SPECIFIC AIMS Protein misfolding and aggregation is a common thread behind many neurodegenerative diseases, including AD, Parkinsons disease (PD), Frontal Temporal Dementia (FTD), Lewy Body Dementia (LBD), and Huntingtons disease (HD) among others. While each disease has been primarily associated with aggregation of a specific protein; beta-amyloid (A) with AD, alpha-synuclein (a-syn) with PD and LBD, tau with various tauopathies including FTD, and huntingtin with HD, more than one protein is likely to misfold and aggregate in brain tissue complicating diagnosis and treatment strategies. While all these proteins can form fibrillar aggregates, they can also form a variety of different smaller soluble aggregate structures as well, and increasing evidence implicates small soluble oligomeric forms of these different proteins as the relevant toxic species in the various diseases rather than the fibrillar aggregates that serve as diagnostic hallmarks. Since cellular stress induced by misfolding and aggregation of one protein such as A may well lead to misfolding and aggregation of other proteins such as tau and a-syn, the presence of multiple misfolded proteins in different diseases should be expected. Therefore characterizing which aggregated protein species are present at different stages of each disease would greatly facilitate identification of suitable biomarkers and development of better diagnostic and treatment strategies for these neurodegenerative diseases. We have developed novel capabilities to isolate monomeric and oligomeric forms of recombinant tau isoforms and to isolate and oligomerize tau from AD brain tissue. We have also developed novel technology that enables us to isolate single chain antibody fragments, or nanobodies, that selectively bind specific morphologies of a target protein. We have isolated nanobodies that selectively recognize several different oligomeric forms of A or asyn and showed that the nanobodies recognize aggregates present in diseased but not healthy tissue [1-7]. Our long term goal is to develop selective reagents that can identify which protein variants of tau are specific for human AD brain tissue and which of these tau species represent the best diagnostic and therapeutic targets. In this proposal we will separate out naturally occurring tau species present in well characterized human AD and control brain tissue, and use these samples to generate antibody based reagents (nanobodies) that selectively bind AD derived tau variants. We will isolate nanobodies that selectively bind AD derived tau variants but not healthy brain variants. We will characterize the binding specificity of the selected nanobodies and identify those nanobodies that have the most promise as diagnostics for AD. Our hypothesis is that generation of specific tau variants is an early event in AD, and that this process can be detected and at early stage to facilitate diagnosis of AD. SPECIFIC AIMS To achieve our goal of developing nanobodies that specifically and selectively recognize the most relevant tau aggregate species associated with AD, we have devised the following specific aims: Aim 1: Immunoprecipitate total tau from age matched post-mortem human AD and cognitively normal brain tissue. Aim 2: Generate and characterize nanobodies that selectively recognize tau variants present in human AD brain tissue but not cognitively normal samples. Aim 3: Validate that tau specific nanobodies distinguish between CSF and brain tissue samples from additional AD and control sources. Identification of tau forms involved in AD and other neurodegenerative diseases, in particular identification of species that are useful for early diagnosis and staging of tauopathies and other neurodegenerative diseases will provide valuable biomarker tools to facilitate early and accurate diagnoses of disease onset and progression, and identify potential therapeutic targets. The morphology specific nanobodies developed here will be valuable reagents to detect specific tau morphologies for diagnostic tests or to follow disease progression, or as reagents to specifically target toxic tau species for intracellular and extracellular therapeutic applications. While several reagents already exist that can recognize monomeric and phosphorylated tau, these reagents cannot distinguish between different aggregated states of tau. The nanobody reagents we will develop here can be used either extra- or intracellularly to identify or target specific tau morphologies involved in the onset and progression of AD. The reagents can also be used in conjunction with other reagents recognizing specific morphologies of A and a-syn to characterize the concentration profiles of these markers in CSF and/or serum samples from patients with various neurodegenerative diseases and to monitor the progression of these diseases.
|Effective start/end date||9/30/13 → 6/30/16|
- HHS: National Institutes of Health (NIH): $431,464.00