Investigating the relationship between DNA methylation, genetic variation, and suicide attempt in bipolar disorder.

Individuals with bipolar disorder are at increased risk for suicide, and this can be influenced by a range of biological, clinical, and environmental risk factors. Biological components associated with suicide include DNA modifications that lead to changes in gene expression. Common genetic variation and DNA methylation changes are some of the most frequent types of DNA findings associated with an increased risk for suicidal behavior. Importantly, the interplay between genetic predisposition and DNA methylation patterns is becoming more prevalent in genetic studies. We hypothesized that DNA methylation patterns in specific loci already genetically associated with suicide would be altered in individuals with bipolar disorder and a history of suicide attempt. To test this hypothesis, we searched the literature to identify common genetic variants (N=34) previously associated with suicidal thoughts and behaviors in individuals with bipolar disorder. We then created a customized sequencing panel that covered our chosen genomic loci. We profiled DNA methylation patterns from blood samples collected from bipolar disorder participants with suicidal behavior (N=55) and without suicidal behavior (N=51). We identified seven differentially methylated CpG sites and five differentially methylated regions between the two groups. Additionally, we found that DNA methylation changes in MIF and CACNA1C were associated with lethality or number of suicide attempts. Finally, we identified three meQTLs in SIRT1 , IMPA2 , and INPP1 . This study illustrates that DNA methylation is altered in individuals with bipolar disorder and a history of suicide attempts in regions known to harbor suicide-related variants.

Blood epigenome-wide association studies of suicide attempt in adults with bipolar disorder.

Suicide attempt (SA) risk is elevated in individuals with bipolar disorder (BD), and DNA methylation patterns may serve as possible biomarkers of SA. We conducted epigenome-wide association studies (EWAS) of blood DNA methylation associated with BD and SA. DNA methylation was measured at >700,000 positions in a discovery cohort of n = 84 adults with BD with a history of SA (BD/SA), n = 79 adults with BD without history of SA (BD/non-SA), and n = 76 non-psychiatric controls (CON). EWAS revealed six differentially methylated positions (DMPs) and seven differentially methylated regions (DMRs) between BD/SA and BD/non-SA, with multiple immune-related genes implicated. There were no epigenome-wide significant differences when BD/SA and BD/non-SA were each compared to CON, and patterns suggested that epigenetics differentiating BD/SA from BD/non-SA do not differentiate BD/non-SA from CON. Weighted gene co-methylation network analysis and trait enrichment analysis of the BD/SA vs. BD/non-SA contrast further corroborated immune system involvement, while gene ontology analysis implicated calcium signalling. In an independent replication cohort of n = 48 BD/SA and n = 47 BD/non-SA, fold changes at the discovery cohort's significant sites showed moderate correlation across cohorts and agreement on direction. In both cohorts, classification accuracy for SA history among individuals with BD was highest when methylation at the significant CpG sites as well as information from clinical interviews were combined, with an AUC of 88.8% (CI = 83.8-93.8%) and 82.1% (CI = 73.6-90.5%) for the combined epigenetic-clinical classifier in the discovery and replication cohorts, respectively. Our results provide novel insight to the role of immune system functioning in SA and BD and also suggest that integrating information from multiple levels of analysis holds promise to improve risk assessment for SA in adults with BD.

Blood epigenome-wide association studies of suicide attempt in adults with bipolar disorder.

