Recombinant Inbred Research Articles

Systems genetics identifies methionine as a high risk factor for Alzheimer's disease.

As a dietary strategy, methionine restriction has been reported to promote longevity and regulate metabolic disorders. However, the role and possible regulatory mechanisms underlying methionine in neurodegenerative diseases such as Alzheimer's disease (AD), remain unexplored. This study utilized the data from BXD recombinant inbred (RI) mice to establish a correlation between the AD phenotype in mice and methionine level. Gene enrichment analysis indicated that the genes associated with the concentration of methionine in the midbrain are involved in the dopaminergic synaptic signaling pathway. Protein interaction network analysis revealed that glycogen synthase kinase 3 beta (GSK-3β) was a key regulator of the dopaminergic synaptic pathway and its expression level was significantly correlated with the AD phenotype. Finally, in vitro experiments demonstrated that methionine deprivation could reduce the expression of Aβ and phosphorylated Tau, suggesting that lowering methionine levels in humans may be a preventive or therapeutic strategy for AD. In conclusion, our findings support that methionine is a high risk factor for AD. These findings predict potential regulatory network, theoretically supporting methionine restriction to prevent AD.

Spontaneous nonsense mutation in the tuftelin 1 gene is associated with abnormal hair appearance and amelioration of glucose and lipid metabolism in the rat.

Recently, we have identified a recessive mutation, an abnormal coat appearance in the BXH6 strain, a member of the HXB/BXH set of recombinant inbred (RI) strains. The RI strains were derived from the spontaneously hypertensive rat (SHR) and Brown Norway rat (BN-Lx) progenitors. Whole genome sequencing of the mutant rats identified the 195875980 G/A mutation in the tuftelin 1 (Tuft1) gene on chromosome 2, which resulted in a premature stop codon. Compared with wild-type BXH6 rats, BXH6-Tuft1 mutant rats exhibited lower body weight due to reduced visceral fat and ectopic fat accumulation in the liver and heart. Reduced adiposity was associated with decreased serum glucose and insulin and increased insulin-stimulated glycogenesis in skeletal muscle. In addition, mutant rats had lower serum monocyte chemoattractant protein-1 and leptin levels, indicative of reduced inflammation. Analysis of the liver proteome identified differentially expressed proteins from fatty acid metabolism and β-oxidation, peroxisomes, carbohydrate metabolism, inflammation, and proteasome pathways. These results provide evidence for the important role of the Tuft1 gene in the regulation of lipid and glucose metabolism and suggest underlying molecular mechanisms.NEW & NOTEWORTHY A new spontaneous mutation, abnormal hair appearance in the rat, has been identified as a nonfunctional tuftelin 1 (Tuft1) gene. The pleiotropic effects of this mutation regulate glucose and lipid metabolism. Analysis of the liver proteome revealed possible molecular mechanisms for the metabolic effects of the Tuft1 gene.

Marginal Zone B (MZB) Cells: Comparison of the Initial Identification of Immune Activity Leading to Dacryoadenitis and Sialadenitis in Experimental Sjögren's Syndrome.

Although multiple mouse strains have been advanced as models for Sjögren's syndrome (SS), which is a human systemic autoimmune disease characterized primarily as the loss of lacrimal and salivary gland functions, the C57BL/6.NOD-Aec1Aec2 recombinant inbred (RI) mouse derived from the NOD/ShiLtJ line is considered one of the more appropriate models exhibiting virtually all the characteristics of the human disease. This mouse model, as well as other mouse models of SS, have shown that B lymphocytes are essential for the onset and development of observed clinical manifestations. Recently, studies carried out in the C57BL/6.IL14α transgenic mouse have provided clear evidence that the marginal zone B (MZB) cell population is directly involved in the early pathological events initiating the development of the clinical SS disease, as well as late-stage lymphomagenesis resulting in B-cell lymphomas. Since MZB cells are difficult to study in vivo and in vitro, we carried out a series of ex vivo investigations that utilize temporal global RNA transcriptomic analyses to profile differentially expressed genes exhibiting temporal upregulation during the initial onset and subsequent development of pathophysiological events within the lacrimal and salivary gland tissues per se or associated with the leukocyte cell migrations into these glands. The initial transcriptomic analyses revealed that while the upregulated gene expression profiles obtained from lacrimal and salivary glands overlap, multiple genetic differences exist between the defined activated pathways. In the current study, we present a concept suggesting that the initial pathological events differ between the two glands, yet the subsequent upregulated TLR4/TLR3 signal transduction pathway that activates the type-1 interferon signature appears to be identical in the two glands and indicates an autoimmune response against dsRNA, possibly a virus. Here, we attempt to put these findings into perspective and determine how they can impact the design of future therapeutic protocols.

