Clinical data suggest an association between elevated levels of Hcy, also known as hyperhomocysteine-mia (HHcy) and stroke. HHcy is generated due to increase in de-methylation of methionine by S-adenosine- homocysteine hydrolase (SAHH) and a decrease in methyltetrahydrofolate reductase (MTHFR) and cystathionine-y-lyase (CSE, an enzyme responsible for Hcy metabolism to H2S, a most potent vasodilator, antioxidant and anti-hypertensive agent) contribute to mitochondria dysfunction (mitophagy) and ischemic stroke. Cytochrome-C transports electrons and facilitates mitochondrial bioenergetics. Interestingly, during HHcy, cytochrome-C becomes homocysteinylated (N-Hcy-cyt-c). However, it's consequence to mitophagy and stroke is unclear. The long-term goal of this project is to understand the mechanism of mitophagy, mitochondrial repair and permeability in brain vasculature during I/R injury. Our preliminary studies suggest that during I/R ,total Hcy levels increases, causes N-Hcy-cyt-C , increases mitochondrial matrix metalloproteinase-9 (mtMMP-9), in-part degradation of mt-matrix (connexin and tight junction protein, TJP) which led to mitophagy and permeability in brain vasculature. Interestingly, THC decreases Hcy level and mitigates brain damage. Tetra hydro-curcumin (THC), a major herbal antioxidant and anti-inflammatory agent, has shown to protect brain against I/R injury. The central hypothesis of this proposal is that HHcy contributes to mitophagy mediated brain damage through N-Hcy-cyt-C in part, by increasing oxidative stress, mtMMP-9, degrades connexin-43 and TJP (Figure 1). The treatment with THC, CSE gene and SAHH shRNA gene transfer attenuates mitophagy and permeability. We will test this hypothesis by following three specific aims:
Specific Aim #1 : To determine whether the Hcy contributes to mitophagy, in part by inducing oxidative stress, exacerbating homocysteinylation of cytochrome-c in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy mitigates these changes.
Specific Aim #2 : To determine whether the homocysteinylation of cytochrome-c activates mt-MMP-9, disruption of collagen/elastin ratio, mtCxn43 and mt-tight junction proteins in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy ameliorate.
Specific Aim #3 : To determine whether Hcy alters mitochondrial (mt) bioenergetics and cerebro-vascular remodeling in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy alleviate. These studies will demonstrate the novel mechanism of cerebrovascular remodeling and have therapeutic ramifications for mitochondrial repair in cerebral ischemic stroke.

Public Health Relevance

These studies will delineate the mechanisms of mitophagy and cerebrovascular remodeling in brain vasculature. The treatment with dietary herbal antioxidant tetrahydrocurcumin, naked CSE DNA and SAHH shRNA gene therapy mediated stabilization of mitochondrial membrane potential by inhibition of mitochondrial DNA methylation;oxidant and proteolysis stress via novel mechanistic pathways will be powerful therapeutic strategy for preventing ischemic stroke.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-VH-J (02))
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Reid, Diane M
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University of Louisville
Schools of Medicine
United States
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Kamat, Pradip K; Kalani, Anuradha; Tyagi, Suresh C et al. (2015) Hydrogen Sulfide Epigenetically Attenuates Homocysteine-Induced Mitochondrial Toxicity Mediated Through NMDA Receptor in Mouse Brain Endothelial (bEnd3) Cells. J Cell Physiol 230:378-94
Kalani, Anuradha; Kamat, Pradip K; Kalani, Komal et al. (2015) Epigenetic impact of curcumin on stroke prevention. Metab Brain Dis 30:427-35
Kalani, A; Kamat, P K; Chaturvedi, P et al. (2014) Curcumin-primed exosomes mitigate endothelial cell dysfunction during hyperhomocysteinemia. Life Sci 107:1-7
Kamat, Pradip K; Kalani, Anuradha; Kyles, Philip et al. (2014) Autophagy of mitochondria: a promising therapeutic target for neurodegenerative disease. Cell Biochem Biophys 70:707-19
Kalani, Anuradha; Tyagi, Alka; Tyagi, Neetu (2014) Exosomes: mediators of neurodegeneration, neuroprotection and therapeutics. Mol Neurobiol 49:590-600
Kalani, Anuradha; Kamat, Pradip K; Givvimani, Srikanth et al. (2014) Nutri-epigenetics ameliorates blood-brain barrier damage and neurodegeneration in hyperhomocysteinemia: role of folic acid. J Mol Neurosci 52:202-15
Kamat, Pradip Kumar; Kalani, Anuradha; Tyagi, Neetu (2014) Method and validation of synaptosomal preparation for isolation of synaptic membrane proteins from rat brain. MethodsX 1:102-107
Kalani, Anuradha; Kamat, Pradip K; Voor, Michael J et al. (2014) Mitochondrial epigenetics in bone remodeling during hyperhomocysteinemia. Mol Cell Biochem 395:89-98
Kalani, Anuradha; Kamat, Pradip K; Familtseva, Anastasia et al. (2014) Role of microRNA29b in blood-brain barrier dysfunction during hyperhomocysteinemia: an epigenetic mechanism. J Cereb Blood Flow Metab 34:1212-22
Vacek, Thomas P; Kalani, Anuradha; Voor, Michael J et al. (2013) The role of homocysteine in bone remodeling. Clin Chem Lab Med 51:579-90

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