Matthew P. Frosch, MD PhD - US grants

Cadherins are cell surface proteins which subserve calcium-dependent cell- cell adhesion; they are responsible for many critical events in development and are modulated in important disease states including malignancy. A novel cadherin-like protein has been cloned from endothelial cells using a strategy designed to detect cDNAs containing trinucleotide repeats. The trinucleotide repeat, (CTG)5, falls within the open reading frame. Expansion of repeats such as this in other cDNAs has been implicated recently as the basis for several diseases. Overlapping endothelial cell cDNAs, corresponding to 4 kb of sequence, contain a single open reading frame with a predicted protein of 882 amino acids. Sequence analysis reveals that the protein contains 6 cadherin-like extracellular domains but it is otherwise not related to any previously identified protein. The mRNA for this protein is approximately 4.5 kb long and is expressed in a wide variety of adult human tissues. The gene for this protein has been localized to chromosome 1p12. The proposed work will study the adhesion function of this novel protein. Molecular cloning methods will be used to generate altered protein molecules in order to map the functionally important domains of this protein and to relate them to other studies on classical cadherins. Additionally, various techniques will be used to map the distribution of the protein (via Western blotting and immunohistochemistry, using antibodies to be developed) and its mRNA (via Northern blotting and in situ hybridization) in a wide variety of normal, abnormal and developing human tissues. These experiments are designed to investigate the function of this novel endothelial cell protein with probable adhesive capacity and to properly place it within the scheme of endothelial cell function in normal tissue and in disease states.

Recent advances in the understanding of certain mutations which underlie familial Alzheimer disease (AD) have opened new approaches to the development of appropriate small animal models for this neurodegenerative disorder. The characterization of the AD genetic loci on chromosomes 14 and 1 has led to the discovery of two highly related proteins, presenilin-1 (PS-1) and presenilin-2 (PS-2), in which numerous mutations have been linked to early-onset AD in some families. The P.I. has successfully generated transgenic mice which express various forms of PS-1 (including mutant and wild type proteins) under the control of the platelet-derived growth factor B-chain promotor. These mice represent an attempt to model the early-onset AD caused by these mutations and to assess the consequences of the over-expression of both wild type and mutant forms of these proteins. One major criterion by which these animals can be evaluated as an adequate model for AD is whether the transgene leads to AD-like pathologic findings. Thus, the first Specific Aim of this project is to investigate the neuropathologic consequences of the over-expression of various forms of PS-1 in the brains of these transgenic mice. These mice also have the potential to serve as a source of neuronal tissue for use in cell culture experiments. Such experiments can elucidate the consequences of these forms of PS-1 on cellular functioning and in response to a wide variety of exogenous influences. It is first critical, however, to determine when, during tissue culture, and from which brain regions the transgene is active. Thus, the second Specific Aim of this project is to characterize the expression of PS-1 transgenes in primary neuronal cultures of various brain regions from these animals. The combined outcome of these experiments will be the characterization of these animals at the intact brain level and as a potential source for cultured primary neurons. Through the detailed analysis of this model system for AD at both of these levels, it may become possible to use this a system to evaluate the effects of possible therapeutic interventions for this devastating neurodegenerative disorder.

Core C: The specific aim of this core is to develop, characterize and maintain transgenic animals which express various forms of proteins relevant to the study of presenilin biology and Alzheimer disease and to provide animals of various ages (from embryos to aged adults) to various projects within this program. Already available to this projects are several lines of transgenic mice which produce wild type and mutant forms of presenilin-1 (PS1) under the control of the platelet-derived growth factor (PDGF) B-chain promoter. This promoter has been shown in these and other animals to drive efficient transgene expression in neurons of the central nervous system. Recent studies with these animals have demonstrated that the transgene is functional, resulting in the production of human PS1 in primary neuronal cultures prepared from these animals. This observation opens the possibility for use of these animals in a wide variety of experiments which are detailed elsewhere in the application. The Transgenic Animal Core will serve to make animals available at a wide variety of ages for use in individual projects. Additionally, timed gestations will be prepared, as appropriate, so that primary neuronal cultures can be generated for similar experimental use. Over the course of the program, additional animals will be generated, incorporating new insight into the biology of the presenilins and their relationship to other processes in the cell and in the development of Alzheimer disease. The Core will serve to characterize these transgenic mice at the DNA, RNA and protein levels, in a manner comparable to that which has already been done for the PDGF B-chain PS1 animals.

