C. elegans hermaphrodites have a comparatively simple DAergic system comprising eight neurons in total: six anterior DAergic neurons, which include four cephalic (CEP) neurons and two anterior deirid (ADE) neurons, and two posterior deirid (PDE) neurons (Figures 1, 2). Males have six additional DAergic neurons located in the tail (Sulston et al., 1975). DA synthesis, storage and transport mechanisms are conserved in C. elegans and DAergic nerve endings and synaptic vesicles have DA levels similar to those in mammalian neurons (Fuxe and Jonsson, 1973; Bargmann, 1998).

The functions of DAergic neurons have been investigated using laser ablation, a technique which can target a specific neuron while leaving neighboring neurons intact. The loss of DAergic neurons revealed that they are important for food searching and the basal slowing response upon sensing food (Sawin et al., 2000; Hills et al., 2004). Exposure to exogenous DA resulted in decreased egg laying, slowed defecation and paralysis (Schafer and Kenyon, 1995; Weinshenker et al., 1995; Hills et al., 2004; McDonald et al., 2006). Studies of mutations in cat-2, the tyrosine hydroxylase which is the rate limiting enzyme in DA synthesis, showed loss of basal slowing response and decreased touch habituation suggesting that DA signaling is necessary for mechanosensation (Sawin et al., 2000; Sanyal et al., 2004). These findings suggest that DAergic neurons are important for locomotion, associative learning, food searching, food sensing, egg-laying and defecation.

Most models of DAergic neurodegeneration in C. elegans are induced through exposure to neurotoxins and some metals, which selectively ablate DAergic neurons. These toxins include 6-hydroxydopamine (6-OHDA), l-methyl-4-phenylpyridinium (MPP+), methylmercury (MeHg), and manganese (Table 2) (Nass et al., 2002; Braungart et al., 2004; Settivari et al., 2009; VanDuyn et al., 2010). When exposed to 6-OHDA, C. elegans show a progressive and selective DAergic neuron degeneration and loss as evidenced by formation of blebs in axonal and dendritic membranes. (Nass et al., 2002; VanDuyn et al., 2010).

Although C. elegans lacks a human α-synuclein homolog, α-synuclein expression has been investigated in transgenic C. elegans. The targeting of transgene expression in C. elegans body wall muscle cells has been used to explore the toxicity of several disease-associated proteins. Body wall muscles run longitudinally along the length of the nematode and are essential for locomotion. Functional disruption of these cells causes a clear and robust paralysis phenotype (McColl et al., 2009, 2012). In PD research, green or yellow fluorescent protein-tagged α-synuclein was expressed in the body-wall muscle of C. elegans to visualize α-synuclein aggregation in vivo (Hamamichi et al., 2008; van Ham et al., 2008). These lines have been used to screen RNAi libraries, revealing 20 neuroprotective genes whose knock down enhanced α-synuclein aggregation. One of these genes was the ortholog of human VSP41, a key lysosomal trafficking protein that protects against toxicity of DA-derived neurotoxins (Hamamichi et al., 2008; Ruan et al., 2010). Another genome-wide RNAi screen revealed 80 genes that when knocked down accelerated formation of α-synuclein inclusions. These genes, which appear to suppress inclusion formation, are predominantly involved in vesicular transport and lipid metabolism (van Ham et al., 2008). These findings suggest that defects in the endosomal-lysosomal and ER-Golgi vesicular trafficking system pathways may be implicated in α-synuclein toxicity.

Additionally, neurodegenerative processes can also be studied directly in C. elegans neurons. Over-expression of wild type and A53T mutant α-synuclein under the control of pan-neuronal promoter, aex-3 and under the DAergic neuron specific promoter, dat-1, caused loss of DAergic neurons (Lakso et al., 2003; Cao et al., 2005). Two neuroprotective endoplasmic reticulum (ER) associated proteins, TorsinA and Rab1 A, were found to ameliorate α-synuclein toxicity and prevent neuron loss (Cao et al., 2005; Cooper et al., 2006), suggesting that α-synuclein toxicity affects the ER-Golgi vesicular trafficking system. Another model over-expressing wild type or mutant α-synuclein under the control of the pan-neuronal promoter, unc-51, was used to screen an RNAi library for genetic modifiers that either suppress or exacerbate α-synuclein toxicity. Knock down of four genes that are involved in synaptic endocytosis enhanced α-synuclein toxicity (Kuwahara et al., 2008), suggesting that impaired endocytosis may contribute to α-synuclein dysfunction seen in PD pathology.

