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Individual differences in cognitive decline during normal aging and Alzheimer's disease (AD) are common, but the molecular mechanisms underlying these distinct outcomes are not fully understood. We utilized a combination of genetic, molecular, and behavioral data from a mouse population designed to model human variation in cognitive outcomes to search for the molecular mechanisms behind this population-wide variation. Specifically, we used a systems genetics approach to relate gene expression to cognitive outcomes during AD and normal aging. Statistical causal-inference Bayesian modeling was used to model systematic genetic perturbations matched with cognitive data that identified astrocyte and microglia molecular networks as drivers of cognitive resilience to AD. Using genetic mapping, we identified "Fgf2" as a potential regulator of the astrocyte network associated with individual differences in short-term memory. We also identified several immune genes as regulators of a microglia network associated with individual differences in long-term memory, which was partly mediated by amyloid burden. Finally, significant overlap between mouse and two different human coexpression networks provided strong evidence of translational relevance for the genetically diverse AD-BXD panel as a model of late-onset AD. Together, this work identified two candidate molecular pathways enriched for microglia and astrocyte genes that serve as causal AD cognitive biomarkers, and provided a greater understanding of processes that modulate individual and population-wide differences in cognitive outcomes during AD.

Substance use disorders are heritable disorders characterized by compulsive drug use, the biological mechanisms for which remain largely unknown. Genetic correlations reveal that predisposing drug‐naïve phenotypes, including anxiety, depression, novelty preference and sensation seeking, are predictive of drug‐use phenotypes, thereby implicating shared genetic mechanisms. High‐throughput behavioral screening in knockout (KO) mice allows efficient discovery of the function of genes. We used this strategy in two rounds of candidate prioritization in which we identified 33 drug‐use candidate genes based upon predisposing drug‐naïve phenotypes and ultimately validated the perturbation of 22 genes as causal drivers of substance intake. We selected 19/221 KO strains (8.5%) that had a difference from control on at least one drug‐naïve predictive behavioral phenotype and determined that 15/19 (~80%) affected the consumption or preference for alcohol, methamphetamine or both. No mutant exhibited a difference in nicotine consumption or preference which was possibly confounded with saccharin. In the second round of prioritization, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two‐bottle choice and ethanol drinking‐in‐the‐dark protocols. We identified 15/401 KO strains (3.7%, which included one gene from the first cohort) that differed most from controls for the predisposing phenotypes. 8 of 15 gene deletions (53%) affected intake or preference for alcohol, methamphetamine or both. Using multivariate and bioinformatic analyses, we observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction‐related processes further. [ABSTRACT FROM AUTHOR]

Background: In Alzheimer's disease (AD), risk factors and clinical progression differ between sexes. Men have lower post‐diagnosis life‐expectancies 1,2, while women display worsened cognitive decline than men with similar neuropathology3‐5 or shared genetic risk6,7. Investigation of sex‐specific genetic contributions to AD has largely focused on the X‐chromosome, but emerging evidence has also linked somatic Y‐chromosomal instability to age‐linked disease outcomes, including AD8‐10. Notably, the Y‐chromosome contains few coding genes, but is enriched with transposable elements (TEs)11, enigmatic DNA sequences capable of introducing variation into the genome12. Differential expression of TEs has recently been reported in AD13‐15. We hypothesize that TEs could contribute to sex‐specific variation observed in AD onset and progression. Method: Here, we utilized bulk hippocampal RNAseq and contextual fear memory (CFM) data from a 28‐strain AD‐BXD dataset comprised of male and female 5xFAD‐carrying animals and non‐transgenic (Ntg) littermates to investigate whether expression of coding genes and/or TEs contribute to AD‐relevant sex differences and cognitive outcomes. Data were stratified by sex and Y‐chromosome haplotype (C57BL/6J‐type vs. DBA/2J‐type). TEs were quantified in RNAseq data using TETranscripts16. For each TE, we predicted genomic localization using DFAM17, and calculated sex‐specific heritability. Result: In 5xFAD animals, C57BL/6J‐type males significantly out‐performed both DBA/2J‐type males and females on a CFM task (Fig.1). TEs identified in this dataset were differentially expressed due to both sex and Y‐chromosome haplotype in 5xFAD animals (Fig.2). Interestingly, Y‐haplotype did not affect Y‐chromosome coding gene expression. We calculated that a large portion of TEs identified in our dataset are highly heritable, with differential heritability associated with sex and 5xFAD‐transgene status (Fig.3) Conclusion: We report that the C57BL/6J Y‐chromosome confers cognitive resilience in 5xFAD males, which may undermine the utility of models maintained on a homogenous C57BL/6J background as tools for investigating age‐related cognitive decline. We speculate that differential TE expression/activity between Y‐haplotypes and between sexes may account for differential resilience, but further investigation is needed to establish this link directly. Finally, our data suggest that not only are TEs likely influencers of AD risk and disease progression, but that TE expression in disease states may be heritable, rather than stochastically induced. [ABSTRACT FROM AUTHOR]

