Ken A. Witt, Ph.D. - US grants

DESCRIPTION (provided by applicant): Ischemia associated pathologies (cardiac arrest, stroke, asphyxiation, etc.) significantly contribute to death and long-term disability in the United States. However, little is understood in regards to how the ischemic event induces a breakdown and increased permeability of the blood-brain barrier (BBB). Although hypoxia associated with stroke and cardiac arrest has been reported to cause increased BBB permeability, effects of re-oxygenation on brain endothelial cells following a hypoxic insult remains unclear, and what is known revolves solely around in vitro modeling. The hypothesis of this proposal is hypoxia / re-oxygenation induces significant alterations in the expression and localization of junctional and cytoskeletal proteins leading to increased BBB permeability. This proposal expands previous work on hypoxic insult to the BBB by focusing on functional, biochemical and molecular changes in brain endothelial cells during hypoxia / reoxygenation, in a whole animal model. Changes in BBB permeability, alterations in cytoskeletal I tight junction proteins, and distribution I localization of those proteins will be examined in this pathology. Data from this research will demonstrate how alterations in the cytoskeletal framework and junctional proteins are related to functional changes (i.e. paracellular permeability) that occur during hypoxia I re-oxygenation. The insight into the cellular mechanism, which occur over the time course of the pathology, will contribute to the development of alternative therapeutic strategies for the treatment of ischemia and associated brain alterations.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is associated with specific pathological features, the most prominent being extracellular deposits of amyloid b-peptide (Ab). The accumulation of Ab in the brain results in the formation of plaques, leading to down-stream neuronal death and dementia. A promising avenue for treatment of AD is through the use of selective somatostatin receptor (SSTR) agonists. Somatostatin has been shown to be decreased in humans suffering from AD and specifically regulates neuronal neprilysin activity, a potent Ab-degrading enzyme. Additionally, the somatostatin receptor subtype-4 (SSTR4) is highly expressed in neocortex and hippocampus, areas significantly affected by Ab accumulation in AD patients. We hypothesize that SSTR4 agonists will increase neprilysin activity in the brain leading to decreased Ab concentrations, enhancing learning and memory. The objectives of this grant are to evaluate our novel SSTR4 agonists in their ability to mitigate the decline in memory and learning observed in SAMP8 and transgenic APPswe mice, as well as the corresponding alterations in neprilysin and Ab. This examination has three aims. (1.) Evaluate brain uptake and regional distribution of our novel compounds within the brain. This will allow us to assess the potential viability of respective compounds to adequately enter the brain, establish which regions of the brain they get to, as well as establish appropriate dosing ranges for subsequent aims. (2.) Based on aim-1, selected SSTR4 agonists will be evaluated for learning and memory behavior in mouse models which display learning and memory deficits associated with increased Ab accumulation. This will allow us to identify treatment associated changes in learning and memory retention. (3.) After behavioral evaluations, molecular analyses will be performed on extracted brain tissues to determine the effect of agonist treatment on changes in Ab, SSTR4, amyloid precursor protein (APP) and neprilysin (NEP). These aims will allow us to directly compare the behavioral outcomes with molecular identifiers of AD pathology, with regards to treatment with our SSTR4 agonists. Taken together these analyses will provide new information into the use of SSTR4 agonists for the treatment of AD, as well as shed additional light on the A2 hypothesis. This study addresses priorities of both the NINDS and NIA. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is associated with elevated levels of Amyloid-b peptide (Ab), which acts as a pathological trigger culminating in neuronal death and dementia. A promising avenue for treating AD is in the use of selective and stable somatostatin receptor (SSTR) agonists, which may act to reduce Ab through enhancement of enzymatic degradative processes. This research will evaluate our novel SSTR agonists as to their ability to mitigate Ab concentrations in the brain in conjunction with learning and memory retention, using established models and assessments of AD pathology.

