Steven E. Arnold, MD - US grants

This is an application for an ADAMHA Scientist Development Award for Clinicians. My overall career goal is to gain expertise in the molecular neuroanatomic and neuropathologic investigation of neuropsychiatric illness, with an emphasis on schizophrenia. During the tenure of the requested award I expect to: 1) acquire further skills in basic neuropathologic diagnosis; 2) develop technical expertise with qualitative and quantitative immunohistochemical methods for characterizing brains of patients with schizophrenia and controls; 3) learn techniques for biochemical analysis of brain tissue and for generating monoclonal antibodies; and 4) expand skills in data analysis and the integration of postmortem and antemortem findings. These goals will be accomplished in the course of a research plan whose aims are to: 1) investigate the cytoarchitecture and molecular character of neurons in limbic temporal structures in schizophrenia; and 2) characterize normal human fetal molecular development of these brain regions by determining the timing of cytoskeletal and synaptic protein expression and establishment of neuronal polarity and laminar position. By this research I hope to extend and clarify the abnormalities that have been suggested by studies using traditional neuropathologic methods and to explore the thesis of schizophrenia as a neurodevelopmental disorder.

Clinical and neuropsychological studies of chronically institutionalized elderly patients with schizophrenia indicate a high prevalence of severe cognitive impairment and functional disability. The biological substrates for this dementia are not known. Our preliminary diagnostic neuropathologic investigations have revealed notably few recognized disease states (e.g. Alzheimer's disease) in more than 30 cases to date, although subtle morphometric abnormalities in the hippocampal region and astrocytosis in hippocampal and frontal regions have been observed. The purpose of the proposed research is to investigate the neuronal and molecular substrates of neurodegeneration and dementia in a clinicopathological correlation study of prospectively accrued, well characterized, chronically institutionalized elderly patients with schizophrenia in comparison to Alzheimer disease and non-neuropsychiatric controls. Because of the severity of illness in this population, it may be particularly suitable for this research as neuropathologic abnormalities might be expected to be more evident than in a less afflicted population. Further, if involutional or neurodegenerative processes are an aspect of the pathophysiology of schizophrenia, then these would be most manifest in the elderly. All schizophrenia cases will have been characterized with standardized chart review, clinical diagnostic examinations, and clinical rating scales including the Brief Psychiatric Rating Scale, the Scale for the Assessment of Negative Symptoms, the Scale for the Assessment of Positive Symptoms, Physical Self-Maintenance Scale, Mini-Mental State Examination, and the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Neuropsychological Battery. Markers of neural injury and degeneration will be identified using histochemical, immunohistochemical and other molecular methods and will be quantified with particle counting and/or optical densitometry using principles of stereology and systematic sampling. Primary regions of interest will be the hippocampus, entorhinal cortex, mid-frontal and orbitofrontal cortices, and primary visual cortex (as an internal control region). Markers will include: a) Alzheimer- related pathology (neurofibrillary tangles and senile plaques identified immunohistochemically); b) astrocytic and microglial proliferation (GFAP immunohistochemistry and RCA-1 lectin histochemistry); c) neuron loss (total Nissl-stained neuron counts in hippocampus, and densities in temporal, frontal and occipital cortices); d) ubiquitination (anti- ubiquitin immunohistochemistry); e) DNA fragmentation (Tdt-mediated dUTP- digoxigenin nick end labeling); and synapse-related protein abnormalities ( synaptophysin, EP-l0, synapsin, chromogranin, GAP-43 immunohistochemistry). Neuropathologic findings will be correlated with antemortem clinical and neuropsychological measures.

