Mutant Mice Research Articles

IFN-γ-producing TH1 cells and dysfunctional regulatory T cells contribute to the pathogenesis of Sjögren's disease.

Sjögren's disease (SjD) is an autoimmune disorder characterized by progressive salivary and lacrimal gland dysfunction, inflammation, and destruction, as well as extraglandular manifestations. SjD is associated with autoreactive B and T cells, but its pathophysiology remains incompletely understood. Abnormalities in regulatory T (Treg) cells occur in several autoimmune diseases, but their role in SjD is ambiguous. We had previously shown that the function and development of Treg cells depend on store-operated Ca2+ entry (SOCE), which is mediated by ORAI1 Ca2+ channels and stromal interaction protein 1 (STIM1) and STIM2. Here, we show that mice with a Foxp3+ Treg cell-specific deletion of Stim1 and Stim2 develop a phenotype that fulfills all classification criteria of human SjD. Mutant mice have salivary and lacrimal gland inflammation characterized by strong lymphocyte infiltration and transcriptional signatures dominated by T helper 1 (TH1) and interferon (IFN) signaling. CD4+ T cells from mutant mice are sufficient to induce SjD-like disease in an IFN-γ-dependent manner. Inhibition of IFN signaling with the JAK1/2 inhibitor baricitinib alleviated CD4+ T cell-induced SjD in mice. These findings are consistent with the transcriptional profiles of CD4+ T cells from patients with SjD, which indicate enhanced TH1 but reduced memory Treg cell function. Together, our study provides evidence for a critical role of dysfunctional Treg cells and IFN-γ-producing TH1 cells in the pathogenesis of SjD.

Alterations of the IKZF1-IKZF2 tandem in immune cells of schizophrenia patients regulate associated phenotypes

Schizophrenia is a complex multifactorial disorder and increasing evidence suggests the involvement of immune dysregulations in its pathogenesis. We observed that IKZF1 and IKZF2, classic immune-related transcription factors (TFs), were both downregulated in patients’ peripheral blood mononuclear cells (PBMCs) but not in their brain. We generated a new mutant mouse model with a reduction in Ikzf1 and Ikzf2 to study the impact of those changes. Such mice developed deficits in the three dimensions (positive–negative-cognitive) of schizophrenia-like phenotypes associated with alterations in structural synaptic plasticity. We then studied the secretomes of cultured PBMCs obtained from patients and identified potentially secreted molecules, which depended on IKZF1 and IKZF2 mRNA levels, and that in turn have an impact on neural synchrony, structural synaptic plasticity and schizophrenia-like symptoms in in vivo and in vitro models. Our results point out that IKZF1-IKZF2-dependent immune signals negatively impact on essential neural circuits involved in schizophrenia.

A sex-specific effect of M4 muscarinic cholinergic autoreceptor deletion on locomotor stimulation by cocaine and scopolamine

