Recent human studies also support neurodegenerative changes resulting from Mn exposure in frontal cortex white matter. Stepens et al. (2010) report that individuals injecting ephedroncontaining Mn express white matter abnormalities based on DTI. The authors describe evidence of diffuse white matter changes reflected by reductions in fractional anisotropy (FA) in the ephedron users. They also find effects specific to white matter underlying the right ventral premotor cortex and the medial prefrontal cortex. The authors indicate that the clinical features of these ephedron users point to a disorder of higher-level motor programming and that the pattern of motor function deficits resemble executive function deficits similar to those displayed by patients with prefrontal cortex lesions (Stepens et al., 2010). Another human study examining white matter ultrastructural integrity in welders also reveal white matter changes measured by DTI (Kim et al., 2011). They show that FA was significantly reduced in the corpus callosum and frontal white matter of welders. The FA values in these white matter regions was significantly associated with blood Mn levels and pallidal index. Importantly, the degree of FA disruption was associated with impaired attention, lower working memory and deficits in executive function tests (Kim et al., 2011).

These findings provided strong evidence that the frontal cortex gray matter and subjacent white matter are vulnerable, but previously unrecognized targets for Mn-induced neurotoxicity despite the fact that Mn accumulates in cortical structures at significantly lower concentrations than in the basal ganglia. These observations suggest that the neurotoxicological effects of Mn are not solely based on the degree to which Mn accumulates in different brain regions but they are also based on the vulnerability of a specific brain region to Mn-induced neurotoxicity. The emerging evidence in humans and non-human primates suggest that future studies on subjects with environmental and occupational exposures to Mn or in patients with medical conditions in which excess brain Mn accumulation occurs should be tested for neuropsychiatric symptoms and cognitive function deficits.

#### **EFFECTS OF MANGANESE EXPOSURE ON WORKING MEMORY**

In the previous section evidence is provided that exposure to elevated levels of Mn results in detrimental effects on cortical structures, specifically the frontal and parietal cortex. Recent human and non-human primates studies suggest that a resulting effect of Mn-induced neuropathology in the frontal cortex is working memory deficits. Chang et al. (2010) report that welders with chronic Mn exposure express increased brain activity measured by functional MRI in working memory networks during the 2-back verbal working memory task. They interpret these findings as the welders requiring more neural resources in working memory networks to compensate for subtle deficits in working memory. In another study, Wasserman et al. (2011) found significant associations between Mn levels in drinking water and reductions in Perceptual Reasoning and Working Memory scores.

Our non-human primate studies were the first to provide initial evidence of Mn effects on working memory under highly controlled experimental conditions (Schneider et al., 2006, 2009). We showed that chronic Mn exposure resulted in deficits in non-spatial and spatial working memory. Non-spatial working memory assessed by delayed matching to sample performance appeared to be more affected than spatial working memory using a variable delayed response task (Schneider et al., 2009). In general, the human and non-human primate studies provide substantial evidence for impairments of cognitive domains that are mediated by the frontal cortex. Further, the non-human primate findings also implicate brain metabolite changes in the parietal cortex, a brain region that is important for working memory performance and plays an important role in integrating sensory information and visuo-spatial processing (Constantinidis and Wang, 2004; Seger, 2006; Linden, 2007).

#### **CAN DOPAMINE NEURON DYSFUNCTION IN THE STRIATUM AND/OR FRONTAL CORTEX EXPLAIN THE WORKING MEMORY DEFICITS OBSERVED IN Mn EXPOSED NON-HUMAN PRIMATES?**

Working memory is closely associated with frontal cortex function (Constantinidis and Wang, 2004; Linden, 2007) and dopamine neurotransmission in the striatum (Rinne et al., 2000; Sawamoto et al., 2008; Jokinen et al., 2009) and the frontal cortex (Brozoski et al., 1979; Rotaru et al., 2007). The dopamine cell bodies located in the SNpc project to the caudate and putamen and this nigrostriatal system is involved in motor control. In addition, there are direct mesolimbic dopaminergic projections from the ventral tegmental area to the frontal cortex (Bjorklund and Dunnett, 2007). The caudate nucleus receives dopaminergic input from the SNpc and it can influence frontal cortex function via well-defined frontostriatal circuits (Alexander et al., 1986; Seger, 2006). Human and non-human primates studies show that the dorsolateral prefrontal cortex (DLPFC) is an important region for the execution of working memory tasks with reciprocal connections to other cortical structures such as the parietal, temporal and cingulate cortex and these combined participate in a cortical network related to working memory (Kubota and Niki, 1971; Petrides et al., 1993; Berman et al., 1995; Cohen et al., 1997).

Lesions or dysfunction of the caudate nucleus has been reported to produce impairment in the delayed response tasks that assesses working memory (Levy et al., 1997; White, 2009). Relevant to our own studies, Mn-exposed animals have impairments of both spatial and non-spatial working memory (Schneider et al., 2009) and they also express a significant impairment of *in vivo* dopamine release in the striatum (Guilarte et al., 2008a). Further, welders exposed to Mn express an early deficit in dopamine neuron function specific to the caudate nucleus and not the putamen (Criswell et al., 2011). These findings suggest that dopamine neuron dysfunction via impairment of dopamine release in the striatum and specifically in the caudate may be associated with the working memory deficits expressed in Mn-exposed non-human primates and in humans. Other studies have shown that the levels of NAA in the DLPFC predict the activation of cortical regions involved in the execution of working memory tasks such as the frontal, parietal and temporal cortices and this network has been found to be affected in mental disorders such as schizophrenia (Bertolino et al., 2000; Castner et al., 2004). Postmortem studies in the frontal cortex of Mn-exposed non-human primates have found a significant degree of neuronal degeneration with diffused β-amyloid plaques and α-synuclein aggregation (Guilarte et al., 2008b; Verina et al., 2013) implicating a potentially important role of this neuropathology in the working memory deficits observed in Mn-exposed non-human primates (Schneider et al., 2009). Imaging studies in welders exposed to Mn support a Mn-induced neuronal cell death or dysfunction in the frontal cortex based on decreased NAA/tCr ratio (Dydak et al., 2011), an effect that was associated with cumulative Mn exposure. Combined these studies provide evidence that several brain regions (i.e., the caudate nucleus, the frontal cortex and the parietal cortex) within the working memory network appear to have substantial neuropathology and/or dysfunction as a result of chronic exposure to Mn.

Experimental animal and human studies have shown that dopamine is a key neurotransmitter in the regulation of working memory in the frontal cortex and caudate nucleus (Levy et al., 1997; Aalto et al., 2005; Cools et al., 2008; Landau et al., 2009; Backman et al., 2011; Cools and D'Esposito, 2011). Microdialysis studies have shown that working memory tasks induce the release of dopamine in the prefrontal cortex of monkeys (Watanabe et al., 1997) and rats (Phillips et al., 2004) and there is increased blood flow to prefrontal and parietal cortex in humans performing working memory tasks (Bertolino et al., 2000; Cabeza and Nyberg, 2000). Other studies have shown that D1-dopamine receptor (D1R) antagonists can impair working memory (Sawaguchi and Goldman-Rakic, 1991) while low doses of D1R agonists can improve working memory (Arnsten et al., 1994). Contrary to using low doses of dopamine receptor agonists, high doses of D1R agonists also impair working memory performance, an effect that is abrogated by pretreatment with a D1R antagonist (Zahrt et al., 1997; Goldman-Rakic et al., 2000). These findings suggest that either low levels or excessive levels of D1R dopamine receptor stimulation can have a negative impact on working memory performance (Goldman-Rakic et al., 2000; Cools and D'Esposito, 2011). Based on this literature, it is likely that the impairment of *in vivo* dopamine release measured in the striatum of Mn-exposed animals may be responsible for their impairment in working memory (see Guilarte et al., 2008a; Schneider et al., 2009). Alternatively, it is possible that chronic Mn exposure may also alter *in vivo* dopamine release in the frontal cortex, and along with deficits of dopamine release in the caudate nucleus may precipitate deficits on working memory performance.

#### **ANALYSIS OF** *In vivo* **DOPAMINE RELEASE IN THE FRONTAL CORTEX: PET IMAGING WITH [11C]-FLB 457**

While the displacement of D2R specific PET ligands such as [11C]-raclopride by an acute amphetamine challenge has been validated and used extensively to measure *in vivo* dopamine release in the striatum (Laruelle, 2000). The use of this methodology is just emerging for the cerebral cortex (Narendran et al., 2009, 2011a,b, 2013). Since dopamine innervation to cortical structures is significantly lower than to the striatum, that is, dopamine terminals and dopamine receptor levels are much lower in the frontal cortex than in the caudate/putamen, *in vivo* dopamine release PET in cortical structures is a much more difficult task to perform. However, the development and use of high affinity D2R-PET ligands such as [11C]-FLB 457 (*Kd* = 0*.*06 nM) and [18F]-fallypride (*Kd* = 0*.*14 nM) have made such studies possible. Several publications have now described the reliability of using [11C]-FLB 457 to measure *in vivo* dopamine release in the cerebral cortex of humans and non-human primates (Narendran et al., 2009, 2011a,b, 2013). Further, a recent study has shown that the degree of [11C]-FLB 457 binding potential reduction measured by PET was directly associated with the amount of extracellular dopamine release induced by the acute amphetamine administration (Narendran et al., 2013). In summary, the ability to measure *in vivo* dopamine release in the cerebral cortex using PET is an extremely valuable approach to understand the molecular basis of the working memory impairments observed in humans and non-human primates exposed to Mn. We are currently performing these types of studies in our Mn-exposed animals in order to make associations between *in vivo* dopamine in cortical regions and working memory performance.

## **SUMMARY**

In the last decade there has been significant progress using state-of-the-art neuroimaging and behavioral methodologies that have opened up a new understanding of Mn neurotoxicology. While historically the focus of Mn neurotoxicity has been associated with parkinsonism as a result of the high levels of exposure that occurred in the mining and processing of Mn ore and in other occupational settings, the last decade has brought about compelling experimental evidence that at lower cumulative doses of Mn that are likely to occur from occupational and environmental exposures, other non-motor neurological effects appear to be more prevalent and these seem to be associated with cognitive function deficits. The later may

#### **REFERENCES**

Physiological activation of a cortical network during performance of the Wisconsin Card Sorting Test: a positron emission tomography study. *Neuropsychologia* 33, 1027–1046. doi: 10.1016/0028-3932(95)00035-2

be the result of Mn producing brain chemistry and structural changes in cortical regions, and the frontal and parietal cortex appear to be sensitive targets. Lastly, because of its relevance to motor and cognitive domains, it is possible that dysfunction of the dopaminergic system could be a common mechanism by which Mn could have an impact on both cognitive and motor function deficits observed in humans and non-human primates.

#### **ACKNOWLEDGMENTS**

The non-human primate work presented in this review was funded by a grant from the National Institute of Environmental Health Sciences ES010975 to Tomás R. Guilarte.

30, 59–64. doi: 10.1001/archneur.1974.00490310061010

*Exp. Neurol.* 202, 381–390. doi: 10.1016/j.expneurol.2006.06.015

2033–2039. doi: 10.1212/01.WNL.<PHONE_NUMBER>.93483.A1

microdialysis and [11C]-FLB 457 PET study. *Mol. Psychiatry*. doi: 10.1038/mp.2013.9. [Epub ahead of print].

Mexico. *Environ. Res.* 85, 90–104. doi: 10.1006/enrs.2000.4108

induces microglia activation and dystrophy in the substantia nigra of non-human primates. *Neurotoxicol*o*gy* 32, 215–226. doi: 10.1016/j.neuro.2010.11.003

prefrontal cortex during a working memory task. *J. Neurophysiol.* 78, 2795–2798.

Zhou, Y., Chen, M.-K., Endres, C. J., Ye, W., Brasic, J. R., Alexander, M., et al. (2006). An extended simplified reference tissue model for the quantification of dynamic PET with amphetamine challenge. *NeuroImage* 33, 550–563. doi: 10.1016/j.neuroimage.2006.06.038

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 07 May 2013; paper pending published: 23 May 2013; accepted: 05 June 2013; published online: June 2013. 24*

*Citation: Guilarte TR (2013) Manganese neurotoxicity: new perspectives from behavioral, neuroimaging, and neuropathological studies in humans and non-human primates. Front. Aging Neurosci. 5:23. doi: 10.3389/fnagi. 2013.00023*

*Copyright © 2013 Guilarte. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

## Biometals in rare neurodegenerative disorders of childhood

## *Sarah J. Parker 1, Jari Koistinaho1, Anthony R. White2 and Katja M. Kanninen1,2\**

*1 Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland 2 Department of Pathology, The University of Melbourne, Parkville, VIC, Australia*

#### *Edited by:*

*Peter Crouch, University of Melbourne, Australia*

#### *Reviewed by:*

*Paula I. Moreira, University of Coimbra, Portugal Junming Wang, University of Mississippi Medical Center, USA*

#### *\*Correspondence:*

*Katja M. Kanninen, Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Yliopistonranta 1, 70211 Kuopio, Finland. e-mail<EMAIL_ADDRESS>
Copper, iron, and zinc are just three of the main biometals critical for correct functioning of the central nervous system (CNS). They have diverse roles in many functional processes including but not limited to enzyme catalysis, protein stabilization, and energy production. The range of metal concentrations within the body is tightly regulated and when the balance is perturbed, debilitating effects ensue. Homeostasis of brain biometals is mainly controlled by various metal transporters and metal sequestering proteins. The biological roles of biometals are vastly reviewed in the literature with a large focus on the connection to neurological conditions associated with ageing. Biometals are also implicated in a variety of debilitating inherited childhood disorders, some of which arise soon following birth or as the child progresses into early adulthood. This review acts to highlight what we know about biometals in childhood neurological disorders such as Wilson's disease (WD), Menkes disease (MD), neuronal ceroid lipofuscinoses (NCLs), and neurodegeneration with brain iron accumulation (NBIA). Also discussed are some of the animal models available to determine the pathological mechanisms in these childhood disorders, which we hope will aid in our understanding of the role of biometals in disease and in attaining possible therapeutics in the future.