This core will serve to collect, store and analyze brain tissue from: (l) patients with relevant neurologic diseases, including Parkinson disease (PD) and dementia with Lewy bodies (DLB); (2) patients with other neurologic disorders, including Alzheimer disease as well as other movement disorders; (3) patients without evidence of neurologic disease; and (4) transgenic mice which seek to model PD, DLB and other synuclein-associated neurodegenerative disorders. The first major component of the core will deal with human tissues. The core -- under the direction of a board-certified neuropathologist, Dr. Matthew Frosch -- will collect material at the time autopsy, and prepare and store samples in a manner compatible with biochemical, histologic and structural studies as considered in the various projects of the Center. The cases will then also be subjected to a detailed neuropathologic analysis, in a standardized manner, in order to ensure the accuracy of clinical diagnosis and the understanding of the pathology specific to each case. All material will be stored in an anonymized manner. The second component of the core will deal with the collection, preparation and histologic analysis of animal tissue, specifically brains from transgenic mice developed elsewhere in the Center. This portion of the core, under the direction of Dr. Cynthia Lemere, will provide experienced histologic services to the other projects of the Center. The histologic methods used in the analysis of these animals will parallel those used in the diagnosis of the human material considered above.

DESCRIPTION (provided by applicant): Cerebral amyloid angiopathy (CAA), the age-related deposition of cerebrovascular ft-amyloid (AH), is a common cause of hemorrhagic stroke, an accompanying pathology in most cases of Alzheimer disease (AD), and a potential cause of adverse responses to anti-All immunotherapeutic approaches to AD. Studies of CAA in the postmortem human brain are limited by the inability to make observations over time as the disease progresses. We have used in vivo multiphoton microscopy in mouse models of CAA to define the spatial and temporal progression of the disease in living animals. We have shown that cerebrovascular AH deposition occurs predictably with readily measured kinetics and that passive immunotherapy alters the progression of CAA and can lead to clearance of amyloid deposits from vessel walls. The consequences of CAA are associated in part with breakdown of the vessel wall. For this reason, we now propose to use novel methods to define the mechanisms that underlie Ali-induced vascular wall injury, including oxidative stress, activation of matrix metalloproteinases and other proteases and injury to vascular smooth muscle cells. In Specific Aim #1, we will perform serial imaging of CAA progression in mouse models and define the spatial and temporal relationship between amyloid deposition and markers of injury to the vessel wall. The goal of this aim will be a precise understanding of the sequence of events in the pathway that lead to vascular wall injury. In Specific Aim #2, we will examine which of the CAA-induced alterations in vessel wall injury can be interrupted or reversed by clearance of Afi using passive immunization and other methods. Because mouse models are only relevant in as much as they speak to human disease, in Specific Aim #3, we will determine which of our validated set of markers of vascular wall injury from mouse models are also found in human CAA. We will make use of our expertise in the clinical and neuropathologic characteristics of CAA and our access to a wide range of human samples (including sporadic CAA, CAA linked to APP mutations, tissue from the AN1792 trial of the Aft vaccine and familial British and Danish dementias. From these studies we will develop a clearer understanding of (1) mechanisms by which AIS deposition results in vascular wall injury and neurologic injury;(2) points suitable for therapeutic intervention;and (3) risks associated with the CAA in patients undergoing anti-amyloid therapies for Alzheimer disease.

