Genetic Screening of the Mitochondrial Rho GTPases MIRO1 and MIRO2 in Parkinson’s Disease

1. INTRODUCTION

Degeneration of dopamine (DA) neurons in substantia nigra (SN) pars compacta causes the typical motor symptoms seen in patients with Parkinson’s disease (PD); resting tremor, rigidity, bradykinesia and postural instability [1,2]. However, neuropathology in PD is widespread, affecting neurons in areas from the gastrointestinal tract to cerebral cortex [3]. In accordance with the systemic nature of the disease, there is increasing evidence that mitochondrial dysfunction may underlie some forms of PD [4-6]. Mitochondria are essential in all eukaryotic cells for generating ATP, calcium buffering and involvement in programmed cell death [7-9]. To adapt to cellular demands mitochondria undergo fusion and fission, two opposing processes that exist in equilibrium [10]. The mitochondrial network is very dynamic in neurons [11,12] and the rate of mitochondrial fusion and fission is high [13]. Recruitment of mitochondria to specific neuronal compartments has been shown to be an active ATP consuming process [14]. In support of a mitochondrial involvement in PD, the metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is 1-methyl-4-phenylpyridinium (MPP⁺), is a neurotoxin and inhibits the mitochondrial respiratory chain complex I, which causes degeneration of DA neurons in SN [15]. Another inhibitor of complex I is the pesticide rotenone. In rodents it causes selective degeneration of DA neurons, as well as other neurons to a lesser extent, thereby resembling the pathology of PD [16]. Complex I activity has been found to be reduced in SN and in platelets from PD patients [17].

Different polymorphisms in mtDNA have been reported to be associated with both increased and decreased risk of PD [18]. We and others have found associations between PD risk and two nuclear genes involved in mitochondrial maintenance, MTIF3 (mitochondrial translation initiation factor 3) and POLG1 (DNA polymerase gamma 1). MTIF3 is part of the initiation complex formation on the mitochondrial 55S ribosome and regulates translation of proteins within mitochondria [19,20]. A synonymous single nucleotide polymorphism (SNP) in MTIF3, rs7669, has been reported to be associated with PD in cohorts with different geographical origin [21-23]. POLG1 is important for replication and repair of the mitochondrial genome [24]. Variations in length of the polyglutamine tract of POLG1 have been found to associate with PD by a number of groups in different geographical regions [25-27].

Several of the PARK genes identified by linkage studies have been reported to influence mitochondria in different ways. Mutations in PTEN induced putative kinase 1 (PINK1) at PARK6, reported to regulate mitochondrial fission, cause early-onset familial PD [28]. Mutations in Parkin at PARK2 have been reported to cause autosomal recessive early-onset Parkinsonism [29]. Parkin acts in a similar way as PINK1 [30] and is believed to be recruited from the cytoplasm by PINK1 to initiate autophagic degradation of impaired mitochondria [31]. DJ-1 at PARK7 causes autosomal recessive early-onset Parkinsonism [32] and is suggested to operate in a parallel pathway to that of PINK1/Parkin to maintain mitochondrial function in oxidative environments [33]. Pink1 has been shown to be part of a mitochondrial multi-protein complex together with the atypical GTPases Miro1 and Miro2 (mitochondrial Ras homolog gene family, member T1 and T2) and the adaptor protein Milton [34]. This suggests that Pink1 also plays a role in mitochondrial trafficking.

Miro1 and Miro2, also referred to as Rhot1 and Rhot2, belong to the mitochondrial Rho GTPase family and are involved in axonal transport of mitochondria [35,36]. Miro1 and Miro2 were first identified in yeast [37] and later described to share similarities with Rho GTPases [38]. Miro proteins have a C-terminal domain locating them to the mitochondrial outer membrane, as well as two GTPase domains and two calcium binding EF-hands [36,37]. Calcium binding to the EF-hands regulates the trafficking of mitochondria along microtubules [39,40].

