Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Published: January 22, 2021

doi:

10.3791/61958

Zelda Z Mendelowitz*^1,2, Adem Idrizi*¹, Itzhak Mano²

¹Cellular, Molecular, and Biomedical Science, CUNY School of Medicine,The City University of New York (CUNY), ²PhD Program in Biology, The CUNY Graduate Center,The City University of New York (CUNY)

Summary

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Abstract

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::GαS(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Introduction

Excitotoxicity is the leading cause of neuronal death in brain ischemia and a contributing factor in multiple neurodegenerative diseases¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹. Disruption of oxygenated blood flow to the brain (e.g., due to a blood clot) results in the malfunction of glutamate transporters, leading to accumulation of glutamate in the synapse. This excess of glutamate over-activates post-synaptic Glutamate Receptors (GluRs) leading to an excessive (catalytic, non-stoichiometric) influx of Ca²⁺into neurons (Figure 1A). This detrimental influx leads to progressive postsynaptic neurodegeneration that morphologically and mechanistically ranges from apoptosis to regulated necrosis¹⁰^,¹¹^,¹². Although they were based on successful interventions in animal models, multiple clinical trials of GluR antagonists that sought to block Ca²⁺ entry and promote cell viability have failed in the clinical setting¹³^,¹⁴^,¹⁵^,¹⁶. A likely critical contributor to these failures is the fact that (in contrast to the animal models) treatment in the clinical setting is administered hours after stroke onset, causing the intervention to block late-acting neuroprotective mechanisms, while failing to interrupt degenerative signaling downstream of GluRs¹⁴^,¹⁶^,¹⁷. An alternative approach, which is based on thrombolysis, can only be administered within a severely restricted time window, leaving many patients (who suffer stroke at home with poorly identifiable time of onset) unable to benefit from it¹⁷. These setbacks emphasize the need to focus excitotoxicity research on the study of events occurring after GluR hyper-stimulation and differentiate subsequent degenerative cascades from concurrent neuroprotective processes. This approach can help prevent cell damage and identify efficient drug targets that can be administered later after damage onset.

One approach to identify subsequent events in excitotoxicity is to study the cell-death signaling mechanisms downstream of GluR hyperstimulation, such as those leading to mitochondrial collapse. Drastic malfunction of mitochondrial physiology and dynamics is a hallmark of neurodegeneration, as seen in excitotoxicity¹⁸^,¹⁹^,²⁰. While all cells depend on mitochondrial function and availability for survival, activity, and cellular maintenance, neurons are particularly dependent on mitochondrial energy production to support signal transmission and propagation. Specifically, neurons spend ~50% of their signaling-related energy consumption to restore resting membrane potential following the activation of postsynaptic receptors/channels²¹, with high dependence on oxygen and glucose. The reduced availability of glucose and oxygen observed in stroke leads to serious mitochondrial alterations, causing further reduction in ATP production¹⁹^,²²^,²³^,²⁴. However, studies to identify the sequence of events that lead to mitochondrial collapse produced controversial results and lacked consensus. Analyzing mitochondrial morphology can help understand these events leading to mitochondrial pathology since it is a good indicator of neuronal health²⁵^,²⁶^,²⁷^,²⁸^,²⁹. Filamentous mitochondria are representative of a healthy neuron, whereas fragmented mitochondria reveal substantial neuronal damage that could lead to cell death. Analyzing mitochondrial morphology in live animals under different genetic conditions can help focus on specific genes and pathways involved in mitochondrial-dependent neurodegeneration in excitotoxicity.

Another approach to identifying subsequent events that might regulate the extent of excitotoxic neurodegeneration is to study the transcriptional neuroprotective mechanisms that mitigate some of the effects of excitotoxicity¹⁴^,¹⁶. However, the lack of specificity of key neuroprotective transcription factors and the divergence of experimental setups impede the success of efforts to clearly identify core neuroprotective programs (especially in regulated necrosis).

Therefore, both the study of downstream death signaling pathways and the study of transcriptional neuroprotection in excitotoxicity encountered great difficulties and disagreements on observed outcomes. Much of this controversy is likely to arise from the use of ex vivo or in vitro models of excitotoxicity, and the variability introduced by the specificity of different experimental setups. It is therefore highly beneficial to focus on identifying core mechanisms that are highly conserved, and study them in vivo. The simple model system of the nematode C. elegans offers a particularly effective option, due to the potent combination of particularly powerful and diversified research tools, the conservation of core cell-death pathways, and the rich information on the structure and connectivity of its nervous system³⁰^,³¹^,³²^,³³. Indeed, the seminal work of the Driscoll lab on the genetic analysis of necrotic neurodegeneration in mechanosensory neurons is an excellent demonstration of the power of this approach³⁴. Importantly for the analysis of excitotoxicity, the conservation of signaling pathways in the nematode includes all major components of glutamatergic neurotransmission³⁵^,³⁶.

