C. elegans and Neurodegeneration In Caenorhabditis Elegans: Anatomy, Life Cycles and Biological Functions

I. Introduction

Parkinson’s Disease (PD), Alzheimer’s Disease (AD), Huntington’s Disease (HD) and Amyotrophic Lateral Sclerosis (ALS) are some of the most prevalent neurodegenerative diseases, with ‘neurodegeneration’ referred to as a conglomeration of pathological hallmarks, such as dying back of neurons and neuronal cell death. The prevalence of neurodegenerative diseases is increasing as the population ages and life span increases, reflecting upon the improvement in lifestyle and healthcare. The molecular mechanisms underlying neurodegenerative diseases remain elusive as exposure to environmental contaminants influencing the neurodegenerative pathology is generally lacking. Nevertheless, improvements in diagnosis have greatly enhanced the understanding of the neuropathology and the relationship between environment, genetics and risk susceptibility.

The complexity of the vertebrate brain has made the study of neurodegenerative disease processes slow, difficult and expensive. The C. elegans model has offered a faster and less expensive alternative, while still serving as a viable in vivo model system. At its inception, the C. elegans platform was developed as a tool to study nervous system development (Brenner, 1974). Subsequently, the organism has been meticulously characterized, with differentiation and migration patterns elucidated (Sulston, 1983; White, 1986), allowing for analysis of changes in nervous system in response to mutations and toxic insults. Due to advances in characterization of C. elegans behaviors and their links to specific neural circuits additional insights into mechanisms have been made, many of which through computerized software quantification of behaviors such as thrashing, chemotaxis, and pharyngeal pumping.

C. elegans offers many advantages over other model systems in deciphering mechanisms of neurodegeneration. Of particular importance for the study of neurodegenerative processes are the nematode’s small genome, anatomical simplicity and availability of a complete three-dimensional map of the 302 cell nervous system (Hobert, 2005). The accessibility of the full sequence of the nematode genome (Consortium TCeS, 1998) and a high-density map of polymorphisms for the wildtype nematode allows for mapping of gene mutations and linking of mechanisms to genetic susceptibility. RNA interference, transgenics and site-directed mutagenesis are also useful tools for generating gene knockdowns and loss-of-function mutants, which are increasingly important in revealing modifier pathways. The C. elegans male has been beneficial in generating lines with multiple mutations and the homozygosity of these mutations is easily sustained by the self-fertilizing hermaphrodites.

Contributing to the investigative value of this species is the transparency of the worm, allowing for in vivo visualization of fluorescently-labeled neurons at the individual process level, as well as any neurodegeneration that has occurred, including puncta, loss of cell bodies or strand breaks in neuronal processes. Green fluorescent protein (GFP) has frequently been employed to visualize specific neurons and synapses in living animals. Additionally, the ease of making reporter gene fusions further facilitates visualization of neuronal morphology and protein expression patterns. Sharing ~60-80% of human genes (Kaletta and Hengartner, 2006), ~40% of human disease-related genes (Culetto and Sattelle, 2000) and conserved neurotransmitter systems; C. elegans have high homology to mammalian systems. In addition to the high level of gene conservation, the processes of synaptic release, trafficking and formation are also conserved. Based on the conservation of genetics and cell differentiation processes between the nematode and mammalian systems, the C. elegans model has emerged as a useful tool in deciphering mechanisms of neurodegenerative damage.

C. elegans has been used as a model for various neurodegenerative disorders (Berkowitz et al., 2008) following exposure to several classes of toxicants including metals and pesticides. Various neurodegeneration endpoints have been assessed including puncta, strand breaks and cell death. Methods to study these neurotoxicologic endpoints have included examining the morphology, behaviors, and gene expression of the C. elegans nervous system. Metals such as aluminum, arsenic, barium, cadmium, copper, lead, mercury, uranium, and zinc have been assessed in the C. elegans model, revealing the mechanisms through with these agents exert their neurodegenerative effects (reviewed in Martinez-Finley et al., 2011). Although the mechanisms by which metals exert neurodegenerative damage is metal- and dose-dependent, they share common mechanisms including free radical production, protein aggregation, bioenergetic dysfunction, calcium dysregulation and metal transport alteration (Gaeta and Hider, 2005; Levenson, 2005; Price, 1999). In addition, the toxicity of pesticides, including paraquat, rotenone, and organophosphates have been investigated using the C. elegans model and assessed by behavioral and morphological alterations upon toxicant exposure (Leung et al., 2008).

Genetics is an important determinant of predisposition to neurodegenerative disease and due to the growing knowledge of environmental influences on genetic background and the outcome of disease, and there has been an increase in the need for a reproducible, high-throughput in vivo approach to address such interactions. The C. elegans model system has provided researchers with such a system. In summary, C. elegans is a model amenable to studying neurodegeneration and revealing mechanisms of toxicity of a wide range of neurodegenerative toxicants by exploiting the many advantages of the model. In this chapter we give an overview of the ways in which the powerful C. elegans model has provided us with insights into PD, AD and other types of neurodegeneration.

II. Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia worldwide, affecting more than 30 million people (Association, 2008; Brookmeyer et al., 2007). It is characterized as a progressive, terminal neurodegenerative disorder and is typically diagnosed in people older than 65. Primary symptomology includes memory loss with secondary symptoms including confusion, irritability, language breakdown and social withdrawal. The pathology underlying the neurodegenerative process of AD is complex and is characterized by synaptic damage and neural loss with presence of senile plaques and neurofibrillary tangles composed of β-amyloid (Aβ) and hyperphosphorylated and aggregated microtubule-associated tau protein, respectively. The exact cause of the deposits is not fully understood nor is their correlation to disease progression. Aβ plaques are produced through the amyloidogenic pathway by cleavage of β-amyloid precursor protein (APP) by β- and γ-secretase (Jacobson and Iverfeldt, 2009). Toxic oligomers are thought to be driven more by Aβ than by neurofibrillary tangles (Crews and Maslian, 2010). Phosphorylated tau protein normally functions to stabilize microtubules and provide a support and transport system for neuronal survival however in the case of AD, tau becomes hyperphosphorylated, which leads to disintegration of the neural transport system due to the contact of the tangles with microtubules (Li et al., 2007).

Disease-linked mutations and neurodegeneration

Late-onset sporadic AD has not been linked to genetic mutations; however familial AD has been linked to several genetic mutations (Campoin et al., 1999; Chartier-Harlin et al., 1991). C. elegans contribution to the literature on AD was based on transgenic models of worms expressing the human betaAPP (Boyd-Kimball et al., 2006; Drake et al., 2003; Gutierrez-Zepeda and Luo, 2004; Wu and Luo, 2005; Wu et al., 2006) or tau protein (Kraemer et al., 2004). These models revealed novel insights into AD pathology. For example, the beta APP model identified two new genes, aph-1 and pen-2, involved in disease progression (Boyd-Kimball et al., 2006; Francis et al., 2002) and identified genes activated following betaAPP induction (Link et al., 2003). C. elegans have an APP gene, apl-1 however the protein lacks the Aβ peptide and the genome also does not encode for a β-secretase (Jacobsen and Iverfeldr, 2009; Daigle and Li, 1993). The apl-1 gene encodes for a single-pass transmembrane domain protein with conserved domains and deletion of apl-1 results in 100% larval lethality (Hornsten et al., 2007), while RNAi knockdown of apl-1 produces animals with reduced body size (Niwa et al., 2008). Wiese and colleagues reported that the lethality and molting observed in apl-1 worms can be rescued by full-length APL-1 C-terminal truncation (Wiese et al., 2010). They also showed that knockdown of small GTPase rab-5 decreases the amount of apl-1 expression in neurons, however loss-of-function of the small GTPase UNM-108 leads to retention of APL-1 in the cell body (Wiese et al., 2010). Overexpression of apl-1 resulted in interference of motor neuron functions including reduced swimming and crawling rates compared to wildtype as well as defects in brood size, and viability which were shown to be correlated with the level of APL-1 (Hornsten et al., 2007).

Beta-amyloid peptide precursor protein and the presenilins, PS1 and PS2 were the first AD-associated proteins identified. C. elegans presenilin orthologs sel-12, hop-1 and spe-4 (Baumeister et al., 1997; Levitan and Greenwald, 1995; Li and Greenwald, 1997; Smialowska and Baumeister, 2006) linked AD to the apoptotic pathway (Kitagawa et al., 2003) and Notch signaling (Berezovska et al., 1998; 1999). Mutations in worm orthologs of the presenilin proteases responsible for cleaving APP through the γ-secretase pathway, favored in the pathogenesis of AD (Nunan and Small, 2000; Selkoe, 1999), such as a sel-12 mutation, have revealed deficits in thermal memory necessary for proper thermotaxis, a behavior that is dependent upon the AFD sensory neurons and AIZ and AIY interneurons. The same model exhibits increased vulnerability to AD as shown by axonal abnormalities in acetylcholine-producing AIY neurons (Wittenburg et al., 2000). The C. elegans beta APP orthologue gave insights into the role of microRNAs in the regulation of AD genes (Niwa et al., 2008). Link and colleagues have engineered C. elegans models expressing Aβ_(1-42) (Link, 1995, 2006; Link et al., 2003; Wu et al., 2006). Worms expressing fluorescently-lableled Aβ_(1-42) under the constitutive unc-54 body-wall muscle promoter show progressive paralysis and intracellular cytoplasmic Aβ plaques (Link, 1995; Link et al., 2001). In another model, the same group expressed a temperature-inducible Aβ transcript driven by the myo-3 muscle-specific promoter and regulated by an incorporated long 3’ untranslated region (3’UTR). These worms display a paralysis phenotype due to the stable Aβ_(1-42) mRNA and protein (Link et al., 2003; Link, 2006). Gene expression changes in these worms have been assessed via DNA microarray with 67 upregulated and 240 down-regulated genes identified following intracellular accumulation of Aβ (Link et al., 2003). The two upregulated families, αB-crystallin and tumor necrosis factor-induced protein 1, have also been shown to be increased in AD brains (Link et al., 2003). Another group, using the same model, demonstrated that overexpression of arsenite-inducible protein (AIP-1) suppressed Aβ accumulation and paralysis via increased protein turnover and clearance (Hassan et al., 2009). An additional model of Aβ toxicity contains intraneuronal deposition paired with hypersensitivity to serotonin and defects in chemotaxis (Link, 2006; Wu et al., 2006). Yet another strain that has been generated is Aβ constitutively expressed under the control of the unc-54 promoter. Cohen and colleagues used the C. elegans Aβ (unc-54/Aβ_(1-42)) strain to delineate the role of the insulin-like signaling pathway in AD and showed that toxicity is dependent on longevity genes (Cohen et al., 2006). DAF-2 knockdown, acting through DAF-16 and heat shock factor-1 (HSF-1) reduced Aβ_(1-42) toxicity. DAF-16 knockdown decreased the amount of toxic Aβ aggregation whereas hsf-1 knockdown increased the amount of toxic Aβ aggregation (Cohen et al., 2006). Another group identified six Aβ-interacting proteins, HSP70A and HSP70C (HSP70 orthologs), HSP-16.1, 16.2, and 16.48 (HSP16 proteins) and the ortholog of the human SGT protein via mass spectrometry (Fonte et al., 2002; Liu et al., 1999). HSP-16 was also found to coimmunoprecipitate with intracellular Aβ (Fonte et al., 2002), an event that was further examined in the temperature-inducible model. HSP-16.2 overexpression in that model had no effect on the total Aβ accumulation but did partially suppress the Aβ paralysis phenotype (Fonte et al., 2008). An additional model is an inducible Aβ model with muscle-specific expression under the control of myo-3. Inducible expression of human Aβ_(1-42) in muscle cells was shown to lead to an accumulation of autophagic vesicles (Florez-Mcclure et al., 2007). They also showed that RNAi knockdown of lysosomal component genes such as asp-1, asp-4, asp-5 and asp-6 (aspartyl proteases), lmp-1 and lmp-2 (lysosomal-associated membrane proteins) and vha-15 (vacuolar proton-translocating ATPase) enhanced the paralysis rate and caused higher Aβ accumulation (Florez-Mcclure et al., 2007).

