The plaques in the brain during Alzheimer's diseases (AD) are primarily composed of amyloid-b (Ab) peptide. Although a strong association between Ab, fibrinogen (Fg), and elevated level of homocysteine (Hcy), i.e. hyperhomocysteinemia (HHcy) during AD is well- documented, the mechanism of amyloid plaque formation is unclear. The long-term goal of this project is to understand the mechanisms of cerebrovascular permeability leading to amyloid plaque formation. We have shown that elevated levels of Hcy and Fg i.e. hyperfibrinogenemia (HFg) increase cerebrovascular permeability and Fg can cross vascular endothelial cell (EC) layer secondary to activation of matrix metalloproteinase-9 (MMP-9). In addition, increased levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), which enhances collagen content in subendothelial matrix (SEM), are associated with AD. In pilot studies, we found that while HHcy enhances vascular permeability through mainly the paracellular pathway, HFg activates mainly the transcellular transport. We also found that blood level of Fg is increased during HHcy. Therefore, Fg can have an additive effect in HHcy-induced cerebrovascular permeability. We found that HHcy increases collagen level in SEM and Fg and Ab depositions in brain vasculature. While HHcy increases MMP-9 activity and enhances the paracellular transport as well as homocyteinylates Fg (making fibrin clots more rigid), Fg enhances the transcellular transport through caveolin-1 (Cav-1) signaling (via phosphorylation of Cav-1). However, the role of Hcy and Fg in Hcy-Fg-Ab-collagen complex formation is unclear. The hypothesis of this proposal is that HHcy increases vascular permeability by primarily affecting the endothelial cell (EC) junction proteins and activating MMP-9, and indirectly by increasing blood content of Fg, which enhances transcellular transport leading to an enhanced Hcy- Fg-Ab-collagen complex formation. We will test this hypothesis with three specific aims: 1) To determine whether the HHcy instigates cerebrovascular permeability via paracellular transport leading to enhanced cerebrovascular crossing of fibrinogen, 2) To determine whether the fibrinogen deposition enhances cerebrovascular permeability mainly through caveolar transcytosis via Cav-1 signaling secondary to MMP-9 activation, 3) To determine whether the HHcy increases fibrinogen-Ab-collagen complex accumulation in mouse brains secondary to increasing tissue inhibitor of metalloproteinase-1. To define specific causative effects of Hcy, Fg, MMP-9, Cav-1, and TIMP-1 in Fg-Ab-collagen complex formation, the studies will be done on pial vessels of wild type (WT), Cystathionine b-Synthase heterozygote (CBS+/-) mice (a model of HHcy), MMP-9 gene knockout (MMP9-/-), CBS and MMP-9 double knockout (CBS+/-/MMP9-/-), Cav-1 gene knockout (Cav1-/-), Fg g-chain-deficient (Fg-/-), CBS+/-/Fg-/-, HFg transgenic (HFg), HFg/MMP9-/-, CBS+/-/Cav1-/-/MMP9-/-, CBS+/- /HFg, CBS+/-/HFg/MMP9-/-, TIMP-1 gene knockout (TIMP1-/-), CBS+/-/TIMP1-/- double knockout, Fg-/-/MMP1-/-, and CBS+/-/Fg-/-/TIMP1-/- mice using a newly developed dual-tracer probing method that allows separation of paracellular from transcellular transport. Formation of Fg-Ab-collagen complex in brain cortex will be assessed with immunohistochemical analysis. The results of the proposed research should uncover the molecular mechanisms regulating Hcy-Fg-Ab-collagen complex formation and lead to effective strategies for the treatment of cerebrovascular complications during diseases such as AD.

Public Health Relevance

Alzheimer's Disease (AD), is the most common form of dementia and it is not a normal part of aging. Up to 5 percent of people with the disease have early onset AD. The plaques in the brain during AD are primarily composed of amyloid-b (Ab) peptide. Although a strong association between Ab, fibrinogen (Fg), and homocysteine (Hcy) is well-documented, the mechanism is unclear. Our findings show that at elevated levels Hcy increases cerebrovascular permeability to proteins leading to Fg deposition in brain cortical vessels. We also found that this Fg is associated with increased levels of Ab and collagen in brain vasculature. These effects of Hcy and Fg were diminished in the absence of matrix metalloproteinase-9 (MMP-9) activity. The long-term goal of this project is to understand mechanisms leading to brain Fg-Ab-collagen complex formation. The knowledge of mechanisms that control the amyloid plaque formation will lead to the design of therapeutic strategies for the control of this devastating neurodegenerative disease. The proposed research is relevant to public health and to the mission of NIH because it will clarify a novel pathogenic causal relationship into the role of Hcy, Fg, and MMPs in development of AD.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Special Emphasis Panel (ZRG1)
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Corriveau, Roderick A
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University of Louisville
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