**Keywords: metals, neurodegeneration, childhood, copper, iron, zinc**

#### **INTRODUCTION**

The significance of biometals for the correct functioning of the human brain has long been discovered. The importance of metals such as iron, zinc, and copper results from the numerous roles that they have, such as stabilization of proteins and transcription factors, acting as co-factors for metallochaperones for cellular transport and their role in enzyme catalysis (Markossian and Kurganov, 2003; Butterworth, 2010). Deviations of metal homeostasis have been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease, Amyotrophic lateral sclerosis, and Huntington's disease (HD) (Dexter et al., 1993; Deibel et al., 1996; Jomova et al., 2010; Skjorringe et al., 2012). A risk factor in the development of many of these disorders appears to be an increase in age. Although the pathological characteristics of the disease are in most cases well-understood, the exact causes are often unknown.

Neurodegeneration is not restricted to an ageing population. Several debilitating neurodegenerative disorders affecting children have been identified. These include neurodegeneration with brain iron accumulation (NBIA), ATPase pathologies such as Wilson's/Menkes disease and neuronal ceroid lipofuscinoses (NCLs). These disorders share several pathological features with more common neurodegenerative diseases, including protein aggregation and oxidative stress. Aberrant biometal homeostasis has also been identified in children suffering from these diseases (**Table 1**). What is not clear is whether the distinct changes in metal levels cause neurodegeneration or occur as a result of the

neurodegeneration. In some cases, the pathology associated with childhood disorders is clearly evident and often fatal at an early age. Changes in metal levels have also been identified in children with Autism spectrum disorder (ASD) which affects the behavior of children more so than hinder them physically. Dissecting the roles that biometals have in these disorders is fundamental for finding potential therapeutics to reduce or inhibit neuropathological changes associated with altered metal homeostasis (Yasuda et al., 2011).

Since biometal homeostasis plays a major role in neurodegenerative disease, the aim of this review is to highlight the effects that metals have on the developing brain. Multiple childhood disorders arise from defects in the concentrations of metals within the body and brain but little is known about how these changes can be corrected or prevented to avoid or ameliorate disease. While most of the childhood disorders are inherited and disease progression can occur not long after birth, it is critical to determine the molecular disease characteristics and metal alterations within the brain and other tissues for the development of therapeutic treatments to provide these children with a chance at life.

#### **WILSON'S DISEASE**

Wilson's disease (WD) is an autosomal recessive disease affecting copper metabolism (Bearn and Kunkel, 1954). Copper is involved in neurotransmitter synthesis, cellular respiration, scavenging of toxic free-radicals, and has an important role in maintaining homeostasis of other trace elements such as iron (Butterworth,

2010). However, in abundance, copper is toxic and thus strict regulation of levels is critical. Free copper ions are virtually nonexistent in human blood (Linder and Hazegh-Azam, 1996) and are usually bound to a multitude of proteins and chaperones for transport around the body. WD presents in patients typically between the ages of 6–20 years, and has an estimated incidence of 1 in 30,000 (Reilly et al., 1993).

The defective gene involved in WD is ATPase Cu(2+) transporting beta polypeptide (*ATP7B*), which encodes for a membrane bound copper-transporting ATPase protein primarily located within the liver cells, as part of the trans golgi network (Cox and Moore, 2002). To date, approximately 300 mutations within *ATP7B* have been associated with WD (Merle et al., 2010). Normal copper metabolism involves the transfer of copper molecules to ceruloplasmin for transport into the plasma so it can circulate to other tissues or when in excess, to the bile for excretion (Terada et al., 1998, 1999). ATP7B is involved in transporting the copper molecules to ceruloplasmin. When ATP7B is non-functional, copper accumulates to toxic levels and severely damages the liver. The liver therefore releases copper directly into the bloodstream where copper continues to cause damage to other tissues, in particular the CNS. In the brain, accumulation of copper is most evident in the basal ganglia, the region which co-ordinates movement (DeLong et al., 1984), making WD primarily a movement disorder. This neuropathology caused by the build up of copper leads to patients suffering tremors of the arms or hands, unpredictable movement, difficulty with speaking, swallowing, and walking as well as confusion, dementia, and various psychological problems (Rosencrantz and Schilsky, 2011). Some cognitive deficits have also been noted (Middleton and Strick, 2000), and could arise due to disruptions of projections of the basal ganglia to the prefrontal cortex (Bolam et al., 2000). Gliosis is also observed in the brains of patients suffering from WD (Anzil et al., 1974). The mechanism for neurological damage caused by excess copper is still not fully understood. However, it is known that copper is involved in the production toxic free radicals (Samuele et al., 2005), and excess copper has been shown to inhibit Inhibitor of apoptosis proteins (IAPs) caused by toxic deposits of intracellular copper (Mufti et al., 2006).

WD is a progressive disorder and was ultimately a fatal disease until the discovery of the first treatment in 1951 (Denny-Brown and Porter, 1951). With the copper-chelating agent, dimercaptopropanol, the disease is now treatable (Denny-Brown and Porter, 1951). The most common treatment to date is the use of zinc salts, which block enteral copper absorption in the stomach. Various drugs are currently available for WD and ongoing compliance with treatment is necessary.

#### **ANIMAL MODELS OF WD**

The Long Evans Cinnamon (LEC) rat is one model used for *in vivo* studies of WD. Since showing symptoms of liver failure in 1987 (Yoshida et al., 1987), the rats were first used as a model for liver cancer and hepatitis. Upon further characterization, accumulation of hepatic copper and decreased ceruloplasmin activity were found. Years later when the gene responsible for WD was discovered, the rat homolog *atp7b* was found to be mutated in the LEC rats, with a significant deletion at the 3 end of the gene (Wu et al., 1994). Although there are similarities in liver pathogenesis between the LEC rat and humans, this rodent model shows minimal evidence of neuropathology (Terada and Sugiyama, 1999). In contrast to the LEC rat model, ATP7B knockout mice exhibit both neuro- and liver pathology. In these mice, copper levels are dramatically low at birth with a gradual increase by 2 months of age, where hepatic copper levels are approximately 50 times higher compared to wild-type controls (Buiakova et al., 1999). Another interesting model for WD is the toxic milk mouse, which also has an autosomal recessive inheritance (Theophilos et al., 1996). Female mice with the *tx* milk mutation produce copperdeficient milk, which is fatal to their pups. The *tx* mice have a single nucleotide change in the *ATP7B* gene, and studies have shown this to cause changes in the ATP7B localization in the mammary gland, therefore leading to impaired copper transport and little to no copper given to the pups. The *tx* mothers retain this copper in the liver (Rauch, 1983; Michalczyk et al., 2000).

Both the LEC rat and *tx* milk mouse models arose from spontaneous mutations. There is only one genetically engineered mouse model for WD, the *ATP7b*−*/*− mouse, which was generated by Buiakova et al. (1999). *ATP7b*−*/*− mice are born copperdeficient and display neurological symptoms such as tremors and abnormal locomotor behavior. If nursed by a homozygous *ATP7b*−*/*−mother, the pups will die at around 2 weeks of age. However, if nursed by a heterozygous mother, copper concentrations begin to rise to toxic levels in the liver by the age of 6 weeks, irrespective of initial birth concentration. Similar to human disease, these mice accumulate copper in the liver due to impaired export of copper into the bile, making these mice an invaluable model for the study of WD (Lutsenko, 2008).

#### **MENKES DISEASE**

Menkes disease (MD) is an inherited disease related to copper metabolism, but in contrast to WD, it is characterized by a significant reduction in normal copper levels in the blood, liver, and brain (Danks et al., 1973). Disease progression is also more rapid than in WD (de Bie et al., 2007). The reduction of copper is due to decreased absorption of copper from the intestine and therefore reduced transfer to copper-requiring proteins and enzymes (Danks, 1988). Symptoms present within the first year of life, generally within 3 months of birth and without treatment, death is likely before the third year of life. MD is a rare childhood disease with an estimated worldwide incidence of 1 in 300,000 (Tonnesen et al., 1991), but can be higher in certain countries such as Australia (1 in 50,000 to 1 in 100,000) (Tumer and Moller, 2010).

MD has an X-linked mode of inheritance and is more prevalent in males than females (Kodama and Murata, 1999). At birth, babies appear normal, although premature labor has been documented (Uriu-Adams et al., 2010). An early sign in a baby with MD is the presence of sparse and abnormal hair, which looks fragile and twisted and appears to have a "kink" in it (Kaler, 2011). MD is thus also referred to as "Kinky Hair Syndrome." The baby begins to exhibit normal cognitive behavior with smiling and normal childhood dialogue; however, these behaviors do not progress further. Babies do not exhibit increases in muscle tone and over time generally become weaker and spasticity of the limbs is observed. Toward the end of the babies' short life, they succumb to blindness and respiratory failure and premature death is often due to infection or the extreme neuronal degeneration observed. A less-severe form of MD is called occipital horn syndrome (OHS) which is also an X-linked recessive inherited disorder with mutations in *ATP7A*. These patients exhibit fewer neurologic abnormalities and have a longer lifespan (Tumer and Moller, 2010). Connective tissue abnormalities tend to be the focus of this disorder with calcification of the occipital bulb, leading to horn-like structures at the base of the brain.

This detrimental disease is caused by a mutation in the gene encoding the Copper transporting ATPase-1 (ATP7A) protein (Fatemi and Sarkar, 2002), which is also a "P-type" ATPase protein similar to ATP7B. To date, there are over 200 known mutations scattered throughout the gene (His and Cox, 2004). Although both ATP7A and ATP7B are involved in the transport of copper to secreted enzymes and metalloproteins, ATP7A is also involved in the absorption of intestinal copper and can transport copper to all cell types and vital structural enzymes (Barry et al., 2011). ATP7B is predominantly involved in the exit of copper from hepatocytes. These differences in protein function are demonstrated by the fact that MD patients exhibit a variable range of symptoms throughout the body as opposed to mainly liver damage seen in patients with WD. The neurological damage caused by mutant *ATP7A* is due to the decreased activity of the copper-dependent enzymes. Decreased cytochrome *c* oxidase leads to a reduction in ATP production by the mitochondria and causes apoptotic cell death (Borm et al., 2004). Dopamineβ-hydroxylase is necessary for neurotransmitter synthesis and its activity is decreased in response to low copper levels (Kaler et al., 1995). Superoxide dismutase 1 (SOD1) is a free-radical scavenger the activity of which has been linked to several neurodegenerative disorders, particularly ALS. Copper is a co-factor imperative for SOD1 activity. Levels of SOD1 are decreased in MD patients, contributing to lower protection against toxic oxidative species within the brain (Kaler, 1994). Collectively, the reduced activity of copper-dependent enzymes is thought to be responsible for the observed neurological damage in MD.

The cellular location of ATP7A is sensitive to the concentration of copper; when copper levels are low ATP7A remains on the membrane of the Golgi but when levels are increased, ATP7A is located to the plasma membrane where it can "pump" copper to other proteins and cuproenzymes (Hasan et al., 2012). In MD, where copper levels are low, it is essentially located to the Golgi and there is a deficiency of copper transport to essential enzymes, and other tissues.

The therapeutic benefit of injection of copper histidine as a copper replacement to humans has provided mixed results and is dependent on the mutation within the ATP7A gene (Kaler et al., 1995; Christodoulou et al., 1998). Some studies have shown that early administration of copper to babies can prevent the observed neurological decline but this is likely to be in patients with mutations in *ATP7A* that still allow for some transfer of copper to copper-requiring enzymes, but not severe mutations affecting initial absorption from the intestine (Tumer et al., 1996).

#### **ANIMAL MODELS OF MD**

Spontaneous mutations in the mouse homolog of *ATP7A*, *atp7a*, have been used for modeling defective ATP7A in humans (Kuo et al., 1997). These mice are called mottled mice and different mutations in the *atp7a* gene cause different neurological and connective tissue anomalies. The two mottled mice mutations mottled brindled (Mobr) and mottled macular (Moml) show severe reductions in copper within the liver (Hunt and Clarke, 1983), and die within 1–3 weeks of birth (Lenartowicz et al., 2012). This premature death can be prevented with injected copper within the first week of birth, as well as transgenic over expression of *atp7a* (Danks, 1986).