ObjectiveAcetylcholine modulates the activity of the direct and indirect pathways within the striatum through interaction with muscarinic M4 and M1 receptors. M4 receptors are uniquely positioned to regulate plasticity within the direct pathway and play a substantial role in reward and addiction-related behaviors. However, the role of M4 receptors on cholinergic neurons has been less explored. This study aims to fill this gap by addressing the role of M4 receptors on cholinergic neurons in these behaviors.MethodsTo investigate the significance of M4-dependent inhibitory signaling in cholinergic neurons we created mutant mice that lack M4 receptors on cholinergic neurons. Cholinergic neuron-specific depletion was confirmed using in situ hybridization. We aimed to untangle the possible contribution of M4 autoreceptors to the effects of the global M4 knockout by examining aspects of basal locomotion and dose-dependent reactivity to the psychostimulant and rewarding properties of cocaine, haloperidol-induced catalepsy, and examined both the anti-cataleptic and locomotion-inducing effects of the non-selective anticholinergic drug scopolamine.ResultsBasal phenotype assessment revealed no developmental deficits in knockout mice. Cocaine stimulated locomotion in both genotypes, with no differences observed at lower doses. However, at the highest cocaine dose tested, male knockout mice displayed significantly less activity compared to wild type littermates (p = 0.0084). Behavioral sensitization to cocaine was similar between knockout and wild type mice. Conditioned place preference tests indicated no differences in the rewarding effects of cocaine between genotypes. In food-reinforced operant tasks knockout and wild type mice successfully acquired the tasks with comparable performance results. M4 receptor depletion did not affect haloperidol-induced catalepsy and scopolamine reversal of catalepsy but attenuated scopolamine-induced locomotion in females (p = 0.04). Our results show that M4 receptor depletion attenuated the locomotor response to high doses of cocaine in males and scopolamine in females, suggesting sex-specific regulation of cholinergic activity.ConclusionDepletion of M4 receptors on cholinergic neurons does not significantly impact basal behavior or cocaine-induced hyperactivity but may modulate the response to high doses of cocaine in male mice and the response to scopolamine in female mice. Overall, our findings suggest that M4-dependent autoregulation plays a minor but delicate role in modulating specific behavioral responses to pharmacological challenges, possibly in a sex-dependent manner.

Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.

Inositol 1,4,5-Trisphosphate Receptor 1 Gain-of-Function Increases the Risk for Cardiac Arrhythmias in Mice and Humans.

Ca2+ mishandling in cardiac Purkinje cells is a well-known cause of cardiac arrhythmias. The Purkinje cell resident inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is believed to play an important role in Ca2+ handling, and ITPR1 gain-of-function (GOF) has been implicated in cardiac arrhythmias. However, nearly all known disease-associated ITPR1 variants are loss-of-function and are primarily linked to neurological disorders. Whether ITPR1 GOF has pathological consequences, such as cardiac arrhythmias, is unclear. This study aimed to identify human ITPR1 GOF variants and determine the impact of ITPR1 GOF on Ca2+ handling and arrhythmia susceptibility. There are a large number of rare ITPR1 missense variants reported in open data repositories. Based on their locations in the ITPR1 channel structure, we selected and characterized 33 human ITPR1 missense variants from open databases and identified 21 human ITPR1 GOF variants. We generated a mouse model carrying a human ITPR1 GOF variant, ITPR1-W1457G (W1447G in mice). We showed that the ITPR1-W1447G± and recently reported ITPR1-D2594K± GOF mutant mice were susceptible to stress-induced ventricular arrhythmias. Confocal Ca2+ and voltage imaging in situ in heart slices and Ca2+ imaging and patch-clamp recordings of isolated Purkinje cells showed that ITPR1-W1447G± and ITPR1-D2594K± variants increased the occurrence of stress-induced spontaneous Ca2+ release, delayed afterdepolarization, and triggered activity in Purkinje cells. To assess the potential role of ITPR1 variants in arrhythmia susceptibility in humans, we looked up a gene-based association study in the UK Biobank data set and identified 7 rare ITPR1 missense variants showing potential association with cardiac arrhythmias. Remarkably, in vitro functional characterization revealed that all these 7 ITPR1 variants resulted in GOF. Our studies in mice and humans reveal that enhanced function of ITPR1, a well-known movement disorder gene, increases the risk for cardiac arrhythmias.

Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease.

Angiopoietin-like 4 (ANGPTL4), a key protein involved in lipoprotein metabolism, has diverse effects. There is an association between Angptl4 and diabetic kidney disease; however, this association has not been well investigated. We show that both podocyte- and tubule-specific ANGPTL4 are crucial fibrogenic molecules in diabetes. Diabetes accelerates the fibrogenic phenotype in control mice but not in ANGPTL4 mutant mice. The protective effect observed in ANGPTL4 mutant mice is correlated with a reduction in stimulator of interferon genes pathway activation, expression of pro-inflammatory cytokines, reduced epithelial-to-mesenchymal transition and endothelial-to-mesenchymal transition, lessened mitochondrial damage, and increased fatty acid oxidation. Mechanistically, we demonstrate that podocyte- or tubule-secreted Angptl4 interacts with Integrin β1 and influences the association between dipeptidyl-4 with Integrin β1. We demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the kidneys and protects diabetic kidneys from proteinuria and fibrosis. Together, these data demonstrate that podocyte- and tubule-derived Angptl4 is fibrogenic in diabetic kidneys.

