Robert E. McCullumsmith - US grants

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this Mentored Research Career Award (K08) application is to provide training in innovative techniques that will permit the applicant to ask and answer the largest possible questions in translational mental health research. This research training plan has two overall objectives. The first is to train the applicant in new cutting-edge techniques to study gene expression in postmortem tissue. The second is for the applicant to learn how to perform and evaluate inducible gene deletion or overexpression in the rodent brain. To achieve these objectives, the applicant has planned a series of complementary educational and research training experiences, including formal coursework in biostatistics, bioinformatics, clinical epidemiology, genetics and signal transduction, as well as extended laboratory experiences with consultants to learn electrophysiology, laser capture microscopy (LCM), quantitative PCR, and gene manipulation techniques with viral vectors. In concert with this application's overall objectives, we have designed a research project that maximizes the applicant's exposure to innovative research methodologies and builds on his previous findings of glutamate transporter transcript abnormalities in schizophrenia. These data suggest abnormalities of glutamate transporter expression in corticothalamic circuits in schizophrenia. Accordingly, we hypothesize that expression and regulation of excitatory amino acid transporters (EAATs) is altered in corticothalamic circuits in schizophrenia, contributing to psychopathology in this illness. To evaluate this hypothesis we will measure EAAT mRNA expression in populations of microdissected cells and examine post-translational modification of EAAT proteins in subjects with schizophrenia and a control group. Guided by these human studies, we will then characterize the electrophysiological and neurochemical consequences associated with corticothalamic alterations of transporter gene expression generated using viral mediated gene transfer and/or the cre-loxP recombinase system. These studies will link identified changes in gene expression in schizophrenia with circuit specific alterations in glutamate synapse composition and function. At the conclusion of this set of experiments, the applicant will have learned innovative, state-of-the-art techniques for investigating abnormalities of gene expression and regulation in severe psychiatric illness, providing the tools needed to develop an independent research program. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): While the treatment of schizophrenia with antipsychotic medications revolutionized the clinical management of this illness, approximately one-third of patients with schizophrenia have persistent positive symptoms despite multiple trials of antipsychotic medicines. Recently, new strategies for the treatment of schizophrenia have emerged, including modulation of glutamate receptors, an approach which was developed, in part, based on an accumulating body of evidence of alterations in glutamate transmission from postmortem, imaging, and preclinical studies. While the initial glutamate hypothesis of schizophrenia was focused on NMDA receptor dysfunction, this hypothesis has been extended to include other glutamate receptors, transporters, and enzymes involved in glutamate transmission. Postmortem findings of changes in the expression of gluta- matergic molecules in schizophrenia may be conceptualized as functional alterations of remodeled glutamate synapses, secondary to the underlying pathophysiology of chronic severe mental illness and a lifetime of treatment with psychotropic medications. We have found decreased expression of glial glutamate transporters in this illness, suggesting that glutamate synapses have alterations in glutamate reuptake capacity. Since glutamate transporters facilitate excitatory neurotransmission by limiting glutamate spillover to adjacent synapses, we postulate that the localization of excitatory amino acid transporters (EAATs) is altered in the prefrontal cortex (PFC) in schizophrenia, and may contribute to psychopathology in this illness. Specifically, we hypothesize that perisynaptic localization of EAATs with asymmetric synapses, which are characteristic of excitatory glutamate transmission, is decreased in schizophrenia. To evaluate this hypothesis, we will assess the ultrastructural localization of EAAT isoforms using electron microscopy in postmortem tissue from subjects with schizophrenia. Our studies will focus on the middle layers of the dorsal lateral prefrontal and anterior cingulate cortices, regions with dense reciprocal thalamic innervation that are implicated in the pathophysiology of this illness. These studies will link identified changes in gene expression in the PFC in schizophrenia with circuit specific alterations in glutamate synapse composition and function. We also plan to assess the effects of chronic typical and atypical antipsychotic treatment on ultrastructural localization of glutamate transporters in the rat PFC. These rodent studies will provide novel data on the effects of chronic antipsychotic treatment on the composition of excitatory synapses, and compliment the interpretation of our postmortem findings, since most of these subjects were treated with antipsychotics. At the conclusion of this set of experiments, we will have tested the hypothesis that perisynaptic localization of glutamate transporters with asymmetric synapses is diminished in schizophrenia, suggesting decreased perisynaptic reuptake of glutamate and increased glutamate spillover. These studies will extend the glutamate hypothesis of schizophrenia beyond the NMDA receptor and provide new substrates for diagnosis and treatment of this often devastating illness. PUBLIC HEALTH RELEVANCE: This project will identify the critical elements of brain function that contribute to the pathophysiology of schizophrenia. Identification of the molecular elements underlying schizophrenia will provide new targets for the development of medicines to treat this illness.

