ROSALINDA C. ROBERTS, PhD - US grants

Rats subjected to chronic treatment with neuroleptics provide a meaningful way to assess the effects of such treatment on the brain. Neuroleptic treatment in rats produces oral dyskinesias called VCMs, which mimic many of the pharmacological and phenomenological characteristics of TD. In an initial study we found that striatal synaptic density decreased in rats treated chronically with haloperidol, was more profoundly and uniquely affected in the subset of these animals with VCMs and recovered following a drug withdrawal period. Our preliminary data suggests that schizophrenics with TD show similar changes to those seen in rats with VCMs, namely fewer striatal symmetric synapses and fewer mitochondria. The overall goal of this proposal is to answer additional questions raised by the data obtained from the animal model. The Specific Aims are as follows: 1) to confirm and extend our initial findings that haloperidol-induced VCMs are correlated with fewer symmetric synapses and mitochondrial abnormalities and to test the hypothesis that these changes are absent in rats treated with the atypical neuroleptic, clozapine, which does not cause TD or VCMs: 2) to determine, using immunocytochemical techniques, which neurochemically defined subsets of striatal terminals are affected in animals with high VCM scores and which subset(s) recover after drug withdrawal; 3) to determine if the ultrastructural correlates of VCMs in the striatum of rats treated chronically with haloperidol are attenuated by the coadministration of two GABA agonists,,just as are the behavioral manifestations; and 4) to test the hypothesis that ultrastructural changes will be present before or at the same time as VCMs by doing a time course behavioral and anatomical study. The proposed experiments will correlate VCMs and ultrastructural changes, thus determining how universal the covariance is. Moreover, the results from SA#4 may show that the ultrastructural changes precede or occur simultaneously with VCMs, suggesting causality. The results will reveal which neurochemically defined subset(s) of striatal terminals are lost in the rats with high VCMs, suggesting which circuitry is disturbed and ultimately the structural substrate of VCMs. These data may guide the discovery of new antipsychotic medications without dyskinetic side effects. These results will have implications for interpreting drug versus disease effect in data obtained from the brains of schizophrenics who died on medication.

[unreadable] DESCRIPTION (provided by applicant): This is a revised competing renewal of a project studying the synaptic organization of postmortem striatum in schizophrenic subjects (SZ) at the ultrastructural level. The striatum, which interacts with other brain areas to affect motor, cognitive and limbic behavior, is one of the regions affected in schizophrenia. The results of the studies in the last grant cycle indicated an increase in cortico-striatal type synapses in the caudate matrix and putamen patches, that was not caused by antipsychotic medication. The higher density of cortical-type synapses in the SZ cases than in controls suggests hyper-stimulation of striatal projection neurons. This could have several important and different downstream effects depending on the precise circuitry involved. The present application seeks to identify the specific striatal circuitry affected in SZ. SA#1) To test the hypothesis that limbic and prefrontal circuitry are perturbed at the level of the striatum, we will examine synaptic density in the subregions of the striatum that process these circuits. SA2 will examine synaptic density of striatonigral and striatopallidal neurons in the patch and matrix in select striatal territories determined in SA1. SA#2A) To test the hypothesis that striatopallidal matrix neurons in the caudate receive more excitatory inputs, the immunocytochemical localization of enkephalin, a marker of these neurons, will be performed; the number of synapses formed onto labeled spines will be compared between groups. SA#2B) Tests the hypotheses that striatonigral matrix neurons in the caudate receive more excitatory inputs, but that striatonigral neurons in the putamen patch receive normal or fewer numbers of synapses. The immunocytochemical localization of substance P, a marker of striatonigral neurons, will be performed; the number of synapses formed onto labeled spines will be compared between groups. SA#3) To test the hypothesis that typical vs atypical APDs have different effects on the patch and matrix compartment, we will treat rats chronically with APDs, process the tissue for calbindin immunocytochemistry to identify the patch and matrix and analyze EM samples obtained from each. In monkey tissue obtained from Dr. Lewis, we will examine the synaptic density (labeled with synaptophysin) within the patch and matrix compartments in chronic haldol treated animals and controls using light microscopy. The proposed experiments will: 1) distinguish between drug effects and disease related alterations in synaptic pathology; 2) will provide insight into the mechanisms of action of antipsychotic drugs; and 3) are an important initial step in identifying putative abnormal striatal circuitry that may underlie some of the psychopathology of schizophrenia. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Schizophrenia is a devastating illness, with unknown pathophysiology, that affects 1% of the world's population. The experiments in the following revised proposal will focus on the basal ganglia and dopamine (DA) pathology in schizophrenia (SA1) and relate these changes to those occurring in rats treated with antipsychotic drugs (APDs) (SA2). Our preliminary data shows abnormalities in morphology of DAergic neurons in the substantia nigra (SN) and in the number of TH+ striatal synapses in electron microscopic (EM) studies of postmortem tissue from subjects with schizophrenia (SZ), similar structural changes and a decrease in number of TH+ cells in rats treated with APD. SA1 tests the hypothesis that the DA system is perturbed in the basal ganglia of SZ, using tissue from normal controls, SZ treated with typical or atypical APDs or off-drug. SA2 tests the hypothesis that anatomical changes observed in SN and ventral tegmental area (VTA) of SZ are the results, in part, of APDs, and will determine the contributing physiological mechanisms. In both aims, we will determine if the morphological alterations seen will show regional variations that are consistent with the differential effects of typical and atypical APDs on the activity of midbrain DA neurons. In this revision, we have modified the EM analysis of the SN and added 3 parallel experiments in both the human tissue and rats (treated with haloperidol or clozapine or controls). In SA1a the synaptic organization of DA labeled profiles will be analyzed in the human striatum at the EM level. In SA1b & SA2a, the number and size of Nissl stained, and TH+ cells double labeled with the DA transporter (DAT), or a selective marker of DA cells, SK3, will be determined using stereological methods in the SN/VTA. In SA1c & SA2b at the EM level, the integrity of subcellular organelles and the synaptic organization to the TH+ neurons (also labeled with DAT or SK3) will be studied. Using in situ hybridization SA1d & SA2e will determine if TH synthesis is affected at the level of transcription. SA1c & SA2d will determine if cytoskeletal proteins are upregulated. SA1f & SA2e will determine if the loss of TH in neurons is due to changes at the translational level by using Western blot analysis. SA2f will study the time course of the anatomical changes observed during APD treatment and relate these changes to the development of depolarization (DP) block. SA2g tests the hypothesis that morphological alterations in SN/VTA neurons will not occur in rats treated with APD if DP block is prevented (with a unilateral striatal lesion).

