Caldwell 2020 Modeling Neurodegeneration in Caeno

© 2020. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2020) 13, dmm046110. doi:10.1242/dmm.
046110
AT A GLANCE
Modeling neurodegeneration in Caenorhabditis elegans

Kim A. Caldwell1,2,*, Corey W. Willicott1 and Guy A. Caldwell1,2
ABSTRACT
amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s
The global burden of neurodegenerative diseases underscores the disease and Huntington’s disease. Transgenic nematodes with
urgent need for innovative strategies to define new drug targets genes encoding normal and disease variants of proteins at the
and disease-modifying factors. The nematode Caenorhabditis single- or multi-copy level under neuronal-specific promoters
elegans has served as the experimental subject for multiple limits expression to select neuronal subtypes. The anatomical
transformative discoveries that have redefined our understanding transparency of C. elegans affords the use of co-expressed
of biology for fluorescent proteins to follow the progression of
∼60 years. More recently, the considerable attributes of C. elegans neurodegeneration as the animals age. Significantly, a completely
have been applied to neurodegenerative diseases, including defined connectome facilitates detailed understanding of the
impact of neurodegeneration on organismal health and offers a
1
Department of Biological Sciences, The University of Alabama, Tuscaloosa, unique capacity to accurately link cell death with behavioral
AL 35487, USA. 2Departments of Neurobiology, Neurology, Center for
Neurodegeneration and Experimental Therapeutics, and Nathan Shock Center of dysfunction or phenotypic variation in vivo. Moreover, chemical
Excellence in the Basic Biology of Aging, University of Alabama at Birmingham, treatments, as well as forward and reverse genetic screening,
Birmingham, AL 35294, USA. hasten the identification of modifiers that alter neurodegeneration.
*Author for correspondence ([email protected]) When combined, these chemical-genetic analyses establish
critical threshold states to enhance or reduce cellular stress
K.A.C., 0000-0003-1580-6122; G.A.C., 0000-0002-8283-9090 for dissecting associated pathways. Furthermore, C. elegans
can rapidly reveal whether lifespan or healthspan factor
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, into neurodegenerative processes. Here, we outline the
distribution and reproduction in any medium provided that the original work is properly
attributed.
Disease Models & Mechanisms
1
AT A GLANCE Disease Models & Mechanisms (2020) 13, dmm046110. doi:10.1242/dmm.046110
methodologies employed to investigate neurodegeneration gene expression patterns, as discerned by the collective efforts of
in C. elegans and highlight numerous studies that exemplify its researchers in the CeNGEN
utility as a pre-clinical intermediary to expedite and inform
mammalian translational research.
KEY WORDS: Aging, Behavior, Genetics, Modeling, Phenotype,

Proteostasis
Introduction
Neurodegenerative diseases represent a significant and growing
burden globally and the identification of factors that reduce their
impact necessitates considerable research effort. Undoubtedly,
rodent models have expanded our understanding of
neurodegenerative disorders over the past few decades. However,
a range of phenotypic variations can manifest in humans; therefore,
it seems prudent to use diverse model organisms to hasten research
into causes and cures for neurodegenerative diseases. In this At a
Glance article, and the accompanying poster, we survey
Caenorhabditis elegans as a model for neurodegenerative disease
research. Many consider this nematode roundworm to be the most
completely understood animal on the planet. Therefore, given the
sense of urgency to accelerate discovery, the assays, molecular tools
and genetic strategies developed through years of collaborative
and collective effort in the worm community should be seriously
considered within the arsenal for eradicating neurodegenerative
diseases from the human population.
Despite the lack of evolutionary complexity, C. elegans shares
many conserved molecular pathways and cellular mechanisms with
mammals, thus allowing for comparative studies. OrthoList 2 is a
searchable compendium of all orthologs and paralogs between
C. elegans and humans and includes 7943 genes, or ∼41% of the
C. elegans protein-coding genome (Shaye and Greenwald, 2011;
Kim et al., 2018a). Moreover, 3357 worm genes possess human
orthologs with Online Mendelian Inheritance in Man (OMIM)
annotations; this is 17% of the worm genome (Kim et al., 2018a;
http: /ortholist.shaye- lab.org/). This conservation provides great
potential for modeling human genetic disease. For example, many
C. elegans neuronal genes are expressed in comparable
mammalian cell types (Hobert, 2013).
While mammalian nervous systems consist of billions of
neurons, the adult C. elegans hermaphrodite has ∼300 neurons
throughout its body, thereby reducing the complexity and
increasing the accuracy of neuronal analyses (Cook et al., 2019).
The major functional components of mammalian synaptic
transmission, such as neurotransmitters, receptors, transporters
and ion channels, are conserved in C. elegans. Based on
differences in function, morphology and connectivity, the

neurons of this animal have been sorted into separate classes:
mechano-, chemo- and thermo-sensory neurons, interneurons,
motor neurons and neuromuscular junction components, among
others. Importantly, all neuron types can be further
subcategorized by neurotransmitter biosynthesis and activity;
neuronal signaling molecules include glutamate (Li et al., 1997),
GABA (McIntire et al., 1993), acetylcholine (Alfonso et al.,
1994), dopamine (Sulston et al., 1975), serotonin and octopamine
(Horvitz et al., 1982), as well as neuropeptides (Li and Kim,
2008). Stereotypical behavioral phenotypes are associated with
distinct neuronal subclasses (see poster, ‘Representative
neurobehavioral assays’). The C. elegans neuronal circuitry has
also been fully mapped since the 1980s (White et al., 1986), and
researchers recently updated the compendium of the neuronal
connectomes of both the male and hermaphrodite using state-of-
the-art technologies (Cook et al., 2019). Moreover, the constituent
2
Project, facilitate enhanced understanding of neuronal signaling 2016; Wang et al., 2018; Fang et al., 2019).
at a level of detail simply not yet possible in other species
(Hammarlund et al., 2018). Furthermore, because C. elegans is
anatomically transparent, neurons are easily visualized by
expressing fluorescent proteins in live animals to study neuronal
properties throughout development and over time.
Genetic models of neurodegeneration

