Juan C. Troncoso, MD - US grants

Neurons in human degenerative disease, such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), develop a conspicuous neurofibrillary pathology in selected neruonal populations. My long-term objective is to understand some of the functional abnormalities occurring in these diseases, specifically the role of changes in axonal transport and neurotransmitters. My immediate goals in this proposal are to investigate the role of axonal transport in the production of cytoskeletal pathology in aluminum chloride (AlCl3) intoxication and to examine the consequences of this type of pathology on neurotransmitter-related properties of nerve cells. I also plan to study three additional animal models with documented changes in the cytoskeleton and in the axonal transport of motor neurons. First, in an extension of my previous investigations, I plan to examine the effects of AlCl3 intoxication on motor neurons, particularly on the axonal transport of slow components a (SCa) and b (SCb) in the sciatic nerve of the rabbit. Second, because the major biological property of neurons is neurotransmission mediated by neurotransmitters, synthesizing enzymes, and receptors, I plan to study cholinergic enzymes and a variety of receptors in the spinal cords of four animal models which are characterized by rearrangement or disorder of cytoskeletal elements. I plan to analyze changes in receptor populations (acetylcholine [ACh], glycine, gamma-aminobutyric acid [GABA], and benzodiazepine) and cholinergic enzymes (choline acetyltransferase [CAT] and acetylcholinesterase [AChE]) in aluminum myelopathy and in three models with documented changes in axonal transport (intoxication with Beta,Beta'-iminodipropionitrile [IDPN], Hereditary Canine Spinal Muscular Atrophy [HCSMA], and regeneration following sciatic nerve axotomy). The results of analysis of axonal transport, transmitter-specific activities, and receptors will be correlated with morphological observations at the light and electron microscopic level. I believe that, by using a variety of neurochemical and anatomical methods, I will gain new insights into the relationship between the appearance of cytoskeletal pathology and the development of dysfunction of motor neurons. This information should prove significant for our understanding of human degenerative disorders like ALS and AD.

The topic of this proposal is the origin of paired helical filaments (PHF), a major pathological feature of Alzheimer's disease (AD). Although several lines of evidence point towards neuronal cytoskeletal proteins as possible constituents of PHF, their chemical composition remains uncertain. The task we now confront is to unravel the mechanisms that lead to the development of PHF and to ascertain their molecular origin. Our general hypothesis is that PHF are derived from neurofilament or tau proteins that are modified during the course of a disease, such as AD, or by aging. To explore this hypothesis, we propose the following strategies: . To study the in vitro assembly of normal neurofilament and tau proteins; . To submit these cytoskeletal proteins to chemical modifications in vitro and to observe the structural changes that ensue (i.e., formation of insoluble polymers or PHF). The effects of multivalent cations and partial proteolysis have been chosen as conditions that are relevant to posttranslational modifications that may lead to pathological changes of the cytoskeleton in vivo, such as occurs in AD; and . To compare the in vitro assembly of neurofilament and tau proteins isolated from the brains of individuals with AD to those of controls.

neuritic plaques; inflammation; pathologic process; Alzheimer's disease; brain mapping; cognition; neurons; microglia; neural degeneration; tumor necrosis factor alpha; interleukin 6; cytokine; aging; hippocampus; dementia; interleukin 1; colony stimulating factor; complement; synapses; astrocytes; clinical research; human subject; human old age (65+);

The chief goals of the Neuropathology Core (Core C) are to arrange and perform autopsies and provide neuropathological diagnoses in subjects enrolled by the Clinical Core for prospective autopsy, and to maintain a repository (Brain Resource Center) of well-characterized postmortem human brain tissue for investigations of the etiology and basic mechanisms of Parkinson's disease (PD) and Lewy body diseases (LBD). These tissues will be essential to validate the hypotheses and observations generated by the cell biology and biochemical studies of genetically engineered mice proposed in Projects 1-4. Core C will also maintain and staff histology/immunocytochemistry and stereology laboratories to support and facilitate the morphological assessment of human and experimental mouse models relevant to the studies of PD delineated in Projects 1-4. Finally, the staff of Core will train basic investigators and clinical neuroscientists in clinical and neuropathology issues relevant to PD. The staff of Core C has experience with the arrangement and conduction of autopsies, dissection and preparation of human brain tissues for research protocols, neuropathological diagnoses, morphological studies, and quantitative morphometry of neurodegenerative disorders. To accomplish its goals, Core C will staff and maintain the Brain Resource Center (BRC), a histology/immunocytochemistry (ICC) laboratory, and a quantitative morphometry/stereology facility. The BRC serves as a repository of fixed and frozen brain tissues prepared for research including cases of PD, other LBD, and normal and diseased controls. Through its many functions and facilities, Core C will support and coordinate the accession of human postmortem brain tissue material critical for studies of the basic mechanisms underlying Parkinson's disease and facilitate the morphological assessments of experimental animals from Projects 1-4.

