Carol A. Miller, MD - US grants

Neuronal degeneration is a major histopathologic finding in Alzheimer's disease (AD). Selective vulnerability of subpopulations of subcortical neurons has previously been described. We recently described retinal ganglion cell loss and optic neuropathy, all in the absence of neuritic plaques, neurofibrillary tangles and amyloid angiopathy. Clinically, some AD patients show changes in visual evoked responses. Although the CNS contains heterogeneous cell populations, a monoclonal antibody marker (MAb 3F12) has identified some of the vulnerable neurons in the neocortex, hippocampus, and possibly the axons of retinal ganglion cells undergoing degeneration. Little is known of the molecular specificities of these cells in normal or AD brain or retina. IN this study we propose to analyze neuronal-specific function at three levels: clinical, histologic, and molecular. For the clinical studies we shall focus on the visual system, integrating the neurologic and psychometric database with specific visual function studies. Patients will be followed to autopsy and retinal ganglion cell loss assessed. A parallel histologic assessment of auditory system neuronal changes will be made. Temporal progression of neuronal loss in AD will be examined. With use of neuron-specific monoclonal probes, the architectonic differences in neuronal changes in AD will also be compared to other dementing diseases (Pick's, Parkinson's). The AD-vulnerable neurons will be further defined by: (1) immunocytochemical identification of their associated neurotransmitter and neuropeptides, (2) molecular characterization of Ag3F12, and (3) development of a neuron-enriched cDNA library. MAb 3F12 will be used to isolate clones from a human brain cDNA expression library, localization confirmed by combined in situ hybridization and immunoperoxidase staining. Isolated 3F12 cDNA clones will be useful as markers of pyramidal cell enrichment in preparation of a new neuron-enriched cDNA library. To enhance isolation of rare or low abundance mRNA species, we shall use subtraction hybridization methods. Transcripts will be screened by Northern and Southern blot analyses, and positives by in situ hybridization in normal and AD neocortex, hippocampus and retina. Proteins and/or transcripts of interest will be sequenced to determine if they are of known molecular specificity. Development of this neuronal subset-specific molecular panel may contribute to the understanding of regulatory mechanisms operative in these cells in AD.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Neuronal degeneration is a major histopathologic finding in Alzheimer's disease (AD). Selective vulnerability of subpopulations of subcortical neurons has previously been described. We recently described retinal ganglion cell loss and optic neuropathy, all in the absence of neuritic plaques, neurofibrillary tangles and amyloid angiopathy. Clinically, some AD patients show changes in visual evoked responses. Although the CNS contains heterogeneous cell populations, a monoclonal antibody marker (MAb 3F12) has identified some of the vulnerable neurons in the neocortex, hippocampus, and possibly the axons of retinal ganglion cells undergoing degeneration. Little is known of the molecular specificities of these cells in normal or AD brain or retina. IN this study we propose to analyze neuronal-specific function at three levels: clinical, histologic, and molecular. For the clinical studies we shall focus on the visual system, integrating the neurologic and psychometric database with specific visual function studies. Patients will be followed to autopsy and retinal ganglion cell loss assessed. A parallel histologic assessment of auditory system neuronal changes will be made. Temporal progression of neuronal loss in AD will be examined. With use of neuron-specific monoclonal probes, the architectonic differences in neuronal changes in AD will also be compared to other dementing diseases (Pick's, Parkinson's). The AD-vulnerable neurons will be further defined by: (1) immunocytochemical identification of their associated neurotransmitter and neuropeptides, (2) molecular characterization of Ag3F12, and (3) development of a neuron-enriched cDNA library. MAb 3F12 will be used to isolate clones from a human brain cDNA expression library, localization confirmed by combined in situ hybridization and immunoperoxidase staining. Isolated 3F12 cDNA clones will be useful as markers of pyramidal cell enrichment in preparation of a new neuron-enriched cDNA library. To enhance isolation of rare or low abundance mRNA species, we shall use subtraction hybridization methods. Transcripts will be screened by Northern and Southern blot analyses, and positives by in situ hybridization in normal and AD neocortex, hippocampus and retina. Proteins and/or transcripts of interest will be sequenced to determine if they are of known molecular specificity. Development of this neuronal subset-specific molecular panel may contribute to the understanding of regulatory mechanisms operative in these cells in AD.