A novel quantitative trait locus implicates Msh3 in the propensity for genome-wide short tandem repeat expansions in mice.

Short tandem repeats (STRs) are a class of rapidly mutating genetic elements typically characterized by repeated units of 1-6 bp. We leveraged whole-genome sequencing data for 152 recombinant inbred (RI) strains from the BXD family of mice to map loci that modulate genome-wide patterns of new mutations arising during parent-to-offspring transmission at STRs. We defined quantitative phenotypes describing the numbers and types of germline STR mutations in each strain and performed quantitative trait locus (QTL) analyses for each of these phenotypes. We identified a locus on Chromosome 13 at which strains inheriting the C57BL/6J (B) haplotype have a higher rate of STR expansions than those inheriting the DBA/2J (D) haplotype. The strongest candidate gene in this locus is Msh3, a known modifier of STR stability in cancer and at pathogenic repeat expansions in mice and humans, as well as a current drug target against Huntington's disease. The D haplotype at this locus harbors a cluster of variants near the 5' end of Msh3, including multiple missense variants near the DNA mismatch recognition domain. In contrast, the B haplotype contains a unique retrotransposon insertion. The rate of expansion covaries positively with Msh3 expression-with higher expression from the B haplotype. Finally, detailed analysis of mutation patterns showed that strains carrying the B allele have higher expansion rates, but slightly lower overall total mutation rates, compared with those with the D allele, particularly at tetranucleotide repeats. Our results suggest an important role for inherited variants in Msh3 in modulating genome-wide patterns of germline mutations at STRs.

Quantitative trait loci on chromosomes 9 and 19 modulate AII amacrine cell number in the mouse retina.

Sequence variants modulating gene function or expression affect various heritable traits, including the number of neurons within a population. The present study employed a forward-genetic approach to identify candidate causal genes and their sequence variants controlling the number of one type of retinal neuron, the AII amacrine cell. Data from twenty-six recombinant inbred (RI) strains of mice derived from the parental C57BL/6J (B6/J) and A/J laboratory strains were used to identify genomic loci regulating cell number. Large variation in cell number is present across the RI strains, from a low of ∼57,000 cells to a high of ∼87,000 cells. Quantitative trait locus (QTL) analysis revealed three prospective controlling genomic loci, on Chromosomes (Chrs) 9, 11, and 19, each contributing additive effects that together approach the range of variation observed. Composite interval mapping validated two of these loci, and chromosome substitution strains, in which the A/J genome for Chr 9 or 19 was introgressed on a B6/J genetic background, showed increased numbers of AII amacrine cells as predicted by those two QTL effects. Analysis of the respective genomic loci identified candidate controlling genes defined by their retinal expression, their established biological functions, and by the presence of sequence variants expected to modulate gene function or expression. Two candidate genes, Dtx4 on Chr 19, being a regulator of Notch signaling, and Dixdc1 on Chr 9, a modulator of the WNT-β-catenin signaling pathway, were explored in further detail. Postnatal overexpression of Dtx4 was found to reduce the frequency of amacrine cells, while Dixdc1 knockout retinas contained an excess of AII amacrine cells. Sequence variants in each gene were identified, being the likely sources of variation in gene expression, ultimately contributing to the final number of AII amacrine cells.

