Bernardino Ghetti, MD, University of Pisa - US grants

The long term objectives of the present proposal are to gain knowledge about the reaction of the central catecholamine systems to pathologic conditions. Specifically, with two models of genetically determined neuronal losses, seen in the Purkinje cell degeneration mutant mouse and the weaver mutant mouse, we will be able to investigate a) the effects on a major catecholamine system when one of its target neuronal populations (the Purkinje cell) degenerates; b) the effects on a major target field (caudo-putamen) when its major and important catecholamine neuronal input (substantia nigra, cells of pars compacta) degenerate. In the Purkinje cell degeneration mutant the Purkinje cells degenerate at the end of cerebellar maturation, but norepinephrine levels in the cerebellum are not reduced. Therefore, we will monitor the plastic changes of the catecholaminergic axons and determine whether the catecholamine turnover is altered by the loss of the postsynaptic neuron. In the weaver mutant a marked deficiency of the dopamine system is present and is due to depletion of dopaminergic neurons in the substantia nigra. We will analyze the effects of the atrophy of the substantia nigra on the postsynaptic neurons of the caudate nucleus. These models will provide insight into afferent and efferent relationships which catecholamine neurons are able to maintain under conditions of degeneration of either their target neurons or cells of the catecholamine nucleus itself. The comprehension of the biology of the systems of the catecholaminergic neurons and their modality of reaction in pathologic conditions is of relevance to neurology, psychiatry and medicine; in fact the importance of the central catecholamine system in the expression of a number of behaviors, as substrates for the mediation for the action of a variety of drugs, and a possible substrate for diseases such as Parkinsonism, schizophrenia and depressive illnesses are well recognized. The proposed goals will be achieved by bringing together numerous techniques including neurohistochemical, electron microscopic, Golgi impregnation techniques, and computerized cytomorphometry that will be correlated with sophisticated neurochemical and neuropharmacological techniques.

Presenile and senile dementia of the Alzheimer's type is presently one of the most important causes of disability. Histopathologically Alzheimer's disease is characterized by distinct features foremost of which is the presence of senile plaques which contain material meeting all the criteria for amyloid. Indeed, fibrillar deposits which stain with Congo red to give green birefringence by polarization microscopy are usually found in the senile plaques in patients with Alzheimer's disease as well as in the walls of intracerebral blood vessels. Whether there is one or more than one amyloid substance in these locations is not known. Furthermore, it is not known whether the amyloid substance is reactive in nature and/or plays a primary role in the pathogenesis of the dementia. The objective of this proposal is to isolate and characterize the protein or proteins which are contained in the intracerebral amyloid deposits in Alzheimer's disease. The working hypothesis is that the amyloid deposits are composed of a small subunit protein which is deposited in a beta pleated sheet configuration as has been shown for immunoglobulin light chains in primary amyloidosis, protein AA in secondary amyloidosis and prealbumin in hereditary amyloidosis. It is proposed to isolate the amyloid fibrils from central nervous system tissues of patients dying with Alzheimer's disease and use these fibril preparations to isolate the subunit protein or proteins. Light microscopy of Congo red stained materials and electron microscopy will be used to follow the extraction procedure as well as to analyze the amount of amyloid material in individual brains and demonstrate the feasibility of extraction procedures. Chemical analysis of the amyloid fibril preparations will be done by solubilizing the amyloid fibrils in denaturing solvents and fractionation by chromatography both on Sepharose gels and by high performance liquid chromatography. Amino acid analysis of isolated subunit proteins and amino acid sequencing of tryptic peptides of the subunit proteins will be used to characterize the isolated products. The chemical characterization by primary structure of amyloid subunit proteins in Alzheimer's disease will allow the determination of whether the amyloid is a product of 1) normal proteins such as neuroendocrine peptide or prealbumin, 2) a variant form of a normal human protein such as has been described for prealbumin variants in hereditary amyloidosis or 3) the product of an infectious agent such as has been suggested in scrapie.