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): Schizophrenia (SZ) is thought to be a disorder due in part to abnormal connectivity within and between brain regions. The symptoms present in schizophrenia are not only related to abnormalities in specific brain regions and neurotransmitters, but also are related to aberrant communication within and between networks of brain regions that are structurally connected by fiber pathways. This abnormal connectivity within and between brain regions in SZ is termed dysconnectivity. Abnormalities in structural or functional connectivity or the coupling of the two could be due to disorders of the white matter, ie the axon bundles connecting distant regions of the brain. Imaging studies have shown abnormalities in white matter in the brains of subjects with SZ. Studies conducted with postmortem tissue, where higher resolution studies can be performed, show different types of pathology. In this proposal we intend to examine major white matter tracts that connect areas of the brain that are abnormal is SZ, including the internal capsule, cingulum bundle, and corpus callosum. We hypothesize that there will be abnormalities in white matter integrity in SZ that could underlie abnormal connectivity, including anomalies in myelin, glia, mitochondria, and/or the cytoskeleton. We will approach this hypothesis using protein studies, histology, immunohistochemistry and electron microscopy in postmortem tissue from SZ subjects on or off APD and a matched comparison group to determine which deficits could result in impaired network activity. SA1) Using western blots we will measure proteins including: myelin basic protein, markers of oligodendrocytes, microtubule associated protein, neurofilament, and markers of mitochondrial function. SA2) will use histology and immunocytochemistry to confirm and further localize changes identified in SA1). SA3) Using electron microscopy (EM), we will count and measure the cross sectional area of axons and myelin sheaths and measure the structural integrity of glial cells. Here we propose a low risk, high throughput study to identify the underpinnings of structural dysconnectivity in major white matter pathways in postmortem SZ brain. The study is novel, as it will combine different techniques and study several white matter tracks in the same brains. In addition, we will use electron microscopy, a rarely used technique in postmortem human brain, to answer questions that can only be answered with electron microscopy. The results of these studies will allow us to more comprehensively understand the white matter pathology in schizophrenia, and hopefully to identify targets for new treatment mechanisms.

