Biochemical characterization of physiological age-dependent aggregates in C. elegans

Abstract

Aging is the most important risk factor for neurodegenerative diseases associated with pathological protein aggregation such as Alzheimer’s disease. Although aging is an important player, it remains unknown which molecular changes are relevant for disease initiation. Recently, we and others demonstrated that several hundred proteins become highly insoluble with age, in the absence of disease. But how these misfolded proteins aggregating with age affect neurodegenerative diseases is not known until today. Importantly, several of these aggregation-prone proteins are found as minor components in disease-associated aggregates such as amyloid-β plaques or neurofibrillary tangles. In this thesis we demonstrate that insoluble protein extracts from aged Caenorhabditis elegans or aged mouse brains are able to seed amyloid-β aggregation in vitro, whereas protein aggregates formed during the early stages of life did not initiate amyloid-β aggregation. The injection of insoluble protein extracts from aged mouse brains into the hippocampus of APP23 transgenic mice lead to a formation of some amyloid-β plaques in three out of five mice. By mass spectrometry analysis of insoluble protein extracts from C. elegans we found late-aggregating proteins that were previously identified as minor components of amyloid-β plaques and neurofibrillary tangles such as 14-3-3, Ubiquitin-like modifier-activating enzyme 1 and Lamin A/C, highlighting these as strong candidates for cross-seeding. Double-transgenic worms overexpressing human amyloid-β in the body-wall muscle together with PAR-5 (C. elegans homolog of 14-3-3) showed an increase in paralysis, which demonstrates that PAR-5 (14-3-3) could be a potential seed for the aggregation of amyloid-β. In conclusion, the results presented here show that physiological protein aggregation with age might constitute a heterologous seed for disease-associated protein aggregation. To find out why these heterologous seeds form with age would have a major impact in advancing our understanding of aging and its influence on pathophysiology. Targeting the seeds before the onset of the disease would be an important prevention strategy.

Recently, widespread protein aggregation as a common feature of aging has become an important topic of research. We already demonstrated that cross-seeding between different age-dependent aggregating proteins is possible in the absence of disease. The investigation of endogenous age-dependent protein aggregation could give insights into molecular and cellular mechanisms that regulate protein aggregation and into the effect of protein insolubility on organisms health. Therefore, we wanted to analyze whether rapidly-aggregating proteins can act as harmful seeds for the aggregation of other proteins in C. elegans. The goal was to crosslink a rapidly-aggregating protein together with its co-aggregating proteins, to purify them and to identify them by mass spectrometry. This thesis presents the establishment of a tandem affinity purification under denaturing conditions to be used to purify rapidly-aggregating proteins tagged with a tandem affinity tag.