1993 — 1997 |
Leblanc, Andrea C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
App Processing in Human Cerebral Primary Cultures @ Case Western Reserve University
Alzheimer's disease (AD) is characterized by the presence of cerebral intraneuronal fibrillary tangles, extracellular neuritic plaques and amyloid fibril-laden vessels. The amyloid fibrils in these three pathological lesions are composed of an abundant amount of a highly insoluble 39-42 amino acid peptide called the beta-amyloid protein (betaAP) which is encoded by the amyloid precursor protein (APP) gene. The primary structure of the APP predicts a single transmembrane domain with a long N-terminal extracellular domain and a short cytoplasmic C- terminal domain. The betaAP domain is an internal peptide which starts in the extracellular domain 99 amino acids from the C-terminus residue and extends for 11-14 amino acids within the transmembrane domain. Proteolytic processing of the APP at amino acids 15-17 of the betaAP domain generates a secreted N-terminal peptide and a cellular C-terminal peptide therefore precluding the formation of the 39-42 amino acid amyloidogenic peptide. Processing of the amyloid precursor protein through the endosomal/lysosomal pathway generates a series of C-terminus peptides, some of which contain full length betaAP and are therefore potentially amyloidogenic. This pathway may be a key element in the formation of betaAP. However, since amyloid precursor protein is expressed in most cells but large amounts of betaAP occurs only in the CNS, there must be specific elements in the CNS which determine betaAP accumulation. In this proposal, we will study amyloid precursor protein post-translational proteolytic processing in the major cell types of the CNS such as astrocytes, neurons, and microglia from human cerebral cortex. Three specific aims are proposed: 1) To compare APP processing in the various cell types of the rat cerebral cortex as well as in meninges in order to identify which cell may have the most potential for the formation of betaAP and to compare with that of human non-AD and AD cerebral cortex to identify differences in processing between human and rat and between human non-AD and AD cerebral cortex. 2) To determine if a shift from the secretory pathway to the endosomal/lysosomal pathway leads to betaAP production. 3) To identify differences in APP processing with age in rat and human cerebral cortex.
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0.958 |
2002 — 2005 |
Leblanc, Andrea C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
17 Beta-Estradiol Induced Caspase Inhibitory Factor
DESCRIPTION (provided by applicant): The primary goal of this application is to isolate a caspase inhibitory factor induced by 17-beta-estradiol in primary cultures of human neurons. A group of mammalian cysteinyl caspases is activated in a cell-, insult- and species-specific manner during apoptosis of various cell types. In human neurons, caspase-6 is active during serum deprivation-mediated neuronal apoptosis. We have previously shown that caspase-6 activity is lethal to human neurons in culture. Now, we find that 17 -beta-estradiol but not 17-alpha-estradiol, testosterone, or epitestosterone delay caspase-6 mediated neuronal cell death (Zhang et al. 2001). 17-beta-estradiol-treated neuronal extracts directly inhibit recombinant active caspase-6 in an in vitro assay. In contrast, 17-beta-estradiol does not induce CIF nor prevent caspase-mediated cell death in astrocytes. We conclude that 17-beta-estradiol induces a caspase inhibitory factor (CIF) that is preventing neuronal apoptosis. CIF is induced through estrogen receptors via a non-genomic pathway. We show that CIP is a broad spectrum caspase inhibitor between 10 and 14 kDa in size that is fairly resistant to boiling and proteinase K in neuronal extracts. Our results indicate that 17-beta-estradiol induces a novel inhibitor of active caspases and provide an additional mechanism for the neuroprotective action of 17-beta-estradiol. In this proposal, the primary goal is to identify CIF and determine its role in neuronal survival and cell death. In aim #1, we will biochemically isolate and sequence CIF. In aim #2, we will clone CIF cDNA and obtain antibodies. We will then confirm the role of CIF in neuronal survival and against caspases (aim #3), and determine the mode of activation of CIF (aim #4) and inactivation of caspase-6 (aim #5). Finally, we will study the regulation of CIF expression in normal and AD brains (aim #6). Given the strong epidemiological link of estrogen against Alzheimer's disease and its possible prophylactic role in neuroprotection, our results suggest a novel mechanism of action of 17-Beta-estradiol that could be exploited to promote neuroprotection in injury or neurodegenerative diseases.