Additional models of AD involve genetic mutations in gene that encodes for the tau protein. In C. elegans the tau ortholog is called ptl-1 (Goedert et al., 1996). Because the ptl-1 gene does not accurately represent tau pathology, C. elegans transgenics expressing human tau have been used to recapitulate the disease conditions. Worms designed to overexpress pseudohyperphosphorylated mutant or human tau protein showed neurodegeneration, locomotor defects as a result of failed neurotransmission and other age-dependent motor neuron dysfunctions (Brandt et al., 2007; Kraemer et al., 2003). Mutations in the tau gene have been associated with the neurodegenerative disorder, frontotemporal dementia with parkinsonism chromosome 17 type (FTDP-17) and not specifically with AD. Human tau mutations associated with FTDP-17 have been introduced into C. elegans, and these strains exhibit uncoordinated locomotion, impaired cholinergic transmission and GABA-ergic axonal degeneration arising from the accumulation of insoluble tau (Kraemer et al., 2003). Tauopathies are the common name for neurodegenerative disorders associated with tau protein aggregates. Kraemer and colleagues examined pan-neural expression, under the control of the aex-3 promoter, of human brain tau isoform (4R1N) and mutant versions of tau (^V337^M and ^P301^L) and reported uncoordinated locomotion, axonal abnormalities, loss of GABAergic motor neurons and accumulation of tau aggregates (Kraemer et al., 2003). Another pan-neural model, under the control of the rgef-1 promoter, to express fetal human tau or pseudohyperphosphorylated (PTP) tau reported highly phosphorylated human neuronal tau in the nematode similar to those isolated from AD patients (Brandt et al., 2009). Aggregates and axonal abnormalities in inhibitory motor neurons associated with age-dependent uncoordinated movement were only observed in the PTP tau strain and not in wildtype worms (Brandt et al., 2009). Miyasaka and colleagues reported another model strain expressing human wildtype or FTDP-17 mutant tau, under the control of the mec-7 promoter in the mechanosensory neurons (Miyasaka et al., 2005). Mutant tau expressing worms showed an age-dependent decline in touch response, swelling in cell bodies, accumulation of phosphorylated tau and disorganized microtubules, not seen in the wildtype worms (Miyasaka et al., 2005). The group also reported in improvement in touch response with overexpression of human HSP70 and a slight increase in neurodegeneration with mammalian GSK-3β kinase overexpression (Miyasaka et al., 2005).

Chemical- and metal-induced neurodegeneration

Pathophysiology of AD has been associated with a number of genetic factors but several lines of evidence suggest that altered metal homeostasis might also be playing a significant role. The presence of copper, zinc, iron or aluminum in senile plaques in AD brains has highlighted metal toxicity in AD pathology (Bolognin et al., 2009). AD brains have enhanced zinc in the neuropil and copper in the rims of the plaques (Lovell et al., 1998). Copper, zinc and iron can reportedly cause conformational changes in Aß allowing stabilization of the oligomeric form and enhancing oxidative stress (Faller, 2009) and both iron and copper have high affinity for Aβ. Additional studies have shown colocalization of the divalent metal transporter (DMT1), important for transporting many divalent metals such as zinc, iron and copper, with Aß plaques in postmortem brain samples from AD patients (Zheng et al., 2009).

Hesse and colleagues examined the relationship between copper neurotoxicity, APP and AD using C. elegans (Hesse et al., 1994). They reported a copper-binding domain (Cu-BD) in the N-terminal region of APP between residues 135 and 156, containing two crucial histidines that are conserved in APP-related protein APLP2 (Hesse et al., 1994). This CuBD both binds copper and through a redox reaction reduces Cu(II) to Cu(I), producing reaction oxygen species (ROS) in the process (Multhaup et al., 1996). Copper and zinc binding sites are also found in the carboxy-terminal of APP, similar to Aß. Overexpression of the Aß-containing fragment leads to significantly decreased Cu and Fe levels that correspond with increased Mn levels (Maynard et al., 2002). Based on these studies, Aß and APP are therefore implicated as regulators of metal homeostasis, as Aß aggregations potentially disrupt proper metal levels in AD. In another experiment, the CuBD of C. elegans APL-1 was injected into rat dorsal hippocampus and conferred protection against Cu²⁺ toxicity in vivo by enhancing Cu²⁺ uptake, showing a direct regulation of extracellular Cu levels (Cerpa et al., 2004). White and colleagues have shown that the tyrosine residue at position 147 and the lysine residue at 151 in the C. elegans APL-1 CuBD are responsible for protection against Cu-mediated toxicity (White et al., 2002).

Point mutations (V18A, E22G) in C. elegans strains overexpressing Aß in their muscle cells result in decreased intracellular amyloid aggregation compared to wildtype Aß. Exposure to Cu²⁺ enhances acceleration of wildtype Aß aggregation in these worms. Despite evidence of increased amyloid deposits following Cu²⁺ exposure, the Aß transgenic worms show decreased sensitivity to toxic CuCl₂ exposures (150-450 μM) compared to control worms (Minniti et al., 2009). The expression of human Aß in C. elegans muscle cells was not found to be in its full-length (1-42) form but a (3-42) truncated Aß product which self-aggregates faster in vitro than Aß_1-42 and is enhanced by the presence of Cu²⁺ (McColl et al., 2009). These results suggest that metal-induced changes on the aggregation state of Aß is complex and may be coupled with protection against Cu²⁺ toxicity from the aggregates themselves.

Additionally, due to the presence of iron in AD plaques, Wan and colleagues measured iron levels in the Aβ-expressing strain (CL2006) and observed increased iron accumulation and oxidative stress (Wan et al., 2011).

Screens identifying modifier genes

Link and colleagues reported a transgenic C. elegans strain encoding Aβ_1-42 gene with the ability to detect overexpression of the protein in vivo (Link et al., 2006). Taking advantage of the behavioral deficit in the mutant tau-induced Unc locomotion strain, a genome-wide 16,000 gene RNAi screen for genetic modifiers that would affect the behavioral defect was run (Kraemer et al., 2006). Kinases, phosphatases, chaperones, and proteases were found that enhanced the tau-induced behavioral defect, some of which corresponded to mammalian GSK-3β, CSTE, CHRNA6 shown to be human tauopathy modifier genes (Kraemer et al., 2006).

The aex-3/tau transgenic model was used in a forward genetic screen identifying sut-1 (Kraemer and Schellenberg, 2007) and sut-2 (Guthrie et al., 2009) as novel candidate genes who loss of function mitigated neurodegeneration.

Testing for efficacy of Pharmacological compounds

Ginkgo biloba extract Egb 761, soy isoflavone glycitein and ginkgolides were first identified in the nematode as potential therapeutic drugs for AD as they were shown to increase lifespan, alleviate paralysis, serotonin hypersensitivity and chemotaxis and decrease toxic ROS levels (Wu and Luo, 2005; Gutierrez-Zepeda et al., 2005; Luo, 2006; Wu et al., 2006). Additionally, an FDA approved antihypertensive drug and VMAT inhibitor, reserpine, was tested in the unc-54/Aβ_1-42 strain and shown to enhance longevity, stress tolerance and delayed paralysis (Arya et al., 2009). The antidepressant fluoxetine (Keowkase et al., 2010), and tetracyclines (Diomede et al., 2010) were found to protect against Aß toxicity in worms, with an increase in thermal stress resistance and lifespan (Keowkase et al., 2010) and decreased oxidative stress (Diomede et al., 2010). In addition, copper chelators, histidine and clioquinol have been shown to diminish the formation of intracellular Aß deposits (Minniti et al., 2009). Choline analogs, JWB1-84-1 and JAY2-22-33 were tested in the Aβ constitutive muscle expression strain and JAY2-22-33 significantly reduced Aβ toxicity by delaying paralysis (Keowkase et al., 2010). The authors showed that the effect of JAY2-22-33 requires the insulin signaling pathway and nicotinic acetylcholine receptors (Keowkase et al., 2010). Wiese and colleagues used RNAi knockdown of apl-1 and reported that loss of apl-1 leads to sensitivity to the acetylcholinesterase inhibitor aldicarb, suggesting that synaptic function is defective in these worms (Wiese et al., 2010). They also showed that the hypersensitivity could be rescued by the application of full length APL-1 and this rescue was shown to be dose-dependent (Wiese et al., 2010).

III. Parkinson’s Disease

Parkinson’s disease (PD) is one of the common neurodegenerative diseases in the population, affecting over 1-2% of those over the age of 60(Lees et al., 2009). This brain disorder is marked by the selective loss of dopaminergic (DAergic) cells in the substantia nigra pars compacta (SNpc) region of the brain. Such distinctive degeneration initially results in emotional and cognitive decline, which is then followed by the hallmark features of bradykinesia, rigidity, tremors and postural instability. Symptoms start arising when approximately 70-80% of DAergic cells in the SNpc have been lost. Later stages may be characterized by the appearance of a masked-face, forward-flexed posture, gait freezing, shuffling steps, as well as gastrointestinal issues (Olanow et al., 2009). Nevertheless, with increasing life expectancy classifying the elderly as a growing proportion of the population, PD will become an even more critical public health concern. However, the mechanisms behind the selectivity in the cell loss underlying this disorder remain a mystery, making the discovery of a cure extremely challenging.

Disease-linked mutations and neurodegeneration

Although most PD cases are sporadic in nature, about 10-20% of cases have been linked to genetic causes. These genes include α-synuclein, leucine-rich repeat kinase 2 (LRRK2), parkin, DJ-1, and phosphatase and tensin homolog (PTEN)-induced novel kinase 1 (PINK1) (Dawson et al., 2010). C. elegans show conservation of all of the dopamine (DA)-related genes required for proper synthesis (CAT-2 homolog of TH (Sawin et al., 2000)), packaging (CAT-1 homolog of VMAT (Duerr et al., 1999), reuptake (DAT-1 homolog of DAT (Jayanthi et al., 1998) and signaling (DOP1-3 homologs of D_1/2 receptors (Suo et al., 2002, 2003, Chase et al., 2004).

Autosomal Dominant Genes

α-synuclein is the major component of cytoplasmic inclusions known as Lewy bodies that are found in the spared DAergic neurons of PD-plagued brains (Spillantini et al., 1997). This pathological hallmark is a feature of familial Parkinson’s disease (FPD) that manifests itself in an autosomal dominant manner, with missense mutations or multiplications leading to the disease. In particular, G209A (Polymeropoulos et al., 1997), A53T (Munoz et al., 1997) and A30P (Kruger et al., 1998) are the most well known FPD-associated point mutations. However, α-synuclein aggregation has also been found in some sporadic PD cases, as well as other neurodegenerative conditions that are collectively termed “synucleinopathies” (Spillantini et al., 1998). Thus, overexpressing α-synuclein specifically in DAergic neurons presents an ideal model for studying its contribution to the etiology of PD-induced neurodegeneration. However, it has been difficult finding a vertebrate model that overexpresses α-synuclein to successfully reproduce this selective neurodegeneration in order to generate DAergic-specific behavioral phenotypes. Despite the fact that worms do not endogenously express α-synuclein, the C. elegans system has become an increasingly popular model to study neurodegeneration generated from exogenous α-synuclein expression.

Using a transgene expressing both mutant and wildtype forms of human α-synuclein under the control of the dat-1 promoter, Kuwahara et al. (2006) successfully generated transgenic (Tg) C. elegans selectively expressing α-synuclein in their DAergic neurons. These Tg worms also show the characteristic accumulation of α-synuclein in both cell bodies and neurites, with the mutant A30P and A53T Tg worms showing degeneration of their DAergic dendrites. Remarkably, the mutant α-synuclein Tg worms show a behavioral phenotype of diminished food-sensing behavior. This is a dopamine (DA)-specific adaptive behavior required for C. elegans food intake: when worms sense bacteria (their food source) near them, they slow down their body bending movements in order to feed more efficiently (Sawin et al., 2000). This failure in body bending corresponds with lowered DA content in the A30P and A53T mutant Tg worms. The decreased behavioral output, however, is rescued upon exposure to exogenous DA (Kuwahara et al., 2006). Conversely, a recent study found using confocal laser scanning that expression of wildtype α-synuclein in worms results in increased intraworm dopamine and dopamine metabolite DOPAC content, in addition to impairment on dopamine synaptic vesicle distribution. These effects correspond with reduced numbers of DAergic neurons in α-synuclein Tg worms compared to wildtype controls (Cao et al., 2010). Moreover, both DAergic neuronal and dendritic degeneration is enhanced in worms expressing both wildtype and A53T α-synuclein under control of a pan-neuronal or the dopamine transporter-1 (dat-1) promoter, but not under a motor neuron promoter, indicating a degree of dependency on neuronal subtype specificity (Lakso et al., 2003). Thus, both the DA-specific neurodegeneration and behavioral decline seen in α-synuclein-linked PD has been successfully reproduced in C. elegans.

Another autosomal dominant gene linked to FPD is LRRK2/PARK8. Mutations in this gene were first connected to late-onset Parkinsonism through candidate gene sequencing in 46 families. Five missense mutations and one putative splice site mutation were found in LRRK2, whose protein product is part of the ROCO protein family (Ras/GTPase superfamily) and contains a leucine-rich repeat domain, MAPKKK-like protein kinase domains, among others (Zimprich et al., 2004). With the most frequent pathogenic mutation found in the kinase domain of LRRK2, recent studies have turned to finding putative inhibitors of LRRK2’s kinase activity, finding that LRRK2-induced neurodegeneration is kinase dependent in vivo (Lee et al., 2010). Additionally, LRRK2 has recently been shown to negatively modulate protein synthesis through interactions with the micro RNA (miRNA) pathway, hinting at the possibility of miRNA-directed therapies (Gehrke et al., 2010). The G2019S heterozygous mutation in the kinase domain and the R1441C/G mutation in the GTPase domain appear to be the most frequently found LRRK2 mutations associated with PD (Di Fonzo et al., 2005).

