1997 — 1999 |
Fortini, Mark E |
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. |
Activity of the Alzheimers Disease Presenilin Protein @ University of Pennsylvania
DESCRIPTION: Research proposed in this application seeks to identify new members of the Presenilin protein family and analyze their functions during development of the retina. The two known human Presenilins are highly related integral membrane proteins with seven putative transmembrane segments, and mutations in the two human presenilin genes are believed to account for over 90% of early-onset Alzheimer's disease. Although the biochemical activities of the Presenilin proteins are unknown, they may regulate the intracellular trafficking or processing of other proteins, such as the B-amyloid precursor protein (B-APP), which is processed to yield the major constituent of the amyloid neuritic plaques found in the brain tissues of Alzheimer's disease patients. Recently, a C elegans Presenilin protein, termed Sel-12, has been identified through genetic screens for modifiers of the Notch/Lin- 12 signaling pathway, which regulates numerous cell fate decisions during nematode development. The applicants preliminary studies have led to the isolation of a new member of the Presenilin protein family that displays 44-52% amino acid sequence identity to the human and nematode Presenilins. Specific aims of this proposal include the determination of the complete sequence and genomic organization of this new presenilin gene, the production of antibodies that recognize the encoded protein, and the isolation of mutations that disrupt the gene. Additional goals include a detailed analysis of the subcellular distribution and function of this new Presenilin in the highly polarized neuroepithelium of the developing retina. Further studies performed in mutant and transgenic retinal tissues will seek to clarify the possible involvement of Presenilin in Notch/Lin-12-mediated signaling and B-APP processing. A more long-term goal of this research is to use genetic strategies to elucidate the molecular mechanisms involving Presenilin protein activity. Screening for modifiers of Presenilin-associated phenotypes may ultimately identify interacting proteins and increase our understanding of the biochemical causes of early-onset Alzheimer's disease.
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0.958 |
2000 — 2002 |
Fortini, Mark E |
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. |
Activity of the Alzheimers Disease Presenil in Protein @ University of Pennsylvania
DESCRIPTION (From the applicant's abstract): Research proposed in this application seeks to continue the applicant's genetic and molecular studies on Presenilin function and its role in the intracellular trafficking and proteolytic processing of its substrate proteins. Mutant human Presenilins influence the proteolysis of amyloid precursor protein (APP), resulting in an accelerated accumulation of the neurotoxic amyloid peptides during Alzheimer's disease. In the model organisms Caenorhabditis and Drosophila, Presenilins are required for Notch/Lin-12 developmental signaling. Presenilins have recently been shown to regulate proteolytic processing events during Notch receptor maturation and signaling that may be analogous to the Presenilin-dependent cleavages of APP in Alzheimer's disease. Finally, Presenilins have also been implicated in the cellular response to apoptotic stimuli in both mammalian cells and Drosophila. Mosaic tissue studies will be performed in vivo using newly isolated Presenilin gene mutants. Preliminary experiments have revealed integrin-like phenotypes in the mutant tissue clones, prompting the applicants to analyze the role of Presenilin in integrin cleavage using the genetic and biochemical approaches that have been used previously to demonstrate the effects of Presenilin on Notch processing. These studies may reveal shared feature of Presenilin substrates and lead to a better understanding of the specific pathway of protein processing controlled by Presenilin. A central goal of this proposal is to develop an extensive collection of new molecular probes to dissect Notch processing at much higher resolution than is currently possible. These reagents, including new antibodies and epitope-tagged constructs that can discriminate among Notch cleavage products, will be combined with mutational and proteolysis inhibition studies to identify the biochemical steps of Notch processing that involve Presenilin. Genetic and molecular screens for Presenilin-interacting factors will also be performed, taking advantage of the applicant's recent finding that the conserved C-terminus of Presenilin is a crucial functional domain. Finally, detailed parallel studies on the trafficking of Notch and other proteins will be undertaken in tissues lacking either Presenilin or another protein with known effect of subcellular trafficking, the SERCA-type Calcium-ATPase. These experiments are made possible by the applicant's recent isolation of Calcium-ATPase mutants, and they will address the unresolved issue of whether Presenilin is required for protein trafficking or only proteolysis. The studies proposed here will clarify the biochemical activity of Presenilin in the processing of Notch, APP and other proteins, and may ultimately increase our understanding of the molecular causes of Alzheimer's disease.