The nematode excitotoxicity model builds on these seminal studies, allowing the researcher to study biochemical processes akin to those that occur in stroke and other neurodegenerative diseases affected by glutamate-dependent neurotoxicity. To induce excitotoxic conditions in C. elegans this experimental approach uses an excitotoxicity strain that is the combination of a knockout of a glutamate transporter gene (glt-3) and neuronal sensitizing genetic background (nuls5 [Pglr-1::GαS(Q227L)]) to produce GluR hyperstimulation and neurodegeneration³⁷. This excitotoxicity strain exposes 30 specific (glr-1-expressing) neurons that are postsynaptic to glutamatergic connections to excitotoxic neurodegeneration. Of these 30 at-risk neurons, individual neurons go through necrosis as the animal progresses through development (with mixed stochasticity and partial preference towards certain specific neurons³⁸), while at the same time cell corpses are also being gradually removed. In combination with the accessibility of many mutant strains, this approach allows the study of multiple pathways that affect neurodegeneration and neuroprotection. These approaches have already been used to analyze some of the downstream death signaling cascades³⁹ and transcriptional regulators of excitotoxic neurodegeneration in excitotoxicity³⁸^,⁴⁰. Like other cases of necrotic neurodegeneration in the worm⁴¹, the nematode's excitotoxic neurodegeneration does not involve classic apoptosis⁴⁰.

This methods paper describes the basic system to induce, quantify, and manipulate excitotoxic necrotic neurodegeneration in C. elegans. Furthermore, it outlines two main protocols that are currently in use to streamline studies of specific aspects of nematode excitotoxicity. By using fluorescent reporters and live in vivo imaging the researcher can study mitochondrial involvement and dynamics in the nematode model of excitotoxic neurodegeneration. To determine the effect of specific neuroprotective transcription factors, the investigator can use cell type-specific expression of fluorescent markers, dissociation of animals into single cells, and FACS to isolate specific neurons that are at risk of necrosis from excitotoxicity. These cell-type specific isolated neurons can then be used for RNA sequencing in strains that harbor mutations in key transcription factors. Put together, these methods can allow researchers to tease out the molecular underpinnings of excitotoxic neurodegeneration and neuroprotection in vivo with great clarity and precision.

Protocol

1. Strains used to Investigate Excitotoxic Neurodegeneration & Neuroprotection Use the nematode excitotoxicity strain ZB1102 as the reference point for standard excitotoxic neurodegeneration. NOTE: Glutamate-dependent excitotoxicity in C. elegans is produced in strain ZB1102 by combining a knockout (ko) of a glutamate transporter with a transgene that sensitizes neurons in these animals to neurotoxicity and is expressed in a subset of neurons postsynaptic to glutamatergic connecti…

Representative Results

Nematode model of excitotoxicity and identification of vacuolated degenerating neurons Data shown here is reproduced from previous publications37,38. To mimic excitotoxic-induced neurodegeneration, a glutamate-transporter gene knockout (glt-3) is combined with a neuronal sensitizing transgenic background (nuls5 [Pglr-1::GαS(Q227L);Pglr-1::GFP)]). The transgenic construct is ex…

Discussion

While the prevalent controversies and failures suggest that excitotoxicity presents an exceptionally hard process to decipher, the analysis of excitotoxicity in the nematode offers a particularly attractive strategy to illuminate conserved neuronal cell death pathways in this critical form of neurodegeneration. The investigator can rely on the rich collection of research tools available in this system, and particularly on the animal's transparency (allowing in vivo analysis) and the large repertoire of viable mutants…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank all members of the Mano Lab and the Li lab (current and recent) for their help and support. We thank Dr. Monica Driscoll (Rutgers Univ.) for pioneering the analysis of necrotic neurodegeneration in nematodes and providing continuous support; Dr. Chris Li (CCNY) for support and advice; Jeffery Walker (CCNY Flow Cytometry Core facility), Dr. Bao Voung (CCNY), and Stanka Semova (Rockefeller Univ. Sorting Faculty Core) for practical support and advise on cell sorting; Dr. Chris Rongo (Rutgers Univ.) for reagents; Drs. David Miller (Vanderbilt Univ.), Coleen Murphy (Princeton Univ.), Shai Shaham, Menachem Katz, & Katherine Varandas (all three from Rockefeller Univ.) for C. elegans dissociation protocols.