The genetic and epigenetic mechanisms involved in the premature loss of specific nerve cells in the central nervous system are for the most part unknown. In the proposed studies an effort is made to determine whether gene-dose-dependent mechanisms operate in the degeneration of neurons of the substantia nigra in the weaver and of the Purkinje cells in the pcd mutant mouse. Weaver and pcd are recessive mutations. Neuronal losses are known to take place in the homozygotes; the demonstration of losses in heterozygotes would support the hypothesis that a gene- dose-dependent mechanism regulates losses either of nigral neurons or of Purkinje cells in the respective mutants. In weaver mutants loss of dopamine neurons of the substantia nigra will be used as a natural model of nigrostriatal degeneration to analyze the neurochemical dysfunction of the cholinergic and GABAergic neurons possibly resulting from loss of presvnaptic cells. Finally, strategies of neural tissue grafting in the cerebellum and in the striatum will be applied in order to obtain a restoration of structure and neurotransmitter function. The comprehension of both the biological phenomena related to nerve cell degeneration and its sequelae as well as of the mechanisms related to the structural and neurochemical interaction between neural elements of host and grafted tissue is a fundamental step in designing strategies for the repair of neural circuitries in degenerative conditions of the central nervous system and in aging. The proposed goals will be achieved by bringing together techniques of genetic crossing and neural grafting as well as methodologies of immunocytochemistry, electron microscopy and computerized morphometry that will be correlated with sophisticated neurochemical and neuropharmacological methods and with behavioral analysis.

The weaver (wv) mutant mouse, the only known rodent model characterized by the association of a genetically determined degeneration of the neurons of the substantia nigra (SN) and of the cerebellar granule cells, lends itself to an analysis of fundamental issues of pathologic neurobiology which are relevant to the understanding of human neurodegenerative disorders. The strength of the proposal has been built from established work of each of the investigators who possess diverse backgrounds representing the fields of neuropathology and neuromorphology, developmental neurobiology, neurochemistry and pharmacology, molecular biology and genetics. The investigators are united by a common goal, i.e. understanding the mechanisms of nerve cell degeneration in the nigrostriatal system damaged by the genetic mutation. The cohesive effort is organized in a multi- disciplinary and highly interactive research program. We plan to investigate: the neuropathologic alterations of the nigrostriatal system using morphometric quantitative approaches combined with new developmental and genetic strategies; the transmitter neurochemistry of the SN and the molecular biological mechanisms which underlie the wv defect. Project #1 will investigate the influences of the strain associated variability of the number of midbrain dopamine neurons upon the expression of a single gene mutation affecting that neuronal population. Project #2 will characterize the embryogenesis of the SN and the postnatal degeneration of both the SN and striatum of the wv mutant. Project #3 plans to investigate the functional neurochemical correlates of the SN deficit and to study the adaptive changes which take place when various neuronal systems attempt to compensate for the deleterious effect of the mutation. Project #4 is directed to isolate and characterize the weaver gene and to study its expression during development.

The Indiana Alzheimer's Center will be established on the facilities at Indiana University School of Medicine. It will comprise four cores: an Administrative Core, a Clinical Core, a Neuropathological Core and an Education and Information Transfer Core. The Director of the Center will be Dr. Bernardino Ghetti, (Professor of Pathology and Psychiatry, Associate Director of the Division of Neuropathology and Associate Director of the Neurobiology Program) and two Associate Directors will be Drs. Hugh C. Hendrie (Albert E. Sterne Professor and Chairman of Psychiatry) and Michael Conneally (Distinguished Professor of Medical and Molecular Genetics and Neurology). The Center will draw upon the strengths of the faculty of Indiana University School of Medicine and its associated schools in particular its existing multi-disciplinary programs, the graduate program in medical neurobiology and the Geriatric Education Center to develop its research and educational agenda. The Principal Investigators in this project have an established record in research in AD and related disorders and have a long collaborative history with each other. The core faculty have already excellent community relationships and teaching programs through the operation of the existing Alzheimer's Disease and Related Disorders Clinic with the co-operation of the ADRDA and other community agencies. These programs will be enhanced and in particular a carefully evaluated educational intervention program for long term facilities will be developed. A major emphasis of the Center will be to reach out to the Afro-American community of Indianapolis and to involve them in the educational, clinical and research aspects of the program. The Clinical Core will allow the development of a cohort of carefully diagnosed patients with AD and related dementias together with normal subjects to participate in the research studies. The Center will combine the two major current research themes of the investigators at Indiana University; the search for the genetic basis of AD and the search for other risk factors into one basic and clinical program.