The objective of this research is to develop a family of fully nonparametric statistical methods and associated software for use in social sciences research. These procedures require minimal modeling assumptions, possess desirable invariance properties, will allow the analysis of all types of ordinal data (continuous, ordered categorical, and data with ties), and will also permit the data to be either independent or dependent (as, for example, in longitudinal studies). The software developed will consist of SAS macros and S-plus functions which will be made available to researchers in the social sciences through several World-Wide-Web sites.

neuropathology; schizophrenia; microtubule associated protein; dendrites; cytoskeleton; protein isoforms; messenger RNA; depression; hippocampus; visual cortex; frontal lobe /cortex; bipolar depression; age difference; in situ hybridization; human old age (65+); immunocytochemistry; human tissue; histopathology; postmortem;

Growing evidence from clinical and postmortem research implicates abnormal neurodevelopment in the pathophysiology of schizophrenia. While neuron birth, migration, differentiation, and connectivity are largely completed by the end of gestation in almost all parts of the CNS, the olfactory epithelium and olfactory bulb are unique in that they undergo continuous regeneration and reinnervation throughout life. Thus, they offer an opportunity to investigate cellular and molecular events of ongoing neuronal and connectional development, even in late life. In addition, cytoarchitectural abnormalities suggestive of abnormal development have been described in olfactory cortices, although findings remain controversial. The importance of examining the olfactory system in schizophrenia is further underscored by the marked impairments in odor detection, identification, and memory that are present in the disorder. The proposed research will characterize neurodevelopmental processes in human olfactory epithelium, olfactory bulb, and primary olfactory cortices in well-characterized individuals with schizophrenia and matched controls. Our overarching hypothesis is that abnormal patterns of molecular, cellular and connectional neurodevelopment are evident in olfactory regions in schizophrenia. In the olfactory epithelium (Aim 1), immunohistochemistry with cell-type-specific antibodies and computer-assisted microscopy will be used to determine the relative proportions and morphologic characteristics of differentiating cell types in the olfactory receptor neuron lineage, as well as their expression of polypeptides that are important for the neuronal cytoskeleton and for neurotrophin mediated signal transduction. In the olfactory bulb (Aim 2), the cellular, dendritic, and axonal composition of the glomerulus will be quantified after immunolabeling for synaptophysin, tyrosine hydroxylase, calretinin, calbindin D28k, NCAMs and GAP- 43. In Aim 3, we will perform a quantitative cytoarchitectural analysis of piriform and entorhinal cortices. The relative densities of immunohistochemically distinct populations of neurons will be determined and the spatial arrangement of neurons will be assessed using spatial point pattern analyses.

The University of Pennsylvania School of Medicine (PENN) has developed a unique and exciting program to prepare promising medical students to become leaders at the cutting edge of clinical neuroscience practice and research. The present proposal is aimed at enhancing this program in such a way as to further encourage its students to enter research careers in mental health. The advent of managed care has placed academic medical centers under severe economic pressures to trim research and education budgets. Medical students are aware of these trends and are discouraged from pursuing research careers. MD/PhD programs encourage students interested in basic research, but few mechanisms exist for nurturing clinical researchers. In an attempt to counter these tendencies, PENN has created the Clinical Neuroscience Track (CNST). This program identifies incoming medical students who are interested in the clinical neurosciences, develops in them an esprit de corps with each other and with the clinical neuroscience faculty, and trains them in the skills of scientific thinking necessary to evaluate and participate in clinically relevant research. Students also perform a research project. The CNST has been very successful and after four years, has grown to over 100 students. Thus it can no longer be run without cost sharing by extramural sources. The existing CNST will now be modified in ways that will further strengthen the exposure of its medical students to mental health research. The following specific aims are proposed: Aim 1. To identify incoming medical students who may be interested in research in mental health, expose them to mental health related research throughout their medical school careers and instill in them the skills of critical thinking necessary to evaluate this research. Aim 2. To provide the students with research experiences during medical school, in order to encourage them to consider careers in mental health research. Aim 3. To maintain contact with these students after they graduate in order to assist them with their career development, as well as to monitor the success of the program. Participation by minorities and women will be strongly encouraged. The CNST will be administered by a director with the assistance of a coordinator. There will be two major committees - an educational planning committee and a research committee. It is expected that each year, 15 of the initial 20-30 students will complete the entire program, including the research project. This program will increase the number of physicians entering careers in mental health research and serve as a model for other medical schools to emulate.

