High levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with cerebral- vascular disease, dementia, stroke, and Alzheimer's disease. The ?-amino butyric acid (GABA) stimulates the inhibitory neurotransmitter GABA-A receptor and decreases vascular dementia and stroke. The novelty of this proposal is that Hcy specifically competes with the GABA-A receptors and acts as an excitotoxic neurotransmitter. Hcy activates cerebral vascular matrix metalloproteinases (MMPs) by inducing redox stress and reactive oxygen species (ROS): The long-term goal of this proposal is to understand the mechanisms of cerebral vascular remodeling in HHcy. The hypothesis of this proposal is that Hcy induces MMPs and suppresses tissue inhibitors of metalloproteinase (TIMPs), in part, by inhibiting the GABA-A receptor. This leads to degradation of the matrix and disruption of the blood brain barrier We will test this hypothesis by three specific aims:
Specific aim #1 : To determine whether Hcy increases levels of NADH oxidase and ROS, and decreases levels of thioredoxin and peroxiredoxin by attenuating the GABA-A receptor. Levels of NADH oxidase activity and thioredoxin in brain cortex of transgenic mouse model of HHcy (cystathionine ? synthetase, CBS -/+) and GABA-A receptor null mice treated with and without muscimol (GABA-A receptor agonist) will be measured. The mRNA levels will be measured by Q-RT-PCR.
Specific aim #2 : To determine whether Hcy increases metalloproteinase activity and decreases TIMP activity by antagonizing the GABA-A receptor. Levels of MMP-2, -9, -13, and TIMP-1, -2, -3, and -4 will be measured by innovative 2-D zymography, functional proteome, Western blots and Q-RT-PCR analyses.
Specific aim #3 : To determine whether Hcy alters brain microvascular reactivity and increases permeability of brain microvessels by augmenting GABA-A receptor. Brain microvascular permeability will be measured by in vivo video fluorography, using fluorescence-labeled albumin. The vascular reactivity will be measured by a topical application of vasoactive agents. These studies will demonstrate a novel mechanism in which brain microvascular permeability changes during HHcy and vascular dementias, and will have therapeutic ramifications for microvascular disease in Alzheimer's patients.
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