Marquis P. Vawter - US grants

DESCRIPTION (provided by applicant): The mRNA profile of postmortem brain shows evidence of neural signatures in certain psychiatric disorders such as bipolar disorder, major depression, schizophrenia, and other neuropsychiatric disorders. This suggests that diagnostic specificity might be present in the reported neural signatures. The neural signature could be further examined using peripheral lymphocytes as a neural probe. The broad goal of this application is to test for biomarkers and stability in postmortem brain using gene expression profiling by microarray. Aims 1 through 4 incorporate novel approaches to fill in existing gaps in scientific knowledge using an accessible tissue source of lymphocytes for a neural probe and organotypic brain culture as an extension to current postmortem studies to study candidate biomarkers. The present scientific gaps will be addressed in: Aim 1. Compare gene expression profiles in post-mortem human brain and outpatient lymphocyte tissue between controls, major depressive disorder (MDD), and bipolar disorder (BPD) groups for unique gene profiles that may act as diagnostic biomarkers for these disorders greatly facilitating better treatments. Aim 2. Simultaneous gene expression from brain and lymphocyte within the same subjects and analysis of lymphocyte expression between controls, MDD, and BPD subjects. Aim 3. Comparison between 2 sources of lymphocytes (outpatient and post-mortem) and effects of transformation on gene expression profiling. Aim 4. Stability testing of potential biomarkers using organotypic brain tissue culture to study the impact of agonal factors on mRNA profiling and biomarkers. The 4 aims of this application are inter-related, i.e. biomarker evaluation and the potential impact of agonal factors on the stability of biomarkers in peripheral and central gene expression. The potential use of biomarkers in lymphocytes will provide clinical researchers a tool to better address questions concerning diagnostic subgroups and treatment responders and non-responders.

DESCRIPTION (provided by applicant): Mitochondria are organelles that provide most of the energy for brain cells by the process of oxidative phosphorylation. Mitochondrial abnormalities and deficiencies in oxidative phosphorylation have been reported in individuals with schizophrenia (SZ) and bipolar disorder (BD). The overarching hypothesis for this grant is that mitochondrial dysfunction is one of the risk factors for SZ and BD based upon evidence of mitochondrial dysfunction in transcriptomic, proteomic, and metabolomic studies, genetic studies of families, in vivo neuroimaging studies, and mitochondrial DNA (mtDNA) sequence variations. Several mildly deleterious mutations in mtDNA have been reported in SZ and BD patients. The investigators found deletion of a large portion of mtDNA was increased in the brain, dorsolateral prefrontal cortex (DLPFC), of BD subjects relative to age-matched controls. The substitution of synonymous base pairs in the entire mtDNA genome was elevated in DLPFC of individuals with SZ compared to controls and subjects with SZ had a significantly decreased expression of 10 mtDNA transcripts. The decreased expression of mtDNA transcripts in SZ might be related to increased mtDNA substitution in the control or coding regions which will be tested. The causes for increased base pair substitutions in SZ might be inherited or accumulated substitutions in brain. Two of the aims for this grant are to study mtDNA substitutions in brain and to compare the substitution rate in the same subjects9 germ line tissue. This grant proposes to examine mtDNA common deletion, copy number, and transcript abundances in brain from individuals with SZ and BD and compare to controls. The accumulation of novel mtDNA substitutions and deletions in brain might be a risk factor for BD and SZ, and has a great potential significance in determining future targets for therapy of chronic mood and psychotic disorders. This study fills a void as there has not been an integrative brain study of the entire mitochondrial genome and transcriptome conducted in the same subjects with psychiatric disorders. A comprehensive integration of data from the genome and transcriptome of brain mitochondria can show whether moderate dysfunction in one or both systems leads to disease threshold. Focusing on the mitochondria, as a target organelle of functional brain deficits, may lead to improvements in integrative treatments that improve mitochondrial health and brain function. PUBLIC HEALTH RELEVANCE: Project Narrative/Relevance The causes of schizophrenia and bipolar disorder have not been discovered. This grant proposes to analyze mitochondrial DNA, contained in brain cells, which might harbor abnormal structure and sequence. Alterations in mitochondrial sequence during the lifespan in brain might contribute to risk of developing a serious mental disorder. By understanding the accumulation of mitochondrial DNA defects in brain, it will advance mitochondrial medicine for earlier diagnosis and treatment of brain related disorders.

