Yogesh Dwivedi - US grants

This proposal for a Mentored Scientist Development Award focuses on elucidating the role of beta adrenergic receptors (ARs) and the adenylyl cyclase-cyclic cAMP (AC-cAMP) pathway in the pathophysiology of depression and suicide. Several studies suggest that betaAR number is increased in the postmortem brain of suicide subjects; however, the precise mechanism, significance and the cellular/molecular nature of events associated with this increase have not been elucidated. The goals of this study are to examine: 1) if upregulation of betaARs is due to increased rnRNA and/or protein expression of beta1 and/or beta2 ARs; 2) whether this increase is associated with increase transcription rate and/or dysregulated HPA axis; 3) functional consequences of increased betaARs in the AC-cAMP signaling pathway at the level of catalytic and regulatory activities of protein kinase A (PKA) and gene transcription of their specific subunits; 4) functional significance of altered PKA by examining functional characteristics and gene expression of transcription factors and target genes; 5) the localization of these changes at the cellular level in discrete brain areas; and 5) whether these changes are specific to depression.To achieve these goals we propose a series of related human postmortem brain and animal studies. We will study mRNA and protein expression of beta1 and beta2ARs by quantitative RT-PCR, in-situ hybridization and gold-immunolabeling. To examine whether changes in betaARs are associated with alterations in components of the AC- cAMP signaling pathway, we will study [3H]cAMP binding to PKA; total, endogenous and betaAR-mediated PKA activity; mRNA and protein expression of PKA (regulatory and catalytic subunits), CREB, BDNF and phospho-CREB; CREB-DNA binding activity in BAs 8, 9, 10 and hippocampus of suicide victims and age-, postmortem interval-, and gender-matched nonpsychiatric control subjects. We will examine changes in mRNA and protein expression in discrete areas of the brain (layers I-VI of prefrontal cortex and areas CA1-4, dentate gyrus subiculum and area entorhinalis of the hippocampal formation). Further, to examine if the changes in the proposed measures are specific to depression, we will examine the effects of major depression on the proposed measures. To consolidate our human postmortem brain findings, we will study the proposed measures in prefrontal cortex and hippocampus of behaviorally depressed rats. Finally, to examine if these changes are associated with abnormal HPA function we will study the proposed measures in prefrontal cortex and hippocampus of corticosterone-treated rats with and without adrenalectomy. With this proposal the candidate seeks training in 1) Clinical and psychological aspects of mental disorders and suicide, 2) molecular biology, 3) neuroanatomy, 4) animal behavior, and 5) specialized statistical analyses. The rigorous training plan, which integrates strong didactics and multidisciplinary expertise, and the research plan will advance the knowledge of molecular mechanisms associated with depression and suicide and will provide the candidate with the skills needed to achieve independence in this highly complex field.

We and several other investigators have previously reported that neurotransmitter receptors, such as serotonergic and adrenergic, and specific effectors of these receptor-mediated signal transduction systems are altered in the postmortem brain of suicide victims. Recent investigations in our laboratory have revealed that the functional characteristics and the expression of transcription factor CREB, a common substrate for many neurotransmitter receptor systems implicated in the pathophysiology of suicide, and the expressions of its target gene BDNF and receptor for BDNF, i.e., TrkB, are significantly altered in the postmortem brain of suicide victims, suggesting that these molecules may be playing an important role in the pathophysiology of suicidal behavior. However, the significance and consequences of these alterations at the functional level in suicide brain are not clear. ERK-1/2 and PI3-kinase cascades are the two most important signaling systems in the CNS that are activated by TrkB and mediate the physiological functions of BDNF. The major objective of the proposed research is to elucidate the cellular and the molecular mechanisms associated with suicide by examining whether abnormalities in BDNF and TrkB are associated with abnormalities further downstream in the ERK-1/2 and the PI3-kinase pathways, both at the level of kinases and at the functional level in the substrate molecules responsible for physiological actions in the brain. More specifically, in postmortem brain of suicide victims and well-matched nonpsychiatric control subjects, we will study the activation and the expression of upstream kinases Raf-1, MEK-1, ERK-1, ERK-2, PI3-kinase, Akt-1, Akt-3, and of downstream substrates, transcription factors Elk-1 and FKHRL1, and regulatory