Nerve cell degeneration and loss are pivotal events in most hereditary diseases of the central nervous system. Among these diseases are the heredo-degenerative ataxias, Parkinson's disease, and Alzheimer's disease. in the heredo-degenerative ataxias, the degeneration of Purkinje cells (PC), which are essential for normal motor functions, leads to incoordination. In Parkinson's disease, degeneration of midbrain dopamine (DA) neurons, that participate in a variety of motor functions and mental processes, leads to tremor, rigidity, bradykinesia and deficit of posture. Neuronal loss triggers a cascade of transsynaptic changes, whose mechanisms are poorly understood. Understanding the mechanisms of such degeneration is best achieved by studying a given neuronal population prior to or early in the degenerative process. An analysis of the cascade of transsynaptic changes can be best achieved in natural models of degenerative diseases in which nerve cell loss and the transneuronal sequelae occur in a well known and predictable time sequence. In the Purkinje cell degeneration (pcd) and weaver (wv) mutant mice, loss of PC and of midbrain (DA) neurons, respectively, are among the major phenotypic events and they occur in a fixed time frame. A long term goal of this project is to investigate the mechanisms leading to cell loss at the cellular and molecular level and to attempt to understand the primary defect. We will construct cDNA libraries from cerebella of pcd mutants at the onset of Purkinje cell degeneration and we will isolate clones that are differentially expressed. These cDNAs will be evaluated by sequencing and their cell of origin determined by in situ hybridization. We will begin molecular/biochemical characterization of the pcd-induced neurodegenerative cascade by following the expression of these clones, PC markers and cytoskeletal markers during the disease and we will attempt to clone the pcd cDNA. For the wv mutant, studies will be carried out in order to determine the effect of DA losses on the GABAergic striatal interneurons and efferents and on the striato-pallidal enkephalin and the striato-nigral dynorphin systems. In addition, we will determine the significance of the increased content of serotonin in the striatum of the wv mutant. Since the trophic interactions between DA neurons and their target cells in the establishment and maintenance of the nigrostriatal DA projection are not understood, we will utilize the three-dimensional reaggregate tissue culture system to investigate whether the loss of DA neurons observed in the wv mutant is due to a defect in the DA neurons themselves, or in their target cells. Embryonic brain cells from wv and normal (non-weaver) mice will be cocultured to test whether target cells from normal mice can promote the survival of the wv DA neuron. Conversely, target cells from wv mice will be assessed for their ability to produce a loss of DA cells from normal mice. In addition, we propose to use the wv mouse as a means of obtaining a selected population of medial DA neurons, cells which project to limbic and cortical areas and are relatively spared in wv mutants, for the purposes of generating an immortalized DA cell line by somatic cell hybridization. The availability of such a DA cell line will be an important model for examining DA neuronal target recognition and neurotoxicity.

Nerve cell degeneration and loss are pivotal events in most hereditary diseases of the central nervous system. Among these diseases are the heredo-degenerative ataxias, Parkinson's disease, and Alzheimer's disease. in the heredo-degenerative ataxias, the degeneration of Purkinje cells (PC), which are essential for normal motor functions, leads to incoordination. In Parkinson's disease, degeneration of midbrain dopamine (DA) neurons, that participate in a variety of motor functions and mental processes, leads to tremor, rigidity, bradykinesia and deficit of posture. Neuronal loss triggers a cascade of transsynaptic changes, whose mechanisms are poorly understood. Understanding the mechanisms of such degeneration is best achieved by studying a given neuronal population prior to or early in the degenerative process. An analysis of the cascade of transsynaptic changes can be best achieved in natural models of degenerative diseases in which nerve cell loss and the transneuronal sequelae occur in a well known and predictable time sequence. In the Purkinje cell degeneration (pcd) and weaver (wv) mutant mice, loss of PC and of midbrain (DA) neurons, respectively, are among the major phenotypic events and they occur in a fixed time frame. A long term goal of this project is to investigate the mechanisms leading to cell loss at the cellular and molecular level and to attempt to understand the primary defect. We will construct cDNA libraries from cerebella of pcd mutants at the onset of Purkinje cell degeneration and we will isolate clones that are differentially expressed. These cDNAs will be evaluated by sequencing and their cell of origin determined by in situ hybridization. We will begin molecular/biochemical characterization of the pcd-induced neurodegenerative cascade by following the expression of these clones, PC markers and cytoskeletal markers during the disease and we will attempt to clone the pcd cDNA. For the wv mutant, studies will be carried out in order to determine the effect of DA losses on the GABAergic striatal interneurons and efferents and on the striato-pallidal enkephalin and the striato-nigral dynorphin systems. In addition, we will determine the significance of the increased content of serotonin in the striatum of the wv mutant. Since the trophic interactions between DA neurons and their target cells in the establishment and maintenance of the nigrostriatal DA projection are not understood, we will utilize the three-dimensional reaggregate tissue culture system to investigate whether the loss of DA neurons observed in the wv mutant is due to a defect in the DA neurons themselves, or in their target cells. Embryonic brain cells from wv and normal (non-weaver) mice will be cocultured to test whether target cells from normal mice can promote the survival of the wv DA neuron. Conversely, target cells from wv mice will be assessed for their ability to produce a loss of DA cells from normal mice. In addition, we propose to use the wv mouse as a means of obtaining a selected population of medial DA neurons, cells which project to limbic and cortical areas and are relatively spared in wv mutants, for the purposes of generating an immortalized DA cell line by somatic cell hybridization. The availability of such a DA cell line will be an important model for examining DA neuronal target recognition and neurotoxicity.

