**4. Paraquat and Parkinson´s disease-related proteins**

As previously described, PD is characterised by the selective degeneration of dopaminergic neurons. The aetiology of PD is unknown but has a multifactorial origin that involves both genetic and environmental factors. The interaction of both factors was, in part, involved in the selective death of dopaminergic neurons observed in PD. Apart from the studies that have identified human mutations as a basis for disease, the high number of individuals with

(R. A. Gonzalez-Polo *et al.*, 2007a, 2007b). Moreover, our group has shown that PQ exposure induced an early reticulum stress response that was correlated with the adaptive activation of autophagy, characterised by the accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3-II, p62 degradation, and mammalian target of rapamycin dephosphorylation (R. A. Gonzalez-Polo *et al.*, 2007a, 2007b; Niso-Santano *et al.*, 2011). This response was increased in cells that overexpressed wild-type (WT) ASK1 (apoptosis signal kinase 1) protein. In this model, the inhibition of autophagy caused an exacerbation of the apoptosis induced by ASK1 WT overexpression with or without PQ. These results suggest that autophagy has an important role in the cell death/survival events produced by PQ and

Therefore, increased autophagy might be a new strategy for the treatment of neurodegenerative diseases (Menzies *et al.*, 2006). It is encouraging to consider enhancing the autophagic capacity as a therapeutic strategy in the prevention of neurodegeneration because studies have shown that the abnormal regulation of autophagic pathways may lead

ASK1 that contribute to neuronal degeneration.

to apoptosis and cell death (Walls *et al.*, 2010).

Fig. 3. Schematic representation of the different types of autophagy

As previously described, PD is characterised by the selective degeneration of dopaminergic neurons. The aetiology of PD is unknown but has a multifactorial origin that involves both genetic and environmental factors. The interaction of both factors was, in part, involved in the selective death of dopaminergic neurons observed in PD. Apart from the studies that have identified human mutations as a basis for disease, the high number of individuals with

**4. Paraquat and Parkinson´s disease-related proteins** 

sporadic PD have an unknown aetiology. These individuals have multifactorial disease in which the environment plays important roles. PQ is an environmental agent that has been associated with PD. A recent study by Caroline Tanner concluded that people using PQ and rotenone were 2.5 times more likely to develop PD than those who were not in contact with them (Tanner *et al.*, 2011). Therefore, there is a relationship between the toxicity of PQ and PD. This interaction is not known; however, several studies directly indicate the interaction of PQ with PARK genes.

The development of PD was attributed to different events, such as mitochondrial dysfunction, oxidative stress or the aggregation of proteins. These events could be important to understanding the relationship between PQ and PARK genes (Table 1).


Table 1. Some characteristics of PARK genes

The increase in oxidative stress has been observed in the substantia nigra of PD brains, as demonstrated by the increased lipid, protein, and DNA oxidation or increased total iron content (Bagchi *et al.*, 1995; Mattson, 2006). This alteration of cellular redox balance may be produced by different mechanisms because of the enzymatic conversion to secondary reactive products and/or ROS by the depletion of antioxidant defences or the impairment of antioxidant enzyme function (Abdollahi *et al.*, 2004). Autosomal recessive PD-associated genes such as parkin, DJ-1 and PTEN-induced putative kinase 1 (PINK), have been shown to be involved in mitochondrial function, which suggests that mitochondrial dysfunction and the generation of ROS were central events in the pathogenesis of PD. Therefore, further study of the implication of these proteins in PQ toxicity would be of interest.

Paraquat, Between Apoptosis and Autophagy 247

Therefore, parkin mutations should lead to an incorrect ubiquitination, blocking the degradation of the protein and leading to protein accumulation. Mutant parkin has been shown to impair mitochondrial function and morphology in human fibroblasts and to sensitise the cells to an insult with PQ, producing higher levels of oxidised proteins in the *Parkin*-mutant samples than in controls (Grunewald *et al.*, 2010). PQ has also been demonstrated to induce alterations in parkin solubility and result in its intracellular