C. elegans is informative for basic studies of neuronal function.
Perhaps more impressively, it can be effectively harnessed to
rapidly and cost effectively identify gene targets and drug
candidates that modify neurodegenerative processes. In contrast,
rodent neurodegeneration studies are time consuming and
economically challenging. Despite the abundance of cellular and
animal models to study neurodegenerative disease, we contend that
C. elegans has been underutilized with respect to its predictive
capacity for translational biology. In this At a Glance article, we
describe common forms of neurodegeneration modeled in C.
elegans (see poster, ‘Neurodegenerative diseases modeled’),
including Alzheimer’s disease (AD), amyotrophic lateral
sclerosis (ALS), frontotemporal dementia (FTD), Huntington’s
disease (HD) and Parkinson’s disease (PD). We discuss the
genetic and environmental modulators that share common
pathways leading to cellular malfunction and eventually
neurodegeneration within the context of these
C. elegans human disease models. There are additional models of
these neurodegenerative diseases that are not covered within the
article, and we apologize for this omission due to space
constraints. Furthermore, we have largely excluded discussions of
any models in which neurodegeneration was not an endpoint
phenotype evaluated.
AD
Two defining characteristics of AD are the presence of insoluble
amyloid β-peptide (Aβ) plaques and of tau-associated neurofibrillary
tangles in the brain. In mammals, Aβ is accumulated into
extracellular plaques, followed by endocytic uptake of these
neurotoxic oligomers. This process induces tau phosphorylation
and aggregation into neurofibrillary tangles. Aβ accumulation is
an outcome of sequential enzymatic processing of the human
amyloid precursor protein (APP) by membrane-bound secretase
enzymes mediated by the subcomponent PS1 and PS2 presenilin
proteins. Although the product of the worm sel-12 gene provided
some of the first evidence for presenilins as functional subunits of
human γ-secretase (Levitan and Greenwald, 1995), the C. elegans
genome lacks both a clear β-secretase ortholog and an ortholog
of APP that would be processed to yield Aβ peptides. The worm
APL-1 protein is an ortholog of the human amyloid beta
precursor-like proteins 1 and 2 (APLP1 and APLP2), and has
71% sequence similarity to the intracellular domain of APP, but
completely lacks an Aβ domain. Thus, AD modeling in C.
elegans has primarily focused on the transgenic expression of
mature human Aβ and tau (Griffin et al., 2017). Nevertheless,
single-copy insertion and expression of human APP results in
neurodegeneration and neurobehavioral dysfunction in
C. elegans (Yi et al., 2017). Likewise, although apl-1 is essential
for
viability, overexpression of apl-1 yields pleiotropic phenotypes
that include neuronal deficits (Hornsten et al., 2007; Ewald et al.,
2012). Owing to spatial constraints, the AD section of this article
will focus on Aβ modeling, while the ALS section will describe
tau expression in
C. elegans. However, we direct the reader to studies in which
transgenic nematodes expressing human tau have been generated
and include the fine work of several groups with interests in AD
modeling (Kraemer et al., 2006; Ayyadevara et al., 2016; Pir et al.,
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Modeling AD in C. elegans has largely been based on the recently, FTD, a dementia syndrome that can present with changes in
‘amyloid cascade hypothesis’ (Hardy and Selkoe, 2002). Here, the behavior or language dysfunction, has become recognized in many
Aβ1-42 peptide is thought to be the most toxic species generated by ALS patients (Caga et al., 2019; Zucchi et al., 2019). Most ALS is
the cleavage of APP. The original and most commonly used C. sporadic,
elegans AD model was a result of pioneering research by Chris
Link (1995). In this model, and related variants, the Aβ peptide is
tagged with a secretion signaling sequence that is expressed from
the unc-54 promoter in the body wall muscles, where it causes
paralysis, thereby allowing for quantitative analysis of modulators.
Aβ1-42-mediated neurodegeneration has been studied primarily in
mechanosensory and in glutamatergic neurons, which are
commonly susceptible to hyperexcitability and are clinically
associated with the early stages of degeneration in AD (Palop and
Mucke, 2009, 2016). Wild-type C. elegans have five
glutamatergic neurons in their tails. Models of AD that
overexpress GFP-fused human Aβ1-42 specifically in these neurons
under the eat-4 glutamate transporter promoter demonstrate
progressive, age-dependent neurodegeneration (Treusch et al., 2011).
Researchers utilized a yeast model expressing Aβ1-42 targeted to
the secretory pathway to identify genetic modifiers of Aβ toxicity.
This genome-wide screen revealed 23 suppressors and 17
enhancers of toxicity, with 12 of these having human homologs.
These modifiers were then tested in C. elegans to determine their
neurotoxicity. Phosphatidylinositol-binding clathrin-assembly
protein (PICALM) protected against Aβ neurotoxicity, which was
further verified in primary rat cortical neurons and corroborated the
human genetic data implicating this gene in AD (Treusch et al.,
2011). In a separate study, a high-throughput compound screen in
yeast and C. elegans models of AD identified a class of metal-
binding compounds that were neuroprotective through the
degradation of Aβ (Matlack et al., 2014). Further investigation
showed that these small molecules were related to clioquinol, which
was previously found to reduce Aβ toxicity in mouse models
(Cherny et al., 2001). Using the same transgenic C. elegans Aβ1-42
model, Lu et al. (2014) showed that Wnt/β-catenin signaling
attenuated glutamatergic neuron loss through regulation of spr-4, a
homolog of the stress regulatory transcription factor REST.
The ε2 isoform of apolipoprotein E (APOE) has been associated
with reduced risk for developing AD, whereas the APOEε4 allele
increases risk, with APOEε3, the most frequent isoform, being
neutral (Spinney, 2014). Our group developed a suite of
transgenic
C. elegans models expressing human APOE alleles, with and
without Aβ1-42 (Griffin et al., 2019). Co-expressing APOEε2 with
Aβ protected glutamatergic neurons from degeneration and
restored normal mechanosensory behavior. In contrast, the
APOEε3 worms displayed an intermediate phenotype, and
expression of APOEε4 did not protect from Aβ neurotoxicity. In
the absence of Aβ, the APOE alleles did not alter glutamatergic
neuron function. These allele-specific neuroprotective effects
could be modulated in response to endoplasmic reticulum (ER)-
associated calcium changes, both pharmacologically and via RNA
interference (RNAi) knockdown. Moreover, lifespan was
reduced in
C. elegans expressing Aβ and APOEε4; it was rescued by
APOEε2 and APOEε3 alleles (Griffin et al., 2019). Thus, transgenic
C. elegans lines recapitulate the established clinical profile of
APOE polymorphism-associated impact on neurodegeneration,
which can be used to discern new mechanistic insights for AD.
ALS and FTD