proteins Bcl-2 and Bad, both at the molecular and the cellular level. These studies will be performed in prefrontal cortical and hippocampal brain areas utilizing quantitative RT-PCR, Western blot, immunoprecipitation, enzymatic assays, in-situ hybridization, and gold-immunolabeling techniques. Furthermore, to examine whether observed changes are restricted to depression, we will compare the findings in the proposed measures between suicide victims with a firm diagnosis of major depression and those diagnosed with other psychiatric disorders. We will also study whether there is coordinated regulation of the proposed measures (upstream kinases, downstream substrates, and regulatory proteins) of the two signaling pathways within the same brain by examining the correlational structure overall and individually within the group and by statistical path analysis. The proposed research is based on the central hypothesis that there may be abnormalities in the components of ERK-1/2 and PI-3 kinase signaling pathways in postmortem brain of suicide victims, which may be associated with abnormalities in activation and/or expression of the substrate molecules responsible for BDNF-elicited neuronal functions, and that these abnormalities may play an important role in the pathophysiology of suicidal behavior. Elucidation of the alterations in ERK-1/2 and PI3-kinase pathways in postmortem brain of suicide victims will yield important information on the neurobiology of suicide and will indicate possible novel sites for therapeutic interventions, which may eventually lead to better treatment and possibly prevention of suicidal behavior.

[unreadable] DESCRIPTION (provided by applicant): The family of short trans-acting antisense RNA transcripts known as the microRNAs regulate target mRNAs via eliciting mRNA degradation or arrested translation. MicroRNAs are important in regulating prenatal brain development, and almost certainly have functions within the mature brain as well. We hypothesize that microRNAs participate in the regulation of human mRNAs, including those that are involved in the pathophysiology of psychiatric disorders. Although postmortem human brain samples have proven to be a valuable and unique resource for studying biochemical abnormalities that occur in depression and major psychoses, no methods for reliably measuring microRNAs have yet been reported for postmortem brain. In order to study the possible role of microRNA pathways in human disease, we aim to: 1) Validate methods of microRNA microarrays for measuring known human microRNAs within postmortem human brain. 2) Compare microRNA expression in prefrontal cortex across two different cohorts of postmortem brain samples including normal controls and a set of matched depressed subjects. The purpose of this proposal is to set the stage for genome-wide analysis of all known human microRNAs in samples of postmortem brain. This is the necessary first step towards our eventual goals: to learn which mRNA targets and neural pathways are regulated by microRNAs, and to learn whether any specific microRNAs show differential expression in subjects with psychiatric disorders. Our proposed study will yield important information on the neurobiology of depression and may indicate possible novel sites for therapeutic interventions, which may eventually lead to better treatment and possibly prevention of depression. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Altered synaptic and structural plasticity play a crucial role in the pathogenesis of major depressive disorder (MDD); however, the precise molecular and cellular nature of events that lead to such altered plasticity remains unclear. Ca2+ is one of the critical molecules that play a decisive role in initiating and regulating synaptic and structural plasticity. A considerable body of evidence points to altered Ca2+ homeostasis and Ca2+ signaling in MDD. The varied effects of Ca2+ are mediated through Ca2+ sensing proteins; one highly characterized member is calmodulin (CaM). Recently, two different subfamilies of Ca2+ sensing proteins have been identified, i.e., neuronal Ca2+ sensing (NCS) and calcium binding (CaBP) proteins, that are solely or highly expressed in neurons. These proteins bind to Ca2+ with a very high affinity, undergo myristoylation and conformational changes, and thereby, interact with specific target proteins to mediate neuronal functions. These Ca2+ sensing/binding proteins and their novel target proteins have become a focus of great interest as potential molecular switches for plasticity phenomena. In a preliminary study, we found that the expression of Ca2+ sensing proteins are not only differentially regulated, but their interaction with specific target proteins are also disturbed in brains of MDD subjects. These changes were quite specific as either opposite or no changes were found in other mental disorders, i.e., bipolar disorder (BPD) or schizophrenia (SCHIZ). We hypothesize that Ca2+ sensing