Cathy Fernandes

Drugs and addiction: an introduction to epigenetics

Addiction is a debilitating psychiatric disorder, with a complex aetiology involving the interaction of inherited predispositions and environmental factors. Emerging evidence suggests that epigenetic alterations to the genome, including DNA methylation and histone modifications, are important mechanisms underlying addiction and the neurobiological response to addictive substances. In this review, we introduce the reader to epigenetic mechanisms and describe a potential role for dynamic epigenetic changes in mediating addictive behaviours via long-lasting changes in gene expression. We summarize recent findings from both molecular and behavioural experiments elucidating the role of epigenetic changes in mediating the addictive potential of various drugs of abuse, including cocaine, amphetamine and alcohol. The implications of these findings for molecular studies of addiction and the future development of novel therapeutic interventions are also discussed.

Cannabis use in young people: The risk for schizophrenia

Cannabis is one of the most commonly used illicit drugs, and despite the widely held belief that it is a safe drug, its long-term use has potentially harmful consequences. To date, the research on the impact of its use has largely been epidemiological in nature and has consistently found that cannabis use is associated with schizophrenia outcomes later in life, even after controlling for several confounding factors. While the majority of users can continue their use without adverse effects, it is clear from studies of psychosis that some individuals are more vulnerable to its effects than others. In addiction, evidence from both epidemiological and animal studies indicates that cannabis use during adolescence carries particular risk. Further studies are warranted given the increase in the concentration of the main active ingredient (Δ(9)-tetrahydrocannabinol) in street preparations of cannabis and a decreasing age of first-time exposure to cannabis.

Genetics of behavioural domains across the neuropsychiatric spectrum: Of mice and men

Family and twin studies have revealed that genetic factors play a major role in psychiatric disorders, however, attempts to find susceptibility genes for these complex disorders have been largely unsuccessful. Therefore, new research strategies are required to tackle the complex interactions of genes, developmental, and environmental events. Here, we will address a behavioural domain concept that focuses on the genetics of behavioural domains relevant to both animal behaviour and across human psychiatric disorders. We believe that interspecies trait genetics rather than complex syndrome genetics will optimize genotype–phenotype relationships for psychiatric disorders and facilitate the identification of biological substrates underlying these disorders.

Copy number variations in neurodevelopmental disorders.

Common neurodevelopmental disorders (including autism, speech and language delay, schizophrenia, epilepsy and intellectual disability) have complex aetiology, which is predominantly genomic, but also environmental in origin. They share a paradox, in that high heritability is matched by lowered fecundity, placing them under negative genetic selection. This implicates variants of recent origin, such as de novo mutations or common, very low-risk polymorphisms that escape negative selection. High or moderate risk variants have been discovered by chromosome analysis, genome sequencing and copy number variant (CNV) detection, including a 3Mb deletion causing 22q11.2 deletion syndrome (Velo-Cardio-Facial Syndrome) that has penetrance of up to 50% for schizophrenia. More recently, rare, recurrent and often de novo pathogenic CNVs, including deletions at NRXN1, 1q21.2, 15q11.2 and 15q13.3, 16p11.2 and duplications at VIPR2 and 16p13.11, have also been discovered. These have several unique features that differentiate them from Mendelian disease mutations in that they have incomplete penetrance, with moderate-to-high odds ratios for risk, and show diagnostic pleiotropy, increasing risk across the neurodevelopmental disorder spectrum. Some are also syndromic, with characteristic features such as facial dysmorphology, and other specific risks such as aortic dissection or obesity, implying that they might be better classified as distinct diagnoses. The discovery of pathogenic CNVs provide new opportunities for translation leading to patent benefit, including improvements in clinical genetic diagnosis and genetic counselling, the possibility of clinician decision-making tools for risk prediction, and the identification of drug targets and implementation of personalised medicine using stratification by genotype.

Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt–PCP–JNK pathway

Although the mechanism of Aβ action in the pathogenesis of Alzheimer’s disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt–planar cell polarity (PCP)–c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt–PCP–JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt–PCP–JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.

Hippocampal gene expression profiling across eight mouse inbred strains: towards understanding the molecular basis for behaviour

Mouse inbred strains differ in many aspects of their phenotypes, and it is known that gene expression does so too. This gives us an opportunity to isolate the genetic aspect of variation in expression and compare it to other phenotypic variables. We have investigated these issues using an eight‐strain expression profile comparison with four replicates per strain on Affymetrix MGU74av2 GeneChips focusing on one well‐defined brain tissue (the hippocampus). We identified substantial strain‐specific variation in hippocampal gene expression, with more than two hundred genes showing strain differences by a very conservative criterion. Many such genetically driven differences in gene expression are likely to result in functional differences including differences in behaviour. A large panel of inbred strains could be used to identify genes functionally involved in particular phenotypes, similar to genetic correlation. The genetic correlation between expression profiles and function is potentially very powerful, especially given the current large‐scale generation of phenotypic data on multiple strains (the Mouse Phenome Project). As an example, the strongest genetic correlation between more than 200 probe sets showing significant differences among our eight inbred strains and a ranking of these strains by aggression phenotype was found for Comt, a gene known to be involved in aggression.