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0.958 |
2000 — 2002 |
Fortini, Mark E |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Alzheimer's Disease Related Presenilins @ University of Pennsylvania
Mutant human Presenilins influence the proteolysis of amyloid precursor protein (APP), causing an accelerated accumulation of neurotoxic amyloid peptides in Alzheimer's disease. In the model organisms Caenorhabditis, Drosophila, and the mouse, Presenilins are required for Notch/Lin-12 developmental signaling. Presenilins regulate key proteolytic processing events during Notch receptor maturation and signaling that may be analogous to the Presenilin-dependent cleavages of APP in Alzheimer's disease. Presenilins have also been implicated in the cellular response to neurodegenerative apoptotic stimuli in both mammalian cells and Drosophila. In this FIRCA application, transgenic Drosophila will be generated that overexpress both wild-type and Alzheimer's disease-associated Presenilins. The functional properties of transgenic Presenilins will be assessed by several criteria, including analysis of developmental and adult phenotypes, interactions with Notch pathway genes, effects on Notch receptor processing, and induction of apoptosis. Human Presenilins (PS1 and 2) as well as human APP will also be functionally characterized in this transgenic model. These studies will be coupled with parallel tests of the wild-type and mutant proteins in mammalian neuronal cell culture to assess the degree of functional similarity between Drosophila and human Presenilins. The possibility that Presenilin genes are transcriptionally regulated by Notch signaling output will also be tested using these models. These studies will determine if Presenilin is a component of the feedback mechanism known to operate on the Notch pathway. Finally, Drosophila Presenilin mutants and other new mutants that interact with Presenilin will be systematically examined for adult learning and memory deficits, extending the developmental analysis of these mutants that is already underway. This well-defined set of proposed studies seeks to evaluate the utility of Drosophila as a transgenic model for functional analysis of human Presenilins, and to uncover conserved activities of fly and human Presenilins in apoptosis, neurogenesis, and neurodegeneration that occur during early development as well as during later learning and memory consolidation. The studies proposed here will clarify the in vivo activity of Presenilin in the processing of Notch, APP, and other proteins, and may ultimately increase our understanding of the molecular causes of Alzheimer's disease and its accompanying memory decline in humans.
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0.958 |
2009 — 2012 |
Fortini, Mark E |
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. |
Trafficking and Proteolysis of Notch and Other Gamma-Secretase Substrates @ Thomas Jefferson University
DESCRIPTION (provided by applicant): Many integral membrane proteins have been identified as cleavage substrates for the intramembrane aspartyl protease complex termed ??secretase. These ??secretase substrates include receptors for signaling pathways, adhesion molecules, and other factors that are localized at the cell surface and whose activities are regulated by ??secretase-mediated cleavage. Understanding the molecular details of ??secretase substrate biology is important due to the prominent role that many substrates play in normal cellular physiology and in human disease. For example, the Notch receptor, a well- characterized ??secretase substrate, is essential for proper cell-fate specification and cellular differentiation during the development of animal and human tissues. Inappropriate activation of the Notch pathway in the human immune system causes T-cell leukemia and dysregulated Notch activity has been implicated in other cancers and developmental diseases. Furthermore, processing of the Amyloid Precursor Protein (APP) by ??secretase contributes directly to the production of secreted amyloid-2 peptide in human brain tissue, a crucial event in the pathogenesis of Alzheimer's disease. A key feature of ??secretase substrate cleavage is its tight control by ligand binding, ectodomain removal, and other regulatory processes. A growing body of data indicates that cleavage also depends upon intracellular membrane trafficking of ??secretase substrates and their sorting within specific trafficking compartments. In the case of both Notch and APP, ??secretase-mediated cleavage is associated with endocytosis of the substrate from the cell surface and its entry into endosomes. We have found that proper biogenesis of the early endosome compartment requires the function of an aquaporin channel protein, defining a specialized endosome route for efficient ??secretase-dependent Notch trafficking and signaling. In Specific Aim 1, we propose to characterize further the role of this aquaporin in endosome biogenesis and Notch trafficking. These studies will shed light on the cell biological aspects of ?? secretase substrate trafficking and their relationship to ??secretase substrate cleavage, recycling, and degradation. In Specific Aim 2, we propose to conduct a forward genetic screen for new factors involved in the intracellular membrane trafficking of Notch and other ??secretase substrates. Preliminary results from this screen have already proven to be very promising, including the recovery of many potentially new genes needed for various steps of Notch intracellular trafficking. Further analysis and molecular cloning of several of these new genes is now underway. The main goal of this project is to advance our understanding of ??secretase substrate trafficking and its relevance to secretase- dependent processes in tumorigenesis and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Proteins cleaved by ??secretase, including the Notch receptor and amyloid precursor protein, are important for developmental signaling and neuronal function, and their dysregulation contributes to cancer and neurodegenerative disease. This research proposal seeks to identify cellular factors that control the production, trafficking, and proteolytic processing of ?? secretase targets. These studies might lead to the identification of new proteins and intracellular pathways that could be therapeutically targeted in cancer and neurodegenerative disorders.
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0.958 |