This Core will maintain existing lines of transgenic (and other) mice and will provide animals of various ages (from embryos to aged adults) to projects within this program. The Core will also work with investigators of the Program Project to to develop new lines of mice relevant to their projects. Animals with transgenic expression of wild type and mutant PS1, animals under development expression aph-1 and pen-1, and animals lacking PS1 (embryos or neuronal cultures) will be used in Project 1 to examine effects on the levels and stability of the other gamma-secretase components (aph-1, pen-2 and nicastrin). In Project 3, these animals will be used to study the effects of these transgenes on the competition between APP and Notch as substrates for gamma-secretase and on the interactions between gamma-secretase and its substrate. In Project 5 and Project 6, these animals will be used to assess the contribution of each of these proteins to the cleavage of other gamma-secretase substrates. Design and generation of additional transgenics will be performed in collaboration with Project 6 (APLP1 and APLP2) and, potentially, with Project 5 (Nectin-1). Additionally, animal husbandry services will be provided for investigators as needed. By consolidation of efforts involving animals, the Program Project seeks to have efficient use of mice, through the coordination of requests from different investigators and sharing of the animals when possible. This will help to keep animal numbers and costs are kept as low as possible while ensuring that, within the limits of colony space, the needs of investigators in all projects are met. Animals from this Core are also made available, as possible, to investigators outside of the Program Project.

This Core will maintain existing lines of transgenic (and other) mice and will provide animals of various ages (from embryos to aged adults) to projects within this program. The Core is organized with two dominant Specific Aims: ¿ Maintaining transgenic mice relevant to the study of presenilin biology and Alzheimer disease and to provide animals of various ages (from embryos to aged adults) to investigators. ¿ Organizing and coordinating animal requirements across the individual projects to make maximal and efficient usage of this important, expensive and limited resource. Animals to be covered in this Core include locally generated mice expressing mutant (M146L) or wild type forms of human PS1, wild type human APLP2, as well as animals generated in other laboratories. The later include several transgenic models in which A_ deposition occurs as the consequence of a mutant form of APP (with or without mutant forms of PS1). These lines include the Tg2576 line of mice [over-expression of APP-Sw alone], the APPswe/PS1dE9 line of mice [with over-expression of both APPSw and the exon 9 deleted form of PS1] and the J20 line of mice [over-expression of APP-Sw,Ind ]. The Core will also work with investigators of the Program Project to develop new lines of mice relevant to their projects should these become relevant. Thus a potential third Specific Aim of the Core is to: ¿ Participate, in collaboration with the relevant investigator(s), in developing and characterizing and utilizing new mouse models. By consolidation of efforts involving animals, the Program Project seeks efficient use of animal resources, through the coordination of requests from different investigators and sharing of the animals when possible. This will help to keep animal numbers and costs are kept as low as possible while ensuring that, within the limits of colony space, the needs of investigators in all projects are met. Animals from this Core are also made available, as possible, to investigators outside of the Program Project.

Seeinstructions): The underlying philosophy of the Core remains a belief that validation of diagnosis is critical to clinical research into neurodegenerative diseases and that tissue-based studies are a critical complement to in vitro studies of the biologic process that underlying these diseases. By providing these two pillars to the clinical and scientific community, the Neuropathology Core is able to make a contribution to the amelioration of suffering associated with AD and related disorders. The Specific Aims of the Neuropathology Coreare: 1. To establish an accurate neuropathological diagnosis on all brains submitted with standardized reporting, including clinicopathological correlation and interpretation of findings, to the Clinical Core, other treating physicians andfamilies; 2. To maintain a source of brain tissue and other samples for investigators studying AD and related disorders, through preparation of tissue in a standardized manner, including determination of RNAquality, with special consideration of investigators within the Massachusetts ADRC; 3. To work with the Clinical Core to develop, store and distribute DNA, cell lines, plasma and serum collected under the Clinical Core's Biomarkers Initiative; 4. To train diagnostic and experimental neuropathologists in the neuropathology of dementing disorders; and 5. To participate in cooperative ventures with other groups studying neurodegenerative diseases, both human and animal models, including other Alzheimer Centers (ADRCs & ADCs), NACC, NINDS- supported Udall Center, as well as other consortia and individual investigators. These broad goals, which are in continuity with the historical activities of the Neuropathology Core, will enhance the value of the collected brain tissue to individual investigators whose specific research projects depend upon receiving carefully prepared and examined tissue. RELEVANCE (See instructions): Brain tissue is a critical resource for many important experiments that are defining the underlying pathology of Alzheimer disease and other neurodegenerative diseases. The ability to use the collect tissue in experiments is essential as new therapies and disease markers are developed.