[unreadable] DESCRIPTION (provided by applicant): Twelve studies to date report significant associations between certain haplotypes in the dysbindin gene and schizophrenia (SZ). Dysbindin was initially identified as a binding partner of the dystrobrevins, members of the dystrophin glycoprotein complex present in muscle and postsynaptic sites in the brain. Subsequent work has shown that dysbindin has additional binding partners, including several members of BLOC-1 (the biogenesis of lysosome-like organelles complex-1). We have found that dysbindin and several BLOC-1 proteins are frequently and significantly reduced in presynaptic fields of intrinsic glutamatergic projections of the hippocampal formation in SZ. The dysbindin reductions are inversely correlated with increased vesicular glutamate transporter-! (VGluT-1) immunoreactivity in the same projections. These alterations occur without apparent synaptic loss, alterations in (3-dystrobrevin, or evidence of neuroleptic effects on dysbindin or [unreadable] VGluT-1 We propose to investigate dysbindin in postmortem SZ brains and in the sandy (sdy) mouse, a mouse strain with a large deletion mutation in the dysbindin gene. Our specific aims are designed to answer key questions about the generality, causes, and consequences of dysbindin reductions in SZ with a focus on its role in glutamate transmission. In Aim 1, we map the regional distribution and isoform specificity of dysbindin reductions in SZ and correlate these with markers of several neurotransmitter systems. In Aim 2, we investigate dysbindin gene expression as well as its relation to high risk haplotypes for SZ. In Aim 3, we test the effects of reduced dysbindin on its BLOC-1 binding partners and hypothesized mechanisms by which reduced dysbindin alters glutamatergic synaptic machinery and glutamate release. In Aim 4, we use high resolution imaging of voltage-sensitive dyes and whole cell patch and field recordings in the sdy mouse hippocampal formation to investigate the effects of the dysbindin negative mutation on neurotransmission, long term potentiation and long term depression in glutamatergic pathways. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Targeted Proteomics of Resilient Cognition in Aging Abstract Cognition in later life deteriorates when age-associated degenerative and other pathological processes overwhelm the brain's capacity to withstand, counteract or compensate for those processes through neuroplastic, reparative responses. Thus, while clinicopathological correlation studies show significant associations of plaques, tangles, infarctions and other lesions with cognitive impairment, the relationships are imperfect, and it is increasingly recognized that some elderly persons may have abundant pathology and yet still are unimpaired. We propose an innovative clinicopathological approach in the richly characterized Religious Orders Study (ROS) cohort to identify candidate proteins and pathways that best confer cognitive resilience despite the presence of neurodegenerative disease pathologies. To accomplish this we apply non-biased stratification in the ROS cohort to identify three distinct test groups of 60 each: 1) AD-Dementia (dementia with abundant plaque and tangle pathology), 2) AD-Resilient (normal cognition despite abundant AD plaque. tangle and other pathology), and 3) Healthy (normal cognition and minimal AD or other pathology). We will measure the expression of >500 proteins that span functional families of apoptosis, synaptic neurotransmission and neuroplasticity, metabolic, inflammatory, cytoskeletal, signal transduction pathways and others. We will test two hypotheses that pro-apoptotic proteins and synaptic plasticity proteins differentiate the clinicopathological category groups and we will conduct machine learning approaches to further identify these and novel analytes that best predict categories. Based on these analyses, we will generate a targeted multi-analyte immunoassay panel. This will be used to validate the association of candidate proteins with cognitive resilience in another set of 480 ROS cases. Our focus on the neurobiology of resilient brain aging will be an important counterpoint and complement to historical and current efforts focused on the neurobiology of disease pathology. PUBLIC HEALTH RELEVANCE: Targeted Proteomics of Resilient Cognition in Aging Cognitive decline in aging arises when age-associated neuropathological processes overwhelm the brain's capacity to withstand or adapt to those processes. We propose an innovative clinicopathological approach to identify candidate proteins and pathways that best confer cognitive resilience despite the presence of neurodegenerative disease pathologies. Using antibody microarrays for discovery and multi-analyte immunoassays for validation in brain tissues from participants of the Religious Orders Study, we will identify potential novel mechanisms of cognitive resilience in aging.

