The objective of our proposed research is to develop a water-soluble molecule that can reduce the levels of amyloid beta (A?) and Drp1 and prevent abnormal interactions between A? and Drp1 in Alzheimer s disease (AD)-affected neurons. Our previous AD studies found: 1) increased production and accumulation of A? and increased expression of Drp1, and 2) an abnormal interaction between A? and Drp1 in AD neurons that was associated with synaptic dysfunction, mitochondrial (mt) damage, and dysfunction of neurons affected by AD. A therapeutic strategy for AD may involve treating AD-affected neurons with molecules designed to reduce A? and Drp1 levels and to inhibit the interaction of A? and Drp1. Very few molecules have been developed to prevent AD, but those that are the most promising are insoluble in water, rendering them problematic treatments due to water insolubility. Therefore, in this project we are proposing to develop a water-soluble molecule capable of reducing A? and Drp1 levels in AD neurons and of inhibiting the interaction between A? and Drp1. To achieve this objective, we have developed technology and have designed and produced 82 molecular crystal structures, including DDQ (diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonate). We selected DDQ for the proposed research to reduce A? and Drp1 interaction at specific interacting sites in the A?-Drp1 complex and exhibits the best docking capabilities of all 82 structures and received the best docking score; DDQ also readily bound to Drp1 independently and while Drp1 interacted with A?. We synthesized DDQ by following retro-synthesis analysis and analyzed its structure spectrally. We tested DDQ in AD-affected neurons, using different methodologies, and measured the production of A? and expression of Drp1 after DDQ docked with Drp1. Our preliminary studies, which included 1) human cells incubated with A? and treated with DDQ and 2) further transfected human cells with mutant APP cDNA and treated with DDQ, and characterized A? and DDQ treated cells and mutant APP transfected and DDQ treated cells for mRNA (using qRT-PCR), A?-Drp1 interactions (using Co-IP, western blot analysis, immunocytochemistry) and ultrastructural changes (using electron microscopy). These studies revealed that DDQ reduced A? and Drp1 levels as well as A?-Drp1 interactions and protect AD-affected cells from synaptic and mt toxicities. The current application seeks: 1) to determine the blood brain barrier-crossing properties and pharmacokinetics of DDQ in wildtype mice, 2) to measure cognitive behavior in the DDQ- treated and -untreated APP transgenic mice (Tg2576 line), and 3) to determine the protective effects of DDQ in terms of reducing the interaction of A? and Drp1. The outcome of our application will determine the drug-delivery characteristics of DDQ and whether it is capable of targeting and binding to AD neurons and protecting them from A?- and Drp1-induced toxicities. The proposed research will also provide new information about DDQ for future AD clinical trials.
Public Health Relevance Statement: Project Narrative The elevated levels of A? and increased expressions of mitochondrial fission protein Drp1, and abnormal interactions between A? and Drp1, have been found to induce synaptic dysfunction and mitochondrial oxidative damage, causing neuronal damage in Alzheimer s disease (AD) neurons. The primary objective of the proposed research is to develop the water-soluble drug molecule DDQ, which, based on our preliminary studies, appears to be capable of reducing the A? and Drp1 levels, and of inhibiting interactions between A? and Drp1. In our proposed experiments, we will determine whether DDQ enhances neuronal function and survival in AD-affected neurons, and through pharmacokinetic and and animal studies of AD, we will characterize drug properties of DDQ, including whether DDQ can cross the blood brain barrier, which will indicate whether DDQ is a promising drug molecule that may slow AD progression.
Project Terms: abeta accumulation; Abeta synthesis; Active Sites; Adverse effects; Affect; Age; Alzheimer's Disease; Amyloid beta-Protein; analog; Animals; base; Behavior; Binding; Binding Sites; Biochemical; Biological Assay; Blood - brain barrier anatomy; Body Weight; Brain; brain tissue; Breeding; Cells; Characteristics; Clinical Trials; Co-Immunoprecipitations; Cognitive; Complementary DNA; Complex; Crystallization; Data; data warehouse; Dendritic Spines; design; Disease Progression; Docking; dosage; Dose; Drug Delivery Systems; Drug Kinetics; Dynamin; Dynamin I; Electron Microscopy; Exhibits; experimental study; Functional disorder; Future; Genetic Crosses; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Heterozygote; High Pressure Liquid Chromatography; Human; immunocytochemistry; Immunofluorescence Immunologic; Immunohistochemistry; Impaired cognition; In Vitro; in vivo; Incubated; Individual; inhibitor/antagonist; innovation; Intention; intraperitoneal; Intraperitoneal Injections; Knockout Mice; Label; Laboratories; Lead; Link; Mass Spectrum Analysis; Measures; Messenger RNA; Methodology; methylphosphonate; Mitochondria; Molecular; Molecular Structure; mouse model; Mus; mutant; Neurons; Outcome; oxidative damage; Patients; Peptides; Permeability; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology Study; Phase; Plasma; prevent; Procedures; Production; Property; protective effect; protein complex; Protein Isoforms; Proteins; Publishing; Quantitative Reverse Transcriptase PCR; Research; Safety; Site; Small Business Technology Transfer Research; small molecule inhibitor; Solubility; Structure; Synapses; tau-1; Techniques; Technology; Testing; Tg2576; Therapeutic; therapeutic target; Toxic effect; Transgenic Mice; Water; Western Blotting; Wild Type Mouse