Lithium normalizes ASD-related neuronal, synaptic, and behavioral phenotypes in DYRK1A-knockin mice.

Dyrk1A deficiency is linked to various neurodevelopmental disorders, including developmental delays, intellectual disability (ID) and autism spectrum disorders (ASD). Haploinsufficiency of Dyrk1a in mice reportedly leads to ASD-related phenotypes. However, the key pathological mechanisms remain unclear and human DYRK1A mutations remain uncharacterized in mice. Here, we generated and studied Dyrk1a-knockin mice carrying a human ASD patient mutation (Ile48LysfsX2; Dyrk1a-I48K mice). These mice display severe microcephaly, social and cognitive deficits, dendritic shrinkage, excitatory synaptic deficits, and altered phospho-proteomic patterns enriched for multiple signaling pathways and synaptic proteins. Early chronic lithium treatment of newborn mutant mice rescues the brain volume, behavior, dendritic, synaptic, and signaling/synapse phospho-proteomic phenotypes at juvenile and adult stages. These results suggest that signaling/synaptic alterations contribute to the phenotypic alterations seen in Dyrk1a-I48K mice, and that early correction of these alterations by lithium treatment has long-lasting effects in preventing juvenile and adult-stage phenotypes.

Early introduction of IL-10 weakens BCG revaccination’s protection by suppressing CD4+Th1 cell responses

BackgroundThe Bacillus Calmette-Guérin (BCG) vaccine, currently the sole authorized vaccine against tuberculosis (TB), demonstrates limited effectiveness in safeguarding adolescents and adults from active TB, even when administered as a booster with either BCG itself or heterologous vaccine candidates. To effectively control the persistent epidemic of adult TB, it is imperative to investigate the mechanisms responsible for the suboptimal efficacy of the BCG prime-boosting strategy against primary Mycobacterium tuberculosis (M.tb) infection.MethodsC57BL/6J mice were immunized with the BCG vaccine either once or twice, followed by analysis of lung tissue to assess changes in cytokine levels. Additionally, varying intervals between vaccinations and detection times were examined to study IL-10 expression across different organs. IL-10-expressing cells in the lungs, spleen, and lymph nodes were analyzed through FACS and intracellular cytokine staining (ICS). BCG-revaccinated IL-10−/− mutant mice were compared with wild-type mice to evaluate antigen-specific IgG antibody and T cell responses. Protection against M.tb aerosol challenge was evaluated in BCG-revaccinated mice, either untreated or treated with anti-IL-10R monoclonal antibody.ResultsIL-10 was significantly upregulated in the lungs of BCG-revaccinated mice shortly after the booster immunization. IL-10 expression peaked in the lungs 3–6 weeks post-revaccination and was also detected in lymph nodes and spleen as early as 2 weeks following the booster dose, regardless of the intervals between the prime and booster vaccinations. The primary sources of IL-10 in these tissues were identified as macrophages and dendritic cells. Blocking IL-10 signaling in BCG-revaccinated mice—either by using IL-10−/− mutant mice or administering anti-IL-10R monoclonal antibody increased levels of antigen-specific IFN-γ+ or IL-2+ CD4+ T cells, enhanced central and effector memory CD4+ T cell responses, and provided better protection against aerosol infection with 300 CFUs of M.tb.ConclusionOur findings are crucial for formulating effective immunization strategies related to the BCG vaccine and for developing efficacious adult TB vaccines.Graphical abstract

Eosinophilic inflammation that begins in the juvenile stage causes hydronephrosis and urothelial cancer in mutant mice