ALS is a neurodegenerative disorder that affects the upper and
lower motor neurons, causing progressive muscle weakness. More
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with only ∼10% of cases displaying a familial inheritance dependent paralysis as a consequence of axonal guidance defects
pattern. A causative mutation has been identified in ∼80% of (Li et al., 2013). Notably, these same two
these kindreds; notably, some of these same mutations also lead to
sporadic ALS and many are autosomal dominant (Al-Chalabi et al.,
2017). C. elegans has been used to model the four most common
ALS-causing mutations in C9orf72-SMCR8 complex subunit
(C9orf72), superoxide dismutase (SOD1), TAR DNA-binding
protein (TARDBP) and RNA-binding protein FUS/TLS (FUS), as
well as a mutation associated with FTD [microtubule-associated
protein tau (MAPT)].
Mutations in C9orf72, where a hexanucleotide repeat
(GGGGCC) within the first intron of C9orf72 undergoes
expansion, are responsible for 10-15% of familial ALS cases
(van Damme et al., 2017). The mechanism associated with this
expansion remains under investigation. One hypothesis is that the
translation of these repeats is AUG independent. This example of
repeat-associated non-AUG (RAN) translation (Cleary and
Ranum, 2017) can result in five separate dipeptide-containing
proteins from the GGGGCC repeat.
C. elegans researchers created transgenic models of four
possible dipeptides that could potentially lead to toxic cellular
consequences. Subsequent characterization revealed that
arginine-containing dipeptides exhibited potent motor neuron
and muscle cell toxicity (Rudich et al., 2017). A distinct effort
involved C. elegans overexpressing nine and 29 GGGGCC
repeats under the broadly active hsp-16 promoter. The phenotypes
were more severe in animals expressing 29 repeats versus nine
repeats and included paralysis followed by lethality (Wang et al.,
2018). A forward genetic screen of the 29-repeat transgenic worms
identified uncharacterized genes, still under analysis, that reversed
this severe phenotype. In addition to overexpressing the
hexanucleotide repeat, the C. elegans genome has an ortholog of
C9orf72 termed alfa-1; a loss-of-function alfa-1 mutant that
exhibits motor neuron degeneration and a motility defect
(Therrien et al., 2013). Non-neuronally, these worms have an
endocytosis defect that can be partially rescued by introducing
wild- type human C9orf72 (Corrionero and Horvitz, 2018). C.
elegans alfa-1 has also been reported to be involved in nutrient
sensing and metabolic control (Ji et al., 2020).
Discovery of SOD1 mutations in 1993 allowed for the initial
modeling of ALS (Rosen et al., 1993), even though SOD1
mutations are now understood to be responsible for only ∼2% of
familial ALS (van Damme et al., 2017). As a cytosolic enzyme,
SOD1 catalyzes the detoxification of superoxide. There are more
than 100 mutant alleles of SOD1 associated with disease, and
most mutations cause a toxic gain of function in motor neurons
(Cleveland and Rothstein, 2001). Some of these mutations, such
as G85R and G93A, misfold and then eventually aggregate in
motor neurons, as well as in vitro (Ghosh et al., 2020). Other
mutations, such as A4V, cause ER stress, but the underlying
mechanism for this stress remains unclear (Perri et al., 2020).
Dysfunctional proteostasis correlates with ER stress in a mouse
model of G93A SOD1 (Kikuchi et al., 2006); moreover,
motor neuron subclasses vulnerable to degeneration also exhibit
increased ER stress upon G93A or G85A SOD1 expression
(Saxena et al., 2009). Pan-neuronal expression of human G85R
SOD1 in
C. elegans leads to locomotor defects, development of
aggregates and axonal abnormalities, such as reduced numbers
and diameter of axons and fewer organelles within the remaining
axons (Wang et al., 2009). Thompson et al. (2014) demonstrated
that co-expression of torsinA, a highly-expressed neuronal
chaperone in humans, can attenuate the ER stress caused by
human G85R SOD1, as well as restore normal locomotion to
transgenic C. elegans. Overexpression of a different human
SOD1 mutation, G93A, specifically in motor neurons led to age-
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mutations (G85R and G93R) were subsequently modeled as single- not aggregate did not cause observable phenotypes
copy insertions, along with three other common SOD-1 mutations
(A4V, H71Y, L84) in C. elegans. The impact on both
glutamatergic and cholinergic neuron degeneration was
examined for all five of these mutations (Baskoylu et al., 2018).
G93R displayed phenotypes consistent with a toxic gain-of-
function phenotype in cholinergic neurons, while G85R caused
glutamatergic neurodegeneration following exposure to the
neurotoxin paraquat (Baskoylu et al., 2018).
Although the associated pathogenic mechanisms have yet to be
understood, TAR DNA-binding protein-43 (TDP-43) (Arai et al.,
2006) and FUS (Kwiatkowski et al., 2009) have been identified in
proteinaceous cytoplasmic inclusions in motor neurons of ALS
patients. ALS-causing mutations within the TDP-43-encoding
gene, TARDBP, are associated with 0.9% of patients, whereas
0.7% of patients harbor mutations in FUS (van Damme et al.,
2017). TDP-43 and FUS, which are DNA/RNA-binding proteins,
regulate transcription, splicing, RNA transport and stress granule
formation (Aksoy et al., 2020), and are predominantly nuclear
under wild-type conditions. Notably, when mutated, both proteins
display cytoplasmic mislocalization, along with protein
aggregates, as a hallmark of ALS (Liu et al., 2017).
Multiple research groups have examined the phenotypic
consequences of TBP-43 expression within C. elegans neurons.
For example, pan-neuronal expression of human wild-type TDP-
43 under control of the snb-1 promoter caused slowed
and uncoordinated movement, as well as defasciculation of the
motor neurons of the transgenic worms (Ash et al., 2010). In a
separate effort, using the same pan-neuronal promoter, motility
defects, aggregation and neurodegeneration specifically
within the GABAergic neurons, resulted from the expression
of mutant variants of TDP-43 (Liachko et al., 2013). These TDP-
43 models have led to mechanistic insights, including the
observation that calcineurin, a phosphatase, removes the
pathological C-terminal phosphate from TDP-43 (Liachko et al.,
2016). A phenotypic drug screen discovered that α-methyl-α-
phenylsuccinimide (MPS) reversed the locomotion defects
associated with TDP-43 mutant worms. MPS is the active
metabolite of methsuximide, a currently used anti-epileptic drug,
suggesting the utility of repurposing this compound in treating
TDP-43 proteinopathies (Wong et al., 2018). The C. elegans
genome encodes an ortholog of TDP-43, called TDP-1, that is
primarily expressed in the nuclei of neurons and body wall muscle
cells (Vaccaro et al., 2012a; Zhang et al., 2012). TDP-1 contains
most motifs found in human TDP-43, including two RNA- binding
motifs, and nuclear import and export signals. However, the
glycine-rich domain of human TDP-43 is missing. Nonetheless,
there is still functional conservation between these two proteins;
specifically, the toxicity phenotypes associated with the
C. elegans tdp-1 mutant can be rescued by expressing wild-
type human TDP-43 (Zhang et al., 2012). Deletion of
endogenous tdp-1 could rescue the neurodegeneration
associated with overexpression of mutant human TDP-43 in
C. elegans GABAergic neurons (Vaccaro et al., 2012a).
FUS variants have also been examined in transgenic C.
elegans. Expressing a FUS variant prone to aggregation in
GABAergic neurons via the unc-47 promoter resulted in
neurodegeneration, synaptic dysfunction, paralysis and
aggregation, whereas expression of wild-type FUS did not cause
these phenotypes (Vaccaro et al., 2012b). In a separate study,
when FUS mutants were expressed pan- neuronally under control
of the rgef-1 promoter, multiple mutations associated with
aggregation resulted in motor dysfunction, while variants that do
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(Murakami et al., 2012). The C. elegans genome has an ortholog uptake a fluorescent dye
of FUS named fust-1. The fust-1 gene product is well conserved
and has been reported to interact with the ortholog of AGO2
in C. elegans, ALG-1, a core microRNA-induced silencing
complex component. Additionally, FUST-1 is required to achieve
maximum microRNA (miRNA)-mediated gene silencing (Zhang et
al., 2018). FTD with parkinsonism linked to chromosome 17
(FTDP-17) is characterized by the accumulation of the
microtubule-associated protein tau. Symptoms include
neurodegeneration and cognitive decline. However, the
underlying mechanisms remain unclear. Researchers have
developed transgenic C. elegans models expressing either the
wild-type human tau or FTDP-17-causing variants (P301L or
V337M) under control of a pan-neuronal promoter (Kraemer et al.,
2003). Expression of the mutant alleles resulted in cholinergic
signaling defects and GABAergic neurodegenerative phenotypes.
Additionally, these animals were uncoordinated, displayed
egg-laying defects and had a reduced lifespan. Western blotting
demonstrated the accumulation of phosphorylated insoluble tau in
these animals (Kraemer et al., 2003). Another study showed that
expression of either normal human tau or
pseudohyperphosphorylated tau within the GABAergic neurons
caused uncoordinated movement without neurodegeneration
(Brandt et al., 2009). Notably, pseudohyperphosphorylated
tau-expressing animals did exhibit a
developmental abnormality with visible gaps in the motor neurons.
The tau mutation A152T is a rare risk factor for FTD and AD.
Worms with pan-neuronal expression of human tau A152T
displayed GABAergic neuron degeneration (Pir et al., 2016). A
subsequent study found that pan-neuronal tau A152T expression
effectuated the loss of glutamatergic neurons significantly more
than GABAergic neurons and attributed this loss to differential
vulnerability to excitotoxicity (Choudhary et al., 2018). Results
from a genome-wide RNAi screen (Kraemer et al., 2006) and a
follow-up study using a C. elegans model overexpressing human
tau pan-neuronally discovered that the unfolded protein response
(UPR) of the ER (UPRER) is a potential modulator of tau
proteostasis (Waldherr et al., 2019). Furthermore, the expression
of the constitutively active X-box binding protein 1 (XBP-1), the
driving transcription factor of the UPRER, improved tau clearance
in the cytoplasm and enhanced neuron survival (Waldherr et al.,
2019).
HD
An autosomal-dominant disorder characterized by the
progressive and age-associated decline in motor control and
cognition, typically beginning in mid-life, HD is a profoundly
tragic neurodegenerative disease because of its strong familial
association and highly predictive mortality, owing to the absence
of any disease- modifying therapy. In humans, HD is
characterized by an increase in polyglutamine repeats ( polyQs)
in the N-terminus of the huntingtin protein (HTT). Repeat length
correlates with the age of onset and severity, with 35 repeats
being the threshold of disease development (Orr and Zoghbi,
2007). In C. elegans, HD has proven archetypical for modeling
the protein misfolding pathology and altered proteostasis shared
among neurodegenerative diseases.
Despite the absence of a C. elegans ortholog of HTT, several
groups have generated transgenic animals expressing polyQ
tracts and human HTT fragments with varying polyQ-repeat
lengths in different neuronal subtypes to study HD pathology
(Satyal et al., 2000; Parker et al., 2001; Morley et al., 2002;
Nollen et al., 2004). One of these is limited to the ASH sensory
neuron, achieved by placing the transgene under the control of
the osm-10 promoter. Degeneration correlates with polyQ length
and is readily detected by the failure of this ciliated neuron to
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(Faber et al., 1999). Expression of HTT with normal and elegans and primary rat neuronal cultures confirming that
expanded polyQ tracts specifically in the touch receptor neurons, increased expression of a key
under control of the mec-3 promoter, caused mechanosensory
neuron dysfunction and degeneration of axons in a polyQ-length-
dependent manner (Parker et al., 2001). These models have been
used to investigate the mechanisms underlying HD and the
therapeutic potential of various compounds.
Because mouse models of neurodegenerative diseases showed that
loss of autophagy enhanced degeneration, researchers tested the
effect of neuron-specific autophagy gene loss to evaluate the role of
protein turnover in an HD C. elegans model. Inactivation of these
genes in the presence of polyQ expansions increased the
accumulation of aggregates and their toxicity, resulting in
augmented neurodegeneration (Jia et al., 2007). Similarly,
researchers observed that glucose was neuroprotective in the 128-
repeat polyQ HD model. This protection was DAF-16 dependent
and glucose operated by reducing misfolded protein levels
(Tauffenberger et al., 2012). As a final example, the protective
activity of rutin, previously shown to be therapeutic in rat models of
HD, was demonstrated through a reduction in polyQ-induced
neuron death and elevated activation of DAF-16 (Cordeiro et al.,
2020).
PD
Following AD in prevalence, PD is the second most common
neurodegenerative disease, afflicting ∼10 million people
worldwide. Unlike HD, PD is primarily idiopathic, with only
∼10% showing familial inheritance (Hernandez et al., 2019).
Although early-onset PD accounts for a small amount of cases,
the vast majority of individuals display symptomality late in life,
typically after age 65, with increased age being the predominantly
accepted risk factor. The primary clinical hallmarks of PD include
postmortem evidence of Lewy bodies and loss of dopaminergic
neurons in the substantia nigra region of the brain. Owing to its
central role in PD pathogenesis, α-synuclein (α-syn), which is
encoded by the PARK1/SNCA locus in humans and is associated
with autosomal-dominant PD (Meade et al., 2019), has been
modeled extensively in C. elegans. α-syn is also a major
component of Lewy bodies and multiplication of the
PARK1/SNCA locus has been linked to enhanced aggregation and
dopaminergic neuron death (Mezey et al., 1998; Singleton et al.,
2003). C. elegans has orthologs of many PARK genes, with the
notable exception of PARK1/SNCA. This conveniently allows C.
elegans researchers to overexpress α-syn without interference from
endogenous α-syn or a dominant-negative effect, as the worm
essentially serves as ‘an α- syn null genetic background’.
Researchers have developed numerous C. elegans models of PD
and, in most, α-syn and GFP are selectively expressed from either
a dopaminergic or pan- neuronal promoter. The dopamine
transporter (dat-1) promoter delivers higher expression levels
and concomitant dopaminergic
neurodegeneration from overexpression of either wild-type or
familial mutant forms of α-syn (Lakso et al., 2003; Cao et al.,
2005; Karpinar et al., 2009). Worm models of α-syn-induced
neurodegeneration have been widely used in combination with
genetic and drug screening in a discovery pipeline comprised of
more than one model system to successfully demonstrate the
evolutionary conservation of function for modifiers and pathways.
Cooper et al. (2006) demonstrated that overexpression of
human α-syn in yeast blocks ER-to-Golgi vesicular trafficking; this
was the first report of cellular malfunction due to α-syn.
Moreover, this study employed multiple model systems to
provide additional support with results from Drosophila, C.
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vesicular trafficking pathway protein, Rab1, could rescue acids, and largely remains a functional enigma underlying PD
dopaminergic neuron loss induced by α-syn (Cooper et al., pathology.
2006). A separate screen of 190,000 compounds for reversal of
α-syn- induced yeast toxicity identified N-aryl benzimidazole
(NAB). Notably, NAB activity was conserved in neurons, as it
protected C. elegans dopaminergic neurons and α-syn-
expressing rat primary neurons from α-syn-induced
neurodegeneration (Tardiff et al., 2013), as it did in
dopaminergic neurons differentiated from patient- derived
induced pluripotent stem cells (Chung et al., 2013). Another
large-scale candidate gene screen conducted in C. elegans
expressing α-syn::GFP in a daf-2 receptor-mutant background, a
pivotal regulator of aging and lifespan, identified the glycolytic
enzyme GPI-1/GPI1 as a neuromodulatory factor. Concurrently,
GPI-1/GPI1 was shown to protect against α-syn proteotoxicity in
Drosophila and mouse primary neuronal cultures (Knight et al.,
2014). These collaborative pipeline-type studies emphasize the
importance of basic cell biology in neurodegeneration.
Furthermore, they exemplify the power of using multiple model
systems to identify evolutionarily conserved pathways and new
targets as functional effectors of neurodegeneration.
One of the enduring mysteries of PD is the selective vulnerability
of
dopaminergic neurons to degeneration. It has been hypothesized
that dopamine itself may be a key contributor to pathogenesis, as it
is more readily oxidized than other neurotransmitters. In vitro
experiments with purified α-syn polypeptides demonstrated that
addition of dopamine enhances and stabilizes the oligomeric
state of α-syn, whereas deletion of five amino acid residues
(125YEMPS129) in the C- terminal region of α-syn abolished this
effect (Herrera et al., 2008). Using transgenic C. elegans
expressing the aggregation-prone A53T α-syn variant, researchers
investigated the interaction between α-syn and dopamine with
and without the dopamine interaction site (Mor et al., 2017). The
animals were crossed into an established strain that overproduces
dopamine by the overexpression of tyrosine hydroxylase, the
product of cat-2 (Cao et al., 2005), to reveal that excess
dopamine exacerbates neurodegeneration. These data
corroborated results showing that enhanced dopamine levels in
mice expressing human A53T α-syn induced nigrostriatal
degeneration (Mor et al., 2017). Importantly, worms expressing
A53T α-syn lacking the 125YEMPS129 sequence exhibited
baseline levels of neurodegeneration. This differential effect
depended on the presence of cat-2 overexpression (Mor et al.,
2017). The use of C. elegans in this study provided mechanistic
clarity to help resolve a long-standing question of PD
neuropathology with clinical implications.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are
associated with autosomal dominant PD. Based on biochemical
experiments, mutations are linked to modifications in the
GTPase and kinase activities of LRRK2 that are likely the basis
of the neuronal toxicity in these PD patients. Transgenic
expression of wild-type or disease- causing mutant LRRK2 under
pan-neuronal or dopaminergic- specific promoters results in
neuronal loss in C. elegans (Saha et al., 2009; Liu et al., 2011;
Johnson et al., 2015). Using the dopaminergic LRRK2 model,
Liu et al. (2011) confirmed the effects of select inhibitors of
LRRK2 kinase activity identified from chemical library
screening. Additionally, the reduced glutaredoxin (Grx1; also
known as GLRX) levels observed in postmortem PD patient
midbrain samples was subsequently validated in a C. elegans
LRRK2 model. Specifically, loss of worm Grx1, combined with
LRRK2 overexpression, resulted in significantly greater levels of
neurodegeneration (Johnson et al., 2015). While its kinase
activity offers an attractive target for drug intervention, LRRK2 is
a complex multidomain protein comprised of over 2500 amino
9
Interestingly, phosphoproteomic analysis has identified Rab Researchers incorporate exogenous DNA into C. elegans for
GTPases as biological substrates for LRRK2-dependent numerous purposes, such as to rescue mutant alleles, analyze gene
phosphorylation (Steger et al., 2016). It is notable that among the function, or to examine gene expression (see poster, ‘Transgenic
Rab proteins modified by LRRK2 (Rab1, Rab3, Rab8, Rab10 and
others), several were identified as modulators of α-syn-dependent
neurodegeneration using C. elegans well over a decade ago
(Cooper et al., 2006; Gitler et al., 2008). More recently, a
convergence of α-syn and LRRK2 dysfunction around
intracellular trafficking has coincided with evidence highlighting
the significance of endolysosomal vesicular fusion and lysosome
function in PD- associated neurodegeneration (Winckler et al.,
2018). In this context, there is increased interest in factors
affecting lipid homeostasis as a potential avenue of therapeutic
target development (Fanning et al., 2020). C. elegans has already
been used to demonstrate the neuroprotective effect of stearoyl-
CoA desaturase inhibition (Fanning et al., 2019), an effect that
translates to human-derived neurons cultured in the presence of
neurotoxic α- syn (Vincent et al., 2018). Likewise,
phosphatidylethanolamine (PE) deficiency enhances
neurodegeneration in transgenic α-syn animals and ethanolamine
supplementation reverses this effect (Wang et al., 2014). These
data correlate with reports of an age-dependent decline in PE
found in PD patients.
The role of the lysosome in misfolded protein degradation
(autophagy) and damaged mitochondrial clearance (mitophagy)
has emerged as a substantial factor in PD. Again, over a decade
ago,
C. elegans RNAi screening to identify modifiers of α-syn
misfolding and neurodegeneration revealed in vivo effects of
proteins that function in autophagy or mitophagy (ATG7, ULK1,
PINK1, PRKN), endolysosomal trafficking (VPS41) and
lysosome function (cathepsin D, ATP13A2/PARK9) on
dopaminergic neurodegeneration (Hamamichi et al., 2008; Gitler
et al., 2009; Qiao et al., 2008). Most significantly, Sidransky et al.
(2009) defined an association between PD and mutations in the
human glucocerebrosidase gene, GBA1 (also known as GBA), a
causal factor in the rare lysosomal storage disorder Gaucher’s
disease. Whereas homozygous mutations in GBA1 cause
Gaucher’s disease, heterozygous carriers are of significantly
higher risk of PD. Thus, a deficit in glucocerebrosidase leads to
accumulation of glycolipids. Using transgenic C. elegans
overexpressing human α-syn in the dopaminergic neurons,
Mazzulli et al. (2011) reported that neuron- specific RNAi
knockdown of gba-1 enhanced dopaminergic neurodegeneration.
This supported an initial hypothesis that α-syn misfolding and
GBA-1 loss of function combine in a pathogenic loop of
exacerbated neurotoxicity. As of writing, GBA1 mutations
comprise the most common class of genetic cases of PD.
These examples are only a small sampling of the numerous
contributions C. elegans models have made to neurodegenerative
disease research. Through the collective efforts of worm
researchers worldwide, functional characterization of previously
uncharacterized or poorly understood disease-associated gene
products has provided mechanistic insights to illuminate the
translational path forward. As discussed below, the toolbox of
resources available in the C. elegans research community
continues to expand, further establishing this model system as a
means to enable and accelerate discovery in this area of urgent
global need.
Tools and approaches for the functional characterization of