Wild type and A53T mutant (human) α-synuclein have been transgenically over-expressed via the C. elegans DAergic neuron specific promoter, dat-1 (Lakso et al., 2003; Cao et al., 2005). A screen of 115,000 compounds in cells and then C. elegans identified four 1,2,3,4-tetrahydroquinolinones antagonists of α-synuclein toxicity (Su et al., 2010). Another larger screen revealed that several 8-hydroxyquinolines could ameliorate α-synuclein aggregation and toxicity in C. elegans (Tardiff et al., 2012). The underlying mechanism of protection is proposed to be via interplay between metal homeostasis and proteotoxicity of aggregation prone proteins. Interestingly another 8-hydroxyquinoline, PBT2, has been found to reduce (the Alzheimer's associated peptide) Aβ toxicity in transgenic C. elegans (McColl et al., 2012). This compound is currently under clinical trial as an AD therapeutic (Lannfelt et al., 2008; Crouch et al., 2011).

PD brains show increased levels of oxidized macromolecules, which can be used as an indirect measure of ROS levels. Malondialdehyde, lipid hydroperoxides and 4-hydroxynonenal, which are lipid peroxidation products, are increased in PD brains (Dexter et al., 1989a, 1994; Yoritaka et al., 1996). PD brains also show increased levels of 8-hydroxydeoxyguanosine (8-OHdG) and protein carbonyls, which are products of DNA and protein oxidation, respectively, (Alam et al., 1997a,b). Another marker of elevated ROS levels in PD brains is increased SOD activity in the substantia nigra (Marttila et al., 1988; Saggu et al., 1989). SOD catalyzes the dismutation of superoxide (O^•−₂) into oxygen and hydrogen peroxide, therefore its activity may increase as a neuroprotective measure to cope with increased ROS levels.

Increased ROS levels not only lead to cellular damage but also to production of oxidation by-products that are also potentially neurotoxic. For example, 4-hydroxynonenal irreversibly modifies α-synuclein aggregation in vitro, potentially leading to formation of protofibrils, which are neurotoxic to cultured DAergic neurons (Qin et al., 2007). Lipid hydroperoxides have been shown to lead to oxidation of DA to 6-OHDA, a known neurotoxin (Sauer and Oertel, 1994; Przedborski et al., 1995; Pezzella et al., 1997; Lotharius and O'Malley, 2000). Additionally, α-synuclein aggregation can be induced in vitro in the presence of hydrogen peroxide (Hashimoto et al., 1999). This suggests that increased ROS levels not only directly cause neuronal damage but also indirectly contribute to DA depletion and α-synuclein aggregation, which can further exacerbate PD progression.

Taken together, these findings suggest that PD brains are under oxidative stress, which leads to neurodegeneration. However, the mechanisms underlying the increase in ROS levels are not clearly understood. Mitochondrial dysfunction, neuroinflammation, DA autoxidation and environmental toxins have been implicated in the increase of ROS in PD brains (Thomas and Beal, 2007; Jomova et al., 2010; Hwang, 2013). Metal ion dyshomeostasis may also lead to increased ROS production in PD. Generally, the substantia nigra has the highest distribution of iron in the central nervous system. However, PD brains have more elevated levels of iron in this region (Dexter et al., 1989b; Riederer et al., 1989; Sofic et al., 1991; Good et al., 1992; Gerlach et al., 1994; Vymazal et al., 1999; Haacke et al., 2007). The infusion of iron into rat brains results in parkinsonism and behavioral changes (Ben-Shachar and Youdim, 1991; Sengstock et al., 1993). Additionally, in mice the 8-hydroxyquinoline metal ion chelator, clioquinol, and over-expression of ferritin, an iron storage protein, both prevent neurodegeneration in PD models (Kaur et al., 2003). These findings suggest that iron may play a significant role in PD neurodegeneration.

