Parkinson's disease brain mitochondria have impaired respirasome assembly, age-related increases in distribution of oxidative damage to mtDNA and no differences in heteroplasmic mtDNA mutation abundance

Abstract

Background: Sporadic Parkinson's disease (sPD) is a nervous system-wide disease that presents with a bradykinetic movement disorder and is frequently complicated by depression and cognitive impairment. sPD likely has multiple interacting causes that include increased oxidative stress damage to mitochondrial components and reduced mitochondrial bioenergetic capacity. We analyzed mitochondria from postmortem sPD and CTL brains for evidence of oxidative damage to mitochondrial DNA (mtDNA), heteroplasmic mtDNA point mutations and levels of electron transport chain proteins. We sought to determine if sPD brains possess any mtDNA genotype-respiratory phenotype relationships.

Results: Treatment of sPD brain mtDNA with the mitochondrial base-excision repair enzyme 8-oxyguanosine glycosylase-1 (hOGG1) inhibited, in an age-dependent manner, qPCR amplification of overlapping ~2 kbase products; amplification of CTL brain mtDNA showed moderate sensitivity to hOGG1 not dependent on donor age. hOGG1 mRNA expression was not different between sPD and CTL brains. Heteroplasmy analysis of brain mtDNA using Surveyor nuclease(R) showed asymmetric distributions and levels of heteroplasmic mutations across mtDNA but no patterns that statistically distinguished sPD from CTL. sPD brain mitochondria displayed reductions of nine respirasome proteins (respiratory complexes I-V). Reduced levels of sPD brain mitochondrial complex II, III and V, but not complex I or IV proteins, correlated closely with rates of NADH-driven electron flow. mtDNA levels and PGC-1alpha expression did not differ between sPD and CTL brains.

Conclusion: PD brain mitochondria have reduced mitochondrial respiratory protein levels in complexes I-V, implying a generalized defect in respirasome assembly. These deficiencies do not appear to arise from altered point mutational burden in mtDNA or reduction of nuclear signaling for mitochondrial biogenesis, implying downstream etiologies. The origin of age-related increases in distribution of oxidative mtDNA damage in sPD but not CTL brains is not clear, tracks with but does not determine the sPD phenotype, and may indicate a unique consequence of aging present in sPD that could contribute to mtDNA deletion generation in addition to mtDNA replication, transcription and sequencing errors. sPD frontal cortex experiences a generalized bioenergetic deficiency above and beyond aging that could contribute to mood disorders and cognitive impairments.

Figure 1

Levels of Complex I subunit proteins in sPD brain mitochondria. Individual protein band intensities were normalized to porin intensity in each sample and expressed in terms of mean CTL levels. A 2-way ANOVA across subunits and disease showed a significant interaction. Mann-Whitney test showed significant reductions for 30 kDa, 15 kDa and 8 kDa subunits in sPD samples.

Figure 2

Levels of representative subunit proteins from Complexes II-V in sPD brain mitochondria. Individual protein band intensities were normalized to porin intensity in each sample and expressed in terms of mean CTL levels. A 2-way ANOVA across subunits and disease showed a significant interaction. Mann-Whitney test showed significant reductions for Complex IV subunit in sPD samples.

Figure 3

Correlations among levels of Complexes II, III and V and NADH-driven electron flux rates in sPD brain mitochondria. NADH-driven electron flux rates for mitochondria isolated from frontal cortex of the same cases were taken from data described in Keeney, et al. Porin-normalized levels of respiratory proteins were determined as described in Methods.

Figure 4

Distributions of heteroplasmic mutations in sPD and CTL brain mtDNA. mtDNA's from six sPD and six CTL brains were amplified using primer pairs A-H. The amplicons were subject to Surveyor Nuclease treatment and separation/analysis of products using Experion 12K DNA chips. Bands from 1000-1900 bp size are included in the Figure. The Y-axes are DNA levels and X-axes are product sizes in bp.

Figure 5

Distribution across mtDNA of inhibition of PCR amplification from treatment with hOGG1 as a function of donor age. mtDNA's from CTL and sPD brains were treated with hOGG1 and amplified with qPCR and primer pairs A-H. Shown are the proportional amplification for each sample and each primer pair. sPD samples showed (with one exception) an age-related in increase of hOGG1 sensitivity not seen in CTL samples. Donor ages for each sample are shown on the right.

Figure 6

Levels of mtDNA and PGC-1α expression in sPD and CTL brain homogenates. mtDNA D-loop was assayed with qPCR in genomic DNA's and PGC-1α expression was assayed with qPCR in cDNA's derived from sPD and CTL brain total homogenates and normalized to 18S rRNA levels. Their relationship was best described by a two-variable rectangular hyperbola shown in C.