Suicide attempt (SA) risk is elevated in individuals with bipolar disorder (BD), and DNA methylation patterns may serve as possible biomarkers of SA. We conducted epigenome-wide association studies (EWAS) of blood DNA methylation associated with BD and SA. DNA methylation was measured at > 700,000 positions in a discovery cohort of n = 84 adults with BD with a history of SA (BD/SA), n = 79 adults with BD without history of SA (BD/non-SA), and n = 76 non-psychiatric controls (CON). EWAS revealed six differentially methylated positions (DMPs) and seven differentially methylated regions (DMRs) between BD/SA and BD/non-SA, with multiple immune-related genes implicated. There were no epigenome-wide significant differences when BD/SA and BD/non-SA were each compared to CON, and patterns suggested that epigenetics differentiating BD/SA from BD/non-SA do not differentiate BD/non-SA from CON. Weighted gene co-methylation network analysis and trait enrichment analysis of the BD/SA vs. BD/non-SA contrast further corroborated immune system involvement, while gene ontology analysis implicated calcium signalling. In an independent replication cohort of n = 48 BD/SA and n = 47 BD/non-SA, fold-changes at the discovery cohort's significant sites showed moderate correlation across cohorts and agreement on direction. In both cohorts, classification accuracy for SA history among individuals with BD was highest when methylation at the significant CpG sites as well as information from clinical interviews were combined, with an AUC of 88.8% (CI = 83.8-93.8%) and 82.1% (CI = 73.6-90.5%) for the combined epigenetic-clinical predictor in the discovery and replication cohorts, respectively. Our results provide novel insight to the role of immune system functioning in SA and BD and also suggest that integrating information from multiple levels of analysis holds promise to improve risk assessment for SA in adults with BD.

Functional connectivity of the cerebellar vermis in bipolar disorder and associations with mood.

Purpose: Studies of the neural underpinnings of bipolar type I disorder have focused on the emotional control network. However, there is also growing evidence for cerebellar involvement, including abnormal structure, function, and metabolism. Here, we sought to assess functional connectivity of the cerebellar vermis with the cerebrum in bipolar disorder and to assess whether connectivity might depend on mood. Methods: This cross-sectional study enrolled 128 participants with bipolar type I disorder and 83 control comparison participants who completed a 3 T magnetic resonance imaging (MRI) study, which included anatomical as well as resting state Blood Oxygenation Level Dependent (BOLD) imaging. Functional connectivity of the cerebellar vermis to all other brain regions was assessed. Based on quality control metrics of the fMRI data, 109 participants with bipolar disorder and 79 controls were included in the statistical analysis comparing connectivity of the vermis. In addition, the data was explored for the potential impacts of mood, symptom burden, and medication in those with bipolar disorder. Results: Functional connectivity between the cerebellar vermis and the cerebrum was found to be aberrant in bipolar disorder. The connectivity of the vermis was found to be greater in bipolar disorder to regions involved in motor control and emotion (trending), while reduced connectivity was observed to a region associated with language production. In the participants with bipolar disorder, past depression symptom burden affected connectivity; however, no effects of medication were observed. Functional connectivity between the cerebellar vermis and all other regions revealed an inverse association with current mood ratings. Conclusion: Together the findings may suggest that the cerebellum plays a compensatory role in bipolar disorder. The proximity of the cerebellar vermis to the skull may make this region a potential target for treatment with transcranial magnetic stimulation.

Neuronal deletion of CaV1.2 is associated with sex-specific behavioral phenotypes in mice.

The gene CACNA1C, which encodes the pore forming subunit of the L-type calcium channel CaV1.2, is associated with increased risk for neuropsychiatric disorders including schizophrenia, autism spectrum disorder, major depression, and bipolar disorder. Previous rodent work identified that loss or reduction of CaV1.2 results in cognitive, affective, and motor deficits. Most previous work has either included non-neuronal cell populations (haploinsufficient and Nestin-Cre) or investigated a discrete neuronal cell population (e.g. CaMKII-Cre, Drd1-Cre), but few studies have examined the effects of more broad neuron-specific deletion of CaV1.2. Additionally, most of these studies did not evaluate for sex-specific effects or used only male animals. Here, we sought to clarify whether there are sex-specific behavioral consequences of neuron-specific deletion of CaV1.2 (neuronal CaV1.2 cKO) using Syn1-Cre-mediated conditional deletion. We found that neuronal CaV1.2 cKO mice have normal baseline locomotor function but female cKO mice display impaired motor performance learning. Male neuronal CaV1.2 cKO display impaired startle response with intact pre-pulse inhibition. Male neuronal CaV1.2 cKO mice did not display normal social preference, whereas female neuronal CaV1.2 cKO mice did. Neuronal CaV1.2 cKO mice displayed impaired associative learning in both sexes, as well as normal anxiety-like behavior and hedonic capacity. We conclude that deletion of neuronal CaV1.2 alters motor performance, acoustic startle reflex, and social behaviors in a sex-specific manner, while associative learning deficits generalize across sexes. Our data provide evidence for both sex-specific and sex-independent phenotypes related to neuronal expression of CaV1.2.