DESCRIPTION (provided by applicant): Metabolic disturbances in particular type 2 diabetes mellitus (T2D), are common and increasing in incidence with contemporary lifestyles. T2D is associated with adverse health consequences, including impaired brain Yet, the causes and effects of insulin resistance in the brain are complex, as there are reciprocal interactions with other hormone signaling systems, in particular the adipokines (e.g., adiponectin and leptin) which strongly affect insulin sensitivity. Characterizing the molecular mechanisms that increase risk of AD for persons with insulin resistance is now of great scientific interest (PAS-11-029) and their elucidation may lead to novel therapeutic strategies. Insulin resistance, a core feature of T2D, may lead to increased brain pathology. T2D is modestly associated with cerebrovascular disease, but perhaps of even greater interest, recent data show brain insulin signaling abnormalities associated with AD pathology, specifically amyloid-¿ and tau-related pathology as well. Furthermore, adipokine receptor expression abnormalities have also been found in AD. The overall goal of the proposed interdisciplinary collaborative project is to establish molecular mechanisms linking insulin, adopinectin, and leptin signaling in human brain, and determine how dysregulation in this network is associated with brain structure, pathology, and function, including AD and cognitive impairment. The proposed study will quantify markers of insulin, adiponectin, and leptin signaling, neurons and synapses, and use existing pathologic and clinical data from 200 community-dwelling women and men, with and without T2D and across a spectrum of cognitive function, who were well-characterized clinically and died and came to autopsy as participants in the Religious Orders Study (P30AG010161; R01AG015819). First, we will describe expression levels of brain insulin, adiponectin, and leptin pathway components in subjects with and without T2D (Aim 1). Then, using a novel ex-vivo stimulation paradigm in human postmortem tissue, we will experimentally test the stimulated responses and interactions of insulin and adipokine signaling in brain tissue (Aim 2). Finally, in the clinicopathologic translational component of the study, we will test the relations of insulin, adiponectin, and lepti signaling to brain structure (synaptic markers), pathology (amyloid-¿ and tau), and function (cognition). Because insulin resistance is a common condition for which therapies are available, this study will break new ground in research of insulin and adipokine mechanisms in human brain, and show insulin resistance and adipokine dysfunction are associated with changes in the human brain at multiple levels, thus providing important data with potential to improve public health.

PROJECT SUMMARY/ABSTRACT Type 2 diabetes mellitus (T2D) is common and results in enormous personal and societal costs. While many complications of T2D are well-established, less is known about the link between T2D and the brain, beyond associations with cerebrovascular disease, cognitive impairment, and dementia. Recent data show that insulin resistance (InsRes), a key component in T2D, may be present in the aging human brain itself and be associated with Alzheimer?s disease (AD) pathology, including amyloid-?, and cognitive impairment. A pressing scientific question is whether peripheral (systemic) InsRes is related to central (brain) InsRes and to AD. In response to PAR-17-031 calling for studies to elucidate metabolic regulation, and insulin signaling in particular, in AD, we propose an interdisciplinary project with the overall goal to examine associations of peripheral with central insulin resistance, and the associations of peripheral and central insulin resistance with AD neuropathology and cognitive function. The proposed study will take advantage of available biospecimens (frozen ante-mortem serum and peripheral blood mononuclear cells, and postmortem skeletal muscle and brain tissues) from which to quantify markers of chronic hyperglycemia and InsRes, and from which to use existing laboratory, clinical, and neuropathologic data, from 200 community-dwelling women and men, with and without T2D, who were well-characterized clinically and died and came to autopsy (R01AG17917). First, we will characterize peripheral (blood, muscle) and central (brain) InsRes in biospecimens, using two complementary approaches, including biochemical assays and an ex vivo stimulation paradigm to directly examine the insulin receptor signaling cascade (Aims 1 and 2). Next, we will test the hypothesis that peripheral InsRes is linked to central InsRes, and conduct additional analyses considering secondary markers of interest and whether other factors, such as sex and others, affect associations (Aim 3). Finally, in a translational aim, we will test the hypotheses that peripheral and central InsRes are associated with neurodegenerative and cerebrovascular neuropathology, and domain-specific cognitive function (Aim 4). Because InsRes is a key component of T2D for which therapies are available, this study which will test the link of peripheral and central InsRes, and neuropathology and cognition in persons with and without T2D, will fill major gaps in scientific knowledge and inform future research in the field of metabolism, insulin signaling, and AD, and provide important data with potential to improve public health.