Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Summary

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Abstract

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington’s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Introduction

Protein homeostasis (proteostasis) is defined as the cellular ability to maintain proper function and folding of the proteome. The inherent challenge to proteostasis is ensuring all proteins are properly folded and maintained in a native conformation, which is further amplified by the varied nature of protein size, amino acid composition, structural conformation, stability, turnover, expression, sub-cellular compartmentalization, and modifications¹. Proteostasis is maintained through the coordinated action of a large proteostatic network, consisting of approximately 2000 unique proteins, which regulate proper synthesis, folding, trafficking, an....

Protocol

1. Preparation of reagents

1. Select genes of interest to be inactivated via feeding-based RNAi. Purchase stocks of HT115 E. coli containing the RNAi clone of interest²⁰. Alternatively, subclone the cDNA of the gene of interest into the multicloning site of the L4440 plasmid.
   NOTE: To prevent degradation of dsRNA within the bacteria, use the HT115 strain. This is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. For proteostasis studies that do not use feeding-based RNAi, either HT115 or OP50 E. coli on standard NGM plates can be used.
2. Prepare 5 x 6 cm plates p....

Results

In C. elegans, the polyglutamine repeat model has been instrumental for the identification of genes that regulate the proteostatic network. For instance, we previously showed that the homeodomain interacting protein kinase (hpk-1), a transcriptional cofactor, influences proteostasis during aging by regulating expression of autophagy and molecular chaperones³¹. We found that loss of hpk-1, either by RNAi silencing or in hpk-1(pk1393) null mutant animals, increase.......

Discussion

Aging is characterized by a gradual decline in proteostasis. Proteostasis is maintained by a complex system, the proteostatic network, for the coordinated, dynamic, stress-responsive control of protein folding, degradation, and translation. Why proteostasis fails in the course of aging is poorly understood, but a decaying epigenome, declining inducibility of stress responses, and loss of compensatory crosstalk all coincide with this breakdown. In C. elegans, the transcriptional inducibility of multiple forms of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 Well Culture Plates	Greiner Bio-One	#662102
2 mL 96-well plates	Greiner Bio-One	#780286
600 µL 96-well plates	Greiner Bio-One	#786261
96-pin plate replicator	Nunc	250520
Air-permeable plate seal	VWR	60941-086
bacteriological agar	Affymetrix/USB	10906
bacto-peptone	VWR	90000-368
C. elegans RNAi clone library in HT115 bacteria- Ahringer	Source Bioscience	C. elegans RNAi Collection (Ahringer)	See also Kamath et. al, Nature 2003.
C. elegans RNAi clone library in HT115 bacteria- Vidal	Source Bioscience	C. elegans ORF-RNAi Resource (Vidal)	See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.
FuDR (5-Fluoro-2'-deoxyuridine)	Alfa Aesar	L16497
Glass microscope cover slips	VWR	48404-455
Glass microscope slides	VWR	160004-422
IPTG (isopropyl beta-D-1-thigalactopyranoside)	Gold Bio	12481C100
Retangular non-treated single-well plate, 128x86mm	Thermo-Fisher	242811
Sodium Azide, CAS #26628-22-8	Sigma-Aldrich	S2002
Zeiss Axio Imager M2m microscope with AxioVision v4.8.2.0 software	Zeiss	unknown
Zeiss StemiSV11 M2 Bio Quad microscope	Zeiss	unknown