Abstract 11608: Differences in Genetic Variants and Common Genetic Regulators between Arrhythmogenic Cardiomyopathy and Arrhythmogenic Dilated Cardiomyopathy Identified Using Systems Genetics Approaches

Introduction: Prominent arrhythmias are features of arrhythmogenic cardiomyopathy (ACM) and arrhythmogenic dilated cardiomyopathy (aDCM). Despite clinicopathological differences, these diseases share common genetic causes. Hypothesis: This study aimed to identify variants in ACM and aDCM and map the candidate genetic regulators using a mouse genetic reference population (GRP). Methods: Whole exome sequencing was performed on 226 individuals with ACM and 102 patients with aDCM. Bioinformatics analysis was applied to identify the pathogenic or likely pathogenic variants followed by direct sequencing confirmation. Expression quantitative trait loci (eQTLs) mapping to heart transcriptomes of GRP of BXD recombinant inbred (RI) mice was conducted in GeneNetwork (http://www.genenetwork.org). Results: In the ACM cohort, a total of 82 variants (MAF<0.01) in 24 genes have been confirmed being positive in 42 probands. Depending on the frequency of the identified variants, the top 10 genes (excluding TTN) with the most pathogenic variants were PKP2, MYH6, FLNC, RYR2, MYH7B, PLEC, JPH2, SYNE1, NRAP and POSTN. In the aDCM cohort, 141 pathogenic variants have been identified and 91 patients harbored at least 1 deleterious variant. The top 10 genes were RYR2, FLNC, MYH7, DSP, SCN5A,MYPN, NEBL, CTNNA3, ACAP6 and SLC22A5. We analyzed the distribution of the first principal component (PC1) of mRNA expression levels of those 10 genes (eigentraits) from each cohort in the heart across BXD strains. Interval mapping for PC1 eigentraits for ACM identified a significant eQTL (P<0.05) in the mouse genome at 24-36 Mb of chromosome (Chr) 6. In aDCM, interval mapping for PC1 eigentraits identified 3 suggestive eQTLs at Chr 5 (44-53 Mb), Chr 6 (32-34 Mb) and Chr 15 (82-84 Mb). Further gene exploration of these eQTLs suggested that Mtpn, Lep, Cyb5r3, Tspo, Slit2, and Ppargc1a could be common regulators that interact with those top ACM and aDCM genes. Conclusions: This study identified diverse genes in ACM and aDCM cases. Our joint human and mouse analysis using a systems genetics approach identified genetic regulators that could affected expression of genes with pathogenic variants in ACM and aDCM patients.

Multi-omics analysis reveals expression complexity and functional diversity of mouse kinome.

Protein kinases are a crucial component of signaling pathways involved in a wide range of cellular responses, including growth, proliferation, differentiation, and migration. Systematic investigation of protein kinases is critical to better understand phosphorylation-mediated signaling pathways and may provide insights into the development of potential therapeutic drug targets. Here we perform a systems-level analysis of the mouse kinome by analyzing multi-omics data. We used bulk and single-cell transcriptomic data from the C57BL/6J mouse strain to define tissue- and cell-type-specific expression of protein kinases, followed by investigating variations in sequence and expression between C57BL/6J and DBA/2J strains. We then profiled a deep brain phosphoproteome from C57BL/6J and DBA/2J strains as well as their reciprocal hybrids to infer the activity of the mouse kinome. Finally, we performed phenome-wide association analysis using the BXD recombinant inbred (RI) mice (a cross between C57BL/6J and DBA/2J strains) to identify any associations between variants in protein kinases and phenotypes. Collectively, our study provides a comprehensive analysis of the mouse kinome by investigating genetic sequence variation, tissue-specific expression patterns, and associations with downstream phenotypes.

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>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.