Nonetheless, most studies have ceased to model overt neurodegeneration as seen in PD, in addition to PD-associated behavioral phenotypes. Consequently, the mechanisms behind LRRK2-induced neurodegeneration still remain unclear, but the use of C. elegans has allowed for the recent in vivo modeling of LRRK2-linked PD. LRK-1, the C. elegans homolog of LRRK2, was first found to impair the proper localization of synaptic vesicle proteins (Sakaguchi-Nakashima et al., 2007). The closer connection to PD came in the form of studying mitochondrial dysfunction in C. elegans, one of the major mechanisms thought to underlie PD. Worms overexpressing LRRK2 show increased resistance to mitochondrial inhibitors, whereas RNAi knockdown of endogenous lrk-1 increases sensitivity to mitochondrial stress. Moreover, using simultaneous expression of GFP under the dat-1 promoter, worms expressing LRRK2 show a loss of DAergic signal in early adulthood that was greater in the G2019S mutants compared to WT LRRK2. A similar pattern is seen in regards to mitochondrial inhibition using rotenone, with G2019S LRRK2-expressing worms showing decreased DAT1::GFP fluorescence compared to WT LRRK2 (Saha et al., 2009). Thus, it appears that loss of lrk-1 produces the opposite effect of WT LRRK2 overexpression in C. elegans. Furthermore, a more recent study found that LRRK2 overexpression specifically in C. elegans DAergic neurons produces adult-onset, progressive DAergic neurodegeneration that was most pronounced in the R144C and G2019S mutants than the WT LRRK2 worms. Additionally, these Tg worms also exhibit DA-specific behavioral deficits, with a progressive loss of their food-sensing behavior that can be rescued with exogenous DA treatment. They also show hyperactive locomotive behavior that can be rescued by exogenous DA, as monitored by a high-speed camera and computer-controlled detection system. These effects could be explained by the decrease in DA content found in worms expressing LRRK2. Interestingly, the loss of lrk-1 antagonizes LRRK2-induced neurodegeneration, as well as the behavioral deficits seen with the food-sensing and locomotor assays (Yao et al., 2010). This confirms that the C. elegans LRK-1 is functionally homologous to human LRRK2, and allows for in vivo evidence of its role in promoting neurodegeneration and DA-specific behavioral decline.

Autosomal Recessive Genes

Homozygous mutations in the PARK2/parkin gene are the most common cause of an early-onset Familial PD (FPD) known as autosomal recessive juvenile parkinsonism (AR-JP), accounting for 40-50% of familial cases (Lucking et al., 2000). Its gene product encodes for an E3 ubiquitin ligase, implicating impairments in the ubiquitin-proteasome system (UPS) and protein degradation as other potential mechanisms behind PD. Parkin is a large protein, consisting of 465 amino acids that make up several domains: a ubiquitin-like domain that recognizes substrates and RING finger and in-between-RING (IBR) domains that interact with other UPS machinery (Kitada et al., 1998). It has been shown to form a giant E3 complex with two other PD-associated proteins, DJ-1 and PINK1 (Xiong et al., 2009). In addition to itself, parkin has many substrates, including the synaptic vesicle-associated protein CDCrel-1 (Zhang et al., 2000), α-synuclein (Shimura et al., 2001), the α-synuclein-interacting protein synphilin-1 (Chung et al., 2001), and the membrane receptor Pael-R (Imai et al., 2001). Parkin mutants exhibit altered intracellular localization, altered substrate binding and enzymatic activity, with mutations primarily occurring in the RING-IBR-RING region (Sriram et al., 2005). The large size of parkin itself potentially explains the high number of mutations associated with this gene. Parkin knockout mice show an increase in extracellular striatal DA concentration (Goldberg et al., 2003), while wildtype parkin seems to increase cell surface expression of the dopamine transporter (DAT) for increased DA reuptake (Jiang et al., 2004). Parkin has also been shown to affect various signaling pathways, including the negative regulation of the JNK pathway in DAergic neurons (Cha et al., 2005).

Moreover, a more recently illuminated role for parkin involves the autophagy of damaged mitochondria as a potential protective mechanism against oxidative stress. Recent studies have found that parkin translocation to damaged mitochondria that have lowered mitochondrial membrane potentials is dependent on PINK1, followed by the accumulation of these mitochondria into the perinuclear region of the cell for autophagic removal (Narendra et al., 2008, Vives-Bauza et al., 2010). The mechanism behind this mitophagy is still being elucidated. However, one of the latest studies focused on parkin’s effects on mitochondria has found that parkin can promote the ubiquitination and subsequent degradation of outer mitochondrial membrane proteins in a proteasome-dependent manner (Yoshii et al., 2011). From the mitophagy studies, it seems that one potential mechanism for the neurodegeneration seen in parkin mutants could be impaired damaged mitochondrial trafficking, producing an overall increased oxidative stress in DAergic cells. However, most studies involving parkin focus on its role as an E3 ligase and its numerous ubiquitination targets. Unfortunately, this diversity in parkin’s protein functions may be further clouding the mystery behind the selectivity in DAergic cell death in all parkin-mediated cases.

Although various animal parkin models have been created to model PD (Lu et al., 2009, Sang et al., 2007), very few have been able to capture all aspects of the disease, in addition to the severe neurodegeneration associated with familial parkin mutations. A C. elegans homolog, pdr-1, was discovered in 2005 in the hopes of studying parkin in a more highly genetically tractable model that would allow for the ease of testing a variety of parkin mutations in vivo. The pdr-1 homolog functions like parkin, with its protein product interacting with conserved degradation machinery as seen in the UPS. Interestingly, pdr-1 deletion mutants associated with impaired intracellular localization and solubility (lg103, tm598, lg101 and tm395) show increased sensitivity to proteotoxic stress compared to loss-of-function mutants (Springer et al., 2005). One study looking at global changes in microRNA (miRNA) expression changes in PD has recently found that in pdr-1 mutants, miR-64, miR-65 and let-7 family members are underexpressed (Asikainen et al., 2010). These data suggest that miRNAs putatively involved with a bHLH transcription factor and miRNA-mediated protective mechanisms could be associated with the pathogenesis behind PD. However, some of these miRNAs do not have direct human orthologs, and thus necessitate further investigation into the role of miRNAs in PD. Nevertheless, the capacity of C. elegans to be a high-throughput model for finding both genetic and environmental modifiers of the neurodegeneration seen in parkin-related PD warrants more in vivo studies using the pdr-1 model system, as mutations in this gene are so commonly linked to a large majority of familial PD cases.

Two other autosomal-recessive genes associated with FPD are PINK1/PARK6 and DJ-1/PARK7. Mutations in PINK1 are the second most common genetic cause of FPD behind parkin. Both a nonsense truncating mutation and a highly conserved G309D missense mutation were first connected to PINK1 in Italian families suffering from early-onset PD. Its protein product encodes for a serine/threonine kinase with an N-terminal mitochondrial targeting sequence (Valente et al., 2004). Recent evidence indicates that PINK1 spans the outer mitochondrial membrane, with its kinase domain facing the cytoplasm (Zhou et al., 2008), although previous data show its localization to be within the mitochondrial intermembrane space (Silvestri et al., 2005). The loss of PINK1 decreases mitochondrial respiration and aconitase activity selectively in the striatum of mice (Gautier et al., 2008), as well as decreasing dopamine release and impairing long-term potentiation and depression (LTP, LTD) (Kitada et al., 2007). Drosophila expression of parkin allows for the rescue of the mitochondrial dysfunction seen in the loss-of-function PINK1 phenotype, with PINK1 functioning upstream of parkin (Park et al., 2006, Clark et al., 2006). The proposed interaction relies on the localization of the two proteins, as PINK1’s cytoplasmic kinase domain makes its biochemical interaction with cytosolic parkin plausible in regards to the Parkin-mediated mitophagy mechanism. This mechanism is thought to be due to direct PINK1-directed phosphorylation of parkin, indicating the necessity of its kinase activity for proper mitochondrial function (Kim et al., 2008).

Meanwhile, DJ-1-related mechanisms of autosomal recessive PD remain more elusive than both Parkin and PINK1. DJ-1 is considered a redox-sensitive protein, lending to its role as a neuroprotective ROS scavenger (Mitsumoto and Nakagawa, 2001). Recent evidence has found that loss of DJ-1 downregulates mitochondrial uncoupling proteins, in addition to increasing the oxidation of mitochondrial matrix proteins specifically in SNpc DAergic neurons (Guzman et al., 2010). Furthermore, DJ-1 also can complex with mutant Mn-superoxide dismutase (MnSOD) in order to attenuate its toxicity in neuronal cells in vitro (Yamashita et al., 2010). DJ’s antioxidant role has been expanded on, with effects on signaling pathways potentially being an indirect mode of action. In fact, DJ-1 has been shown to protect against oxidative stress by regulating the thioredoxin(Trx)/MAP3 kinase apoptosis signal-regulating kinase 1 (ASK1) pathway. ASK1 is normally bound to and inhibited by Trx. Interestingly, wildtype DJ-1 has been shown to prevent the dissociation of ASK1 from Trx, subsequently blocking the activation of JNK pathway-induced apoptosis (Im et al., 2010). Moreover, DJ-1 is also necessary for the phosphorylation and subsequent activation of AKT, a neuroprotective pathway induced by oxidative stress (Aleyasin et al., 2010). Thus, DJ-1’s antioxidant mechanism may be indirect via modulation of cell signaling pathways that function to protect DAergic cells from oxidative stress. Regardless, the true mechanism(s) of DJ-1’s ability to protect DAergic cells in PD remains unclear and support the need for further examination.

Regardless, few groups have decided to use invertebrate C. elegans as an alternative to mice and rat models in an attempt to better model PD. The C. elegans genome encodes two homologs of DJ-1: djr-1.1 and djr-1.2 (Bandyopadhyay and Cookson, 2004). Tg C. elegans lines expressing a pdr-1 deletion, or knockdown of pdr-1 or djr-1 all show similar sensitivity to mitochondrial complex I inhibitors like rotenone. Furthermore, this sensitivity is partially rescued by the antioxidant probucol, the mitochondrial complex II activator D-β-hydroxybutyrate, or anti-apoptotic bile acid tauroursodeoxycholic acid. However, complete rescue of mitochondrial dysfunction can be achieved with a combination of the complex II activator and the anti-apoptotic compound (Ved et al., 2005). This confirms the role of these proteins in PD-linked mitochondrial dysfunction in another model system in an in vivo manner. Additionally, using the benefit of in vivo live imaging in C. elegans, α-synuclein overexpression in worms induces mitochondrial fragmentation in motor neurons compared to wildtype worms, as seen using cyan fluorescent protein (CFP) fused to the outer mitochondrial membrane protein TOM70. Mitochondrial fragmentation is also shifted to an earlier time point in worms expressing α-synuclein compared to wildtype worms, indicating that α-synuclein can accelerate the mitochondrial fragmentation phenotype associated with aging. This fragmentation is independent of the typical mitochondrial fusion (Mfn1/2, Opa1) and fission (Drp1) proteins, and is rescued by expression of wildtype parkin, DJ-1 and PINK1 in vitro (Kamp et al., 2010). A separate study focused on innate immunity signaling found that in the absence of djr-1, worms show heightened phosphorylation of p38 mitogen-activated protein kinase 1 (PMK-1) and higher induction of PMK-1’s target genes (Cornejo Castro et al., 2010). Thus, using C. elegans, a new neuroprotective mechanism by DJ-1 on cell signaling via modulation of innate immunity was revealed (Cornejo Castro et al., 2010). The C. elegans model system is also the first to elucidate an antagonistic role between the autosomal dominant LRRK2 and the autosomal recessive PINK1 genes. Mutations in the worm homolog of PINK1, known as pink-1, lead to reduced mitochondrial cristae length and increased sensitivity to the mitochondrial inhibitor paraquat. These effects were reversed in worms lacking lrk-1. Similarly, lrk-1 loss-of-function mutant worms show a hypersensitivity to the endoplasmic reticulum (ER) stress inducer tunicamyin that is absent in pink-1 mutants (Samann et al., 2009). Although all of these studies have augmented the PD knowledge base concerning mechanisms behind neurodegeneration, we still have not been able to truly decipher the basis for the selectivity in this degeneration.

Chemical- and metal-induced neurodegeneration

Studies routinely use the expression of GFP under the control of the C. elegans DA transporter (dat-1) promoter, commonly noted as P_dat-1::GFP Tg worms (Nass et al., 2002). A typical hermaphrodite contains eight DAergic neurons: four cephalic (CEP) and two anterior deirid (ADE) neurons in the head, as well as two posterior deirid (PDE) neurons in tail. Male worms contain additional DAergic neurons that facilitate mating behavior, including three pairs located in the tail, and four male-specific spicule socket cells.