Modeling Human Cerebellar Development In Vitro in 2D Structure.

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, disruption in cerebellar development has been implicated in many neurodevelopmental disorders, including autism, attention deficit-hyperactivity disorder (ADHD), and schizophrenia. Investigations of cerebellar development in humans have previously only been possible through post-mortem studies or neuroimaging, yet these methods are not sufficient for understanding the molecular and cellular changes occurring in vivo during early development, which is when many neurodevelopmental disorders originate. The emergence of techniques to generate human-induced pluripotent stem cells (iPSCs) from somatic cells and the ability to further re-differentiate iPSCs into neurons have paved the way for in vitro modeling of early brain development. The present study provides simplified steps toward generating cerebellar cells for applications that require a 2-dimensional (2D) monolayer structure. Cerebellar cells representing early developmental stages are derived from human iPSCs via the following steps: first, embryoid bodies are made in 3-dimensional (3D) culture, then they are treated with FGF2 and insulin to promote cerebellar fate specification, and finally, they are terminally differentiated as a monolayer on poly-l-ornithine (PLO)/laminin-coated substrates. At 35 days of differentiation, iPSC-derived cerebellar cell cultures express cerebellar markers including ATOH1, PTF1α, PAX6, and KIRREL2, suggesting that this protocol generates glutamatergic and GABAergic cerebellar neuronal precursors, as well as Purkinje cell progenitors. Moreover, the differentiated cells show distinct neuronal morphology and are positive for immunofluorescence markers of neuronal identity such as TUBB3. These cells express axonal guidance molecules, including semaphorin-4C, plexin-B2, and neuropilin-1, and could serve as a model for investigating the molecular mechanisms of neurite outgrowth and synaptic connectivity. This method generates human cerebellar neurons useful for downstream applications, including gene expression, physiological, and morphological studies requiring 2D monolayer formats.

Distinct patterns of altered quantitative T1ρ and functional BOLD response associated with history of suicide attempts in bipolar disorder.

Despite the high risk for suicide, relatively few studies have explored the relationship between suicide and brain imaging measures in bipolar disorder. In addition, fewer studies have explored the possibility that altered brain metabolism may be associated with suicide attempt. To begin to fill in these gaps, we evaluated functional (task based fMRI) and metabolic (quantitative T1ρ) differences associated with suicide attempt in participants with bipolar disorder. Thirty-nine participants with bipolar disorder underwent fMRI during a flashing checkerboard task and 27 also underwent quantitative T1ρ. The relationship between neuroimaging and history of suicide attempt was tested using multiple regression while adjusting for age, sex, and current mood state. Differences between two measures of suicide attempt (binary: yes/no and continuous: number of attempts) were quantified using the corrected Akaike Information Criterion. Participants who had attempted suicide had greater fMRI task-related activation in visual areas and the cerebellum. The number of suicide attempts was associated with a difference in BOLD response in the amygdala, prefrontal cortex, and cerebellum. Increased quantitative T1ρ was associated with number of suicide attempts in limbic, basal ganglia, and prefrontal cortex regions. This study is a secondary analysis with a modest sample size. Differences between measures of suicide history may be due to differences in statistical power. History of suicide was associated with limbic, prefrontal, and cerebellar alterations. Results comparing those with and without suicide attempts differed from results using number of suicide attempts, suggesting that these variables have different neurobiological underpinnings.

