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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.
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® 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-1α expression did not differ between sPD and CTL brains.
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.
Parkinson's disease (PD) is the most commonly occurring neurodegenerative movement disorder of adults and occurs sporadically (sPD) in >90% of cases. sPD is now recognized to be a nervous system-wide disorder with a temporal progression of protein aggregation pathology that begins in gut enteric neurons, then enters vagal and olfactory neurons before appearing in midbrain substantia nigra [
Currently five monogenic causes of parkinsonism are known that produce clinical phenotypes variably similar to, and in some cases virtually identical with sPD [
Assessing mitochondrial bioenergetic function and consequences of bioenergetic inefficiency is limited in frozen postmortem sPD brain. Traditionally, maximal catalytic activities of individual ETC complexes have been assayed, yielding a frequent finding that complex I activity is reduced. However, these assays of isolated complex activities do not necessarily provide insight into respiratory capacity, coupling of respiration to ATP synthesis and rate of production of reactive oxygen species (ROS). Freezing disrupts mitochondrial capacity to generate proton gradients across the inner membrane, which are necessary for ATP synthesis and are inversely related to ROS production.
In our earlier work we used complex I immunocapture antibodies to show that sPD brain mitochondria had elevated oxidative damage to several complex I proteins, and that the levels of these oxidized complex I subunit proteins inversely correlated with NADH-driven electron flow [
In the present study we have extended our analysis to measure in sPD brain mitochondria the levels per unit mitochondrial mass of multiple ETC proteins that are believed to functionally associate with each other in a respiratory unit known as the "respirasome" [
Clinical histories, cause of and age at death, sex, and postmortem interval between death and dissected sample placement into -80°C freezer are shown in Additional Files
Figure
Figure
In our previous work we made mitochondrial preparations from many of the same sPD postmortem cortex samples used in the present study and incubated them with NADH to initiate electron flow through complex I [
We used a Surveyor® mismatch-cleaving nuclease approach to estimate relative burdens of low abundance heteroplasmic mutations in sPD and CTL brain mtDNA samples [
Since all of our PCR products were ~2 kbases in size, we report results from Surveyor-cleaved products of 1000-1900 bp. In this manner we hoped both to minimize inclusion of bands that could represent the same mutation and heteroplasmies in the ~50-100 bp primer overlaps. By varying the proportion of heteroplasmy in plasmid sequences provided by the manufacturer, we found that our system could reliably detect heteroplasmic mutation abundances at the 2% level (Additional File
With this technique we found in their mtDNA's covered by the A-H primers an average of 19.0 heteroplasmies/brain in six sPD brains and 19.5 heteroplasmies/brain in six CTL brains. Figure
We estimated in mtDNA from our brain samples the distribution of 8-oxoG residues, a most commonly formed oxidized nucleoside derivative that is increased in mtDNA of vulnerable PD nigral neurons [
Figure
To search for evidence of alterations in mitochondrial biogenesis we assayed with qPCR levels of mtDNA copy number in genomic DNA and PGC-1α expression in cDNA from sPD and CTL brain homogenates (Figure
A fundamental limitation of postmortem brain studies in a neurodegenerative disease such as sPD is that the primary tissues analyzed result from "uncontrolled experiments". Individuals develop sPD at different ages for unclear reasons, are treated with variable doses of symptomatic drugs for varying time intervals, have different additional environmental exposures and lifestyles, possess individualized genetic backgrounds and die across a spectrum of ages from processes that may or may not be physiologically related to their sPD. What joins these specimens together in a study such as this one is the appearance of a similar clinical phenotype and the presence of common neuropathological abnormalities such as nigral cell death and Lewy body aggregates. However, that those similar clinical and pathological phenotypic characteristics exist does not require common etiologies either across individuals or disease stages. The recent discoveries of multiple monogenic parkinsonisms that share phenotypic properties with sPD and derive from mutations in seemingly disparate genes provide excellent examples of how similar neurodegenerative phenotypes can be causally distinctive. There is no reason to assume that the causes of sPD will be any less diverse.