Obstructive hydronephrosis is caused by various factors such as chronic inflammation and tumors. Eosinophils and chitinase-like proteins (CLPs) are involved in the pathogenesis of hydronephrosis in mice; however, the specific mechanisms remain unknown. In this study, we morphologically analyzed a novel mouse model of obstructive hydronephrosis from onset to progression to clarify the effects of eosinophils and CLP on the development of hydronephrosis and tumorigenesis. The primary change was slight eosinophil infiltration of the ureteropelvic junction, even at 1 week of age, followed by a significant increase in CLP expression in the urothelium at 5 weeks of age, which led to proliferation of the urothelium. At 8 weeks of age, polyps with eosinophilic inflammation and urothelial hyperplasia expressing high levels of CLP formed at the ureteropelvic junction, leading to hydronephrosis. At 60 weeks of age, all mice with hydronephrosis exhibited chronic eosinophilic inflammation and adenomas that progressed to adenocarcinomas with high CLP expression. In summary, inflammation and epithelial proliferation at the ureteropelvic junction began with a single infiltration of eosinophils at the juvenile stage in mice. Eosinophilic inflammation is associated with the development of hydronephrosis and urothelial hyperplasia, which may progress to urothelial adenocarcinoma due to increased CLP expression.

Behavioral decline in Shank3Δex4–22 mice during early adulthood parallels cerebellar granule cell glutamatergic synaptic changes

BackgroundSHANK3, a gene encoding a synaptic scaffolding protein, is implicated in autism spectrum disorder (ASD) and is disrupted in Phelan-McDermid syndrome (PMS). Despite evidence of regression or worsening of ASD-like symptoms in individuals with PMS, the underlying mechanisms remain unclear. Although Shank3 is highly expressed in the cerebellar cortical granule cells, its role in cerebellar function and contribution to behavioral deficits in ASD models are unknown. This study investigates behavioral changes and cerebellar synaptic alterations in Shank3Δex4–22 mice at two developmental stages.MethodsShank3Δex4–22 wildtype, heterozygous, and homozygous knockout mice lacking exons 4–22 (all functional isoforms) were subjected to a behavioral battery in both juvenile (5–7 weeks old) and adult (3–5 months old) mouse cohorts of both sexes. Immunostaining was used to show the expression of Shank3 in the cerebellar cortex. Spontaneous excitatory postsynaptic currents (sEPSCs) from cerebellar granule cells (CGCs) were recorded by whole-cell patch-clamp electrophysiology.ResultsDeletion of Shank3 caused deficits in motor function, heightened anxiety, and repetitive behaviors. These genotype-dependent behavioral alterations were more prominent in adult mice than in juveniles. Reduced social preference was only identified in adult Shank3Δex4–22 knockout male mice, while self-grooming was uniquely elevated in males across both age groups. Heterozygous mice showed little to no changes in behavioral phenotypes in most behavioral tests. Immunofluorescence staining indicated the presence of Shank3 predominantly in the dendrite-containing rosette-like structures in CGCs, colocalizing with presynaptic markers of glutamatergic mossy fiber. Electrophysiological findings identified a parallel relationship between the age-related exacerbation of behavioral impairments and the enhancement of sEPSC amplitude in CGCs.LimitationsOther behavioral tests of muscle strength (grip strength test), memory (Barnes/water maze), and communication (ultrasonic vocalization), were not performed. Further study is necessary to elucidate how Shank3 modulates synaptic function at the mossy fiber-granule cell synapse in the cerebellum and whether these changes shape the behavioral phenotype.ConclusionsOur findings reveal an age-related exacerbation of behavioral impairments in Shank3Δex4–22 mutant mice. These results suggest that Shank3 may alter the function of glutamatergic receptors at the mossy fiber-cerebellar granule cell synapse as a potential mechanism causing cerebellar disruption in ASD.