Epidermolysis Bullosa (EB) is a group of rare genetic disorders that compromise the structural integrity of the skin such that blisters and subsequent erosions occur after minor trauma. While primary genetic risk of all subforms of EB adhere to Mendelian patterns of inheritance, their clinical presentations and severities can vary greatly, implying genetic modifiers. The Lamc2jeb mouse model of non-Herlitz junctional EB (JEB-nH) demonstrated that genetic modifiers can contribute substantially to the phenotypic variability of JEB and likely other forms of EB. The innocuous changes in an 'EB related gene', Col17a1, have shown it to be a dominant modifier of Lamc2jeb. This work identifies six additional Quantitative Trait Loci (QTL) that modify disease in Lamc2jeb/jeb mice. Three QTL include other known 'EB related genes', with the strongest modifier effect mapping to a region including the epidermal hemi-desmosomal structural gene dystonin (Dst-e/Bpag1-e). Three other QTL map to intervals devoid of known EB-associated genes. Of these, one contains the nuclear receptor coactivator Ppargc1a as its primary candidate and the others contain related genes Pparg and Igf1, suggesting modifier pathways. These results, demonstrating the potent disease modifying effects of normally innocuous genetic variants, greatly expand the landscape of genetic modifiers of EB and therapeutic approaches that may be applied. [ABSTRACT FROM AUTHOR]

Cocaine use and overdose deaths attributed to cocaine have increased significantly in the United States in the last 10 years. Despite the prevalence of cocaine use disorder (CUD) and the personal and societal problems it presents, there are currently no approved pharmaceutical treatments. The absence of treatment options is due, in part, to our lack of knowledge about the etiology of CUDs. There is ample evidence that genetics plays a role in increasing CUD risk but thus far, very few risk genes have been identified in human studies. Genetic studies in mice have been extremely useful for identifying genetic loci and genes, but have been limited to very few genetic backgrounds, leaving substantial phenotypic, and genetic diversity unexplored. Herein we report the measurement of cocaine-induced behavioral sensitization using a 19-day protocol that captures baseline locomotor activity, initial locomotor response to an acute exposure to cocaine and locomotor sensitization across 5 exposures to the drug. These behaviors were measured in 51 genetically diverse Collaborative Cross (CC) strains along with their inbred founder strains. The CC was generated by crossing eight genetically diverse inbred strains such that each inbred CC strain has genetic contributions from each of the founder strains. Inbred CC mice are infinitely reproducible and provide a stable, yet diverse genetic platform on which to study the genetic architecture and genetic correlations among phenotypes. We have identified significant differences in cocaine locomotor sensitivity and behavioral sensitization across the panel of CC strains and their founders. We have established relationships among cocaine sensitization behaviors and identified extreme responding strains that can be used in future studies aimed at understanding the genetic, biological, and pharmacological mechanisms that drive addiction-related behaviors. Finally, we have determined that these behaviors exhibit relatively robust heritability making them amenable to future genetic mapping studies to identify addiction risk genes and genetic pathways that can be studied as potential targets for the development of novel therapeutics. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disorder. Although most cases of ALS are sporadic, 5–10% of cases are familial, with mutations associated with over 40 genes. There is variation of ALS symptoms within families carrying the same mutation; the disease may develop in one sibling and not in another despite the presence of the mutation in both. Although the cause of this phenotypic variation is unknown, it is likely related to genetic modifiers of disease expression. The identification of ALS causing genes has led to the development of transgenic mouse models of motor neuron disease. Similar to families with familial ALS, there are background-dependent differences in disease phenotype in transgenic mouse models of ALS suggesting that, as in human ALS, differences in phenotype may be ascribed to genetic modifiers. These genetic modifiers may not cause ALS rather their expression either exacerbates or ameliorates the effect of the mutant ALS causing genes. We have reported that in both the G93A-hSOD1 and G59S-hDCTN1 mouse models, SJL mice demonstrated a more severe phenotype than C57BL6 mice. From reciprocal intercrosses between G93A-hSOD1 transgenic mice on SJL and C57BL6 strains, we identified a major quantitative trait locus (QTL) on mouse chromosome 17 that results in a significant shift in lifespan. In this study we generated reciprocal intercrosses between transgenic G59S-hDCTN1 mice on SJL and C57BL6 strains and identified survival QTLs on mouse chromosomes 17 and 18. The chromosome 17 survival QTL on G93A-hSOD1 and G59S-hDCTN1 mice partly overlap, suggesting that the genetic modifiers located in this region may be shared by these two ALS models despite the fact that motor neuron degeneration is caused by mutations in different proteins. The overlapping region contains eighty-seven genes with non-synonymous variations predicted to be deleterious and/or damaging. Two genes in this segment, NOTCH3 and Safb/SAFB1, have been associated with motor neuron disease. The identification of genetic modifiers of motor neuron disease, especially those modifiers that are shared by SOD1 and dynactin-1 transgenic mice, may result in the identification of novel targets for therapies that can alter the course of this devastating illness. [ABSTRACT FROM AUTHOR]