In addition, most postmortem sPD brain samples are from persons who died in relatively advanced disease stages. Abnormalities uncovered in these specimens cannot be assumed to represent "genesis" events in disease origin. Further, sPD is an anatomically progressive disease across the entire neuraxis that is beginning to involve limbic and frontal cortex at the time nigral degeneration has progressed to yield the earliest clinical symptoms [
In this context, and considering these caveats, our results provide additional support to a view of sPD that considers mitochondrial dysfunction to be a substantial component of pathological progression [
In our analysis of brain mitochondria isolated from PD frontal cortex, the normalized abundances of multiple subunits of Complex I examined were significantly reduced in comparison to CTLs. By normalizing to a mitochondrial mass marker (porin), we demonstrated that the 8 kDa, 15 kDa, and 30 kDa subunits were significantly decreased in sPD brain mitochondria. In our earlier work, only the 8 kDa subunit was significantly reduced, but no normalization to mitochondrial mass was utilized. In the present study we normalized each individual sample's ETC protein levels to the porin signal in that specific sample. In this manner, the relative ETC protein levels could be analyzed as a function of an abundant marker for mitochondrial mass.
The deficiency of the nuclear encoded 30 kDa subunit of Complex I, known as NADH deydrogenase (ubiquinone) iron-sulfur protein 3 (NDUFS3), is especially intriguing since this subunit is known to be catalytically active in the iron sulfur moiety of Complex I and critically involved in Complex I assembly [
Our quantitative immunoblot study of representative members of the respirasome from mitochondria isolated from the same frontal cortex samples indicated a more widespread reduction of complexes II-V. When each complex was analyzed individually versus CTLs, the cytochrome c oxidase subunit 2 of Complex IV was significantly reduced (p = 0.027). This subunit is encoded by mtDNA and is one of three catalytic units composing the inner core of Complex IV.
The widespread reduction of multiple members of complexes I-V in sPD frontal cortex, combined with the highly correlated relationships among levels of complexes II, III and V with NADH-driven electron flux rates, suggest that the entire respirasome is deficient. Respirasomes are viewed as macromolecular assemblies of individual ETC complexes, that themselves are assemblies of multiple ETC protein subunits [
We admit that our assessment is based on indirect measurements of respirasome function, since coupled respiration is absent in frozen postmortem mitochondria. If indeed present, dysfunction of the respirasome may lead to inefficient formation of the proton gradient across the inner mitochondrial membrane needed by ATP synthase (Complex V) to create ATP, the cell's major metabolic fuel.
Our analysis of 8-oxoG distribution within the mitochondrial genome in sPD frontal cortex showed that the older cases had extensive 8-oxoG oxidation in each ~2 kbase amplicon from primers A-H (Figure
In human cell DNA replication 8oxoG:A mismatches are repaired more slowly than 8oxoG-C, resulting in increased mutagenicity from G-T transversions [
Our study also showed that the prevalence of oxidatively damaged 8-oxoG residues increased with age in PD and not in CTL mtDNA. The presence of 8-oxoG damage did not act as a biomarker of sPD however, because many of the CTL samples had a considerable proportion of mtDNA containing 8-oxoG lesions. Several sPD samples also had relatively low distributions of 8-oxoG damage. Increased age and increased 8-oxoG levels in both mtDNA and nDNA have been linked in a study by Lezza et al. who examined cerebral cortex from normal human brain, and found that 8-oxoG levels were significantly increased in the cohort over the age of 70 as compared to cases below 70 years of age [
Our findings complement those of Zhang et al [
Taken together, these findings suggest a complicated interplay between mtDNA oxidation, aging, and the development of sPD; increased aging and 8-oxoG damage may be two of many factors involved in the etiology of this disease. Furthermore, the increases in hOGG1-2a in PD nigral neurons and hMUTYH in PD nigral mitochondria may demonstrate a neural compensatory mechanism when faced with increased oxidative stress damage to mtDNA.
Our studies showed that sPD brain mitochondria from frontal cortex have a generalized deficiency of respirasome protein components. This finding implies a more diffuse mitochondrial bioenergetic deficit than proposals that discuss sPD as primarily possessing a deficiency at one ETC complex. We do note, however, that the greatest losses of respirasome proteins were several Complex I subunits. Thus, the functional, catalytic deficiencies found in sPD brain may be greatest at complex I, even though the larger respirasome assembly is impaired more generally at the molecular level.
Could sPD brain mtDNA be causally responsible for a more generalized respirasome deficit? Although our study does not provide any supportive evidence for this possibility, we note that our studies of sPD cybrid models have shown generalized deficiencies of multiple respirasome proteins apparently due to reduced mitochondrial biogenesis signaling [
Finally, we note that we studied mitochondria purified from total brain homogenates; thus, the majority of mitochondria are likely from non-neuronal cells. It will be of value to attempt similar studies in isolated sPD brain neurons, once proteomic sensitivity has improved to allow single cell analyses.