neurodegenerative mechanisms
Transgenic nematodes
10
nematode production’). In all cases, the gene of interest is performed, based on individual
engineered to be controlled by an endogenous C. elegans
promoter. This promoter permits expression in all or in selected
tissues. Additionally, for strain construction, a fluorescent or
phenotypic marker (GFP, mCherry, rol-6) is also used to
distinguish transgenic progeny. For many years, these expression
constructs were microinjected, via the germ line, into C. elegans,
where they become linearized and naturally form concatemers
that result in overexpression in the progeny of successfully
injected animals (Mello et al., 1991; Berkowitz et al., 2008). A
caveat to this method is that it is not possible to control the
amount of transgene incorporated into separate transgenic lines.
To correct for this, researchers typically analyze multiple lines of
transgenic animals and determine a consensus phenotype for
transgenic overexpression. More recently, worm researchers
have turned to CRISPR/Cas9-guided genome engineering
approaches for the generation of transgenic animals. This
method can achieve single- copy insertion of transgenes, thus
circumventing the issues of overexpression described above.
Additionally, if one were to express a human disease mutation in
a C. elegans strain that was concomitantly null mutant for the
same gene, this would result in a ‘humanized’ worm. As an
example, Zhu et al. (2020) sought to functionally validate
epilepsy-related mutations and generated a strain in which a null
mutation was introduced into the C. elegans ortholog of
STXBP1, unc-18, via CRISPR/Cas9, which resulted in paralysis.
They found that the human ortholog functionally replaced the
worm gene and that the epilepsy-associated variants caused
seizure-like activity. Although CRISPR/Cas9 and other means
by which single-copy insertion or editing can be achieved are
often desirable approaches, in the case of neurodegenerative
disease modeling, multicopy transgene expression can actually
be preferable. For example, dose-dependent neurotoxicity of α-
syn and Aβ are established characteristics of human pathology
that can be recapitulated in transgenic C. elegans harboring
tandem multicopy arrays. Lower copy number arrays or less robust
promoters can result in different levels of neurodegeneration, which
is useful for threshold- based genetic screening of suppressors and
enhancers, or a complete lack of degenerative phenotype in vivo
(Harrington et al., 2010; Therrien and Parker, 2014; Wang et al.,
2016).
Forward genetic screening

