Automating Aggregate Quantification in <em>Caenorhabditis elegans</em>

Abstract

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissuespecific polyglutamine (polyQ) tracts exhibit age-and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegansbased image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Oh my gut! Is the microbial origin of neurodegenerative diseases real?

“…Transgenic C. elegans that express fluorescent polyQ reporters exhibit quantifiable aggregates in response to proteotoxic stress ( 71 , 162 , 188 , 189 ). Using these strains as sensors capable of detecting changes in polyQ aggregation has allowed for the discovery of microbes and microbial products that affect cellular proteostasis ( 71 , 72 , 190 ). P. aeruginosa was among the microbes that robustly disrupted host proteostasis and led to polyQ aggregation ( 71 ).…”

C. elegans as an Animal Model to Study the Intersection of DNA Repair, Aging and Neurodegeneration

“…Systems like this allow us to determine parameters like locomotion, lifespan and behavior ( SenGupta et al, 2021 ). Other groups have developed methods to automatize the quantification of aggregate formation ( Molenkamp et al, 2021 ; Vaziriyan-Sani et al, 2021 ) or the number of head thrashes ( Zhang et al, 2022 ); both methods that have been described previously as traditional ways to analyze ND models ( Figure 5 ). The continuous development and improvement of the existing methods and the automatization of traditionally laborious assays will allow for more precise and wide analysis; doing possible the inclusion of new variables or scopes in this kind of assays.…”