SCN2A channelopathies in the autism spectrum of neuropsychiatric disorders: a role for pluripotent stem cells?

Efforts to identify the causes of autism spectrum disorders have highlighted the importance of both genetics and environment, but the lack of human models for many of these disorders limits researchers' attempts to understand the mechanisms of disease and to develop new treatments. Induced pluripotent stem cells offer the opportunity to study specific genetic and environmental risk factors, but the heterogeneity of donor genetics may obscure important findings. Diseases associated with unusually high rates of autism, such as SCN2A syndromes, provide an opportunity to study specific mutations with high effect sizes in a human genetic context and may reveal biological insights applicable to more common forms of autism. Loss-of-function mutations in the SCN2A gene, which encodes the voltage-gated sodium channel NaV1.2, are associated with autism rates up to 50%. Here, we review the findings from experimental models of SCN2A syndromes, including mouse and human cell studies, highlighting the potential role for patient-derived induced pluripotent stem cell technology to identify the molecular and cellular substrates of autism.

Risk Factors Associated With Antidepressant Exposure and History of Antidepressant-Induced Mania in Bipolar Disorder.

OBJECTIVE: Despite their widespread use in bipolar disorder, there is controversy surrounding the inclusion of antidepressant medications in the disorder's management. We sought to identify which demographic, socioeconomic, and clinical factors are associated with antidepressant exposure in bipolar disorder and which bipolar disorder patients are most likely to report a history of antidepressant-induced mania (AIM) when exposed to antidepressants. METHODS: Our study included subjects with bipolar I disorder (n = 309), bipolar II disorder (n = 66), and bipolar disorder not otherwise specified (n = 27) and schizoaffective disorder, bipolar type (n = 14), from a longitudinal, community-based study. Subjects were evaluated using the Diagnostic Interview for Genetic Studies, modified for DSM-IV criteria. We applied multivariate logistical regression modeling to investigate which factors contribute to antidepressant exposure in bipolar disorder patients. We also used a logistic regression modeling approach to determine which clinical factors in bipolar disorder patients are associated with a history of AIM. Data were gathered from February 2006 through December 2010. RESULTS: Our results suggest that the risk factors most strongly associated with antidepressant exposure are female sex (OR = 2.73, P = .005), older age (OR = 1.03, P = .04), greater chronicity of illness (OR = 2.29, P = .04), and, to a lesser extent, white race (OR = 0.44, P = .051). Factors associated with reduced antidepressant exposure include history of affective psychosis (OR = 0.36, P = .01) and a greater number of previous manic episodes (OR = 0.98, P = .03). In subjects who reported a history of AIM, regression analysis revealed that the only statistically significant factor associated with AIM history was female sex (OR = 3.74, P = .02). CONCLUSIONS: These data suggest that there are certain identifiable factors associated with antidepressant exposure in bipolar disorder patients, and some of these, specifically female sex, are also associated with a history of AIM. These data may be useful in designing prospective trials to identify interventions that can reduce the risk of this adverse outcome.

Atypical psychotic symptoms and Dandy-Walker variant.

New-onset psychotic symptoms often respond well to antipsychotic treatment; however, symptoms may be difficult to treat when an underlying brain malformation is present. Here, we present a case of atypical psychotic symptoms in the context of a congenital cerebellar malformation (Dandy-Walker variant). The patient ultimately improved with paliperidone palmitate after multiple antipsychotic medication trials (both oral and one long-acting injectable) were ineffective. Neuroimaging may provide valuable diagnostic and prognostic information in cases of new-onset psychosis with atypical features and treatment resistance, even in the absence of neurologic signs and symptoms.

Deletion of fibroblast growth factor 22 (FGF22) causes a depression-like phenotype in adult mice.

