Differential mtDNA damage patterns in a transgenic mouse model of Machado-Joseph disease (MJD/SCA3)

Mitochondrial dysfunction has been associated with late onset neurodegenerative disorders, among which is Machado-Joseph disease (MJD/SCA3). In a previous study, using a transgenic mouse model of MJD, we reported a decrease in mitochondrial DNA (mtDNA) copy number and an accumulation of the 3876-bp deletion with age and with phenotype development. We extended this study by analyzing the pattern of mtDNA depletion and the accumulation of the 3876-bp deletion in 12 older transgenic (TG) and 4 wild-type (wt) animals, and by investigating the accumulation of somatic mutations in the D-loop region in 76 mice (42 TG and 34 wt). mtDNA damage was studied in TG and wt mice at different ages and tissues (blood, pontine nuclei, and hippocampus). Results for older mice demonstrate an accumulation of the mtDNA 3867-bp deletion with age, which was more pronounced in TG animals. Furthermore, the tendency for mtDNA copy number decrease with age, in all analyzed tissues of TG and wt animals, was also confirmed. No point mutations were detected in the D-loop, neither in TG nor wt animals, in any of the tissues analyzed. Due to the absence of mtDNA somatic mutations, we can suggest that mtDNA point mutation accumulation cannot be used to monitor the development and progression of the phenotype in this mouse model and likely in any MJD mice model. The present results further confirm not only the association between mtDNA alterations (copy number and deletions) and age, but also between such alterations and the expression of the mutant ataxin-3 in TG mice.NK and AR are a Fundo Regional para a Ciencia postdoctoral fellow (M3.1.7/F/002/2008 and M3.1.7/F/031/2011). This work was partially supported by Generalitat de Catalunya (SGR 2009-566)

Patterns of mitochondrial DNA damage in blood and brain tissues of a transgenic mouse model of Machado-Joseph disease

BACKGROUND: Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by a CAG tract expansions in the ATXN3 gene. Patterns of mitochondrial damage associated with pathological findings of brain tissues could provide molecular biomarkers of this disorder. OBJECTIVE: The potential of mitochondrial DNA (mtDNA) damage as a biomarker of MJD progression was investigated using a transgenic mouse model. METHODS: DNA was obtained from affected (pontine nuclei) and nonaffected tissues (hippocampus and blood) of transgenic animals of three distinct age groups: 8 weeks, before onset of the phenotype; 16 weeks, at onset, and 24 weeks, at well-established phenotype. Wild-type littermate mice, serving as controls, were analyzed for the same tissues and age groups. mtDNA damage was studied by fluorescence-based quantitative PCR in 84 transgenic and 93 wild-type samples. RESULTS: A clear pattern of decrease in mtDNA copy number with age and accumulation of 3,867-bp deletions at the initial stages (both being more pronounced in transgenic mice) was observed. Pontine nuclei, the affected tissue in transgenic mice, displayed 1.5 times less copies of mtDNA than nonaffected brain tissue hippocampus (odds ratio = 1.21). Pontine nuclei displayed the highest percentage of mtDNA deletions (6.05% more in transgenic mice). CONCLUSION: These results suggest that mtDNA damage is related to the initiation of the phenotype in transgenic mice; mtDNA 3,867-bp deletions may be a biomarker of the initial stages of the disease.This study was supported by the following grants: DRCT Postdoctoral fellowship to N.K. (M3.1.7/F/002/2008), FCT Postdoctoral fellowship to T.C. (SFRH/BPD/38659/2007) and C.B. (SFRH/BPD/63121/2009), FCT research grants to S.S. (PTDC/SAU-GMG/64076/2006) and A.S.F. (PIC/IC/83013/2007)

The APOE ε2 allele increases the risk of Earlier Age at onset in Machado-Joseph disease

Background. Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder of late onset, caused by a (CAG)n expansion at the ATXN3 gene (14q32.1). Variation in age-at-onset is partially explained by the size of the (CAG)n tract in expanded alleles. The remaining variation should be the product of other factors, namely modifier genes. The genotype at the APOE locus has been described as a possible modifier in different neurological disorders, namely Parkinson (PD) and Huntington disease (HD). In the CNS, apolipoprotein E constitutes an important mediator of cholesterol transport/metabolism, which is essential for synaptic integrity and neuronal function. Objective. To investigate a modulating effect of the APOE polymorphism on age-at-onset of MJD. Design and Subjects. The APOE polymorphism was typed in a series of 192 MJD patients. Results. Cases with the ε2/ε3 genotype presented an earlier onset, when compared with those with ε3/ε3 or ε3/ε4. In this series of patients, the presence of an APOE ε2 allele implies a decrease of nearly 5 years in the age-at-onset. When combining, in a general linear model, several other predictors, namely the presence/absence of the APOE ε2 allele, with the size of the (CAG)n in expanded alleles, the model was significantly improved and the explanation of onset variance was raised from 59.8% to 66.5%. Furthermore, the presence of the ε2 allele was associated with an onset below 39 years (OR=5.00; 95% CI: 1.18-21.14). Conclusions. These findings indicate that the polymorphism at the APOE gene plays a role as a genetic modifier of MJD phenotype.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BPD/63121/2009, SFRH/BPD/38659/2007, M3.1.3/F/004/2009, “Secretaria Regional da Ciência, Tecnologia e Equipamentos”.Fundação para a Ciência e a Tecnologia (FCT) - “Transcriptional variation of the ATXN3 gene as modulator of the clinical heterogeneity in Machado-Joseph disease (MJD)” (PIC/IC/83074/2007)Institute of Biotechnology and Biomedicine (IBBA) - “High prevalence diseases in the Azores Islands” (M2.1.2/I/026/2008

