DBA/2J Research Articles

Ninein, a candidate gene for ethanol anxiolysis, shows complex exon-specific expression and alternative splicing differences between C57BL/6J and DBA/2J mice.

Ethanol's anxiolytic actions contribute to increased consumption and the development of Alcohol Use Disorder (AUD). Our laboratory previously identified genetic loci contributing to the anxiolytic-like properties of ethanol in BXD recombinant inbred mice, derived from C57BL/6J (B6) and DBA/2J (D2) progenitor strains. That work identified Ninein (Nin) as a candidate gene underlying ethanol's acute anxiolytic-like properties in BXD mice. Nin has a complex exonic content with known alternative splicing events that alter cellular distribution of the NIN protein. We hypothesize that strain-specific differences in Nin alternative splicing contribute to changes in Nin gene expression and B6/D2 strain differences in ethanol anxiolysis. Using quantitative reverse-transcriptase PCR to target specific Nin splice variants, we identified isoform-specific exon expression differences between B6 and D2 mice in prefrontal cortex, nucleus accumbens and amygdala. We extended this analysis using deep RNA sequencing in B6 and D2 nucleus accumbens samples and found that total Nin expression was significantly higher in D2 mice. Furthermore, exon utilization and alternative splicing analyses identified eight differentially utilized exons and significant exon-skipping events between the strains, including three novel splicing events in the 3' end of the Nin gene that were specific to the D2 strain. Additionally, we document multiple single nucleotide polymorphisms in D2 Nin exons that are predicted to have deleterious effects on protein function. Our studies provide the first in-depth analysis of Nin alternative splicing in brain and identify a potential genetic mechanism altering Nin expression and function between B6 and D2 mice, thus possibly contributing to differences in the anxiolytic-like properties of ethanol between these strains. This work adds novel information to our understanding of genetic differences modulating ethanol actions on anxiety that may contribute to the risk for alcohol use disorder.

RNA-seq analysis highlights DNA replication and DNA repair associated with early-onset hearing loss in the cochlea of DBA/2J mice

AimsAge-related hearing loss (ARHL) is a significant health concern, and DBA/2J (D2) and C57BL/6 (B6) mouse strains serve as valuable models for its study. B6 mice, harboring a homozygous ahl allele in Cdh23, manifest high-frequency hearing loss at 3 months. In contrast, D2 mice, carrying the R109H variant of the Fascin-2 gene (Fscn2), experience early-onset hearing loss by 3 weeks. Yet, the underlying molecular mechanisms driving early-onset hearing loss in D2 mice remain elusive. This study aimed to identify novel genes and regulatory pathways as therapeutic targets for early deafness. Main methodsThis study employs RNA-sequencing (RNA-seq) to analyze cochlear mRNA expression at two different ages in D2 and B6 mice, respectively. The differentially expressed genes (DEGs) are uniquely associated with D2 mice by Venn diagram analysis. A protein–protein interaction (PPI) network is further constructed, followed by module analysis utilizing MCODE. Enrichment analysis of GO and KEGG pathways revealed biological functions and molecular pathways. The PPI network and VarElect analysis are conducted for genes within these pathways, facilitating the identification of pivotal genes based on scoring criteria. Subsequently, five genes are meticulously selected and validated through qRT-PCR. Key findingsNotably, 1181 DEGs are uniquely associated with D2 mice by Venn diagram analysis. GO and KEGG pathway enrichment analyses shed light on distinctive pathways in D2 mice, encompassing DNA replication, mismatch repair, base excision repair, and nucleotide excision repair, which are associated with apoptosis. Five genes involved in these pathways were finally selected and validated by qRT-PCR. Their down-regulation with age is consistent with RNA-seq result. SignificanceOur study underscores the potential implication of down-regulated genes associated with DNA replication and DNA damage repair in the early-onset hearing loss observed in D2 mice.

Generation and screening of mouse models of ADRD with multiple genetic manipulations to model dementia with mixed brain pathologies