Background: A strong predictor for the development of alcohol use disorder (AUD) is altered sensitivity to the intoxicating effects of alcohol. Individual differences in the initial sensitivity to alcohol are controlled in part by genetic factors. Mice offer a powerful tool to elucidate the genetic basis of behavioral and physiological traits relevant to AUD, but conventional experimental crosses have only been able to identify large chromosomal regions rather than specific genes. Genetically diverse, highly recombinant mouse populations make it possible to observe a wider range of phenotypic variation, offer greater mapping precision, and thus increase the potential for efficient gene identification. Methods: We have taken advantage of the Diversity Outbred (DO) mouse population to identify and precisely map quantitative trait loci (QTL) associated with ethanol sensitivity. We phenotyped 798 male J:DO mice for three measures of ethanol sensitivity: ataxia, hypothermia, and loss of the righting response. We used high‐density MegaMUGA and GigaMUGA to obtain genotypes ranging from 77,808 to 143,259 SNPs. We also performed RNA sequencing in striatum to map expression QTLs and identify gene expression–trait correlations. We then applied a systems genetic strategy to identify narrow QTLs and construct the network of correlations that exists between DNA sequence, gene expression values, and ethanol‐related phenotypes to prioritize our list of positional candidate genes. Results: We observed large amounts of phenotypic variation with the DO population and identified suggestive and significant QTLs associated with ethanol sensitivity on chromosomes 1, 2, and 16. The implicated regions were narrow (4.5–6.9 Mb in size) and each QTL explained ~4–5% of the variance. Conclusions: Our results can be used to identify alleles that contribute to AUD in humans, elucidate causative biological mechanisms, or assist in the development of novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Drugs of abuse, including alcohol and stimulants like cocaine, produce effects that are subject to individual variability, and genetic variation accounts for at least a portion of those differences. Notably, research in both animal models and human subjects point toward reward sensitivity and impulsivity as being trait characteristics that predict relatively greater positive subjective responses to stimulant drugs. Here we describe use of the eight collaborative cross (CC) founder strains and 38 (reversal learning) or 10 (all other tests) CC strains to examine the heritability of reward sensitivity and impulsivity traits, as well as genetic correlations between these measures and existing addiction‐related phenotypes. Strains were all tested for activity in an open field and reward sensitivity (intake of chocolate BOOST®). Mice were then divided into two counterbalanced groups and underwent reversal learning (impulsive action and waiting impulsivity) or delay discounting (impulsive choice). CC and founder mice show significant heritability for impulsive action, impulsive choice, waiting impulsivity, locomotor activity, and reward sensitivity, with each impulsive phenotype determined to be non‐correlating, independent traits. This research was conducted within the broader, inter‐laboratory effort of the Center for Systems Neurogenetics of Addiction (CSNA) to characterize CC and DO mice for multiple, cocaine abuse related traits. These data will facilitate the discovery of genetic correlations between predictive traits, which will then guide discovery of genes and genetic variants that contribute to addictive behaviors. [ABSTRACT FROM AUTHOR]

Abstract Background miRNAs are short single-stranded non-coding RNAs involved in post-transcriptional gene regulation that play a major role in normal biological functions and diseases. Little is currently known about how expression of miRNAs is regulated. We surveyed variation in miRNA abundance in the hippocampus of mouse inbred strains, allowing us to take a genetic approach to the study of miRNA regulation, which is novel for miRNAs. The BXD recombinant inbred panel is a very well characterized genetic reference panel which allows quantitative trait locus (QTL) analysis of miRNA abundance and detection of correlates in a large store of brain and behavioural phenotypes. Results We found five suggestive trans QTLs for the regulation of miRNAs investigated. Further analysis of these QTLs revealed two genes, Tnik and Phf17, under the miR-212 regulatory QTLs, whose expression levels were significantly correlated with miR-212 expression. We found that miR-212 expression is correlated with cocaine-related behaviour, consistent with a reported role for this miRNA in the control of cocaine consumption. miR-31 is correlated with anxiety and alcohol related behaviours. KEGG pathway analysis of each miRNA’s expression correlates revealed enrichment of pathways including MAP kinase, cancer, long-term potentiation, axonal guidance and WNT signalling. Conclusions The BXD reference panel allowed us to establish genetic regulation and characterize biological function of specific miRNAs. QTL analysis enabled detection of genetic loci that regulate the expression of these miRNAs. eQTLs that regulate miRNA abundance are a new mechanism by which genetic variation influences brain and behaviour. Analysis of one of these QTLs revealed a gene, Tnik, which may regulate the expression of a miRNA, a molecular pathway and a behavioural phenotype. Evidence of genetic covariation of miR-212 abundance and cocaine related behaviours is strongly supported by previous functional studies, demonstrating the value of this approach for discovery of new functional roles and downstream processes regulated by miRNA.