proteins, via their altered expression in a distinct manner, will affect their interactions with specific target proteins, which will lead to modulation in neural network/pathways, implicated in neural plasticity; these alterations will contribute to the pathogenesis of MDD. To test this, we propose a series of experiments examining Ca2+ sensing and their specific target proteins at molecular and cellular levels, in well-characterized and well-matched brain samples obtained from MDD and nonpsychiatric normal control subjects. We will examine the specificity of these changes by determining the proposed measures in brains of BPD or SCHIZ subjects. In addition, we will determine the consistency of changes in MDD by examining brain samples obtained from a different cohort. These studies will be performed in two brain areas, implicated in affective illnesses, namely, PFC and hippocampus. More specifically, we will examine: 1) expression and/or functional characteristics of a) NCS proteins NCS-1, VILIP1, VILIP2, VILIP3, hippocalcin, neurocalcin 4, and DREAM; b) CaBP proteins CaBP1 and CaBP4; and c) calmodulin; 2) the interactions of these Ca2+ sensing proteins with specific targets, i.e., PI 4-kinase 2, inositol trisphosphate receptors, CREB, neuronal apoptotic inhibitory protein, CaM kinase II, CaM kinase IV, and calcineurin; 3) the expression and/or functional characteristics of these target proteins. Our proposed study could lead to a fundamental breakthrough in our understanding of the molecular mechanisms associated with MDD, and also provide critical new insight for novel target-based drug discoveries for the treatment of MDD.

DESCRIPTION (provided by applicant): Suicide is a major public health concern. Although attempts have been made to investigate the neurobiology of suicide, the precise molecular mechanisms associated with this disorder are still unclear. It has been proposed that mood disorders/suicide result from an inability of the brain to make appropriate adaptive responses to environmental stimuli. Epigenomic processes are essential not only for normal cellular development and differentiation, but also critical for regulation of gene function through non-mutagenic mechanisms. DNA methylation is the major epigenetic approach for modulating the gene-environment interaction through alterations of promoter regulatory sequences and is associated with stable and adaptive changes in brain functions. In our previous studies, we have consistently shown that the expression of genes involved in neural and structural plasticity is significantly down-regulated in the postmortem brain of suicide subjects, suggesting that altered gene expression may be crucial in the etiology of suicide. Interestingly, a large number of genes exhibit an inverse correlation between the degree of methylation and the magnitude of gene expression. Therefore, the possibility that epigenetic modifications of DNA causing such alterations in the expression of certain genes in brain of suicide subjects, cannot be ruled out. In fact, research in the area of epigenomic regulation of gene expression has led to the hypothesis that the polygenic nature of complex psychiatric disorders might indicate that a common pathway is involved in the dysregulation of multiple genes through an epigenomic mechanism. Although still in infancy, recent evidence suggests that epigenetic factors may play a key role in the pathogenic mechanisms of psychiatric illnesses. To investigate whether epigenetic modifications of DNA play any role in suicidal behavior, we are proposing a pilot study to investigate large- scale epigenetic profiling in the prefrontal cortex of a cohort comprising depressed suicide, non-suicide depressed and well-matched non-psychiatric healthy normal comparison subjects. More specifically, we will examine the following: 1) DNA methylation pattern at the promoters of all known protein-coding genes;2) confirm microarray results using sodium bisulfite modification-based mapping of metC/CAEC/T ratios in the CpG sites;3) use a network-based approach to test the modularity of the epigenomic data;and 4) combined analysis of epigenomic profiles and global gene expression data. To further replicate the DNA methylation study, in another cohort of depressed suicide and healthy comparison subjects, we propose to follow-up 10 to 15 promoters of genes that exhibit the largest epigenomic differences between depressed suicide and normal control subjects. To our knowledge, the proposed research will be the first of its kind in suicide research, and will likely identify major epigenetic modifications in the suicide brain. The proposed studies will also pave the way for our future epigenetic studies and will be crucial for identifying the etiological and pathogenic mechanisms of suicide. PUBLIC HEALTH RELEVANCE: Our proposed study will yield important information on the neurobiology of suicide, which may eventually lead to better treatment and possibly prevention of suicide.