## **NEURONAL CEROID LIPOFUSCINOSIS**

The NCLs are a group of fatal autosomally-inherited neurodegenerative diseases occurring in childhood. The NCLs are also classified as lysosomal storage disorders since one of the main pathological characteristics is the accumulation of autofluorescent granules which contain lipids and proteins (lipopigments) in the lysosomes. Currently at least ten forms of NCLs have been described. Each form is distinguished by different defective genes (CLNs) and the age of onset (Cooper, 2003; Mitchison et al., 2004). Collectively, it is the most common neurodegenerative disorder of childhood with an estimated incidence of up to 1:12,500 (Rider and Rider, 1999). Although mutations in different genes cause different forms of the disease, all NCLs share common clinical symptoms; progressive loss of vision leading to blindness, motor and mental retardation, seizures, and finally premature death (Siintola et al., 2006). The exact function of these genes is still unknown (Tyynela et al., 2004). These genes encode soluble proteins and membrane-bound proteins, for example, CLN6 is a transmembrane protein which is localized to the endoplasmic reticulum (Heine et al., 2004). Children with defective CLN6 present with symptoms at the age of 3–5 years and tend to only live until 8–12 years of age. Currently there is no cure for any form of NCL (Sharp et al., 2003).

In NCLs, early reports have described some changes to metal levels, including iron and zinc, in blood cells of patients with NCL (Johansson et al., 1990). In a larger study of NCL patients, no association between "loosely bound" iron or copper levels in cerebrospinal fluid could be found (Heiskala et al., 1988). There is some evidence that altered metal homeostasis may also occur in other NCL models. A recent study reported changes in expression of the metal transporter, ZnT6, in both CLN3 and CLN6 mouse cerebellar neuron precursor cell lines (Cao et al., 2011). Furthermore, reduction in expression of the gene causing CLN7 NCL disease was observed in the brains of mice fed a high-iron diet (Johnstone and Milward, 2010), suggestive of a heightened sensitivity of NCL brains to fluxes in metal concentrations.

Recently, a link between brain biometal homeostasis and CLN6 disease was discovered. Sheep harboring a natural mutation in the CLN6 gene show significant increases in manganese and more so, zinc levels in all brain regions except the cerebellum and brain stem (Kanninen et al., 2013). In the CLN6 sheep model, the accumulation of biometals was evident at the symptomatic age of 12–14 months. Early studies on the metal contents of lipopigments from CLN6 sheep revealed no obvious association between metal contents and disease progression (Palmer et al., 1988). Metal contents were variable between lipopigments from different tissues in line with the normal role of lysosomes as storage depots for metals, indicating the lysosomal origin of the lipopigments. Changes in the metal contents of retina and retinal pigment epithelial cells of CLN6 sheep have been reported, and an association with lipopigment accumulation and altered manganese levels with increasing photoreceptor cell loss suggested (Jolly et al., 1989).

#### **ANIMAL MODELS OF NCLs**

To understand the pathogenesis of NCLs, gene-specific mutant mice have been engineered which contain mutations in *Cln1, Cln2, Cln3, Cln5, Cln7,* and *Cln10* genes (Cooper et al., 2006). Naturally occurring mutations in *Cln6* and *Cln8* have also been identified (Lonka et al., 2000; Gao et al., 2002). Many of these mouse models share phenotypic features in common with human patients such as neurological and motor deficits, seizures and neuronal loss, and as such, are valuable tools to study disease mechanisms and potential therapeutic approaches. Like the human form, the mouse models display variance in timing and sequence of events toward disease progression, highlighting the genetic differences between the NCLs (Cooper et al., 2006). As the function of the majority of these genes is still unknown, using these animal models will prove invaluable in elucidating the function and mechanism behind NCL pathologies. The naturally occurring mutation in the CLN6 gene of sheep mentioned above share a similar disease progression and phenotypic features similar to that of the human CLN6 NCL form (Jolly et al., 1989; Kay et al., 2011), making them an excellent model of NCL disease due to their complex CNS. However, with large animal models it may be challenging to obtain sufficient numbers of animals for assessment due to difficulties in breeding and housing such large animals.

#### **NEURODEGENERATIVE DISORDERS WITH BRAIN IRON ACCUMULATION**

As with copper and zinc mentioned above, levels of iron within the brain need to be tightly regulated. Iron homeostasis is regulated by proteins involved in absorption, transport, influx, and storage in tissues (Zhang and Enns, 2009). Increases in brain iron levels are typically associated with ageing and is highly prevalent in AD and HD. Neurodegenerative disorders with brain iron accumulation (NBIA) refers to a wide group of disorders affecting children, with similar outcomes: neurodegeneration and the accumulation of iron. The disease is caused by mutations in seven genes (Ke and Ming Qian, 2003). At birth, brain iron is not present, but levels rapidly increase until 20 years of age with an uneven distribution over time (Zorzi et al., 2012). The most recognized gene defects are mutations in pantothenate kinase (PANK2), which is an enzyme located within the mitochondria and regulates the formation of Coenzyme A, which is essential for deriving cellular energy via the tricarboxylic acid cycle (Leoni et al., 2012). PANK2 phosphorylates pantothenate and requires N-pantothenoyl-cysteine and pantetheine. The exact mechanism how mutations in *PANK2* lead to iron accumulation is not fully understood, but one hypothesis is that if cysteine cannot be utilized within the cell by PANK2, it accumulates over time. Cysteine can bind iron, and over time this may explain the observed accumulation of iron (Gregory and Hayflick, 2005). Although the mutation in *PANK2* directly does not cause neurodegeneration, it is the increased iron which contributes to the production of toxic and tissue-damaging reactive oxygen species (Ke and Ming Qian, 2003). Increases in iron have also been linked to several neurological disorders such as AD and PD (Batista-Nascimento et al., 2012). There are over 100 mutations in this gene and PANK2 disorders account for over half of all NBIA cases (Gregory and Hayflick, 2005). PANK2 specific NBIA, referred to as pantothenate kinase-associated neurodegeneration (PKAN) was formally called Hallervorden–Spatz syndrome before the name was changed to divert any association with the unethical euthanization of mentally ill patients during World War II by Dr. Hallervorden (Dooling et al., 1974). PKAN disease is autosomally inherited and presents in childhood as either the classical, early onset or atypical, late onset form (Hayflick et al., 2003). The extent of iron accumulation can be easily visualized and quantified using magnetic resonance imaging (MRI). The pattern exhibited by MRI-T2 weighted images shows the increased iron as "darkened spots" and provides a novel "eye of the tiger" image, making MRI an important diagnostic tool for NBIA (Schenck and Zimmerman, 2004).

The symptoms of classical PKAN disease become apparent within the first 10 years of life, with symptoms usually starting between 3 and 4 years of age. Affected children begin to have difficulty with walking and other movements, which is attributed to the build up of iron in the basal ganglia (Gregory and Hayflick, 2005), a region of the brain highly important in movement. Rigidity, spasticity, and speech difficulties soon follow (Dooling et al., 1974). Children become wheelchair bound within only a few years of diagnosis. Atypical PKAN disease begins later in life, with an average age of onset of 13–14 years and disease progression unfolds slowly over many years (Chan et al., 2008). Patients present firstly with behavioral problems such as depression and personality changes with movement and speech impediments following later. The symptoms of atypical PKAN can be highly variable from case to case.

Treatment for NBIA had been aimed at symptoms rather than the genetic defect causing the iron build up and severe neurodegeneration. Drugs aimed at reducing abnormal movement are widely prescribed, but to date, there is no preventative for NBIA disorders. Recently, reducing the accumulation of iron has been the target for treatment using iron-chelators, however this is met with varying efficacy (McNeill and Chinnery, 2011). Deferiprone is an iron chelator which acts to redistribute iron within the brain, by donating built-up iron to transferrin for transport elsewhere (Brittenham, 1992). The promising feature of this drug is the ability to cross the blood-brain barrier, necessary for targeting the build up of iron in CNS. However, no conclusive results can be drawn from these clinical studies due to the small number of patients trialed (Forni et al., 2008; Zorzi et al., 2011).

#### **ANIMAL MODELS OF NBIA**

Unlike the other childhood disorders discussed in this review, PKAN-NBIA does not have a suitable murine model of disease. A PANK2 knockout mouse was generated which showed retinal degeneration and growth retardation, but no defects on movement and gait were evident (Kuo et al., 2005; Brunetti et al., 2012). However, *Drosophila melanogaster* have been a powerful

#### **REFERENCES**

three affected males in a single family. *J. Pediatr.* 145, 119–121.

tool for understanding the biochemical basis of PANK2 mutations. Mutations in the homolog of PANK, *fumble (fbl*) exhibited a shortened lifespan, progressive locomotion defect, and degenerations in both central nervous system and retina (Gregory and Hayflick, 2005). Interestingly, mutations in PANK2 in both the *Drosophila* and mouse led to male sterility (Wu et al., 2009).

#### **CONCLUSIONS**

The importance of metals in neurological disease has been outlined in numerous reviews over many decades, however, the exact mechanisms leading to aberrant metal homeostasis and why this is so detrimental to the brain remain poorly understood. While the brain is still developing during childhood, it is highly susceptible to slight changes in metal levels as outlined above with discussion of various childhood neurodegenerative disorders. Although certain therapeutics exist for some of these disorders, they mainly target disease symptoms and prolong the disease, rather than being preventative. It is the work forthcoming from *in vitro*, animal and clinical studies, which aim to add insight into the role of metal changes and consequences so potential therapeutics can be tested and hopefully one day applied to prevent disease progression.

chelation therapy in a case of neurodegeneration with brain iron accumulation. *Mov. Disord.* 23, 904–907.

(batten's disease): pathogenesis and sequential neuropathological changes in the ovine model. *Neuropathol. Appl. Neurobiol.* 15, 371–383.

by dimethyldithiocarbamate (DMDTC). *PLoS ONE* 7:e40400. doi: 10.1371/journal.pone.0040400

bearing WND cDNA. *J. Biol. Chem.* 273, 1815–1820.

in the human neuronal ceroidlipofuscinoses: distinct patterns of storage deposition, neurodegeneration and glial activation. *Brain Pathol.* 14, 349–357.

**Conflict of Interest Statement:** 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.

*Received: 25 January 2013; accepted: 05 March 2013; published online: 25 March 2013.*

*Citation: Parker SJ, Koistinaho J, White AR and Kanninen KM (2013) Biometals in rare neurodegenerative disorders of childhood. Front. Aging Neurosci. 5:14. doi: 10.3389/fnagi.2013.00014*

*Copyright © 2013 Parker, Koistinaho, White and Kanninen. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Metalloproteomics: principles, challenges, and applications to neurodegeneration

## *Amber Lothian1, Dominic J. Hare1,2, Rudolf Grimm3, Timothy M. Ryan1, Colin L. Masters1 and Blaine R. Roberts1\**

1 The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia

2 Elemental Bio-Imaging Facility, University of Technology Sydney, Broadway, NSW, Australia

3 Life Sciences Group, Agilent Technologies, Santa Clara, CA, USA

#### *Edited by:*

Peter Crouch, University of Melbourne, Australia

#### *Reviewed by:*

William Griffith, Texas A&M Health Science Center, USA Robert B. Petersen, Case Western Reserve University, USA

#### *\*Correspondence:*

Blaine R. Roberts, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, At Genetics Lane, Royal Parade, Parkville, VIC 3010, Australia e-mail<EMAIL_ADDRESS>
Trace elements are required for a variety of normal biological functions. As our understanding of neurodegenerative disease advances we are identifying a number of metalloenzymes involved in disease process. Thus, the future of metals in neurobiology will rely more on detailed information regarding what metalloenzymes are present and how they are involved in the pathophysiology of disease. To gain this detailed information, we will rely less on bulk measures of the amount of a trace elements in a particular tissue and turn to metalloproteomic techniques to help elucidate both metalloprotein structure and function. Recent advances in metalloproteomics will translate to a richer understanding of the mechanism and precise role of metalloenzymes and proteins in the brain.

**Keywords: metalloproteomics, LC-ICP-MS, metals in neurodegeneration, quantitative methods, hyphenated ICP-MS techniques**

#### **INTRODUCTION**

The brain is an incredibly complex organ that has an equally unique metabolic need. In humans, although the brain only composes about 2% of body mass, it consumes approximately 25% of the energy output. The excessive proportion of metabolic activity occurring in the brain, compared to the whole body is seen in numerous examples of metal-mediated cellular function. Metalloenzymes are important for all aspects of physiology, including mitochondrial function, transcriptional regulation, catabolism, and, for the brain, the production of the important secondary messenger nitric oxide (NO) by NO synthase, which depends on Fe and Zn (Mayer et al., 1991; Li et al., 1999). In line with the brain's tendency for excess, the production of NO is about 20 times greater for the central nervous system compared to the vasculature (Salter et al., 1991; Garthwaite and Boulton, 1995; Pacher et al., 2007). Further, the actual signaling pathway for NO is dependent on Fe bound to a heme in soluble guanylate cyclase (Gerzer et al., 1981; Ignarro et al., 1982). The role of metals in NO production and signaling is just one example of the vital role trace elements have in biology. Indeed, life itself would not exist without oxygen produced by chloroplasts, and transport through our body by hemoglobin, both of which require a metal ion for function.