Background: Interindividual variation in voluntary ethanol consumption and ethanol response is partially influenced by genetic variation. Discovery of the genes and allelic variants that affect these phenotypes may clarify the etiology and pathophysiology of problematic alcohol use, including alcohol use disorder. Genetically diverse mouse populations, which demonstrate heritable variation in ethanol consumption, can be utilized to discover the genes and gene networks that influence this trait. The Collaborative Cross (CC) recombinant inbred strains, Diversity Outbred (DO) population and their 8 founder strains are complementary mouse resources that capture substantial genetic diversity and can demonstrate expansive phenotypic variation in heritable traits. These populations may be utilized to discover candidate genes and gene networks that moderate ethanol consumption and other ethanol‐related traits. Methods: We characterized ethanol consumption, preference, and pharmacokinetics in the 8 founder strains and 10 CC strains in 12‐hour drinking sessions during the dark phase of the circadian cycle. Results: Ethanol consumption was substantially heritable, both early in ethanol access and over a chronic intermittent access schedule. Ethanol pharmacokinetics were also heritable; however, no association between strain‐level ethanol consumption and pharmacokinetics was detected. The PWK/PhJ strain was the highest drinking strain, with consumption substantially exceeding that of the C57BL/6J strain, which is commonly used as a model of "high" or "binge" drinking. Notably, we found strong evidence that sex moderated genetic effects on voluntary ethanol drinking. Conclusions: Collectively, this research serves as a foundation for expanded genetic study of ethanol consumption in the CC/DO and related populations. Moreover, we identified reference strains with extreme consumption phenotypes that effectively represent polygenic models of excessive ethanol use. [ABSTRACT FROM AUTHOR]

Substance use disorders are prevalent and present a tremendous societal cost but the mechanisms underlying addiction behavior are poorly understood and few biological treatments exist. One strategy to identify novel molecular mechanisms of addiction is through functional genomic experimentation. However, results from individual experiments are often noisy. To address this problem, the convergent analysis of multiple genomic experiments can discern signal from these studies. In the present study, we examine genetic loci that modulate the locomotor response to cocaine identified in the recombinant inbred (BXD RI) genetic reference population. We then applied the GeneWeaver software system for heterogeneous functional genomic analysis to integrate and aggregate multiple studies of addiction genomics, resulting in the identification of Rab3b as a functional correlate of the locomotor response to cocaine in rodents. This gene encodes a member of the RAB family of Ras-like GTPases known to be involved in trafficking of secretory and endocytic vesicles in eukaryotic cells. The convergent evidence for a role of Rab3b includes co-occurrence in previously published genetic mapping studies of cocaine related behaviors; methamphetamine response and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) transcript abundance; evidence related to other addictive substances; density of polymorphisms; and its expression pattern in reward pathways. To evaluate this finding, we examined the effect of RAB3 complex perturbation in cocaine response. B6;129- Rab3btm1Sud Rab3ctm1sud Rab3dtm1sud triple null mice (Rab3bcd –/–) exhibited significant deficits in habituation, and increased acute and repeated cocaine responses. This previously unidentified mechanism of the behavioral predisposition and response to cocaine is an example of many that can be identified and validated using aggregate genomic studies. [ABSTRACT FROM AUTHOR]

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice (Mus musculus) enables data-driven discovery of biological network components and mechanisms of host--microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of Odoribacter (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg-Dock7m +/+ Leprdb/J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by Odoribacter, treatment with antibiotics altered sleep in a genotypedependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms. [ABSTRACT FROM AUTHOR]

Background: The frontal cortex is critical to many cognitive processes and is vulnerable to pathology and neurodegeneration in Alzheimer's disease. Cortical dysfunction and impaired connectivity in the frontal cortex have also been observed in "normal" age‐related cognitive decline, suggesting common mechanisms contributing to both age‐related and AD‐related cognitive decline. The divergence that may lead to AD versus normal aging, however, is unclear. Method: Here, we have used genetically diverse mouse populations modeling either AD or normal aging, the AD‐BXDs and their nontransgenic littermates (Neuner et al, Neuron, 2019). We measured cognitive function from early (6mo) to middle (14mo) adulthood to capture presymptomatic and advanced AD stages and early aging processes. We performed bulk RNAseq from the frontal cortex, followed by age‐ and AD‐related differential gene expression analysis and Weighted Gene Coexpression Network Analysis (WGCNA) to identify genes and gene networks whose expression is associated with normal and pathological aging. Result: In both differential gene expression analysis and WGCNA, we found common mechanisms underlying normal aging and AD (e.g., upregulation of inflammation‐related gene pathways). We also found that downregulation of genes and gene networks associated with synaptic function specifically occurred in AD and not in normal aging, suggesting that early synaptic dysfunction and/or synaptic loss is a unique feature of AD that distinguishes it from normal aging processes. Based on differential gene expression, network analyses, presence of high‐impact variants across the BXD panel, and association with cognitive function, we identified one gene candidate, B230209E15Rik, a lncRNA, as a potential regulator of synaptic gene networks in the frontal cortex that promotes resilience to cognitive decline in AD. Conclusion: Overall, our data indicate that cognitive aging and AD may share common neuroimmune activation and dysregulation of neuronal maintenance, but that AD, and particularly late AD, is uniquely characterized by synaptic dysfunction. We nominate an intriguing candidate, B230209E15Rik, as a potentially key driver of synaptic gene regulation in the frontal cortex. In order to elucidate the mechanism by which this gene may promote cognitive resilience in AD, we are currently validating this finding in vivo. [ABSTRACT FROM AUTHOR]

The pairwise interaction of aromatic amino acids with anionic amino acids which were explored by using small molecule mimics is presented.

