Analysis and therapy of age-dependent proteostasis failure in neurodegeneration

神经退行性变中年龄依赖性蛋白质稳态失败的分析和治疗

• 批准号: 10474260
• 负责人: Robert Joseph Shmookler Reis
• 金额: --
• 依托单位: CENTRAL ARKANSAS VETERANS HLTHCARE SYS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474260
• 关键词: Affect; Affinity; Age; Alzheimer' s Disease; Alzheimer' s disease patient; Amyloid; Amyloid beta-42; Amyotrophic Lateral Sclerosis; Binding; Biological Assay; Brain; Caenorhabditis elegans; Cell Culture Techniques; Cells; Chemical Agents; Chemistry; Chronic; Combined Modality Therapy; Complex; Computer Simulation; Computer software; Craniocerebral Trauma; Data; Dementia; Development; Diagnosis; Disease; Disease model; Docking; Drug Design; Drug Screening; Drug Targeting; Elderly; Environmental Risk Factor; Experimental Designs; Exposure to; Failure; Free Energy; Genetic; Heart Diseases; Hippocampus (Brain); Human; Huntington Disease; Hypertension; Individual; Knowledge; Lead; Learning; Libraries; Link; Mass Spectrum Analysis; Mechanics; Mediating; Modeling; Molecular; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neuropathy; Oxidation-Reduction; Oxidative Stress; Oxides; Parkinson Disease; Pathology; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phosphorylated Peptide; Post-Translational Protein Processing; Process; Property; Protective Agents; Proteins; Proteomics; Protocols documentation; Psyche structure; RNA Interference; Rare Diseases; Reagent; Research Proposals; Resistance; Risk; Role; Sampling; Signal Pathway; Site; Substantia nigra structure; Testing; Therapeutic Agents; Tissue Banks; Tissue Sample; Tissues; Trypsin; Veterans; active duty; age related; alpha synuclein; base; brain cell; brain dysfunction; cohesion; crosslink; design; drug candidate; drug efficacy; effective therapy; experience; first-in-human; genome-wide; high risk; human old age (65+); human tissue; in silico; in vivo; in vivo evaluation; inhibitor; inhibitor therapy; insight; knock-down; novel; novel therapeutics; prevent; protective effect; protein aggregation; protein protein interaction; proteostasis; quantum; rational design; screening; simulation; small hairpin RNA; tau Proteins; therapeutically effective; tool

The previous period of Merit support allowed us to develop novel protocols for immuno-affinity isolation and characterization of very pure protein aggregates. We began with C. elegans models of neurodegenerative diseases, and then applied the same protocols to purify Aβ42- and tau-containing aggregates from affected hippocampal tissue of Alzheimer's patients, seeking aggregate proteins that differentiate them from age-matched controls. We now propose to apply our experience with these models to develop novel therapeutic agents that might delay, prevent, or even reverse Alzheimer's and Parkinson's pathology, through the following Aims: Aim 1. We have access through Dr. Sue Griffin, our collaborator, to a tissue bank of tissue samples from Alzheimer's Disease (caudal hippocampus) and Parkinson's Disease (substantia nigra), from which we will isolate aggregates by immuno-affinity for Aβ42, tau and α-synuclein. We propose to cross-link these aggregates, thoroughly digest them with trypsin, and identify cross-linked peptide pairs that will reveal the protein-protein interactions that mediated their conglomeration. We will use state-of-the-art “click chemistry” reagents to create, tag and recover cross-link sites, and Xlink Identifier software to analyze mass-spectrometry data. To visualize, integrate and interpret the protein-protein interactions thus revealed, we will create protein-interaction networks and analyze them with tools adapted to the nonfunctional interactions that predominate in protein aggregation. Aim 2. Proteomics data from Aim 1 will define critical protein-protein interactions within aggregates containing tau, Aβ42 or α-synuclein, which are highly enriched in Alzheimer or Parkinson brain samples. These protein- protein interactions will be ranked on the basis of their predicted propensity (by ΔG estimation) and stability (by molecular-dynamic simulations). The top 10 protein interfaces from each pulldown will then serve as targets for in silico screening of drug libraries developed to disrupt protein-protein interactions. Such libraries, constructed in diverse ways, include many `PPII' drugs designed to block specific protein-pair interactions, but which have frequently found applications beyond their original targets. For each target, we will perform initial screens of PPII-library drugs in silico, from which the top candidates will be retested in molecular-dynamic simulations. Aim 3. The drugs predicted in silico to most effectively disrupt key protein interactions (Aim 2) will be tested in vivo for reduction of aggregate formation―first in human neuronal-cell cultures expressing APPSwe (forming amyloid aggregates) or split-GFP::tau (fluorescing upon tau oligomerization). For drugs that are protective in either assay, tests will also be conducted in C. elegans aggregation models expressing Aβ42, tau, or α-synuclein. Biotinylated versions of 4–6 top candidates will be assessed for activity, and active biotinylated drugs will be used to pull down proteins to which the drug attaches. This will be done initially in C. elegans, using an in-house `genomewide' RNAi-knockdown library, and will then be cross-checked by introducing shRNA constructs into cultured neuronal cells. Targets producing maximal protection will then be tested to ask whether drug efficacy is partially redundant with RNAi knockdown, as expected for a target that mediates the drug's protective effects. By discovery of novel drugs designed specifically to reduce aggregation, we expect to find far more effective therapeutic and preventative agents than the limited drugs currently available. Based on our observations that many aggregate components are shared among diverse neurodegenerative-disease models, at least some of these drugs may also prove effective against a variety of other neurodegenerative diseases including orphan diseases.

前一时期的功绩支持使我们能够开发新的免疫亲和分离方案和

项目成果

Physiological Consequences of Targeting 14-3-3 and Its Interacting Partners in Neurodegenerative Diseases.

• DOI: 10.3390/ijms232415457
• 发表时间: 2022-12-07
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Ganne, Akshatha;Balasubramaniam, Meenakshisundaram;Mainali, Nirjal;Atluri, Paavan;Reis, Robert J. Shmookler J.;Ayyadevara, Srinivas
• 通讯作者: Ayyadevara, Srinivas