The elevated iron levels in the substantia nigra are proposed to directly and indirectly contribute to increased ROS production. Increased unbound iron levels can produce ROS, such as superoxide, via Fenton chemistry (Halliwell and Gutteridge, 1986). Additionally, ferric ions can precipitate oxidation of DA to 6-OHDA in the presence of hydrogen peroxide (Pezzella et al., 1997). Superoxide and 6-OHDA have the ability to release iron stored in ferritin and [4Fe-4S] cluster-containing enzymes (Liochev and Fridovich, 1994). This can potentially lead to a vicious cycle in which unbound iron increases levels of superoxide and 6-OHDA causing release of more unbound iron. This may contribute to the progressive neurodegeneration observed in PD.

Iron dyshomeostasis not only contributes to ROS production but also negatively impacts the function of proteins that use iron as a co-factor. For example, tyrosine hydroxylase, the rate-limiting enzyme in DA synthesis, depends on iron (Nagatsu, 1995; Ponting, 2001). Therefore, an increase in iron as seen in PD brains may increase DA synthesis, causing excess DA to be released into the cytoplasm, which may lead to increased ROS production. This iron-induced DA dysfunction not only inhibits the normal function of DA but may also lead to increased DA oxidation into the neurotoxin 6-OHDA (Pezzella et al., 1997; Jiang et al., 2013).

In addition to increased ROS production and iron dyshomeostasis, PD brains also exhibit a reduction in metal ion storage capacity and antioxidant activity. Ferritin is a key iron storage protein and disruption of its function perturbs iron homeostasis. PD brains have decreased ferritin levels (Dexter et al., 1991). This potentially leads to iron storage deficiency, which allows unbound reactive iron to accumulate in the substantia nigra, facilitating ROS production (White and Munro, 1988; Dexter et al., 1991; Connor et al., 1995). Neuroferritinopathy is a condition caused by a genetic mutation of the ferritin light chain which disrupts ferritin assembly, leading to iron accumulation and neurodegeneration in the basal ganglia, resulting in severe motor disorders (Curtis et al., 2001; Vidal et al., 2003). In addition to decreased ferritin levels, PD patients have decreased concentration and activity of Cp (Kristinsson et al., 2012). Cp is a multi-copper oxidase that oxidizes ferrous ions (Fe²⁺) to less reactive ferric ions (Fe³⁺). This oxidation is essential for cellular iron uptake and efflux by ferroportin and transferrin. Aceruloplasminaemia, a heritable condition resulting from Cp deficiency, leads to iron accumulation in the basal ganglia, neurodegeneration and motor problems including dystonia and tremors (Harris et al., 1998).

PD brains have approximately 40% lower reduced glutathione (GSH), an antioxidant enzyme that catalyzes the reduction of ROS (Sofic et al., 1992). GSH also forms complexes with other enzymes, such as glutathione peroxidase and glutathione S-tranferases, to facilitate ROS reduction (Smeyne and Smeyne, 2013). Decreased antioxidant capacity likely contributes to the oxidative stress seen in PD brains. These findings suggest that defective metal ion transport and storage, decreased antioxidant activity and increased reactive metal ion accumulation contribute to oxidative stress leading to neurodegeneration.

Another link between PD and oxidative stress is gleaned from studies of mutations in DJ-1 and PINK1, which are associated with early onset PD (Bonifati et al., 2003; Valente et al., 2004). DJ-1 and PINK1 have been shown to protect against oxidative stress (Junn et al., 2005; Pridgeon et al., 2007). This was confirmed in C. elegans by studying the nematode homologs, djr1.1 and pink-1. The djr-1.1 knock-down and pink-1 mutant strains showed increased sensitivity to toxin-induced oxidative stress (Ved et al., 2005; Samann et al., 2009). These C. elegans models complement the familial PD studies and strengthen the hypothesis that oxidative stress contributes to PD pathology.