Contemporary mouse genetic reference populations are a powerful platform to discover complex disease mechanisms. Advanced high-diversity mouse populations include the Collaborative Cross (CC) strains, Diversity Outbred (DO) stock, and their isogenic founder strains. When used in systems genetics and integrative genomics analyses, these populations efficiently harnesses known genetic variation for precise and contextualized identification of complex disease mechanisms. Extensive genetic, genomic, and phenotypic data are already available for these high-diversity mouse populations and a growing suite of data analysis tools have been developed to support research on diverse mice. This integrated resource can be used to discover and evaluate disease mechanisms relevant across species. High-diversity mouse populations with known and reproducible genetic variation make complex trait genetics tractable in a mammalian system. Together, these populations are a valuable integrated and scalable tool for discovery genetics in complex trait studies. The Collaborative Cross (CC), its founders, and the heterozygous CC-RIX derived from crosses of the CC strains are a fully reproducible population for exact genome-matched correlational and controlled studies. The Diversity Outbred (DO) population displays high genetic and phenotypic variability and enables precise genetic mapping. Cross-species genomic analysis of mouse-derived results allows comparative and translational applications. [ABSTRACT FROM AUTHOR]

Across the population, individuals exhibit a wide variation of susceptibility or resilience to developing Alzheimer's disease (AD). Identifying specific factors that promote resilience would provide insight into disease mechanisms and nominate potential targets for therapeutic intervention. Here, we use transcriptome profiling to identify gene networks present in the pre-symptomatic AD mouse brain relating to neuroinflammation, brain vasculature, extracellular matrix organization, and synaptic signaling that predict cognitive performance at an advanced age. We highlight putative drivers of these observed relationships, including Itgb2 , Fcgr2b, Slc6a14 , and Gper1 , which represent prime targets through which to promote resilience prior to overt symptom onset. In addition, we identify a genomic region on chromosome 2 containing variants that directly modulate resilience network expression. Overall, work here highlights new potential drivers of resilience to AD and contributes significantly to our understanding of early, potentially causal, disease mechanisms. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD), the leading cause of dementia, affects millions of people worldwide. With no disease‐modifying medication currently available, the human toll and economic costs are rising rapidly. Under current standards, a patient is diagnosed with AD when both cognitive decline and pathology (amyloid plaques and neurofibrillary tangles) are present. Remarkably, some individuals who have AD pathology remain cognitively normal. Uncovering factors that lead to "cognitive resilience" to AD is a promising path to create new targets for therapies. However, technical challenges discovering novel human resilience factors limit testing, validation, and nomination of novel drugs for AD. Method: In this study, we use single‐nuclear transcriptional profiles of postmortem cortex from human individuals with high AD pathology who were either cognitively normal (resilient) or cognitively impaired (susceptible) at time of death, as well as mouse strains that parallel these differences in cognition with high amyloid. Result: Our cross‐species discovery approach highlights a novel role for excitatory layer 4/5 cortical neurons in promoting cognitive resilience to AD, and nominates several resilience genes that include ATP1A1, GABRB1, PTK2, and ROCK2. Nominated resilience genes were tested for replication in orthogonal data sets and confirmed to be correlated with cognitive resilience. Additionally, we identified several potential mechanisms of resilience, including regulation of membrane potential, axonal and dendritic growth, and general increase of protein cycle, potentially of membrane proteins. Conclusion: Because our discovery of resilience‐associated genes in layer 4/5 cortical neurons originates from an integrated human and mouse transcriptomic space from susceptible and resilient individuals, we are positioned to test causality and perform mechanistic, validation, and pre‐clinical studies in our human‐relevant AD‐BXD mouse panel. [ABSTRACT FROM AUTHOR]

Background: 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. Method: Here, 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. Result: 5XFAD‐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. Conclusion: C57BL/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. [ABSTRACT FROM AUTHOR]

