High doses of radiation (HDR) are clearly detrimental to human health, but relatively little is known about the health consequences following exposure to low doses of radiation (LDR, <10cGy). Understanding the risks associated with LDR is of great importance to the general public due to the recent dramatic increase in diagnostic radiological imaging. While HDR clearly suppress immune function, there is evidence that LDR can be immunostimulatory. Within the organism, defining the consequences of LDR is further complicated by the impact of genetic background, particularly in systems such as the immune system for which both radiosensitivity and genetic effects are profound. We addressed the issue of genetic susceptibility to LDR using the immune system as a target system and treated the LDR response as a complex trait analyzed using a systems genetics framework. Using the BXD recombinant inbred strain mouse panel as a genetic reference population allowed us to address the radiation response within the context of natural genetic variation. Our overarching hypothesis is that, within a population, the immunological effects of LDR exposure depend in part on the individual’s baseline immunoprofile and gene expression which are ultimately dependent upon genetic background. We began by establishing the immunophenotypic variation (i.e., T:B cell ratio, CD4:CD8 ratio) within the BXD panel and used baseline spleen transcriptome profiling to identify putative candidate genes controlling these traits, specifically Acp1 and Ptprk for CD4:CD8 ratio. The same set of BXD strains was exposed to LDR (10cGy gamma radiation) to determine effects on immune function and oxidative stress. LDR significantly enhanced neutrophil phagocytosis in a manner that was independent of genetic background. In contrast, genetic background significantly impacted LDR-induced changes in spleen superoxide dismutase activity. By integrating these results with our previous analyses of BXD RI strains, we have demonstrated that baseline expression of Sod2 correlates with LDR-induced SOD activity, and baseline CD4:CD8 ratio is inversely correlated with LDR-induced neutrophil phagocytosis. In addition, spleen transcriptomic data from the BXD parental strains further highlighted the impact of genetic background on LDR responses. These data provide the groundwork for predicting LDR responses using baseline expression, immunophenotypes, and/or genotype.

Yes / In this article we briefly review the Boxed Molecular Dynamics (BXD) method, which allows analysis of thermodynamics and kinetics in complicated molecular systems. BXD is a multiscale technique, in which thermodynamics and long-time dynamics are recovered from a set of short-time simulations. In this article, we review previous applications of BXD to peptide cyclization, diamond etching, solution-phase organic reaction dynamics, and desorption of ions from self-assembled monolayers (SAMs). We also report preliminary results of simulations of diamond etching mechanisms and protein unfolding in AFM experiments. The latter demonstrate a correlation between the protein’s structural motifs and its potential of mean force (PMF). Simulations of these processes by standard molecular dynamics (MD) is typically not possible, since the experimental timescales are very long. However, BXD yields well-converged and physically meaningful results. Compared to other methods of accelerated MD, our BXD approach is very simple; it is easy to implement, and it provides an integrated approach for simultaneously obtaining both thermodynamics and kinetics. It also provides a strategy for obtaining statistically meaningful dynamical results in regions of configuration space that standard MD approaches would visit only very rarely. / DRG is grateful for funding from a Royal Society Research Fellowship. JB and DVS acknowledge the support of EPSRC (Grant No EP/E009824/1). E.M.-N. and S.A.V. are grateful to the “Centro de Supercomputación de Galicia (CESGA)” for the use of its computational resources, as well as to “Ministerio de Economía y Competitividad” (Grant No. CTQ2009-12588) for financial support. DS and E.M.-N. acknowledge the Leverhulme Trust for funding the E.M.-N. visit to Leeds by the grant “Accelerated classical and quantum molecular dynamics and its applications” (Grant No. VP1-2012-013).