A systems genetics approach delineates the role of Bcl2 in leukemia pathogenesis

Leukemia is a group of malignancies of the blood forming tissues, and is characterized by the uncontrolled proliferation of blood cells. In the United States, it accounts for approximately 3.5% and 4% of all cancer-related incidences and mortalities, respectively. The current study aimed to explore the role of Bcl2 and associated genes in leukemia pathogenesis using a systems genetics approach. The transcriptome data from BXD Recombinant Inbred (RI) mice was analyzed to identify the expression of Bcl2 in myeloid cells. eQTL mapping was performed to select the potential chromosomal region and subsequently identify the candidate gene modulating the expression of Bcl2. Furthermore, gene enrichment and protein-protein interaction (PPI) analyses of the Bcl2-coexpressed genes were performed to demonstrate the role of Bcl2 in leukemia pathogenesis. The Bcl2-coexpressed genes were found to be enriched in various hematopoietic system related functions, and multiple pathways related to signaling, immune response, and cancer. The PPI network analysis demonstrated direct interaction of hematopoietic function related genes, such as Bag3, Bak1, Bcl2l11, Bmf, Mapk9, Myc, Ppp2r5c, and Ppp3ca with Bcl2. The eQTL mapping identified a 4.5 Mb genomic region on chromosome 11, potentially regulating the expression of Bcl2. A multi-criteria filtering process identified Top2a, among the genes located in the mapped locus, as the best candidate upstream regulator for Bcl2 expression variation. Hence, the current study provides better insights into the role of Bcl2 in leukemia pathogenesis and demonstrates the significance of our approach in gaining new knowledge on leukemia. Furthermore, our findings from the PPI network analysis and eQTL mapping provide supporting evidence of leukemia-associated genes, which can be further explored for their functional importance in leukemia. Data availabilityThe myeloid cell transcriptomic data of the BXD mice used in this study can be accessed through our GeneNetwork (http://www.genenetwork.org) with the accession number of GN144.

English

Bean bruchids are among the most devastating insect pests of common bean that can inflict huge losses in storage. To identify potential resistance to these pests, screening was performed at Sokoine University of Agriculture. Two resistant landraces were identified, viz Kalubungula and KK25. Recombinant inbred (RI) KSy, KSw and ML populations were created from crosses between Soya × Kalubungula, Soworo × Kalubungula and Nagaga × KK25, respectively. Seed storage proteins were characterized and sequenced in RI population progenies to determine if phenotypic resistance was associated with α-amylase inhibitor – phytohemagglutinin – arcelin (APA) storage proteins. We found no association between the seed storage proteins observed in Kalubungula and its recombinant inbred lines with an APA protein. KK25 and its progenies had Arcelin-5, Leucoagglutinin, Erythroagglutinin and a hypothetical seed storage protein that conditions antibiosis effects as a resistance mechanism. The hypothetical seed storage protein observed in these lines may contribute to enhanced resistance.   Key words: α-amylase, phytohemagglutinin, arcelin, Acanthoscelides obtectus, Zabrotes subfasciatus, common beans.

Identification of cyclin D1 as a major modulator of 3-nitropropionic acid-induced striatal neurodegeneration