Performance deficit of α7 nicotinic receptor knockout mice in a delayed matching-to-place task suggests a mild impairment of working/episodic-like memory

Patients with schizophrenia exhibit deficits in a range of cognitive functions, particularly working and episodic memory, which are thought to be core features of the disorder. Memory dysfunction in schizophrenia is familial and thus a promising endophenotype for genetic studies. Both human and animal studies suggest a role for the neural nicotinic acid receptor family in cognition and specifically the α7‐receptor subunit in schizophrenia and its endophenotypes. Consequently, we tested mice lacking the α7 subunit of the neural nicotinic receptor (B6.129S7‐Chrna7tm1Bay/J) in the delayed matching‐to‐place (DMP) task of the Morris water maze, a measure of working/episodic memory akin to human episodic memory. We report that a minor impairment in α7 knockout mice was observed in the DMP task, with knockout mice taking longer to find the hidden platform than their wildtype controls. This suggests a role for the α7 subunit in working/episodic memory and a potential role for the α7 neural nicotinic receptor gene (CHRNA7) in schizophrenia and its endophenotypes.

Maternal separation is associated with strain-specific responses to stress and epigenetic alterations to Nr3c1, Avp, and Nr4a1 in mouse

Stressful events early in life have been widely linked to behavioral phenotypes and have been implicated in the development of psychiatric disorders. Using a maternal separation paradigm, we investigated phenotypic and epigenetic changes following early life stress in two inbred strains of mice, C57BL/6J and DBA/2J. We found an increase in the corticosterone response to stress in male, C57BL/6J mice that had undergone maternal separation compared to controls. In addition, early life stress induced a number of mild but significant behavioral changes, many of which were sex and strain dependent. Following maternal separation anxiety was decreased in males but increased in DBA/2J females, DBA/2J males displayed reduced exploration of a novel object, and baseline activity was altered in males of both strains. Finally, we examined DNA methylation levels in the hippocampus across promoter regions of Nr3c1, Avp, and Nr4a1, and found altered levels at several CpG sites in maternally separated male mice compared to controls. This study contributes to a growing body of recent literature suggesting that epigenetic changes may mediate the impact of early life stress on behavior. In particular, we establish that the phenotypic and epigenetic responses to an adverse environment differ as a function of genetic background.

Cross-region reduction in 5-hydroxymethylcytosine in Alzheimer's disease brain

Epigenetic processes play a key role in the central nervous system and altered levels of 5-methylcytosine have been associated with a number of neurologic phenotypes, including Alzheimers disease (AD). Recently, 3 additional cytosine modifications have been identified (5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine), which are thought to be intermediate steps in the demethylation of 5-methylcytosine to unmodified cytosine. Little is known about the frequency of these modifications in the human brain during health or disease. In this study, we used immunofluorescence to confirm the presence of each modification in human brain and investigate their cross-tissue abundance in AD patients and elderly control samples. We identify a significant AD-associated decrease in global 5-hydroxymethylcytosine in entorhinal cortex and cerebellum, and differences in 5-formylcytosine levels between brain regions. Our study further implicates a role for epigenetic alterations in AD.

Genotyping DNA pools on microarrays: Tackling the QTL problem of large samples and large numbers of SNPs

BackgroundQuantitative trait locus (QTL) theory predicts that genetic influence on complex traits involves multiple genes of small effect size. To detect QTL associations of small effect size, large samples and systematic screens of thousands of DNA markers are required. An efficient solution is to genotype case and control DNA pools using SNP microarrays. We demonstrate that this is practical using DNA pools of 100 individuals.ResultsUsing standard microarray protocols for the Affymetrix GeneChip® Mapping 10 K Array Xba 131, we show that relative allele signal (RAS) values provide a quantitative index of allele frequencies in pooled DNA that correlate 0.986 with allele frequencies for 104 SNPs that were genotyped individually for 100 individuals. The sensitivity of the assay was demonstrated empirically in a spiking experiment in which 15% and 20% of one individuals DNA was added to a DNA pool.ConclusionWe conclude that this approach, which we call SNP-MaP (SNP m icroarrays a nd p ooling), is rapid, cost effective and promises to be a valuable initial screening method in the hunt for QTLs.