Despite the intense amount of research into cellular mechanisms metalloenzymes have largely been overlooked, yet the "metalloproteome" dictates much of the reactivity within a cell. The targeted investigation of metalloproteins in the central nervous system will provide mechanistic insights into how reported changes in total levels of trace elements translates to specific proteins.

## **WHAT IS "METALLOPROTEOMICS"?**

Over a decade ago, Glen Evans commented on the "omic" science revolution, stating that the advent of post-genomic sciences was brought about by a need to "analyze the components of a living organism in its entirety" (Evans, 2000). As systems biology has become more integrated into the modern laboratory the traditional streams of "omic" sciences have diversified to include specific fields of study examining the functional components of biomolecules, rather than simply their presence or structure.

Metalloproteomics is one such newly established area of study, which amalgamates proteomic and metallomic approaches to biology (Barnett et al., 2012). Proteomics is the large-scale investigation of the structural and functional properties of proteins (Anderson and Anderson, 1998), whereas metallomics encompasses the "comprehensive analysis of the entirety of metal and metalloid species within a cell or tissue type" (Szpunar, 2005). We believe the term "metalloproteomics" is more suited to the field than metallomics alone, as it recognizes the important relationship between biometals and proteins, rather than focusing solely on the presence of an individual metal species. In 2004, Hiroki Haraguchi first described metallomics as "integrated biometal science" (Haraguchi, 2004), though it is only in recent years that the potential of integrating high-end atomic spectrometry techniques into typical proteomics workflows is beginning to gain attention.

It is estimated that around one-third of all proteins in the human body require a metal cofactor for functionality (Andreini et al., 2008; Waldron et al., 2009; Barnett et al., 2012). Redox properties of metal ions mediate a plethora of cellular processes, from the electron transport mechanisms within mitochondria to the formation of myelin in developing nerve cells. Metals have

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the ability to interact with multiple proteins, all with varying functions, located in every cell of the human body. Due to the abundance of proteins that are estimated to require a metal cofactor for function, this is an area that requires extensive work to be done in order to characterize the vital role metals may play in the molecular basis of disease.

Unlike glycosylation and phosphorylation, which do not always have a one-to-one relationship with protein function (Jensen, 2006), the presence of a metal cofactor is intimately linked with enzymatic function. For example the function of Cu,ZnSOD is dependent on the presence of both metals. The Zn-only containing enzyme does occur in transgenic animal models overexpressing the enzyme (Lelie et al., 2011; Rhoads et al., 2011), though it does not pose any superoxide scavenging ability without Cu. SOD can even produce superoxide rather than scavenge it in the absence of Zn (Estévez et al., 1999). Bottom-up proteomics neglect information on non-covalent cofactors, including metals. The overall goal of systems biology or proteomics is to measure how proteins change to help elucidate function, hence the interest in glycosylation and phosphorylation and other post-translational modifications. However, the functional importance of non-covalent cofactors has the promise to determine the functional output of proteins, and cannot be overlooked.

## **HOW BIG IS THE "METALLOPROTEOME"?**

The proportion of metalloproteins in the proteome is still widely unknown. Even in relatively simple single-cellular organisms, the number of metalloproteins that have been comprehensively identified is only a fraction of the one-third of all proteins predicted to bind metals, most likely due to technical limitations. This suggests that studying the human metalloproteome will encounter significant difficulties, as the number of metalloproteins that it will encode for is larger then that of relatively simple prokaryotes.

Relatively few research groups are applying new technologies to metalloproteomics. One such group that has been actively conducting research in this field and has confirmed the lack of characterization of metalloproteins is that lead by John Tainer and Michael Adams. Their recent study of metalloproteins in *Pyrococcus furiosus* illustrates the difficulties involved in identifying metalloproteins found in even the most basic of life forms (Cvetkovic et al., 2010). It is estimated that there are around 2,000 encoded proteins in the *P. furiosus* genome (Lee et al., 2009), and if, as stated previously, one-third of them are expected to be metalloproteins, around 600 metalloproteins should be present. However, experiments conducted by the Tainer and Adams group demonstrated that only 50% of the metal peaks they analyzed contained a protein that could be linked to a known metalloprotein (Cvetkovic et al., 2010). This study demonstrates the large gap in our knowledge of what proteins even utilize a metal ion.

A conservative interpretation of these results is that for any organism yet to have it's metalloproteome mapped, 50% of these metalloproteins will have the metal association incorrectly predicted or it will not yet be known. In the human proteome, which consists of around 20,000 protein-encoding genes, an estimated number of unknown or misidentified metalloproteins can

be predicted. About 6,600 protein-encoding genes will encode for metalloproteins (Waldron et al., 2009). Using *P. furiosus* as a guide, 3,300 of these will have a predicted metal association, of which a further three-quarter will exhibit a correctly predicted metal association. This leaves an estimated 4,125 metalloprotein-encoding genes that will have an incorrect metal association predicted, by current bioinformatic tools, or display no metal interactions (**Figure 1**). The complexity of a multi-cellular complex organism like the human body is expected to have a wider range of metal–protein interactions than those observed in *P. furiosus.* It should, however, serve as an indication of the complex task at hand that faces scientists embarking on the next phase of systems biology that encompasses this functional component of an organism.

## **WHAT ARE THE ANALYTICAL CHALLENGES TO METALLOPROTEOMICS?**

The central issue that hampers the characterization of metalloproteins is difficulty in preserving their native state during analysis. Traditional proteomic approaches are generally incompatible for studying metal–protein interactions, as they tend to require denaturing conditions and enzymatic digestion, leading to disruption of the comparatively weak ionic interactions governing most metalloprotein bonds. A targeted metalloproteomics approach that acknowledges the importance of retaining native conditions is the answer to these issues, as it will provide the capability to determine the roles that metals play in the functional properties of proteins in biological systems, whilst ensuring that the detailed structural analysis of proteins is still obtainable.

The central tenet to characterizing metalloproteins is that in order to correctly identify a species the metal must still be bound to the protein. Once metalloproteins are no longer in their native state, misincorporation of metals or complete loss of metal becomes a significant problem. To accurately quantify metalloproteins it is vital that their native state is kept intact and is not altered by denaturing conditions. The use of strong acids/bases, concentrated inorganic salt, organic solvents and heat all contribute to the loss of native folded states. Thus, chromatographic separation for metalloproteomics should endeavor to use buffers that are of physiological pH, as this will help to prevent alterations to the secondary and tertiary structures that lead to the loss of metal binding. A metalloproteomics workflow must ensure that each possible source of experimental error is mitigated to a point where its influence is negligible.

Experimental error may be encountered even prior to analysis at the point of sampling. Metalloprotein integrity may be disrupted by reagents and buffers used in collection or sample preparation, and even storage conditions (Manley et al., 2009) such as repeated freeze-thaw cycles, that have an uncharacterized effect on metal–protein interactions. The post-mortem stability of SOD shows that the time in which it takes to freeze the sample post-collection does not have any significant effect on the concentration of the protein (Brooksbank and Balazs, 1984). However, the post-mortem stability of other metalloproteins is an area for further investigation. Error during this initial step of the experimentation causing loss of bound metals will impact

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all of the conclusions that are drawn from the experiment. Even relatively inert chemicals, such as acetate buffers used in native size-exclusion chromatography (SEC), may impart unwanted effects on metal binding through the presence of a relatively strong complexing anion (Inagaki et al., 2000; Wang et al., 2007; El Balkhi et al., 2010).

Characterizing an unknown metalloprotein within an environment that is rich in proteins is exceptionally challenging. Metalloproteomic techniques have been used to characterize known metalloproteins, such as metallothionein isoforms (Chassaigne and Lobi ´nski, 1998). Targeted proteomic approaches where the specific nature of the protein in question is already known does not present as many difficulties as a *de novo* approach, since these proteins can be targeted for isolation. There are multiple ways protein can be targeted and measured using a combination of chromatography techniques (del Castillo Busto et al., 2005) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) and more recently with mass spectrometry. For example, mass spectrometry including top-down techniques have been used to characterize both apo- and metallated-metallothionein in both rabbit and dolphin liver as well as horse kidney (Chassaigne and Lobinski, 1998; Chassaigne and Lobi´nski, 1998; Ryvolova et al., 2011; Pedrero et al., 2012). These studies highlight the kind of detailed information that can be obtained by using a combination of mass spectrometry and chromatography to study a particular protein.

### **HOW DO WE OVERCOME THESE ANALYTICAL LIMITATIONS?**

The limitations to current metalloproteomics outlined above are not insurmountable; rather they simply require some lateral thinking regarding how we integrate modern analytical technology into systems biology. A variety of individual techniques can be combined for metalloproteomic experimental procedures, and these can generally be easily integrated into traditional proteomic workflows (**Figure 2**; Lancaster et al., 2011). By not reinventing the wheel, integration of atomic mass spectrometry (specifically ICP-MS) will allow for metal quantification and direct correlation between the presence of metal species and the function of associated proteins (Lelie et al., 2011; Alvarez et al., 2012). This comprehensive approach will greatly supplement the solely sequence information normally obtained through mass spectrometry independent of any other complementary technique.

Protein peaks identified from chromatographic separation can be correlated to the metal concentration peaks identified by online ICP-MS detection, allowing for the likely position of the metalloprotein to be determined, providing not only selectivity but also sensitivity (Gercken and Barnes, 1991; del Castillo Busto et al., 2005; Lopez-Avila et al., 2006; Manley et al., 2009). 2D separation offers a powerful tool for resolving complex mixtures of metalloproteins, and the multi-elemental capacity of ICP-MS produces a hyperspectral snapshot of metal–protein interactions in a single sample. Like traditional proteomics, this approach will use the vast arsenal of protein databases available to determine which of the proteins identified is most likely to exhibit metal–protein interaction. The 20,000+ proteins predicted in human samples will produce complex fractions highlighting the large dynamic range needed and requirement for fractionation.

Although there are inherent difficulties in investigating proteins in their native state, advances in mass spectrometry and

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#### **FIGURE 2 | Proposed workflow for integrated metalloproteomics.**

Hyphenating native separation techniques to ICP-MS is the key to unlocking the secrets of metals and protein function. Rather than relying solely on bulk measures, directly associating metals with specific proteins provides new insight into how metals carry out biochemical processes in the cell. At the current state of our knowledge about the level of metalloproteins in biology the coupling of size exclusion to ICP-MS have the promise of being quantitative thus allowing the comparison of different samples and detail about the global or metalloprotein specific changes that occur. Despite SEC being a low resolution technique it can allow researchers to make educated guesses about the ID of proteins of interests based on their MW. Continued evolution of hyphenated LC-ICP-MS will only increase the arsenal of tools at our disposal to discover, identify, and characterize metalloproteins. The

techniques used for proteomics [such as SILAC (stable isotope labeling by amino acids in cell culture) and SISCAPA (stable isotope standards and capture by anti-peptide antibodies)] to include the addition of isotopically enriched metal salts, providing new opportunities to probe the direct relationship metal cofactors have with protein function and allowing simultaneous analysis of both metals and proteins in individual experimental groups. Highly sensitive, isotope-specific ICP-MS detection is used to align metal distribution with quantitative proteomics, directly associating the presence of a protein species with a specific, metal-mediated function. This approach is extremely cost-effective, and can be seamlessly integrated into existing workflows with minimal disruption to the standard laboratory process.

analytical techniques are now making it possible to carry out investigations on intact proteins. Groundbreaking work from Joe Beckman's laboratory has shown that using Fourier transformion cyclotron resonance MS it is possible to directly quantify and determine the metal status of a single protein from a specific cell type directly from tissue (Rhoads et al., 2011, 2013). This demonstrates that quantitative determination of both the amount of protein and the metal status can be achieved from biological tissue. Providing a new level of detail for a disease where it is clear that the metal status of a protein is key in the disease process (such as amyotrophic lateral sclerosis; Estévez et al., 1999), this technology is invaluable. The advent of top-down proteomics will be an increasingly valuable tool for metalloproteins identification.

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## **HOW DOES METALLOPROTEOMICS RELATE TO NEURODEGENERATION?**

The brain is the most complex organ in the human body. Some of the proteins in the brain, just like the proteins in other tissues, require metal cofactors for function. Variations of the amount of metal that is present, or defects in the way a protein associates with a metal ion may cause disease states, particularly with regard to neurodegeneration.