DESCRIPTION (provided by applicant): While the treatment of schizophrenia with antipsychotic medications revolutionized the clinical management of this illness, approximately one-third of patients with schizophrenia have persistent positive symptoms despite multiple trials of antipsychotic medicines. Recently, new strategies for the treatment of schizophrenia have emerged, including modulation of glutamate receptors, an approach which was developed, in part, based on an accumulating body of evidence of alterations in glutamate transmission from postmortem, imaging, and preclinical studies. While the initial glutamate hypothesis of schizophrenia was focused on NMDA receptor dysfunction, this hypothesis has been extended to include other glutamate receptors, transporters, and enzymes involved in glutamate transmission. Postmortem findings of changes in the expression of glutamatergic molecules in schizophrenia may be conceptualized as functional alterations of remodeled glutamate synapses, secondary to the underlying pathophysiology of chronic severe mental illness and a lifetime of treatment with psychotropic medications. We have found decreased expression of glial glutamate transporters in subjects with schizophrenia, suggesting that glutamate synapses have alterations in glutamate buffering and reuptake capacity. Glutamate transporters facilitate excitatory neurotransmission by limiting glutamate spillover to adjacent synapses, and we postulate that the localization of excitatory amino acid transporters (EAATs) is altered in corticothalamic circuits in schizophrenia, contributing to the psychopathology of this disease. Specifically, we hypothesize that cell-specific localization of EAATs is altered in schizophrenia. We also hypothesize that there are defects of trafficking and subcellular localization of EAATs in this illness. To evaluate these hypotheses, we will assess the localization of EAAT isoforms using immunofluorescence, subcellular fractionization, and Western blot analysis in postmortem tissue from subjects with schizophrenia and a comparison group. Our studies will focus on the dorsomedial nucleus of the thalamus and the anterior cingulate cortex, regions with dense reciprocal innervation that are implicated in the pathophysiology of this illness. We also plan to assess the effects of chronic typical and atypical antipsychotic treatment on localization of glutamate transporters in the rat brain. These rodent studies will provide novel data on the effects of chronic antipsychotic treatment on the composition of excitatory synapses, and compliment the interpretation of our postmortem findings, since most of these subjects were treated with antipsychotics. At the conclusion of this set of experiments, we will have tested the hypothesis that cellular and subcellular localization of glutamate transporters is altered in schizophrenia, suggesting decreased perisynaptic buffering and reuptake of glutamate and increased glutamate spillover. These studies will extend the glutamate hypothesis of schizophrenia beyond the NMDA receptor and provide new substrates for diagnosis and treatment of this often devastating illness. PUBLIC HEALTH RELEVANCE: This project will identify the critical elements of brain function that contribute to the pathophysiology of schizophrenia. Identification of the molecular elements underlying schizophrenia will provide new targets for the development of medicines to treat this illness.