PINK1 is a serine/threonine kinase capable of autophosphorylation. This protein has an Nterminal mitochondrial targeting signal (MTS), is synthesised as a full-length version (FL) and is processed into at least two cleaved forms (∆1 and ∆2) (W. Lin & Kang, 2008). PINK1 is considered to be a mitochondrial protein with a role in protecting against oxidative stress and apoptosis in *in vitro* models (Valente *et al.*, 2004). Mutations in *PINK1* have been associated with autosomal recessive PD (Valente *et al.*, 2004) and with *PINK1* KO flies with motor deficits and disorganised mitochondrial morphology (Clark *et al.*, 2006). For the link between PINK1 and the toxicity of PQ, studies using silencer PINK1 have shown an increase in oxidative stress and ATP depletion and a higher sensitivity to PQ (Gegg *et al.*, 2009). Similar results have been observed in studies that examined PINK1 nonsense and missense

DJ-1 is a small protein that belongs to the ThiJ/PfpI protein superfamily (S. Bandyopadhyay & Cookson, 2004) that was initially identified as an oncogene that interacted with H-Ras (Nagakubo *et al.*, 1997). The involvement of DJ-1 in neurodegeneration was found when it was discovered that the DJ-1 gene (*PARK7*) was the cause of autosomal recessive PD in a Dutch family (Bonifati *et al.*, 2003). Different pathogenic mutations have been identified in the *PARK7* gene, including truncation, exonic deletions and homozygous and heterozygous missense mutations (Hague *et al.*, 2003). L166P is the most dramatic point mutation, whereas other mutations, such as A104T and M26I, have a weaker destabilising effect on the protein structure. The L166P mutation is located in the centre of α-helix 7, which is a major part of the hydrophobic patch. This mutation has been shown to destabilise the dimeric structure of DJ-1 by promoting the unfolding of its C-terminal region, resulting in rapid degradation (Miller *et al.*, 2003; Moore *et al.*, 2003). However, the frequency of DJ-1 mutations was low, with it being estimated at approximately 1-2 % in early onset PD. The physiological function of DJ-1 is unclear, but it may have a role in protecting against mitochondrial damage in

The link between DJ-1 and PQ exposure has been correlated with autophagy and the apoptotic process. An active role for DJ-1 in the autophagic response produced by PQ has been suggested. In a study using transfected cells exposed to PQ and DJ-1-specific siRNA, an inhibition of the autophagic events induced by the herbicide, the increased sensitisation during PQ-induced apoptotic cell death and the exacerbation of apoptosis in the presence of the autophagy inhibitor 3-methyladenine (R. A. Gonzalez-Polo *et al.*, 2009) had been shown. Interestingly, PQ-induced toxicity and proteasome dysfunction was potentiated in a DJ-1 deficiency (Lavara-Culebras & Paricio, 2007; Menzies *et al.*, 2005). In another study using DJ-1 null cells from the DJ-1(-/-) mouse embryos, DJ-1 null cells showed a resistance to PQinduced apoptosis, including reduced poly (ADP-ribose) polymerase and procaspase-3. Therefore, DJ-1 could be important to maintain mitochondrial complex I, and complex I could be a key target in the interaction of PQ toxicity and DJ-1 in PD (Kwon *et al.*, 2011). In

aggregation (C. Wang *et al.*, 2005).

mutations (Grunewald *et al.*, 2009).

**4.3 DJ-1 (PARK7) and paraquat** 

response to oxidative stress (Canet-Aviles *et al.*, 2004).

In contrast, the misfolding and aggregation of proteins is another pathway of cell toxicity in PD. The failure of α-synuclein (PD-related protein) clearance by the ubiquitin-proteasome system UPS (ubiquitin proteosome system) led to its accumulation over time and to the formation of fibrillar aggregates and Lewy bodies. In this vein, there is a relationship between PQ toxicity and PD because exposure to PQ has been shown to induce proteasome dysfunction and α-synuclein aggregation (Ding & Keller, 2001; Fei *et al.*, 2008; Goers *et al.*, 2003; Manning-Bog *et al.*, 2002; Yang & Tiffany-Castiglioni, 2007).

Therefore, there is a relationship between the toxicity exerted by PQ and different *PARK* genes.