Based on the finding that mitochondrial dysfunction has been implicated in PD [4,41], the importance of Miro1 and Miro2 in mitochondrial transport [35,36] and the link between Miro and Pink1 [34], we analyzed SNPs in MIRO1 and MIRO2 with regard to PD. We hypothesized that genetic variations in functionally important regions of these two genes might lead to disturbed mitochondrial trafficking, fusion and/or fission disturbances in neurons and hence increase the risk of neurodegenerative events, such as those observed in PD. We therefore investigated the possible association of five SNPs located in functional regions of the proteins MIRO1 and MIRO2. The SNPs were selected from the NCBI database and screened in a Swedish Parkinson case-control material (see Table 1). Three SNPs were selected in MIRO1, one synonymous SNP in the GTPase domain (rs16967164) and two non-synonymous SNPs in each of the two EF-hands (rs28630420, rs34538349). The other two SNPs were non-synonymous SNPs located in one of the EF-hand domains of MIRO2 (rs1139897 and rs3743912).

2. MATERIALS AND METHODOLOGY

3. RESULTS

The results from the TaqMan genotyping of the five SNPs in MIRO1 and MIRO2 are presented in Table 2. We did not find any genotypic or allelic (data available upon request) association of the MIRO1 or MIRO2 SNPs screened in our Swedish Parkinson case-control material. The observed frequencies of the controls were in agreement with the Hardy-Weinberg equilibrium (data not shown). For two of the SNPs in MIRO1 (rs28630420, rs34538349) we only observed the wild-type genotype.

To investigate if any of the SNPs had a possible effect on age of onset we stratified the material into early disease onset (≤50 years) or late disease onset (>50 years) and compared genotype and allele frequencies. However, such stratification did not reveal any significant associations with disease at genotype or allele (data available upon request) levels, as shown in Table 3.

4. DISCUSSION

Dysfunctional mitochondria may be the cause or the consequence of DA neuron degeneration in PD. This is the first genetic study analyzing possible links between the two GTPases MIRO1 and MIRO2 and disease in general and neurodegenerative disease in particular. Two functionally important regions in the two Miro proteins are the two GTPase domains and two calcium binding EF-hands [36,37]. Another protein with a GTPase domain linked to PD is leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains, including functional Roc GTPase and a protein kinase domain. Several mutations in the kinase domain of LRRK2 are associated with both sporadic and familial PD [44]. Recently, a new mutation (N1437S) localized within the GTPase domain of LRRK2 (PARK8) was found to cosegregate with PD [45]. Furthermore, it has been suggested that the GTPase domain may contribute to the toxicity of LRRK2 [46]. Interestingly, knockdown of lrk-1, the ortholog of LRRK2 in C. elegans, has been reported to lead to reduced survival and is associated with dysfunctional mitochondria [47]. There are no reports on proteins with EF-hand domains being linked to PD today.

It has been shown that all domains of Gem1P (i.e. yeast Miro) are needed for proper mitochondrial morphology [37]. Loss-of-function mutations in the Miro GTPase in Drosophila melanogaster lead to impaired locomotion and premature death of the flies [48]. Based on these findings we selected polymorphisms that are located in functional regions in MIRO1 and MIRO2 to analyze possible association with risk for neurodegeneration. The three selected MIRO1 SNPs are localized in one of the GTPase domains (rs16967164) and in each of the two EF-hands (rs28630420, rs34538349). The two SNPs in MIRO2 (rs1139897 and rs3743912) are situated in one of the EF-hand domains. We did not find association with any of the selected SNPs in our Swedish case-control material. For two of the SNPs in MIRO1 (rs28630420 and rs34538349) we only detected the wild-type genotype in our material of cases and controls. Thus these two mutations tend to be very rare in the area from which our DNA samples have been collected. The results after stratification of the material regarding age of onset did not reveal any age-of-onset association with any of the detected polymorphisms. Further genetic analysis and functional studies in disease are however needed before one can exclude MIRO1 and MIRO2 as potential candidate genes for PD.

CONCLUSION

Genetic variation in MIRO1 and MIRO2 might influence transport of mitochondria along microtubules, leading to reduced local energy production which can lead to degeneration of DA neurons. Although we did not find a significant association with PD and the selected polymorphisms in MIRO1 and MIRO2 in our Swedish case-control material, we cannot exclude these Rho GTPases as candidate genes for PD or other neurodegenerative disorders.

CONFLICT OF INTEREST

None declared.

REFERENCES