The level at which metals are present within the brain is generally higher then the levels of the same metals in the rest of the body, and concentrations of metals in the brain is highly compartmentalized (Harrison et al., 1968; Hare et al., 2012b; Roberts et al., 2012). When metal homeostasis in these regions is altered, brain pathologies and increased oxidative stress is observed. As we age metals accumulate in the brain (Zecca et al., 2004) and this along with any other alterations in metal homeostasis can lead to neuronal damage, death, oxidative stress and may even cause misfolding and aggregation of proteins. However, it is important to point out that the accumulation of metals in tissues is not necessarily labile metals, but also metalloproteins. For example, it is estimated that there is less than one free Cu ion per cell (Rae et al., 1999). Thus an observed accumulation in bulk levels of Cu will have concomitant accumulation of Cu-metalloproteins. This shift in thinking about the general accumulation of metals to the change in functional metalloproteins is the basis of metalloproteomics. Bulk analysis only indicates a global change and still poses the question: do all metalloproteins change or are there specific targets? Our investigations suggest the latter.

In order to fully understand the roles that these metalloproteins play in age-related diseases it is first important to understand the role they play in the aging brain. Studies of trace elements and aging have shown consistent changes in trace elements such as a decrease in Rb and K and increase in metals Fe, Cu, and Co (Ehmann et al., 1984; Takahashi et al., 2001). The identification and characterization of metalloproteins is essential to understanding their functions and elucidating the specific disease pathways they are involved in, as well as assisting with the diagnosis of specific conditions (Gercken and Barnes, 1991; Muñiz et al., 2001; Lopez-Avila et al., 2006; El Balkhi et al., 2010). Characterized metalloproteins such as hemoglobin, transferrin, SOD, and ceruloplasmin are used in clinical laboratories as markers for specific disease states (Swart, 2013), such as anemia, inflammation (Ahluwalia,1998), Down's syndrome (Brooksbank and Balazs, 1984), and Wilson's disease (Mak et al., 2008), respectively.

Metals have long been thought to play a role in pathophysiology of Alzheimer's disease (AD) and Parkinson's disease (PD). In AD, the concentration of three of the most abundant biochemically functional metals (Cu, Zn, and Fe) are altered in respect to their locations (i.e., metal redistribution in response to plaque formation). These metals are also thought to play a

#### **REFERENCES**

Ahluwalia, N. (1998). Diagnostic utility of serum transferrin receptors measurement in assessing iron status. *Nutr. Rev.* 56, 133–141. doi: 10.1111/j.1753- 4887.1998.breaktb01738.x

Alvarez, L., Gonzalez-Iglesias, H., Garcia, M., Ghosh, S., Sanz-Medel, A., and Coca-Prados, M. (2012). The stoichiometric transition from Zn6Cu1-metallothionein to Zn7-metallothionein underlies the up-regulation of metallothionein role in β-amyloid aggregation, which in turn will cause plaque formation, leading to the neurodegenerative effects seen in AD (reviewed in Roberts et al., 2012). The level of Cu in AD brain tissue is decreased, as it is hypothesized that Cu is removed from the tissue and associates itself with the senile plaques that are forming (Deibel et al., 1996; Hung et al., 2010). Zn is also thought to be associated with β-amyloid plaques (Bush et al., 1994), indicating that it has also been redistributed within the brain. Fe is also thought to be associated with plaques, but the overall change of Fe levels within the brain tissue surrounding fibril inclusions has yet to be described conclusively (Lovell et al., 1998; Schrag et al., 2011).

In PD, the levels of Fe and Zn have been suggested to be elevated, while the level of Cu may be decreased. Cu and Fe have been shown to interact with the protein alpha synuclein (Davies et al., 2011; Camponeschi et al., 2013), which is involved in proteinaceous Lewy body formation. Their interaction with alpha synuclein leads to crosslinking and protein aggregation (Barnham and Bush, 2008). Initial research into post-mortem changes in metals in PD dating back to the 1920s indicated that normal Fe became diminished from cells that are present in the globus pallidus and abnormal deposits of large quantities of Fe werefound in globules in the same region of the brain (Lhermitte et al., 1924). Continued pursuit of elucidating the role of metals in PD pathogenesis has still only provided limited insight into the direct metal binding proteins involved, and limitations to analytical technology still present considerable problems for accurate quantification of metal changes (Hare et al., 2012a).

#### **CONCLUDING REMARKS**

Perhaps the biggest impediment to the study of the metalloproteome has been technical limitations. However, the rapid advances in mass spectrometry and analytical chemistry in general over the past decade has helped, in some part, to overcome the shortfalls in the tools available to routinely study incredibly complex matrices. The ubiquitous nature of metal ions and metalloproteins coupled with our relatively limited knowledge about the metalloproteome presents an exciting frontier of new discoveries for the modern biochemist. As the continued growth in analytical technology finds new footholds in the life sciences, we can expect a transition from bulk analysis of trace elements to a detailed investigation of the metalloproteins. With this transition the interest in metalloproteomics will grow exponentially.

#### **ACKNOWLEDGMENTS**

We would like to acknowledge funding from the Victorian Government's Operational Infrastructure Support Program, the Australian Research Council Linkage Projects Scheme (with Agilent Technologies and Kenelec Scientific), and the Australian National Health and Medical Research Council.

(MT) expression quantitative analysis of MT-metal load in eye cells. *J. Biol. Chem*. 287, 28456–28469. doi: 10.1074/jbc.M112.365015

Anderson, N. L., and Anderson, N. G. (1998). Proteome and proteomics: new technologies, new concepts, and

new words. *Electrophoresis* 19, 1853– 1861. doi: 10.1002/elps.1150191103 Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L., and Thornton, J. M. (2008). Metal ions in biological catalysis: from enzyme databases to general principles. *J. Biol. Inorg. Chem.*

"fnagi-05-00035" — 2013/7/16 — 20:21 — page 5 — #5

13, 1205–1218. doi: 10.1007/s00775- 008-0404-5

143, 137–142. doi: 10.1016/S0022- 510X(96)00203-1

in parkinsonism with special reference to its localisation. *J. Neurol. Psychopathol.* 5, 195. doi: 10.1136/jnnp.s1-5.19.195

"fnagi-05-00035" — 2013/7/16 — 20:21 — page 6 — #6

101, 9843–9848. doi: 10.1073/pnas.<PHONE_NUMBER>

**Conflict of Interest Statement:** Rudolf Grimm is an employee of Agilent Technologies. Dominic J. Hare and Blaine R. Roberts receive material support from Agilent. The remaining 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.

*Received: 03 May 2013; paper pending published: 12 June 2013; accepted: 25 June 2013; published online: 18 July 2013.*

*Citation: Lothian A, Hare DJ, Grimm R, Ryan TM, Masters CL and Roberts BR (2013) Metalloproteomics: principles, challenges, and applications to neurodegeneration. Front. Aging Neurosci. 5:35. doi: 10.3389/fnagi.2013.00035*

*Copyright © 2013 Lothian, Hare, Grimm, Ryan, Masters and Roberts. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

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## Metal-induced neurodegeneration in *C. elegans*

## *Pan Chen1, Ebany J. Martinez-Finley1, Julia Bornhorst 1, Sudipta Chakraborty1 and Michael Aschner 1,2\**

*1 Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA*

*2 Department of Pharmacology, the Kennedy Center for Research on Human Development, and the Center for Molecular Toxicology, Vanderbilt University Medical Center, Nashville, TN, USA*

#### *Edited by:*

*Anthony R. White, The University of Melbourne, Australia*

#### *Reviewed by:*

*Marcelo Farina, Universidade Federal de Santa Catarina, Brazil Gawain McColl, The Florey Institute of Neuroscience and Mental Health, Australia*

#### *\*Correspondence:*

*Michael Aschner, Division of Pediatric Toxicology, Vanderbilt University Medical Center, 11425 MRB IV, 2215B Garland Avenue, Nashville, TN 37232-0414, USA. e-mail: michael.aschner@ vanderbilt.edu*

#### **INTRODUCTION**

The simplicity of the small nematode *Caenorhabditis elegans* (*C. elegans*) makes it a suitable model organism for biomedical research. Compared with mammalian models, it has a number of advantages that makes it a powerful tool for studies on human disease. First, 60–80% of human genes have corresponding genes in *C. elegans*, depending on the specific bioinformatic methods used (Titus and Michael, 2006). Many genetic factors causing human diseases have corresponding worm homologs, allowing for mechanism-based studies. Second, the small size and the fast life-cycle of the worm allow for easy maintenance. The adult worm is only 1.3 mm long and it takes 3 days for an embryo to reach adulthood and reproduce. A 100 mm petri-dish can accommodate thousands of self-fertilizing nematodes within couple of days. This is particularly important for pharmaceutical drug screens. Third, *C. elegans* are transparent, which allows *in vivo* study with fluorescent reporters, such as green and red fluorescent proteins. This has been widely used to study axon guidance, neurodegeneration, endocytosis and fat metabolism in living worms. Fourth, the simple, but complete nervous system makes it easy to investigate neurological function in *C. elegans*. Although a worm has only 302 neurons, it shares similar neurotransmitters with humans, including dopamine (DA), acetylcholine (ACh), serotonin (5-HT), *gamma*-aminobutyric acid (GABA), glutamate, and others. Fifth, a variety of tools are available in *C. elegans.* The genome and cell line lineage have been completely mapped, RNA interference (RNAi) libraries are able to knock down 90% of genes, a large pool of knockout mutants are available from the *C. elegans* genetic center (CGC) and commercial worm-specific microarray probes have been developed.

A big advantage afforded by *C. elegans* as a model for studies on human neurological diseases is the simplicity of its nervous system: the worm contains 302 neurons and about 5,000 synapses

The model species, *Caenorhabditis elegans*, has been used as a tool to probe for mechanisms underlying numerous neurodegenerative diseases. This use has been exploited to study neurodegeneration induced by metals. The allure of the nematode comes from the ease of genetic manipulation, the ability to fluorescently label neuronal subtypes, and the relative simplicity of the nervous system. Notably, *C. elegans* have approximately 60–80% of human genes and contain genes involved in metal homeostasis and transport, allowing for the study of metal-induced degeneration in the nematode. This review discusses methods to assess degeneration as well as outlines techniques for genetic manipulation and presents a comprehensive survey of the existing literature on metal-induced degeneration studies in the worm.

**Keywords: neurodegeneration, behavior, metals,** *C. elegans***, neurotransmitters**

(White et al., 1986). Despite this simplicity, *C. elegans* has a complete nervous system with four functional categories of neurons based on their circuitry: (1) motor neurons, which pass synaptic signals to muscle cells; (2) sensory neurons, which convert environmental signals into internal stimuli; (3) interneurons, which receive and deliver signals between neurons; and (4) polymodal neurons, which have two or more of the functions described above. Most commonly, studies are performed on different types of neurons based on their neurotransmitter profile.

The biosynthesis and transport of neurotransmitters are conserved in the nematode and human nervous system. Among different types of neurons, dopaminergic (DAergic) and gammaaminobutyric acid (GABA-ergic) neurodegeneration are the two best characterized. DAergic neuron death is a well-known feature of Parkinson's disease (PD).  Since their genome has been fully characterized it is also known that they contain all genes responsible for DA biosynthesis, packaging and reuptake (Sulston et al., 1975; Jayanthi et al., 1998). Male *C. elegans* have additional DAergic neurons (Sulston et al., 1975). DA signaling is important in mediating learning and locomotion behavior (Sawin et al., 2000). DA is also a requirement for olfactory adaptation (Colbert and Bargmann, 1995). The role of DA in modulation of locomotor activity has been exploited to study DAergic degeneration. Using green fluorescent protein (GFP) (Nass et al., 2002; Cao et al., 2005) or mCherry protein (Harrington et al., 2012) to visualize DAergic neurons in *C. elegans*, researchers are able to monitor neurodegenerative processes in living animals. Many known causes of PD also result in DAergic neurodegeneration in *C. elegans*, including aberrant proteins [alpha-synuclein (Cao et al., 2005; Harrington et al., 2012)], heavy metals [i.e., manganese (Mn) (Benedetto et al., 2010), methylmercury (MeHg) (VanDuyn et al., 2010), aluminum (VanDuyn et al., 2013)], and environmental contaminants [i.e., fungicides (Harrison Brody et al., 2013), pesticides (Negga et al., 2012), insecticides (Mocko et al., 2010), 1-methyl-4-phenylpyridinium (MPP+) (Pu and Weidong, 2008; Mocko et al., 2010), and 6-hydroxydopamine (6-OHDA) (Cao et al., 2005; Ruan et al., 2010)]. The DAergic neurons are mechanosensory and defects or loss of these neurons results in behavioral changes in response to environmental stimuli (i.e., food sensing).