Many neurotoxins have been employed in attempts to model the neurodegeneration seen in PD. One such toxin is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This chemical was first discovered as a neurotoxin when illicit drug users accidentally produced MPTP and trace amounts of 1-methyl-4-phenyl-4-propionoxy-piperidine (MPPP) as a by-product of creating the opiate meperidine that was self-administered intravenously. As a result, these patients presented symptoms that strikingly resembled Parkinsonian features (Langston et al., 1983). This is thought to occur due to the conversion of MPTP to its metabolite N-methyl-4-phenylpyridine (MPP+), and its selective uptake by DAergic neurons via the dopamine transporter (DAT) (Javitch et al., 1985), upon which MPP+ can inhibit mitochondrial complex I to inhibit ATP synthesis and subsequently cause DAergic degeneration (Mizuno et al., 1988). It is well known for producing PD-like symptoms in humans, primates and mice by generating selective lesions in nigrostriatal DAergic neurons, making it an ideal neurotoxin to model neurodegeneration. In C. elegans exposed to 340 μM MPP+, mobility defects, such as slow movements, twitching and coiling, are seen after 2 days of treatment, effects which are not present in untreated worms. Higher concentrations (1-1.5 mM) of MPTP result in a higher number of immobile worms, in addition to a development delay in reaching the gravid adult stage. Using a Tg cat-2::GFP strain, 1.0 and 1.5 mM MPP+-exposed worms show weaker or total loss of GFP expression in DAergic neurons, respectively. This degeneration is selective for DAergic neurons, as AIY interneurons, serotonergic neurons and AWB sensory neurons in show no change upon exposure to MPP+. The ability of DAT to uptake MPP+ is conserved in C. elegans, as concurrent exposure to a transporter inhibitor rescues the immobility produced by MPP+ treatment alone (Braungart et al., 2004). MPP+-induced ATP depletion has also been confirmed as the mechanism behind the DAergic cell death in C. elegans (Wang et al., 2007). Thus, MPP+/MPTP-induced selective neurodegeneration is reproducible in C. elegans and consistent with vertebrate models, making it a suitable chemically induced PD model in worms.

Another neurotoxin, 6-hydroxydopamine (6-OHDA), is transported via DAT and can subsequently cause selective ablation of DAergic neurons (Uretsky and Iversen, 1970, Perese et al., 1989, Redman et al., 2006). The 6-OHDA rat model presents with selective nigrostriatal neurodegeneration, with near complete depletion of striatal dopamine, movement deficits and hypersensitive D2 receptors (Perese et al., 1989). One mode of action of 6-OHDA’s ability to ablate DAergic neurons arises from dopamine autoxidation that subsequently leads to the production of damaging quinones that result in cell death (Heikkila and Cohen, 1973). Additionally, 6-OHDA has been shown to cause cell death via both apoptotic and necrotic mechanisms (Ochu et al., 1998, Walkinshaw and Waters, 1994, Woodgate et al., 1999). Recent work has shown that lysosomal cysteine protease cathespin L may play a role in 6-OHDA-induced apotosis (Xiang et al., 2011). In the C. elegans model system, the effects of 10 mM 6-OHDA In P_dat-1::GFP Tg worms are seen as soon as two hours post-treatment, with the presence of blebbing along CEP and ADE processes; by 72 hours post-treatment, there is complete loss of GFP expression in most of the DAergic neurons. Similar to vertebrates, 6-OHDA toxicity is mediated by uptake by DAT-1 in C. elegans, with DAT-1 inhibitors rescuing the loss of GFP expression. This was further confirmed using dat-1 deletion mutants that exhibit resistance to 6-OHDA-induced DAergic neurodegeneration. Moreover, CED-3 (worm homolog for caspase-9) and CED-4 (worm homolog for APAF-1) are not necessary for 6-OHDA-mediated neurotoxicity to occur (Nass et al., 2002). Thus, 6-OHDA is now commonly used for the total ablation of DAergic neurons in C. elegans permitting analysis of mechanisms associated with the selectivity in PD-associated DAergic neurodegeneration. However, despite which animal model is used, the exact mechanism, be it apoptotic and/or necrotic or neither, behind 6-OHDA-induced toxicity remains a current target in PD research.

Similar to MPP+, another neurotoxin that inhibits mitochondrial complex I is rotenone, a conventional insecticide and pesticide. Unlike MPTP, rotenone is hydrophobic and does not require the DAT for cytoplasmic entry. Compared to a shorter treatment paradigm (1 week), chronic, systemic rotenone exposure (5 weeks) of 2.5 mg/kg in rats produces complete and selective nigrostriatal DAergic neurodegeneration that is associated with hypokinetic movement deficits and rigidity. Moreover, degenerating neurons contain cytoplasmic inclusions that are comprised of α-synuclein and ubiquitin (Betarbet et al., 2000, Cannon et al., 2009). In C. elegans, exposure to rotenone induces oxygen hypersensitivity associated with enhanced oxidative damage (Ishiguro et al., 2001). Furthermore, treatment of adult worms expressing P_cat-2::DsRed2 with 1.5 μM rotenone leads to gradual loss of the DsRed2 expression after one week, with a complete loss on the ninth day post-treatment. Degeneration initially presents with fragmentation of ADE projections, followed by loss of DsRed2 expression in PDE neurons, then ADE neurons, and ending with the CEP neurons on the ninth day (Mocko et al., 2010). However, rotenone’s effects on DAergic neurodegeneration seem to be exposure length-dependent, as after two days of rotenone treatment, DAergic neuronal morphology is unchanged, but the presence of thioflavin, a biomarker for protein aggregation, can be seen within inclusions inside the DAergic neurons (Ved et al., 2005). Thus, rotenone exposure in C. elegans produces consistent neurodegenerative changes analogous to those seen in vertebrate models, making it another ideal choice for an in vivo chemically induced PD model.

Outside of these commonly used pharmacological agents, more current research has turned to investigations of other environmental factors shown to cause neurodegeneration and PD-associated genes. One such factor is the essential heavy metal, manganese (Mn), which in excess can produce an irreversible, early-onset condition known as manganism that highly resembles PD. Although there are several differences (Couper, 1837, Mena et al., 1967), the fact that this condition shares many of the same molecular mechanisms and behavioral phenotypes as PD permits a convergence between a genetic predisposition and exposure to an environmental factor on the same neural circuits, coming together to produce the disease instead of individually. This could also explain the reason why many of the current genetic animals expressing mutations in PD genes alone are unable to reproduce as an overt neurodegeneration as there is in actual PD cases. This could also explain alterations in the age of onset of actual symptoms. Consequently, this has provoked much of the latest research to focus on several potential gene-environment interactions that involve PD-associated genes. C. elegans exposed to Mn-containing Maneb fungicide exposure on its own is sufficient in inducing neuronal degeneration and increased lethality compared to wildtype (Negga et al., 2011). Moreover, Mn itself can induce DAergic-specific neurodegeneration in worms, with this selectivity thought to occur from the presence of extracellular dopamine (Benedetto et al., 2010). However, there are worsened effects in combination with genetic mutations, such as in the parkin gene. Drosophila parkin mutants have a significantly shortened lifespan compared to wildtype flies upon exposure to environmental pesticides (Saini et al., 2010). Additionally, combination of parkin polymorphisms with exposure to organic solvents produces a much stronger effect in lowering the age of onset compared to one element on its own (Ghione et al., 2007). Parkin also protects against Mn-induced DAergic cell death in vitro (Higashi et al., 2004), which has been expanded to in vivo analysis in C. elegans expressing loss-of-function mutations in pdr-1 (unpublished data). It is quite possible that a genetic susceptibility is insufficient in producing enough DAergic neurodegeneration to present with symptoms; it could be the integrated effect of combined exposure to the multitude of selective neurotoxins that induces PD-specific DAergic neurodegeneration.

Screens identifying modifier genes

The ease in following the effects of quick genetic manipulation in C. elegans has made genetic screening for modifiers of neurodegeneration a popular, high-throughput approach. For example, suppressors of 6-OHDA-induced toxicity have been identified in p_dat-1::GFP Tg worms. In this screen, upon exposure to 50 mM 6-OHDA, worms maintaining GFP expression in all four CEPs are isolated, and if 40% of their progeny retain this resistance, the lines are then analyzed. This screen discovers three dat-1 alleles: two alleles containing point mutations in conserved glycine residues (G55 and G90) within DAT-1 transmembrane domains (TM1 and TM2, respectively), and a third dat-1 allele with disrupted translation of its COOH terminus. These point mutations and altered C-terminus translation lead to improper DAT-1 biosynthesis and trafficking both in vitro and in vivo. Thus, this screen critically identifies particular residues and changes in structure that modify normal DAT-1 function in C. elegans, explaining the connection of the transporter to 6-OHDA-induced neurodegeneration (Nass et al., 2005).

Moreover, a genome-wide yeast screen finds that the most effective suppression of α-synuclein toxicity involved the overexpression of genes involved in ER-to-Golgi trafficking. One suppressor, Rab guanosine triphosphatase (GTPase), is expressed along with α-synuclein under the dat-1 promoter in C. elegans. Compared to 60% of animals showing DA neuron loss when expressing α-synuclein alone, co-expression with Rab1 significantly rescues this neurodegeneration. This screen thereby reveals the mechanism of α-synuclein-mediated inhibition of ER-golgi trafficking behind its toxicity, as well as identifying Rab1 as being sufficient in protecting against this toxicity in DAergic neurons (Cooper et al., 2006). Similarly, a targeted RNAi screen looking at modifiers of α-synuclein-induced toxicity utilizes C. elegans overexpressing α-synuclein in all neurons (pan-neuronal expression). This rapid method involves feeding C. elegans bacteria that produce target-specific dsRNA (Kamath et al., 2003). Using a list of 1673 genes, ten genes are identified that cause significant growth and motor deficits upon knockdown in α-synuclein Tg worms. Four of these genes are involved in the endocytic pathway, including subunits of the AP-2 complex, which recruits clathrin and cargo receptors to the endocytic pits before being internalized into the cytosol via vesicles. This is confirmed using RNAi knockdown and deletion mutants of the worm homologs of these subunits, apa-2 and aps-2, in α-synuclein Tg worms, exhibiting severe growth defects and locomotor dysfunction. Subsequent studies examining neurotransmission show that worms overexpressing both wildtype and mutant forms of α-synuclein display defective neurotransmission of acetylcholine (Ach) compared to non-Tg worms, as determined by resistance to aldicarb, an Ach esterase inhibitor that produces a hyperactive cholinergic synapse. These worms, however, exhibit no difference from wildtypes in sensitivity to levamisole, an Ach receptor agonist (Kuwahara et al., 2008). This suggests that α-synuclein Tg worms have defective presynaptic release of neurotransmitters due to impaired endocytosis of synaptic vesicles, but with no effects on the postsynaptic side. Additionally, this systematic screen is the first to identify the novel involvement of the endocytic pathway in α-synuclein-induced neurotoxicity in C. elegans.

An additional large-scale RNAi screen focused on α-synuclein misfolding uses a candidate gene list based on C. elegans homologs of genes associated with FPD. More specifically, this screen employs adult worms expressing both α-synuclein::GFP and TOR-2, a chaperone protein that stabilizes α-synuclein overexpression at the level of misfolding. Twenty gene targets that enhanced α-synuclein aggregation are found, including FPD-associated worm homologs of DJ-1, PINK1, Parkin, NURR1, and ATP13A2, with both DJ-1 and PINK1 appearing again as hits in a secondary screen looking at younger, L3 worms. Furthermore, the screen also identifies five novel neuroprotective worm homologs: trafficking proteins (VPS41 and Sec22p), a scaffolding protein involving in G-protein signaling (GIPC), an autophagy-related protein (ATG7), as well as a protein with unknown function. These gene products show protection against α-synuclein-induced DAergic degeneration in C. elegans in both an age- and dose-dependent manner (Hamamichi et al., 2008). Additionally, a separate, genome-wide genetic screen in worms expressing wildtype and A53T α-synuclein has also found evidence of alterations in PD-associated genes, with upregulation of seven genes that function in the ubiquitin-proteasome system, including the PD-associated gene pdr-1 (Vartiainen et al., 2006).

In a more recent study of VPS41’s functional role in neuroprotection, Tg worms are created to express human VPS41 (hVPS41) in addition to GFP and α-synuclein in DAergic neurons. VPS41 has been previously connected to lysosomal trafficking, a mechanism thought to be both dysfunctional in PD and linked to α-synuclein toxicity (Chu and Kordower, 2007, Gitler et al., 2009). Compared to wildtype controls expressing α-synuclein alone, C. elegans overexpressing hVPS41 show attenuation of α-synuclein-induced DAergic neurodegeneration. These results are consistent in worms exposed to 6-OHDA, with hVPS41 overexpression rescuing the DAergic ablation seen in 6-OHDA-treated worms expressing α-synuclein alone. Subsequently, overexpression of hVPS41 in SH-SY5Y cells inhibits caspase-3, caspase-9 and PARP cleavage, in addition to reduction of the destructive insoluble forms of α-synuclein upon rotenone treatment (Ruan et al., 2010). Thus, genetic screening in C. elegans, both RNAi-based on genome-wide, allows for the identification of potential neuroprotective targets like VPS41 in a high-throughput fashion, with the hopes of hastening the discovery of novel therapeutic strategies.