Background: It is estimated that a third of the elderly population is not diagnosed with Alzheimer's disease (AD) due to the absence of cognitive impairment despite carrying risk variants of AD or presenting AD pathology post‐mortem. This suggests that at‐risk individuals may carry protective mechanisms that promote resilience to cognitive impairment, however the underlying molecular processes that promote resilience remain unknown. Method: To determine transcriptional changes associated with resilience, we profiled the hippocampal transcriptome at the single cell level in 7 resilient and 7 susceptible strains from the AD‐BXD mouse reference panel1, a genetically diverse mouse model of AD that better mimics human AD. Here, we used contextual fear memory paradigm to assess short‐term memory function in AD‐BXDs carrying the 5XFAD mutation. Resilience was defined based on age‐related change in cognitive function relative to that of the entire AD‐BXD population, where strains showing no or lower than average decline were considered resilient. Result: Using single nucleus RNA‐sequencing, we profiled ∼220 K nuclei from the hippocampal formation and identified 32 cell clusters representing the major cell types in the hippocampus including glutamatergic neurons, GABAergic neurons, astrocytes, oligodendrocytes and microglia. With the exception of GABAergic neurons, transcriptional changes associated with the 5XFAD mutation were greater in susceptible AD‐BXDs. Gene expression changes associated with cognitive resilience were primarily observed in excitatory neurons, specifically in the CA1 and dentate gyrus and were enriched for ribosomal genes and nuclear encoded mitochondrial genes. In attempt to infer potential ligands that regulate resilience‐associated transcriptional changes we used a ligand‐target interaction analysis and predicted potential ligands regulating resilience programs in excitatory neurons. Conclusion: Our findings demonstrate that the rate of cognitive decline is concomitant with increased transcriptional changes associated with the 5XFAD mutation across cell types in the hippocampus. We show that molecular programs associated with resilience in excitatory neurons are enriched in protein metabolism, cellular respiration and translation, possibly indicating a response to energy demand and maintenance of cellular homeostasis. Neuner SM, Heuer SE, Huentelman MJ, O'Connell KMS, Kaczorowski CC. Harnessing Genetic Complexity to Enhance Translatability of Alzheimer's Disease Mouse Models: A Path toward Precision Medicine. Neuron. 2019. [ABSTRACT FROM AUTHOR]

Recent technical and methodological advances have greatly enhanced genome-wide association studies (GWAS). The advent of low-cost, whole-genome sequencing facilitates high-resolution variant identification, and the development of linear mixed models (LMM) allows improved identification of putatively causal variants. While essential for correcting false positive associations due to sample relatedness and population stratification, LMMs have commonly been restricted to quantitative variables. However, phenotypic traits in association studies are often categorical, coded as binary case-control or ordered variables describing disease stages. To address these issues, we have devised a method for genomic association studies that implements a generalized LMM (GLMM) in a Bayesian framework, called Bayes- GLMM. Bayes-GLMM has four major features: (1) support of categorical, binary, and quantitative variables; (2) cohesive integration of previous GWAS results for related traits; (3) correction for sample relatedness by mixed modeling; and (4) model estimation by both Markov chain Monte Carlo sampling and maximal likelihood estimation. We applied Bayes-GLMM to the whole-genome sequencing cohort of the Alzheimer's Disease Sequencing Project. This study contains 570 individuals from 111 families, each with Alzheimer's disease diagnosed at one of four confidence levels. Using Bayes-GLMM we identified four variants in three loci significantly associated with Alzheimer's disease. Two variants, rs140233081 and rs149372995, lie between PRKAR1B and PDGFA. The coded proteins are localized to the glial-vascular unit, and PDGFA transcript levels are associated with Alzheimer's disease-related neuropathology. In summary, this work provides implementation of a flexible, generalized mixed-model approach in a Bayesian framework for association studies. [ABSTRACT FROM AUTHOR]

Background: Cognitive resilience to familial Alzheimer's Disease (FAD) is a phenomenon whereby cognitive functioning is better than predicted based on highly penetrant mutations in APP and PS1. This resilience is likely mediated by unidentified genetic factors, and we hypothesize that these factors provide key targets for treatment and prevention of AD. We previously discovered that the most commonly used background strain in AD model research (C57BL/6J, B6) is resilient to AD‐related cognitive decline. Method: Using genetic mapping, we determined that cognitive resilience is associated with a variant in the Apoe receptor binding domain and utilized CRISPR to generate a novel Apoe knockin mouse (Apoe KIB6/E163D). Contextual fear conditioning (CFC), tau pathology, and single‐nuclear RNAseq hippocampi analyses were performed in male and female Apoe KIB6/E163D x 5XFAD progeny that differed by only a single variant in Apoe leading to the E163D base pair change in the protein. Result: We found that, indeed, cognitive resilience to the 5XFAD transgene was conferred in ApoeB6/B6 mice compared to Apoe KIB6/E163D mice tested on CFC. Although minimal differences in hyperphosphorylated tau abundance were observed between 5XFAD‐ApoeWT/WT mice compared to 5XFAD‐Apoe KIB6/E163D, we uncovered robust differences in the transcriptomes of hippocampal excitatory and inhibitory neurons and microglia using single‐nuclear sequencing. Gene enrichment and pathway analyses of differentially expressed genes suggest that the 5XFAD‐ApoeB6/B6 genotype promotes cation transport and suppression of amyloid fibril formation in excitatory neurons, promotes genes involved in ribosome assembly and protein translation in inhibitory neurons, and enhances gene expression associated with stress response in microglia. Conclusion: Our results suggest that 5XFAD‐ApoeB6/B6 homozygotes exhibit cognitive resilience to causal FAD mutations that involve dynamic regulation of gene networks across multiple cell types, emphasizing the need for well‐aligned cell culture models of cognitive resilience to validate targets and screen resilience‐based interventions. Future work leveraging in vivo electrophysiology and imaging will establish mechanisms whereby Apoe variants alter neural and synaptic function. Understanding the relationship between protective genetic factors and cognitive resilience are crucial for determining mechanisms of resilience and uncovering novel targets for intervention. [ABSTRACT FROM AUTHOR]