? DESCRIPTION (provided by applicant): A critical property of the blood-brain barrier (BBB) is the unique complex of tight junctions (TJs) that exist between adjacent endothelial cells, which impede paracellular diffusion and help maintain brain homeostasis. Numerous lines of evidence implicate BBB TJ dysfunction as a primary contributor to central nervous system (CNS) disease. While it has become increasingly evident that pro-inflammatory high-fat Western diets contribute to CNS disease, the impact of such diets on the BBB TJs has not been fully elucidated. Likewise, the potential benefit of omega-3 (?3) polyunsaturated fatty acids (PUFAs) on BBB TJ regulation also remains unclear. The goal of this examination is to elucidate the effects of dietary fats on BBB TJ integrity and protein regulation, respective to inflammatory contributions. This study is divided into two aims: 1) Determine the effect of Western vs. omega-3 based diets on BBB TJ integrity and protein regulation over time. Evaluations of BBB TJ protein regulation (microvascular isolate) and paracellular integrity (permeability marker) corresponding with high and low fat diets, both with and without primary ?-3 PUFAs (EPA and DHA), will be assessed over 14- and 28-weeks of dietary treatment (C57BL/6 mice). Supportive evaluations will assess primary dietary variables, inflammatory components, and PPAR? alterations. 2) Determine the effect of Western vs. omega-3 based diets on BBB TJ integrity and protein regulation against an acute inflammatory challenge. Herein, 14-week dietary treatments will be run against an acute inflammatory challenge via lipopolysaccharide (LPS), followed by evaluation sets identified in aim-1. Assessing BBB TJs at key inflammatory time-points, post-LPS injection, will allow for the delineation of BBB TJ susceptibility and resilience respective to dietary treatments. This exploratory study will provide novel insight into the impact of dietary fas on the health and resilience of the BBB TJs, as well as a greater understanding of inflammatory influences. Additionally, it will serve as the foundation for future examinations respective to BBB regulation and disease outcomes. The aims of this study address priorities of National Institutes of Health, respective to the National Institute of Neurological Disorders and Stroke (NINDS), National Institute on Aging (NIA), National Center for Complementary and Alternative Medicine (NCCAM), and the Office of Dietary Supplements (ODS).

? DESCRIPTION (provided by applicant): The goal of this proposal is to advance an orally bioavailable first-in-class somatostatin receptor subtype-4 selective drug candidate with disease-modifying attributes for the treatment of Alzheimer's disease (AD). The hit compound has been validated to enhance learning and memory in mouse models of AD and cognitive decline, with a reduction of beta-amyloid (Aß) oligomer levels within the brain. Our program has now advanced to lead optimization, which is accomplished through enhancement of drug-like properties of the lead series in tandem with well-defined advancement criteria, respective to modeling, in vitro screens and in vivo/ex vivo validation. This study will be accomplished via three specific aims. Based on outcomes within each aim, additional design adjustments and testing may be performed. Aim-1: Design and synthesis. Rational drug design strategies employing iterative in silico modeling, synthesis, and structure-activity relationship (SAR) studies will be conducted to enhance potency and selectivity, reduce potential toxicity, and enhance physiochemical properties for oral bioavailability. Synthetic methods will be further developed and applied to iterative and parallel medicinal chemistry where appropriate. Aim-2: In vitro screens. Using established in vitro methods, critical properties (i.e. solubility, receptor affinity and selectivity, activity, plasma binding, stability, permeability, and toxicity potential will be assessed in a sequential manner to delineate compound viability. Aim-3: In vivo/ex vivo assessments. Compounds meeting necessary criteria will be advanced to pharmacokinetic evaluations (i.v. and p.o.) for further delineation and identification of a primary lead. This lead will be tested via chronic p.o. administration in the 3xTg mouse model of AD at age-dependent intervals that coincide with critical periods of neuropathological development and learning/memory decline. Dosing range will be determined from pharmacokinetic data. Delineation of lead compound impact on learning and memory will be assessed via Morris water-maze, T-maze, and object recognition testing. Following final behavioral testing, cortical and hippocampal tissues will be evaluated to delineate changes in critical proteins/enzymes associated with AD pathology and proposed drug mechanism. This study will culminate in the advancement of a first-in-class AD drug candidate to the next stage of development. The aims of this study address priorities of National Institutes of Health, specific to drug discovery for nervous system disorders respective to drug-candidate lead optimization: FOA number: PAR-13-048.