Specific growth factors induce formation and differentiation of excitatory and inhibitory synapses, and are essential for brain development and function. Fibroblast growth factor 22 (FGF22) is important for specifying excitatory synapses during development, including in the hippocampus. Mice with a genetic deletion of FGF22 (FGF22KO) during development subsequently have fewer hippocampal excitatory synapses in adulthood. As a result, FGF22KO mice are resistant to epileptic seizure induction. In addition to playing a key role in learning, the hippocampus is known to mediate mood and anxiety. Here, we explored whether loss of FGF22 alters affective, anxiety or social cognitive behaviors in mice. We found that relative to control mice, FGF22KO mice display longer duration of floating and decreased latency to float in the forced swim test, increased immobility in the tail suspension test, and decreased preference for sucrose in the sucrose preference test, which are all suggestive of a depressive-like phenotype. No differences were observed between control and FGF22KO mice in other behavioral assays, including motor, anxiety, or social cognitive tests. These results suggest a novel role for FGF22 specifically in affective behaviors.

The best-laid plans go oft awry: synaptogenic growth factor signaling in neuropsychiatric disease.

Growth factors play important roles in synapse formation. Mouse models of neuropsychiatric diseases suggest that defects in synaptogenic growth factors, their receptors, and signaling pathways can lead to disordered neural development and various behavioral phenotypes, including anxiety, memory problems, and social deficits. Genetic association studies in humans have found evidence for similar relationships between growth factor signaling pathways and neuropsychiatric phenotypes. Accumulating data suggest that dysfunction in neuronal circuitry, caused by defects in growth factor-mediated synapse formation, contributes to the susceptibility to multiple neuropsychiatric diseases, including epilepsy, autism, and disorders of thought and mood (e.g., schizophrenia and bipolar disorder, respectively). In this review, we will focus on how specific synaptogenic growth factors and their downstream signaling pathways might be involved in the development of neuropsychiatric diseases.

JosD1, a membrane-targeted deubiquitinating enzyme, is activated by ubiquitination and regulates membrane dynamics, cell motility, and endocytosis.

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic "Josephin" domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.

The Machado-Joseph disease-associated mutant form of ataxin-3 regulates parkin ubiquitination and stability.

Machado-Joseph disease (MJD), the most common dominantly inherited ataxia worldwide, is caused by a polyglutamine (polyQ) expansion in the deubiquitinating (DUB) enzyme ataxin-3. Interestingly, MJD can present clinically with features of Parkinsonism. In this study, we identify parkin, an E3 ubiquitin-ligase responsible for a common familial form of Parkinson's disease, as a novel ataxin-3 binding partner. The interaction between ataxin-3 and parkin is direct, involves multiple domains and is greatly enhanced by parkin self-ubiquitination. Moreover, ataxin-3 deubiquitinates parkin directly in vitro and in cells. Compared with wild-type ataxin-3, MJD-linked polyQ-expanded mutant ataxin-3 is more active, possibly owing to its greater efficiency at DUB K27- and K29-linked Ub conjugates on parkin. Remarkably, mutant but not wild-type ataxin-3 promotes the clearance of parkin via the autophagy pathway. The finding is consistent with the reduction in parkin levels observed in the brains of transgenic mice over-expressing polyQ-expanded but not wild-type ataxin-3, raising the intriguing possibility that increased turnover of parkin may contribute to the pathogenesis of MJD and help explain some of its parkinsonian features.

In vivo suppression of polyglutamine neurotoxicity by C-terminus of Hsp70-interacting protein (CHIP) supports an aggregation model of pathogenesis.