Transgenic C. elegans expressing α-synuclein in neurons exhibited mitochondrial fragmentation attributed to α-synuclein interaction with mitochondrial membranes, affecting membrane fusion (Kamp et al., 2010). Alpha-synuclein is localized in mitochondria, suggesting that α-synuclein dysfunction potentially contributes to mitochondrial dysfunction in PD (Li et al., 2007). In turn, mitochondrial dysfunction leads to ROS overproduction in the substantia nigra of PD brains, which leads to cellular damage and cell death.

A recent study using electron paramagnetic resonance demonstrated that unbound reactive iron levels increased during oxidative stress in C. elegans (Rangel et al., 2012). Increased iron levels in C. elegans resulted in increased protein oxidation, suggesting that iron triggers increased ROS production. Iron chelation using deferoxamine and over-expression of ferritin (ftn-1) reduced protein oxidation (Valentini et al., 2012). Knock down or deletion of C. elegans SMF-1/2/3 (orthologs of human iron transporter, DMT-1) partially inhibits DAergic neuronal death (Settivari et al., 2009; VanDuyn et al., 2013). PD brains have increased iron levels, decreased ferritin levels and increased DMT-1 levels, consistent with defective iron transport and storage systems in PD brains (Dexter et al., 1991; Salazar et al., 2008).

Knock down of SKN-1 (a C. elegans ortholog of Nrf2), a transcription factor that regulates expression of glutathione S-transferase, increased susceptibility to metal-induced neurodegeneration in DAergic neurons (VanDuyn et al., 2010; Settivari et al., 2013). This finding and the observation that PD brains show decreased levels of glutathione suggests that decreased antioxidant activity contributes to PD pathology (Sofic et al., 1992).

Metal dyshomeostasis and oxidative stress may represent an important component underlying idiopathic PD. C. elegans possesses homologs of some of the iron homeostasis proteins (Table 3) therefore the nematode can be used to further our understanding of metal homeostasis in relation to PD. Even more broadly, any findings can potentially be extended to familial autosomal PD because C. elegans also has homologs for the majority of genes implicated in familial PD (Table 1). These features may be exploited to investigate these genes and their interactions with metal homeostasis.

Tau is a MAP predominantly expressed in axons and is thought to regulate the assembly of microtubules (Weingarten et al., 1975; Kosik and Finch, 1987). Neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau aggregates are a pathological hallmark of AD (Kidd, 1963; Wischik et al., 1988). Although not often emphasized, tau has also been implicated in PD pathology. Some PD patients have NFTs and in older people with parkinsonian symptoms, the severity of gait impairment appears to correlate with the degree of NFT accumulation (Joachim et al., 1987; Bancher et al., 1993; Schneider et al., 2006). Certain single-nucleotide polymorphisms in the tau gene pose an increased risk factor for PD (Zabetian et al., 2007; Edwards et al., 2010). Tau KO mice have recently been reported to exhibit neuronal iron accumulation, substantia nigra neuronal loss, parkinsonism and cognitive deficits (Lei et al., 2012). Anti-psychotic DA D2 receptor antagonists, such as azaperone, suppress insoluble tau aggregation in C. elegans (McCormick et al., 2013), suggesting an interplay between tau and DA.

Increasing evidence highlights the importance of tau and α-synuclein in PD pathology and indicates that the two proteins significantly interact. Tau is co-localized with α-synuclein in Lewy bodies (Arima et al., 1999). Tau and α-synuclein can seed and promote each other's polymerization to form insoluble aggregates (Giasson et al., 2003; Geddes, 2005). Alpha-synuclein has been shown to directly facilitate tau phosphorylation and also to mediate glycogen synthase kinase 3 (GSK-3β, a serine/threonine protein kinase) catalyzed tau phosphorylation, which is increased in PD brains (Jensen et al., 1999; Muntane et al., 2008; Duka et al., 2009). This indicates that α-synuclein may contribute to the increased GSK-3β activity, which leads to tau hyperphosphorylation.