Testing for efficacy of pharmacological compounds

In addition to screening for modifiers of neurotoxicity in C. elegans, this model system is also valuable for high-throughput investigation of the efficacy of potentially neuroprotective pharmacological compounds. One example involves low-dose bafilomycin, an antibiotic that functions to maintain the integrity of the autophagy-lysosomal pathways. Exposure to 50-150 μg/mL bafilomycin B1 attenuates DAergic neurodegeneration induced by α-synuclein overexpression in C. elegans (Pivtoraiko et al., 2010). This could prove to be a promising treatment option, considering the prominent role of mitophagy as a potential mechanism for the increased oxidative stress seen in PD. Similarly, the antioxidant phenothiazine also protects against DAergic cell death induced by MPP+ in C. elegans without any adverse DA receptor antagonist effects. Although not as potent as free phenothiazine, mitochondrial targeted antioxidants, such as methylene blue, the hydroquinone MitoQ and the dimethylphenol-carryingtetrapeptide SS-31, are better at ameliorating the DAergic degeneration in worms than untargeted antioxidants, like α-tocopherol, catechin and epigallocatechin gallate (Mocko et al., 2010). Testing compounds aimed at combating oxidative stress-induced DAergic neurodegeneration in C. elegans is common practice. For example, xyloketal B is another compound that shows protection against C. elegans DAergic degeneration in worms exposed to MPP+. Interestingly, xyloketal B is a novel marine compound that is extracted from the mangrove fungus the mangrove fungus Xylaria sp. (no. 2508) that scavenges free radicals to protect against oxidative stress. It is also thought to be neuroprotective be restoring glutathione (GSH) levels in vitro (Lu et al., 2010). Moreover, a class of small compounds is able to rescue ER-to-golgi trafficking defects established by α-synuclein expression in C. elegans, attenuating the DAergic neurodegeneration seen in these Tg worms. These compounds seem to be structurally similar, with only differences found in their phenol groups, indicating that they may share the same target (Su et al., 2010). Identification of this target and how it relates to mitochondrial function may be beneficial in potentially establishing neuroprotective therapy.

Forward chemical genetic screens represent a faster, more modern avenue towards testing the efficacy of other neuroprotective agents. C. elegans can aid in the speed component of combining genetics and the chemistry of compounds in one screen. For example, cyclic peptides are bioactive compounds that are more natural than other drugs that have been shown to provide neuroprotection. By genetically expressing both α-synuclein in DAegic neurons and constructs expressing cyclic peptides within one worm, researchers find attenuation of DAergic neurodegeneration compared to worms overexpressing α-synuclein alone (Kritzer et al., 2009). However, synthetically derived compounds have also shown to protect against C. elegans DAergic degeneration. Acetaminophen, a common household pain reliever and fever reducer, shows amelioration of oxidative stress-induced DAergic neurodegeneration, but with no effects on protein misfolding (Locke et al., 2008). The idea that this commonly used drug could combat PD may sound eccentric, but upon confirmation in mammalian systems, could potentially represent a new look at acetaminophen as a preventative compound. Meanwhile, DA receptor pharmacology has always been a high contender in the targets of neuroprotective pharmaceutical compounds.

In a high-throughput drug screening using MPP+-treated C. elegans, the dopamine receptor agonists apomorphine and lisuride and the protein kinase C inhibitor rottlerin are all able to reduce lethality and behavioral defects seen in MPP+-treated worms not exposed to these compounds. The antioxidants ascorbic acid and Coenzyme Q-10 and the antiparkinsonian drug amantadine also reduce these DA-specific effects in C. elegans, but only at higher doses (Braungart et al., 2004). Considering worms possess a thick cuticle that requires higher concentrations of drugs to see effects, finding drugs that can provide neuroprotection at lower doses would be most practical in testing in mammalian systems. Interestingly, it has been found in C. elegans that two DA D₂ receptor agonists, bromocriptine and quinpirole, protect against 6-OHDA-induced DAergic neurodegeneration in a receptor-independent manner (Marvanova and Nichols, 2007). Thus, C. elegans can also be used to study the mechanistic explanations for why and how certain pharmacological compounds may or may not be the ultimate PD treatment option.

IV. Huntington’s Disease

Huntington’s disease (HD) is caused by CAG expansion in the huntingtin gene (htt). HD is dominantly inherited with symptoms including chorea, memory deficits, alterations in personality, and depression manifesting in mid-life. Death generally occurs 10-15 years after symptom onset. The most prominent neuropathological findings in HD patients are caudate atrophy followed by cortical atrophy. The GABA-ergic medium spiny neurons in the caudate are the most sensitive (Zuccato et al., 2010). There are nine distinct polyglutamine (polyQ) diseases caused by expansion of an unstable CAG trinucleotide repeat within the coding region of a specific gene. When the gene is expressed, the CAG expansion is translated to lengthy polyQ tracts in the resultant protein. Although the CAG expansions occur in distinct genes, each polyQ disease is caused by CAG expansion, and has unique symptomatic and pathological progression. There are many features common to all polyQ diseases. The most striking similarity is the inverse relationship between polyQ length and age of disease onset, with longer polyQ expansions resulting in earlier onset. In addition, although expression of the polyQ protein is ubiquitous, all the diseases are characterized by progressive neuronal degeneration, including the formation of protein aggregates (Zoghbi and Orr, 2000). The strictly genetic origin of polyQ disease is markedly different from the idiopathic nature of most of the other neurodegenerative diseases discussed in this chapter and therefore research has been focused largely on understanding the cellular consequences of polyQ protein.

Polyglutamine diseases include Spinobulbar Muscular Atrophy (SBMA), or Kennedy disease, which arises from CAG expansion in the androgen receptor gene. SMBA is X-linked, recessively inherited and characterized by muscle cramps followed by muscle weakness and eventually wasting and hypogonadism. SBMA results in degeneration of the anterior horn cells, bulbar neurons, and dorsal root ganglia and is also characterized by chronic muscle denervation (La Spada et al., 1991; Biancalana et al., 1992). Also included in polyQ diseases are Spinal Cerebellar Ataxias-1, 2, 3, 6, 7, 17 (SCA-1, 2, 3, 6, 7, 17) and Dentatorubral-Pallidoluysian Atrophy (DRPLA). These diseases are dominantly inherited and difficult to distinguish clinically because they are all characterized by mid-life onset of ataxia, tremor and dysarthria. Cerebellar atrophy and loss of Purkinje cells are hallmarks of these diseases. Despite the similarities, the genes implicated in SCA and DRPLA are largely unrelated. CAG repeats in ataxin-1, −2, −3, −6, and −7 genes cause SCA-1, −2, −3, −6, and −7 respectively, while SCA6 is caused by an expansion in the α-subunit of the voltage gated calcium channel (CACNA1A), SCA17 results from an expansion in the TATA binding protein (TBP) gene, and DRPLA from a CAG expansion within atrophin-1 (Zoghbi and Orr, 2000).

Disease-linked mutations and neurodegeneration

Despite sharing many symptomatic and pathological characteristics, the only common genetic link between polyQ diseases is CAG expansion and subsequent polyQ expansion. As such, the polyQ tract is thought to be the primary contributing factor to disease pathology by conferring a toxic gain of function to the affected protein (Zoghbi and Orr, 2000). The clinical and pathological differences between polyQ diseases are attributed to the specific proteins containing the polyQ expansion due to the influence of the structure and localization on polyQ interactions and to loss of the normal function of the protein (Orr, 2001; Gatchel and Zoghbi, 2005). The use of C. elegans in polyQ disease research has primarily focused on understanding the toxic mechanisms of polyQ expression.

Expression of isolated polyQ

Expression of polyQ proteins in C. elegans provides in vivo models to assess mechanisms and modifiers of polyQ toxicity that may be relevant to all polyQ diseases. The most commonly used polyQ model was developed by Morimoto and colleagues by transgenic expression of different lengths of fluorescently labeled polyQ proteins in muscle cells of the body wall and the pharynx using tissue specific promoters (Satyal et al., 2000). This approach has been very useful for visualizing protein aggregation and protein-protein interactions through the use of several distinct fluorescent proteins, including GFP, YFP, and CFP. In addition, Morimoto and colleagues adapted this model to study polyQ expression in neurons using a pan-neuronal promoter (Brignull et al., 2006; Gidalevitz et al., 2006).

Protein aggregation is observed in all polyQ diseases (Zoghbi and Orr, 2000), and expression of polyQ in C. elegans muscle cells and neurons causes length dependent aggregate formation. In muscle cells Q82-GFP, but not Q19-YFP forms aggregates, and in fact Q82-CFP is able to sequester Q19 YFP into the aggregates (Satyal et al., 2000). Using a broader range of polyQ-YFP proteins, including Q0, Q19, Q29, Q33, Q35, Q40, Q44, Q64, and Q82, the threshold polyQ length that caused aggregate formation in muscle cells was Q35-Q40 (Morley et al., 2002). A similar threshold length was also observed in neuronal cells expressing Q0, Q19, Q35, Q40, Q67, and Q86-CFP (Brignull et al., 2006).

In humans, disease progression is correlated with both CAG repeat length and aging. Findings from C. elegans models show polyQ length-dependent impairment of behavior in an age-dependent manner. PolyQ expression in muscle cells results in developmental delay and reduced motility phenotypes (Satyal et al., 2000). Motility was reduced in young animals expressing fusion proteins with greater than Q35, coincident with aggregate formation (Morley et al., 2002). Furthermore, as the animals aged, the incidence and severity of the behavioral phenotype increased, an effect which was delayed in age-1 mutant worms which have a longer lifespan and delayed aging (Morley et al., 2002). In worms expressing polyQ in neurons, body thrashing, pharyngeal pumping, and defecation rates (behaviors mediated by distinct subsets of neurons) were impaired by expression of greater than Q35 (Brignull et al., 2006).

Given the presence of protein aggregation in many polyQ and diseases, C. elegans models have been used to investigate the effects of isolated polyQ expression on the protein processing environment. The heat shock response occurs following increased heat and cellular stressors and results in the expression of heat shock proteins (hsp) which act as chaperones that aid in protein folding. Hsp expression in body wall muscle cells were elevated by Q82-GFP but not Q19-GFP expression (Satyal et al., 2000). This effect was reduced in the Q82-GFP animals through exogenous expression of the yeast hsp104 (Satyal et al., 2000). Temperature sensitive (ts) mutants have been combined with muscle cell and neuronal polyQ expression to further examine the effect of polyQ on protein processing. Ts mutations disrupt protein folding and confirmation only at high, restrictive temperatures. Ts mutant worms therefore appear normal at lower, permissive temperatures but demonstrate distinct behavioral phenotypes at restrictive temperatures when the mutated protein is not processed properly. Q40-YFP expression in a paramyosin ts mutant background, which shows embryonic lethality and slow movement at restrictive temperatures, unmasked the restrictive phenotypes at permissive temperatures (Gidalevitz et al., 2006). Similarly, neuronal expression of Q40-YFP in adynamin-1 ts background, which is paralyzed at restrictive temperatures, unmasked the paralyzation phenotype at restrictive temperatures (Gidalevitz et al., 2006). The appearance of ts phenotypes at permissive temperatures suggests that in both muscle cells and neurons, polyQ disrupts the folding environment in a length-dependent manner.

These models of polyQ expression have recapitulated findings from humans and other experimental models, specifically that the appearance of protein aggregates and behavioral phenotypes is dependent on the presence of a pathogenic length of polyQ expansion and age.

Expression of Htt N-terminal fragments

While expression of isolated polyQ proteins in C. elegans has confirmed the length and age dependent toxic nature of polyQ expression, the contribution of protein context to polyQ toxicity has also been addressed in C. elegans. There is no known htt homolog in C. elegans, however several groups have generated transgenic models that express N-terminal fragments of the htt protein with varying lengths of polyQ expansions (Voisine and Hart, 2004).

Similar to isolated polyQ expression, Htt-Q results in length- and age-dependent aggregate formation, behavioral deficits, and neuronal degeneration. Hart and colleagues created the first htt transgenic strain by expressing the first 171aa of the N-terminal of the human htt gene containing Q2, Q23, Q95, or Q150 in a subset of sensory neurons including the ASH neurons, which mediate the nose-touch response (Faber et al., 1999). They observed protein aggregation and ASH degeneration in the 171htt-Q150 worms at 8 days of age, and when 171htt-150Q was co-expressed with GFP there observed degeneration, nose-touch deficits, and caspase mediated cell death of the ASH neurons at day 8 (Faber et al., 1999). There was no degeneration observed in the worms expressing 171htt-Q2, Q23, or Q95, which suggests that the pathogenic threshold in this model is higher than that observed with isolated polyQ expression, in addition, appearance of the phenotype is dependent on age because the effects of 171htt-Q150 expression did not appear until 8 days, again later than expression of polyQ alone (Faber et al., 1999). In a different model, a smaller 57aa fragment of human htt with Q19, Q88, or Q128 was expressed in PLM sensory neurons, which mediate response to gentle touch. In this model, there was no cell death, however protein aggregation and altered gentle touch response was evident in 57htt-Q88 and Q128 worms but not control or 57htt-Q19 worms (Parker et al., 2001). In yet another model developed by the Monteiro group, htt-Q28, Q55 or Q74 was expressed in body wall muscle cells; they saw a length dependent aggregate formation and reduction in body movement at one day of age in htt-Q55 and Q74 worms but not htt-Q28 worms (Wang et al., 2006). These findings are consistent with findings from isolated polyQ expression in muscle cells, however aggregates and behavioral phenotypes appear earlier and with shorter polyQ lengths than with htt-Q expression in neurons (Faber et al., 1999; Parker et al., 2001).

