Genetic Screening of the Mitochondrial Rho GTPases MIRO1 and MIRO2 in Parkinson’s Disease
Anna Anvret1, Caroline Ran1, Marie Westerlund1, 2, Olof Sydow3, Thomas Willows3, Lars Olson1, Dagmar Galter1, Andrea Carmine Belin*, 1
Identifiers and Pagination:Year: 2012
First Page: 1
Last Page: 5
Publisher Id: TONEUJ-6-1
Article History:Received Date: 13/12/2011
Revision Received Date: 20/2/2012
Acceptance Date: 20/2/2012
Electronic publication date: 3/4/2012
Collection year: 2012
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
MIRO1 and MIRO2 (mitochondrial Ras homolog gene family, member T1 and T2) also referred to as RHOT1 and RHOT2, belong to the mitochondrial Rho GTPase family and are involved in axonal transport of mitochondria in neurons. Because mitochondrial dysfunction is strongly implicated in Parkinson’s disease (PD), MIRO1 and MIRO2 can be considered as new candidate genes for PD. We analyzed two non-synonymous polymorphisms and one synonymous polymorphism in MIRO1 and two non-synonymous polymorphisms in MIRO2, in a Swedish Parkinson case-control material consisting of 241 patients and 307 neurologically healthy controls. None of the analyzed polymorphisms in MIRO1 and MIRO2 were significantly associated with PD. Although we did not find a significant association with PD in our Swedish case-control material, we cannot exclude these Rho GTPases as candidate genes for PD or other neurodegenerative disorders.
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 . 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 . The mitochondrial network is very dynamic in neurons [11,12] and the rate of mitochondrial fusion and fission is high . Recruitment of mitochondria to specific neuronal compartments has been shown to be an active ATP consuming process . 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 . 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 . Complex I activity has been found to be reduced in SN and in platelets from PD patients .
Different polymorphisms in mtDNA have been reported to be associated with both increased and decreased risk of PD . 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 . 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 . Mutations in Parkin at PARK2 have been reported to cause autosomal recessive early-onset Parkinsonism . Parkin acts in a similar way as PINK1  and is believed to be recruited from the cytoplasm by PINK1 to initiate autophagic degradation of impaired mitochondria . DJ-1 at PARK7 causes autosomal recessive early-onset Parkinsonism  and is suggested to operate in a parallel pathway to that of PINK1/Parkin to maintain mitochondrial function in oxidative environments . 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 . 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  and later described to share similarities with Rho GTPases . 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 , 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).
Investigated Polymorphisms in MIRO1 and MIRO2, their Nucleotide Position, Consequence, Genomic Location and Functional Protein Region
|Reference Sequence||Nucleotide Position and Change||Amino Acid Change||Genomic Location||Protein Domain|
|rs16967164||c.1458A>G||Glu486Glu||Exon 17||GTPase domain|
2. MATERIALS AND METHODOLOGY
A Swedish PD case-control material of Caucasian origin was genotyped for three SNPs in MIRO1 and two in MIRO2 (see Table 1). The PD material consisted of 241 individuals from the Stockholm area (mean age 67.0 years, 60.9% men). The DNA was obtained after informed oral and written consent and approval of the local ethics committee, Regionala etikprövningsnämnden, Stockholm, Sweden. All PD subjects met the United Kingdom Parkinson's Disease Society Brain Bank Criteria for PD except that more than one affected first, second or third degree relative was allowed . Control subjects consisted of 307 neurologically healthy spouses of PD patients and individuals from the SNAC-K project (The Swedish National Study on Aging and Care in Kungsholmen) from the Stockholm area (mean age 64.2 years, 40.4% men). DNA was extracted from blood according to standard protocols.