#### **4.1 α-synuclein (PARK1, PARK4) and paraquat**

Lewy bodies (LBs) are abnormal aggregates of protein that develop inside the nerve cells in PD. The presence of α-synuclein in these aggregates has been shown to play an important role in the formation of LBs (Masliah *et al.*, 2000; Spillantini *et al.*, 1997). The mechanisms that promote intraneuronal α-synuclein assembly remain poorly understood. Missense mutations (A53T, A30P and E46K) or multiplications (duplications and triplications) in the *α-synuclein* gene (*PARK1/4*) caused autosomal-dominant parkinsonism (Polymeropoulos *et al.*, 1997), but it is still unclear whether fibrils of aggregated α-synuclein, as found in LBs, have a causative role in the more common forms of PD or could be a marker for the underlying pathogenetic process. α-synuclein has three common forms, monomers, dimers, and protofibrils, and it is thought that an excess of the protofibril forms inhibited UPS *in vitro* (McNaught *et al.*, 2001) and *in vivo* (Dyllick-Brenzinger *et al.*, 2010). α-synuclein protofibrils have been shown to directly lead to oxidative stress that could further impair UPS by reducing ATP levels, inhibiting the proteasome and by the oxidation of parkin.

Studies have indicated that the interaction of environmental factors with alterations in αsynuclein might be involved in the aetiology of PD. The interaction of α-synuclein with PQ toxicity has been extensively examined. PQ has been shown to potentiate α-synucleininduced toxicity (Norris *et al.*, 2007). PQ preferentially binds to the partially folded αsynuclein intermediate because PQ has been shown to induce a conformational change in αsynuclein and significantly increase the rate of the formation of α-synuclein fibrils *in vitro* (Uversky *et al.*, 2001). *In vivo*, rodent studies have shown that the administration of PQ induced an increase in α-synuclein levels in the brain. These results suggest that the upregulation of α-synuclein as a result of toxic insult and the direct interactions between the protein and environmental agents are potential mechanisms leading to α-synuclein pathology in neurodegenerative disorders (Manning-Bog *et al.*, 2002).

#### **4.2 PINK1/PARKIN (PARK6/PARK2) and paraquat**

Another hallmark PD characteristic is mitochondrial dysfunction. In *post-mortem* analysis in the substantia nigra, some patients with PD showed complex I deficiency (Schapira *et al.*, 1989). In addition, the oxidative stress was higher in patients with parkinsonism (Jenner, 2003). In this sense, *PINK1* (*PARK6*) and *Parkin* (*PARK2)* are 2 genes related to PD that may be involved in the regulation of mitochondrial homeostasis.

Parkin mutations were first linked to an autosomal recessive juvenile-onset form of PD in Japanese families (Kitada *et al.*, 1998; Matsumine *et al.*, 1997). Numerous parkin mutations have been described, including deletions, multiplications and missense mutations (Hattori & Mizuno, 2004). Parkin protein acts as an E3 ubiquitin protein ligase in the UPS (Shimura *et al.*, 2000). Ubiquitination of proteins is essential to start to proteasomal protein degradation.

In contrast, the misfolding and aggregation of proteins is another pathway of cell toxicity in PD. The failure of α-synuclein (PD-related protein) clearance by the ubiquitin-proteasome system UPS (ubiquitin proteosome system) led to its accumulation over time and to the formation of fibrillar aggregates and Lewy bodies. In this vein, there is a relationship between PQ toxicity and PD because exposure to PQ has been shown to induce proteasome dysfunction and α-synuclein aggregation (Ding & Keller, 2001; Fei *et al.*, 2008; Goers *et al.*,

Therefore, there is a relationship between the toxicity exerted by PQ and different *PARK*