Project Summary: This is a revised R01 application to investigate abnormalities of signaling networks in pyramidal neurons in schizophrenia. It is an understatement to say that the treatment of schizo-spectrum disorders has not progressed in the past 25 years, since the development of atypical antipsychotics. However, there is broad consensus that these newer medications do not extend the efficacy of pharmacological treatments to cognitive and negative/deficit symptoms, which lead to profound disability in a large number of persons afflicted with schizophrenia. While effective for the psychotic, or positive symptoms, all antipsychotic medications are associated with significant side effects, and have high rates of discontinuation. We posit that abnormalities of cortical pyramidal neurons underlie many of the cognitive deficits observed in the schizophrenia phenotype, including abnormalities of working memory, executive function, and motivation. Pyramidal neurons typically project to other cortical regions (superficial pyramidal cells, in layers II and III) or subcortically (deep pyramidal cells, in layers V and VI) to the thalamus, striatum, and other basal ganglia. Divergent abnormalities of superficial and deep pyramidal neurons in schizophrenia may arise from neurodevelopmental insults that reflect differences in the circuitry of these cell types, resulting in altered gene expression profiles, neuronal migration, and/or aberrant connectivity of these neurons. We hypothesize that abnormalities of pyramidal neurons extend well beyond simple measures of gene expression, and include disease- and lamina- specific changes in functionally related signaling networks. To address this problem, we adapted a novel ?omics? bioinformatics approach for analysis of serine/threonine sub-kinomes in postmortem brain tissue, and identified high-yield protein kinase targets for further study. We propose to focus in this application on two of our high- yield ?hits? from these hypothesis generating preliminary studies: AKT and PKA. Specifically, we will test the hypothesis that these kinases are differentially regulated in superficial and deep pyramidal neurons in schizophrenia. We will use an innovative approach that combines standard techniques, including laser capture microdissection and biochemical kinase activity assays, to measure pyramidal neuron-specific kinase expression and activity in schizophrenia. We will follow up on these studies by measuring expression of factors downstream from AKT and PKA, including phosphoproteins and related networks of mRNAs. Finally, we propose two discrete in silico studies, one focused on developing pyramidal neuron-specific signaling models in schizophrenia, and the other focused on integration of our data with cutting-edge bioinformatics databases, to identify pathways associated with standardized pathophysiological disease-drug dyads. These innovative studies will identify pyramidal neuron-specific signaling pathways disrupted in schizophrenia and provide new ideas regarding the pathophysiology and the development of novel treatment strategies for this often devastating illness.

? DESCRIPTION (provided by applicant): This is an innovative R21 application to investigate abnormalities of protein-protein interactions in schizophrenia. It is an understatement to say that the treatment of schizo-spectrum disorders has not progressed in the past 20 years since the development of atypical antipsychotics. There is broad consensus that these newer medications do not extend the efficacy of pharmacological treatments to cognitive and negative/deficit symptoms, which lead to profound disability in persons afflicted with schizophrenia. Thus, there is a pressing need to develop a more sophisticated understanding of the pathophysiology of this illness in order to develop new treatment strategies. Converging evidence suggests that schizophrenia is a disorder of neuroplasticity, involving pathophysiological changes in synaptic function that lead to cognitive deficits. Synapses throughout the brain contain microdomains called postsynaptic densities, which are dynamic aggregations of receptor, structural, and signaling proteins. Postsynaptic densities in excitatory synapses contain ionotropic glutamate receptors, including NMDA and AMPA receptors, and multipotent scaffolding molecules, such as postsynaptic density-95 (PSD-95). PSD-95 regulates trafficking and assembly of postsynaptic density constituents via protein-protein interactions. Co-localization of receptors in the postsynaptic density via scaffolding proteins underlies molecular correlates of learning and memory, such as long-term potentiation (LTP) and long-term depression (LTD). Accumulating evidence implicates abnormalities of postsynaptic density content and function in schizophrenia, but studies on key elements of the postsynaptic density in schizophrenia have not been performed. We postulate that there are profound changes in the constituents of postsynaptic protein complexes in this illness. We specifically hypothesize that abnormalities of the PSD-95 protein-protein interactome underlie the neuroplastic defects found in chronic schizophrenia. We propose to affinity purify PSD-95 protein complexes from the dorsolateral prefrontal cortex in subjects with chronic schizophrenia (n = 20) and a control group (n = 20), and examine the PSD-95 interactome using liquid chromatography-mass spectrometry (LCMS/MS). We will follow up these studies with targeted LCMS/MS and western blot analyses to confirm changes in protein expression and concentration from our initial studies. We will use pathway analyses and network modeling to identify biological processes and upstream modulators involved in the pathophysiology of schizophrenia. Finally, we propose confirmation studies that include assessing the same dependent measures in an animal model of broken synapses, as well as antipsychotic treated rodents. This application proposes experiments that will extend our understanding of the pathophysiology of schizophrenia and identify novel substrates that may be targeted for the treatment of this often devastating illness.