The htt models have been used to investigate secondary effects of polyQ expansion that may contribute to neurodegeneration. Monteiro and colleagues examined mitochondrial dynamics in HeLa cells and found that polyQ expression increased mitochondrial fragmentation, decreased ATP, and reduced mitochondrial motility and fusion. These effects were prevented by promoting mitochondrial fusion (Wang et al., 2009). In the C. elegans model of muscle cell htt-Q expression they used RNAi to knockdown dynamin related protein-1 (drp-1), a mitochondrial fission protein. Knockdown of drp-1 impairs mitochondrial fission and promotes fusion, and increased motility of Htt-Q74 worms (Wang et al., 2009). Reduction of oxidative metabolism using the drug meclizine in the 57htt-Q PLM neuron model prevented neuronal dystrophy and sensory deficits characteristic of the PLM model, however meclizine did not alter protein aggregation (Gohil et al., 2011). Together, these studies provide evidence that polyQ induced alteration of mitochondrial dynamics and function may contribute to toxicity and disease progression.

Mutant huntingtin protein associates with the histone acetyltransferase domain of CREB binding protein (CBP-HAT), suggesting that mutant htt may be modified by acetylation. Indeed, Jeong et al. confirmed that mutant htt is acetylated at lysine 444 (K444) by CBP-HAT in COS-7 cells, as well as in mouse and human brain tissue (Jeong et al., 2009). In this study, they also generated several C. elegans models to determine the effect of K444 acetylation on neuronal toxicity. A larger 564htt-Q150 fragment with either intact L444, or acetylation resistant 564htt-Q150-KR was expressed in ASH neurons. Both forms of 564htt-Q150 caused neuronal degeneration and cell death, as in previous models, however co-expression of CBP-HAT was protective and reduced neuronal degeneration and cell death in 563htt-Q150 worms but not in the acetylation resistant 564htt-150Q-KR worms (Jeong et al., 2009). The protective effect of L444 acetylation is likely mediated through increased clearance of mutant htt.

While there is no C. elegans htt homolog, hipr-1 is a homolog of huntingtin interacting protein-1 (HIP-1), an endocytic protein that binds htt and is involved in trafficking of ionotrophic glutamate receptors (Kalchman et al., 1997; Parker et al., 2007). Inhibition of hipr-1 through mutation and RNAi in worms expressing 57htt-Q in PLM neurons, resulted in neuronal dysfunction in 57htt-128Q but not 57htt-Q19 worms (Parker et al., 2007). Together, these findings demonstrate the usefulness of C. elegans models of HD for addressing the role of post translational modification and endocytosis in polyQ toxicity.

Screens identifying modifier genes

A whole genome RNAi screen to identify factors that accelerate aggregate formation was performed in worms expressing Q35-YFP in muscle cells, which form age-dependent protein aggregates at 5 days (Morley et al., 2002; Nollen et al., 2004). RNAi knock down of 186 genes resulted in formation of visible protein aggregates on day 3, two days earlier than expression of Q35-YFP alone. These genes were further screened using worms expressing Q0, Q24, and Q33-YFP. All of the genes tested demonstrated length dependent aggregated formation at day 3 in Q33 and Q35-YFP strains but not Q0, or Q24-YFP strains, indicating a role for these genes in polyQ toxicity. The genes represented several broad functional categories including RNA metabolism, protein synthesis, protein folding, protein degradation, protein trafficking, and ATP synthesis (Nollen et al., 2004). In a mutagenesis screen using the same model of polyQ-YFP expression in muscle cells (Morley et al., 2002), several non-functional alleles of unc-30 caused premature aggregate formation in muscles. Unc-30 is a neuron specific transcription factor regulating GABA synthesis, providing evidence that neuronal activity can affect protein homeostasis in postsynaptic cells (Garcia et al., 2007). The 171htt-Q ASH neuron model was also used in a mutagenesis screen to identify genes that protect ASH neurons from PolyQ mediated degeneration and cell death. The screen identified pqe-1 (polyQ enhancer-1), a protein which has a glutamine/proline rich domain. In pqe-1 knock out animals expressing 171htt-Q150, there is a marked increase in toxicity with cell death and degeneration occurring by 3 days, while overexpression of pqe-1 is protective (Faber et al., 2002).

In addition to RNAi and mutagenesis screens, yeast-two-hybrid screens of the C. elegans genome have also been used to identify proteins that interact with human N-terminal-htt (Holbert et al., 2001; Holbert et al., 2003). This approach has identified novel C. elegans htt binding proteins (ZK1127.9 and K08E3.3b), with respective human homologs transcriptional co-activator CA150 (Holbert et al., 2001), and cdc42 interacting protein 4 (CIP4) (Holbert et al., 2003). Using human brain tissue, expression of both CA150 and CIP4 was elevated in HD patients compared to age-matched controls. CA150 levels account for a small portion of the variability in age of disease onset, and expression of CIP4 in straital neuron models results in cell death, suggesting a role for these proteins in disease (Holbert et al., 2001; Holbert et al., 2003).

Testing for efficacy of pharmacological compounds

The pqe-1 Q150 model with accelerated toxicity grows slower than expression of 171htt-Q150 alone (Faber 02). Consequently the pqe-1 171htt-Q150 strain consumes food more slowly than wild type animals (Voisine et al., 2007). This slower rate of food consumption was used to develop a highthroughput assay in which optical food density can be measured over time in combination with drug treatment to identify compounds which alter food consumption rates and indicating an effect on polyQ toxicity. Nine compounds previously shown to alter polyQ toxicity were tested for an effect on food clearance pqe-1 worms expressing 171htt-Q150 proteins in ASH neurons. Two of the compounds, mithramycin and lithium chloride, were able to restore food clearance with in a dose-dependent manner. Furthermore, previously identified mutant strains were also used to rule out aging and growth as the basis for the benefical effects of these compounds (Voisine et al., 2007).

A large drug screen in HD neuronal cultures identified several mitochondrial inhibitors that were able to rescue polyQ induced cell death (Varma et al., 2007). Consistent with mitochondrial findings discussed above, treatment of pqe-1 171htt150Q worms with rotenone, one of the identified compounds, resulted in a dose-dependent rescue of ASH neuron cell death (Varma et al., 2007).

V. Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s Disease, is an idiopathic and fatal neurodegenerative disease of motor neurons caused by the degeneration of the upper and lower motor neurons of the ventral horn of the spinal cord and the cortical neurons that provide their afferent input. It is the most common motor neuron disease, affecting 5 out of 100,000 people worldwide. ALS is characterized by progressive muscle weakness, muscle atrophy and fasciculations, spacity, dysarthria, dysphagia, and respiratory compromise. Disease progression is rapid and progressive, with patients losing the ability to walk, breathe, swallow, and death occurs within 2-3 years after initial diagnosis (Wijesekera and Leigh 2009; Kiernan, Vucic et al. 2011). 90-95% of ALS cases are sporadic, idiopathic, only 5-10% cases of ALS are due to genetics (Bruijn, Miller et al. 2004). Clinical phenotypes have been observed in patients who have mutations in the SOD1 (Cu/Zn superoxide dismutase), TARDBP (TAR DNA binding protein, TDP-43), FUS (fusion in sarcoma), ANG (the angiogenin ribonuclease), VAPB (VAMP (vesicle-associated membrane protein)-associated protein B) and OPTN (optineurin) genes (Wijesekera and Leigh 2009; Kiernan, Vucic et al. 2011). Humans commonly develop ALS between the ages of 40-70, suggesting a role for aging in its etiology. The mechanisms that lead to death of the neurons in ALS are unknown. Prior to death, neurons acquire proteinaceous inclusions that contain ubiquitin but not amyloid, implicating the involvement of the protein processing pathway (Bruijn, Miller et al. 2004). Whether these inclusions are neurotoxic or cytoprotective is being debated. Furthermore, glutamate excitotoxicity, inflammation, oxidative stress, and mitochondrial dysfunction have been implicated in ALS (Bruijn, Miller et al. 2004).

Disease-linked mutations and neurodegeneration

The use of C. elegans in ALS research has revolved around understanding how mutations in genes involved in familial ALS contribute to motor dysfunction. C. elegans models have been developed expressing human wild-type and mutant forms of SOD1, TDP-43, and VAPB.

SOD1 is ubiquitously expressed in the cytoplasm, where it catalyzes the conversion of O₂⁻ into O₂ and H₂O₂. SOD1 acts as a homodimer, with each monomer complexed to a Zn and a Cu ion, capable of enzymatic activity (Fridovich 1986). Mutations in SOD1 account for 20% of familial ALS and 5% of sporadic ALS, with more than 70 different mutations identified, with the majority displaying dominant inheritance (Rosen 1993; Majoor-Krakauer, Willems et al. 2003). Whether the mutation is sporadic or familial, mutated SOD1 results in both clinical and pathological presentations of ALS, which has prompted researchers to use mutant SOD1 in mammalian and non-mammalian models. Interestingly, there are several types of mutations that result in the ALS phenotype; these include SOD1 incapable of binding metal ions and less stable SOD1 protein (Valentine, Doucette et al. 2005). Exactly how SOD1 contributes to developing ALS remains unclear. Studies have shown mutated SOD1 to have aberrant gain of function enzymatic activity such as peroxynitrite catalysis, nitration of tyrosine, and generation of hydroxyl radical (Beckman, Carson et al. 1993; Yim, Kang et al. 1996). Indeed increased oxidative stress has been observed in tissues of ALS patients (Bowling, Schulz et al. 1993). It has also been shown that mutant SOD1 forms cytoplasmic and some mitochondrial aggregates in vitro and in vivo in SOD1 transgenic mice (Gurney, Pu et al. 1994; Matsumoto, Stojanovic et al. 2005). Aggregation may be due to unstable mutant monomers of SOD1; however, how the aggregates contribute to disease is not known.

C. elegans strains have been generated that express wild-type or mutant SOD1 that show phenotypes associated with motor neuron disease. C. elegans expressing a G85R mutant, an inactive metal binding mutant, human SOD1 in neurons under the control of the synaptobrevin (snb-1) promoter had slower forward movement and thrashing than worms expressing wild-type human SOD1 (Wang, Farr et al. 2009). Worms expressing point mutations in SOD1 at G85R or G93A (a wild-type like phenotype that have mild loss of thermal stability), or a truncated and highly unstable SOD1in the body wall muscle by using the tissue-specific pUnc-54 promoter had reduction in motility, but no major changes in organization of myofilaments, embryonic lethality or growth arrested larvae as compared to worms expressing wild-type human SOD1 (Gidalevitz, Krupinski et al. 2009). Expression of wild-type or mutant human SOD1 has not been shown to reduce lifespan or cause neurodegeneration in C. elegans, however expression of the SOD1 A4V, G93A, or G37R point mutants in non-germ line cells had increased lethality in response to paraquat (Oeda, Shimohama et al. 2001). Both soluble SOD1 and insoluble SOD1 aggregates have been observed in mutant SOD1 expressing worms (Witan, Kern et al. 2008; Gidalevitz, Krupinski et al. 2009; Wang, Farr et al. 2009). Using both cell culture in vitro and C. elegans in vivo, Witan et al have demonstrated that aggregates of SOD1 can form between G85R mutant SOD1homodimers and G85R mutant-wild-type SOD1heterodimers (Witan, Kern et al. 2008). These mutant homodimers were also found to have reduced enzymatic activity than the wild-type homodimers or the mutant-wild-type heterodimer and worms expressing the heterodimer had increased movement impairment and reduced survival in response to paraquat-induced oxidative stress, suggesting that mutant SOD1 toxicity may be due its ability to dimerize with wild-type SOD1(Witan, Kern et al. 2008). Interestingly, the ability to form aggregates and the distribution of the aggregates varied depending on the specific mutation expressed, with the G85R mutant showing dispersed small aggregates while a truncated SOD1 mutant, 127X, contained irregular, non-spherical aggregates (Gidalevitz, Krupinski et al. 2009).

In addition to forming aggregates and causing locomotor dysfunction, Wang et al. have found G85R mutant SOD1had reduced number of processes, reduced diameter, number of organelles including mitochondria and vesicles in the ventral nerve cord while innervated body wall muscles appeared normal (Wang, Farr et al. 2009). Crossing the mutant SOD1 with Punc-129::GFP-synaptobrevin, a strain that shows a continuous pattern of puncta in dopaminergic motor neurons at the sites of neuromuscular junctions, revealed that the mutant SOD1 expressing worms had reduced synaptic vesicles (Wang, Farr et al. 2009). Electron microscopy showed mutants had fewer presynaptic vesicles, and those present were trapped in aggregates (Wang, Farr et al. 2009). This observation is similar to a G85R SOD transgenic mouse model where vesicle accumulation, decreased vesicle density, and loss of presynaptic markers were observed (Pun, Santos et al. 2006). Post synaptic receptors were not affected by the expression of mutant SOD1 as mutants were resistant to aldicarb, a cholinesterase inhibitor, paralysis, showing a loss of cholinergic synaptic transmission, but was not resistant to levamisole, a cholinergic receptor agonist (Wang, Farr et al. 2009).

TAR DNA-binding protein (TDP-43) is a ubiquitously expressed RNA and DNA-binding protein involved in repressing gene transcription, alternative exon splicing, and mRNA stability (Buratti and Baralle 2001; Strong, Volkening et al. 2007). Mutations in TDP-43 account for 5-10% of familial ALS. Additionally, it has been identified in ubiquitinated inclusions in affected neurons of 95% of ALS patients (Wijesekera and Leigh 2009). TDP-43 is primarily a nuclear protein, containing a nuclear localization sequence, but in ALS it is found in the cytoplasm. Cytoplasmic inclusions of TDP-43 may contain truncated forms of TDP-43 consisting of C-terminal fragments that may arise from caspase cleavage (Zhang, Xu et al. 2007). It is thought that caspase cleavage may make TDP-43 more prone to aggregate. Surprisingly both loss of function and overexpression of TDP-43 can lead to neuronal pathology in cell culture (Kabashi, Lin et al. 2010).