In vertebrates, GABA is the most abundant neurotransmitter in inhibitory synapses in the central nervous system (CNS), while in nematodes, GABA is used as both an inhibitory and excitatory neurotransmitter primarily at neuromuscular synapses, as worms do not have a CNS. GABAergic neurons in the nematode consist of 26 neurons that are categorized under six classes, DD, VD, RME, RIS, AVL, and DVB, based on their synaptic outputs. DD and VD innervate the dorsal and ventral body muscles, RME innervate the head and AVL and DVB innervate the enteric muscles and RIS are interneurons (White et al., 1986). Worms defective in GABAergic function exhibit "shrinking" behavior, whereby the nematode simultaneously hypercontracts both ventral and dorsal body muscles, as well as abnormal defecation and foraging. *unc-25* encodes the GABA enzyme glutamic acid decarboxylase and *unc-47* encodes the GABA vesicular transporter (McIntire et al., 1997; Jin et al., 1999). *unc-30* encodes a homeodomain protein of the Pitx family and regulates the differentiation of the D-type GABAergic neurons (Jin et al., 1994). Overexpression of genetic risk factors associated with Alzheimer's disease (AD) [tau (Kraemer et al., 2003; Miyasaka et al., 2005; Brandt et al., 2009)] and Amyotrophic lateral sclerosis (ALS) [TDP-43 and FUS (Vaccaro et al., 2012)], as well as exposure to several heavy metals [lead (Pb), mercury (Hg), copper (Cu), chromium (Cr), and Mn (Du and Wang, 2009; Xing et al., 2009b)] results in GABAergic neurodegeneration in nematodes. The loss or injury of GABAergic neurons alters locomotion (McLntire et al., 1993), foraging (White et al., 1986) and defecation (McLntire et al., 1993) behaviors, which may be used to monitor the integrity of GABAergic neurons.

In addition to DAergic and GABAergic neurons, neurodegeneration has also been studied in cholinergic neurons. In *C. elegans*, ACh is the major excitatory neurotransmitter, which directly or indirectly regulates locomotion (crawling and swimming) (Mullen et al., 2007), egg laying (Bany et al., 2003), pharyngeal pumping (McKay et al., 2004), defecation cycling (Thomas, 1990), and male mating (Liu and Sternberg, 1995). Gain-offunction nicotinic ACh receptor *acr-2* (Barbagallo et al., 2010) and selenium (Se) (Estevez et al., 2012) are both able to induce cholinergic motor neuron degeneration and paralysis in worms.

Another neurotransmitter, serotonin (5-HT), is synthesized in eight types of neurons and regulates locomotion, defecation, egg laying, male mating and pharyngeal pumping in worms (Mendel et al., 1995; Segalat et al., 1995; Weinshenker et al., 1995; Niacaris and Avery, 2003). The basal slowing response and egg laying assay have been well-characterized in studies on 5-HT signaling. Mutants for 5-HT enzymes, *bas-1* and *cat-4* (both involved in 5-HT and DA biosynthesis), have been used to analyze serotoninmediated behaviors (Loer and Kenyon, 1993; Waggoner et al., 1998; Sawin et al., 2000). The locomotor behaviors have been used most often for analysis of neurodegeneration and application of exogenous 5-HT inhibits locomotion, but stimulates egg-laying and pharyngeal pumping (Horvitz et al., 1982; Segalat et al., 1995; Weinshenker et al., 1995; Sawin et al., 2000; Rogers et al., 2001; Niacaris and Avery, 2003). In many of the worm behavioral assays it is difficult to determine the effects of DA vs. 5-HT. Degeneration of other types of neurons induced by metal exposure is less studied in worms, thus not discussed here.

Finally, glutamate is important in synaptic transmission, plasticity and disease, with excitotoxicity, inflammation, oxidative stress, and mitochondrial dysfunction implicated in ALS. Excitatory and inhibitory ionotropic glutamate receptors (iGluRs) mediate some behavior and approximately 10 putative iGluR subunits are expressed in *C. elegans (glr 1-8 and nmr 1-2)* (Brockie et al., 2001). Six of the subunits, including *glr-1*, *glr-2*, *glr-4*, *glr-5*, *nmr-1*, and *nmr-2*, are expressed in many of the *C. elegans* interneurons. GLR-1 in particular has been linked to mediating the avoidance of hyperosmotic stimuli (Mellem et al., 2002), control of forward and backward movement (Brockie et al., 2001), foraging behavior (Hills et al., 2004), and long-term memory (Rose et al., 2003). There is also conserved sequence and function of the vesicular glutamate transporter (*eat-4*) (Lee et al., 1999), and other glutamate transporters (*glt-1; glt-3-7*) (Mano et al., 2007).

In summary, *C. elegans* provides a valuable research tool to study the mechanisms of metal induced human neurological disorders by permitting the visualization of different types of neurons and assessment of their function by florescence labeled cell bodies and functional behavior assays, respectively.

## **TECHNIQUES FOR STUDYING NEURODEGENERATION IN** *C. elegans*

Conserved neurotransmitter biology and high homology with mammalian systems make *C. elegans* a unique system for studies on neurodegenerative disease. The processes of synaptic release, trafficking and formation of neurotransmitters are also conserved. Methods to study neurotoxicologic endpoints have included examining the morphology, behaviors, and changes in gene expression and neurotransmitters.

## **TOOLS FOR ASSESSING NEURONAL STRUCTURE** *Fluorescently tagged neurons and microscopy*

Contributing to the investigative value of this species is the transparency of the worm, allowing for*in vivo* visualization of fluorescently labeled neurons. Cell bodies and individual processes can be visualized *in vivo* in the worm. Morphological changes, characterized by puncta, blebs, neuronal absence or shrinkage, presence of vacuoles, dorsal or ventral cord gaps, loss of cell bodies or strand breaks in neuronal processes can be used as indicators of neurodegeneration (Nass et al., 2002; Martinez-Finley and Aschner, 2011). GFP has frequently been employed as a reporter construct to visualize specific neurons and synapses in *C. elegans*. DAergic neurons have been tagged for visualization most frequently through the *dat-1*::GFP reporter (Helmcke et al., 2010; VanDuyn et al., 2010), serotonergic neurons through *tph-1*::GFP (Sze et al., 2000; Nass et al., 2002), GABAergic neurons through *unc-25*::GFP (Cinar et al., 2005), and cholinergic neurons through *unc-1*::GFP (Winnier et al., 1999; Nass et al., 2002), and glutamatergic neuron through *eat-4*::GFP (Lee et al., 1999; Earls et al., 2010). Reporter gene fusions also allow for visualization of neuronal morphology and protein expression patterns. Visualization of the neurons is useful, but it is important to correlate the structural damage with cellular and molecular changes as quenching of GFP fluorescence is a possibility and can result in false data interpretation. Both confocal and standard fluorescence microscopy have been used to visualize fluorescently tagged neurons. Electron microscopy has also been used historically to measure structural integrity and fluorescence-activated cell sorting (FACS) has been utilized for purification.

#### **TOOLS FOR ASSESSING NEURONAL FUNCTION**

#### *Behavioral assays*

*C. elegans* behavior is used as a reliable and sensitive measure of the function of neurons as the behaviors are sensitive to underlying cellular and molecular alterations. There are a large variety of behavioral tests in *C. elegans* to assay neurodegeneration. There are nematode equivalents of mammalian DAergic, serotonergic, cholinergic, GABAergic and glutamatergic neurons (see above) and high homology exists between the two systems. A number of behaviors can be assayed to determine the function of the cholinergic and glutamatergic neurons, but these behaviors have not been extensively studied in regards to metals and neurodegeneration, and therefore are not included in this review [for a review see (WormBase, 2013)]. Many of the assays listed below have been designed to assess DA, 5-HT and GABA subtypes.

#### *Basal slowing response*

Bacterial mechanosensation induces the DA-mediated slowing of locomotion in the presence of food (bacteria), and can be measured by counting the number of body bends per 20-s interval (Sawin et al., 2000). Locomotor rates are compared between the well-fed worms placed on plates of food vs. those placed on plates without food and the measurement is referred to as the change (-) in body bends over a set period of time. A lower value represents less slowing on food and therefore deficits in DAergic function as worms deficient in DA do not slow their locomotion in the presence of food. The *cat-2* strain is tyrosine hydroxylase (TH)-deficient and therefore defective in bacterial mechanosensation, making it a positive control (Sawin et al., 2000). Proper controls for the assay include well-fed nematodes and consistency in the size and thickness of the bacterial lawn in all plates used. *Bas-1* and *cat-4* mutants also do not slow in response to the bacterial lawn (Sawin et al., 2000). Application of exogenous serotonin can rescue the behavior in *bas-1* and *cat-4* mutants. In this assay the key to parsing out 5-HT effects from DA effects is the state of the worms; the 5-HT version of this task requires starved worms, while the DA version requires that the worms be well-fed.

#### *Ethanol preference*

Ethanol preference in *C. elegans* requires DA and serotonin. This has been shown using *cat2* and *tph-1* mutants as these worms do not show a normal wildtype response in the ethanol preference task (Lee et al., 2009). In this assay young adult worms are preincubated on a seeded control or ethanol plate. After an incubation period the worms are removed from the preincubation plates and placed on assay plates. Assay plates are divided into quadrants, with two quadrants laced with ethanol and the other without. Worms are placed at an origin point of an assay plate and allowed to move for 30 min at which point they are scored for their quadrant of preference. A preference index (PI) is calculated as [(number of worms in ethanol quadrants) − (number of worms in control quadrants)]/Total number of worms tested (Lee et al., 2009). Worms with functional DA and 5-HT develop ethanol preference whereas mutant worms do not.

#### *Area-restricted searching (ARS)*

This task capitalizes on the fact that worms must have functional DA in order to search for food. Wildtype worms that have consumed and exhausted the immediate food source will expand their search to surrounding areas. This behavior is measured by time-dependent reduction in turning frequency after the last food encounter. After a short time if the worm cannot find more food in the immediate area, the frequency of high-angled turns is reduced and the animal begins to explore more distant areas.  The glutamatergic neurons also participate in this behavior as glutamate receptor mutants exhibit problems with the ARS behavior (Hills et al., 2004).

#### *Habituation task/tap withdrawal response*

Functional DA allows worms to alter their behavior in the tapping task. Nematodes respond to a mechanical stimulus (plate tapping) by accelerating their forward locomotion rate or by swimming backwards. Repeatedly tapping of the plate results in worms habituating to the stimulus and decreasing the frequency of their reversals. What is measured is the time that it takes to habituate to the tapping rather than whether or not the worm responds to the tapping. All worm strains tested in this task are able to reduce their reversal rates but the distinguishing factor of whether DAergic neurons are functional or dysfunctional is the lag time in response. Intertapping interval is also an important consideration in this assay. *cat-2* mutant worms habituate to tap faster than wildtype worms and this response can be rescued by pre-exposing mutants to exogenous DA (Groves and Thompson, 1970; Broster and Rankin, 1994; Rose and Rankin, 2001; Sanyal et al., 2004).

#### *Pharyngeal pumping and thrashing behaviors*

Pharyngeal pumping and thrashing rates are controlled by various mechanisms in the nematode. The pharynx is responsible for feeding via rhythmic contractions. Age-related decline in pharyngeal pumping rate has been described in *C. elegans* and is correlated with a decline in survival probability and body movement decline. Pumps per minute are manually counted using a dissecting microscope to assess pharyngeal pumping rate. The maximum pumping rate of the pharynx is ∼300 pumps per minute (in adults that are at least 2 days old). Thrashing behavior is a common test in *C. elegans* for measuring motility. Thrashing behavior is assessed by videotaping nematodes that are placed in ∼10μL of water on a transparent plate with shallow, concave wells. A thrash is a change in direction in midbody bending. A body bend is counted as a change in the direction of the posterior bulb of the pharynx. There are multiple programs used to assess this behavior including Worm Tracker, Nemo, Parallel Worm Tracker, OptoTracker, Multimodal illumination, and tracking system, CoLBeRT, many of which use MATLAB, JAVA, or LabVIEW software to quantify movement (WormBase, 2013). This assay, along with visualization of the neurons, has been used to assess GABAergic function after lead or mercury exposure (Helmcke et al., 2009; Xing et al., 2009b).

 Thousands of worms (over 30 ul of packed worms) must be used for HPLC analysis and the worms must be washed several times to avoid bacterial contamination (Sulston et al., 1975; Horvitz et al., 1982; Sanyal et al., 2004; Alkema et al., 2005).

#### **USING** *C. elegans* **AS A MODEL OF NEURODEGENERATION: MODIFYING GENETICS**

The *C. elegans* model system has become a popular and innovative tool in advancing mechanistic studies concerning various neurodegenerative processes. The generation of genetic knockouts and knockdowns can be achieved rapidly in nematodes due to their quick life cycle and short lifespan. Classical forward genetics studies involve treating organisms with mutagens to induce DNA mutations followed by isolation of individuals with atypical phenotypes of interest. Reverse genetics allows the opposite strategy by altering a specific gene in order to study its function and the role it may play in various processes and pathways. RNA interference (RNAi) and transgenesis, which can be accomplished using microinjection and gene bombardment techniques are also tools for achieving gene knockdown.