DESCRIPTION (provided by applicant): Mitochondrial Deletions in Mood Disorders Mood disorders (bipolar disorder, BD; major depressive disorder, MDD) account for a high percentage of life- time disability on a world-wide basis, shortened life span, and devastating personal impacts. The pathophysiology of mood disorders, implicates major abnormalities in energy metabolism and mitochondrial function. Mitochondrial dysfunction and disease can be inherited from genetic mutation and can affect multiple target organs, including the brain. We believe that part of the risk for developing mood disorders is genetic variation involving mitochondria DNA (mtDNA). We have replicated our initial findings that a large common somatic deletion of 4,977 base pairs of mtDNA is increased in mood disorders compared to controls. It is important to note that these large deletions of mtDNA appear more frequently in tissues with high metabolic rates, such as brain and muscle and accumulate in an age dependent manner. We believe the study of the full spectrum of large deletions and possible translation into protein will provide substantial evidence supporting the role of mitochondrial dysfunction in mood disorders. In this proposal we hypothesize that large somatic mtDNA deletions accumulate in brain to high levels in mood disorders leading to abnormalities in mitochondrial function. We propose three Specific Aims focusing on the accumulation of large deletions in MDD and BD. 1) Collect fresh mitochondria from human brain (10 MDD, 10 BD, and 10 controls) from which we will sample fourteen brain regions implicated in cognition, affective regulation, and anhedonia in mood disorders. 2) Generate a spectrum of large deletion sequences in mtDNA and cDNA in human brain. 3) Determine the impact of large deletion sequences on proteins and on mitochondria function. We have already discovered novel large somatic mtDNA deletions in postmortem human brains, and are now ready to screen the full spectrum of mtDNA deletions that might cause mitochondrial dysfunction in psychiatric disorders. These novel deletions occur in brain samples at even higher levels compared to the known common deletion of mtDNA. Currently, there are no reports of either protein translation or functional effects of these large deletions i human brain. In the interest of collaborative research, we will share all mtDNA deletion sequences at NCBI. These results could lead to novel treatments that target mitochondrial functional deficits and reduce the accumulation of somatic deletions, thereby improving therapy of mood disorders.