The Mano lab received funding from NIH NINDS (NS096687, NS098350, NS116028) to I.M., and through a NIH U54 CCNY-MSKCC partnership (CA132378/CA137788).

Materials

Agar	VWR	AAA10752-0E
Bactopeptone	VWR	90000-264
BD FACSAriaIII	BD
Bleach	Any household
CaCl₂	VWR	97062-586
CaCl₂·2H₂O	BioExpress	0556-500G
Cell Strainer, PluriStrainer mini 70um	PluriSelect	43-10070-40
Cell Strainer, PluriStrainer mini 5um	PluriSelect	43-10005-60
Centrifuge – 15-50 mL Sorval benchtop LEGENDX1R TC	Fisher Sci	75618382
Centrifuge – microfuge ; Ependorff 5424	VWR	MP022629891
Chloroform	VWR	97064-680
Cholesterol	Sigma	C8667-25G
DAPI	Fisher Sci	EN62248
Dry ice	United City Ice Cube
DTT	VWR	97061-340
E. coli OP50	CGC	OP50
Ethanol (100%)	VWR	EM-EX0276-1S
Ethanol (90%)	VWR	BDH1160-4LP
FACS tubes	USA Sci	1450-2810
Filter tips	USA Sci	1126-7810
Glass 10 mL serological pipettes	USA Sci	1071-0810
Heating block	BioExpress	D-2250
Hepes	VWR	97061-824
Immersion Oil – Carl Zeiss Immersol	Fisher Sci	12-624-66A
Isopropanol	VWR	EM-PX1830-4
KCl	VWR	BDH9258-2.5KG
KH₂PO₄	VWR	BDH9268-2.5KG
Low bind 1.5mL tubes	USA Sci	4043-1021
Metamorph Imaging Software	Molecular Devices
MgCl₂	VWR	97063-152
MgCl₂·6H₂O	BioExpress	0288-500g
MgSO₄	VWR	97061-438
Microscope, Confocal, for Fluorescence Imaging	Zeiss	LSM 880
Microscope, Inverted, for Fluorescence Imaging	Zeiss	Axiovert 200 M
Microscope Camera	Q-Imaging	Retiga R1
Microscope Light Source for Fluorescence Imaging	Lumencor	SOLA SE Light Engine
Microscope, Nomarski DIC	Zeiss	Axiovert Observer A1
Microscope, Nomarski DIC	Nikon	Eclipse Ti-S
Na2HPO4	VWR	97061-588
NaCl	VWR	BDH9286-2.5KG
NaOH	VWR	97064-476
Petri dishes, 100mm	Fisher Sci	FB0875712
Petri dishes, 60mm	TriTech	T3308
Pipet Controller	TEquipment	P2002
Pipettor P10 Tips	USA Sci	1110-3000
Pipettor P1000 Tips	USA Sci	1111-2020
Pipettor P200 Tips	USA Sci	1110-1000
Pronase	Sigma	P8811-1G
RNAse away spray	Fisher Sci	7000TS1
RNAse free serological pipettes	USA Sci	1071-0810
RNAse-free 50 mL tubes	USA Sci	5622-7261
RNeasy micro	Qiagen	74004
SDS	VWR	97064-496
Streptomycin sulfate	Sigma	S6501-100G
Sucrose	VWR	AAJ63662-AP
SUPERase·in RNase inhibitor	Fisher Sci	AM2694
Quality Control automated electrophoresis system: Tapestation – High Sensitivity RNA ScreenTape	Agilent	5067-5579
Tapestation – High Sensitivity RNA ScreenTape Ladder	Agilent	5067-5581
Tapestation – High Sensitivity RNA ScreenTape Sample Buffer	Agilent	5067-5580
Tapestation – IKA MS3 vortexer	Agilent/IKA	4674100
Tapestation – IKA vortexer adaptor at 2000 rpm	Agilent/IKA	3428000
Tapestation – Loading tips	Agilent	5067- 5152 or 5067- 5153
Tapestation – Optical Cap 8x Strip	Agilent	401425
Tapestation – Optical Tube 8x Strip	Agilent	401428
Quality Control automated electrophoresis system: TapeStation 2200	Agilent	G2964AA
Tetramisole	Sigma	L9756-10G
Tris base	Fisher Sci	BP152-500
Tris hydrochloride	Fisher Sci	BP153-500
Trizol-LS	Fisher Sci	10296-010
Wescor Vapro 5520 Vapor Pressure Osmometer	Fisher Sci	NC0044806
Wheaton Unispense μP Dispenser	VWR	25485-003

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