The rate and spectrum of new mutations in mice inferred by long-read sequencing

All forms of genetic variation originate from new mutations, making it crucial to understand their rates and mechanisms. Here, we use long-read PacBio sequencing to investigate de novo mutations that accumulated in 12 inbred mouse lines derived from three commonly used inbred strains (C3H, C57BL/6, and FVB) maintained for 8-15 generations in a mutation accumulation (MA) experiment. We built chromosome-level genome assemblies based on the MA line founders' genomes, and then employed a combination of read and assembly-based methods to call the complete spectrum of new mutations. On average, there are ~45 mutations per haploid genome per generation, about half of which (54%) are insertions and deletions shorter than 50 bp (indels). The remainder are single nucleotide mutations (SNMs, 44%) and large structural mutations (SMs, 2%). We found that the degree of DNA repetitiveness is positively correlated with SNM and indel rates, and that a substantial fraction of SMs can be explained by homology-dependent mechanisms associated with repeat sequences. Most (90%) indels can be attributed to microsatellite contractions and expansions, and there is a marked bias towards 4 bp indels. Among the different types of SMs, tandem repeat mutations have the highest mutation rate, followed by insertions of transposable elements (TEs). We uncover a rich landscape of active TEs, and notable differences in their spectrum among MA lines and strains, and a high rate of gene retroposition. Our study offers novel insights into mammalian genome evolution, and highlights the importance of repetitive elements in shaping genomic diversity.

R183Q GNAQ Sturge–Weber Syndrome Leptomeningeal and Cerebrovascular Developmental Mouse Model

Objective(s): Sturge–Weber syndrome (SWS), a rare neurovascular malformation disorder, is usually caused by the R183Q GNAQ somatic mosaic mutation enriched in brain endothelial cells. A developmental mouse model of SWS brain involvement is needed to investigate mutation impact upon brain vascular development and to facilitate preclinical drug studies. Methods: A new Tet-ON R183Q GNAQ transgenic mouse line was paired with rtTA tet transactivator mice under the Tie2 promoter to generate mice expressing endothelial R183Q GNAQ in the presence of doxycycline. Litters were perfused at P14-17; half received a subseizure dose (1.5 mg/kg; intraperitoneal) of kainate an hour before perfusion. A subset was perfused with Evans blue. Fixed mouse brains were stained with X-gal, DAPI, and antibodies for Gαq, Tie2, phosphorylated-S6, and claudin-5. Images were scored for vessel staining intensity. Results: X-gal staining was seen only in mutant mice; leptomeningeal endothelial X-gal staining was more frequent in kainate-treated mice (P < 0.001). When perfused with Evans blue, only mutant brains showed severe staining (P = 0.028). Median phosphorylated-S6 vessel scores were significantly higher in the leptomeninges of mutant mice (P = 0.035). Mutant cortical microvessels demonstrated discontinuous claudin-5 and phosphorylated-S6 staining as well as increased vessel length in kainate-treated mice (P = 0.024). Conclusions: The new R183Q GNAQ Tet-ON developmental mouse brain model of SWS demonstrates endothelial expression of mutant Gαq associated with blood–brain barrier breakdown, altered vascular mammalian target of rapamycin activity, and abnormal cortical microvessel structure. This new translational model can be used to develop new drug targets and treatments for SWS.

Deletion of AT1a receptors selectively in the proximal tubules of the kidney alters the hypotensive and natriuretic response to atrial natriuretic peptide via NPRA/cGMP/NO signaling.