The Indiana Alzheimer's Center will be established on the facilities at Indiana University School of Medicine. It will comprise four cores: an Administrative Core, a Clinical Core, a Neuropathological Core and an Education and Information Transfer Core. The Director of the Center will be Dr. Bernardino Ghetti, (Professor of Pathology and Psychiatry, Associate Director of the Division of Neuropathology and Associate Director of the Neurobiology Program) and two Associate Directors will be Drs. Hugh C. Hendrie (Albert E. Sterne Professor and Chairman of Psychiatry) and Michael Conneally (Distinguished Professor of Medical and Molecular Genetics and Neurology). The Center will draw upon the strengths of the faculty of Indiana University School of Medicine and its associated schools in particular its existing multi-disciplinary programs, the graduate program in medical neurobiology and the Geriatric Education Center to develop its research and educational agenda. The Principal Investigators in this project have an established record in research in AD and related disorders and have a long collaborative history with each other. The core faculty have already excellent community relationships and teaching programs through the operation of the existing Alzheimer's Disease and Related Disorders Clinic with the co-operation of the ADRDA and other community agencies. These programs will be enhanced and in particular a carefully evaluated educational intervention program for long term facilities will be developed. A major emphasis of the Center will be to reach out to the Afro-American community of Indianapolis and to involve them in the educational, clinical and research aspects of the program. The Clinical Core will allow the development of a cohort of carefully diagnosed patients with AD and related dementias together with normal subjects to participate in the research studies. The Center will combine the two major current research themes of the investigators at Indiana University; the search for the genetic basis of AD and the search for other risk factors into one basic and clinical program.

The aims of the Neuropathology Core are to provide technical resources, laboratory facilities and professional expertise for the collection, diagnosis and storage of brain tissue obtained at autopsy from patients and control subjects studies in the Clinical Core of the Indiana ADC, as well as from referral cases from other institutions. The Neuropathology Core will provide information about pathologic data to families, referring physicians and basic researchers. In addition, the Core will be involved in continuing education to the community about new developments emerging from Alzheimer disease (AD) research. Our understanding of the molecular, biochemical, histochemical, pathologic and clinical aspects of AD has advanced rapidly during the last five years. Thus, brain tissue of demented individuals must be studied for diagnostic and research purposes using a multidisciplinary approach. We have established a Dementia Laboratory, developed a laboratory for morphometric studies of degenerative brain diseases and expanded our Brain Bank. The Core has contributed to the investigations of hereditary presenile dementias with neurofibrillary tangles by (i) characterizing familial AD with a mutation in APP gene, (ii) characterizing the neuropathologic phenotype of Gerstmann-Straussler- Sckeinker (GSS) disease in the Indiana kindred, (iii) studying tau pathology in neurodegenerative disease, (iv) expanding our knowledge on the Neuropathology of diseases caused by mutations in the APP and PRNP genes, (v) discovering new forms of presenile dementias. We are expanding our mission integrating modern molecular technology to assist in the diagnosis of dementias, in particular the cerebral amyloidoses. In fact, we are combining data obtained by neuromorphology, immunohistochemistry, and immunocytochemistry to recognize and characterize the localization and the antigenic profile of molecules that are important to the pathogenesis of dementia of the adult. This analysis is fundamental to explore phenotypic heterogeneity. Furthermore, by using brain tissue obtained at autopsy as well as from paraffin embedded material, we will define the molecular genetics of individual cases. Specifically, we will use technology that allows us to recognize mutations described in genes currently known to cause AD and other dementias. We consider that this multidisciplinary approach required for an ADC Neuropathology Core to provide diagnosis at the histological, biochemical and DNA level, taking full advantage of the collected material.