In the early 1960s, Nobel laureate Sydney Brenner began a
nascent interest in developing a model to understand an
organism’s nervous system in its entirety. He chose C. elegans to
uncover cellular details of the mechanistic foundation of the
nervous system. This nematode was a wise choice because of its
ease of genetic manipulation. While males exist among
populations as a result of chromosomal non- disjunction events,
C. elegans is predominantly a hermaphrodite in nature. Thus, for
example, if a hermaphrodite has one mutation and is crossed with a
male that has a different mutation, the next generation
hermaphrodite will inherit both mutations as a heterozygote.
Achieving homozygosity is simply a matter of ‘selfing’ this
individual hermaphrodite, which will produce up to 300
offspring.
Brenner began understanding the nervous system in C.
elegans by identifying synaptic transmission genes via a forward
genetic screen (see poster, ‘Genetic modifier screens’). He
treated worms with ethyl methanesulfonate (EMS) and screened
for uncoordinated (or ‘Unc’) worms, identifying 77 Unc genes
(Brenner, 1974). Mapping these genes was arduous, and cloning
mutations had no guarantee of success. Now, with advances in
genome sequencing techniques, there has been a renaissance in
the approach. Countless forward genetic screens have been
11
phenotypes or as modifier screens. As examples, an EMS screen available from multiple sources, enabling large-scale screening
of 80,000 haploid C. elegans genomes expressing GFP in the
dopaminergic neurons followed by fluorescence-activated sorting
of individual worms with defective dopaminergic neuron
differentiation, identified 22 alleles organized into six
complementation groups. It was estimated that the screen reached
∼78% saturation based on the allele recovery rate (Doitsidou et
al., 2008). In a separate effort, following the development of C.
elegans models of tauopathy by pan-neuronally expressing wild-
type or FTD-associated mutant tau (tau-FTDP-17) (Kraemer et
al., 2003), forward genetic screens identified two new molecular
factors, SUT- 1 and SUT-2, that participate in the activation of tau
(Kraemer and Schellenberg, 2007; Guthrie et al., 2009). SUT2 is
now an established susceptibility factor for tau pathology in the
mammalian brain (Wheeler et al., 2019). Worms harboring
deletions and point mutations that have been isolated from
genetic screens and carefully annotated are deposited in the
Caenorhabditis Genetics Center, with over 20,000 strains
available to the research community. These include mutations in
many genes associated with neurodegenerative diseases.
Reverse genetic screening

Neurodegeneration-associated phenotypes have also served as the
basis of reverse genetic screens (see poster, ‘Genetic modifier
screens’). The discovery and application of RNAi has
revolutionized phenotypic analysis in C. elegans. Unlike in most
animals, RNAi is typically performed by feeding parental
C. elegans with bacteria that produce double-stranded RNA
(dsRNA) against the target of interest (Timmons et al., 2001).
The progeny exhibit altered phenotypes that are scored. RNAi
feeding of individually cloned dsRNA of nearly every predicted
open-reading frame has been used in high-throughput screening
of embryonic and post-embryonic phenotypes in C. elegans
(Kamath et al., 2001, 2003). As an example, an RNAi screen
covering 85% of the genome was performed in transgenic worms
that express normal and FTDP-17 mutant human tau display
uncoordinated phenotypes (Unc). Notably, 75 genes enhanced the
Unc phenotype. Among these, the 46 candidates with human
homologs were preferentially examined further and could be
broadly classified as genes involved in dysregulated cell signaling
(Kraemer et al., 2006).
RNAi in neurons
Historically, experimental and anecdotal reports suggested that
RNAi was less effective in neurons (Asikainen et al., 2005),
although mutant worm strains with enhanced sensitivity to RNAi,

such as rrf-3, lin-15b or eri-1, bypass this limitation (Simmer et al.,
2002; Kennedy et al., 2004). However, when performing RNAi in
these strains, the silencing is systemic and occurs in all tissues of
the animal. An alternative method for neuronal RNAi involves
selective expression of sid-1, which encodes the ATP-independent
dsRNA transporter (see poster, ‘Genetic modifier screens’). This is
accomplished by generating sid mutant animals expressing sid-1
under the control of a neuron-specific promoter that restricts RNAi to
neurons. Calixto et al. (2010) first used this approach to
demonstrate successful selective pan-neuronal knockdown of
targets. Similar strains have been developed for dopaminergic or
glutamatergic neuron-specific RNAi (Harrington et al., 2012;
Griffin et al., 2019). Using a neuronal-specific RNAi strain (versus eri-
1 or rff-3) is an important strategy for studying the effects of depleting
essential gene targets that would be lethal for C. elegans if
knocked down systemically.
Comprehensive libraries of individual RNAi ‘feeding vectors’ are
12
efforts in neuronal contexts (Kamath et al., 2003; Rual et al.,
2004). For example, a genome-wide RNAi screen for
neurodegenerative phenotypes utilized a transgenic C. elegans
strain in which human mutant TDP-43 was expressed pan-
neuronally and caused motor neuron dysfunction. The authors
identified 46 genes (out of 16,767 screened) that, when knocked
down, partially suppressed the TDP- 43-induced motor
phenotype (Liachko et al., 2019). They then focused their efforts
on the 24 candidates with human homologs. Knocking down one
of these candidates, glucuronic acid epimerase (GLCE), in
human cultures protected against the phosphorylated TDP-43
accumulation caused by oxidative stress. Additionally, GLCE
expression was significantly decreased in the brains of patients
with frontotemporal lobar degeneration with TDP-43 inclusions
(FTD-TDP) relative to normal controls (Liachko et al., 2019).
This exemplifies the value of C. elegans as a means to accelerate
the translational path through defining previously undiscovered
genetic modifiers, susceptibility factors, or potential therapeutic
targets with direct disease relevance.
Strategies to examine neurodegeneration

The pathological loss of neurons in neurodegenerative diseases
is recapitulated in C. elegans models, where conditions that
promote either neuron survival or loss can be assessed in live
animals that express fluorescent proteins in the neuronal class of
interest (see poster, ‘Strategies to examine neurodegeneration’).
Neurodegeneration is induced by expressing pathogenic proteins
within specific the neuronal class(es), followed by scoring subtle
attributes of neuronal health, including cell body rounding (a sign
of apoptosis or necrosis), the disappearance of axons and/or
axonal blebbing, broken neurites and/or retreat of dendritic
terminals. Given the limited numbers of neurons comprising
specific classes in C. elegans, researchers can score
neurodegeneration with unparalleled accuracy in hundreds of
animals per condition to provide robust and rigorous analyses.
Genetic crosses to examine neuronal phenotypes