Human cerebral frontal cortex samples were obtained from the Brain Resource Facility at the University of Virginia Health System. Mitochondrial isolation was performed on sPD samples (diagnosis confirmed histologically; N = 8) and age-matched controls (CTL, N = 10). Gradient purified mitochondria were obtained via a modified protocol developed by Lai and Clark [
The pellet was resuspended in 5 mL of gradient purified mitochondrial isolation buffer (GPMIB; 0.15 M KCl, 20 mM monopotassium phosphate, 1 mM EDTA, 5.5 mM 2-mercaptoethanol, 1 mM phenylmethanesulphonyl fluoride, 10% v/v of 100 × protease inhibitor cocktail from CalBioChem, pH 7.6) and homogenized with a Teflon glass homogenizer. A Ficoll solution gradient was constructed via pouring 10 mL of a 7.5% Ficoll w/w solution over 12 mL of a 10.0% Ficoll w/w solution. The crude mitochondrial fraction homogenate was poured over the Ficoll gradient and centrifuged for 37 min at 28,000 RPM at 4°C. The Ficoll solution gradient was decanted from the gradient purified mitochondrial pellet. The pellet was divided and stored at -80°C in modified radioimmunoprecipitation buffer (RIPA; 50 mM Tris-HCl pH 7.4, 1% NP 40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethanesulphonyl fluoride, 10% v/v of 100 × protease inhibitor cocktail from CalBioChem) for Western Blots and in GPMIB for subsequent DNA analyses.
Gradient purified mitochondrial pellets were sonicated until completely dissolved before nucleic acid and protein isolation. Nucleic acids were isolated from gradient purified mitochondria using the AllPrep DNA/RNA Mini Kit from Qiagen using 600 μL Buffer RLT Plus. mtDNA quantification was performed using the Quant-iT dsDNA Assay Kit by Invitrogen and measured on a TECAN Genios Pro 96-well plate optical reader. For protein isolation, the mitochondrial pellets were sonicated until dissolved and vortexed every 5 min for 30 min while held on ice. The samples were centrifuged at 15,000 × g for 10 min at 4°C and supernatants containing the soluble protein fractions were collected. Protein quantification was performed using the Detergent Compatible Protein Assay Kit by Bio-Rad with BSA standards and measured using an OptiMax 96-well plate optical reader at 750 nm.
~2 kbase amplicons of the mtDNA coding region used primers A-H as described in Bannwarth, et al. [
The concentration of DNA in amplicons from primers A-H was estimated after separation on a 0.8% E-gel and comparing with the Gel-Doc (BioRad) their band intensity to the intensity of the bands in the 1 kb ladder whose concentration is known. 400 ng of amplicon DNA was mixed with 2 ul of "Enhancer" and 2 ul Surveyor Nuclease® to 0.2 mL PCR tubes on ice that were then mixed and incubated at 42°C for 60 min. Tubes were then transferred to ice and 2 ul of Stop Solution added. Controls (undigested amplicons) were run alongside their corresponding digests on 12K DNA chips (Experion®, BioRad). The Experion® software calculated both bp size of DNA bands and their concentrations.