The Neuropathology Core C, serves as a focal point to address the central question of this program project: What are the pathogenic cerebrovascular mechanisms in the aging brain that determine the relationships of cerebrovascular amyloidosis to age-dependent Chronic vascular activation and injury, and to the initiation of brain injury and/or stroke? The Core will prepare post-mortem human CNS tissues and accession non-human primate CNS tissues from the animal core . Four of the 4 projects will directly receive tissues from the Core. Brains with clinically and neuropathologically confirmed Alzheimer's disease (AD), will be compared to normal, young, and aged control brains, and those with other non-AD dementias. Parallel studies will be done on aged and adult squirrel and rhesus monkey brains as non-human primate models for vascular and parenchymal amyloid deposits respectively. The amyloid-related vasculopathy will be defined as to regional distribution, vessel sizes and type. Fresh brain tissues will serve as a source of endothelial and smooth muscle cells for cultures and for ApoE isotyping. Vasculopathy with amyloid burden assessed histochemically, and immunocytochemically for Abeta peptide type, will be correlated with distribution of receptors such as gp330/megalin, a member of the LDL receptor family that binds Abeta/APO-J complex; RAGE, for Abeta binding; macrophage markers )CD11b, CD68); cell surface adhesion molecules (PECAM-1 and PAF receptors), and hemostasis markers (CPA, thrombomodulin). Electron microscopy, including immunocytochemistry with the above antibodies will identify disease-related translocations of these receptors. Our database will track tissue distribution from each patient to every investigator. Results of studies with human tissues with human tissues will be compared among the projects including project 3, which examines a murine transgenic model of RAGE. These studies are complementary. and underlie functional relationships of Abeta and the four cellular components, endothelial, smooth muscle, pericytes and astrocytes, which participate in the blood-brain barrier.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Signal-transduction pathways in the CNS provide key mechanisms for cellular responses to environmental stresses. Oxidant-stresses are operative in the aging brain with infarction and Alzheimer's disease. MAP kinases mediate these inputs to downstream targets, such as transcription factors. The result is either regeneration and protection, or cell death. DENN-MADD, a GDP/GTP exchange factor expressed in neurons has basal functions mediating release of neurotransmitters from presynaptic terminals. DENN-MADD, identified by two-hybrid screening of a human brain cDNA library using JNK3 as bait, is expressed in neurons as a 200KD protein. Functional domains include a nuclear localization signal,JNK binding domain, leucine-zipper and a death domain. Under oxidant stress conditions, DENN-MADD is translocated from the cytoplasm to the nucleolus, and the cell ultimately dies. To compare basal and stress-induced effects, we will first examine, interactions of JNK and other MAPKs with DENN-MADD, including binding and phosphorylation. Using cDNA deletion constructs transfected into neuronal cultures, mechanisms of nuclear translocation, including masking of the putative nuclear localization signal and interactions with other nucleolar-associated proteins will be examined. Second, the DENN-MADD death domain is known to bind to a death domain of the TNF receptor alpha-1, part of an apoptosis pathway. Interactions of DENN-MADD with TNFR and other death domain proteins will be compared under stress (hypoxia, A-beta). Third, effects of nuclear translocation of DENN-MADD on basal neuronal functions, specifically synaptic vesicle release will be tested to determine if stress results in reduced acetylcholine release. Fourth, possible synergistic stress effects of A-beta and hypoxia on DENN-MADD mediated cell death will be compared immunocytochemically in primary CNS tissue cultures and directly in brain tissues from AD, CNS infarction and normal, aged patients. Neurons vulnerable or resistant to oxidant stress will be compared for DENN-MADD nuclear translocation. This pivotal role of DENN-MADD provides a model for selective neuronal vulnerability in response to oxidant stress.

ABSTRACT: NEUROPATHOLOGY CORE Alzheimer's disease (AD) is multifactorial and complex and includes the impact of the cerebrovascular system on the induction and progression of the dementia. Correlation of experimental findings in disease models with the changes in the human CNS is needed at the cellular, molecular, genetic and system connectivity levels. In AIM 1, the Neuropathology Core serves these functions by providing at autopsy, CNS tissues, for accurate diagnoses and in AIM 2, individual and multiple investigators with tissues, blood and CSF for research. Contribution to a common database in AIM 3 serves to correlate neurological, psychometric, neuroimaging and behavioral data with the neuropathological and molecular findings. It also coordinates tissues, blood and cerebrospinal fluid (CSF) for national initiatives defining genomic data, such as ApoE and other risk factors, and biomarker information in AIMS 4-6, highlighting those functional in the neurovascular unit and in the blood- brain-barrier. These latter functions serve both Projects 1 and 2. In AIM 7, training and education of neuropathologists,neurologists, and neuroscientists bring AD and related dementia further into focus by defining the potential translational approaches leading to new therapies.

? DESCRIPTION (provided by applicant): Bridging an understanding of neuroanatomy from the cellular level of microns to the systems level of millimeters is both challenging and important at this time. We are uniquely poised to undertake this multidisciplinary research topic applied to the human postmortem brain, because we have two PI's: Dr. Carol Miller, M.D., with expertise in the neuropathology and cell and molecular biology of neurodegenerative diseases, such as tauopathies, especially Alzheimer's disease (AD), and FTLD-tau variants, using the new CLARITY preparation method for large tissue volume; and Kristi Clark, Ph.D., a neuroscientist with expertise in developing novel neuroimaging methods of high angular and spatial resolution diffusion MRI (dMRI). AIM 1 Examines tau protein detection and effects of axonal aggregates in the human postmortem corpus callosum in AD, and in disease controls, e.g. progressive supranuclear palsy (PSP) a tauopathy, and in neurologically normal, age-matched controls. First, tissues (~1cm blocks) are imaged with dMRI followed by three dimensional (3D) 2-photon microscopy imaging of CLARITY processed tissue immunostained for tau, phospho-tau and neurofilament. Then, we will spatially co-register the dMRI data with the 3D 2-photon microscopy and superimpose the maps to evaluate the sensitivity and specificity of several dMRI metrics for the detection of tau. In AIM 2, cross-sections of hippocampus, one of the earliest sites affected in AD, will be analyzed by dMRI models followed by CLARITY-processed tissues to determine effects of tau aggregation on neuronal connectivity. With the CLARITY protocol applied to formalin-fixed, hippocampal cross-sections, we have already achieved immunolabeling with neuronal subtype markers in 500?m tissue sections. Similarly, we have successfully acquired our dMRI acquisition on the ex vivo human hippocampus, achieving delineation of hippocampal sub-regions and the connectivity among the sub-regions. These studies could potentially lead to development of an in vivo, non-invasive imaging protocol for clinical use in the early diagnosis and monitoring of treatment responses in a myriad of neurodegenerative diseases where protein aggregates form. In future studies, companion, unfixed tissue samples could be examined for molecular correlates pinpointing changes in RNA processing and post-translational modifications, which cannot be done in disease model systems.