To tackle familial forms of neurodegenerative disease, C.
elegans investigators often take a genetic approach by examining
mutant variants of endogenous proteins. As previously discussed,
a form of familial AD results from mutations in PSEN1 or
PSEN2. When mutated, the C. elegans presenilin ortholog, sel-
12, causes ER Ca2+ dysregulation similar to that found in
mammals (Sarasija and Norman, 2015). Notably, further
research with the sel-12 mutant revealed mitochondrial
morphological and metabolic phenotypes that fostered
neurodegeneration (Sarasija et al., 2018). Another sel- 12 mutant
phenotype, an egg-laying defect, can be rescued by mutating the
worm REST ortholog, spr-4 (Lakowski et al., 2003). These spr-4
mutations, originally isolated from a forward genetic screen for
egg-laying defects, have since revealed insights into pathways
regulating neurodegeneration. For example, mutations in SPR-
4/REST enhance Aβ-induced glutamatergic neurodegeneration in
transgenic C. elegans; this result correlated with those from
companion studies in mammals (Lu et al., 2014).
Notably, mutations in C. elegans pink-1 and pdr-1, which are
homologous to the recessive familial PD proteins PINK1 and
PRKN, do not display neurodegenerative phenotypes even
though they are null and strong loss-of-function mutations,
respectively. However, these genetic lesions do cause dopamine-
dependent behavioral deficiencies (Cooper et al., 2017) that
could potentially be used in screens for gene-by-gene or gene-
by-environment interactions. As examples, exposing pdr-1
mutant worms to either a secondary stressor such as α-syn, a
proteasome inhibitor (i.e. MG-132) or the neurotoxic dopamine
analog 6-hydroxydopamine (6-OHDA)
13
enhances neurodegeneration (Martinez et al., 2015; Hartman et ARF-like GTPase gene product, ARL-8 (Griffin et al., 2018).
al., 2019). Conversely, pink-1 mutant worms in which α-syn is Given the high conservation of endocytic components among cells
expressed in the dopaminergic neurons have significantly and species, the
enhanced neurodegeneration, but no sensitivity to the proteasome
inhibitor MG-132, and exhibit reduced neurodegeneration upon 6-
OHDA exposure (Martinez et al., 2015; Hartman et al., 2019).
These studies highlight the ability to dissect mechanisms in a
genetically tractable organism as an informative prelude to more
expensive and time- consuming validation studies in mammalian
models.
Modeling protein misfolding

Many researchers also want to establish whether neurodegeneration
is associated with alterations in protein handling. However, the
small size of nematode neurons render visual inspection of
protein misfolding a challenge and impractical for screening
or serial analyses. To circumvent this limitation, models of
pathogenic proteins conjugated to fluorescent molecules have
been expressed in the large body wall muscle cells in C.
elegans. This enables researchers to assess changes due to
alterations in protein misfolding, apparent aggregate density or
count following RNAi or small- molecule exposures, in distinct
genetic backgrounds and over the course of aging. Although these
are not neurodegenerative readouts, the ease with which rapid
visual screening can be conducted in muscles has afforded
researchers a means toward preliminary identification of
putative functional modifiers of pathogenic proteins that can
be subsequently investigated in neurons and/or mammalian cells.
As outlined in the following sections, this strategy has identified
multiple gene and drug modifiers of neurodegeneration that have
successfully predicted outcomes in mammalian models.
As many neurodegenerative diseases have concomitant defects
in protein handling, C. elegans researchers have developed
models of pathogenic protein misfolding (i.e. Aβ 1-42, polyQn, tau
and α-syn) in the nematode body wall muscle cells (see poster,
‘Strategies to examine neurodegeneration’). Misfolded protein
density and/or count of aggregated pathogenic proteins can be
assayed in the α-syn models (Hamamichi et al., 2008; van Ham et
al., 2008). Following hypothesis-based bioinformatics approaches
to systematically define candidate targets potentially associated
with PD, RNAi was used to identify genetic modifiers of α-
syn::GFP misfolding and accumulation in screens of hundreds of
candidate targets, initially in body wall muscle cells (Hamamichi
et al., 2008; Knight et al., 2014). Upon a winnowing of
candidates, the significantly shorter lists of hits were then
examined for knockdown or overexpression in dopaminergic
neurons (Hamamichi et al., 2008; Knight et al., 2014). Using this
tiered strategy in screens for α-syn- related phenotypes,
neuroprotective candidates identified in
C. elegans have been repeatedly validated in mammals and/or in
human genetic studies. For example, overexpression of the
lysosomal trafficking protein VPS41 (VPS-41 in C. elegans)
protects cells against several PD-related neurotoxins, including 6-
OHDA and rotenone (Ruan et al., 2010). Moreover,
overexpression of VPS41 decreases the levels of α-syn protein
levels in human neuroglioma cells (Harrington et al., 2012).
Considering the pathological overlap between PD and AD, VPS-
41 was examined for therapeutic potential using C. elegans Aβ
paralysis and neurodegeneration models. Notably, VPS-41
protected in both diseases, but with notable functional distinctions
between the modes of neuroprotection between PD and AD.
Specifically, in the α-syn model, neuroprotection is mediated via
RAB-7 and AP-3, while in the Aβ model, it occurred through an
14
ability to dissect such mechanistic distinctions in neuroprotective candidates that reduce
pathways using C. elegans warrants further consideration when
prioritizing targets for in vivo modeling.
In the Aβ models of AD, Aβ peptide accumulation in muscle
cells induces paralysis, which can be readily quantified. Two
different muscle expression models are widely used in the field;
one constitutively expresses Aβ3-42 and is utilized for studies of
toxicity and metabolism (Link, 1995), while the other provides
inducible expression of Aβ1-42 (Link et al., 2003). RNAi screens
can identify the targets of drugs previously shown to inhibit Aβ
aggregation. Studies of the antioxidant resveratrol in C. elegans
have demonstrated that it is neuroprotective and prevents the
formation of Aβ aggregates (Regitz et al., 2016). Following
verification of this protection in
C. elegans constitutively expressing Aβ3-42 in the body wall
muscles, RNAi was performed on proteostasis-related targets.
Notably, UBL- 5, part of the mitochondrial UPR (UPRmt), was
identified as a critical component of resveratrol-mediated aggregate
prevention and XBP-1, a key regulator in the UPRER, was also
necessary (Regitz et al., 2016). The polyQn HD body wall muscle
expression models can provide both quantitative and
behavioral information. Aggregate accumulation
positively correlated with adverse effects on motility,
which worsened with increasing polyQ lengths (Q29 versus
Q35 versus Q82) (Satyal et al., 2000). These models have been
used in concert with either α-syn or Aβ models in comparative
RNAi screens to parse gene candidates into subcategories for
those that have a common basis in proteostasis malfunction
versus those that drive a more pathogenic-protein specific
misfolding event (Nollen et al., 2004; van Ham et al., 2008;
Knight et al., 2014). For example, a genome-wide RNAi
screen performed on animals expressing polyQ expansions
identified genes involved in RNA processing and in the
synthesis, folding, trafficking and degradation of proteins as
important in polyQ aggregate formation (Nollen et al., 2004). A
subsequent screen using animals expressing misfolded α- syn
identified ER/Golgi vesicle-trafficking and quality control
genes (van Ham et al., 2008).
Chemical modulation of neurodegeneration

A variety of chemical compounds can alter the extent of
neurodegeneration in C. elegans (see poster, ‘Strategies to
examine neurodegeneration’). Neurodegeneration enhancers such
as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-
OHDA induce PD-like phenotypes in C. elegans, rodents and
other model organisms (Betarbet et al., 2002; Braungart et al.,
2004; Cao et al., 2005). Conversely, chemical modulators can
also attenuate neurodegeneration through distinct mechanisms.
Bafilomycin, for example, reduces neurodegeneration by
inhibiting autophagic vesicle maturation (Pivtoraiko et al.,
2010), dantrolene reduces neurodegeneration through inhibition
of intracellular calcium release in the ER (Xu et al., 2001), and
probucol attenuates neurodegeneration via its antioxidant
properties (Ray et al., 2014). Diverse modulators, applied alone
or in tandem with transgenes, coupled with medium- or high-
throughput screening, facilitate the discovery of drugs to combat
neurodegenerative diseases.
In addition to the aforementioned examples, a transgenic
C. elegans ALS model expressing mutant TARDBP was an
initial platform for screening chemical compounds; the hits,
neuroleptics, were then validated in a zebrafish model, and the
most potent molecules were subsequently examined in mice and
in a small clinical trial (Patten et al., 2017). This screen is among
the first in the neurodegenerative disease class to realize the true
translational potential for target engagement in humans. In
another example, after screening >14,000 chemical
15
aggregation and fibril formation in a human neuroblastoma cell relevance to neurodegenerative disorders that we describe here (see
line (SH-SY5Y), a hit compound also reduced α-syn-induced poster, ‘Representative neurobehavioral assays’). For many,
neurodegeneration in C. elegans (Pujols et al., 2018). The automated image software programs assist with accuracy. It
predictive nature of these screening results, based on genomic and should be noted
cellular homology across species, demonstrates the utility of this
nematode as part of a comprehensive discovery scheme.
Monitoring neuronal cell health

