ROCKEFELLER UNIVERSITY, THE
Grant: $603,457 - National Institutes of Health - Jun. 15, 2009
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Award Description: Alzheimer's disease (AD) is a neurodegenerative disorder that leads to profound cognitive decline and eventually death. There are no effective treatments or preventative measures available, and the incidence and prevalence of the disease are increasing. New insights and tractable therapeutic targets are sorely needed. Genetic evidence indicates that a major cause of AD is the production of the amyloid-beta (A-beta) peptide, which is proteolytically derived from the amyloid-beta precursor protein (APP). The A_ peptide can oligomerize and be deposited as extracellular plaques in the brain and blood vessels, but the mechanism of how it leads to neuronal death is not known. There is increasing evidence of a vascular contribution in AD: patients suffer from brain hypoperfusion, the cerebral vasculature is damaged, and abnormal hemostasis is present. Circulatory deficiencies could therefore play an important role in the pathogenesis of this disease. During the last grant period, we demonstrated an increase in blood brain barrier (BBB) permeability and neurovascular damage in AD mice, and we showed that fibrin(ogen) deposition potentiates these processes. We have now found that A-beta binds to fibrinogen and has a dramatic effect on fibrin clot formation. Clots formed in the presence of A-beta have an abnormal structure and are resistant to degradation by fibrinolytic enzymes. Therefore, in the presence of A-beta, any fibrin deposits formed would be more persistent and would exacerbate BBB damage, neuroinflammation, and neuronal death. In keeping with the known genetic interaction between AD and the ApoE genotype, we have also demonstrated that the different ApoE isoforms differentially affect the structure of the fibrin clot formed in the presence of A-beta. To further study the role of A-beta in fibrin clot formation, we will combine in vitro and in vivo techniques to analyze and characterize the interaction between A-beta and fibrinogen. The role of the various ApoE isoforms in this process will also be examined. The proposed experiments will define the role of fibrin(ogen) in AD and could lead to new therapeutic strategies for preventing or retarding progression of the disease.PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) affects a large and growing portion of the population and has been studied for over a century, yet there are few available therapies to aid in the loss of cognition and no effective preventative measures. We have found that the build-up of fibrin in the brains of AD mouse models is deleterious and contributes to their cognitive decline. The proposed studies will provide a new way to understand how this disease progresses from the perspective of disrupted blood flow to the brain, which will also explain how the nature of the fibrin clot is altered in the presence of amyloid-beta to allow for this persistence and how other genes can influence progression.
Project Description: Fibrinogen, the inactive precursor of fibrin, is a large molecule of 340 kDa. We examined which fibrinogen domains may be involved in the interaction with A©¬. Identification of the regions of fibrinogen that interact with A©¬ will be critical in defining the nature of the complex. In order to refine the functional element within fibrinogen responsible for A-beta binding, we examined whether A-beta42 or A-beta40 binds fragment D or fragment E of fibrinogen. These are not only major proteolytic fragments of fibrinogen via plasmin cleavage, but also play a very important function in fibrin clot formation. During clotting, fibrinopeptide release via thrombin exposes A and B 'knobs' in the central region of fibrinogen (fragment E), and these exposed 'knobs' interact with beta or psi 'holes' in the terminal region of fibrinogen (fragment D). Pull-down assays with fragments D and E were performed using biotinylated A-beta42 or A-beta40. A-beta42 interacts with fragment D, but A-beta40 has very low affinity for this fragment. Fragment E did not bind with either A-beta42 or A-beta40. To confirm the result from our pull down assay, we also analyzed the interaction between A-beta42 and two fibrinogen domains using ELISA. This ELISA consists of adding biotin-labeled A-beta42 to streptavidin-coated multiwell plates at room temperature. After washing, fragment D or E is added to the plate wells, and the amount of fragment D or E bound to A-beta42 is detected by an HRP-conjugated anti-fibrinogen antibody. We observed an A-beta42-dependent increase in absorbance, consistent with the peptide's binding to the fragment D. However, in case of fragment E, there is no A-beta42-dependent increase.
Jobs Summary: Professor, Postdoctoral Associate, Research Assistant Professor, Laboratory Manager (Total jobs reported: 3)
Project Status: Less Than 50% Completed
This award's data was last updated on Jun. 15, 2009. Help expand these official descriptions using the wiki below.
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