Mitochondria dysfunction occurs in the aging brain as well as in several neurodegenerative disorders and predisposes neuronal cells to enhanced sensitivity to neurotoxins. 3-nitropropionic acid (3-NP) is a naturally occurring plant and fungal neurotoxin that causes neurodegeneration predominantly in the striatum by irreversibly inhibiting the tricarboxylic acid respiratory chain enzyme, succinate dehydrogenase (SDH), the main constituent of the mitochondria respiratory chain complex II. Significantly, although 3-NP-induced inhibition of SDH occurs in all brain regions, neurodegeneration occurs primarily and almost exclusively in the striatum for reasons still not understood.In rodents, 3-NP-induced striatal neurodegeneration depends on the strain background suggesting that genetic differences among genotypes modulate toxicant variability and mechanisms that underlie 3-NP-induced neuronal cell death. Using the large BXD family of recombinant inbred (RI) strains we demonstrate that variants in Ccnd1 - the gene encoding cyclin D1 - of the DBA/2 J parent underlie the resistance to 3-NP-induced striatal neurodegeneration. In contrast, the Ccnd1 variant inherited from the widely used C57BL/6 J parental strain confers sensitivity. Given that cellular stress triggers induction of cyclin D1 expression followed by cell-cycle re-entry and consequent neuronal cell death, we sought to determine if the C57BL/6 J and DBA/2 J Ccnd1 variants are differentially modulated in response to 3-NP. We confirm that 3-NP induces cyclin D1 expression in striatal neuronal cells of C57BL/6 J, but this response is blunted in the DBA/2 J. We further show that striatal-specific alternative processing of a highly conserved 3′UTR negative regulatory region of Ccnd1 co-segregates with the C57BL/6 J parental Ccnd1 allele in BXD strains and that its differential processing accounts for sensitivity or resistance to 3-NP. Our results indicate that naturally occurring Ccnd1 variants may play a role in the variability observed in neurodegenerative disorders involving mitochondria complex II dysfunction and point to cyclin D1 as a possible therapeutic target.

BXD Recombinant Inbred Mice as a Model to Study Neurotoxicity.

BXD recombinant inbred (RI) lines represent a genetic reference population derived from a cross between C57BL/6J mice (B6) and DBA/2J mice (D2), which through meiotic recombination events possesses recombinant chromosomes containing B6 or D2 haplotype segments. The quantitative trait loci (QTLs) are the locations of segregating genetic polymorphisms and are fundamental to understanding genetic diversity in human disease susceptibility and severity. QTL mapping represents the typical approach for identifying naturally occurring polymorphisms that influence complex phenotypes. In this process, genotypic values at markers of known genomic locations are associated with phenotypic values measured in a segregating population. Indeed, BXD RI strains provide a powerful tool to study neurotoxicity induced by different substances. In this review, we describe the use of BXD RI lines to understand the underlying mechanisms of neurotoxicity in response to ethanol and cocaine, as well as metals and pesticide exposures.

Abstract P414: Systems Genetics Analysis Reveals Significant Eqtls Associated With Echocardiography Parameters In Genetic Reference Population Of Bxd Strains.

Background: Causal and modifier genes, genetic background and environment underlie clinical heterogeneity in cardiomyopathy (CM). The BXD recombinant inbred (RI) family represents a murine genetic reference population (GRP) that are descendants from crosses between C57BL/6J (B6) and DBA/2J (D2) mice. The parental D2 mouse is a natural model of hypertrophic CM (HCM). The study aimed to dissect genetic architecture of cardiac traits in BXD GRP using systems genetics analysis. Methods: Echocardiography was performed in 88 strains of male (M) and female (F) BXDs (N>5/sex) at 4-5 months of age. Cardiac traits were then associated with heart transcriptome, and expression quantitative trait loci ( eQTL) mapping was performed. Results: More than 2-fold variance in ejection fraction (EF%), fractional shortening (FS%), left ventricular (LV) volumes at end-systole and end-diastole (Vol;s and Vol;d), internal dimensions (ID;s and ID;d), posterior wall (PW), and interventricular septum (IVS) thickness was found among BXDs. Traits seen in dilated CM (DCM) patients such as reduced EF%, FS%, and LVPW and increased Vol;s and ID;s are found in BXD78 (M, F), BXD32, 111, and 68 (F) strains. Strains D2, BXD90 and 155 (M, F), BXD44 and 65 (M), and BXD113, 16, 77 (F) had significantly greater LV mass, LVPW and IVS thickness compared to sex-matched controls, suggestive for traits seen in HCM patients. A 6.4 Mb QTL (peak LRS=18.50) was identified on chromosome (Chr) 8 to be significantly associated with ID;s, ID;d, Vol;s and Vol;d among male BXDs. eQTL mapping for each of 131 genes on Chr8 QTL identified 6 genes ( Coq9 , Ndrg4 , Crnde, Irx3, Rpgrip1l, and Rbl2 ) being cis -regulated and Ndrg, Slc6a2 and Ces1d being significantly (p < 0.05) correlated with LV volumes. In female BXDs, a significant QTL on Chr7 (40.2 Mb) with 9 genes that significantly correlated with LVPW;d was identified. A suggestive 92.6 Mb QTL on Chr3 with Snapin , Tpm3 , and Wars2 correlated with EF% and FS% (p < 0.05). Conclusions: Our study found cardiomyopathy-associated traits are segregated among BXD family and these traits vary among BXD lines. Multiple associated QTLs demonstrate that the BXD family is suitable to map gene variants and identify genetic factors and modifiers that influence cardiomyopathy phenotypes.