AbstractBackgroundAlzheimer’s disease‐related dementias (ADRD) encompass a wide range of neurodegenerative diseases including Alzheimer’s disease, vascular dementia, frontotemporal dementia (FTD), Lewy body dementia (LBD), and mixed dementia [1‐4]. ADRDs are distinguished by the presence of pathologies including amyloid, tau, TDP‐43 inclusions, alpha‐synuclein aggregates, cerebral amyloid angiopathy, and/or hippocampal sclerosis [4‐9]. Consequently, the heterogeneity of ADRDs presents challenges for animal modeling of disease mechanisms. Our laboratory has demonstrated that genetic diversity in disease modeling is critical to recapitulate the molecular and cognitive features of AD [1,9, 10]. Here, we introduce multiple genetic manipulations to develop novel translational models of ADRD.MethodPrecision models of ADRD on multiple genetic backgrounds were generated via combinatorial CRISPR editing to knock‐in human disease‐causing mutations onto the C57BL/6J (B6) strain and crossed to either B6, DBA/2J (D2), FVB, or WSB to generate a set of 20 unique ADRD models (Fig.1). The effect of homozygous mutations were evaluated on the B6 background. Each model was assessed using a high‐capacity multi‐domain go/no‐go screen to prioritize candidate strains for future ADRD modeling of drug discovery, preclinical testing, and disease pathology.ResultAssays included frailty, open field, y‐maze, rotarod, and contextual fear conditioning. Models with one or more late‐onset behavioral deficits (12 mo) were a “go”; whereas, early behavioral deficits (6 and 9 mo), aggression, high attrition, and low fecundity and additional factors were considered unsuitable (“no‐go”). We identified several promising models of ADRD that exhibited late‐onset frailty and motor deficits as function of mutation burden and genetic background (e.g., App/Psen1/Tardbp/Mapt on the D2 background). The FVB background strain exhibited early frailty deficits (6 mo), and thus considered unsuitable (“no‐go”). The WSB background strain exhibited reserve across multiple frailty and behavioral assays up to 12 mo.ConclusionOur results demonstrate that combining ADRD mutations in genetically distinct backgrounds is a promising strategy to generate new mouse models that better model the human mid and late‐life onset of deficits associated with ADRD. The resulting model(s) will be translationally relevant and provide important insight into disease mechanisms for the future of ADRD research and drug discoveries.

ApoE variant associated with cognitive resilience alters AD associated hyperexcitability in dentate granule neurons

AbstractBackgroundCognitive decline in Alzheimer’s disease (AD) differs based on background genetics even with similar pathology. Using genetic linkage analysis, we identified a coding variant in the receptor binding domain of ApoE that leads to changes in cognitive performance across a genetically diverse population harboring familial (FAD) mutations known as the AD‐BXDs (Neuner et al., 2019). Using CRISPR‐based gene editing, we developed a C57BL/6J (B6) mouse carrying the ApoE allele from the DBA/2J (D2) strain (ApoEE163D) and determined that ApoEE163D mice with 5XFAD transgene (ApoEE163D‐5XFAD) are susceptible to cognitive decline compared to B6‐5XFAD mice (Kaczorowski, et al., 2022).Analysis of hippocampal RNA sequencing profiles from the hippocampus indicates robust transcriptomic changes in dentate gyrus (DG) granule cells associated with cognitive resilience to FAD mutations in the presence of the B6 ApoE genotype. Here we aim to evaluate the functional implications.MethodWe adapted the Patch‐Seq method (Cadwell et al., 2017) to investigate transcriptional and electrophysiological changes within the DG associated with both FAD mutations and ApoE genotype. Whole‐cell patch clamp was used to evaluate intrinsic excitability of granule cells from 14month old B6 mice carrying FAD mutations and ApoEE163D, compared to age matched controls. The cellular contents, including the nucleus, were collected after each recording to characterize the transcriptome, and the cells were filled and stained for downstream analysis of neuronal morphology.ResultWe observed a significant interaction of the 5XFAD and ApoE genotypes on DG granule cell excitability. Specifically, cells from ApoEE163D‐5XFAD required less current stimulus to both elicit a single action potential, and to fire in response to a current ramp (rheobase). Although input resistance was comparable across all 4 groups, the sag ratio of ApoEE163D‐5XFAD was also reduced.ConclusionEnhancement of DG excitability of ApoEE163D‐5XFAD mice may underlie increased seizure susceptibility that has been reported previously in FAD animal models on D2 genetic background and associated with increased risk of seizures in AD patients. Ongoing work will integrate DG excitability with single‐cell RNAseq, morphology, and behavior to dissect mechanisms underlying the modifier effect of ApoE genotype on susceptibility and resilience to cognitive deficits in FAD mutation carriers.

Genetic background influences the 5XFAD Alzheimer's disease mouse model brain proteome.