TDP-43 is highly conserved; TDP-1, the C. elegans homolog, contains the RNA recognition motifs and nuclear localization sequences present in human TDP-43 (Ayala, Misteli et al. 2008). However TDP-1 lacks the glycine rich C-terminal region found in aggregates (Ayala, Misteli et al. 2008). Because of this protein structure, transgenic lines have been developed where wild-type or mutant human TDP-43 under the control of the synaptobrevin promoter is expressed in worms (Ash, Zhang et al. 2010; Liachko, Guthrie et al. 2010; Zhang, Mullane et al. 2011). Worms expressing human TDP-43 have an uncoordinated phenotype consistent with motor neuron dysfunction (Ash, Zhang et al. 2010; Liachko, Guthrie et al. 2010; Zhang, Mullane et al. 2011).. These worms have also been shown to have decreased thrashing and crawl speed (Zhang, Mullane et al. 2011). Expression of G290A, A315T, or M337V mutant human TDP-43, mutations observed in ALS patients, had a more severe uncoordinated phenotype (Liachko, Guthrie et al. 2010). The uncoordinated phenotype was also seen in worms overexpressing endogenous TDP-1 (Ash, Zhang et al. 2010). Using deletion mutants, Ash et al. found that the uncoordinated phenotype required both RNA recognition motifs and the glycine-rich C terminal domain, but not the nuclear localization sequences (Ash, Zhang et al. 2010). Interestingly, the uncoordinated phenotype did not depend on caspase cleavage, as expression of TDP-43 in worms with knocked-down ced-3 and ced-4 caspase showed uncoordinated phenotype (Ash, Zhang et al. 2010; Liachko, Guthrie et al. 2010). Expression of human TDP-43 causes reduced life span in C. elegans (Liachko, Guthrie et al. 2010). Liachko et al have found that worms expressing wild-type human TDP-43 showed no neurodegeneration, but the expression of G290A, A315T, or M337V mutant TDP-43 showed degeneration of dopaminergic and GABAergic neurons (Liachko, Guthrie et al. 2010). This agrees with Ash et al, who have found decreased GABAergic motor neuron synapses in wild-typeTDP-43 expressing worms but no neuronal loss (Ash, Zhang et al. 2010). Additionally, worms expressing TDP-43 have been shown to be resistant to aldicarb, a cholinesterase inhibitor, suggesting reduced efficiency of synaptic transmissions (Zhang, Mullane et al. 2011).

TDP-43 forms nuclear inclusions in neurons of head and ventral nerve cord (Liachko, Guthrie et al. 2010). In patients with ALS, TDP-43 has been found to be phosphorylated and ubiquitinated (Neumann, Sampathu et al. 2006; Hasegawa, Arai et al. 2008; Bodansky, Kim et al. 2010). This has also been observed in worms, where both wild-type and mutant TDP-43 expressing worms show ubiquitination, but only mutated TDP-43 is phosphorylated (Liachko, Guthrie et al. 2010). Mutation of the phosphorylation residues SS409/410AA in the G290A, A315T, or M337V mutant human TDP-43 expressing worms resulted in increased motor functioning and less uncoordinated phenotype, suggesting that phosphorylation may contribute to TDP-43 neurotoxicity (Liachko, Guthrie et al. 2010).

VAMP (vesicle-associated membrane protein)-associated protein B (VAPB) is a protein that is associated with the endoplasmic reticulum of unknown function. A single point mutation (P56S) causes an atypical form of ALS which is slowly progressive, with age of onset between 25-52 years and a speed of progression from 2-30 years (Nishimura, Mitne-Neto et al. 2004). The P56S is quite rare, with the only reported cases occurring in a small population in Brazil. Additionally, it has been reported that VAPB levels are significantly decreased in the spinal cord of human patients with sporadic ALS (Teuling, Ahmed et al. 2007). Human VAPB is homologous to C elegans VPR-1(Miller, Nguyen et al. 2001). VAPB and VPR-1 both contain an N-terminal major sperm protein (MSP) domain, which as its name implies, bears homology to the major sperm protein of C. elegans required for fertilization of eggs. The MSP domain is cleaved from VAPB and secreted, while the P56S mutant leads to a failure to secrete as well as ubiquitination, accumulation of inclusions in the endoplasmic reticulum, and an unfolded protein response in Drosophila (Tsuda, Han et al. 2008). Using the fog-2(q71) strain, which lacks sperm. Tsuda et al. showed that injected VPR-1 can cause oocyte maturation and sheath contraction rates due to its MSP domain, acting like a signaling molecule (Tsuda, Han et al. 2008). MSP binds to Eph receptors (VAB-1), which are involved in axon guidance, cell migration, and angiogenesis. In C. elegans VAB-1 regulates the embryonic migration of amphid neurons in the head, which is visualized by Dil dye and differential-interference contrast microscopy (Zallen, Yi et al. 1998). In worms lacking either VAB-1 or VPR-1 the location of the amphid neurons is less posterior position than wild-type worms. Worms that have both VAB-1 and VPR-1 knocked down show even a potentiated effect, suggesting that the two proteins interact in vivo (Tsuda, Han et al. 2008). How Eph receptors and VAMP signaling are involved in ALS remains to be investigated.

It is important to note that one of the key features of ALS, death of motor neurons, have not been observed in many of the nematode models.

Screens identifying modifier genes

C. elegans have also been used to identify genes that potentially can be involved in ALS. Wang et al. has performed a RNAi screen to find genes whose inactivation strongly increases SOD inclusions. Hits were then confirmed using worms expressing mutant G85R SOD1 and knock down for the candidate gene. Proteins identified are involved in chaperone/quality control (hsf-1, C30C11.4, F08H9.4, stc-1, and dnj-19), protein turnover (sel-10, rbx-1, and W07G4.4), sumoylation (uba-2, ubc-9, and gel-17), redox enzymes (bli-3, pdi-2, and C30H7.2), TGFβ signal transduction (dbl-1), dopamine metabolism (dat-1), DNA replication and repair (top-1 and div-1), lifespan (pha-4) and transcription (H43I07.2) (Wang, Farr et al. 2009). How these proteins are involved in SOD-1 aggregation is not known.

How TDP-43 contributes to the ALS phenotype is not known. Recently RNAi screens in C. elegans have identified the heat shock factor transcription factor (HSF-1) and the insulin signaling pathway as being modifiers of TDP-43 pathology. Worms expressing TDP-43 and have knock down in DAF-2 (Insulin/IGF-1 receptor homolog) had increased locomotor activity and less TDP-43 aggregates than worms expressing TDP-43 alone (Zhang, Mullane et al. 2011), suggesting signaling through the DAF-2 receptor may be involved in TDP-43 mediated neurotoxicity. Conversely, expression of TDP-43 in worms with HSF-1 knocked down had worse locomotor activity than worms expressing TDP-43 alone (Zhang, Mullane et al. 2011), suggesting that the unfolded protein response may be important in the management of protein aggregates in ALS.

VI. Spinal Muscular Atrophy (SMA)

Spinal muscular atrophy (SMA) is an inherited autosomal disease characterized by muscle weakness and atrophy resulting from the degeneration of the lower motor neurons of the anterior horn of the spinal cord and the brainstem nuclei (Lorson, Rindt et al. 2010; Wee, Kong et al. 2010). There are four types of SMA which are classified by severity and age of onset (Lorson, Rindt et al. 2010; Wee, Kong et al. 2010). Type I SMA, or Werdnig-Hoffmann disease, is the most severe form of SMA, which manifests before 6 months of age. Patients are unable to sit unaided and death occurs before 2 years of age. Type II SMA is an intermediate form of SMA where symptoms start before 18 months of age and patients can sit upright unaided but are not able stand or walk. The progressiveness and the mortality of this type are highly variable. Type III SMA, or Kugelberg-Welander disease, manifests in adolescence. Patients may be able to walk and survive into adulthood, but the disease may progress in life leading to loss of mobility. Type IV SMA is adult onset SMA. Muscle weakness observed in all four types of SMA is symmetrical with proximal muscles more affected than distal; legs are more affected than arms, while there is little involvement of facial muscles. Truncal muscles are affected in severe cases of SMA, requiring respiratory aid for breathing. Muscle weakness and atrophy results from denervation of muscles from either the loss of axons or the loss of the entire motor neuron.

Disease-linked mutations and neurodegeneration

SMA is the most common genetic cause of death for infants, with an incidence of 1 in 6,000 live births (Emery 1991). All cases of SMA result from the deletion of the Survival Motor Neuron-1 (SMN1) gene. Frequency for this mutation is 1:80, with 1 in 35 people being a carrier for the disease. SMN is a small ubiquitously expressed protein which is involved in small nuclear ribonucleoprotein (snRNP) biogenesis, mRNA transport, and transcription (Pellizzoni, Baccon et al. 2001; Whitehead, Jones et al. 2002). Humans are unique in possessing SMN2, a nearly identical gene to SMN1, whose only difference is a cytosine to thymine transition in exon 7 that results in an alternatively spliced protein that is truncated and nonfunctional (Lefebvre, Burglen et al. 1995; Lorson, Hahnen et al. 1999). In milder SMA cases there is an increased copy number of SMN2 (Feldkotter, Schwarzer et al. 2002), which thought to give rise to a small amount of functional SMN protein. Smn-1 (also called CeSMN), the C. elegans ortholog of SMN1, is expressed throughout all stages of development in all cells of the worm (Talbot, Miguel-Aliaga et al. 1998; Miguel-Aliaga, Culetto et al. 1999). Worms fed RNAi targeting smn-1 caused a significant amount of lethality, with those surviving displaying an uncoordinated phenotype, paralysis, lack of muscle tone, vulval abnormalities, moulting defects and sterility (Miguel-Aliaga, Culetto et al. 1999). Interestingly overexpression of smn-1 decreases broodsize (Miguel-Aliaga, Culetto et al. 1999), suggesting that smn-1 levels are tightly regulated. Recently two strains have been developed that have deleted smn-1. Sleigh et al. have developed a strain, smn-1(cb131) which contains a point mutation in exon 2 that alters a highly conserved aspartic acid (D27N) that is present in a patient with type III SMA (Sleigh, Buckingham et al. 2011). This line shows reduction in body length, egg laying, and thrashing, as well as shorter lifespan (Sleigh, Buckingham et al. 2011). Smn-1(cb131) worms also show resistance to paralysis by pyridostigmine bromide, an acetylcholinesterase inhibitor, suggesting that the synaptic transmission of the cholinergic system is disrupted in the worms (Sleigh, Buckingham et al. 2011). The smn-1(ok355) line arises from the deletion of 975 bp comprising 246 bp of the upstream intergenic region and all but the terminal 87 bp of the smn-1 gene (Briese, Esmaeili et al. 2009). Homozygotes containing the smn-1(ok355) deletion have larval arrest, defects in gonadogenesis and germline differentiation, and become pale due to loss of intestinal pigments, giving a starved appearance (Briese, Esmaeili et al. 2009). Thrashing and pharyngeal pumping progressively decrease in these worms as they age, which ultimately leads to paralysis and a reduced lifespan, 6 days post L1 stage as compared to 17.7 days post L1 for wild-type N2 strain (Briese, Esmaeili et al. 2009). Heterozygotes carrying this deletion mutation are not significantly different from N2 wild-type in any of the parameter tested, and neuronal expression of smn-1 in homozygotes partially restored size, pigmentation, and lifespan, suggesting that the phenotypic changes were due to loss of the smn-1 protein (Briese, Esmaeili et al. 2009). Crossing the smn-1(ok355) worms with either worms expressing the pan neuronal marker F25B3.3::GFP or worms expressing GFP in cholinergic neurons (unc-17::GFP), revealed that there was no gross abnormalities in any of the neurons or in the number of neurons over the course of 5 days post L1 stage, suggesting that deletion of smn-1 does not produce neurodegeneration in the worms, but may affect neural functioning (Briese, Esmaeili et al. 2009). Both of these strains have been useful in screens for potential drugs to treat SMA and to understand the roles of the SMN protein in cells.

SMN has been shown to form complexes with Gemin family of proteins to assemble, recycle, and maintain snRNP components of the spliceosome. Components of the splicosome have been identified in worms (Talbot, Miguel-Aliaga et al. 1998). Deletion of SMN results in abnormal RNA splicing. Many of the microRNAs that have been associated with the SMN complex in human neuronal cell lines are conserved in C. elegans (Dostie, Mourelatos et al. 2003), suggesting that SMN complex may function similarly in worms.