Schizophrenia (SZ) is a devastating mental illness with genetic and environmental risk factors that affects 1% of the world population. Pathology exists in multiple grey and white matter areas and neurotransmitter systems, making the search for a cause(s) and effective treatment elusive. Exploring new pathological mechanisms is paramount in trying to advance our understanding of SZ. Copper, which is required for proper monoamine metabolism, neurotransmission, mitochondrial activity, and myelination, is implicated in SZ but so far not studied in the brain. There is substantial evidence that copper is increased in the blood of patients with SZ. Interestingly, experimental manipulations that decrease copper produce demyelination, increases in dopamine, and behavioral impairments reminiscent of some SZ symptoms. Dysbindin is an upstream modulator of copper via the dysbindin/BLOC-1-copper metabolism interactome. It is encoded by the gene DTNBP1, which is a top candidate gene for SZ. A consequence of dysfunctional dysbindin in mice is a decrease in the copper transporters ATP7A and CTR1, which facilitate copper transport between the blood and the brain. To date, in spite of compelling evidence for a role of dysbindin in SZ, no one has linked a decrease in dysbindin function with abnormal copper homoeostasis in SZ. My overall hypothesis is that copper homeostatic and transport system alterations contribute to SZ pathology, potentially through decreased dysbindin expression. In support of this, we have observed decreased ATP7A and CTR1 protein in postmortem SZ substantia nigra (SN). SA1) In postmortem brain, we will test the hypothesis that SZ cases have lower levels of brain copper, ATP7A and/or CTR1, and dysbindin. We will study the SN and hippocampus, because these areas are implicated in SZ and have decreased dysbindin levels. We will use western blot protein analyses, quantitative immunohistochemistry to localize copper transporters, copper and dysbindin. SA2) In SZ patients and controls, we will test the hypothesis that medication naïve subjects with SZ at first psychotic break will show markers of decreased dysbindin function, lower levels of ATP7A and/or CTR1, and higher levels of copper in blood or saliva. To do this we will get saliva/and or blood from SZ subjects in two different first episode clinics before and after treatment, and healthy controls. SA3) In an animal model with a knockout of the dysbindin gene, we will test the hypothesis that rescue of impairments in behaviors relevant to SZ, via antipsychotic drugs (APD), will require an increase in ATP7A and/or CTR1. We will treat dysbindin KO, heterozygotes and WT littermates with APDs, test behavior before and after treatment, then analyze the brain for levels of copper, ATP7A and CTR1. These studies will provide the first evidence of the state of copper in SZ brain. Although no one single abnormality will be the cause or cure of SZ, our studies will provide new data about the effects of copper transporters (a previously unstudied pathway in SZ), the role of dysbindin and its and a potential mechanism of antipsychotic rescue of copper starvation deficits, which could yield novel targets for drug development.

Schizophrenia (SZ) is a devastating mental illness with genetic and environmental risk factors that affects 1% of the world population. Pathology exists in multiple grey and white matter areas and neurotransmitter systems, making the search for a cause(s) and effective treatment elusive. Exploring new pathological mechanisms is paramount in trying to advance our understanding of SZ. The hippocampus is considered to play a pivotal role in the neuropathology and physiology of schizophrenia. Though widely studied, there have been inconsistencies in which subregions, neurotransmitters and cell populations are affected; thus how specific subregion abnormalities contribute to disease expression remain to be fully determined. This proposal aims to identify the circuitry that contribute to region-specific imbalances in excitation and inhibition in the hippocampus in schizophrenia. Here, we propose to test whether there are region-specific differences in the excitatory and inhibitory synapses in the trisynaptic pathway in postmortem tissue of patients with schizophrenia compared to a comparison group using quantitative electron microscopy. My overall hypothesis is two fold: 1) synaptic efficacy of excitatory synapses is enhanced by increased number or size of synapses or increased mitochondrial content in the terminals forming these synapses; and 2) the synaptic efficacy of inhibitory synapses in diminished by decreased number or size of synapses or decreased mitochondrial content in the terminals forming these synapses. Preclinical and clinic data indicate hyperactivity in the hippocampus that precedes the onset of psychosis, and which is correlated with the severity of symptoms. In our preliminary data, we have observed increased numbers of excitatory synapses and decreased number of inhibitory synapses, identified by morphological criteria. SA1) In postmortem brain, we will test the hypothesis that SZ cases have decreased efficacy of inhibitory synapses and an increased efficacy of excitatory synapses in the trisynaptic pathway by counting, measuring and categorizing the morphology of synapses and the mitochondria within the terminals forming them. SA2) To determine the synaptic density on the neuronal somata in the trisynaptic pathway. By examining the exact laminar location of excitatory inputs we will be able to determine the sources of abnormal circuitry. Electron microscopy offers the unique opportunity to measure hippocampal subfields in a layer and cell specific way at the level of the synapse. The source of afferent inputs to specific subfields and layers, the density of specific inputs, and the ultrastructural features of the axon terminals, the experiments in this application have the potential to reveal specific circuitry underlying hyperactivity in the hippocampus in schizophrenia and guide future tests of region and/or cell-specific interventions. Quantitative electron microscopic studies of the human postmortem hippocampus in schizophrenia currently are performed in only one other lab in the world. Our results on the ultrastructural results will have distinct functional implications for schizophrenia as well as for normal human hippocampal circuitry.