DESCRIPTION (provided by applicant): Suicide is a major public health concern. Suicidal behavior occurs in the context of a diathesis that is characterized by traits in multiple domains: behavioral, clinical, personality, and biologic. While the complexity of suicidal behavior requires a multi-faceted prevention approach, the identification of neurobiological dysfunction is critical for the pharmacological interventions that may have protective effects against suicidal behavior. In this context, we have shown reduced BDNF gene expression and less activation of its cognate receptor TrkB in the brain of suicide subjects. In addition, we have found that p75NTR, a low affinity BDNF receptor is upregulated in the brain of these subjects. BDNF, which plays a critical role in neural plasticity and cell survival, essentially mediates its action via TrkB-mediated activation of extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide 3 kinase (PI3K) signaling pathways. Abnormalities in these two signaling systems at the upstream levels were also found in the same brain areas of suicide subjects in which abnormalities were noted in BDNF/TRKB/p75NTR. These changes were present in all suicide subjects regardless of psychiatric diagnosis. To better understand the functional significance of altered BDNF signaling at molecular and cellular levels and their implications in the neurobiology of suicide, we propose to test the hypothesis that hypoactive BDNF/TrkB-mediated ERK1/2 and PI3K/Akt and overexpressed p75NTR will lead to modifications in the interaction and activation of downstream scaffolding/regulatory proteins, translational machinery, chromatin remodeling, and structural plasticity in the suicide brain. To test our hypothesis, in brain areas implicated in suicidal behavior, i.e., dlPFC and hippocampus (cerebellum as negative control brain region) from well-characterized and well-matched depressed suicide and non-psychiatric control subjects (n = 30 in each group), we aim to examine whether: 1) hypoactive ERK1/2 will lead to altered activation of substrates p90 ribosomal S6 kinase (RSK) and mitogen- and stress-activated kinase (MSK) and their mediated transactivation of transcription factors and chromatin remodeling; 2) hypoactive ERK1/2 and PI3K will lead to less active translational machinery, translation of postsynaptic genes, and altered dendritic morphology; and 3) altered PI3K/Akt and p75NTR will be associated with altered interactions of scaffolding proteins leading to c-Jun kinase (JNK) activation and altered expression and functional characteristics of downstream apoptotic regulatory proteins and neuronal apoptosis. To make sure that the effects are suicide specific, we will perform these studies in the same brain areas of an additional group of well-matched subjects who were depressed (no previous suicide attempt and no family history of suicide) and died by causes other than suicide (n = 30). Our proposed study will precisely and mechanistically assess the complexity of cellular signaling at the molecular, cellular, and functional levels in suicide brain and will have a significant impact in understanding not only the neurobiological basis of suicide but in designing more efficacious treatment strategies.

