistically this dysfunction arises due to lowered levels of cone-specific opsin (opsin1mw) in Cask (KI) mice. To the best of our knowledge, this is the first description of retinal dysfunction in an animal model with CASK gene suppression. We infer that like ONH and cerebellar hypoplasia, retinopathy also may represent CASK LOF.

The purpose of this study was to determine the role of MUC4, a corneal membrane-associated mucin, on ocular surface health and corneal wounding healing using a Muc4 knockout (KO) mouse model. Complete ophthalmic examinations were performed on wildtype (WT), Muc4 heterozygous (Het) and Muc4 knockout (KO) mice, including slit lamp biomicroscopy, phenol red thread test (PRTT), intraocular pressure (IOP), and fluorescein staining. The mice were also assessed using optical coherence tomography (OCT), an advanced imaging technique. Dynamic contact angle goniometry was performed on ex vivo globes of WT, Muc4 Het and Muc4 KO mice to calculate contact angle hysteresis as a novel measure of the adherence properties of the corneal epithelium. To determine the effect of Muc4 in corneal wound healing, a phototherapeutic keratectomy (PTK) was performed on the right eye. After PTK wounding, the corneas were fluorescein stained, imaged, and the wound size was quantified using ImageJ at 0-, 24-, 36-, and 48-h post-wounding. There were no phenotypic differences identified between WT, Muc4 Het and Muc4 KO mice on clinical examination, diagnostic testing, advanced imaging, and histology. While there was no difference in mucin 1 (Muc1) mRNA expression between WT and KO mice, there was a compensatory upregulation of a previously unreported murine corneal mucin, Muc20 mRNA expression, in corneal epithelium of Muc4 KO mice. No differences were detected between WT and Muc4 KO mice using dynamic contact angle goniometry. The Muc4 KO mice had significantly slower healing rates at 24 and 36 h post-wounding when compared with WT mice (P < 0.05) and all healed by 48 h post-wounding. These results support further investigation into compensatory roles of glycoproteins on the ocular surface, namely Muc4 and Muc20, and the role of Muc4 in epithelial cell migration in corneal wound healing.

Eukaryotic translation initiation factor 3 subunit D (eIF3d) is a noncanonical cap binding protein implicated in selective mRNA translation under stress conditions. Here, we investigate the contribution of eIF3d to pain processing using a heterozygous eIF3d knockout (eIF3d+/−) mouse model. We first validated this model, confirming substantial reductions in eIF3d mRNA and protein levels in dorsal root ganglia. Baseline assessments revealed no differences in mechanical, thermal, cold, or spontaneous pain behaviors between eIF3d+/− (HET) and eIF3d+/+ (WT) mice, indicating intact basal nociceptive function. In pain models involving peripheral inflammation and metabolic stress, including methylglyoxal injection, IL-6 administration and paw incision, HET mice displayed significantly reduced mechanical and cold hypersensitivity. In contrast, HET mice exhibited increased second phase nocifensive behavior in the formalin test, possibly indicating enhanced central sensitization. Hyperalgesic priming was comparable between HET and WT mice following IL-6 exposure. Experimental autoimmune encephalomyelitis (EAE) induced mice were unaffected by eIF3d reduction. These findings demonstrate that eIF3d selectively modulates nociceptive plasticity under defined stress conditions and suggests a context dependent role in the regulation of inflammatory and central pain sensitization.