There is an urgent need to improve the translational validity of Alzheimer's disease (AD) mouse models. Introducing genetic background diversity in AD mouse models has been proposed as a way to increase validity and enable the discovery of previously uncharacterized genetic contributions to AD susceptibility or resilience. However, the extent to which genetic background influences the mouse brain proteome and its perturbation in AD mouse models is unknown. In this study, we crossed the 5XFAD AD mouse model on a C57BL/6J (B6) inbred background with the DBA/2J (D2) inbred background and analyzed the effects of genetic background variation on the brain proteome in F1 progeny. Both genetic background and 5XFAD transgene insertion strongly affected protein variance in the hippocampus and cortex (n = 3,368 proteins). Protein co-expression network analysis identified 16 modules of highly co-expressed proteins common across the hippocampus and cortex in 5XFAD and non-transgenic mice. Among the modules strongly influenced by genetic background were those related to small molecule metabolism and ion transport. Modules strongly influenced by the 5XFAD transgene were related to lysosome/stress responses and neuronal synapse/signaling. The modules with the strongest relationship to human disease-neuronal synapse/signaling and lysosome/stress response-were not significantly influenced by genetic background. However, other modules in 5XFAD that were related to human disease, such as GABA synaptic signaling and mitochondrial membrane modules, were influenced by genetic background. Most disease-related modules were more strongly correlated with AD genotype in the hippocampus compared with the cortex. Our findings suggest that the genetic diversity introduced by crossing B6 and D2 inbred backgrounds influences proteomic changes related to disease in the 5XFAD model, and that proteomic analysis of other genetic backgrounds in transgenic and knock-in AD mouse models is warranted to capture the full range of molecular heterogeneity in genetically diverse models of AD.

Vanadium Pentoxide Exposure Causes Strain-Dependent Changes in Mitochondrial DNA Heteroplasmy, Copy Number, and Lesions, but Not Nuclear DNA Lesions.

Interstitial lung diseases (ILDs) are lethal lung diseases characterized by pulmonary inflammation and progressive lung interstitial scarring. We previously developed a mouse model of ILD using vanadium pentoxide (V2O5) and identified several gene candidates on chromosome 4 associated with pulmonary fibrosis. While these data indicated a significant genetic contribution to ILD susceptibility, they did not include any potential associations and interactions with the mitochondrial genome that might influence disease risk. To conduct this pilot work, we selected the two divergent strains we previously categorized as V2O5-resistant C57BL6J (B6) and -responsive DBA/2J (D2) and compared their mitochondrial genome characteristics, including DNA variants, heteroplasmy, lesions, and copy numbers at 14- and 112-days post-exposure. While we did not find changes in the mitochondrial genome at 14 days post-exposure, at 112 days, we found that the responsive D2 strain exhibited significantly fewer mtDNA copies and more lesions than control animals. Alongside these findings, mtDNA heteroplasmy frequency decreased. These data suggest that mice previously shown to exhibit increased susceptibility to pulmonary fibrosis and inflammation sustain damage to the mitochondrial genome that is evident at 112 days post-V2O5 exposure.

Exploring Early-Stage Retinal Neurodegeneration in Murine Pigmentary Glaucoma: Insights From Gene Networks and miRNA Regulation Analyses.

Glaucoma is a group of heterogeneous optic neuropathies characterized by the progressive degeneration of retinal ganglion cells. However, the underlying mechanisms have not been understood completely. We aimed to elucidate the genetic network associated with the development of pigmentary glaucoma with DBA/2J (D2) mouse model of glaucoma and corresponding genetic control D2-Gpnmb (D2G) mice carrying the wild type (WT) Gpnmb allele. Retinas isolated from 13 D2 and 12 D2G mice were subdivided into 2 age groups: pre-onset (1-6months: samples were collected at approximately 1-2, 2-4, and 5-6months) and post-onset (7-15months: samples were collected at approximately 7-9, 10-12, and 13-15months) glaucoma were compared. Differential gene expression (DEG) analysis and gene-set enrichment analyses were performed. To identify micro-RNAs (miRNAs) that target Gpnmb, miRNA expression levels were correlated with time point matched mRNA expression levels. A weighted gene co-expression network analysis (WGCNA) was performed using the reference BXD mouse population. Quantitative real-time PCR (qRT-PCR) was used to validate Gpnmb and miRNA expression levels. A total of 314 and 86 DEGs were identified in the pre-onset and post-onset glaucoma groups, respectively. DEGs in the pre-onset glaucoma group were associated with the crystallin gene family, whereas those in the post-onset group were related to innate immune system response. Of 1329 miRNAs predicted to target Gpnmb, 3 miRNAs (miR-125a-3p, miR-3076-5p, and miR-214-5p) were selected. A total of 47 genes demonstrated overlapping with the identified DEGs between D2 and D2G, segregated into their time-relevant stages. Gpnmb was significantly downregulated, whereas 2 out of 3 miRNAs were significantly upregulated (P < 0.05) in D2 mice at both 3-and 10-month time points. These findings suggest distinct gene-sets involved in pre-and post-glaucoma in the D2 mouse. We identified three miRNAs regulating Gpnmb in the development of murine pigmentary glaucoma.