Alzheimer's disease (AD) progresses over many years from a preclinical stage to mild cognitive impairment (MCI) to dementia. The notion of preclinical AD has emerged from rigorous clinical and pathological correlations conducted in several longitudinal studies, including ours on the Baltimore Longitudinal Study of Aging (BLSA). Neuropathologically, preclinical AD can be generally defined by the presence of substantial neuritic plaques and neurofibrillary/tau changes in subjects with documented normal cognition. In this project, we propose studies to achieve a more rigorous definition of preclinical AD in terms of structural and biochemical parameters. Specifically, we will measure changes in the number of neurons and synaptic markers in specific regions of the hippocampus and entorhinal cortex (ERG) of individuals from the BLSA and Clinic cohorts, including controls, preclinical AD, and MCI. We will also examine how Abeta and neurofibrillary/tau changes interact and contribute to neuronal and synaptic degeneration in the hippocampus and entorhinal cortex in preclinical AD. Specifically, we will focus on the dendritic field of CA1 pyramidal neurons and on the projections of layer II ERC neurons via the perforant pathway onto the molecular layer of granule cells. A thorough understanding of the preclinical stage of AD and the early mechanism of neurodegeneration is critical since this stage represents and ideal temporal window for the use of emerging preventive and therapeutic strategies in AD.

Alzheimer's Disease; Antibodies; Archives; Autopsy; brain tissue; Cerebral cortex; Clinical; Clinical Data; Collaborations; computerized; Data; Databases; Development; Diagnosis; Diagnostic; Diagnostics Research; disease phenotype; disorder control; Evaluation; Freezing; Genetic; Globus Pallidus; Histology; human Huntingtin protein; Human Resources; Huntington Disease; immunocytochemistry; Location; member; Morbidity - disease rate; Neurobiology; Neurons; Protocols documentation; putamen; repository; Research; Research Personnel; Structure of subthalamic nucleus; Testing; Tissue Banking; Tissue Banks; Tissue Stains; Tissues; Trauma; Trinucleotide Repeats; Work

DESCRIPTION (provided by applicant): There is a shortage of physician-scientists who can bridge basic research and clinical neurosciences. These are the individuals who can take the discoveries of basic science and use them to understand the mechanisms of neurological disorders such as Alzheimer's or Parkinson's disease or stroke, and to develop novel treatments and biomarkers. Neuropathologists play a key role in this process, as they are specifically trained to study the microscopic and molecular manifestations of disease. They can provide critical insights into the changes present in human neural tissues, as well as those of animal models and based on their unique perspective can generate novel hypotheses or form an essential part of multidisciplinary teams studying neurological disease. The goal of the Johns Hopkins Research Education Program in Experimental Neuropathology is to provide neuropathology residents and fellows with an intensive, mentored research experience during their residency or fellowship. This early immersion in research will help them to develop a research career and prepare them to successfully apply for NIH career development awards (KO8 and K23). To attain this goal, we have assembled a group of highly qualified mentors, both from neuropathology and other neurosciences departments, who will guide the research activities of the trainees. These mentors are physician-scientists and basic researchers with strong track records of independent funding and research, scholarship, and previous training of physician-scientists. Trainees will be selected based on their potential for becoming independent investigators and will be admitted to the program while in their neuropathology residency or fellowship. The trainees will then work for one or two years in the laboratory of a mentor, who will guide and supervise them. The plan for research will be jointly developed by the trainee and mentor and approved by the Program Executive Committee. The trainees will also attend courses in the ethical conduct of research and professional development (grant writing). They will also be able to apply for an Administrative Supplement of the award to extend their research experience once they complete their clinical training. While in the program, the trainees will start writing applications for KO8 or K23 awards. The success of the program will be evaluated by the number of trainees that apply for and succeed in obtaining career development awards (i.e. KO8 and K23) or other major research grants (i.e. R01), pursue research and academic careers, and author significant peer-reviewed publications. PUBLIC HEALTH RELEVANCE: Public Health Diseases of the nervous system such as dementia, stroke, epilepsy, and traumatic brain injury are common causes or morbidity and mortality. There is an urgent need to find new ways to diagnose and cure these conditions. New advances in basic sciences, including cell imaging, molecular biology and genetics, have created great opportunities that need to be translated into diagnostic tests and therapies for these neurological disorders. This R25 Neuropathology Research Education Program aims to train physician-scientists focused on Neuropathology who will be able to bridge the gap between basic research and clinical medicine, and to develop the next generation of diagnostic tools and therapies for neurological disorders.

