PROJECT SUMMARY Vascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia along with Alzheimer’s disease (AD) and approximately 60% of patients with AD also present with VCID. Despite its common clinical manifestation, the mechanism underlying VCID remains elusive. Based on extensive data analysis in humans and animal models, a specific family of proteinases that degrade essential components of the blood brain barrier (BBB), in particular matrix metalloproteinase 9 (MMP9), were identified as being strongly associated with progression of VCID. MMP9 degrades tight junctions between endothelial cells and the anchoring proteins located at the astrocytic end feet. I hypothesize that upregulation of MMP9 results in the dissociation of perivascular astrocytes from their vessels, leading to BBB dysfunction and the progression toward VCID. To test this hypothesis, I propose in vivo studies in a hyperhomocysteinemia (HHCy) mouse model of VCID in both wild type (WT) and MMP9 null mice (Aim 1) alongside astrocyte-specific, in vitro studies using WT and MMP9 null primary astrocytes (Aim 2). We have shown that induction of HHCy in WT mice is associated with microhemorrhages, reduced blood flow, neuroinflammation and cognitive impairment indicating its relevance in recapitulating the clinical manifestations of VCID. Preliminary data shows our HHCy model of VCID induces decreased contact of astrocytic end-feet with the vasculature. Aim 1 will assess the role of MMP9 in the progression of VCID pathology in response to HHCy in vivo by examining changes in neurovascular coupling and associations between astrocyte end feet and the cerebrovasculature using two- photon imaging. Aim 2 will investigate the astrocyte-specific role of MMP9 in disruption of the BBB. Using primary cells obtained from both WT and MMP9 null mice, I will use trans-endothelial electrical resistance measurements (TEER) in an astrocyte/endothelial cell co-culture model to assess BBB integrity in response to HHCy. By elucidating the cell specificity and mechanism of MMP9 mediated vascular pathology we can systematically target the various stages of disease progression and ultimately prevent the progression of VCID. Through completion of this project, I will receive training in both in vitro and in vivo models of VCID, which, coupled with my previous training in human subjects will expose me to all aspects of translational research. Additionally, I will regularly present data at both international and national meetings, I will be involved in experimental design and ethics workshops and will gain experience mentoring junior scientists. These experiences, coupled with the mentorship of the renowned scientists on my committee, will ensure that I am well prepared to successfully secure a post-doctoral fellowship in the field of neurodegeneration.