It has been reported that an individual’s initial level of response to a drug might be predictive of his or her future risk of becoming dependent, thus basal gene expression profiles underlying those drug responses may be informative for both predicting addiction susceptibility and determining targets for intervention. This dissertation research aims to elucidate genetic risk factors underlying acute alcohol and nicotine dependence phenotypes using mouse genetic models of addiction. Phenotyping, brain region-specific mRNA expression profiling, and genetic mapping of a recombinant inbred panel of over 25 mouse strains were performed in order to identify quantitative trait loci (QTL) harboring candidate genes that may modulate these phenotypes. Previous BXD (B6 x D2) behavioral studies performed in our laboratory identified an ethanol-induced anxiolysis-like QTL (Etanq1) in the light dark box (LDB). We hypothesized that genetic variation within Nin (a gene within the Etanq1 support interval involved in microtubule-anchoring) may modulate anxiolytic-like responses to acute ethanol in the LDB as well as other preclinical models of anxiety, the elevated plus maze (EPM), and marble burying (MB) task. Molecular studies have allowed us to confirm cis regulation of Nin transcript levels in the NAc. To elucidate potential mechanisms mediating Etanq1, the pharmacological tools, diazepam and HZ166 (a benzodiazepine derivative) were utilized to interrogate whether GABAA receptor activation modulates ethanol’s anxiety-like behaviors in the LDB. We show that the LDB phenotype, percent time spent (PTS) in the light following a brief restraint stress, is not being modulated through direct activation of GABAA α2/α3 receptor subunits. To genetically dissect Etanq1 as well as parse the ethanol anxiolytic-like phenotype, we have assayed 8 inbred strains, selected based on genotypes at Nin, in various preclinical models of anxiety. Principal components analysis of these behavioral data suggests that the gene(s) modulating the ethanol anxiolytic-like component in the LDB do not overlap with similar phenotypes in the elevated plus maze (EPM), nor the MB phenotype. Furthermore, site-specific delivery of an sh-Nin lentivirus into the NAc of D2 mice revealed that Nin may modulate one LDB endophenotype, latency to enter the light side of the LDB, which loaded as a part of the “anxiolysis” principal component. These data strongly imply that basal neuronal Nin expression in the NAc is important for acute ethanol anxiolytic-like behavior, perhaps through a novel mechanism involving synaptic remodeling. In separate behavioral QTL mapping studies, we hypothesized that genetic variation regulating expression of Chrna7 modulates the reward-like phenotype, conditioned place preference (CPP), for nicotine. We provide evidence for genetic regulation of Chrna7 across the BXD panel of mice and through pharmacological and genetic behavioral studies, confirm Chrna7 as a quantitative trait gene modulating CPP for nicotine in mice. Microarrays, followed by network analyses, allowed us to identify a genetically co-regulated network within the nucleus accumbens (NAc), differentially expressed in mice null for Chrna7, which was similarly correlated in the BXD panel of mice. Our network and molecular analyses suggest a putative role for Chrna7 in regulating insulin signaling in the NAc, which together, may contribute to the enhanced sensitivity to nicotine observed in strains of mice that lack or have low mRNA levels of Chrna7 in the NAc. Overall, this research has elucidated and confirmed new genetic risk factors underlying alcohol and nicotine dependence phenotypes and has enabled a better understanding of the neurogenomic bases of alcohol and nicotine addiction. Future studies that further investigate the signaling pathways and/or gene interactions involving Nin and Chrna7 may lead the field to new candidates for pharmacotherapies that may be tailored for use in individuals with susceptible genotypes. Supported by NIAAA grants P20AA017828 and R01AA020634 to MFM, NIDA T32DA007027 to WLD, and NIDA R01DA032246 to MFM and MID.

Adult hippocampal neurogenesis is regulated at various levels and by various factors. Genetic influence is an important key determinant of adult neurogenesis and exerts its effects at all levels. In vivo studies have suggested that adult hippocampal neurogenesis is highly variable and heritable among different laboratory strains of mice. To dissect the genetic effect from other contributing factors, it is necessary to study adult neurogenesis under highly controlled environment conditions. We extracted adult hippocampal precursor cells (AHPCs) from 20 strains of the BXD set of recombinant inbred mice, cultured them and studied the effect of genetic background on neurogenesis. The BXD panel consists of mouse lines derived from an intercross between inbred parentals C57BL/6J and DBA/2J. Both of the parentals are fully sequenced and all the strains are well characterized in terms of genotypic and phenotypic characteristics. This allows us to use advanced genetic techniques to identify novel genomic loci and gene-gene interactions important in adult neurogenesis. Comparison of the AHPCs from 20 BXD strains, with respect to cell proliferation and neuronal and astrocytic differentiation in vitro, revealed a large variation for these traits across the strains. Proliferation, as measured by BrdU incorporation, showed over two- fold differences between the extremes. Similar differences were observed for neurogenic (4-fold) and astrogenic differentiation (2-fold). These three traits all showed strong heritability values indicating that the differences were mainly