NEUROPATHOLOGY CORE PROJECT SUMMARY/ABSTRACT The underlying philosophy of the Neuropathology Core is the belief that validation of diagnosis is critical to clinical research into neurodegenerative diseases and that tissue- and other biospecimen-based studies are a vital complement to in vitro studies of the biologic processes underlying these diseases. From these two pillars to the clinical and scientific community, the Core is able to make a contribution to the amelioration of suffering associated with AD and related disorders. The Core therefore supports the overall mission of the MADRC through the collection, characterization, storage and distribution of brain tissue and other biospecimens (CSF, plasma, DNA, RNA, etc) from subjects who have been well-characterized clinically. Samples from the Core have contributed to significant advances in our understanding of the biologic processes that characterize neurodegenerative diseases, both at early and later stages. The Core will serve to catalyze research into AD through collection, characterization and distribution of samples to individual investigators, larger consortia and collaborative groups and through data sharing with NACC (Aims 1-4). There are components of the Core's activities which are innovative, focused on developing novel tissue handling methods for high resolution microscopy which is important for understanding of early events in AD, on developing novel methods for imaging sharing and analysis and on harnessing opportunities provided by large epidemiologic studies to gather samples from control and early stage subjects (Aims 5-7). Finally, there is an educational component of the Core: training neuropathologists to be expert in diagnosis of neurodegenerative diseases and in the operations of tissue repositories in support of research; and teaching other health professionals about the importance of autopsy and tissue donation for research (Aims 8 & 9). The Core is integrated into all Center activities with active interactions with Administrative, Clinical and Outreach Cores regarding autopsy permission and education, with the Data Core regarding data storage and communication with NACC, and with the projects. Projects 2 and 3 both depend on neuropathological samples from the Core, and our routine evaluation of brains includes attention to the white matter, which complements Project 1's Connectome studies. We also support the local research community and national initiatives, with 309 requests for materials over the past 4 years (including >3600 frozen tissue samples and >17,000 unstained section). Thus, there is an ever-increasing role for the activities of the Core in contributing to research in AD and other dementing illnesses.

Massachusetts Alzheimer?s Disease Research Center: Neuropathology Core The Neuropathology Core serves both the Massachusetts ADRC (MADRC) and the Alzheimer research community by providing diagnostic validation of clinical assessments and providing tissue to investigators. Neuropathologic assessment has contributed to understanding heterogeneity associated with AD/ADRD, both across diseases and in terms of concurrent pathologic processes. The availability of tissue from the Core has been widely used by investigators working to accelerate toward a cure for AD/ADRC. To complete these missions, we have three sets of Aims. The first group (Aims 1-3), which are intended to marshal resources, is focused on the collection, diagnosis, collection of data, full reporting of findings ? to families and relevant physicians with a CLIA-compliant autopsy report through the Clinical Core, and to the Data Core for research purposes -- and the wide distribution of samples in support of the research community. Issues of heterogeneity are directly addressed in these efforts. The second group (Aims 4-6), which are intended to develop new strategies, incorporate innovate methods to determine the neuropathologic basis of imaging and fluid biomarkers in AD/ADRD, define the neuropathologic findings in subjects from clinical trials for AD/ADRD and develop new cellular resources from autopsy tissue including iPSCs (to be deposited at NCRAD) and isolated cell-type specific nuclei. As these three aims develop, we will be sharing insights and collaborating with other ADCs for validation and broad implementation. Disease heterogeneity will be addressed in both the assessment of the basis of biomarker signals and in the assessment of clinical trials subjects. The final group (Aims 7 & 8), which are intended to build the future, focus on training. First, there is a component of training basic and clinical researchers in aspects of the neuropathology of AD/ADRD as well as the intricacies and issues around tissue-based research. These efforts will be done in close collaboration with the REC. There are also efforts, done in collaboration with the ORE Core, in broad education about the importance of autopsy and brain donation to enhance our efforts to tackle AD/ADRD. The Neuropath Core interacts with all other components of the MADRC and with the broader scientific community to address critical problems to accelerate towards a cure for AD/ADRD.