Modification of Experimental Design and Statistical Method for Mapping Imprinted QTLs Based on Immortalized F2 Population.

Genomic imprinting is an epigenetic phenomenon, which plays important roles in the growth and development of animals and plants. Immortalized F2 (imF2) populations generated by random cross between recombinant inbred (RI) or doubled haploid (DH) lines have been proved to have significant advantages for mapping imprinted quantitative trait loci (iQTLs), and statistical methods for this purpose have been proposed. In this paper, we propose a special type of imF2 population (R-imF2) for iQTL mapping, which is developed by random reciprocal cross between RI/DH lines. We also propose two modified iQTL mapping methods: two-step point mapping (PM-2) and two-step composite point mapping (CPM-2). Simulation studies indicated that: (i) R-imF2 cannot improve the results of iQTL mapping, but the experimental design can probably reduce the workload of population construction; (ii) PM-2 can increase the precision of estimating the position and effects of a single iQTL; and (iii) CPM-2 can precisely map not only iQTLs, but also non-imprinted QTLs. The modified experimental design and statistical methods will facilitate and promote the study of iQTL mapping.

Genetic Variation in CNS Myelination and Functional Brain Connectivity in Recombinant Inbred Mice

Myelination greatly increases the speed of action potential propagation of neurons, thereby enhancing the efficacy of inter-neuronal communication and hence, potentially, optimizing the brain’s signal processing capability. The impact of genetic variation on the extent of axonal myelination and its consequences for brain functioning remain to be determined. Here we investigated this question using a genetic reference panel (GRP) of mouse BXD recombinant inbred (RI) strains, which partly model genetic diversity as observed in human populations, and which show substantial genetic differences in a variety of behaviors, including learning, memory and anxiety. We found coherent differences in the expression of myelin genes in brain tissue of RI strains of the BXD panel, with the largest differences in the hippocampus. The parental C57BL/6J (C57) and DBA/2J (DBA) strains were on opposite ends of the expression spectrum, with C57 showing higher myelin transcript expression compared with DBA. Our experiments showed accompanying differences between C57 and DBA in myelin protein composition, total myelin content, and white matter conduction velocity. Finally, the hippocampal myelin gene expression of the BXD strains correlated significantly with behavioral traits involving anxiety and/or activity. Taken together, our data indicate that genetic variation in myelin gene expression translates to differences observed in myelination, axonal conduction speed, and possibly in anxiety/activity related behaviors.

Exploring the Role of Chemokine Receptor 6 (Ccr6) in the BXD Mouse Model of Gulf War Illness.