The SNPs were genotyped with predesigned TaqMan SNP Genotyping Assays: C_1630552_10 (rs16967164), C_60583023_10 (rs28630420), C_25937296_10 (rs3743912), C_2463996_1_ (rs1139897) and a custom designed assay (rs34538349) using a fast real-time PCR instrument (ABI 7500 FAST Real-Time PCR, Applied Biosystems, Foster City, CA, USA). The TaqMan assay contained primers and 5´ fluorescently labeled (FAM and VIC) minor groove binding probes (20×) for detection of the SNPs. Allelic discrimination was run with pre- and custom-designed primers and probes, genotyping master mix (TaqMan®, Applied Biosystems, Foster City, CA, USA) and 10-20 ng of genomic DNA in a total reaction volume of 10 µl mixed in transparent 96-well plates. The polymerase chain reaction (PCR) conditions followed the recommendations of default settings for the SNP assay, except that the number of cycles were set to 55 and run at 92°C for 15 s. The ramp speed was set to standard. A post-PCR read was done for allelic discrimination using appropriate software (SDS version 2.0.4) supplied with the instrument. To test for genotyping errors we used water as negative controls and re-genotyped randomly chosen samples to confirm the results. Case-control analysis between individual sequence variants in MIRO1 and MIRO2 were performed using a Chi-square (χ2) test . Three PD patients previously known to carry the pathogenic G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) were excluded from the association analysis. Distribution of genotypes in controls was tested for consistency with the Hardy-Weinberg equilibrium. Statistical significance was defined as p< 0.05.
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.
Genotype Frequencies of MIRO1 and MIRO2 Variants in Swedish Patients with Parkinson´s Disease (PD) Compared to Matched Neurologically Healthy Controls Analyzed using a Two-sided Chi-square (χ2) test
|Genotype Frequency % (n)||χ2||P-value|
|PD||66.1 (152)||30.9 (71)||3 (7)||0.09||0.96|
|Control||66.3 (203)||31.1 (95)||2.6 (8)|
|PD||100 (235)||0 (0)||0 (0)||-||-|
|Control||100 (300)||0 (0)||0 (0)|
|PD||100 (236)||0 (0)||0 (0)||-||-|
|Control||100 (306)||0 (0)||0 (0)|
|PD||57.3 (133)||34.9 (81)||7.8 (18)||2.03||0.36|
|Control||51.1 (155)||40.3 (122)||8.6 (26)|
|PD||73.5 (169)||24.8 (57)||1.7 (4)||0.87||0.65|
|Control||76.9 (233)||21.8 (66)||1.3 (4)|
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.
Stratification of Parkinson’s Disease (PD) Cases into Early (≤50 years) and Late Onset (>50 years), Comparing MIRO1 and MIRO2 Genotype Frequencies Analyzed using a Two-sided Chi-square (χ2) Test
|Genotype Frequency % (n)||χ2||P-value|
|≤50 PD||62.3 (33)||40.0 (18)||3.8 (2)||0.45||0.80|
|>50 PD||66.7 (118)||31.1 (55)||2.2 (4)||0.06||0.97|
|Controls||66.3 (203)||31.1 (95)||2.6 (8)|
|≤50 PD||49.1 (26)||45.3 (24)||5.7 (3)||0.79||0.67|
|>50 PD||59.6 (106)||32.0 (57)||8.4 (15)||3.51||0.17|
|Controls||51.1 (155)||40.3 (122)||8.6 (26)|
|≤50 PD||77.4 (41)||22.6 (12)||0||-||-|
|>50 PD||72.2 (127)||25.6 (45)||2.3 (4)||1.63||0.44|
|Controls||76.9 (233)||21.8 (66)||1.3 (4)|
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 . Recently, a new mutation (N1437S) localized within the GTPase domain of LRRK2 (PARK8) was found to cosegregate with PD . Furthermore, it has been suggested that the GTPase domain may contribute to the toxicity of LRRK2 . 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 . 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 . Loss-of-function mutations in the Miro GTPase in Drosophila melanogaster lead to impaired locomotion and premature death of the flies . 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.
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
We would like to thank Professor Laura Fratiglioni for providing us with control samples from the Swedish National Study on Aging and Care in Kungsholmen (SNAC-K) project and Doctor Fengqing Xiang for excellent technical assistance. The study was supported by the Swedish Research Council, the Swedish Brain Foundation, Karolinska Institutet Funds, Magnus Bergvalls stiftelse, the Swedish Parkinson Foundation, Åke Wibergs stiftelse and Swedish Brain Power.