Jill R. Glausier - US grants

DESCRIPTION (provided by applicant): Dopaminergic neurotransmission at the D1 family of dopamine receptors (D1 R) is critical for normal cognitive functioning, and impairments in this system are linked with schizophrenic symptomology and cocaine abuse. However, the exact mechanism by which D1R signaling affects these functions is not clear. Studies on individual neurons show that there are specific and complex circuit effects of D1R drugs in the PFC, suggesting a structural component for the actions of dopamine at the D1 and D5 receptors. Thus, the goal of this proposal is to utilize immunohistochemical electron microscopy and co-immunoprecipitation to examine how dopamine acting at D1 and D5 receptors can modulate activity in the PFC. The first aim will determine the localization of D1 and D5 receptors within specific components of PFC circuitry. Because the effects of D1R activation rely on a complex signal transduction pathway, the second aim will determine if key proteins in this pathway are available to each D1R in specific components of PFC circuitry. The third aim will extend the neuroanatomical findings by identifying D1R downstream targets such as NMDA, AMPA and GABA receptors that specifically interact with D1 and D5 and two critical D1R signal transduction proteins.

DESCRIPTION (provided by applicant): Schizophrenia is a debilitating disorder which affects 1% of the population at a substantial cost to the individual, their family and society. Cognitive ability is the best predictor of employment, social integration and relapse in schizophrenia, but current pharmacotherapies do not address cognitive dysfunction. A core component of cognition is working memory (WM), which depends upon proper activation of dorsolateral prefrontal cortex (DLPFC) circuitry, and both WM and DLPFC activation are impaired in schizophrenia. To identify therapeutic targets for improving WM, the underlying nature of DLPFC circuitry abnormalities must be identified. Parvalbumin (PV) basket cells synapse onto the perisomatic region of pyramidal cells and are critical for synchronizing neural populations to oscillate in the gamma range (30-90 Hz). Gamma oscillations are thought to underlie WM ability, and schizophrenia patient show impaired gamma oscillations during WM tasks. Recent evidence demonstrates that PV basket cell axonal arbors and synapses are significantly diminished if adequate levels of GAD67 are not available during development In schizophrenia DLPFC, GAD67 mRNA is undetectable in 45% of PV interneurons, suggesting these GAD67-negative, PV-positive interneurons form fewer connections with pyramidal cells. Thus, alterations in PV basket cell connections may contribute to the circuitry abnormalities underlying impaired WM in schizophrenia. To test the hypothesis that PV basket cell inputs are diminished in schizophrenia, pre- and postsynaptic markers are examined in DLPFC tissue from schizophrenia relative to comparison subjects. In Aim 1, a novel confocal microscopic method is used to test the hypothesis that DLPFC pyramidal somata receives fewer PV basket cell inputs in schizophrenia. PV basket cells synapsing onto pyramidal somata signal via GABAa receptors containing alpha subunits. Thus, in Aim 2, the same method is used to test the hypothesis that the number of alpha subunit clusters on pyramidal somal membranes is lower in schizophrenia. Recent evidence indicates that PV basket cell inputs onto pyramidal cells, but not interneurons, are important for gamma ocsillations. Thus, in Aim 3 the hypothesis that alpha subunits are exclusively lower in pyramidal cells is tested using dual-label in situ hybridization to determine the percentage of pyramidal cells and interneuons containing alpha mRNA in schizophrenia. Relevance: Schizophrenia affects over 3 million Americans at a cost of $62 billion a year, and cognitive ability is the best predictor of employment and relpase in these patients. No treatments which improve cognition in schizophrenia are currently available. The proposed project examines circuitry abnormalities which likely contribute to cognitive impairment in schizophrenia.