Gulf War illness (GWI) is a chronic and multi-symptomatic disorder with persistent neuroimmune symptomatology. Chemokine receptor 6 (CCR6) has been shown to be involved in several inflammation disorders in humans. However, the causative relationship between CCR6 and neuroinflammation in GWI has not yet been investigated. By using RNA-seq data of prefrontal cortex (PFC) from 31 C57BL/6J X DBA/2J (BXD) recombinant inbred (RI) mouse strains and their parental strains under three chemical treatment groups – saline control (CTL), diisopropylfluorophosphate (DFP), and corticosterone combined with diisopropylfluorophosphate (CORT+DFP), we identified Ccr6 as a candidate gene underlying individual differences in susceptibility to GWI. The Ccr6 gene is cis-regulated and its expression is significantly correlated with CORT+DFP treatment. Its mean transcript abundance in PFC of BXD mice decreased 1.6-fold (p < 0.0001) in the CORT+DFP group. The response of Ccr6 to CORT+DFP is also significantly different (p < 0.0001) between the parental strains, suggesting Ccr6 is affected by both host genetic background and chemical treatments. Pearson product-moment correlation analysis revealed 1473 Ccr6-correlated genes (p < 0.05). Enrichment of these genes was seen in the immune, inflammation, cytokine, and neurological related categories. In addition, we also found five central nervous system-related phenotypes and fecal corticosterone concentration have significant correlation (p < 0.05) with expression of Ccr6 in the PFC. We further established a protein-protein interaction subnetwork for the Ccr6-correlated genes, which provides an insight on the interaction of G protein-coupled receptors, kallikrein-kinin system and neuroactive ligand-receptors. This analysis likely defines the heterogeneity and complexity of GWI. Therefore, our results suggest that Ccr6 is one of promising GWI biomarkers.

The BXD21/TyJ recombinant inbred strain as a model for innate inflammatory response in distinct brain regions

Oxidative stress and inflammatory cytokines affect the human brain, increasing the risk for mood and cognitive disorders. Such risk might be selective to brain-specific regions. Here, we determined whether BXD recombinant inbred (RI) mice strains are more suitable than C57BL/6J mice for the understanding of the relationship between antioxidant response and inflammatory responses. We hypothesized that inflammatory responses could be independent of antioxidant response and be inherent to brain-specific regions. This hypothesis will be addressed by the analyses of mRNA expression. We explored, at 7-months-of-age, the innate activation of proinflammatory cytokines (tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6), as well as Kelch-like ECH-associating protein 1 (Keap1), nuclear factor erythroid 2 related factor 2 (Nrf2) and glutathione peroxidase 1 (Gpx1) mRNA in both male and female BXD84/RwwJ RI, BXD21/TyJ RI and control strain (C57BL/6J mice). We report that: (1) The cerebellum is more sensitive to antioxidant response in the BXD21/TyJ RI strain; (2) The cerebellum, hippocampus and striatum show increased levels of cytokines in the BXD21/TyJ RI strain; (3) The BXD RI strain has lower brain weight relative to control strain (C57BL/6 mice). In conclusion, our novel data show the utility of the BXD21/TyJ RI strain mice in offering mechanistic insight into Nrf2’s role in the inflammatory system.

A genetic screen identifies Crat as a regulator of pancreatic beta-cell insulin secretion

ObjectivesGlucose-stimulated insulin secretion is a critical function in the regulation of glucose homeostasis, and its deregulation is associated with the development of type 2 diabetes. Here, we performed a genetic screen using islets isolated from the BXD panel of advanced recombinant inbred (RI) lines of mice to search for novel regulators of insulin production and secretion. MethodsPancreatic islets were isolated from 36 RI BXD lines and insulin secretion was measured following exposure to 2.8 or 16.7 mM glucose with or without exendin-4. Islets from the same RI lines were used for RNA extraction and transcript profiling. Quantitative trait loci (QTL) mapping was performed for each secretion condition and combined with transcriptome data to prioritize candidate regulatory genes within the identified QTL regions. Functional studies were performed by mRNA silencing or overexpression in MIN6B1 cells and by studying mice and islets with beta-cell-specific gene inactivation. ResultsInsulin secretion under the 16.7 mM glucose plus exendin-4 condition was mapped significantly to a chromosome 2 QTL. Within this QTL, RNA-Seq data prioritized Crat (carnitine O-acetyl transferase) as a strong candidate regulator of the insulin secretion trait. Silencing Crat expression in MIN6B1 cells reduced insulin content and insulin secretion by ∼30%. Conversely, Crat overexpression enhanced insulin content and secretion by ∼30%. When islets from mice with beta-cell-specific Crat inactivation were exposed to high glucose, they displayed a 30% reduction of insulin content as compared to control islets. We further showed that decreased Crat expression in both MIN6B1 cells and pancreatic islets reduced the oxygen consumption rate in a glucose concentration-dependent manner. ConclusionsWe identified Crat as a regulator of insulin secretion whose action is mediated by an effect on total cellular insulin content; this effect also depends on the genetic background of the RI mouse lines. These data also show that in the presence of the stimulatory conditions used the insulin secretion rate is directly related to the insulin content.