Other than SMN1 and SMN2, there have been very little other genes shown to modify SMA disease in humans. Plastin-3 may be a modifier of SMA, as increased Plastin-3 protein levels in females results in less severe SMA (Oprea, Krober et al. 2008). Plastin 3 is a calcium binding, actin bundling/stabilizing protein, and may be involved with SMN-mediated anterograde axonal transport of β-actin mRNA (Bowerman, Anderson et al. 2009). Using both homozygotes and heterozygotes for the smn-1(ok355) mutation, Dimitriadi et al have performed a screen for genes that may modify the effects of loss of smn-1 function in C. elegans. Knock down of plst-1, the Plastin-3 ortholog, increased the size of the heterozygotes but had no effect on the homozygote population (Dimitriadi, Sleigh et al. 2010). The reason for this is not clear. Using an RNAi library, 4 genes were found to be enhancers for the smn-1(ok355) body length defect. Ncbp-2 (Cap Binding Protein 20), T02G5.3 (protein of unknown function with no human ortholog), grk-2 (G-protein coupled receptor kinase), and flp-4 (an FMRFamide family neuropeptide) further reduced body length in the smn-1(ok355) animals, however only ncbp-2 was the only one to also enhance the pharyngeal pumping defect (Dimitriadi, Sleigh et al. 2010). Both loss of ncbp-2 and flp-4 orthologs in Drosophila resulted in enhancement of the loss of bouton number in flies lacking Smn (fly ortholog to SMN1), suggesting these genes may be conserved modifiers of SMN function (Dimitriadi, Sleigh et al. 2010). Recently genes in Drosophila have been identified as modifiers of SMN (Chang, Dimlich et al. 2008). To test whether these genes act in a conserved manner in C. elegans, RNAi for the worm orthologs were fed to the smn-1(ok355) animals. Of the enhancers identified in Drosophila, nhr-85 (orphan nuclear hormone receptor RevErb), egl-15 (FGF receptor), kncl-2 (small conductance calcium-activated potassium channel (SK channel) KCNN3), uso-1 (general vesicular transport factor p115 (USO1)), and nekl-3 (NIMA family kinase NEK7) exacerbated the body length defect, while daf-4 (TGFβ receptor BMPRII) was the only one to enhance the pharyngeal pumping in C. elegans (Dimitriadi, Sleigh et al. 2010). Kncl-2 and nhr-25 (liver receptor homolog-1 (LRH-1)) suppressed the pharyngeal pumping defect, but not body length (Dimitriadi, Sleigh et al. 2010). Exactly how these genes interact with smn-1/SMN is not known and whether they play a role in human disease remains to be determined.

Screens identifying modifier genes

Through the use of yeast two-hybrid screens, SMN has been shown to be able to dimerize and is able to bind directly to Gemin2 and Fibrillarin. SMN-1 interacts with both the worm orthologs of Gemin 2 and fibrillarin (Miguel-Aliaga, Culetto et al. 1999; Burt, Towers et al. 2006). SMI-1, the worm orthologue of Gemin 2, is expressed uniformly throughout development and localized to the gut, body wall muscle cells, and neurons in the head and ventral nerve cord (Burt, Towers et al. 2006). Surprisingly, worms fed RNAi for smi-1 did not have any of the paralysis or abnormalities observed in feeding worms RNAi for smn-1 (Burt, Towers et al. 2006). SMI-1 and SMN-1 have been shown to interact through a yeast two hybrid screen. The same screen identified 16 potential binding partners for SMN-1. These include proteins involved in RNA processing (F08F8.9c, K05C4.5, K07F5.14) or contained domains involved in RNA processing and metabolism, such as RNA-binding domains (F56D12.5a, ran-2, ZK1320.7) or DEAD box helicase domains (let-418, mog-4). F56D12.5a, let-418, Y55F3AM.13, spe-11, mog-4, ran-2, F08F8.9c, K1320.7, ZK05C4.5 co-immunoprecipitated with SMN-1, suggesting that these gene products may be components of the SMN complex (Burt, Towers et al. 2006). Whether human orthologs to these genes interacts with SMN1 remains to be determined.

Testing for efficacy of pharmacological compounds

Current treatment for SMA consists of management of symptoms, experimental treatments under development including gene replacement and stem cell replacement of motor neurons (Lorson, Rindt et al. 2010). Patients can live into adulthood and old age if they have a manageable disease phenotype. Using the smn-1(cb131) strain, Sleigh et al. screened the National Institute of Neurological Disorders and Stroke (NINDS) chemical compound library, which consists of 1040 chemicals in a wide variety of drug classes that have been approved by the FDA, for compounds that could relieve the thrashing motor defect (Sleigh, Buckingham et al. 2011). Of the 64 compounds that increased thrashing, 6 compounds showed dose-dependent improvement in thrashing with a non-linear regression coefficient (R²) > 0.1 and a point along the line of regression ≥ 7.3 for two different treatment regimens (Sleigh, Buckingham et al. 2011). The compounds identified were aklavin hydrochloride, 4-aminopyridine (4-AP), gaboxadol hydrochloride (GH), metaraminol bitartrate, N-acetylneuraminic acid (Neu5Ac) and zidovudine (Sleigh). The compounds were also used to treat smn-1(ok355) and unc-63(x26) worms, which has impaired expression of a nicotinic acetylcholine receptor subunit found in both muscles and neurons that results in locomotor dysfunction. 4-AP was the only compound able to significantly increase thrashing in the unc-63(x26) worms (Sleigh, Buckingham et al. 2011). Neu5Ac GH, and 4-AP were able to increase the body length of smn-1(ok355) worms, however only 4-AP and Neu5Ac were the only compounds that increased pharyngeal pumping in smn-1(ok355) worms (Sleigh, Buckingham et al. 2011). 4-AP is a potassium channel blocker that has been shown to restore conductance along focally demyelinated neurons and enhance synaptic transmission by prolonging action potential duration (Sherratt, Bostock et al. 1980; Thomsen and Wilson 1983; Targ and Kocsis 1985). It has shown positive results in humans for multiple sclerosis and spinal cord injury (van Diemen, Polman et al. 1993; Wolfe, Hayes et al. 2001). As synaptic transmission is impaired in several models of SMA, it may prove to be effective in treating SMA. GH is an agonist of the α₄β₃δ GABA_A receptor (Adkins, Pillai et al. 2001), how it may be improving thrashing and body length defects is unclear as GABA is both stimulatory and inhibitory in C. elegans (McIntire, Jorgensen et al. 1993). Neu5Ac is a negatively charged monosaccharide found as a component of glycoproteins and glycolipids on the surface of cells (Schauer 2009). Cleavage of glycolipids releasing monosaccharides like Neu5Ac has been shown to be neuroprotective and display neurotrophic effects (Hadjiconstantinou and Neff 1998; Iijima, Takahashi et al. 2004; Mocchetti 2005), partially due to the ability to scavenge H₂O₂ (Iijima, Takahashi et al. 2007). Oxidative stress may play a role in SMA neuronal cell death (Roy, Mahadevan et al. 1995; Soler-Botija, Ferrer et al. 2002), which may be relieved by the use of Neu5Ac. Further research will be needed to validate the use of these compounds in the treatment of SMA.

VII. Conclusions

In summary, given the ease of manipulability, the conservation of neurotransmitter biology and the high homology with mammalian systems the C. elegans platform offers a unique perspective on the etiology of neurodegenerative disease. By exploiting these advantages and the various genetic strains that can be generated in C. elegans we have novel perspectives on neurodegenerative diseases, their etiologies and the contribution of gene-environment interactions. Although many of the strains fail to fully reproduce all symptoms or pathologies of the diseases they are designed to mimic they do offer a platform for the determination of genetic modifiers of disease. And while there are limitations to testing of pharmaceuticals in the C. elegans model, they offer a high throughput platform for rapid screening in multiple genetic backgrounds. Future studies can use this amenable platform to further the exploration into etiology, mechanisms and therapeutic efficacy of novel treatments of neurodegenerative diseases.

Figure 1. Degeneration of dopaminergic neurons in C. elegans following Mn exposure.

CEP and ADE degeneration upon Mn exposure is dependent on Mn concentration. White arrowheads indicate neuronal processes exhibiting abnormal discontinuous GFP signal. White stars indicate the position of degenerated neuron cell bodies. (From Benedetto et al., 2010)

Table 1.

C. elegans orthologs of key mammalian genes associated with disease-specific neurodegeneration

Mammalian Gene/Role	C. elegans ortholog	Reference
Alzheimer’s Disease (AD)
Amyloid precursor protein family APP/APLP1/APLP2	apl-1	Daigle and Li, 1993
γ –secretase complex Presenilins PSEN1 or 2	sel-12 hop-1 spe-4	Levitan and Greenwald, 1995 Li and Greenwald, 1997 Li and Greenwald, 1997
TAU	ptl-1	Gordon et al., 2008
Parkinson’s Disease (PD)
α-synuclein	None	N/A
LRRK2/PARK8	lrk-1	Sakaguchi-Nakashima et al., 2007
Parkin/PARK2	pdr-1	Springer et al., 2005
PINK1/PARK6	pink-1	Samann et al., 2009
DJ-1/PARK7	djr-1.1, djr-1.2	Bandyopadhyay & Cookson, 2004
Huntington’s Disease (HD) and polyQ Diseases
Huntingtin/HTT	None	N/A
Dynamin related protein-1/drp-1	Drp-1	Wang et al., 2009
CREB binding protein/CBP	None	Jeong et al., 2009
Huntingtin interacting protein/HIP-1	hipr-1	Parker et al., 2007
Amyotrophic Lateral Sclerosis (ALS)
SOD1	sod-1	Oeda et al., 2001
VAPB	vpr-1	Tsuda et al., 2008
Spinal Muscular Atrophy (SMA)
SMN1	smn-1/ CeSMN	Talbot et al., 1997
Gemin complex members: Gemin 2	smi-1	Burt et al., 2006
Fibrillarin	fib-1	Miguel-Aliaga et al., 1999

Table 2.

Neurodegenerative Disease Characteristics

Neurodegenerative Disease	Characteristics
Alzheimer’s Disease (AD)	Most common form of dementia worldwide, affecting more than 30 million people (Association, 2008; Brookmeyer et al., 2007). Characterized as a progressive, terminal neurodegenerative disorder and is typically diagnosed in people older than 65. Primary symptomology includes memory loss with secondary symptoms including confusion, irritability, language breakdown and social withdrawal. Complex pathology characterized by synaptic damage and neural loss with presence of senile plaques and neurofibrillary tangles composed of β-amyloid (Aβ) and hyperphosphorylated and aggregated microtubule-associated tau protein, respectively.
Parkinson’s Disease (PD)	One of the most common neurodegenerative diseases in the population, affecting over 1-2% of those over the age of 60 (Lees et al., 2009). Characterized by the selective loss of dopaminergic (DAergic) cells in the substantia nigra pars compacta (SNpc) region of the brain. Symptomology emotional and cognitive decline, which is then followed by the hallmark features of bradykinesia, rigidity, tremors and postural instability. Symptoms start arising when approximately 70-80% of DAergic cells in the SNpc have been lost. Later stages characterized by the appearance of a masked-face, forward-flexed posture, gait freezing, shuffling steps, as well as gastrointestinal issues (Olanow et al., 2009).
Polyglutamine Disease (PolyQ)	Nine distinct PolyQ diseases, each caused by expansion of a CAG trinucleotide repeat within the coding region of a distinct gene Symptom onset typically occurs in mid-life and is inversely related to the number of CAG repeats Characterized by motor and cognitive abnormalities resulting from progressive neurodegeneration
Amyotrophic Lateral Sclerosis (ALS)	Also known as Lou Gehrig’s Disease Idiopathic and fatal disease of motor neurons caused by the degeneration of the upper and lower motor neurons of the ventral horn of the spinal cord and the cortical neurons that provide their afferent input. Most common motor neuron disease, affecting 5 out of 100,000 people worldwide. Characterized by progressive muscle weakness, muscle atrophy and fasciculations, spacity, dysarthria, dysphagia, and respiratory compromise. Disease progression is rapid and progressive, with patients losing the ability to walk, breathe, swallow, and death occurs within 2-3 years after initial diagnosis. 90-95% of ALS cases are sporadic, idiopathic, only 5-10% cases of ALS are due to genetics. People usually develop ALS between the ages of 40-70, suggesting a role for aging in its etiology.
Spinal Muscular Atrophy (SMA)	Inherited autosomal disease characterized by muscle weakness and atrophy resulting from the degeneration of the lower motor neurons of the anterior horn of the spinal cord and the brainstem nuclei. Four types of SMA which are classified by severity and age of onset. Type I SMA, or Werdnig-Hoffmann disease, is the most severe form of SMA, which manifests before 6 months of age. Patients are unable to sit unaided and death occurs before 2 years of age. Type II SMA is an intermediate form of SMA where symptoms start before 18 months of age and patients can sit upright unaided but are not able stand or walk. The progressiveness and the mortality of this type are highly variable. Type III SMA, or Kugelberg-Welander disease, manifests in adolescence. Patients may be able to walk and survive into adulthood, but the disease may progress in life leading to loss of mobility. Type IV SMA is adult onset SMA. Muscle weakness observed in all four types of SMA is symmetrical with proximal muscles more affected than distal; legs are more affected than arms, while there is little involvement of facial muscles. Truncal muscles are affected in severe cases of SMA, requiring respiratory aid for breathing. Muscle weakness and atrophy results from denervation of muscles from either the loss of axons or the loss of the entire motor neuron.