NEUROPATHOLOGY CORE - CORE G: ABSTRACT The Neuropathology Core (Core G) is responsible for: (1) maintaining a registry of participants who have provided ante-mortem autopsy approval, (2) coordinating autopsy procedures at the time of death, (3) providing neuropathologic diagnoses and entering the autopsy data into the central database maintained by the Informatics Core, (3) preparing, storing, and distributing brain tissues for research, (4) working with other members of the research team to integrate the neuropathological data with data obtained from other cores, and (5) conducting targeted studies relevant to preclinical AD pertaining to gene expression and quantitative morphometry.

CORE SUMMARY Neuropathology Core C The Neuropathology Core (Core C) of the Johns Hopkins Udall Parkinson's Disease Research Center has three overarching goals. The first is to conduct postmortem neuropathological assessments in subjects from the Clinical Core and to distribute autopsy brain tissues for research to Projects 1, 2 & 4 and the Proteomic Core. The second is to obtain a comprehensive genetic profile of all the postmortem tissues available in the Udall Center collection. The third is to provide support for the morphological evaluation of genetically engineered mouse models by investigators in Projects 1, 2 & 4. The specific aims of Core C are as follows: (1) to arrange and perform neuropathological autopsies of cases of Parkinson's disease (PD) and Lewy body disease (LBD), and control subjects followed by the Clinical Core, and to formulate pathological diagnoses; (2) to accession, prepare, catalog, and assist in the analysis of human postmortem tissues from cases of PD/LBD and related disorders, as well as age-matched and also younger controls for studies proposed in Projects 1, 2 & 4 and the Proteomic Core; (3) to characterize the molecular genetic profiles of PD/LBD postmortem tissues available in the BRC through a collaboration with the Laboratory of Neurogenetics at NIA (A. Singleton, Ph.D.); and (4) to use CLARITY technology for preparation of mouse brain tissues in support of studies delineated in Projects 1, 2 & 4. Despite recent advances in neuroimaging postmortem brain examination remains indispensable. For accurate diagnosis of PD (Parkinson's disease) and LBD (Lewy body disease) in our experience, ~20% of cases clinically diagnosed as PD have other etiologies, and >30% of the cases have coexisting morbidities. Moreover, since autopsies are the essential source of tissues for studying molecular/biochemical abnormalities of the human brain associated with PD/LBD, a thorough pathological characterization is important before tissues can be used in studies proposed in Projects 1, 2 & 4 and by the Proteomic Core. Core C is expanding its postmortem tissue collection to include a large number of specimens from younger subjects (16 to 65 years) suitable to examine the very early stages of PD/LBD pathology and its pathogenesis. These tissues are accessioned through collaboration with the Lieber Institute for Developmental Disorders at Johns Hopkins. Finally, by using CLARITY, a state-of-the-art morphological technique implemented by Core C, investigators in Projects 1, 2 & 4 will be able to produce translucent preparations of whole mouse brains that allow interrogation of molecular events not only of single neuronal populations but of networks relevant to PD and LBD.

