SBIR-STTR Award

Linkage specific ubiquitylation patterns as highly sensitive markers for neurodegenerative disease Abstract: Despite an intensive effort by the government, pharmaceutical companies and academic groups, the diagnosis and treatment of Alzheimer's disease remains elusive. The incidence of Alzheimer's disease is predicted to increase, reaching more than 10 million cases in USA by 2025. Thus, the development of quantitative, simple and reproducible diagnostic markers is extremely important. Current methods for diagnosis of Alzheimer's disease are dependent on clinical and neuropsychological assessment, cerebrospinal fluid (CSF) analysis, and brain- imaging procedures, all of which have significant cost- and access-to-care barriers. Thus the need for simple blood-based biomarkers has never been greater. We hypothesize that dysfunction in the ubiquitin proteasome pathway occurs decades before the clinical manifestation of Alzheimer's disease symptoms. Neurons generally do not divide, and their survival depends heavily on the removal of misfolded proteins. The first step in neurodegenerative disease begins with a diminished response to unfolded protein removal, and proteasomal dysfunction. Therefore, neuronal diseases are marked by the appearance or change in pattern of ubiquitylated proteins -- the main hypothesis of this proposal, which describes a unique combination of affinity purification of ubiquitylated proteins and mass spectroscopy approaches to detect ubiquitylated proteins and also determine the nature of the poly-ubiquitin chain linkage. Although the roles of the ubiquitin pathway in cell physiology and pathology have been recognized for the last three decades, surprisingly there are no reliable and sensitive methods available to monitor patterns of ubiquitylation. The development of this technology will enable clinicians to make early diagnoses of Alzheimer's disease, facilitate the discovery of disease modifying drugs, and open doors for cell biologists to rapidly identify patterns of poly-ubiquitylated proteins in tissues, cells or body fluids.

Public Health Relevance Statement:
PUBLIC HEALTH SIGNIFICANCE: The proposed studies are based on the hypothesis that changes in the pattern of ubiquitylation of certain proteins, both in extent and in the structure of the polyubiquitin chains appear early on in the onset of neurodegenerative diseases, especially Alzheimers Disease (AD). Identification of these changes by a combination of linkage specific polyubiquitin affinity-based purification matrices and high-resolution, quantitative mass spectrometry should provide lead markers for the development of diagnostic tests for the early detection of AD and new therapeutic approaches.

Project Terms:
abstracting; Affinity; Affinity Chromatography; Age; Aging; alpha synuclein; Alzheimer's Disease; Amyloid beta-Protein; Amyotrophic Lateral Sclerosis; Appearance; Autophagocytosis; Autophagosome; base; biological adaptation to stress; blood-based biomarker; Body Fluids; Brain imaging; Cell Line; Cell model; Cell physiology; Cells; Cerebrospinal Fluid; Cessation of life; Clinical; clinical efficacy; cost; Detection; Development; Diagnosis; diagnostic biomarker; Diagnostic tests; Disease; Disease Marker; disease phenotype; DNA Repair; Down-Regulation; Early Diagnosis; enzyme activity; Enzymes; Excision; Functional disorder; Generations; Goals; Government; Health Services Accessibility; Homeostasis; Imaging Techniques; Incidence; infancy; inhibitor/antagonist; Lead; link protein; Lysine; Lysosomes; Mammalian Cell; Mass Spectrum Analysis; Mediating; meetings; Methods; Molecular Chaperones; Monitor; multicatalytic endopeptidase complex; mutant; N-terminal; Nature; Neurodegenerative Disorders; Neurofibrillary Tangles; Neuronal Dysfunction; Neurons; neuropsychological; novel; novel therapeutic intervention; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; patient population; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Play; Polyubiquitin; Proteasome Inhibitor; protein aggregate; protein biomarkers; protein misfolding; protein TDP-43; protein transport; Proteins; Proteome; Public Health; Resolution; response; Ribosomes; Role; Serum; Societies; Structure; Study models; Symptoms; tau Proteins; Techniques; Technology; technology development; Tissues; tool; tool development; Ubiquitin

While sensitive imaging tools to detect ? 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.

Thesaurus Terms:
Affinity; Age; Alpha Synuclein; Alzheimer's Disease; Amyloid; Amyloid Beta-Protein; Autophagosome; Base; Binding; Biological Assay; Biological Markers; Biomarker Panel; Blood Tests; Brain Tissue; Cell Line; Cell Physiology; Cells; Cerebrospinal Fluid; Clinic; Clinical Efficacy; Clinical Trials; Commercialization; Complex; Databases; Detection; Development; Diagnosis; Diagnostic Assay; Disease; Disease Progression; Dna Repair; Dosage; Drug Targeting; Engineering; Enzymes; Etiology; Event; Failure; Fda Approved; Functional Disorder; Goals; Grant; Hyperactive Behavior; Imaging Device; Individual; Link Protein; Lysine; Lysosomes; Mass Spectrum Analysis; Mediating; Medicine; Methods; Misfolded Protein; Molecular Profiling; Monitor; Multicatalytic Endopeptidase Complex; N-Terminal; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurology; Neurons; New Therapeutic Target; Novel; Pathogenicity; Pathologic; Pathway Interactions; Patient Population; Patient Stratification; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Phase; Plant Roots; Play; Polymers; Polyubiquitin; Population; Positioning Attribute; Programs; Progress Reports; Protein Aggregate; Protein Biomarkers; Protein Tdp-43; Protein Transport; Proteins; Proteomic Signature; Proteomics; Proteostasis; Research; Response; Ribosomes; Risk; Role; Sampling; Serum; Serum Proteins; Signal Transduction; Stable Isotope Labeling; Success; Symptoms; System; Targeted Biomarker; Tau Proteins; Techniques; Technology; Testing; Tool; Translating; Treatment Strategy; Ubiquitin; Universities;