A mouse model of the 3-hit effects of stress: Genotype controls the effects of life adversities in females

Helplessness is a dysfunctional coping response to stressors associated with different psychiatric conditions. The present study tested the hypothesis that early and adult adversities cumulate to produce helplessness depending on the genotype (3-hit hypothesis of psychopathology). To this aim, we evaluated whether Chronic Unpredictable Stress (CUS) differently affected coping and mesoaccumbens dopamine (DA) responses to stress challenge by adult mice of the C57BL/6J (B6) and DBA/2J (D2) inbred strains depending on early life experience (Repeated Cross Fostering, RCF). Three weeks of CUS increased the helplessness expressed in the Forced Swimming Test (FST) and the Tail Suspension Test by RCF-exposed female mice of the D2 strain. Moreover, female D2 mice with both RCF and CUS experiences showed inhibition of the stress-induced extracellular DA outflow in the Nucleus Accumbens, as measured by in vivo microdialysis, during and after FST. RCF-exposed B6 mice, instead, showed reduced helplessness and increased mesoaccumbens DA release. The present results support genotype-dependent additive effects of early experiences and adult adversities on behavioral and neural responses to stress by female mice. To our knowledge, this is the first report of a 3-hit effect in an animal model. Finally, the comparative analyses of behavioral and neural phenotypes expressed by B6 and D2 mice suggest some translationally relevant hypotheses of genetic risk factors for psychiatric disorders.

Expression Levels of the Tnni3k Gene in the Heart Are Highly Associated with Cardiac and Glucose Metabolism-Related Phenotypes and Functional Pathways.

Troponin-I interacting kinase encoded by the TNNI3K gene is expressed in nuclei and Z-discs of cardiomyocytes. Mutations in TNNI3K were identified in patients with cardiac conduction diseases, arrhythmias, and cardiomyopathy. We performed cardiac gene expression, whole genome sequencing (WGS), and cardiac function analysis in 40 strains of BXD recombinant inbred mice derived from C57BL/6J (B6) and DBA/2J (D2) strains. Expression quantitative trait loci (eQTLs) mapping and gene enrichment analysis was performed, followed by validation of candidate Tnni3k-regulatory genes. WGS identified compound splicing and missense T659I Tnni3k variants in the D2 parent and some BXD strains (D allele) and these strains had significantly lower Tnni3k expression than those carrying wild-type Tnni3k (B allele). Expression levels of Tnni3k significantly correlated with multiple cardiac (heart rate, wall thickness, PR duration, and T amplitude) and metabolic (glucose levels and insulin resistance) phenotypes in BXDs. A significant cis-eQTL on chromosome 3 was identified for the regulation of Tnni3k expression. Furthermore, Tnni3k-correlated genes were primarily involved in cardiac and glucose metabolism-related functions and pathways. Genes Nodal, Gnas, Nfkb1, Bmpr2, Bmp7, Smad7, Acvr1b, Acvr2b, Chrd, Tgfb3, Irs1, and Ppp1cb were differentially expressed between the B and D alleles. Compound splicing and T659I Tnni3k variants reduce cardiac Tnni3k expression and Tnni3k levels are associated with cardiac and glucose metabolism-related phenotypes.

Voluntary wheel running and lithium supplementation promotes fatigue resistance, fat oxidation, and improves insulin tolerance in D2 mdx mice

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle wasting and premature death. Many DMD patients will also exhibit signs of metabolic dysfunction such as insulin resistance that predisposes them to type 2 diabetes and worsened cardiovascular disease. Most treatments for DMD aim to lower muscle inflammation and improve strength, however, none have addressed the complication of insulin resistance. The current standard of treatment is corticosteroids, which prolongs ambulation, however, chronic use increases susceptibility to obesity, insulin resistance, and type 2 diabetes. It is well-established that regular aerobic exercise enhances insulin sensitivity. Further, we have recently found that inhibiting glycogen synthase kinase 3 with lithium (Li), a known insulin mimetic, improves force production and fatigue resistance in wildtype (WT) mice. Here, we tested whether voluntary wheel running (VWR) and Li supplementation, together, could improve whole-body fatigue, metabolism, and insulin sensitivity in the DBA/2J (D2) mdx mouse model of DMD. METHODS: 5-week old male D2 WT and mdx mice were separated into four groups: WT, mdx sedentary (SED), mdx VWR, and mdx Li+VWR (n=11/group). The mdx VWR and mdx Li+VWR mice had unlimited access to a cagewheel, and mdx Li+VWR mice were given a low dose of Li (50 mg/kg/day) via their drinking water throughout the study. A treadmill time-to-fatigue test was performed to assess whole-body fatigue. Mice were housed for 48-hours in metabolic cages to measure energy expenditure. Glucose handling was measured using glucose and insulin tolerance tests. RESULTS: As expected, WT mice had ~2-fold greater total cage activity compared to all mdx groups. When examining cagewheel distance, we found that mdx Li+VWR mice ran half the total distance in kilometres than mdx VWR mice (39.1 ± 9.6 km vs. 74.8 ± 13.6 km). Despite this, VWR with and without Li improved whole-body fatigue as only mdx SED mice (22.8 ± 0.9 min) had a shorter time to exhaustion than WT mice (32.6 ± 0.9 min). All mdx groups had higher energy expenditure than WT mice, suggestive of a hypermetabolic phenotype. When examining the respiratory exchange ratio (RER) among mdx mice, mdxLi+VWR (0.87) mice had a lower RER than mdx SED mice (0.91), indicative of enhanced fat oxidation. Glucose tolerance was equally impaired among mdx groups compared to WT mice. However, insulin tolerance was notably improved in mdx VWR and mdx Li+VWR mice, with a greater improvement observed in the mdx Li+VWR mice. CONCLUSION: Despite running less than mdx VWR mice, mdx Li+VWR mice had enhanced fatigue resistance, fat utilization, and insulin sensitivity, suggesting Li may be a viable treatment to improve muscle and metabolic function in mdx mice. CIHR CRC (Tier II) to VAF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single amino acid change in the receptor binding domain of mouse APOE results in altered protein dynamics and worsened AD‐related cognitive impairment