? DESCRIPTION (provided by applicant): Mitochondria provide nearly all of the energy for synaptic transmission, maintenance of ionic homeostasis in synaptic terminals, development of neuronal spines, and long term potentiation which are all essential for brain function. In addition to these neuronal roles, mitochondria regulate cell survival, react to oxidative stressors, and provide ATP for most cellular functions. It is estimated that a single cortical neuron utilizes 109 molecules of ATP each second. In our initial grant, we found strong evidence of mitochondria dysfunction in schizophrenia, compared to weaker evidence for dysfunction in bipolar disorder. We conservatively estimated mitochondria DNA content heritability (h2 = 0.37), and found a highly significant decrease of mitochondria DNA in schizophrenia compared to controls and bipolar disorder. Our studies have also shown decreased gene expression and common deletion, in schizophrenia, but not in bipolar disorder, compared to controls. We have postulated a polygenic model that nuclear and mitochondrial genes interact to alter mitochondria content in periphery and in brain. This decrease in mitochondria in brain, could account for significant decreases in density of spines, especially prominent in layer III. We will further test this model, that mtDNA content is heritable, and under genetic control. We will also test association of mtDNA content with symptoms of schizophrenia. Since our initial studies have been conducted in chronic subjects with schizophrenia, we are renewing our grant to study subjects with first episode psychosis for evidence of the mitochondria dysfunction before onset of antipsychotic drug treatments. The overarching hypothesis is that the pathophysiology of SZ is associated with a reduction in mitochondrial function specifically in the localization and copy number of mitochondria in dendrite and axon locations compared to controls. The cause of the dysfunction postulated in our model is polygenic variation in both nuclear and mitochondrial genes that contribute to mitochondria copy number trait and function. We will test for association of mitochondria DNA copy number in both nuclear and mitochondrial SNPs in first episode psychosis and chronic psychosis, using blood and postmortem brain, and extend our investigations of postmortem brain to medication free individuals with schizophrenia, including bipolar disorder subjects for specificity of findings. To be confident that chronic treatment with antipsychotic drugs (APDs) does not alter mitochondria localization, we propose to study a cohort of non-human primate brains at cellular resolution, already chronically treated with APDs. Since synaptic terminal activity is dependent upon mitochondria function, we will compare the numbers of mitochondria located in postsynaptic spines in individuals with schizophrenia and controls, and APD treated rhesus monkeys compared to controls. Finally, we will assess whether APD treatment alters mitochondrial copy number, intraneuronal motility, and membrane potential in fibroblast-induced pluripotent stem cells reprogrammed into neurons from first episode psychosis subjects compared to controls. The outcomes of this study will expand our knowledge of genetic basis for mitochondria dysfunction in schizophrenia, and association of that dysfunction with psychiatric symptoms.

Project Summary Alterations of synaptic function in individuals with schizophrenia have been found in transcriptomics, proteomics, and genome wide association studies. Impaired synaptic glutamatergic (excitatory) and GABAergic (inhibitory) neurotransmission in affected brain regions (e.g. dorsolateral prefrontal cortex; DLPFC) is thought to be involved in the core symptoms of schizophrenia. However, there are no quantitative measurements of synaptic function in the human DLPFC, therefore concrete and specific functional alterations of ion fluxes of glutamate and GABA receptors are lacking. We have begun to address this problem by directly measuring AMPA- and GABA receptor-mediated synaptic currents in postmortem brains from subjects with schizophrenia and contrasting to controls. We demonstrate in our preliminary work that the function of synaptic receptors is maintained in postmortem brains and is significantly decreased in schizophrenia compared to controls. Our overarching hypothesis is that reductions in both inhibitory and excitatory currents underlie synaptic deficits in schizophrenia. We will test rigor and reproducibility of this hypothesis in two larger independent case-control cohorts. These deficits in synaptic currents can be statistically modeled with proteomic and transcriptomic data which will be useful in downstream studies that pharmacologically challenge activation of these currents. In our model, we suggest that an unequal loss of GABAergic and glutamatergic transmission potentially biases circuits towards producing increased inhibition by dual complementary mechanisms. Our novel molecular evidence will be tested in three complementary aims. Aim 1 will test whether there are electrophysiological alterations of the excitatory (E) to inhibitory (I) balance (E/I ratio) in the DLPFC of subjects with schizophrenia compared to controls, by using microtransplantation of synaptic membranes, a novel method that allows for electrophysiological studies of synaptic receptors from postmortem human brain. Aim 2 will test the hypothesis that integration of proteomic, transcriptomic, and electrophysiological data in the same subjects predicts synaptic dysfunction and E/I ratio alterations at the molecular level in SZ. We will use mRNA-Seq in conjunction with label free liquid chromatography-MS (LC-MS/MS) to characterize major synaptic elements with modulatory capacity on GABA and glutamate receptors. Aim 3 will test rigor and reproducibility of electrophysiological data across different brain banks, by using an independent cohort from the NIHM Human Brain Collection Core. Understanding the relationships between parallel transcriptomic and proteomic data sets with the actual dysfunction of synaptic receptors would greatly facilitate targeted pharmacological interventions that help persons suffering with schizophrenia. This approach could benefit other neuropsychiatric disorders involving mood, behavior, and cognition by targeting potential alterations in synaptic function.