The transparent nature of C. elegans is useful for gauging cellular
health via fluorescent reporters (see poster, ‘Strategies to examine
neurodegeneration’). As both autophagy and mitochondrial
maintenance or impairment have emerged as pivotal to disease
and aging (Chan, 2006; Malpass, 2013), several such reporters
inform cellular health within the context of neurodegenerative
diseases.
The autophagy marker LGG-1 [the worm homolog of LC3
(also known as MAP1LC3A)] is expressed in the muscles,
intestine, pharynx, vulva, hypodermis, somatic gonad and nervous
system of C. elegans, and allows detection of autophagosomes using
the fluorescent reporter GFP::LGG-1 (Palmisano and Melendez,
2016). This marker shows diffuse intracellular expression under
baseline conditions, with increased expression becoming evident in
conditions that induce autophagy, such as starvation. Monitoring
the localization and/or intensity of GFP::LGG-1 can query gene
targets that functionally affect autophagy. Another transgenic line
to evaluate mitophagy consists of the worm ortholog of human
mitophagic regulator BNIP3, DCT-1, tagged with GFP along with
dsRed::LGG-1 (Palikaras et al., 2015). DCT-1::GFP localizes to
the outer mitochondrial membrane and dsRed::LGG-1 labels
autophagosomes. Mitophagy-inducing stimuli can be visualized
by the colocalization of these markers. Likewise, mitochondrial
fragmentation can be visualized by expressing TOMM-
20::mCherry in C. elegans (Jiang et al., 2015). The localization of
the mitochondrial import receptor TOMM-20 to the outer
mitochondrial membrane allows researchers to study conditions
that interrupt the fusion/fission balance.
Assays can quantify mitochondrial stress response through the
activation of the UPRmt. This mitochondrial quality control
program is activated during periods of stress to promote survival
and recovery of mitochondrial function. One response of this
pathway is the induction of the chaperone HSP-6. By tracking the
fluorescence intensity of an hsp-6::GFP transcriptional reporter,
researchers can measure the activation of UPRmt (Haynes et al.,
2007). This readout has proven key to deciphering the impact of
the UPRmt in neurodegenerative disease. Notably, in transgenic C.
elegans PD models, α-syn overexpression appears to co-opt this
pathway from a normally protective response to transient stressors
into a potential contributor to proteostatic collapse and
neurodegeneration when chronically activated (Martinez et al.,
2017).
C. elegans research ushered in the revolution in live imaging
with the Nobel prize-winning report of GFP by Marty Chalfie and
colleagues (Chalfie et al., 1994) and the toolbox of in vivo
reporters of bioactivity for worm research has always been
substantial, as the examples discussed here highlight. With the
advent of CRISPR/ Cas9 editing, novel optogenetic reporters,
increasingly diverse indicators and high-resolution microscopy
methods, C. elegans is poised to remain at the forefront of
technologies that can evaluate neuronal function and health in
disease modeling.
Evaluation of neurobehavior
Researchers have developed several common assays with
16
that this section is not a comprehensive survey, as we are unable
to describe all the variations and related assays in this space. For
a complete review and applications of worm behavioral assays
and detailed protocols go to www.wormbook.org.
General neuronal dysfunction assay

The thrashing assay is a basic assay for generalized neuronal
dysfunction. Worms placed in liquid rapidly initiate a response
termed ‘thrashing’; a thrash is defined as a directional change in
mid-body bending. During the thrashing assay, the frequency of
lateral swimming is measured over a short time (30-60 s) for
each animal. This assay can be used as a functional readout of
neuronal health of transgenic and drug-treated worms. Kraemer
et al. (2003) used thrashing assays to discern behavioral
distinctions between normal and mutant forms of human tau
linked to FTDP-17 expressed pan-neuronally in C. elegans. The
FTDP-17 variants exhibited decreased thrashing independent of
tau expression levels. Similarly, another C. elegans model in
which neurodegeneration was induced via GABAergic neuron
expression of arginine-rich dipeptide repeat proteins designed to
functionally mimic the effects induced by such repeats in the
human C9orf72 protein, a prevalent cause of ALS and FTD. The
altered motility in these animals was associated with dipeptide-
repeat expression and correlated with morphological changes in
neuron structure indicative of neurodegeneration (Rudich et al.,
2017).
Motor neuron dysfunction

One convenient tool for the identification of genes that code for
synaptic transmission regulators is the quantification of
sensitivity to aldicarb, an acetylcholinesterase inhibitor.
Beginning with the seminal research of Rand and Russell (1984),
aldicarb has been used to define many conserved components of
cholinergic neurotransmission. Aldicarb causes paralysis
in wild-type
C. elegans, while animals with mutations in acetylcholine
neuromuscular signaling at the synaptic cleft are resistant. In
contrast, some animals might be more sensitive to aldicarb,
indicating increased acetylcholine release. Worms are exposed to
aldicarb on a Petri dish, and populations are assessed for
paralysis every 10-30 min for a total of 150 min. A study of the
neurotoxic selectivity of perfluorooctane sulfonate, a chemical
widely used in industry, showed that exposure causes
dopaminergic neuron deficits and, through the use of aldicarb,
found that this was independent of acetylcholine function
(Sammi et al., 2019). In another study, an aldicarb-based assay
showed that DNJ-14, a worm homolog of the human cysteine-
string protein (DNAJC5), a co-chaperone necessary for synaptic
maintenance, was not required for neuromuscular transmission
in aging (Mulcahy et al., 2019). Conversely, in an ALS/FTD
model, Vaccaro et al. (2012a,b) showed that transgenic worms
expressing either mutant or wild-type human TDP-43 and FUS
differed in their response to aldicarb, with the mutants exhibiting
hypersensitivity to paralysis. In this example and others, aldicarb
assays provide a means to correlate behavioral deficits with
neurodegeneration.
Dopaminergic neuron dysfunction

Wild-type C. elegans display a dopamine-mediated slowing of
locomotion upon entry into a bacterial lawn, owing to a
mechanosensory process that detects textural change (Sawin et
al., 2000). This dopamine-regulated behavior is termed the basal
slowing response (BSR) and is measured by counting the number
of body bends in 20-s intervals. Rates are compared between
worms placed on plates with food (Escherichia coli) versus
rates of the
17
same worms on plates without food. This method is a phenotypic intersection of aging and neurodegeneration
readout to assess functional changes in dopaminergic neurons.
Experiments typically compare the change in body bends over
time to positive control worms that do not slow in the presence of
food, such as cat-2 mutants, which lack tyrosine hydroxylase, the
rate- limiting enzyme for dopamine synthesis. The BSR assay can
elucidate pathways leading to the dysfunction and death of
dopaminergic neurons when exposed to toxic substances. As
examples, lead (Pb2+) exposure increases dopaminergic
neurotoxicity in wild-type C. elegans, but protein kinase ( pkc)
mutant animals are resistant, an effect that can be quantified via
the BSR assay (Akinyemi et al., 2019). Similarly, BSR assays
have revealed that Akt signaling provides resistance to
manganese- induced dopaminergic neurotoxicity (Peres et al.,
2019). This assay is also informative when analyzing the
contributions of candidate genes to dopaminergic function. For
example, the BSR assay was used to reveal that depletion of the
C. elegans RAC1 GTPase CED- 10, in the presence of α-syn
overexpression in dopaminergic neurons, contributes to
dopaminergic dysfunction (Kim et al., 2018b). Importantly, BSR
defects precede neurodegeneration in C. elegans PD models
(Martinez et al., 2017); the influence of temporal factors
mediating the aging process can thus be evaluated for their
progressive impact on dopaminergic neurons over time (Gaeta et
al., 2019).
Glutamatergic neuron dysfunction

A wild-type hermaphrodite C. elegans has six sensory neurons
spanning its length that detect light touch as administered by
stroking the animal with an eyebrow hair glued to a toothpick.
This is a mechanosensory response where, normally, animals will
exhibit either forward or backward reversal in locomotion when
stroked toward the posterior or anterior, respectively (Goodman
and Sengupta, 2019). Touch neuron degeneration results in a
mutant mechanosensory (Mec) locomotive phenotype with
defective forward or backward, or both, movement indicative of
glutamatergic dysfunction. As previously described, this assay
was used as a readout to examine phenotypic rescue of Aβ-
induced degeneration of glutamatergic neurons in the presence of
different isoforms of human APOE (Griffin et al., 2019). As
another example, animals treated with quinolinic acid exhibited
neurodegeneration due to glutamatergic neurotransmission
defects (da Silveira et al., 2018). Similarly, in single-copy knock-
in SOD1 models of ALS, loss of sod-1 function produced defects
in light touch response indicative of a disruption in glutamate
signaling (Baskoylu et al., 2018).