In the proximal tubules of the kidney, angiotensin II (ANG II) binds and activates ANG II type 1 (AT1a) receptors to stimulate proximal tubule Na+ reabsorption, whereas atrial natriuretic peptide (ANP) binds and activates natriuretic peptide receptors (NPRA) to inhibit ANG II-induced proximal tubule Na+ reabsorption. These two vasoactive systems play important counteracting roles to control Na+ reabsorption in the proximal tubules and help maintain blood pressure homeostasis. However, how AT1a and NPRA receptors interact in the proximal tubules and whether natriuretic effects of NPRA receptor activation by ANP may be potentiated by deletion of AT1 (AT1a) receptors selectively in the proximal tubules have not been studied previously. The present study used a novel mouse model with proximal tubule-specific knockout of AT1a receptors, PT-Agtr1a-/-, to test the hypothesis that deletion of AT1a receptors selectively in the proximal tubules augments the hypotensive and natriuretic responses to ANP. Basal blood pressure was about 16 ± 3 mmHg lower (P < 0.01), fractional proximal tubule Na+ reabsorption was significantly lower (P < 0.05), whereas 24-h urinary Na+ excretion was significantly higher, in PT-Agtr1a-/- mice than in wild-type mice (P < 0.01). Infusion of ANP via osmotic minipump for 2 wk (0.5 mg/kg/day ip) further significantly decreased blood pressure and increased the natriuretic response in PT-Agtr1a-/- mice by inhibiting proximal tubule Na+ reabsorption compared with wild-type mice (P < 0.01). These augmented hypotensive and natriuretic responses to ANP in PT-Agtr1a-/- mice were associated with increased plasma and kidney cGMP levels (P < 0.01), kidney cortical NPRA and NPRC mRNA expression (P < 0.05), endothelial nitric oxide (NO) synthase (eNOS) and phosphorylated eNOS proteins (P < 0.01), and urinary NO excretion (P < 0.01). Taken together, the results of the present study provide further evidence for important physiological roles of intratubular ANG II/AT1a and ANP/NPRA signaling pathways in the proximal tubules to regulate proximal tubule Na+ reabsorption and maintain blood pressure homeostasis.NEW & NOTEWORTHY This study used a mutant mouse model with proximal tubule-selective deletion of angiotensin II (ANG II) type 1 (AT1a) receptors to study, for the first time, important interactions between ANG II/AT1 (AT1a) receptor/Na+/H+ exchanger 3 and atrial natriuretic peptide (ANP)/natriuretic peptide receptor (NPRA)/cGMP/nitric oxide signaling pathways in the proximal tubules. The results of the present study provide further evidence for important physiological roles of proximal tubule ANG II/AT1a and ANP/NPRA signaling pathways in the regulation of proximal tubule Na+ reabsorption and blood pressure homeostasis.

Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

Macroautophagy (autophagy) involves the formation of phagophores that mature into autophagosomes. The impact of inhibiting autophagosome closure remains unclear. Here, we report the generation and analysis of mice with impaired autophagosome closure by targeting the ubiquitin E2 variant-like (UEVL) β strands of the endosomal sorting complex required for transport (ESCRT) I subunit VPS37A. The VPS37A UEVL mutation (Δ43-139) impairs bulk autophagic flux without disrupting ESCRT-I complex assembly and endosomal function. Homozygous mutant mice exhibit signs of autophagy impairment, including p62/SQSTM1 and ubiquitinated protein accumulation, neuronal dysfunction, growth retardation, antioxidant gene upregulation, and tissue abnormalities. However, about half of the mutant neonates survive to adulthood without severe liver injury. LC3 proximity proteomics reveals that the VPS37A UEVL mutation leads to active TANK-binding kinase 1 (TBK1) accumulation on phagophores, resulting in increased p62 phosphorylation and inclusion formation. These findings reveal a previously unappreciated role of LC3-conjugated phagophores in facilitating protein aggregation and sequestration, potentially alleviating proteotoxicity.

Retinal pigment epithelial cells reduce vascular leak and proliferation in retinal neovessels

In multiple neurodegenerative diseases, including age-related macular degeneration, retinitis pigmentosa, and macular telangiectasia type 2 (MacTel), retinal pigment epithelial (RPE)-cells proliferate and migrate into the neuroretina, forming intraretinal pigment plaques. Though these pigmentary changes are hallmarks of disease progression, it is unknown if their presence is protective or detrimental.Here, we first evaluated the impact of pigment plaques on vascular changes and disease progression in MacTel. In a retrospective, longitudinal study, we analyzed multimodal retinal images of patients with MacTel and showed that pigment plaques were associated with decreased vascular leakage and stabilized neovascular growth. We then modeled the underlying pathomechanisms of pigment plaque formation in aberrant neovascular growth using the very-low-density lipoprotein receptor mutant (Vldlr−/−) mouse. Our data indicated that during RPE-proliferation, migration and accumulation along neovessels RPE-cells underwent epithelial-mesenchymal transition (EMT). Pharmacologic inhibition of EMT in Vldlr−/− mice decreased pigment coverage, and exacerbated neovascular growth and vascular leakage.Our findings indicate that the proliferation, migration and perivascular accumulation of RPE-cells stabilize vascular proliferation and exudation, thereby exerting a protective effect on the diseased retina. We conclude that interfering with this “natural repair mechanism” may have detrimental effects on the course of the disease and should thus be avoided.