Novel candidate blood-based transcriptional biomarkers of Machado-Joseph disease

BACKGROUND: Machado-Joseph disease (or spinocerebellar ataxia type 3) is a late-onset polyglutamine neurodegenerative disorder caused by a mutation in the ATXN3 gene, which encodes for the ubiquitously expressed protein ataxin-3. Previous studies on cell and animal models have suggested that mutated ataxin-3 is involved in transcriptional dysregulation. Starting with a whole-transcriptome profiling of peripheral blood samples from patients and controls, we aimed to confirm abnormal expression profiles in Machado-Joseph disease and to identify promising up-regulated genes as potential candidate biomarkers of disease status. METHODS: The Illumina Human V4-HT12 array was used to measure transcriptome-wide gene expression in peripheral blood samples from 12 patients and 12 controls. Technical validation and validation in an independent set of samples were performed by quantitative real-time polymerase chain reaction (PCR). RESULTS: Based on the results from the microarray, twenty six genes, found to be up-regulated in patients, were selected for technical validation by quantitative real-time PCR (validation rate of 81% for the up-regulation trend). Fourteen of these were further tested in an independent set of 42 patients and 35 controls; 10 genes maintained the up-regulation trend (FCGR3B, CSR2RA, CLC, TNFSF14, SLA, P2RY13, FPR2, SELPLG, YIPF6, and GPR96); FCGR3B, P2RY13, and SELPLG were significantly up-regulated in patients when compared with controls. CONCLUSIONS: Our findings support the hypothesis that mutated ataxin-3 is associated with transcription dysregulation, detectable in peripheral blood cells. Furthermore, this is the first report suggesting a pool of up-regulated genes in Machado-Joseph disease that may have the potential to be used for fine phenotyping of this disease.Fundação para a Ciência e a Tecnologia (FCT) - project FCOMP-01-0124-FEDER-028753 (PTDC/DTP/PIC/0370/2012)Operational Competitiveness Programme—COMPETEUK Medical Research Council (MRC)Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BPD/33611/2009Fundo Regional para a Ciência (FRC), Governo dos Açores - M3.1.2/F/006/2011; M3.1.7/F/031/2011 ; M3.1.3/F/004/200

Sequence analysis of 5' regulatory regions of the Machado-Joseph Disease gene (ATXN3)

Machado–Joseph disease (MJD) is a late-onset autosomal dominant neurodegenerative disorder, which is caused by a coding (CAG)n expansion in the ATXN3 gene (14q32.1). The number of CAG repeats in the expanded alleles accounts only for 50 to 75 % of onset variance, the remaining variation being dependent on other factors. Differential allelic expression of ATXN3 could contribute to the explanation of different ages at onset in patients displaying similar CAG repeat sizes. Variation in 5′ regulatory regions of the ATXN3 gene may have the potential to influence expression levels and, ultimately, modulate the MJD phenotype. The main goal of this work was to analyze the extent of sequence variation upstream of the ATXN3 start codon. A fragment containing the core promoter and the 5′ untranslated region (UTR) was sequenced and analyzed in 186 patients and 59 controls (490 chromosomes). In the core promoter, no polymorphisms were observed. In the 5′ UTR, only one SNP (rs3814834) was found, but no improvements on the explanation of onset variance were observed, when adding its allelic state in a linear model. Accordingly, in silico analysis predicted that this SNP lays in a nonconserved position for CMYB binding. Therefore, no functional effect could be predicted for this variant.Institute of Biotechnology and Biomedicine of the Azores - “High Prevalence Diseases in the Azores Islands” M2.1.2/I/026/2008,Fundação para a Ciência e a Tecnologia (FCT) - “Transcriptional variation of the ATXN3 gene as modulator of the clinical heterogeneity in Machado–Joseph disease (MJD)Secretaria Regional da Ciência, Tecnologia e Equipamentos - M3.1.3/F/004/2009CNP

Genotype analysis and studies of pyrethroid resistance of the oilseed rape (Brassica napus) insect pest - pollen beetle (Meligethes aeneus)

Oilseed Brassicas are vulnerable to attack from many insects and pathogens, calling for an extensive use of pesticides to secure crop yields; this can cause increased resistance in pests. During recent years, one of the main oilseed insect pests—the pollen beetle (Meligethes aeneus), resistant to pyrethroid insecticides—has emerged in southern Sweden. This, because of its frequency and geographic range, provides an excellent source of material for analysis of genetic variation among pollen beetle populations, for study of insecticide resistance and for testing new sources of plant protection. For genotyping pollen beetles, we modified the amplified fragment length polymorphism (AFLP) technique (chosen because it does not depend on prior sequence information when no genome information is available for pollen beetles), and applied it to 133 Swedish populations (susceptible and resistant), collected in different years, and to 14 European populations. AMOVA showed high levels of genetic variation within populations and gene flow among populations, and no evidence of expected regional and resistance-susceptibility to insecticide diversification (clear diversification by time and generations instead) for Swedish populations. European populations showed a clear pattern of regional diversification and a low level of gene flow. To identify possible point mutations associated with pollen beetles resistance to pyrethroids, the primary target sites for pyrethroids—voltage-sensitive sodium channels (VSSC) and metabolic resistance sites—Cytochrome P450, were studied using RT-PCR in resistant and susceptible insects. Two CYP450 partial cDNAs and four cDNA fragments composing VSSC domains I and II were amplified (using primers designed for homologue sequences) and sequenced showing point mutations, which can confer pyrethroid resistance