Correlation between neurodegeneration and aging
Assays that assess whether a treatment or condition affects
lifespan and/or healthspan are well established in the field (see
poster, ‘Neurodegeneration and aging’). Lifespan consists of
healthspan and gerospan. Long-lived animals experience a frailty
period termed gerospan, whereas healthspan is defined as the
period when an animal has greater than 50% maximal functional
capacity (Bansal et al., 2015). Considering that aging is the main
risk factor for AD, PD and other neurodegenerative diseases,
lifespan and healthspan benchmarks of animal health are
appropriate companion assays. Aging-associated molecular
signatures, such as epigenetic changes, telomere shortening,
proteostasis inhibition, mitochondrial dysfunction, altered lipid
metabolism and nutrient sensing dysregulation, among others, can
be assessed (Lopez-Otin et al., 2013). Notably, neurodegeneration
is associated with many of these cellular processes, and the
18
can often be efficiently examined using C. elegans models
(Knight et al., 2014).
In mammals, the lengthy time requirements to evaluate age-
associated changes can be considered a hindrance to higher-
throughput analyses. In contrast, the average ∼20-day lifespan
of the C. elegans wild-type strain Bristol N2 accelerates the
discovery of fundamental modulators of neurodegenerative
processes within the context of lifespan. A common
misconception is that the short survival of C. elegans precludes
its utility in translating lifespan modifiers in this animal to
mammals. A variety of mutants and chemical modifiers of aging
have been identified that directly affect highly conserved
metabolic pathways and mechanisms shared among metazoans.
The most notable example is the daf-2/insulin- like signaling
pathway (Martins et al., 2016), whereby C. elegans research has
led to the discovery of critical modulators of cellular
and organismal health (i.e. mTOR, Nrf2, FOXO/DAF-16).
Among the biological modulators that can be readily examined in
C. elegans are genetic and epigenetic modifiers, chemical
modifiers, microbiome exposures and putative environmental
toxicants, in addition to exercise and diet.
Lifespan is assessed by the cohort survival assay (Lionaki and
Tavernarakis, 2013), in which at least 120 synchronized L4-
stage worms are grown at 20°C and scored for survival/death
every 24 h by gently tapping with a platinum wire at both head
and tail. A log- rank (Mantel–Cox) test is used to compare
survival between strains. Investigation of the role of
nicotinamide-N-methyltransferase (NNMT) found that
expression of the C. elegans homolog, ANMT-1, in
dopaminergic neurons increased neuron survival and overall
lifespan through the regulation of autophagy (Schmeisser and
Parker, 2018). A second study showed that the transcription
factor SPR-4/REST regulates aging by reducing neural activity
and maintaining neural homeostasis (Zullo et al., 2019).
Reproducibility of lifespan data is contingent on rigorous
experimental design and repetition. To evaluate variability in
quantifying lifespan, Lucanic et al. (2017) reported results from
a coalition of multiple laboratories termed the Caenorhabditis
Intervention Testing Program that assessed longevity for 22
strains, inclusive of three Caenorhabditis species. Multiple
replicates were collected from three independent laboratories to
reveal that diversity in replicate data within a single laboratory
for a given strain was more common than variation in lifespan
determined across different laboratories. This study highlights
the importance of tightly controlled experiments and internal
consistency in the replication of lifespan data.
Healthspan
Like humans, C. elegans exhibits a decline in physical ability
with age and loss of ability to recover from stress, which
manifest as reduced body movement and increased sensitivity to
heat and oxidative stress, respectively (Bansal et al. 2015).
When searching for genetic modifiers, increased healthspan
should be prioritized because long-lived mutants also have
increased gerospan (Bansal et al., 2015), which is
counterintuitive to enhancing the quality of life. Multiple assays
can be performed to assess healthspan. For a comprehensive
review, see Bansal et al. (2015). For example, an automated
digital video microscopy system examines the average distance
C. elegans travel on solid agar (Hahm et al., 2015). In a second
assay, healthspan can be examined in liquid media. Using this
method, researchers learned that C. elegans models of
neurodegenerative disease had better outcomes following swim
training exercise, which improved their neuronal healthspan
(Laranjeiro et al., 2019). A third healthspan assay consists
of
19
oxidative stress analysis. Worms are transferred to plates Acknowledgements

We are grateful to Dan Shaye for providing us OrthoList 2 data and to Steve Cook
containing paraquat, and stress-resistant survival is recorded every
for his insights into C. elegans neuroanatomy described in the Introduction section
5 days until death (Bansal et al., 2015). Using this assay, it was of this article. To our colleagues whose C. elegans models and/or assays have
discovered that a gene product associated with cholesterol not
metabolism that was neuroprotective in a C. elegans PD model
maintained healthspan but did not extend lifespan (Zhang et al.,
2017).
Addressing lifespan and healthspan, along with other cellular
signatures such as mitochondrial and/or ER function and stress
responses, as well as alterations in proteostasis and transcriptional
or macromolecule profiling (RNA/DNA/miRNA/lipids), provides
the
C. elegans researcher with an arsenal of complementary
approaches with which to investigate neurodegenerative diseases.
As noted below, the ‘big data’ increasingly emerging from human
genomic analyses provides a greater impetus for the application
of a more rapid and cost-effective system whereby the
significance of genetic variation can be discerned for its impact
not only on disease, but also on quality of life.
Concluding remarks
Among the largely untapped potential avenues of C. elegans
models of neurodegeneration is the functional annotation of
genomic variation among humans to discern factors that
confer either susceptibility or resilience. The ever-expanding
databases of human sequence information have resulted in an
informational overload of variants of uncertain significance
(Cooper, 2015). Characterizing these and other changes via
functional analyses with the bioassays outlined in this article
could greatly advance our understanding of therapeutic options.
While it may superficially appear illogical to use an invertebrate
such as C. elegans for human personalized medicine, there are
successful examples of highly specialized drug discovery tailored
to the genomic composition of cancer patients (Bangi et al.,
2019). C. elegans is also being used in the drug discovery
efforts for rare glycosylation disorders, N-glycanase 1 (NGLY1)
deficiency and phosphomannomutase 2 (PMM2) deficiency
(Iyer et al., 2019a,b).
In closing, this article prompted us to reflect on a Primer that we
had the privilege of co-authoring for the inaugural issue of Disease
Models & Mechanisms. At the inception of this journal as a
platform to communicate the increasingly important role of model
systems in advancing disease research, we titled our article
‘Traversing a wormhole to combat Parkinson’s disease’ (Caldwell
and Caldwell, 2008). The choice of this celestial analogy now
appears prescient in capturing the exceptional contributions of C.
elegans models in rapidly advancing discoveries from the
experimental space through to the other side of clinical
investigation. We contend that the expedience, accuracy,
molecular tools and mechanistic capacity of C. elegans present an
attractive, bioethical alternative to more costly, time- consuming
and sometimes redundant in vitro or mammalian models. With an
increasingly strong track record of translational outcomes that
continue to emerge from C. elegans research, this microscopic
nematode is positioned to help fill the remaining black holes in
our understanding of neurodegeneration. Indeed, the gravity of the
societal burden from these diseases is worldwide, and weighs on
tens of millions of individuals each day. A sense of urgency and
innovative strategies that coalesce into model systems research
are essential to accelerate discovery and progress. In the words of
an admired and fearless explorer known to go where no one (or
no worm) has gone before, “Warp speed ahead. Engage!” (Jean-
Luc Picard).
20
been covered, we apologize for the space limitations that necessitated these
omissions. We further acknowledge the collective contributions of the C. elegans
research community in developing shared resources that have generously made
the advancements outlined in this article possible.
Funding
K.A.C. was funded by National Institutes of Health (NIH) grant R15NS106460;
G.A.C. was funded by NIH grant R15NS104857.
At a glance
A high-resolution version of this poster is available for downloading in the
online version of the article at
http://dmm.biologists.org/content/13/10/dmm046110/F1. poster.jpg.
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© 2020. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2020) 13, dmm046110. doi:10.1242/dmm.

Modeling neurodegeneration in Caenorhabditis elegans

Disease Models & Mechanisms

KEY WORDS: Aging, Behavior, Genetics, Modeling, Phenotype,

Disease Models & Mechanisms

Genetic models of neurodegeneration

ALS and FTD

Tools and approaches for the functional characterization of

Forward genetic screening

Reverse genetic screening

Disease Models & Mechanisms

Strategies to examine neurodegeneration

Genetic crosses to examine neuronal phenotypes

Modeling protein misfolding

Chemical modulation of neurodegeneration

Monitoring neuronal cell health

General neuronal dysfunction assay

Motor neuron dysfunction

Dopaminergic neuron dysfunction

Glutamatergic neuron dysfunction

Disease Models & Mechanisms

oxidative stress analysis. Worms are transferred to plates Acknowledgements

Caldwell, G. A. and Caldwell, K. A. (2008). Traversing a wormhole to combat

Disease Models & Mechanisms

Disease Models & Mechanisms

Disease Models & Mechanisms

Disease Models & Mechanisms

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