Identification of Protein Biomarkers for Aging and Alzheimer's Disease

Alzheimer’s disease is a leading cause of dementia and aging is a strong risk factor. This study will identify the proteins that go awry in aging and Alzheimer’s disease, using an advanced technology that can screen thousands of proteins at a time. The ultimate goal of the project is to identify new protein biomarkers of aging and Alzheimer’s disease and provide new treatment targets.

Maintaining the proteome integrity is essential to organismal health, stress survival and lifespan. This is achieved by the proteostasis network, of which the major components are molecular chaperones for protein folding, refolding and targeting for degradation and the degradative machineries. It is hypothesized that the proteostasis is challenged by a metastable subproteome that is constantly at risk for misfolding, premature degradation and dysfunction. Proteome mismanagement represents a feature of aging biology that contributes to the basis of cellular failure common to all degenerative diseases including Alzheimer’s disease. However, there is a lack of systematic knowledge on which proteins are metastable during aging and whether proteome instability is accelerated by mutant Tau. By identification of the specific proteins that become metastable and aggregate in aging, I propose to establish the first quantitative view of the cascade events of folding failure of metastable proteins during aging and develop new sensors to test the genetic approaches to reset the proteostasis network in aging to suppress AD and related dementias. The results of these studies will provide new insights on metastability as an inherent property of aging that determines which proteins misfold, and amplifies the proteotoxicity of mutant Tau in AD.

Aim 1. To determine how mutant Tau relates to age-dependent changes in proteome metastability and aggregation in C. elegans.

Aim 1.1. To employ LiP-TMT-MS2 proteomics to identify age-dependent metastable proteins in WT animals. (1st – 9th month)

Aim 1.2. To test whether mutant Tau accelerates proteome instability in aging. (4th – 18th month)

I have used C. elegans as the animal model system to determine whether protein metastability is a predictable feature of proteome mismanagement in aging that foreshadows the subsequent risk for age-associated protein aggregation and failure in AD. To obtain a global measure of proteome instability in a living animal, I have optimized limited proteolysis coupled with tandem mass tag-based quantitative proteomics (LiP-TMT-MS2) to identify the metastable sub-proteome that subsequently converts during aging to insoluble aggregates (collaboration with Profs. D. Finley and S. Gygi of Harvard Medical School). These tools will be used to establish the effects of chronic expression of mutant Tau and polyQ in neurons at different ages on global proteome instability. These studies will identify the characteristics of sub-proteome metastability that can be used to predict which proteins fail with aging and AD and related dementias.

Aim 2. To develop new endogenous folding sensors to monitor proteostasis status of WT and mutant Tau expressing animals using live animal imaging.

Aim 2.1. To generate fluorescently tagged endogenous folding sensors in WT and mutant Tau expressing animals. (10th – 20th month)

Aim 2.2. To use genetic approaches to reset proteostasis to prevent mutant Tau-induced protein instability. (20th – 24th month)

I will complement global proteomics analysis with direct live animal imaging by using CRISPR-cas9 system to fluorescently tag a set of endogenous folding sensors identified by LiP-TMT-MS2. These include prion-like proteins pqn-22 and pqn-53 that are metastable in day-1 WT, and aggregate in mutant Tau and polyQ expressing animals. These experiments will provide precise spatial and temporal information on the folding failure of sensors. These sensors will then be used to test genetic approaches to restore the decline of the aging proteostasis network, and thus prevent the effects of mutant Tau on proteome instability. This will be carried out by overexpressing stress response pathways regulators daf-16 and hsf-1 that are known to functionally decline in aging and AD and related dementias.

Using structural proteomics, this study is the first to explore the large-scale folding metastability of the proteome in an organismal model of aging and Alzheimer’s disease.

It will provide the most comprehensive mechanistic insights into how the organism fails in maintaining its proteome folding stability under the proteotoxicity caused by mutant Tau. This project will provide a rich resource of protein metastability and develop new endogenous proteostasis sensors that will be valuable to the field of AD and a wider community of neurodegenerative diseases and protein folding.