The coexistence of PrP amyloidosis with neurofibrillary tangles is the central pathologic feature of Gerstmann-Straussler-Scheinker disease (GSS), in the Indiana Kindred (IK). This large kindred has been the object of extensive neurological, neuropathologic, biochemical, genetic, and molecular investigations. GSS in the IK (GSS-IK), is a dominant disorder characterized by ataxia, Parkinsonism and dementia, and is caused by a mutation at codon 198 of the PRNP gene. During the past three years, we have expanded our studies of PrP amyloidosis with neurofibrillary tangles and found that the co-existence of these two lesions is present in other genetic forms of GSS. We propose to study the prodromal stage, the evolution, and the natural history of GSS-IK in individuals at-risk, by examining them neuropsychologically, by eye movement studies, and by positron emission tomography. Extensive neuropathologic analysis on ten autopsy cases will be carried out by analyzing the amyloid and pre-amyloid deposits and their distribution in the central nervous system. We will correlate this neuropathologic data with biochemical studies directed to further characterize the amyloidogenic peptides and the pathologic chaperone proteins involved in prion protein (PrP) amyloidosis. We will search for regional differences in PrP isoforms that may correlate with pathologic topography. Furthermore, neurofibrillary tangles will be studied and compared with those of Alzheimer disease in an attempt to define the abnormal phosphorylation sites in NFT of GSS. Finally, to further our understanding of the possible mechanisms whereby PRNP mutations lead to disease, we will study PrP expression and processing at the cellular level.

The Indiana Alzheimer Disease Center is established on the facilities of Indiana University School of Medicine. It will comprise six cores: an Administrative Core, a Clinical Core, a Neuropathology Core, an Education and Information Transfer Core, a National Cell Repository, and a Genetic Counseling and Testing Core. The strength of the Indiana ADC lays in its multidisciplinary faculty, that has continued significantly in the fields of Psychiatry, Neurology, Neuropathology, Medical and Molecular Genetics, and in basic neuroscience. The Indiana ADC interacts with other multidisciplinary clinical and research programs at Indiana University, the graduate programs in Medical Neurobiology and Genetics, and the Geriatric Education Center. The Core Leaders have an established record in medical care and research related to Alzheimer Disease (AD) and Related Disorders; they have a long history of collaborative interactions with each other. The Indiana ADC has developed and will continue to foster community relationships and educational programs in cooperation with the Alzheimer's Association, Central Indiana Chapter (AA Chapter) and other community agencies. The major strengths of the Indiana ADC are represented by the clinical studies of AD patients within the African American Community of Indianapolis, the clinical and neuropathologic studies of familial forms of AD and other hereditary degenerative adult onset dementias, the trials for new drug therapy for AD, the availability of the National Cell Repository for genetic research on AD, the Educational activities, and the newly developed Genetic Counseling and Testing Core. The Indiana ADC will continue promoting and strengthening existing and new research endeavors in AD and related dementias by facilitating research interactions, sharing knowledge of faculty and their laboratory facilities for the study of AD, offering educational opportunities to faculty and students of Indiana University School of Medicine.

DESCRIPTION (provided by applicant): The Indiana Alzheimer Disease Center (IADC) is established on the facilities of Indiana University School of Medicine. It will comprise five cores: an Administrative Core, a Clinical Core, a Neuropathology Core, an Education and Information Transfer Core, and a National Cell Repository Core. The Director and three Associate Co-Directors of the Center are Dr. Bernardino Ghetti (Distinguished Professor of Pathology & Laboratory Medicine, Psychiatry, Medical & Molecular Genetics, Neurology), Dr. Hugh C. Hendrie (Professor of Psychiatry) and Dr. P. Michael Conneally (Distinguished Professor of Medical & Molecular Genetics and Neurology). The strength of the IADC lays in its multidisciplinary faculty that has contributed significantly in the fields of Psychiatry, Neurology, Neuropathology, Medical and Molecular Genetics. The IADC interacts with other multidisciplinary clinical and research programs at Indiana University, the graduate programs in Medical Neurobiology and Genetics, and the Indiana University Center for Aging Research. The Core Leaders have an established record in medical care and research related to Alzheimer disease (AD) and related dementias, with particular emphasis on hereditary dementias. The IADC has developed and will continue to foster community relationships and educational programs in cooperation with the Alzheimer?s Association, Central Indiana Chapter and other community agencies. The major strengths of the IADC are the clinicopathological and molecular studies on atypical and hereditary dementias, the clinical studies of AD patients within the African-American community of Indianapolis, the trials for new drug therapy for AD, the availability of cell lines from families affected by familial AD and other hereditary dementias, and the educational activities. The IADC will continue to support research endeavors in dementia by facilitating research interactions, sharing knowledge of faculty and their laboratory/facilities, and providing an environment to faculty, graduates and students of Indiana University School of Medicine that generates interest in dementia research.