#### *Microinjection*

This approach uses a microinjection needle to introduce a plasmid carrying the gene of interest into the cytoplasmic core of distal gonad arms in a hermaphrodite worm, allowing the delivery of the plasmid to several germ cell nuclei and the production of extrachromosomal DNA arrays (Ahringer, 2006). The simplest approach to transgenesis typically involves microinjecting a plasmid that contains a cloned 5 regulatory sequence of the gene of interest fused to a reporter gene whose activity can then be assayed; a 3 UTR should also be included (Praitis and Maduro, 2011). As the worm is fully transparent, it is usually desired to fuse genes of interest to a fluorescent reporter whose intensity can then be imaged, such as GFP, yellow fluorescent protein (YFP), or m-cherry (Miller et al., 1999). There are also sequences specific for subcellular targeting that can be included in the plasmids,

such as a nuclear localization signal (NLS) for nuclear targeting or a mitochondrial targeting sequence (MTS) for mitochondrial localization. Similarly, tissue or cell-specific expression can also be studied using transgenes that are directed by tissue or cellspecific promoters (Praitis and Maduro, 2011). Additionally, it is typical to include a coinjection marker (antibiotic resistance, tissue-specific fluorescent promoter, etc.) to follow the transgene through the transformation process in larvae and subsequent crossing. However, this is not always necessary if the transgene containing the gene of interest includes a marker itself that can be conveniently distinguished from non-transgenic worms (e.g., a bright GFP reporter).

Following microinjection of transgenes, the DNA arrays produced are extrachromsomal and are not fully integrated into the genome (Ahringer, 2006). Without integration, the arrays remain unstable and can be lost through successive generations. Gamma or UV irradiation or exposure to chemical mutagens can induce integration of the arrays into chromosomes. A small population of the transformed nematodes is mutagenized, resulting in several F1 animals that produce the F2 generation that can then be assayed for 100% transmission of the transgene. Integration of the original transgene is vital for any further genetic manipulation of the transformed lines, eventually allowing multiple transgenes to be incorporated into the same strain (Praitis and Maduro, 2011). As with most genetic manipulations, integrated strains should be backcrossed several times in order to remove any mutations introduced into the genome from the integration process.

However, high copies of the transgene in the extrachromosomal array can lead to undesirable expression of the transgene, ranging from increased transgene expression relative to the targeted endogenous gene to gene silencing from tandem repeat sequences present in the array. For this reason, another method of transgenesis uses microparticle bombardment. Biolistic transformation allows for direct insertion of transgenes into the chromosome, especially with a low-copy number that becomes fully integrated. Bombardment is usually done with the DNAcoated gold beads that are then bombarded onto L4 and adult hermaphrodites. This technique revolves around the ability of DNA to form a complex with gold particles in the presence of CaCl2, where the DNA itself is protected from degradation by using cationic polyamines (e.g., spermidine) (Isik and Berezikov, 2013). Researchers have also found that this "gene gun" technique not only reproducibly allows for invariable expression levels and patterns that are difficult to obtain with extrachromosomal arrays, but that low-copy transgene expression does not get silenced in the germline (Praitis et al., 2001). Newer techniques to allow single- or low-copy transgene expression without bombardment are in the process of development, including the use of ultraviolent trimethylpsoralen (Kage-Nakadai et al., 2012).

#### *RNA interference (RNAi)*

This methodology involves the exogenous introduction of double-stranded RNA (dsRNA) that is complementary to a specific sequence of the gene of interest into the model organism, resulting in the activation of an endogenous cellular pathway that causes significantly decreased expression of that gene. This technique is advantageous in the *C. elegans* system and is dependent on RNA-dependent RNA polymerases (RdRPs) (Simmer et al., 2002); unlike mammals, RNAi is actually heritable in nematodes, as the systemic gene knockdown can persist in the progeny.

RNAi effect can be achieved in three ways: (1) by microinjection of a dsRNA sequence complementary to the gene of interest into the body cavity, including the gonad and intestine, (2) by soaking worms in a dsRNA-containing solution, and (3) by feeding worms with *E. coli* expressing the dsRNA of interest (Tabara et al., 1998).

The unique, highly systemic nature of RNAi in *C. elegans* is thought to occur due to a rapid transport of dsRNA from cell to cell through the SID-1 channel. This transmembrane protein allows for passive, bidirectional transport to cells that do not initially receive the dsRNA delivery, and is selectively gated by the presence of dsRNA (Shih and Hunter, 2011). Wildtype N2 strains are sufficient in producing knockdowns using RNAi; however, some RNAi-hypersensitive strains can be necessary to increase the knockdown strength and phenotype penetrance. These strains include *rrf-3*, *eri-1*, and *lin-15B*. *rrf-3* is a *C. elegans* RNA-dependent RNA polymerase (Simmer et al., 2002), while *eri-1* (enhanced RNAi) is a 3 -5 exoribonuclease that negatively regulates RNAi (Kennedy et al., 2004) and *lin-15B* is important in cell differentiation and negatively regulates vulval development, as well as RNAi (Wang et al., 2005). There are currently two commercially available RNAi feeding libraries. The Ahringer lab library contains about 16,757 clones that were constructed from cloning gene-specific genomic sequences between two T7 promoters. The Vidal lab library contains 11,511 clones and was made by the Gateway cloning system that clones full-length open reading frames (ORF) templates into double T7 vectors. Together, these libraries cover approximately 94% of the *C. elegans* genome (Ahringer, 2006; Antoshechkin and Sternberg, 2007).

#### *Gene knockout*

In comparison to RNAi-mediated gene knockdown, knockout in *C. elegans* was made simple by the development of the MosDel system, or Mos-mediated deletion. A plasmid containing Mos1 is delivered to a DNA strand that is next to the target gene, causing a break in that strand. Upon induction of cellular DNA damage repair mechanisms, a DNA template that lacks the targeted gene of interest is introduced and matches the sequence of the broken DNA strand (Frokjaer-Jensen et al., 2010). Thus, this technique tricks the cell into knocking out the gene by repairing the original double-strand break with a DNA template that does not contain the gene of interest. Large-scale projects have aimed to utilize the MosDel system to create large libraries of transposon-tagged alleles (Bazopoulou and Tavernarakis, 2009). Prior to the development of this method, knockouts were primarily generated using random chemical mutagenesis (i.e., EMS mutagenesis) to create loss-of-function mutants that were then screened using gene-specific primers for random deleted regions in targeted genes. However, with this technique there is the possibility of random background mutations and it is not as specific as the MosDEL system. The commercial availability of deletion mutants is possible through the *C. elegans* Gene Knockout Consortium (GKC) and the National BioResource Project of Japan (NBRP), both of whom send their isolated deletion mutants to the *Caenorhabditis* Genetics Center (CGC) for distribution (Antoshechkin and Sternberg, 2007).

## **MECHANISMS OF NEURODEGENERATION INDUCED BY METAL** *Aluminum (Al)*

Al is one of the most abundant metal elements in the Earth's crust and exposure to bioavailable Al may be significant owing to its ubiquitous presence in soil and fertilizers, cookware, and pharmaceutical and cosmetic preparations (Verstraeten et al., 2008). Although the physiological requirement for Al has yet to be defined, it has been posited that Al may have an etiopathogenic role in neurodegenerative diseases (Halatek et al., 2005). Studies with Al in the form of AlCl3, Al(NO3)3, or Al2(SO4)3 identified phenotypic abnormalities, including toxic effects on lifespan, body size, development, reproduction, locomotion, behavioral plasticity, and memory in the worm (Wang et al., 2009; Page et al., 2012). Additionally, Page et al. showed changes in elemental composition of whole worms exposed to Al, noting a significant increase in multiple metals and ensuing oxidative stress (Ba, Fe, Cr, and Cu), hypothesizing that altered levels of these elements contributed to the aforementioned phenotypes seen in chronic Al toxicity (Page et al., 2012).

VanDuyn et al. addressed molecular attributes of Al-induced DA neurodegeneration in *C. elegans* (VanDuyn et al., 2013). A brief exposure to Al decreased mitochondrial membrane potential and cellular ATP levels and led to DAergic neurodegeneration, which was dependent upon SMF-3, a homolog to the human divalent metal transporter (DMT1). Al exposure significantly decreased SMF-3 protein levels and exacerbated DAergic neurodegeneration in the presence of human PD-associated protein α-synuclein, Nrf2/SKN-1 and Apaf1/CED-4. Deletion of SMF-3 conferred resistance to Al due to sequestration of Al in an intracellular compartment (VanDuyn et al., 2013). Neuroprotection was also reported by Ye et al. showing that a treatment with trace vitamin E could ameliorate memory deficits in Al exposed worms (Ye et al., 2008).

The acute and chronic effects of Al2O3 nanoparticles (NPs) in *C. elegans* have been recently addressed (Yu et al., 2011; Li et al., 2012). Acute toxicity was associated with increased lethality and altered growth, reproduction and stress responses, whereas chronic toxicity led to increased oxidative stress. Li et al. noted decreased locomotion behaviors in nematodes chronically exposed to Al2O3-nanoparticles (NPs) concomitant with increased reactive oxygen species (ROS) generation and disruption of ROS defense mechanisms, secondary to the suppression of Mn-SODs (Li et al., 2012).

#### *Copper (Cu)*

The essential trace element Cu serves as a cofactor in many critical biological processes such as in iron (Fe) homeostasis (ceruloplasmin), catecholamine biosynthesis (tyrosinase, dopamine-b-hydroxylase), oxidative phosphorylation (cytochrome c oxidase), and oxidative stress protection (superoxide dismutase) (Arredondo and Nunez, 2005). Therefore, Cu deficiency or overload may result in multiple pathologies, including irreversible CNS damage (Prohaska, 2000; Taly et al., 2007; Diaz-Veliz et al., 2008). In *C. elegans*, Cu deficiency led to decreased Cu/Zn superoxide dismutase (SOD), reducing defenses against oxidative stress (Yang et al., 1998). Exposure to excess CuSO4 induced detrimental effects on brood size and life span, an increase in generation time and impaired development (Harada et al., 2007; Calafato et al., 2008). PD worms expressing α-synuclein or lacking parkin do not show increased sensitivity to Cu toxicity (Ved et al., 2005). In the context of AD, Cu has been shown to increases amyloid deposits and Aß oligomer aggregation, and decrease the amount of soluble Aß oligomer (Rebolledo et al., 2011). The increased Aß aggregation is associated with improvement in behavioral deficits and synaptic function. This protective effect is not attributable to the activation of the SKN-1/NRF2 phase II detoxification pathway (Rebolledo et al., 2011). Luo et al. noted that high Cu concentrations significantly increased the paralysis rate of the Aß(1–42) transgenic worms, whereas lower Cu concentrations decreased paralysis rate. ROS were identified to be responsible for the paralysis and the ROS induced by Aß(1–42) and Cu was mediated through *sod-1*, *prdx-2*, *skn-1*, *hsp-60*, and *hsp-16.2* genes (Luo et al., 2011). The amyloid precursor protein (APP) of AD has a copper-binding domain (CuBD) located in the N-terminal cysteine-rich region that can strongly bind Cu (II) and reduce it to Cu (I). The CuBD of *C. elegans*, APL-1, protected against Cu-mediated lipid peroxidation and neurotoxicity, therefore the CuBD of APP is predicted to play a role in detoxification of neuronal Cu (White et al., 2002; Cerpa et al., 2004).

#### *Cadmium (Cd)*

Cd is an environmental pollutant that has been classified as a category 1 human carcinogen (IARC, 1993). Cd exposure is associated with teratogenic and mutagenic responses (WHO, 1996). The major routes of human exposure include diet and cigarette smoke (EFSA, 2009). In *C. elegans*, Cd has been shown to alter behavior. It also resulted in decreased growth, life span and reproduction and affected feeding and movement (Popham and Webster, 1979; Boyd et al., 2010; Hoss et al., 2011; Hsu et al., 2012). Cd exposure was also shown to cause GABAergic neurodegeneration in worms. At low Cd concentration neuronal loss was observed, while at high Cd concentration axonal degeneration and neuronal loss, as well as reduced size of AVL, RMEs and RIS neurons was noted in fluorescently labeled GABAergic neurons (Du and Wang, 2009).

#### *Iron (Fe)*

The essential trace element Fe exists abundantly in the environment and is present in various enzymes and proteins. It has a central role in many essential cellular processes such as DNA synthesis, mitochondrial respiration, oxygen transport, and neurotransmitter synthesis (Cairo et al., 2002). Fe homeostasis has to be maintained within a small range because a dysregulation caused by Fe deficiency or overload leads to hematological, neurodegenerative and metabolic diseases (Dusek et al., 2012; Tandara and Salamunic, 2012). Genes involved in Fe and energy homeostasis in vertebrates are conserved in the nematode. These include aconitase, ferritin, divalent metal transporter-1 (DMT-1), frataxin, and Fe sulfur cluster assembly proteins. The Fe regulating protein-1 (IRP-1) homolog (ACO-1) of *C. elegans* has aconitase activity and is post-translationally regulated by Fe. Although ACO-1 is predicted to be the IRP-1 homolog in *C. elegans*, it fails to bind to the ferritin mRNAs and no conserved Fe responsive elements have been found on ferritin mRNAs in *C. elegans* (Gourley et al., 2003).