Incubation of mtDNA containing oxidized 8-oxoG residues with hOGG1 is predicted to produce an apurinic site at the site of oxidation due to hOGG1's N-glycosylase activity. When PCR is initiated, the Taq polymerase is unable to amplify past the 8-oxoG cleavage site where there is a single-stranded break in the DNA sugar-phosphate backbone, thus interrupting PCR amplification. Due to this phenomenon, it would be expected that undigested mtDNA would appear to have a larger starting quantity after qPCR when compared to the mtDNA from the same sample treated with hOGG1 prior to qPCR, if it is believed that the mtDNA does indeed suffer from 8-oxoG oxidation. Enzymatic incubation occurred in a 20 μL reaction volume containing 100 ng of mtDNA, 1 unit of hOGG1, 1 × Reaction Buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 at 25°C), and 100 μg/mL bovine serum albumin obtained from New England BioLabs. Incubation occurred at 37°C for 1 hour followed by 20 min of heat inactivation in a water bath at 90°C. All PD and CTL samples underwent incubation conditions with and without hOGG1 where matched undigested samples replaced the 1 unit of hOGG1 with a matching volume of 1 × Reaction Buffer. Quantitative PCR was performed in a 25 μL reaction volume using 5 ng of ± hOGG1 treated mtDNA sample, 0.25 μM sense/anti-sense primers, and 1 × iQ SYBR Green Supermix (2 × reaction buffer with dNTPs, iTaq DNA polymerase, 6 mM MgCl2, SYBR Green I, fluorescein, and stabalizers) from Bio-Rad. Eight primer sets developed by Bannwarth et. al labeled A-H were used and each contained ~2000 bp encircling the sense-coding portion of the mitochondrial genome (Figure
In a 30 μL reaction volume, 25 μg of purified mitochondrial protein was added to 10 μL XT working sample buffer (XT Reducing Agent, XT Sample Buffer from Bio-Rad) and heated for 5 min at 95°C. Electrophoresis was performed using a 12-well 12% polyacrylamide Criterion XT Bis-Tris Precast Gel with 1 × XT MES Running Buffer. Precision Plus Dual Color Protein Standard containing 10 MW markers (10 kDa - 250 kDa) from Bio-Rad was used to monitor the progression of the sample down the electrophoretic gel. The iBlot Dry Blotting System from Invitrogen was used in the Western Blotting transfer of protein from the polyacrylamide gel to nitrocellulose. The nitrocellulose was washed for 3 min with phosphate buffered saline (PBS) and incubated at room temperature for 1 hour in Li-Cor Blocking Buffer. The blot was then placed in the primary antibody solution for 1 hour at room temperature. The primary antibody solution for the subunits of Complex I was made of 20 μL Li-Cor Blocking Buffer and contained 0.1% Tween 20 (polyoxyethylenesorbitan monolaurate from Bio-Rad), 0.5 μg/mL rabbit anti-SOD2 antibody (Abcam), and the following mouse monoclonal antibodies from MitoSciences: 1 μg/mL Complex I subunit 8 kDa, 0.5 μg/mL Complex I subunit NDUFB4 (15 kDa), 0.5 μg/mL Complex I subunit NDUFB8 (20 kDa), 0.5 μg/mL Complex I subunit NDUFS3 (30 kDa), 1.125 μg/mL Complex I subunit NDUFA9 (39 kDa). The primary antibody solution for Complex I-V was made of 20 μL Li-Cor Blocking Buffer and contained 0.1% Tween 20 (Bio-Rad), 0.5 μg/mL rabbit anti-SOD2 antibody (Abcam), and the following mouse monoclonal antibodies from MitoSciences: 0.5 μg/mL Complex I subunit NDUFB8 and 1:570 dilution of the MitoProfile Total OXPHOS Human WB Antibody Cocktail containing Complex I subunit NDUFB8, Complex II-FeS subunit 30 kDa, Complex III subunit Core 2, Complex IV subunit II, ATP synthase subunit alpha. After 1 hour exposure to the primary antibody solution the blot was washed 4 times for 5 min each in PBS with 0.1% Tween 20 (T-PBS). Next, the nitrocellulose was incubated for 30 min at room temperature in 20 mL of secondary antibody solution made of Li-Cor Blocking Buffer which contained 0.1% Tween 20, 33 μL 12% SDS, 1:7500 dilution of IRDye CW 800 goat anti-mouse antibody (Li-Cor) and 1:4200 dilution of IRDye CW 680 goat anti-rabbit antibody (Li-Cor). Blots were washed 4 times for 5 min with T-PBS. The Western Blots were repeated using the same protocol, but with a primary antibody solution containing 1 μg/mL mouse anti-Porin antibody (MitoSciences) and 0.5 μg/mL rabbit anti-SOD2 antibody (Abcam). Western blots were visualized using a Li-Cor Odyssey Infrared Imaging System, which contained software to quantify protein amounts based on calculated integrated intensities.
This assay was described in our earlier publication [
Statistical analyses were performed using InStat software from GraphPad and JMP8 software from SAS Institute Inc.
8-oxoG: 8-oxyguanine; CTL: control; hOGG1: human oxyguanine glycosylase-1; mtDNA: mitochondrial DNA; sPD: sporadically occurring Parkinson's disease.
The authors declare that they have no competing interests.
CRA and JPB designed experiments; CRA, SKM, LDD, PMK and JPB carried out experiments and analyzed data; CRA and JPB wrote the manuscript. All authors read and approved the manuscript.
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This research was supported by NIH grant NS39788 and the D. Loy Stewart Research Fund. We thank Caitlin Quigley for assistance in providing the data shown in Additional File