Linkage-specific ubiquitylation patterns as highly sensitive markers for neurodegenerative disease

点击翻译按钮获取中文摘要

While sensitive imaging tools to detect β amyloid and tau proteins and other biomarkers for established Alzheimer’s disease (AD) and other neurodegenerative diseases are available, similar tools for detecting pre- symptomatic, etiologic biomarkers of these diseases are not. Among the earliest steps in AD is a failure of the ubiquitin proteasome pathway to respond to misfolded proteins by causing their degradation, thereby promoting aggregation. This early pathological event is marked in part by changes in protein ubiquitylation patterns in neuronal cells. “Ubiquitylation patterns” encompasses multiple ubiquitin chains and lysine positions, and this information is far more complex than a simple ubiquitylation event. In addition, ubiquitylation patterns in neuronal cells of AD patients change as the disease progresses. No non-invasive blood test for pre-symptoms or etiology of Alzheimer ’s disease has been approved by the FDA. In addition to diagnosis, the ability to identify molecular signatures of AD could help stratify the patient population, and changes in ubiquitylation patterns could be used to monitor the clinical efficacy of drugs that target the ubiquitin proteasome system. The aim of this project is to capture the initial step in neurodegenerative disease establishment as well as stages in disease progression by identifying appropriate ubiquitylation pattern signatures that can be detected routinely in a diagnostic assay. Toward this end, in Phase I, LifeSensors developed a highly sensitive method to isolate poly-ubiquitylated proteins based on Ub chain selectivity using TUBES – an affinity matrix that can bind selectively to various distinct poly-Ub chains -- in concert with mass spectrometry (MS) to identify molecular signatures based on ubiquitylation. Conditions were simulated in neuronal cells that mimic neurodegeneration, and marker proteins were identified; in addition, methods were developed to identify low levels of ubiquitylated proteins and observe changes in ubiquitylation patterns from cerebrospinal fluid (CSF) of normal and AD patients. In Phase II, it is proposed to extend these studies to examine CSF and serum ubiquitylated protein molecular signatures from approximately 100 normal and AD patients. LifeSensors will collaborate with Drs. Nicholas Seyfried and Allan Levey of the Alzheimer’s Disease Research Center, leaders in AD proteomics, to translate the tools developed in Phase I to monitor ubiquitylation pattern changes in CSF of normal controls and AD patients and to establish biomarker panels that distinguish controls from AD patients at various disease stages, validating the markers using existing databases and predictive studies. Success in Phase II will lead to the discovery of novel ubiquitylation signatures for various stages of AD. LifeSensors will commercialize stable isotope labeled versions of these proteins to quantify the markers by mass spec in the serum and/or CSF. These simple tests will stratify patient populations to monitor efficacy in clinical trials and guide the choice and dosage of drug to treat AD.