SBIR-STTR Award

There is no organ in the body as dependent on a continuous supply of blood as the brain. If cerebral blood flow (CBF) is interrupted, brain function ceases within seconds and irreversible damage to its cellular constituents ensues within minutes. In elderly patients, increasing evidence suggests that vascular risk factors and lowered CBF have a major role in the development of Alzheimer's disease (AD) and possibly even trigger the onset of dementia. We have discovered an over-abundance of two interactive transcription factors in vascular smooth muscle cells (VSMC) from pial cerebral arteries of patients with AD which critically regulate CBF. The factors, serum response factor (SRF) and myocardin (MYOCD), are potent inducers of a contractile gene program linked to VSMC function. Consequently, AD-VSMC display elevated expression of several contractile proteins that effect a hypercontractile state in these cells. Based on these novel findings, we hypothesize that elevated SRF/MYOCD activity in VSMC of small cerebral arteries leads to a hypercontractile phenotype, which contributes to CBF reductions and neurovascular uncoupling in AD. In this Phase I application, we endeavor to further validate these findings and optimize a high-throughput screening assay for SRF/MYOCD-dependent gene transcription. To this end, we have delineated three milestones we are confident we can achieve as a critical foundation for a Phase II application that we have simultaneously submitted in this Fast Track proposal. Milestone 1 will assess the ability of MYOCD to elicit an AD-VSMC hypercontractile phenotype in age-matched control VSMC. Milestone 2 will ascertain whether the AD-VSMC hypercontractile phenotype can be normalized with an RNAi to SRF. In Milestone 3, we will optimize a recently developed large-scale luciferase assay for human cells in preparation for studies described in Phase II. The finding that two interactive transcription factors may underlie the known brain hypoperfusion and cognitive decline in AD represents a new paradigm for therapeutic interventions. The milestones to be addressed here will provide a vital platform for aims delineated in Phase II of this Fast Track Application in which a diverse small molecule library of compounds is to be screened for organic chemicals that specifically disrupt the SRF/MYOCD interaction thereby normalizing VSMC hypercontraction and, by extension, brain hypoperfusion and dysregulated CBF in AD.

Thesaurus Terms:
Alzheimer's disease, blood protein, brain circulation, cytoskeletal protein, gene induction /repression, microcirculation, pathologic process, technology /technique development, transcription factor, vascular smooth muscle genetic transcription, muscle contraction, neuroregulation, phenotype, reporter gene, vasoconstriction, vasoconstrictor Adenoviridae, HeLa cell, RNA interference, high throughput technology, human tissue, luciferin monooxygenase, transfection

Alzheimer's disease (AD) is a multi-factorial neurodegenerative disorder. According to the prevailing amyloid cascade hypothesis, cognitive decline and distinct pathogenic features in AD relate to abnormal accumulation of amyloid beta-peptide (A?) in the brain. While efforts are underway to enhance A? clearance, prevent its formation, or to develop anti- A? small molecules, recent evidence implicates neurovascular dysfunction and compromised cerebral blood flow (CBF) in the pathogenesis of AD. Based on published data derived from our Phase I application and novel preliminary data provided herein, we seek to pharmacologically undermine enhanced activity of two transcription factors, serum response factor (SRF) and myocardin (MYOCD), in cerebral vascular smooth muscle cells (VSMC) derived from patients with AD. SRF/MYOCD constitute a potent transcriptional switch for a VSMC contractile gene program, which is exaggerated in AD-VSMC and coincides with a hypercontractile phenotype leading to reduced CBF in mouse models of AD. Moreover, new preliminary data support SRF/MYOCD in defective A? clearance by VSMC and the expression of LRP1, which is a major mediator of A? elimination via the circulation. Together, these data lead us to formulate the following hypothesis: elevated SRF/MYOCD activity in AD VSMC leads to a hypercontractile phenotype in small cerebral arteries and the accumulation of A? which contribute to CBF reductions and neurovascular uncoupling as seen in A?, whereas drugs which specifically block MYOCD interaction with SRF will "unlock" the hypercontractile/ A? AD VSMC phenotype, improve CBF and alleviate symptoms of dementia. The specific aims designed to test this hypothesis include (1) validating novel small molecule inhibitors of SRF/MYOCD we have identified through library screening; (2) evaluating lead compounds for their ability to normalize VSMC hypercontractility and A? clearance in vitro; and (3) evaluating lead compounds for their ability to normalize VSMC hypercontractility, A? clearance, and behavioral deficits in a novel mouse model of AD phenotype we have recently developed. These innovative and highly robust studies are expected to uncover a new class of potential AD therapeutics that should be poised well for further toxicity and preclinical trials, thus providing impetus for their assessment in normalizing the cerebrovascular dysregulation and neurovascular uncoupling associated with AD and dementia. Currently, there are no effective therapies to prevent or reverse the inexorable course of Alzheimer's disease (AD) and associated dementias. A common thread amongst such neurodegenerative diseases is a compromise in blood flow to the brain. This application seeks to evaluate a potential new class of therapeutics designed to disrupt two proteins shown to be hyperactive in blood vessels of the brain of AD patients.

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