Perturbations in neuronal protein homeostasis likely contribute to disease pathogenesis in polyglutamine (polyQ) neurodegenerative disorders. Here we provide evidence that the co-chaperone and ubiquitin ligase, CHIP (C-terminus of Hsp70-interacting protein), is a central component to the homeostatic mechanisms countering toxic polyQ proteins in the brain. Genetic reduction or elimination of CHIP accelerates disease in transgenic mice expressing polyQ-expanded ataxin-3, the disease protein in Spinocerebellar Ataxia Type 3 (SCA3). In parallel, CHIP reduction markedly increases the level of ataxin-3 microaggregates, which partition in the soluble fraction of brain lysates yet are resistant to dissociation with denaturing detergent, and which precede the appearance of inclusions. The level of microaggregates in the CNS, but not of ataxin-3 monomer, correlates with disease severity. Additional cell-based studies suggest that either of two quality control ubiquitin ligases, CHIP or E4B, can reduce steady state levels of expanded, but not wild-type, ataxin-3. Our results support an aggregation model of polyQ disease pathogenesis in which ataxin-3 microaggregates are a neurotoxic species, and suggest that enhancing CHIP activity is a possible route to therapy for SCA3 and other polyQ diseases.

Polyglutamine neurodegeneration: protein misfolding revisited.

Polyglutamine diseases are a major cause of neurodegeneration worldwide. Recent studies highlight the importance of protein quality control mechanisms in regulating polyglutamine-induced toxicity. Here we discuss a model of disease pathogenesis that integrates current understanding of the role of protein folding in polyglutamine disease with emerging evidence that alterations in native protein interactions contribute to toxicity. We also incorporate new findings on other age-related neurodegenerative diseases in an effort to explain how protein aggregation and normal aging processes might be involved in polyglutamine disease pathogenesis.

The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains.

Ubiquitin chain complexity in cells is likely regulated by a diverse set of deubiquitinating enzymes (DUBs) with distinct ubiquitin chain preferences. Here we show that the polyglutamine disease protein, ataxin-3, binds and cleaves ubiquitin chains in a manner suggesting that it functions as a mixed linkage, chain-editing enzyme. Ataxin-3 cleaves ubiquitin chains through its amino-terminal Josephin domain and binds ubiquitin chains through a carboxyl-terminal cluster of ubiquitin interaction motifs neighboring the pathogenic polyglutamine tract. Ataxin-3 binds both Lys(48)- or Lys(63)-linked chains yet preferentially cleaves Lys(63) linkages. Ataxin-3 shows even greater activity toward mixed linkage polyubiquitin, cleaving Lys(63) linkages in chains that contain both Lys(48) and Lys(63) linkages. The ubiquitin interaction motifs regulate the specificity of this activity by restricting what can be cleaved by the protease domain, demonstrating that linkage specificity can be determined by elements outside the catalytic domain of a DUB. These findings establish ataxin-3 as a novel DUB that edits topologically complex chains.

Redox modifier genes in amyotrophic lateral sclerosis in mice.

Amyotrophic lateral sclerosis (ALS), one of the most common adult-onset neurodegenerative diseases, has no known cure. Enhanced redox stress and inflammation have been associated with the pathoprogression of ALS through a poorly defined mechanism. Here we determined that dysregulated redox stress in ALS mice caused by NADPH oxidases Nox1 and Nox2 significantly influenced the progression of motor neuron disease caused by mutant SOD1(G93A) expression. Deletion of either Nox gene significantly slowed disease progression and improved survival. However, 50% survival rates were enhanced significantly more by Nox2 deletion than by Nox1 deletion. Interestingly, female ALS mice containing only 1 active X-linked Nox1 or Nox2 gene also had significantly delayed disease onset, but showed normal disease progression rates. Nox activity in spinal cords from Nox2 heterozygous female ALS mice was approximately 50% that of WT female ALS mice, suggesting that random X-inactivation was not influenced by Nox2 gene deletion. Hence, chimerism with respect to Nox-expressing cells in the spinal cord significantly delayed onset of motor neuron disease in ALS. These studies define what we believe to be new modifier gene targets for treatment of ALS.