Lewy bodies (LBs) are abnormal aggregates of protein that develop inside the nerve cells in PD. The presence of α-synuclein in these aggregates has been shown to play an important role in the formation of LBs (Masliah *et al.*, 2000; Spillantini *et al.*, 1997). The mechanisms that promote intraneuronal α-synuclein assembly remain poorly understood. Missense mutations (A53T, A30P and E46K) or multiplications (duplications and triplications) in the *α-synuclein* gene (*PARK1/4*) caused autosomal-dominant parkinsonism (Polymeropoulos *et al.*, 1997), but it is still unclear whether fibrils of aggregated α-synuclein, as found in LBs, have a causative role in the more common forms of PD or could be a marker for the underlying pathogenetic process. α-synuclein has three common forms, monomers, dimers, and protofibrils, and it is thought that an excess of the protofibril forms inhibited UPS *in vitro* (McNaught *et al.*, 2001) and *in vivo* (Dyllick-Brenzinger *et al.*, 2010). α-synuclein protofibrils have been shown to directly lead to oxidative stress that could further impair UPS by reducing ATP levels, inhibiting the proteasome and by the oxidation of parkin. Studies have indicated that the interaction of environmental factors with alterations in αsynuclein might be involved in the aetiology of PD. The interaction of α-synuclein with PQ toxicity has been extensively examined. PQ has been shown to potentiate α-synucleininduced toxicity (Norris *et al.*, 2007). PQ preferentially binds to the partially folded αsynuclein intermediate because PQ has been shown to induce a conformational change in αsynuclein and significantly increase the rate of the formation of α-synuclein fibrils *in vitro* (Uversky *et al.*, 2001). *In vivo*, rodent studies have shown that the administration of PQ induced an increase in α-synuclein levels in the brain. These results suggest that the upregulation of α-synuclein as a result of toxic insult and the direct interactions between the protein and environmental agents are potential mechanisms leading to α-synuclein

2003; Manning-Bog *et al.*, 2002; Yang & Tiffany-Castiglioni, 2007).

pathology in neurodegenerative disorders (Manning-Bog *et al.*, 2002).

Another hallmark PD characteristic is mitochondrial dysfunction. In *post-mortem* analysis in the substantia nigra, some patients with PD showed complex I deficiency (Schapira *et al.*, 1989). In addition, the oxidative stress was higher in patients with parkinsonism (Jenner, 2003). In this sense, *PINK1* (*PARK6*) and *Parkin* (*PARK2)* are 2 genes related to PD that may

Parkin mutations were first linked to an autosomal recessive juvenile-onset form of PD in Japanese families (Kitada *et al.*, 1998; Matsumine *et al.*, 1997). Numerous parkin mutations have been described, including deletions, multiplications and missense mutations (Hattori & Mizuno, 2004). Parkin protein acts as an E3 ubiquitin protein ligase in the UPS (Shimura *et al.*, 2000). Ubiquitination of proteins is essential to start to proteasomal protein degradation.

**4.2 PINK1/PARKIN (PARK6/PARK2) and paraquat** 

be involved in the regulation of mitochondrial homeostasis.

**4.1 α-synuclein (PARK1, PARK4) and paraquat** 

genes.

Therefore, parkin mutations should lead to an incorrect ubiquitination, blocking the degradation of the protein and leading to protein accumulation. Mutant parkin has been shown to impair mitochondrial function and morphology in human fibroblasts and to sensitise the cells to an insult with PQ, producing higher levels of oxidised proteins in the *Parkin*-mutant samples than in controls (Grunewald *et al.*, 2010). PQ has also been demonstrated to induce alterations in parkin solubility and result in its intracellular aggregation (C. Wang *et al.*, 2005).

PINK1 is a serine/threonine kinase capable of autophosphorylation. This protein has an Nterminal mitochondrial targeting signal (MTS), is synthesised as a full-length version (FL) and is processed into at least two cleaved forms (∆1 and ∆2) (W. Lin & Kang, 2008). PINK1 is considered to be a mitochondrial protein with a role in protecting against oxidative stress and apoptosis in *in vitro* models (Valente *et al.*, 2004). Mutations in *PINK1* have been associated with autosomal recessive PD (Valente *et al.*, 2004) and with *PINK1* KO flies with motor deficits and disorganised mitochondrial morphology (Clark *et al.*, 2006). For the link between PINK1 and the toxicity of PQ, studies using silencer PINK1 have shown an increase in oxidative stress and ATP depletion and a higher sensitivity to PQ (Gegg *et al.*, 2009). Similar results have been observed in studies that examined PINK1 nonsense and missense mutations (Grunewald *et al.*, 2009).