AbstractBackground APOE genotype is the strongest genetic modifier of late‐onset Alzheimer’s disease (AD) in humans. In genetically diverse mice, natural variation in the Apoe locus predicts AD‐related cognitive decline, with C57BL/6J (B6) mice harboring a protective variant compared to DBA/2J (D2) (Neuner 2019, Neuron). The mechanisms by which protective and detrimental Apoe/APOE variants affect cognitive decline in AD are not fully understood yet, but likely could provide an opportunity for personalized treatment and prevention strategies.MethodHere, we have used CRISPR/Cas9 gene editing to introduce the D2 Apoe variant into the B6 genome. We crossed these heterozygous APOEB6/E163D mice to B6 congenic heterozygous 5XFAD carriers in order to explore the effects of this single variant on AD‐related cognitive function, pathology, cell‐type specific gene dysregulation, and hippocampal and cortical network function. We also performed molecular dynamics modeling to ask how this variant affects APOE protein biophysics.Result5XFAD‐APOEB6/E163D mice showed exacerbated cognitive dysfunction compared to 5XFAD‐APOEB6/B6 mice, indicating that the single D2 variant in Apoe significantly contributes to AD‐related cognitive decline. We did not observe differences in amyloid pathology or phosphorylated tau across Apoe genotypes, suggesting that the effect on cognitive function is not mediated by pathological protein aggregation. However, snRNAseq revealed that excitatory neuron gene expression was substantially dysregulated in 5XFAD‐APOEB6/E163D mice compared to other cell types; we then performed in vivo electrophysiology to characterize how this translates to circuit‐level neuronal dysfunction. Finally, computational modeling of protein structure and function showed that the APOEE163D variant results in protein states most closely resembling human APOE4, particularly in the presence of lipids, suggesting that APOEE163D may show impaired lipid binding and transport function.ConclusionC57BL/6J mice harbor a protective Apoe variant which confers resiliency to AD‐related cognitive dysfunction. This protection, and–by the same measure–the detrimental effects of the DBA/2J variant of Apoe (APOEE163D), is likely ultimately mediated by altered neuronal gene expression and activity. We are currently exploring the intermediate mechanisms between APOEE163D and neuronal function; altered APOE protein states hint at impairments in lipid binding and transport, which may result in pathological lipid and protein accumulation affecting neuron function.

Cardiac Troponin I-Interacting Kinase Affects Cardiomyocyte S-Phase Activity but Not Cardiomyocyte Proliferation.

Identifying genetic variants that affect the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity after permanent coronary artery ligation compared with infarcted DBA/2J (D2J) mice. Cardiomyocyte cell cycle activity after infarction was monitored in D2J, (D2J×B6N)-F1, and (D2J×B6N)-F1×D2J backcross mice by means of bromodeoxyuridine or 5-ethynyl-2'-deoxyuridine incorporation using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus analysis, fine scale genetic mapping, whole exome sequencing, and RNA sequencing analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype. (D2J×B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar compared with D2J mice (0.798±0.09% versus 0.056±0.004%; P<0.001). Quantitative trait locus analysis of (D2J×B6N)-F1×D2J backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of chromosome 3 (logarithm of the odds score=6.38; P<0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, Tnni3k (troponin I-interacting kinase) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of TNNI3K in a D2J genetic background results in elevated cardiomyocyte S-phase activity after injury. Cardiomyocyte S-phase activity in both Tnni3k-expressing and Tnni3k-nonexpressing mice results in the formation of polyploid nuclei. These data indicate that Tnni3k expression increases the level of cardiomyocyte S-phase activity after injury.