Infection with the helminth Schistosoma mansoni results in hepatointestinal granulomatous inflammation mediated by CD4 T cells directed against parasite eggs. The severity of disease varies greatly in humans and mice; however, the genetic basis of such a heterogenous immune response remains poorly understood. Here we show that, despite their close genetic relationship, C57BL/10SnJ (B10) mice developed significantly more pronounced immunopathology and higher T helper 17 cell responses than C57BL/6J (B6) mice. Similarly, live egg-stimulated B10-derived dendritic cells (DCs) produced significantly more IL-1β and IL-23, resulting in higher IL-17 production by CD4 T cells. Gene expression analysis disclosed a heightened proinflammatory cytokine profile together with a strikingly lower expression of Ym1 in B10 versus B6 mice, consistent with failure of B10 DCs to attain alternative activation. To genetically dissect the differential response, we developed and analyzed congenic mouse strains that capture major regions of allelic variation, and found that the level of inflammation was controlled by a relatively small number of genes in a locus mapping to chromosome 4 117-143 MB. Our study has thus identified novel genomic regions that regulate the severity of the schistosome infection by way of controlling the mode of DC activation and consequent CD4 T-cell subset development. [ABSTRACT FROM AUTHOR]

Background Transgenesis by random integration of a transgene into the genome of a zygote has become a reliable and powerful method for the creation of new mouse strains that express exogenous genes, including human disease genes, tissue specific reporter genes or genes that allow for tissue specific recombination. Nearly 6,500 transgenic alleles have been created by random integration in embryos over the last 30 years, but for the vast majority of these strains, the transgene insertion sites remain uncharacterized. Results To obtain a complete understanding of how insertion sites might contribute to phenotypic outcomes, to more cost effectively manage transgenic strains, and to fully understand mechanisms of instability in transgene expression, we've developed methodology and a scoring scheme for transgene insertion site discovery using high throughput sequencing data. Conclusions Similar to other molecular approaches to transgene insertion site discovery, high-throughput sequencing of standard paired-end libraries is hindered by low signal to noise ratios. This problem is exacerbated when the transgene consists of sequences that are also present in the host genome. We've found that high throughput sequencing data from mate-pair libraries are more informative when compared to data from standard paired end libraries. We also show examples of the genomic regions that harbor transgenes, which have in common a preponderance of repetitive sequences. [ABSTRACT FROM AUTHOR]

Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2jeb mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2jeb mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2jeb mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder. [ABSTRACT FROM AUTHOR]

To characterize temporal patterns of transcriptional activity during normal lung development, we generated genome wide gene expression data for 26 pre- and post-natal time points in three common inbred strains of laboratory mice (C57BL/6J, A/J, and C3H/HeJ). Using Principal Component Analysis and least squares regression modeling, we identified both strain-independent and strain-dependent patterns of gene expression. The 4,683 genes contributing to the strain-independent expression patterns were used to define a murine Developing Lung Characteristic Subtranscriptome (mDLCS). Regression modeling of the Principal Components supported the four canonical stages of mammalian embryonic lung development (embryonic, pseudoglandular, canalicular, saccular) defined previously by morphology and histology. For postnatal alveolar development, the regression model was consistent with four stages of alveolarization characterized by episodic transcriptional activity of genes related to pulmonary vascularization. Genes expressed in a strain-dependent manner were enriched for annotations related to neurogenesis, extracellular matrix organization, and Wnt signaling. Finally, a comparison of mouse and human transcriptomics from pre-natal stages of lung development revealed conservation of pathways associated with cell cycle, axon guidance, immune function, and metabolism as well as organism-specific expression of genes associated with extracellular matrix organization and protein modification. The mouse lung development transcriptome data generated for this study serves as a unique reference set to identify genes and pathways essential for normal mammalian lung development and for investigations into the developmental origins of respiratory disease and cancer. The gene expression data are available from the Gene Expression Omnibus (GEO) archive (GSE74243). Temporal expression patterns of mouse genes can be investigated using a study specific web resource (http://lungdevelopment.jax.org).

