**5. Conclusion**

248 Toxicity and Drug Testing

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

In 2002, *PARK8* gene mutations were discovered as a major genetic cause associated with hereditary parkinsonism (Paisan-Ruiz *et al.*, 2004). The *PARK8* gene was associated with PD in studies of a Japanese Sagamihara family who responded positively to treatment with L-DOPA, which had parkinsonism that presented with an unknown aetiology of the disease (Funayama *et al.*, 2002). Other studies examined two additional families (German and Canadian) who also had an autosomal dominant, late-onset parkinsonism (Zimprich *et al.*,

In the LRRK2 structure, two functional domains, kinase and GTPase domains, were shown to be present. The G2019S mutation was present in the kinase domain specific to the binding site for Mg2+ (Kachergus *et al.*, 2005). This mutation facilitates the access of the kinase domain to its substrates, which increases autophosphorylation 2.5-fold the phosphorylation of other substrates, such as myelin basic protein (MBP), 3-fold for the LRRK2 autophosphorylation without the presence of this mutation (Jaleel *et al.*, 2007; West *et al.*, 2005), which is responsible for the increased toxicity of this molecule (Greggio *et al.*, 2006). In the GTPase domain, the R1441C has been the most studied mutation, and there is controversy as to the influence of GTPase mutations on the kinase activity that was observed in some studies in which the increase was similar (Guo *et al.*, 2007) or had no

LRRK2 has been shown to play different roles in the cell; however, little information is available. Based on the data we found from the protein interactions, there was a relationship between LRRK2 and cytoskeletal reorganisation (Gandhi *et al.*, 2008), maintenance functions and cell morphology (Plowey *et al.*, 2008), protein transport through synaptic vesicles (Shin *et al.*, 2008), and the ubiquitination process (Ko *et al.*, 2009). There have also been studies that relate LRRK2 and apoptosis (Ho *et al.*, 2009). Previous studies have shown a relationship between LRRK2 and other PD-related proteins, such as parkin (Ng *et al.*, 2009; W. W. Smith *et al.*, 2005), PINK-1 and DJ-1 (Venderova *et al.*, 2009) or α-synuclein (X. Lin *et al.*, 2009). The interaction of LRRK2 with PQ is not clear. Studies in *Drosophila melanogaster* in which the deletion of kinase domain of LRRK2 did not induce a higher sensitivity to the PQ stimulus has been shown (D. Wang *et al.*, 2008). In contrast, in *Caenorhabditis elegans* studies, the expression of human LRRK2 protein protected against PQ, which increased nematode survival in response to agents that cause mitochondrial dysfunction. However, protection by G2019S, R1441C, or kinase-dead LRRK2 was less effective than wild-type LRRK2 (Saha *et al.*, 2009). In another study with *Caenorhabditits elegans, PINK1* mutant genes have been observed in a minor mitochondrial length and increased PQ sensitivity of the nematode. Moreover, the mutants also displayed defects in axonal outgrowth of a pair of canalassociated neurons. We demonstrated that in the absence of lrk-1 (the *C. elegans* homologue

dopaminergic neuronal loss (Lavara-Culebras & Paricio, 2007)

**4.4 LRRK2 (PARK8) and paraquat** 

change (Jaleel *et al.*, 2007).

2004).

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.