DESCRIPTION (provided by applicant): MDD is one of the most severe and debilitating illnesses that affects millions of individuals worldwide. Despite considerable advances over the past decades, a clear understanding of the etiology of MDD is still lacking. Accumulating evidence suggests that MDD may arise from impairments in cellular cascades, which lead to aberrant information processing in the circuits that regulate mood, cognition, and neurovegetative functions. Recently, microRNAs (miRNAs) have emerged as an important class of small non-coding RNAs that by binding to 3' UTR of mRNAs, suppress the translation and/or stability of specific mRNA targets. Since miRNAs show a highly regulated expression, they contribute in the development and maintenance of a specific transcriptome and thus have the unique ability to influence physiological and disease phenotypes. Our recent studies show that the expression of a group of miRNAs is altered in PFC of MDD subjects and that they are involved in coping response to stress. In addition, accumulative evidence points to the involvement of miRNAs in neural plasticity. These studies suggest a strong possibility that miRNAs may contribute significantly to the etiopathogenesis of MDD. We hypothesize that subsets of miRNAs and their variants, regulated in a coordinated fashion, will show differential co-expression in MDD brain, which by affecting specific neural/synaptic mRNA targets and cellular pathways, will participate in MDD pathogenesis. In well characterized postmortem brain samples obtained from MDD and control subjects, we propose to: 1) profile miRNA expression by small RNA sequencing, identify novel miRNAs, and analyze differentially expressed miRNAs; 2) profile mRNA expression by RNA sequencing and identify regulatory relationships between mRNA and miRNA by mapping co-expression network modules; 3) analyze miRNA-mRNA pairs, validate altered miRNAs and specific target genes experimentally, and localize these changes at the cellular level; 4) analyze pathway associated with differentially co-expressed modules in MDD; 5) examine miRNA biogenesis by determining pri-/pre-miRNAs, RISC complexes, and components of biogenesis machinery; and 6) examine the role of synaptic miRNAs in MDD pathogenesis by determining miRNA enrichment via small RNA sequencing in synaptosomes, analyzing target genes and co-expression modules of miRNA-mRNA specifically altered in the synaptic fraction, and examining pri-/pre-miRNAs and components of miRNA biogenesis machinery at synaptic level. By using a combination of the state-of-the-art high throughput small RNA and RNA sequencing, analyzing data by novel bioinformatics tools and validation, identifying changes in miRNAs and their targets in specific cell type(s), and examining the role of miRNAs at the synaptic level in brain regions implicated in mood and cognition, our proposed study is uniquely positioned to advance the field of MDD research at the molecular level. These investigations will provide novel avenues for the development of miRNAs as ''molecular tools'' with the potential to generate new molecular-based therapies to treat this devastating disorder.

? DESCRIPTION (provided by applicant): Suicide is the 3rd leading cause of death in the US ages between 15-34 years and about one million people commit suicide every year world-wide. Thus, there is a desperate need for identifying risk factors and for non- invasive, reliable biomarkers that can be used for early detection of suicidality and treatment response. Recently, microRNAs (miRNAs) have emerged as an important class of small non-coding RNAs that bind to 3' UTR of mRNAs and suppress the translation and/or stability of specific target genes. Since miRNAs show a highly regulated expression, they contribute in the development and maintenance of a specific transcriptome and thus have the unique ability to influence physiological and disease phenotypes. Our recent studies show that the expression of a group of miRNAs is altered in brain of depressed subjects and that they are involved in coping response to stress. In addition, our preliminary data indicate that a subset of miRNAs is specifically altered in brain of suicide subjects regardless of psychopathology, suggesting that miRNAs can distinguish suicidality. Recently, circulating miRNAs are under intense investigation and have been extremely useful in detecting and following the course of various diseases. Neural miRNAs are responsive to environmental, synaptic, and pathological changes and can be actively secreted by cells such as exosomes from brain into blood. These exosomes bear cell-type specific surface markers. Using a neural specific surface marker, we successfully isolated neural-derived exosomes and found that these exosomes are enriched with miRNAs/mRNAs that are expressed in brain. Using this novel approach we aim to examine whether neural- derived exosomal miRNAs are differentially expressed that are specific to suicidal ideation or behavior, and which by affecting specific mRNA targets and pathways, are associated with suicidal behavior and response to ketamine. We will examine the following groups of subjects: 1) major depressive disorder (MDD) with a recent suicide attempt (in past 2 weeks), 2) MDD with serious ideation without recent suicide attempt (in the past 6 months), 3) MDD without clinically significant suicidal ideation or suicide attempt in the past 6 months, and 4) healthy controls. Both suicidal and non-suicidal MDD will be given ketamine (0.5 mg/kg, IV) and blood drawn at 30 and 180 min post-infusion to measure changes in miRNAs. We also propose a parallel human postmortem brain study to examine whether changes in miRNAs in suicidality correspond to miRNA changes in brain by comparing dlPFC and hippocampus from MDD suicide, MDD non-suicide, and control subjects. With this we attempt to achieve: 1) whether suicidal ideation or behavior is associated with differences in the expression of specific miRNAs, 2) whether anti-suicidal/antidepressant effects of ketamine is associated with miRNAs changes, and 3) whether miRNA/mRNA-regulatory pathways contribute to suicide pathogenesis and treatment response. Our study will provide a novel avenue for the development of miRNAs as ''molecular tool'' to identify suicidality and treatment response and in generating target based therapies to treat this devastating disorder.