In contrast, Kim et al. reported that *aco-1* and *ftn-1* expression levels are regulated by Fe. Both aco-1 and *ftn-1* gene expression is inversely correlated. *ftn-1* and *ftn-2* are encoding ferritins in *C. elegans* (Kim et al., 2004). In mutant animals lacking ACO-1 and FTN-1, reduced lifespan has been observed, indicating that *aco-1* and *ftn-1* are important for regulating Fe homeostasis. *daf-16* mutants show decreased lifespan upon Fe treatment, suggesting that DAF-16 signaling might be also involved in Fe homeostasis (Kim et al., 2004). HIF-1 (hypoxia-inducible factor-1) is a negative regulator of ferritin transcription, inhibiting *ftn-1* and *ftn-2* transcription during Fe deficiency. Furthermore, the activation of the Fe uptake gene smf-3 (a homolog of DMT-1) by HIF-1 during Fe-deficiency (Romney et al., 2011) provides a mechanism to maintain sufficient Fe stock for growth and survival when Fe is limited in *C. elegans*.

Fe overload in worms causes phenotypic and behavioral defects as well as alteration the resistance to oxidative stress, characterized by reduced lifespan, body size, generation time, brood size, head thrash and body bend frequencies, as well as chemotaxis plasticity (Hu et al., 2008; Valentini et al., 2012). Several of these defects (body bend frequency and life span) were transferred from Fe-exposed *C. elegans* to their progeny (Hu et al., 2008). The adverse effects of Fe on locomotive behavior suggest that Fe might be involved in disruption of synaptic function between neurons and muscle cells. In *C. elegans* models of Aß toxicity, Fe was shown to possess high affinity for Aß. Aß accumulation in the Aß-expressing strain CL2006 resulted in Fe homeostasis disruption. In addition to increasing Fe content, Aß has also been shown to increase ROS generation (Wan et al., 2011).

## *Lead (Pb)*

Lead is a pervasive environmental neurotoxicant; it is particularly toxic to developmental brains, causing long-term detrimental effects on learning, memory and behavior in children (Neal and Guilarte, 2010). Pb exposed *C. elegans* show severe abnormalities in their lifespan, development, locomotion, learning and memory behaviors (Ye et al., 2008; Zhang et al., 2010). Younger larva (L1, L2, L3) nematodes exhibited increased lethality and reproductive toxicity (brood size, generation time) compared to L4 stage or adult nematodes (Guo et al., 2009; Xing et al., 2009a). L3 larvae showed higher sensitivity in transgenerational behavioral and growth effects than older life stages (Yu et al., 2013). Neurodegeneration, as shown by neuronal loss and dorsal/ventral cord gaps is more pronounced in L1 larvae compared to older nematodes (Du and Wang, 2009; Xing et al., 2009b).

Pb exposure decreases thermotaxis behaviors in adult *C. elegans*. This behavior is mainly controlled by the AFD sensory neurons and it has been shown that Pb caused severe deficits in the structural properties of these neurons. Pre-treatment with antioxidants inhibited ROS elevation and mitochondrial dysfunction caused by Pb and prevented the induction of oxidative damage, severe deficits in thermotaxis and damage to the AFD sensory neurons (Wu et al., 2012). Vitamin E and mild UV radiation or a pre-treatment with heat shock increase the resistance of nematodes to Pb toxicity, ameliorating the effects on locomotion behaviors, stress response and memory deficits (Ye et al., 2008; Wang et al., 2010; Ye et al., 2010).

#### *Methylmercury (MeHg)*

Mercury (Hg) and methylmercury (MeHg) are of greatest concern as MeHg is an ubiquitous environmental contaminant (Sanfeliu et al., 2003) and a major source of human exposure is associated with the consumption of seafood (Fitzgerald and Clarkson, 1991). McElwee et al. compared the toxicity of HgCl2 and MeHgCl, noting the latter is significantly more toxic than HgCl2 when assessing feeding, movement and reproduction, and in its ability to increase the steady-state levels of stress response genes (McElwee and Freedman, 2011). Helmcke et al. showed that while lethality, pharyngeal, pumping, growth and development were affected in *C. elegans*, brood size, lifespan, trashing rate, and nervous system morphology were largely unaffected in response to MeHg exposure. Concerning Hg uptake in *C. elegans*, its concentrations are as high as those found in mammalian systems (Helmcke et al., 2009; Helmcke and Aschner, 2010). It is also confirmed that the involvement of oxidative stress in MeHg toxicity is through alterations in GSH levels, increasing expression of HSP and glutathione-S-transferase (Helmcke and Aschner, 2010). In mammals, Nrf-2 regulates oxidative stress response.

#### *Manganese (Mn)*

The naturally abundant micronutrient Mn is an essential trace element and is of crucial importance as co-factor for a wide variety of enzymes, including arginase, pyruvate carboxylase, the antioxidant enzyme SOD, as well as enzymes involved in neurotransmitter synthesis and metabolism (Santamaria and Sulsky, 2010). An imbalance in Mn homeostasis caused by either Mn deficiency or overload is well known to cause severe CNS dysfunction, whereas Mn deficiency is because of its ubiquitous presence in food extremely rare. Therefore, several mechanisms are involved in maintaining the Mn homeostasis. The NRAMPs/divalent metal transporters play distinct roles in Mn transport and the identified *C. elegans* homologs (SMF-1, SMF-2, and SMF-3) have been characterized with corresponding roles in Mn homeostasis and sensitivity (Au et al., 2009; Bandyopadhyay et al., 2009; Settivari et al., 2009). The three SMFs differ in localization and function, and SFM-3 has to be shown as the major Mn transporter. A deletion of the *smf-1* or *smf-3* increased Mn tolerance against Mn-induced DAergic neurodegeneration with *smf-3* as the most resistant mutant, whereas the deletion of *smf-2* increases Mn sensitivity indicating the protective role of *smf-2* against Mn (Au et al., 2009).  In *C. elegans* PMR1, a P-type Ca2+/Mn2+ ATPase is involved in the regulation of Ca and Mn ions and PMR-1 knockdowns can render resistance to stresses, such as oxidative stress (Cho et al., 2005; Bandyopadhyay et al., 2009). McColl et al. provide the first quantitative subcellular visualization of endogenous Mn concentrations in *C. elegans* identifying Mn enriched within specific cell types, especially intestinal cells (McColl et al., 2012).

Concerning Mn toxicity in *C. elegans*, it has been investigated that the anti- or pro-oxidative role for Mn is dependent on the Mn-concentration worms are exposed to (Lin et al., 2006). In case of Mn overload accelerated development, an increase in total fertility, reduced body and brood size and life span was observed (Lin et al., 2006; Xiao et al., 2009).  Benedetto et al. found that Mn-induced oxidative stress was dependent on the extracellular levels of DA, which was confirmed by using strains lacking functional DA receptors (DOP-1, 2, 3) and the DA transporter (DAT-1) that regulate extracellular DA, and strains lacking vesicular monoamine transporter 2 (CAT-1 in worms) and tyrosine hydroxylase (CAT-2 in worms) that regulate intracellular DA levels. Decreased extracellular DA levels are protective against Mn toxicity (Benedetto et al., 2010). They identified NADPH dual-oxidase, BLI-3, potentiates the formation of RONS from DA-derived species obtained by the Mn (II) and extracellular DA exposure, while the bli-3 mutant a hyper-resistant phenotype of Mn toxicity showed which could be a potential therapeutic target against Mn toxicity (Benedetto et al., 2010). Consistent with the oxidative stress associated with Mn exposure, SKN-1 (antioxidant transcription factor contributing to protection against ROS; homolog of mammalian Nrf2) overexpression or nuclear relocalization of SKN-1 protects from Mn neurotoxicity indicating a beneficial strategies in limiting Mn toxicity (Benedetto et al., 2010). Prominent theories on neurodegeneration implicate further protein aggregation, including α-synuclein. Recently suppression of α-synuclein-induced toxicity has been demonstrated by expression of PARK9 encoding a putative P-type transmembrane ATPase (ATP13A2) protein (Gitler et al., 2009).

## *Zinc (Zn)*

Zn is one of the most prevalent essential elements. It is a cofactor in many enzymes and transcription factors involved in several cellular processes and cellular signaling pathways (Shuttleworth and Weiss, 2011). Zinc deficiency as well as excess causes a wide spectrum of defects in multiple organ systems. Zn's role in memory, learning, neurogenesis, processes related to brain aging and neurological diseases have been extensively reviewed (Szewczyk et al., 2011).

In *C. elegans*, families of the CDFs (cation diffusion facilitators), ZIPs (Zrt- and Irt-like proteins) and MTs (metallothioneins) are involved in Zn metabolism. Deletion of MT-1 and MT-2 results in increased Zn accumulation, whilst *mtl-1* knockout worms show heightened sensitivity to increased Zn level (Leszczyszyn et al., 2011). *cdf-1* and *sur-7* are both members of the CDF family, and mutations in either gene result in increased sensitivity to Zn, indicating that both genes are necessary for Zn tolerance. However, *sur-7* mutants were more tolerant than a *cdf-1* mutant to increased Zn concentrations, suggesting functional differences between these two proteins (Yoder et al., 2004). In addition, mutations of haly-1, which encodes an enzyme converting histidine to uric acid, cause elevated levels of histidine and protect against Zn toxicity (Murphy et al., 2011). A lossof-function study of *cdf-1*, *cdf-2*, *sur-7*, and *haly-1* showed that *haly-1* promotes Zn resistance in the genetic backgrounds characterized by Zn sensitivity and functions in parallel to *cdf* genes in modulating Zn sensitivity. Zn exposure in *C. elegans* results in multiple biological defects affecting life span, reproduction, locomotion behavior (head trash, body bend) and chemotaxis plasticity (Wang et al., 2007). The phenotypic and behavioral toxicities could be transferred from Zn-exposed nematodes to their progeny (Yoder et al., 2004; Wang et al., 2007). While defects such as life span, generation time, brood size, and chemotaxis plasticity could be partially rescued in the progeny, no rescue phenomena could be observed for body sizes and the locomotion behaviors. In addition, it has been reported that the pesticide Mn/Zn-ethylenebis-dithiocarbamate promotes neurodegeneration in DAergic and GABAergic neurons (Guven et al., 1999; Negga et al., 2011, 2012; Harrison Brody et al., 2013).

### **CONCLUSION**

*C. elegans* is a valuable model to study human neurological diseases due to conserved genome, low culture cost, short generation

### **REFERENCES**

cycle, transparent tissue and simple nervous system, together with well-developed tools. A solid number of nematode models related to human neurological disorders have already been developed, including AD (Miyasaka et al., 2005; Brandt et al., 2009), Parkinson's disease (Cao et al., 2005; Hamamichi et al., 2008), Huntington's disease (Parker et al., 2001; Caldwell et al., 2003), ALS (Vaccaro et al., 2012), and early-onset torsion dystonia (Cao et al., 2005; Chen et al., 2010). These disease models facilitate the study of metal-induced neurological toxicity and many novel discoveries have already been made in *C. elegans* to uncover the mechanisms of metals related human neurological disorders. Despite of these advantages, limitations of the nematode system need to be considered. For example, some genes and signaling pathways inherent to mammals (such as α-synuclein and Aβ discussed above) are missing in this model. In sum, *C. elegans* provide a valuable platform for exploratory study of human neurological diseases by providing mechanistic information on processes inherent to neurodegenerative disorders.

## **ACKNOWLEDGMENTS**

The authors acknowledge partial support for this review from the National Institute of Environmental Health Sciences (R01 ES 10563, P30 ES 00267, T32 ES 007028), The Josef Schormüller Award and the DFG (BO 4103/1-1).

by transgenic overexpression of Alzheimer's disease-like modified tau. *Neurobiol. Aging* 30, 22–33.

cellular stress in the dopaminergic neurons of *Caenorhabditis elegans*. *J. Neurosci.* 25, 3801–3812.

pathways generate olfactory plasticity in *C. elegans*. *Neuron* 14, 803–812.

developmental stages. *Reprod. Toxicol.* 28, 90–95.

in the nematode *Caenorhabditis elegans*. *Science* 216, 1012–1014.

genes in *C.elegans*. *J. Mol. Biol.* 342, 421–433.

future directions. *Biochem. Med. (Zagreb)* 22, 311–328.

and D. H. Hall. Available online at: http://www.wormatlas.org

cross-adaptation response to neurobehavioral toxicity from lead and mercury exposure in nematodes. *PLoS ONE* 5:e14052. doi: 10.1371/journal.pone.0014052

**Conflict of Interest Statement:** 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.

*Received: 26 February 2013; paper pending published: 11 March 2013; accepted: 05 April 2013; published online: 20 May 2013.*

*Citation: Chen P, Martinez-Finley EJ, Bornhorst J, Chakraborty S and Aschner M (2013) Metal-induced neurodegeneration in C. elegans. Front. Aging Neurosci. 5:18. doi: 10.3389/fnagi. 2013.00018*

*Copyright © 2013 Chen, Martinez-Finley, Bornhorst, Chakraborty and Aschner. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

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