NEUROPATHOLOGY CORE ? CORE D: PROJECT SUMMARY/ABSTRACT Postmortem examination remains critical to elucidate the etiology in cases of dementia and to confirm the diagnosis of AD, despite recent advances in neuroimaging and fluid biomarkers. Autopsy is also critical for confirming the clinical diagnosis and identifying comorbidities, which are common in older subjects. The Neuropathology Core (Core D) of the Johns Hopkins Alzheimer's Disease Research Center (JHADRC) has three overarching goals. The first is to conduct postmortem neuropathological assessments in subjects enrolled in the JHADRC Clinical Core. Second, to prepare, store, and distribute autopsy brain tissues for research. The third goal is the training of young physicians and neuroscientists in the neuropathology of dementias and neurodegenerative diseases. The specific aims of Core D are as follows: (1) to arrange and perform autopsies on clinically well-characterized subjects enrolled through the JHADRC and assist with consensus diagnoses on JHADRC subjects, (2) to store optimally prepared tissues from the autopsies and to make these specimens available to investigators associated with the JHADRC and at other collaborating institutions, (3) to integrate the neuropathologic data from these cases with the clinical and biomarker data acquired in the same participants, using the JHADRC database, and to submit the neuropathology data to the National Alzheimer's Coordinating Center (NACC), working with the Data Core (4) to support the assessment of genetically engineered mouse models relevant to Alzheimer's disease (AD) and related disorders, and (5) to train basic investigators and clinical neuroscientists in the morphological and diagnostic concepts relevant to AD, to other types of dementias and neurodegenerative disorders (including trainees supported by the REC). In addition, the availability of human brain postmortem tissues has become increasingly important for validating research hypotheses and for investigations and development of biomarkers of AD. Core D will therefore be expanding its postmortem tissue collection to include a large number of specimens from younger subjects (30 to 65 years) suitable to examine the very early stages of AD pathology and its pathogenesis.

PROJECT SUMMARY There is substantial evidence that soluble A? oligomers are neurotoxic and that impaired clearance of soluble A? is a critical pathologic mechanism in late-onset AD, a concept supported by the observation of decreased passage of A? into the cerebrospinal fluid (CSF) in AD6 and the decreased levels of A? in the CSF of AD patients. The mechanisms for clearance of extracellular A? from the brain are multiple and include local enzymatic degradation, transport across the blood brain barrier (BBB), or via the CSF. The relative contributions of these various systems to A? clearance are not fully understood, although the BBB system appears to be predominant. However, recently, the glymphatic system has been implicated in this process, possibly in addition to BBB mechanism. The glymphatic system is proposed as a brain waste clearance pathway consisting of perivascular tunnels lined by astrocytes that drain into CSF and eventually into cervical lymphatic nodes. This system allows for the elimination of soluble proteins and metabolites from the central nervous system and it may be implicated in A? clearance and the development of AD. The clearance efficiency of the glymphatic system depends on CSF flow, cerebrovascular pulsation, and the aquaporin-4 (AQP4) water channels of perivascular astrocytes. In mice, the efficiency of the glymphatic pathway appears compromised in aging due to impairment of AQP4 dependent bulk flow. The role of the glymphatic system in the clearance of A? and its relevance to AD is based mostly on studies in rodents, which is one of the reasons why it remains controversial. Therefore, the relevance of the glymphatic system to AD needs to be examined in the human brain, which is the overall goal of this proposal. Moreover, we believe that the best time to conduct this examination is before and during the early stages of A? deposition. To this end, we have available a large number of human postmortem brains from individuals 30 to 65 years of age which are fully characterized for AD lesions and tissue integrity, and genotyped for ApoE. The exploratory and observational studies proposed here are the basis for future mechanistic examination of the glymphatic system and its relevance to AD to be conducted in cultured cells and tissues, and animal models.

NEUROPATHOLOGY CORE ? CORE G: PROJECT SUMMARY/ABSTRACT The BIOCARD study is a longitudinal, observational study of 349 individuals who were cognitively normal and primarily middle aged (mean age=57.1) at enrollment. The subjects have now been followed for up to 27 years. The overall objectives of the project are to further advance the study of preclinical Alzheimer?s disease by: (1) clarifying the pattern and rate of change in AD biomarkers (including those based on CSF, blood, MRI, and PET imaging) and cognition; the biomarkers to be studied include several promising novel biomarkers derived from blood, CSF and brain imaging. (2) maximizing our data by working collaboratively with several research groups who have comparable data, and (3) providing a publicly accessible data, brain scans, and biological specimens, for researchers in the field. To accomplish these goals we established 7 Cores and, with this application, are also including 2 projects. The Neuropathology Core (Core G) is responsible for overseeing the neuropathological assessment of brain tissue from participants in the BIOCARD study and for assuring the integration of these findings with relevant data collected by the other Cores and Projects. The specific aims include: (1) to maintain a registry of prospective brain donors, (2) to arrange and perform neuropathologic autopsies, (3) to provide neuropathologic diagnoses, (4) to integrate neuropathology data with data from other Cores and Projects, (5), to prepare, store and distribute autopsy brain tissue for research, and (6) to continue investigations of the early pathogenesis of Alzheimer?s disease.