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0.958 |
2002 — 2006 |
Leblanc, Andrea C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Prion Protein in Neuronal Survival
DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this application is to identify the function of prion protein (PrP) in the central nervous system. Four octapeptide repeats in the N-terminus of PrP are homologous to the BH2 domain of Bcl2 family of proteins. Based on preliminary observations that PrP interacts with pro-apoptotic Bax proteins and protects yeast and primary human neurons against Bax-mediated cell death, the investigator proposes the hypothesis that interaction between PrP and Bax mediates neuronal survival, and that this mechanism may be implicated in prion disease associated neuronal loss. This hypothesis will be tested in the following Specific Aims. Aim 1 will determine if PrP interaction with Bax is necessary for the neuroprotective function of PrP. PrP mutants will be generated that prevent PrP-Bax interaction and determining if co-expression of these mutants abolishes PrP's ability to inhibit Bax-mediated cell death. The function of these mutants will be tested in yeast by using galactose inducible constructs and micro-injection of eukaryotic expression constructs in human neurons. Aim 2 will determine if human PrP mutations associated with prion diseases dysregulate PrP-Bax interaction and PrP neuroprotective function. Mutations of PrP that are known to cause disease will be tested for their ability to interact with Bax and protect yeast or neurons against Bax-mediated cell death. Aim 3 will determine the location of neuroprotective PrP. The functional location of PrP and Bax will be determined by using mutant PrP and protein trafficking blocks. Primary cultures of human neurons will be used to examine protein localization by subcellular fractionation, beta-galactosidase complementation, pulse-chase experiments, immunofluorescence and confocal microscopy, and immuno-EM. Aim 4 will determine the molecular mechanism of neuronal protection by PrP. Specific Aim 5 will determine if PrP interacts with other pro-apoptotic proteins. In Aim 6, the neuroprotective role of PrP and the possible elimination of this function in PrP mutations associated with familial prion diseases will be assessed in mice models of familial prion diseases. The result of these experiments will clearly define the neuroprotective role of PrP against Bax mediated cell death.
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0.958 |
2009 — 2010 |
Leblanc, Andrea C |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Phosphorylation of Prion Protein as a Novel Mechanism For Conversion
DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine if phosphorylation is involved in normal or pathological prion protein (PrP) biology. Endogenously expressed normal cellular prion protein converts into a proteinase K resistant (PKRES) and fibrillar form when neurons in brain are exposed to prion transmissible material or express a familial mutant form of prion protein. The reason for this conversion is unclear. We show here new preliminary evidence that purified recombinant human, mouse and hamster prion protein can be phosphorylated by the neuronal specific and proline-directed cyclin dependent kinase-5 (cdk5) in vitro. A fragment of the phosphorylated prion protein becomes resistant to proteinase K as it often does in prion diseases. Furthermore, electron microscopy shows the formation of fibrillar and globular aggregate structures in phosphorylated prion protein. Therefore, phosphorylation could be a logical mechanism for the conversion of prion protein observed in disease states and could even be involved in the transmissible nature of the prion protein. While in vitro phosphorylation is easily tested, it is much more difficult to determine if phosphorylation of a specific protein occurs in vivo. Therefore, the goal of this exploratory application is to determine if phosphorylation exists in vivo. We will assess phosphorylation with a three-prong approach: using phospho-columns and western blot analyses, generating phospho-PrP specific antibodies to assess phospho-PrP by western blotting or immunohistochemistry, and immunoprecipitating prion protein from gamma-32P- orthophosphate-labeled cells. We will perform these techniques in systems known to be sensitive to transmissible prions and capable of generating protease-resistant prion protein: normal or mutant prion protein- transfected mouse neuroblastoma N2A cell lines, and human and mouse brains affected by prion diseases. Our preliminary evidence indicates cdk5-independent phosphorylation of PrP in N2A cells and in scrapie- infected mouse brains. Therefore, another goal of this application is to determine which other kinases are involved in prion protein phosphorylation, whether phosphorylation of prion protein at different sites generates identical or different protease resistant fragments, and whether phosphorylated prion protein is transmissible or induced by scrapie exposure. If positive, the result of the present application would not only add novel and potentially important mechanisms to the current state of knowledge of prion protein conversion in vivo, but could have a very high impact on how we view and treat prion diseases. PUBLIC HEALTH RELEVANCE: Prion diseases are transmitted through the conversion of the normally inoffensive prion protein of a host (human, bovine, Elk) into a disease-causing protein. The mechanism for the conversion of this protein is unknown. The present application examines one possible mechanism that would explain the conversion of prion protein and offer new avenues for the prevention or treatment of prion diseases.
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0.958 |