? DESCRIPTION (provided by applicant): Schizophrenia is a complex disorder lacking an effective treatment option for the pervasive and debilitating cognitive impairments experienced by patients. I have been acutely interested in identifying the underlying circuitry alterations tha contribute to these impairments, so that they may be treated, since the start of my doctoral training. Though my focus has been singular, the approaches and conceptual framework used to study this problem have evolved over my scientific career, and this K01 application represents the next major step in that evolution. This K01 application includes research, clinical, and career development, along with teaching/training opportunities to maximize my ability to successfully transition to independence. My long term career goals are to 1) identify and describe the molecular and cellular alterations that contribute to cortical dysfunction in schizophrenia, 2) determine the possible causes of these observed pathologies using animal models, 3) use these findings to develop pathophysiology-based pharmacological treatments for cognitive impairment in schizophrenia patients, 4) establish an independent research laboratory at a top-tier university to accomplish these goals, and 5) mentor students and postdoctoral fellows to contribute to the next generation of scientists and innovators. My short term career goals include 1) master the research techniques proposed, including laser capture microdissection of single cell populations from human and rodent cortex, electron microscopic mitochondrial analysis of human prefrontal cortical tissue, and shRNA and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) paradigms for rodent studies; 2) master experimental design to determine whether disease findings are a cause, compensation, consequence or confound; and 3) successfully transition as a faculty member from Instructor to Assistant Professor at the University of Pittsburgh. Importantly, completing these short terms goals via this K01 award begins to address long term goals 1 - 3, and puts me on a trajectory to accomplish the remaining long term objectives. The current training plan is also augmented with [[formal courses that address each of the major training goals]], interactive workshops focused on scientific career development, and teaching experiences to further broaden my career skill set. The Department of Psychiatry at the University of Pittsburgh's School of Medicine is an ideal environment in which to accomplish these short and long term goals. This department is a national leader in clinical research, treatment and training. Under the primary mentorship of Dr. David Lewis, Chair of Psychiatry, I will have full access to his laboratory and all of the infrastructure support required for the current application. Working memory is a core cognitive function impaired in schizophrenia that depends upon activation of prefrontal cortex (PFC) circuitry. Accordingly, individuals diagnosed with schizophrenia show reduced PFC activation while performing working memory tasks. This lower PFC activation appears to be an integral part of the disease pathophysiology, and not simply a reflection of poor performance. Thus, the cellular and circuitry alterations that underlie lower PFC neuronal activity in schizophrenia must be determined in order to identify appropriate therapeutic targets. This research proposal focuses on determining which of two discrete possible molecular/physiological disturbances is a likely upstream event leading to PFC impairments in schizophrenia. Supporting neuronal excitation represents the largest energy-consuming activity in the brain, supplied by ATP synthesis in mitochondria via oxidative phosphorylation (OXPHOS). Accumulating evidence indicates that expression of the terminal and rate-limiting OXPHOS enzyme, cytochrome c oxidase (COX), is lower in the PFC of schizophrenia subjects. Thus, the research goal of this K01 application is to determine the underlying mechanism contributing to lower levels of COX in the PFC of schizophrenia subjects. Lower COX could be a consequence of chronic reductions in neuronal excitation that lower ATP demand in the affected neurons (Hypothesis 1), or due to deficient COX expression that impairs metabolic capacity in all neurons (Hypothesis 2). Distinguishing between these alternatives has important implications for identifying appropriate therapeutic targets for cortical dysfunction in schizophrenia, as H1 indicates targeted enhancement of excitation, whereas H2 indicates enhancing COX expression to recover mitochondrial respiration. In order to test which hypothesis is most supported by findings in affected individuals, laser microdissection is used to dissect samples of three distinct neuronal populations and quantitative PCR is used to measure the expression of COX-related transcripts (Aim 1.1), and stereological electron microscopy is used to quantify mitochondrial abundance and morphology (Aim 1.2) in the PFC of schizophrenia and healthy comparison subjects. However, because cause- and-effect relationships cannot be determined using human postmortem tissue, experimental animal models are used in Aims 2 and 3 to directly test the mechanisms of H1 and H2. In Aim 2, DREADD pharmacogenetic technique is used to induce long-term reductions in PFC pyramidal cell excitation, and each measure from Aim 1 is assessed. In Aim 3, viral delivery of shRNA approach is used to impair COX availability in the PFC, and each measure from Aim 1 is assessed. Together, these Aims provide a definitive characterization of the disease phenomenon, and extend to proof-of-concept studies in animal models to provide compelling, convergent and conclusive data regarding which mechanism is operative in the illness.

7. PROJECT SUMMARY/ABSTRACT Psychosis is a core clinical feature of schizophrenia (SZ) associated with elevated dopamine (DA) synthesis and release in the associative striatum (AST), but not the limbic striatum (LST). Determining the molecular and subcellular substrates of this region-specific presynaptic pathophysiology of psychosis in the disease state requires studies at the site of DA release, DA axonal boutons. This level of resolution in humans can only be achieved in postmortem brain studies. Elevated presynaptic DA signaling in the AST in SZ could be due to molecular changes within DA boutons. Specifically, greater protein levels of tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2) or the DA transporter (DAT), which govern DA synthesis, packaging for vesicular release and reuptake into the bouton, respectively, could represent molecular substrates of this pathophysiology. These molecular changes would be accompanied by ultrastructural alterations reflecting greater DA vesicular release. Alternately, or additionally, a greater density of DA boutons could be the substrate for elevated presynaptic DA signaling. Thus, in Aim 1 we test each of these possibilities by using triple-label immunofluorescence and confocal microscopy in SZ and unaffected comparison (CON) subjects to simultaneously quantify the abundance of TH, VMAT2 and DAT within DA boutons and the density of DA boutons in the AST and LST (Exp 1.1), and DA vesicle density is quantified via serial section transmission electron microscopy (Exp 1.2). Studies are repeated in monkeys chronically exposed to antipsychotic drugs (Exp 1.3). The findings in Aim 1 may result from alterations to local striatal cholinergic interneurons (ChIs) that affect DA boutons. Indeed, because markers of presynaptic DA signaling are normally greater in the AST than LST, an exaggeration of the factors that contribute to this regional specificity might underlie the AST-specific finding in SZ. ChIs are a compelling candidate as they exhibit greater functional and anatomical measures in the AST than LST and potently regulate DA synthesis and release by providing direct axo-axonic inputs to DA boutons. Activation of nicotinic acetylcholine receptors containing the ?2 subunit (nAChR?2) located on DA boutons induces DA release independent of DA neuron firing in the midbrain. Measures reflecting elevated presynaptic DA signaling in the AST in SZ might be the consequence of a greater proportion of DA boutons receiving ChI inputs and/or higher levels of molecular determinants of ChI synaptic strength, such as levels of ACh transferase (ChAT) and/or nAChR?2. Thus, in Aim 2 we utilize quadruple-label immunofluorescence and confocal microscopy to simultaneously quantify the proportion of DA boutons receiving a ChI axo-axonic input and the abundance of ChAT and nAChR?2 at these inputs in the AST and LST. As in Aim 1, studies are repeated in monkeys chronically exposed to antipsychotic drugs. These subcellular, molecular and ultrastructural analyses will provide the first comprehensive, region-specific analysis of the neural substrates and upstream factors of elevated presynaptic DA signaling in the AST of SZ and form the foundation of a future R01 application.