Contemporary genetic studies are revealing the genetic complexity of many traits in humans and model organisms. Two hallmarks of this complexity are epistasis, meaning gene-gene interaction, and pleiotropy, in which one gene affects multiple phenotypes. Understanding the genetic architecture of complex traits requires addressing these phenomena, but interpreting the biological significance of epistasis and pleiotropy is often difficult. While epistasis reveals dependencies between genetic variants, it is often unclear how the activity of one variant is specifically modifying the other. Epistasis found in one phenotypic context may disappear in another context, rendering the genetic interaction ambiguous. Pleiotropy can suggest either redundant phenotype measures or gene variants that affect multiple biological processes. Here we present an R package, R/cape, which addresses these interpretation ambiguities by implementing a novel method to generate predictive and interpretable genetic networks that influence quantitative phenotypes. R/cape integrates information from multiple related phenotypes to constrain models of epistasis, thereby enhancing the detection of interactions that simultaneously describe all phenotypes. The networks inferred by R/cape are readily interpretable in terms of directed influences that indicate suppressive and enhancing effects of individual genetic variants on other variants, which in turn account for the variance in quantitative traits. We demonstrate the utility of R/cape by analyzing a mouse backcross, thereby discovering novel epistatic interactions influencing phenotypes related to obesity and diabetes. R/cape is an easy-to-use, platform-independent R package and can be applied to data from both genetic screens and a variety of segregating populations including backcrosses, intercrosses, and natural populations. The package is freely available under the GPL-3 license at http://cran.r-project.org/web/packages/cape. [ABSTRACT FROM AUTHOR]

Albuminuria is an important marker of nephropathy that increases the risk of progressive renal and chronic cardiovascular diseases. The genetic basis of kidney disease is well-established in humans and rodent models, but the causal genes remain to be identified. We applied several genetic strategies to map and refine genetic loci affecting albuminuria in mice and translated the findings to human kidney disease. First, we measured albuminuria in mice from 33 inbred strains, used the data for haplotype association mapping (HAM), and detected 10 genomic regions associated with albuminuria. Second, we performed eight F2 intercrosses between genetically diverse strains to identify six loci underlying albuminuria, each of which was concordant to kidney disease loci in humans. Third, we used the Oak Ridge National Laboratory incipient Collaborative Cross subpopulation to detect an additional novel quantitative trait loci (QTL) underlying albuminuria. We also performed a ninth intercross, between genetically similar strains, that substantially narrowed an albuminuria QTL on Chromosome 17 to a region containing four known genes. Finally, we measured renal gene expression in inbred mice to detect pathways highly correlated with albuminuria. Expression analysis also identified Glcci1, a gene known to affect podocyte structure and function in zebrafish, as a strong candidate gene for the albuminuria QTL on Chromosome 6. Overall, these findings greatly enhance our understanding of the genetic basis of albuminuria in mice and may guide future studies into the genetic basis of kidney disease in humans. [ABSTRACT FROM AUTHOR]

Genetic reference populations in model organisms are critical resources for systems genetic analysis of disease related phenotypes. The breeding history of these inbred panels may influence detectable allelic and phenotypic diversity. The existing panel of common inbred strains reflects historical selection biases, and existing recombinant inbred panels have low allelic diversity. All such populations may be subject to consequences of inbreeding depression. The Collaborative Cross (CC) is a mouse reference population with high allelic diversity that is being constructed using a randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains. Five of the eight founders are common laboratory strains, and three are wild-derived. Since its inception, the partially inbred CC has been characterized for physiological, morphological, and behavioral traits. The construction of this population provided a unique opportunity to observe phenotypic variation as new allelic combinations arose through intercrossing and inbreeding to create new stable genetic combinations. Processes including inbreeding depression and its impact on allelic and phenotypic diversity were assessed. Phenotypic variation in the CC breeding population exceeds that of existing mouse genetic reference populations due to both high founder genetic diversity and novel epistatic combinations. However, some focal evidence of allele purging was detected including a suggestive QTL for litter size in a location of changing allele frequency. Despite these inescapable pressures, high diversity and precision for genetic mapping remain. These results demonstrate the potential of the CC population once completed and highlight implications for development of related populations. [ABSTRACT FROM AUTHOR]

Commercial SNP microarrays now provide comprehensive and affordable coverage of the human genome. However, some diseases have biologically relevant genomic regions that may require additional coverage. Addiction, for example, is thought to be influenced by complex interactions among many relevant genes and pathways. We have assembled a list of 486 biologically relevant genes nominated by a panel of experts on addiction. We then added 424 genes that showed evidence of association with addiction phenotypes through mouse QTL mappings and gene co-expression analysis. We demonstrate that there are a substantial number of SNPs in these genes that are not well represented by commercial SNP platforms. We address this problem by introducing a publicly available SNP database for addiction. The database is annotated using numeric prioritization scores indicating the extent of biological relevance. The scores incorporate a number of factors such as SNP/gene functional properties (including synonymy and promoter regions), data from mouse systems genetics and measures of human/mouse evolutionary conservation. We then used HapMap genotyping data to determine if a SNP is tagged by a commercial microarray through linkage disequilibrium. This combination of biological prioritization scores and LD tagging annotation will enable addiction researchers to supplement commercial SNP microarrays to ensure comprehensive coverage of biologically relevant regions. [ABSTRACT FROM AUTHOR]