Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Summary

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Abstract

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., α-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Introduction

Over the last two decades, C. elegans has been widely used as a model organism to investigate the molecular mechanisms of necrotic cell death. C. elegans offers an exceptionally well-characterized and mapped nervous system, transparent body structure and a diverse repertoire of genetic and imaging methods to monitor in vivo cellular function and survival throughout ageing. Thus, several C. elegans genetic models of neurodegeneration have been already developed to assess neuronal viability. In particular, well-described and used nematode models include the hyperactive ion channel-induced necrosis^1,

Protocol

1. Necrotic cell death-induced by hyperactive ion channels

NOTE: Gain-of-function mutations in the gene family of degenerins, including mec-4 and deg-3 among others, results in the generation of hyperactive ion channels triggering necrotic cell death of six touch receptor neurons required for mechanosensation in worms³. Necrosis induced by the aberrant stimulation of degenerins displays several mechanistic and morphological similarities to excitotoxicity.......

Discussion

Here, we introduce and describe widely accessible methodologies for growth, synchronization and microscopic examination of some versatile C. elegans models investigating age-dependent neurodegeneration. Particularly, we assess and dissect the cellular and molecular underpinnings of age-related neuronal breakdown by using hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity^1,2,3,

Materials

Name	Company	Catalog Number	Comments
Agar	Sigma-Aldrich	5040
Agarose	Biozym	8,40,004
AM101: rmsIs110[prgef-1Q40::YFP]	Caenorhabditis Genetics Center (CGC)
Calcium chloride dehydrate (CaCl2∙2H2O)	Sigma-Aldrich	C5080
Cholesterol	SERVA Electrophoresis	17101.01
deg-3(u662)V or deg-3(d)	Caenorhabditis Genetics Center (CGC)		Maintain animals at 20 °C
DIC microscope (Nomarsky)	Zeiss		Axio Vert A1
Dissecting stereomicroscope	Nikon Corporation		SMZ645
Epifluorescence microscope	Thermo Fisher Scientific		EVOS Cell Imaging Systems
Escherichia coli OP50 strain	Caenorhabditis Genetics Center (CGC)
Greiner Petri dishes (60 mm x 15 mm)	Sigma-Aldrich	P5237
image analysis software	Fiji		https://fiji.sc
KH2PO4	EMD Millipore	1,37,010
K2HPO4	EMD Millipore	1,04,873
Magnesium sulfate (MgSO4)	Sigma-Aldrich	M7506
mec-4(u231)X or mec-4(d)	Caenorhabditis Genetics Center (CGC)		Maintain animals at 20 °C
Microscope slides (75 mm x 25 mm x 1 mm)	Marienfeld, Lauda-Koenigshofen	10 006 12
Microscope cover glass (18 mm x 18 mm)	Marienfeld, Lauda-Koenigshofen	01 010 30
Microsoft Office 2011 Excel software package	Microsoft Corporation, Redmond, USA
Na2HPO4	EMD Millipore	1,06,586
Nematode growth medium (NGM) agar plates
Nystatin stock solution	Sigma-Aldrich	N3503
Peptone	BD, Bacto	211677
Phosphate buffer
Sodium chloride (NaCl)	EMD Millipore	1,06,40,41,000
Standard equipment for preparing agar plates (autoclave, Petri dishes, etc.)
Standard equipment for maintaining worms (platinum wire pick, incubators, etc.)
statistical analysis software	GraphPad Software Inc., San Diego, USA		GraphPad Prism software package
Streptomycin	Sigma-Aldrich	S6501
Tetramisole hydrochloride	Sigma-Aldrich	L9756
UA44: Is[baIn1; pdat-1α-syn, pdat-1GFP]			Upon request: G. Caldwell (University of Alabama, Tuscaloosa AL)