The premise of this research is that: (1) both early life stress (ELS) and recent stressors increase risk for a wide range of mental disorders including major depressive disorder, but many people are resilient to both; and (2) micro RNAs (miRNAs) mediate shorter- and longer-term effects of environmental stressors on gene expression and cell function. The effects of both ELS and recent stressors involve the induction of shorter- and longer-term coordinated changes across networks of genes, mRNAs, and proteins. miRNAs are a subclass of non-coding RNAs that regulate gene expression via post-transcriptional mechanisms. miRNAs are responsive to environmental cues and function as transducers of environmental effects on gene expression. Further, single miRNAs can regulate a range of mRNA translation and tend to effect coordinated networks of genes. In addition, miRNAs are subject to epigenetic regulation. There is strong evidence that brain miRNAs are responsive to environmental stressors, and that they are adaptively regulated by early life stress. miRNAs directly and indirectly regulate components of both stress response (e.g., glucocorticoid receptor, corticotropin-releasing hormone receptor 1) and neuroplasticity (e.g., BDNF), and regulate stress- and fear-related neural mechanisms such as fear conditioning, extinction memory, and fear memory consolidation. However, there has been no research on the effects of either acute stress or ELS on miRNA expression in humans. Exosomes containing both miRNAs and mRNAs are released into the peripheral circulation. Using specific surface markers, we have successfully isolated neural-derived blood exosomes, which are enriched with miRNAs expressed in brain. The proposed study aims to examine the involvement of neural-specific exosomal miRNAs in both acute stress response and longer-term processes associated with ELS. Our primary outcome will compare people with either PTSD or major depression (MDD) with a history of ELS (that we will combine into an ELS-stress sensitive group [ELS-SS]) with a group of people with ELS but no history of mental disorder (ELS-SS). Participants will undergo an acute experimental stress, the Trier Social Stress Test (TSST); blood will be drawn at baseline and at times 0, 15, 30, 60, and 90 min following the test. miRNA sequencing will be conducted on the baseline and TSST+15 min samples and ELS-SS and ELS-SR groups will be compared. We anticipate that: (1) there will be significant differences between ELS-SS and ELS-SR groups in miRNA expression; (2) the TSST will alter the expression of set of neural-derived exosomal miRNAs that regulate components of neuroplasticity and stress response, which will be differentially expressed in ELS-SS and ELS- SR groups; (3) the alterations in miRNAs will be associated with differential methylation of miRNA sequences. This pilot and feasibility project has a high likelihood of leading to research that will change our understanding of how the environment modulates stress response, and why some people are sensitive to stress while others with similar stressors are resilient. This may lead to the development of novel treatments or prevention.