Influence of Sex and Strain on Hepatic and Adipose Tissue Trace Element Concentrations and Gene Expression in C57BL/6J and DBA/2J High Fat Diet Models.

The objective of this study was to determine the influence of sex and strain on the dysregulation of trace element concentration and associative gene expression due to diet induced obesity in adipose tissue and the liver. Male and female C57BL/6J (B6J) and DBA/2J (D2J) were randomly assigned to a normal-fat diet (NFD) containing 10% kcal fat/g or a mineral-matched high-fat diet (HFD) containing 60% kcal fat/g for 16 weeks. Liver and adipose tissue were assessed for copper, iron, manganese, and zinc concentrations and related changes in gene expression. Notable findings include three-way interactions of diet, sex, and strain amongst adipose tissue iron concentrations (p = 0.005), adipose hepcidin expression (p = 0.007), and hepatic iron regulatory protein (IRP) expression (p = 0.012). Cd11c to Cd163 ratio was increased in adipose tissue due to HFD amongst all biological groups except B6J females, for which tissue iron concentrations were reduced due to HFD (p = 0.002). Liver divalent metal transporter 1 (DMT-1) expression was increased due to HFD amongst B6J males (p &lt; 0.005) and females (p &lt; 0.004), which coincides with the reduction in hepatic iron concentrations found in these biological groups (p &lt; 0.001). Sex, strain, and diet affected trace element concentration, the expression of genes that regulate trace element homeostasis, and the expression of macrophages that contribute to tissue iron-handling in adipose tissue. These findings suggest that sex and strain may be key factors that influence the adaptive capacity of iron mismanagement in adipose tissue and its subsequent consequences, such as insulin resistance.

Loss of Retinogeniculate Synaptic Function in the DBA/2J Mouse Model of Glaucoma.

Retinal ganglion cell (RGC) axons comprise the optic nerve and carry information to the dorsolateral geniculate nucleus (dLGN), which is then relayed to the cortex for conscious vision. Glaucoma is a blinding neurodegenerative disease that commonly results from intraocular pressure (IOP)-associated injury leading to RGC axonal pathology, disruption of RGC outputs to the brain, and eventual apoptotic loss of RGC somata. The consequences of elevated IOP and glaucomatous pathology on RGC signaling to the dLGN are largely unknown yet are likely to contribute to vision loss. Here, we used anatomic and physiological approaches to study the structure and function of retinogeniculate (RG) synapses in male and female DBA/2J (D2) mice with inherited glaucoma before and after IOP elevation. D2 mice showed progressive loss of anterograde optic tract transport to the dLGN and vGlut2 labeling of RGC axon terminals while patch-clamp measurements of RG synaptic function showed that synaptic transmission was reduced in 9-month and 12-month D2 mice because of the loss of individual RGC axon inputs. TC neuron dendrites had reduced Sholl complexity at 12 months, suggestive of delayed reorganization following reduced synaptic input. There was no detectable change in RGC density in 11- to 12-month D2 retinas, quantified as the number of ganglion cell layer-residing somata immuno-positive for NeuN and immuno-negative for the amacrine marker choline acetyltransferase (ChAT). Thus, observed synaptic defects appear to precede RGC somatic loss. These findings identify glaucoma-associated and IOP-associated deficits in an important subcortical RGC projection target, shedding light on processes linking IOP to vision loss.

Genomic Basis for Individual Differences in Susceptibility to the Neurotoxic Effects of Diesel Exhaust.

Air pollution is a known environmental health hazard. A major source of air pollution includes diesel exhaust (DE). Initially, research on DE focused on respiratory morbidities; however, more recently, exposures to DE have been associated with neurological developmental disorders and neurodegeneration. In this study, we investigated the effects of sub-chronic inhalation exposure to DE on neuroinflammatory markers in two inbred mouse strains and both sexes, including whole transcriptome examination of the medial prefrontal cortex. We exposed aged male and female C57BL/6J (B6) and DBA/2J (D2) mice to DE, which was cooled and diluted with HEPA-filtered compressed air for 2 h per day, 5 days a week, for 4 weeks. Control animals were exposed to HEPA-filtered air on the same schedule as DE-exposed animals. The prefrontal cortex was harvested and analyzed for proinflammatory cytokine gene expression (Il1β, Il6, Tnfα) and transcriptome-wide response by RNA-seq. We observed differential cytokine gene expression between strains and sexes in the DE-exposed vs. control-exposed groups for Il1β, Tnfα, and Il6. For RNA-seq, we identified 150 differentially expressed genes between air and DE treatment related to natural killer cell-mediated cytotoxicity per Kyoto Encyclopedia of Genes and Genomes pathways. Overall, our data show differential strain-related effects of DE on neuroinflammation and neurotoxicity and demonstrate that B6 are more susceptible than D2 to gene expression changes due to DE exposures than D2. These results are important because B6 mice are often used as the default mouse model for DE studies and strain-related effects of DE neurotoxicity warrant expanded studies.

