The blood-brain barrier (BBB) restricts the passive diffusion of many drugs into the brain and constitutes a significant obstacle in the pharmacological treatment of central nervous system (CNS) disorders. Approximately 98% of new small-molecule drugs and almost 100% of large-molecule drugs such as recombinant proteins and gene-based medicines with proven CNS activity have BBB penetration problems. Therefore, our long term goal is to elucidate the regulatory mechanisms associated with the maintenance of the BBB function as a prerequisite to the development of protocols that can be used to facilitate therapeutic drugs entering the CNS for the treatment of neurological disease. We hypothesize that low concentrations of a prototypic BBB disruptor (the lipophilic cholinesterase inhibitor, chlorpyrifos, CPF) facilitate passage of exogenous substances across the BBB by short-term (temporary and reversible) alteration of BBB integrity. The hypothesis is based on our following observations from our preliminary studies: 1). low, nontoxic concentrations of CPF and its metabolites transverse the BBB. 2). CPF and its metabolites contribute to the alteration of BBB integrity and structure, as evidenced by altered tight junction proteins and electric resistance in vitro. 3). CPF disrupts BBB tight junction protein and transien receptor potential channels gene expression in a short-term and reversible way in vitro. Based on these observations, the focus of this R03 proposal is on the molecular and biochemical mechanisms by which the CPF affects the disruption of the BBB in vivo. There are 2 specific aims in the proposal: (1) To determine the time-related effects of low dose CPF treatment on the alteration of BBB-tight junction (BBB-TJ) proteins. (2) To identify the signaling pathways involved in the disruption of BBB-TJ following low dose CPF treatment. The methods to be employed will include three levels of experiments to achieve our goal and to prove our hypothesis. (1) At the functional level, Evan blue dye will be used to evaluate BBB disruption. (2) At the cellular level, Western blot, immunofluorescence assays, ELISA, and enzymatic activities will be used to analyze BBB protein profiles after CPF treatment. (3) At the molecular level, quantitative real-time PCR will be used to verify BBB protein level changes correlating to gene changes. In addition, MR imaging technique will be used to evaluate brain water content after CPF treatment. The data obtained from the proposed studies will significantly advance our understanding of the underlying molecular mechanisms associated with the maintenance of BBB function, and also will be very useful for devising strategies associated with use of low dose, safe lipophilic compounds as an effective pretreatment to allow therapeutic drug delivery to the CNS for treatment of Alzheimer's disease, Parkinson's disease, stroke, schizophrenia, epilepsy and other conditions of the CNS.

Public Health Relevance

The blood-brain barrier functions to keep foreign substances out of the brain. This is beneficial, as passage of nutrients can occur without passage of certain toxicants. This is also detrimental as the barrier prevents useful drugs from entering the brain to treat diseases (e.g. brain cancer, Alzheimer's disease, etc). There is significant value in determining conditions that can transiently and reversibly change the blood-brain barrier, as the information may be used to develop more effective methods for drug delivery to brain for treatment of brain diseases.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Small Research Grants (R03)
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Gene and Drug Delivery Systems Study Section (GDD)
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Bosetti, Francesca
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Virginia Polytechnic Institute and State University
Veterinary Sciences
Schools of Veterinary Medicine
United States
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