Developing a behavioral model of Restless Legs Syndrome utilizing mice with natural variances in ventral midbrain iron

BackgroundThe primary symptoms of Restless Legs Syndrome (RLS) are circadian-dependent, leading to increased activity or decreased rest, especially at night. The primary pathology in RLS is brain iron insufficiency despite normal systemic iron stores. Natural variances in brain and peripheral iron concentrations across recombinant inbred (RI) murine strains provide a biological model of RLS. The question is whether these RI mice strains show a behavioral analog to circadian-dependent clinical phenotype of RLS. MethodsThe home cage activity of eight female RI strains was measured over a 72-h period. The ratio of the average activity in the last 2 h of the active period relative to that in the total 12-h active period (late active period activity ratio, LAPAR) was the primary outcome variable. The relation of average LAPAR scores to measures of ventral midbrain (VMB) iron was evaluated across strains in this study. ResultsRI strain 40 (LAPAR = 1.28) and RI strain 21 (LAPAR = 1.02) were the only strains to show an increased activity in the last part of the active period. ANOVA showed the increased activity was significantly greater during the last 2 h compared to the preceding 10 h of the active phase only for the RI strain 40. Average LAPAR across the eight strains did not significantly correlate with the VMB iron content (r = −0.27, p < 0.10) but did correlate with changes in VMB iron with iron deficiency (r = 0.71, p < 0.05) and diurnal change in VMB iron (r = 0.65, p < 0.05). ConclusionThe female RI strain 40 mice exhibited a distinct end-of-active-period behavior analogous to circadian-dependent clinical phenotype of RLS.

A Cross-Species Systems Genetics Analysis Links APBB1IP as a Candidate for Schizophrenia and Prepulse Inhibition.

Background: Prepulse inhibition (PPI) of the startle response is a highly conserved form of sensorimotor gating, disruption of which is found in schizophrenia patients and their unaffected first-degree relatives. PPI can be measured in many species, and shows considerable phenotypic variation between and within rodent models. This makes PPI a useful endophenotype. Genome-wide association studies (GWAS) have been carried out to identify genetic variants underlying schizophrenia, and these suggest that schizophrenia is highly polygenic. GWAS have been unable to account for the high heritability of schizophrenia seen in family studies, partly because of the low power of GWAS due to multiple comparisons. By contrast, complementary mouse model linkage studies often have high statistical power to detect variants for behavioral traits but lower resolution, producing loci that include tens or hundreds of genes. To capitalize on the advantages of both GWAS and genetic mouse models, our study uses a cross-species approach to identify novel genes associated with PPI regulation, which thus may contribute to the PPI deficits seen in schizophrenia.Results: Using experimental data from the recombinant inbred (RI) mouse panel BXD, we identified two significant loci affecting PPI. These genomic regions contain genetic variants which influence PPI in mice and are therefore candidates that may be influencing aspects of schizophrenia in humans. We next investigated these regions in whole-genome data from the Psychiatric Genomics Consortium (PGC) schizophrenia GWAS and identify one novel candidate gene (ABPP1IP) that was significantly associated with PPI in mice and risk of schizophrenia in humans. A systems genetics approach demonstrates that APBB1IP coexpresses with several other genes related to schizophrenia in several brain regions. Gene coexpression and enrichment analysis shows clear links between APBB1IP and the immune system.Conclusion: The combination of human GWAS and mouse quantitative trait loci (QTL) from some of the largest study systems available has enabled us to identify a novel gene, APBB1IP, which influences schizophrenia in humans and PPI in mice.