Sarco(endo)plasmic reticulum Ca2+-ATPase function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice

SummaryThe DBA/2J (D2) mdx mouse is a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. Both D2 and C57 mdx mice exhibited signs of impaired Ca2+ uptake in the gastrocnemius, diaphragm, and left ventricle; however, the level of impairment was more severe in D2 mdx mice. Reductions in maximal SERCA activity were also more prominent in the D2 mdx gastrocnemius and diaphragm when compared to those from C57 mdx mice; however, there were no differences detected in the left ventricle. Across all muscles, D2 mdx mice had the highest levels of oxidative stress as indicated by protein nitrosylation and/or nitration. In conclusion, our study shows that SERCA function is more impaired in young D2 mdx mice compared with age-matched C57 mdx mice.

Kynurenine metabolism is altered in mdx mice: a potential muscle to brain connection.

What is the central question in this study? Promoting muscle health with regular aerobic exercise can improve mental health through a kynurenine metabolic pathway: do conditions of muscle disease such as muscular dystrophy negatively influence this pathway? What is the main finding and its importance? The DBA/2J mdx model of Duchenne muscular dystrophy exhibits altered kynurenine metabolism with less kynurenic acid and peroxisome proliferator-activated receptor-γ coactivator 1-α and higher levels of tumour necrosis factor α mRNA- results associated with anxiety-like behaviour. Regular exercise can direct muscle kynurenine (KYN) metabolism toward the neuroprotective branch of the kynurenine pathway thereby limiting the accumulation of neurotoxic metabolites in the brain and contributing to mental resilience. However, the effect of muscle disease on KYN metabolism has not yet been investigated. Previous work has highlighted anxiety-like behaviours in approximately 25% of patients with Duchenne muscular dystrophy (DMD), possibly due to altered KYN metabolism. Here, we characterized KYN metabolism in mdx mouse models of DMD. Young (8-10week old) DBA/2J (D2) mdx mice, but not age-matched C57BL/10 (C57) mdx mice, had lower levels of circulating kynurenic acid (KYNA) and lower KYNA:KYN ratio compared with their respective wild-type (WT) controls. While both C57 and D2 mdx mice displayed signs of anxiety-like behaviour, spending more time in the corners of the arena during a novel object recognition test, this effect was more prominent in D2 mdx mice. Correlational analysis detected a significant negative association between KYNA:KYN levels and time spent in corners in D2 mice, but not C57 mice. In extensor digitorum longus muscles from D2 mdx mice, but not C57 mdx mice, we found lowered protein levels of peroxisome proliferator-activated receptor-γ coactivator 1-α and kynurenine amino transferase-1 enzyme when compared with WT. Furthermore, D2 mdx quadriceps muscles had the highest level of tumour necrosis factor α expression, which is suggestive of enhanced inflammation. Thus, our pilot work shows that KYN metabolism is altered in D2 mdx mice, with a potential contribution from altered muscle health.

Genotypic Differences in the Effects of Menthol on Nicotine Intake and Preference in Mice

Menthol has been shown to exacerbate elements of nicotine addiction in humans and rodents; however, the mechanisms mediating its effects are not fully understood. This study examined the impact of genetic factors in menthol’s effects on oral nicotine consumption by comparing two inbred mouse strains with differing sensitivities to nicotine. C57BL/6J (B6J) mice are nicotine-preferring, while DBA/2J (D2J) mice are not. While the effects of menthol on oral nicotine consumption have been highlighted in B6J mice, it is unknown if they extend to the D2J strain as well. Consequently, adolescent (PND 21) and adult (PND 63), male and female D2J mice were subjected to the nicotine two-bottle choice (2BC) paradigm with orally and systemically administered menthol. Then, we evaluated its impact on nicotine pharmacological responses in conditioned reward and nociception after systemic administration and, lastly, investigated the potential involvement of the TAAR1 gene and α7 nAChRs in menthol’s effects. Menthol failed to enhance oral nicotine consumption in adult and adolescent female and male D2J mice. Moreover, this lack in effect was not due to nicotine concentration, oral aversion to menthol, or basal preference for nicotine. Menthol also failed to augment nicotine reward or enhance nicotine-induced antinociception in D2J mice, demonstrating that genetic background plays a significant role in sensitivity to menthol’s effects on nicotine. Furthermore, TAAR1 or α7 nAChRs did not seem to mediate menthol’s differential effects in D2J mice. These findings support the existence of genotype-specific mechanisms that may contribute to the variable effects of menthol in different populations.