Based on the interaction between tau and α-synuclein, the dysfunction of the two proteins may disrupt two key functions of microtubules: axonal transport and maintaining neuronal morphology. Microtubule dysfunction precedes impaired axonal transport (Cartelli et al., 2013). This was deduced from altered mitochondria distribution and neurodegeneration in DAergic neurons of mice exposed to MPTP. MPTP is known to destabilize microtubules and impair axonal transport specifically in DAergic neurons (Cappelletti et al., 2005; Ren et al., 2005; Morfini et al., 2007). Administration of a microtubule stabilizer, Epothilone D, attenuated further nigrostriatal neurodegeneration (Cartelli et al., 2013), highlighting a potential link between axonal transport disruption, microtubule dysfunction and neurodegeneration.

As a MAP, tau not only stabilizes microtubules but also regulates transport by serving as a physical barrier and by interacting with transport motor proteins, dynein and kinesin, to regulate microtubule attachment and detachment (Jancsik et al., 1996; Trinczek et al., 1999; Stamer et al., 2002; Mandelkow et al., 2003; Magnani et al., 2007; Dixit et al., 2008). Tau over-expression disrupts the transport of mitochondria and vesicles leading to accumulation of mitochondria in distal parts of the neuron (Ebneth et al., 1998; Stamer et al., 2002; Mandelkow et al., 2003). Hyperphosphorylated tau filaments have been shown to phosphorylate the kinesin light chain thereby triggering the dissociation of kinesin from its cargo (Lapointe et al., 2009). Phosphorylation of tau at the amino terminus can also impact its inhibitory effect on axonal transport (Kanaan et al., 2012). Mutant tau has been shown to cause “traffic jams” which inhibit axonal transport (Shemesh et al., 2008). The tau dysfunction observed in PD may negatively impact axonal transport, contributing to neurodegeneration.

Alpha-synuclein is co-localized with tubulin in Lewy bodies and co-purifies with microtubules. Additionally, when incubated with tubulin, α-synuclein polymerizes tubulin into microtubules (Alim et al., 2002, 2004). Immunofluorescence staining of α-synuclein transfected COS-1 cells with α-synuclein and tubulin antibodies, showed that α-synuclein co-localized predominantly with microtubules (Alim et al., 2004). Alpha-synuclein binds synaptic vesicles via its amino terminus and is involved in vesicle trafficking (Jensen et al., 1999; Cooper et al., 2006). These findings suggest that α-synuclein, like tau, is a MAP and is involved in axonal transport of vesicles. Alpha-synuclein dysfunction likely leads to impaired axonal transport.

Protein with tau-like repeats (PTL-1) is the only known tau/MAP2 ortholog in C. elegans (Goedert et al., 1996). PTL-1 is important for maintaining C. elegans neuronal morphology (Chew et al., 2013). Null mutants for ptl-1 show accelerated neurite branching and microtubule bundle disorganization in mechanosensory and GABAergic neurons (Chew et al., 2013). Microtubule changes in these neurons suggest a link between tau deficiency and compromised neuronal integrity. In addition, transfection of ptl-1 into non-neuronal cells promotes microtubule assembly and bundling (Goedert et al., 1996).

Loss of function of tau and α-synuclein may result in significant microtubule disruption that leads to neurodegeneration seen in PD. Although the downstream effect of microtubule dysfunction in neurons remains to be elucidated, we can speculate that cellular functions which rely upon microtubules will be disrupted. The function of metal ion regulating proteins, such as ferroportin and copper transporter (ATP7A), are dependent on axonal transport (Cobbold et al., 2004; Moos and Rosengren Nielsen, 2006). Microtubule disruption would likely lead to disrupted trafficking of these metal ion homeostasis proteins. This in turn disrupts metal ion homeostasis leading to accumulation of unbound reactive metal ions and metal ion deficiency, resulting in oxidative stress followed by neuronal loss.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.