#### **4.3 DJ-1 (PARK7) and paraquat**

DJ-1 is a small protein that belongs to the ThiJ/PfpI protein superfamily (S. Bandyopadhyay & Cookson, 2004) that was initially identified as an oncogene that interacted with H-Ras (Nagakubo *et al.*, 1997). The involvement of DJ-1 in neurodegeneration was found when it was discovered that the DJ-1 gene (*PARK7*) was the cause of autosomal recessive PD in a Dutch family (Bonifati *et al.*, 2003). Different pathogenic mutations have been identified in the *PARK7* gene, including truncation, exonic deletions and homozygous and heterozygous missense mutations (Hague *et al.*, 2003). L166P is the most dramatic point mutation, whereas other mutations, such as A104T and M26I, have a weaker destabilising effect on the protein structure. The L166P mutation is located in the centre of α-helix 7, which is a major part of the hydrophobic patch. This mutation has been shown to destabilise the dimeric structure of DJ-1 by promoting the unfolding of its C-terminal region, resulting in rapid degradation (Miller *et al.*, 2003; Moore *et al.*, 2003). However, the frequency of DJ-1 mutations was low, with it being estimated at approximately 1-2 % in early onset PD. The physiological function of DJ-1 is unclear, but it may have a role in protecting against mitochondrial damage in response to oxidative stress (Canet-Aviles *et al.*, 2004).

The link between DJ-1 and PQ exposure has been correlated with autophagy and the apoptotic process. An active role for DJ-1 in the autophagic response produced by PQ has been suggested. In a study using transfected cells exposed to PQ and DJ-1-specific siRNA, an inhibition of the autophagic events induced by the herbicide, the increased sensitisation during PQ-induced apoptotic cell death and the exacerbation of apoptosis in the presence of the autophagy inhibitor 3-methyladenine (R. A. Gonzalez-Polo *et al.*, 2009) had been shown. Interestingly, PQ-induced toxicity and proteasome dysfunction was potentiated in a DJ-1 deficiency (Lavara-Culebras & Paricio, 2007; Menzies *et al.*, 2005). In another study using DJ-1 null cells from the DJ-1(-/-) mouse embryos, DJ-1 null cells showed a resistance to PQinduced apoptosis, including reduced poly (ADP-ribose) polymerase and procaspase-3. Therefore, DJ-1 could be important to maintain mitochondrial complex I, and complex I could be a key target in the interaction of PQ toxicity and DJ-1 in PD (Kwon *et al.*, 2011). In

Paraquat, Between Apoptosis and Autophagy 249

of human LRRK2), all phenotypic aspects of *PINK1* loss-of-function mutants were

PQ has been suggested as a potential aetiological factor for the development of PD. We have demonstrated that PQ was able to induce cell death by activating apoptotic machinery. However, PQ also displayed characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Finally, the cells suffered apoptotic death when the PQ remained. Whereas caspase inhibition retarded cell death, autophagy inhibition increased apoptotic cell death induced by PQ. These findings suggest a relationship between autophagy and apoptotic cell death following paraquat exposition and allows us to further investigate and increase our knowledge regarding the toxicity of paraquat and its relationship with the origin of PD.

Jose M. Bravo-San Pedro was supported by a Junta de Extremadura predoctoral fellowship. Mireia Niso-Santano was supported as a postdoctoral contract of the University of Extremadura. Ruben Gómez-Sánchez was supported by a Spanish Ministerio de Educación predoctoral fellowship. Rosa-Ana González-Polo was supported by a "Miguel Servet" contract (ISCIII, Ministerio de Ciencia e Innovación, Spain). Dr. González-Polo receives research support from ISCIII (Ministerio de Ciencia e Innovación, Spain (CP08/00010, PI11/00040). Dr. José M. Fuentes receives research support from the Ministerio de Ciencia e Innovación, Spain (SAF2010-14993), FUNDESALUD (PRIS10013) and Consejería, Economía, Comercio e

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**5. Conclusion** 

**6. Acknowledgments** 

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DJ-1-deficient mice treated with PQ, decreased proteasome activities and increased ubiquitinated protein levels were found, and these pathologies were not observed in brain regions of normal mice treated with PQ (Yang *et al.*, 2007). In another mouse study, the loss of DJ-1 increased the sensitivity to oxidative insults but did not produce neurodegeneration. Similar results have been found when analysing *Drosophila melanogaster* mutants for the DJ-1 orthologous genes, DJ-1alpha and DJ-1beta, that resulted in increased sensitivity to PQ insults, reduced lifespan and motor impairments. However, these mutations did not lead to dopaminergic neuronal loss (Lavara-Culebras & Paricio, 2007)