attributed to the genetic component. QTL mapping, with these phenotypic data, revealed that there was no major contribution from single loci controlling these traits. Instead, we found many loci with smaller effects associated with these traits. Gene expression profiling using RNA samples from proliferating cultures of the 20 BXD mice strains yielded two cis eQTL candidates that directly regulated proliferation, LRP6 and Chchd8. LRP6 is well known as a co-receptor of Wnt signaling, but the function of Chchd8 is not known. Further experimentation, using over expression and gene silencing demonstrated that LRP6 negatively regulates AHPCs proliferation. Thus, from this study using a system genetics approach, we were able to identify, LRP6 as a novel regulator of adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is regulated at various levels and by various factors. Genetic influence is an important key determinant of adult neurogenesis and exerts its effects at all levels. In vivo studies have suggested that adult hippocampal neurogenesis is highly variable and heritable among different laboratory strains of mice. To dissect the genetic effect from other contributing factors, it is necessary to study adult neurogenesis under highly controlled environment conditions. We extracted adult hippocampal precursor cells (AHPCs) from 20 strains of the BXD set of recombinant inbred mice, cultured them and studied the effect of genetic background on neurogenesis. The BXD panel consists of mouse lines derived from an intercross between inbred parentals C57BL/6J and DBA/2J. Both of the parentals are fully sequenced and all the strains are well characterized in terms of genotypic and phenotypic characteristics. This allows us to use advanced genetic techniques to identify novel genomic loci and gene-gene interactions important in adult neurogenesis. Comparison of the AHPCs from 20 BXD strains, with respect to cell proliferation and neuronal and astrocytic differentiation in vitro, revealed a large variation for these traits across the strains. Proliferation, as measured by BrdU incorporation, showed over two- fold differences between the extremes. Similar differences were observed for neurogenic (4-fold) and astrogenic differentiation (2-fold). These three traits all showed strong heritability values indicating that the differences were mainly attributed to the genetic component. QTL mapping, with these phenotypic data, revealed that there was no major contribution from single loci controlling these traits. Instead, we found many loci with smaller effects associated with these traits. Gene expression profiling using RNA samples from proliferating cultures of the 20 BXD mice strains yielded two cis eQTL candidates that directly regulated proliferation, LRP6 and Chchd8. LRP6 is well known as a co-receptor of Wnt signaling, but the function of Chchd8 is not known. Further experimentation, using over expression and gene silencing demonstrated that LRP6 negatively regulates AHPCs proliferation. Thus, from this study using a system genetics approach, we were able to identify, LRP6 as a novel regulator of adult hippocampal neurogenesis.

Complex phenotypes are traits which are influenced by many factors, and not just a single gene, as for classical Mendelian traits. The brain, and its resultant behaviour, gives us a large subset of complex phenotypes to examine. Variation in these traits is affected by a range of different influences, both genetic and environmental, including social interactions and the effects of parents. Systems-genetics provides us with a framework in which to examine these complex traits, seeking to connect genetic variants to the phenotypes they cause, through intermediate phenotypes, such as gene expression and protein levels. This approach has been developed to exploit and analyse massive data sets generated for example in genomics and transcriptomics. In the first half of this thesis, I combine genetic linkage data from the BXD recombinant inbred mouse panel with genome-wide association data from humans to identify novel candidate genes, and use online gene annotations and functional descriptions to support these candidates. Firstly, I discovered MGST3 as a novel regulator of hippocampus size, which may be linked to neurodegenerative disorders. Secondly, I identified that CMYA5, MCTP1, TNR and RXRG are associated with mouse anxiety-like phenotypes and human bipolar disorder, and provide evidence that MCTP1, TNR and RXRG may be acting via inter-cellular signalling in the striatum. The second half of this thesis uses different cross-fostering designs between genetically variable BXD lines and the genetically uniform C57BL/6J strain to identify indirect genetic effects and the loci underlying them. With this, I have found novel loci expressed in mothers that alter offspring behaviour, novel loci expressed in offspring affecting the level of maternal care, and novel loci expressed in offspring, which alter the behaviour of their nestmates, as well as the level of maternal care they receive. Further I provide evidence of co-adaptation between maternal and offspring genotypes, and a positive indirect genetic effect of offspring on their nestmates, supportive of a role for kin selection. Finally, I demonstrate that the BXD lines can be used to investigate genes with parent-of-origin dependent expression, which have an indirect genetic effect on maternal care. In conclusion, this thesis identifies a number of novel loci, and in some cases genes, associated with complex traits. Not only are these techniques applicable to other phenotypes and other questions, but the candidates I identify can now be examined further in vitro or in vivo.