Major depressive disorder (MDD) currently remains one of the leading causes of global disability. Despite the rise in treatment options, remission rates in MDD patients are very low. Thus, there is critical need to identify the biological substrates that precipitate in MDD in order to develop effective therapy. It is widely known that MDD involves short- and long-term maladaptive processes to external stimuli, impairing the ability of individuals to appropriately interact with the environment. So far, there is no coherent hypothesis that can fully explain this phenomenon. Increasing evidence suggests that fine-tuning of transcriptional regulation by gene-environment interaction is central to the etiology of MDD. In this regard, a paradigm shifting phenomenon has recently been introduced with the unique concept of post-transcriptional gene regulation through epitranscriptomic mechanism (most prominently being N6-methyladenosine [m6A]) which is not only involved in the regulation of transcript abundance but has the profound ability to impact maturity, stability, localization, and most importantly, availability of ?select? gene transcripts to protein translation despite varying transcription rates in a highly ?dynamic? and ?reversible? fashion. This mechanism facilitates quick response to external stimuli, fine-tunes protein accessibility, and executes localized control, which is critical to stimulus-adaptive gene expression. Their roles have recently been shown in synaptic plasticity as well as in the stress coping behavior of mice. Our own preliminary data demonstrate that not only is m6A mRNA methylation machinery differentially expressed in various brain areas, but their expression and functions in manipulating m6A methylation and subsequent expression of specific transcripts are aberrant in the MDD brain. This has led us to propose an overarching hypothesis that m6A methylation-based epitranscriptomic modification of mRNAs may act as a dynamic regulator of a subset of genes in a brain region specific manner, which, by affecting specific molecular pathways in a coordinated fashion, will participate in MDD pathogenesis. To test this, in dlPFC and hippocampus from healthy controls and well-matched MDD subjects, we propose the following aims: 1) Examine whether MDD is associated with differential regulation of m6A methylation machinery and distinctive m6A methylation profile at the epitranscriptomic level in brain region specific manner; 2) Define MDD associated role of YTH family of reader proteins in epitranscriptomic turnover of protein coding genes; 3) Examine the impact of m6A mRNA methylation on dendritic availability of local epitranscriptomic pool and their contribution to MDD pathogenesis. By using highly innovative molecular approaches, by precisely identifying the fate of the transcripts in translatable and non-translatable pools mediated through specific m6A reader proteins, by examining the role of m6A methylation at the synapse, and by analyzing data using novel bioinformatics tools, our study is highly innovative; it has the potential to discover unique epitranscriptome-based gene regulation as a mechanism in MDD etiopathogenesis and identify novel targets for therapeutic intervention.

The purpose of this project is to determine if the relationship between a history of childhood maltreatment (CM) and suicide risk is associated with alterations in the expression and epigenetic modification of specific microRNAs (miRNAs), thereby providing a molecular signature of suicide risk in people with a history of CM. We propose that whereas both major depressive disorder (MDD) and suicidality are complex phenotypes, CM alters the risk threshold for both. Epigenetic changes caused by early stressful events can induce long-term alterations affecting networks of genes. miRNA expression represents one of the central mechanisms for environmental regulation of gene expression. miRNA sequences themselves are epigenetically modified. The sum of these effects may explain long-term cellular (mal)adaptations which may lead to suicide vulnerability in the CM population. Using a specific cell surface marker, we isolated neural-derived exosomes from blood plasma and found that these exosomes were not only enriched with brain expressed miRNAs, but also showed a unique set of miRNAs that were associated with CM and suicidality. Changes in the same set of miRNAs were also noted in the brain of suicide subjects with a history of CM. In addition, suicidal subjects with and without CM showed differential regulation of miRNAs in response to acute stress, a short-term risk for suicidal behavior, particulay in the context of CM. Moreover, miRNA expression changes were highly correlated with exosomal miRNA promoter methylation. Based on our preliminary data, we propose an overarching hypothesis that there are multiple paths to suicidal behavior, and CM represents a unique path that is associated with altered expression and epigenetic modification of a specific set of miRNAs and concomitant downstream specific target genes and network(s). To test this, we will: 1) identify a set of neural-derived exosomal miRNAs that are associated with the interaction of suicidality and CM severity while controlling for the independent effects of suicidality, CM, and MDD; 2) examine whether an acute experimental stressor differentially impacts the expression of neural-derived exosomal miRNAs in suicidal patients with and without CM; 3) use bioinformatic tools to examine potential mechanisms by which altered neural-derived exosomal miRNAs may contribute to CM-associated suicidal behavior; and 4) examine if changes in CM-associated miRNAs are explained by modifications in their DNA methylation. In participants (n=450; replicated in a cohort of 350 subjects) across the spectrum of MDD, suicide, and CM severity, we will test for the main effects of each, and identify a subset of miRNAs that are associated with suicidality and CM severity. We will also test for the independent main effects of CM or suicidality on miRNAs and the interactions among those factors. Altogether, using a distinct study population, unique neural-derived plasma exosomes, and innovative molecular, biostatistical, and bioinformatic tools, our study will identify: 1) neural-derived plasma exosomal miRNAs as a novel biosignature of suicide risk in the context of CM that can be tested in longitudinal studies, and 2) potential mechanisms by which CM can act as a risk factor for suicidality.