Instrumented swim test for quantifying motor impairment in rodents.

Swim tests are highly effective for identifying vestibular deficits in rodents by offering significant vestibular motor challenges with reduced proprioceptive input, unlike rotarod and balance beam tests. Traditional swim tests rely on subjective assessments, limiting objective quantification and reproducibility. We present a novel instrumented swim test using a miniature motion sensor with a 3D accelerometer and 3D gyroscope affixed to the rodent's head. This setup robustly quantifies six-dimensional motion-three translational and three rotational axes-during swimming with high temporal resolution. We demonstrate the test's capabilities by comparing head movements of Gpr156-/- mutant mice, which have impaired otolith organ development, to their heterozygous littermates. Our results show axis-specific differences in head movement probability distribution functions and dynamics that identify mice with the Gpr156 mutation. Axis-specific power spectrum analyses reveal selective movement alterations within distinct frequency ranges. Additionally, our spherical visualization and 3D analysis quantifies swimming performance based on head vector distance from upright. We use this analysis to generate a single classifier metric-a weighted average of an animal's head deviation from upright during swimming. This metric effectively distinguishes animals with vestibular dysfunction from those with normal vestibular function. Overall, this instrumented swim test provides quantitative metrics for assessing performance and identifying subtle, axis- and frequency-specific deficits not captured by existing systems. This novel quantitative approach can enhance understanding of rodent sensorimotor function including enabling more selective and reproducible studies of vestibular-motor deficits.

Differential expression of keratin and keratin associated proteins are linked with hair loss condition in spontaneously mutated inbred mice

Hair loss condition is heritable and is influenced by multifactorial inheritance. In the present study, spontaneously mutated mice showed hair loss phenotype with defect in the first cycle of hair follicle formation leading to cyclic alopecia. These mutant mice follow autosomal recessive inheritance pattern. The transcriptomic profile and differential gene expression analysis of skin tissues by RNA-sequencing at different stages of hair cycle formation was performed. The genes with significant differential genes expression levels in each stage of hair cycle formation were identified and most of these genes were shown to be associated with keratinization process and hair follicle formation. Transcriptome profiling followed by QPCR validation revealed that mRNA levels of Krt16, Alox15, Fetub (upregulated) and Msx2 (downregulated) were significantly differentially expressed in mutant skin tissues during late anagen and catagen stages. Krt6b mRNA and protein levels were significantly higher in the mutant mice during all stages of first hair cycle formation. The present study provides basis for understanding the differential gene expression of hair-related genes, including keratinization-associated proteins and its relevance. These mutant mice can serve as a model for studying hair loss condition that can be further used in the identification, evaluation and treatment strategies for alopecia condition.

Synaptic and cognitive impairment associated with L444P heterozygous glucocerebrosidase mutation.

