Somatic mutation rates scale with lifespan across mammals.

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1-7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined-including variation of around 30-fold in lifespan and around 40,000-fold in body mass-the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Nrg1 deficiency modulates the behavioural effects of prenatal stress in mice.

Little is known about the exact genes that confer vulnerability or resilience to environmental stressors during early neurodevelopment. Partial genetic deletion of neuregulin 1 (Nrg1) moderates the neurobehavioural effects of stressors applied in adolescence and adulthood, however, no study has yet examined its impact on prenatal stress. Here we examined whether Nrg1 deficiency in mice modulated the impact of prenatal stress on various behaviours in adulthood. Male heterozygous Nrg1 mice were mated with wild-type female mice who then underwent daily restraint stress from days 13 to 19 of gestation. Surprisingly, prenatal stress had overall beneficial effects by facilitating sensorimotor gating, increasing sociability, decreasing depressive-like behaviour, and improving spatial memory in adulthood. Such benefits were not due to any increase in maternal care, as prenatal stress decreased nurturing of the offspring. Nrg1 deficiency negated the beneficial behavioural effects of prenatal stress on all measures except sociability. However, Nrg1 deficiency interacted with prenatal stress to trigger locomotor hyperactivity. Nrg1 deficiency, prenatal stress or their combination failed to alter acute stress-induced plasma corticosterone concentrations. Collectively these results demonstrate that Nrg1 deficiency moderates the effects of prenatal stress on adult behaviour, but it does so in a complex, domain-specific fashion.

Genetic deletion of P-glycoprotein alters stress responsivity and increases depression-like behavior, social withdrawal and microglial activation in the hippocampus of female mice.

P-glycoprotein (P-gp) is an ABC transporter expressed at the blood brain barrier and regulates the brain uptake of various xenobiotics and endogenous mediators including glucocorticoid hormones which are critically important to the stress response. Moreover, P-gp is expressed on microglia, the brain's immune cells, which are activated by stressors and have an emerging role in psychiatric disorders. We therefore hypothesised that germline P-gp deletion in mice might alter the behavioral and microglial response to stressors. Female P-gp knockout mice displayed an unusual, frantic anxiety response to intraperitoneal injection stress in the light-dark test. They also tended to display reduced conditioned fear responses compared to wild-type (WT) mice in a paradigm where a single electric foot-shock stressor was paired to a context. Foot-shock stress reduced social interaction and decreased microglia cell density in the amygdala which was not varied by P-gp genotype. Independently of stressor exposure, female P-gp deficient mice displayed increased depression-like behavior, idiosyncratic darting behavior, age-related social withdrawal and hyperactivity, facilitated sensorimotor gating and altered startle reactivity. In addition, P-gp deletion increased microglia cell density in the CA3 region of the hippocampus, and the microglial cells exhibited a reactive, hypo-ramified morphology. Further, female P-gp KO mice displayed increased glucocorticoid receptor (GR) expression in the hippocampus. In conclusion, this research shows that germline P-gp deletion affected various behaviors of relevance to psychiatric conditions, and that altered microglial cell activity and enhanced GR expression in the hippocampus may play a role in mediating these behaviors.

The Differential Binding of Antipsychotic Drugs to the ABC Transporter P-Glycoprotein Predicts Cannabinoid-Antipsychotic Drug Interactions.

Cannabis use increases rates of psychotic relapse and treatment failure in schizophrenia patients. Clinical studies suggest that cannabis use reduces the efficacy of antipsychotic drugs, but there has been no direct demonstration of this in a controlled study. The present study demonstrates that exposure to the principal phytocannabinoid, Δ9-tetrahydrocannabinol (THC), reverses the neurobehavioral effects of the antipsychotic drug risperidone in mice. THC exposure did not influence D2 and 5-HT2A receptor binding, the major targets of antipsychotic action, but it lowered the brain concentrations of risperidone and its active metabolite, 9-hydroxy risperidone. As risperidone and its active metabolite are excellent substrates of the ABC transporter P-glycoprotein (P-gp), we hypothesized that THC might increase P-gp expression at the blood-brain barrier (BBB) and thus enhance efflux of risperidone and its metabolite from brain tissue. We confirmed that the brain disposition of risperidone and 9-hydroxy risperidone is strongly influenced by P-gp, as P-gp knockout mice displayed greater brain concentrations of these drugs than wild-type mice. Furthermore, we demonstrated that THC exposure increased P-gp expression in various brain regions important to risperidone's antipsychotic action. We then showed that THC exposure did not influence the neurobehavioral effects of clozapine. Clozapine shares a very similar antipsychotic mode of action to risperidone, but unlike risperidone is not a P-gp substrate. Our results imply that clozapine or non-P-gp substrate antipsychotic drugs may be better first-line treatments for schizophrenia patients with a history of cannabis use.

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.