Methods to Study Changes in Inherent Protein Aggregation with Age in <em>Caenorhabditis elegans</em>

Abstract

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans

“…C. elegans DCD146 expressing RHO-1::Venus, DCD242 expressing KIN-19::HisAvi and DCD243 expressing RHO-1::HisAvi were grown to confluency on high growth medium plates and bleached to obtain a synchronized population of worms as previously described (Sulston, 1988). L1s were transferred into a liquid culture with complete S basal supplemented with OP50-1 (OP50 with Streptomycin resistance) and grown at 20°C or 25°C (to induce sterility of DCD242 and DCD243) as previously described (Groh et al, 2017a; Groh et al, 2017b). At day 1 of adulthood (DCD146) or at day 7 of adulthood (DCD242 and DCD243), worms were allowed to sediment in a separation funnel and washed with cold M9.…”

Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report

“…59 Protein homeostasis in aging A decline in protein homeostasis and the resulting protein aggregation is a feature of both normal aging and age-related neurodegenerative diseases. [61][62][63] Della C. David from Eberhard Karls Universität Tübingen presented work on understanding the mechanisms that regulate proteostasis with the hope that preventing aggregation can promote healthy aging. David focused on protein aggregation control mechanisms in the intracellular and extracellular space in C. elegans.…”

“…A decline in protein homeostasis and the resulting protein aggregation is a feature of both normal aging and age‐related neurodegenerative diseases 61–63 …”

Quantification of Insoluble Protein Aggregation in <em>Caenorhabditis elegans</em> during Aging with a Novel Data-Independent Acquisition Workflow

“…While studying the insolublome has been invaluable in progressing the understanding of the aging process, it has been hindered by the requirement for collecting large amounts of starting sample material. Groh et al recently introduced a label-free proteomic quantification workflow to study inherent protein aggregation changes in C. elegans with aging; however, it required large amounts of starting material (350 mg of ground worms) 12 . In the present report,…”