Cognitive impairment is a common but poorly understood non-motor aspect of Parkinson's disease, negatively affecting patient's functional capacity and quality of life. The mechanisms underlying cognitive impairment in Parkinson's disease are still elusive, limiting treatment and prevention strategies. This study investigates the molecular and cellular basis of cognitive impairment associated with heterozygous mutations in GBA1, the strongest risk gene for Parkinson's disease that encodes glucocerebrosidase (GCase), a lysosome enzyme that degrades the glycosphingolipid glucosylceramide into glucose and ceramide. Using a Gba1L444P/+ mouse model, we provide evidence that L444P heterozygous Gba1 mutation (L444P/+) causes hippocampus-dependent spatial and reference memory deficits independently of α-synuclein (αSyn) accumulation, GCase lipid substrate accumulation, dopaminergic dysfunction and motor deficits. The mutation disrupts hippocampal synaptic plasticity and basal synaptic transmission by reducing the density of hippocampal CA3-CA1 synapses, a mechanism that is dissociated from αSyn-mediated presynaptic neurotransmitter release. Using a well-characterized Thy1-αSyn pre-manifest Parkinson's disease mouse model overexpressing wild type human αSyn, we find that the L444P/+ mutation exacerbates hippocampal synaptic αSyn accumulation, synaptic and cognitive impairment in young Gba1L444P/+:Thy1-αSyn double mutant animals. With age, Thy1-αSyn mice manifest motor symptoms, and the double mutant mice exhibit more exacerbated synaptic and motor impairment than the Thy1-αSyn mice. Taken together, our results suggest that heterozygous L444P GBA1 mutation alone perturbs hippocampal synaptic structure and function, imposing a subclinical pathological burden for cognitive impairment. When co-existing αSyn overexpression is present, heterozygous L444P GBA1 mutation interacts with αSyn pathology to accelerate Parkinson's disease-related cognitive impairment and motor symptoms.

Mitochondrial Redox Status Regulates Glycogen Metabolism via Glycogen Phosphorylase Activity.

Mitochondria and glycogen are co-distributed in skeletal muscles to regulate the metabolic status. Mitochondria are also redox centers that regulate the muscle function during exercise. However, the pathophysiological relationship between the mitochondrial redox status and glycogen metabolism in the muscle remains unclear. In the present study, we examined the pathological effects of mitochondrial dysfunction induced by mitochondrial superoxide dismutase (SOD2) depletion on glycogen metabolism. We found that muscle glycogen was significantly accumulated in association with motor dysfunction in mice with a muscle-specific SOD2 deficiency. Muscle glycogen phosphorylase (GP-M) activity, which is a key enzyme for glycogen degradation at times when energy is needed (e.g., during exercise), was significantly decreased in the mutant muscle. Moreover, the GP-M activity on normal muscle sections decreased after treatment with paraquat, a superoxide generator. In contrast, treatment with antioxidants reversed the GP-M activity and motor disturbance of the mutant mice, indicating that GP-M activity was reversibly regulated by the redox balance. These results demonstrate that the maintenance of the mitochondrial redox balance regulates glycogen metabolism via GP-M activity.

Templating of Monomeric Alpha-Synuclein Induces Inflammation and SNpc Dopamine Neuron Death in a Genetic Mouse Model of Synucleinopathy.

While the etiology of most cases of Parkinson's disease (PD) are idiopathic, it has been estimated that 5-10% of PD arise from known genetic mutations. The first mutations described that leads to the development of an autosomal dominant form of PD are in the SNCA gene that codes for the protein alpha-synuclein (α-syn). α-syn is an abundant presynaptic protein that is natively disordered and whose function is still unclear. In PD, α-syn misfolds into multimeric b-pleated sheets that aggregate in neurons (Lewy Bodies/neurites) and spread throughout the neuraxis in a pattern that aligns with disease progression. Here, using IHC, HC, HPLC, and cytokine analysis, we examined the sequelae of intraparenchymal brain seeding of pre-formed fibrils (PFFs) and monomeric α-syn in C57BL/6J (WT) and A53T SNCA mutant mice. We found that injection of PFFs, but not monomeric α-syn, into the striatum of C57BL/6J mice induced spread of aggregated α-syn, loss of SNpc DA neurons and increased neuroinflammation. However, in A53T SNCA mice, we found that both PFFs and monomeric α-syn induced this pathology. This suggests that the conformation changes in α-syn seen in the A53T strain can recruit wild-type α-syn to a pathological misfolded conformation which may provide a mechanism for the induction of PD in humans with SNCA duplication/triplication.