Project Summary: This is a revised R01 application to investigate abnormalities of signaling networks in pyramidal neurons in schizophrenia. It is an understatement to say that the treatment of schizo-spectrum disorders has not progressed in the past 25 years, since the development of atypical antipsychotics. However, there is broad consensus that these newer medications do not extend the efficacy of pharmacological treatments to cognitive and negative/deficit symptoms, which lead to profound disability in a large number of persons afflicted with schizophrenia. While effective for the psychotic, or positive symptoms, all antipsychotic medications are associated with significant side effects, and have high rates of discontinuation. We posit that abnormalities of cortical pyramidal neurons underlie many of the cognitive deficits observed in the schizophrenia phenotype, including abnormalities of working memory, executive function, and motivation. Pyramidal neurons typically project to other cortical regions (superficial pyramidal cells, in layers II and III) or subcortically (deep pyramidal cells, in layers V and VI) to the thalamus, striatum, and other basal ganglia. Divergent abnormalities of superficial and deep pyramidal neurons in schizophrenia may arise from neurodevelopmental insults that reflect differences in the circuitry of these cell types, resulting in altered gene expression profiles, neuronal migration, and/or aberrant connectivity of these neurons. We hypothesize that abnormalities of pyramidal neurons extend well beyond simple measures of gene expression, and include disease- and lamina- specific changes in functionally related signaling networks. To address this problem, we adapted a novel ?omics? bioinformatics approach for analysis of serine/threonine sub-kinomes in postmortem brain tissue, and identified high-yield protein kinase targets for further study. We propose to focus in this application on two of our high- yield ?hits? from these hypothesis generating preliminary studies: AKT and PKA. Specifically, we will test the hypothesis that these kinases are differentially regulated in superficial and deep pyramidal neurons in schizophrenia. We will use an innovative approach that combines standard techniques, including laser capture microdissection and biochemical kinase activity assays, to measure pyramidal neuron-specific kinase expression and activity in schizophrenia. We will follow up on these studies by measuring expression of factors downstream from AKT and PKA, including phosphoproteins and related networks of mRNAs. Finally, we propose two discrete in silico studies, one focused on developing pyramidal neuron-specific signaling models in schizophrenia, and the other focused on integration of our data with cutting-edge bioinformatics databases, to identify pathways associated with standardized pathophysiological disease-drug dyads. These innovative studies will identify pyramidal neuron-specific signaling pathways disrupted in schizophrenia and provide new ideas regarding the pathophysiology and the development of novel treatment strategies for this often devastating illness.

Project Summary Schizophrenia is a devastating illness with no cure, affecting about 1% of the population worldwide, costing billions of dollars annually. The scientific premise for this proposal is based on accumulating imaging, postmortem, animal model, genetic, and bioinformatics data converging on alterations in the production of bioenergetic molecules in myriad brain regions in this illness. We previously reported abnormally high levels of lactate in living patients with schizophrenia that were strongly associated with poor cognitive function. This finding complements our induced pluripotent stem cell (iPSC) and postmortem work showing higher lactate levels in schizophrenia in iPSC-derived cortical neurons and postmortem anterior cingulate cortex in subjects with schizophrenia. Based on this evidence, we hypothesize that diminished cognitive functioning in schizophrenia is due to impaired bioenergetic metabolism in limbic circuits with increased pathological generation or utilization of lactate in schizophrenia. Specifically, we posit that there is increased production and release of lactate from astrocytes, coupled with increased uptake and utilization of lactate, in lieu of glucose uptake and oxidative phosphorylation, to produce ATP in support of neuronal plasticity in limbic circuits. This new R01 project uses complementary, but distinct approaches, to examine abnormalities of bioenergetic function in schizophrenia. For SA1, we will use magnetic resonance spectroscopy (MRS) to quantify lactate levels and comprehensively characterize patients using neuroimaging, clinical, cognitive, functioning, and metabolic assessments. For Aim 2, cultured human neurons/astrocytes derived from iPSCs obtained in SA1 to assess lactate production and utilization challenges. We will further delineate the functional consequences of lactate production on cellular energy metabolism and neuronal development/function at molecular and cellular levels in cultured human iPSC-derived neurons/astrocytes. In Aim 3, we will use a bioinformatics approach to identify lactate-associated targets for cell-subtype specific studies of biochemical/lactate changes in postmortem brain. Taken together, our aims will comprehensively assess perturbations of lactate and lactate associated pathways across clinical, tissue culture, and postmortem substrates in schizophrenia. By developing a more sophisticated understanding of the pathophysiology of schizophrenia, this project will help identify targets in bioenergetic pathways for development of treatment interventions for this debilitating illness.