Epigenetic Clocks for Mice Based on Age-Associated Regions That are Conserved Between Mouse Strains and Human.

Aging of mice can be tracked by DNA methylation changes at specific sites in the genome. In this study, we used the recently released Infinium Mouse Methylation BeadChip to compare such epigenetic modifications in C57BL/6 (B6) and DBA/2J (DBA) mice. We observed marked differences in age-associated DNA methylation in these commonly used inbred mouse strains, indicating that epigenetic clocks for one strain cannot be simply applied to other strains without further verification. In B6 mice age-associated hypomethylation prevailed with focused hypermethylation at CpG islands, whereas in DBA mice CpG islands revealed rather hypomethylation upon aging. Interestingly, the CpGs with highest age-correlation were still overlapping in B6 and DBA mice and included the genes Hsf4, Prima1, Aspa, and Wnt3a. Notably, Hsf4 and Prima1 were also top candidates in previous studies based on whole genome deep sequencing approaches. Furthermore, Hsf4, Aspa, and Wnt3a revealed highly significant age-associated DNA methylation in the homologous regions in human. Subsequently, we used pyrosequencing of the four relevant regions to establish a targeted epigenetic clock that provided very high correlation with chronological age in independent cohorts of B6 (R 2 = 0.98) and DBA (R 2 = 0.91). Taken together, the methylome differs extensively between B6 and DBA mice, while prominent age-associated changes are conserved among these strains and even in humans. Our new targeted epigenetic clock with 4 CpGs provides a versatile tool for other researchers analyzing aging in mice.

Analysis of cardiac function and morphology in genetic reference population of BXD strains reveals associated eQTLs and candidate causal and modifier genes

IntroductionThe murine recombinant inbred (RI) family of BXD strains derived from crosses between C57BL/6J (B6) and DBA/2J (D2) mice are the largest genetic reference population (GRP). The parental D2 mouse is a natural model of hypertrophic cardiomyopathy (HCM). Thus, the BXD family represents an applicable rodent model for phenotype‐genotype studies of complex cardiomyopathy traits.HypothesisGenetic background affects an expression of cardiomyopathy traits.MethodsEchocardiography, electrocardiography (ECG) and blood pressure measurements were performed in 44 BXD strains (N&gt;5/sex) at 4‐5 months of age. Cardiac traits (heart function, mass, rhythm, and arrhythmias) and blood pressure values were then associated with heart transcriptome, and expression quantitative trait loci (eQTL) mapping was performed.ResultsMore than 2‐fold variance in ejection fraction (EF%), fractional shortening (FS%), left ventricular (LV) volumes, internal dimensions (ID) and mass, as well as in thickness of LV posterior wall (LVPW), and interventricular septum (IVS) was found by echocardiography among BXDs. Varied abnormal heart rhythms including bradycardia, atrioventricular block, premature atrial or ventricular complexes, ventricular tachycardia and sick sinus syndrome were recorded among BXD strains using ECG. Parental D2 strains had significantly prolonged QT interval (62.82±9.42 ms and 53.42±1.73 ms, respectively) compared to B6 parental strains. A significant variability in systolic and diastolic BP between BXD strains was also recorded.Among male BXDs, a significant QTL of 91.0 to 97.4 Mb (LRS=18.50) was identified on chromosome (Chr) 8 that is associated with LV volumes and IDs during systole and diastole. eQTL mapping for each of the 131 genes at the QTL interval of Chr 8 identified 6 genes (Coq9, Ndrg4, Crnde, Irx3, Rpgrip1l, and Rbl2) being cis‐regulated. Among those genes, Ndrg4was found to be positively correlated with LV volumes at systole and diastole and ID traits, and negatively correlated with the cardiomyocyte size (r = ‐0.588, p = 0.025). In female BXDs, a significant QTL with LRS=20.1 was found on Chr 7 of 40.2 Mb that is associated with LVPW at diastole, where Josd2is a strong candidate gene that is cis‐regulated and significantly correlated with LVPW trait.ConclusionsOur study found variable traits of echocardiographic, electrocardiographic and blood pressure parameters are segregated among the BXD family. The cardiovascular traits significantly vary between BXD lines suggesting an influence of genetic background on expression of those traits. Traits such as reduced EF%, FS%, and LVPW and increased LV volumes are seen in patients with dilated cardiomyopathy (DCM). Increased LV mass, LV thickness, LVPW, and IVS mimic traits seen in patients with HCM. Varied abnormal heart rhythms detected in BXD strains simulate cardiac conduction abnormalities in humans. Multiple associated eQTLs defined in BXDs demonstrate that the BXD family of RI strains is a suitable model to map gene variants and identify causal genes and genetic modifiers that influence cardiovascular phenotypes including cardiomyopathies.