Mitochondrial abnormalities in the putamen in Parkinson's disease dyskinesia

Abstract

Prolonged treatment of Parkinson's disease (PD) with levodopa leads to disabling side effects collectively referred to as 'dyskinesias'. We hypothesized that bioenergetic function in the putamen might play a crucial role in the development of dyskinesias. To test this hypothesis, we used post mortem samples of the human putamen and applied real time-PCR approaches and gene expression microarrays. We found that mitochondrial DNA (mtDNA) levels are decreased in patients who have developed dyskinesias, and mtDNA damage is concomitantly increased. These pathologies were not observed in PD subjects without signs of dyskinesias. The group of nuclear mRNA transcripts coding for the proteins of the mitochondrial electron transfer chain was decreased in patients with dyskinesias to a larger extent than in patients who had not developed dyskinesias. To examine whether dopamine fluctuations affect mtDNA levels in dopaminoceptive neurons, rat striatal neurons in culture were repeatedly exposed to levodopa, dopamine or their metabolites. MtDNA levels were reduced after treatment with dopamine, but not after treatment with dopamine metabolites. Levodopa led to an increase in mtDNA levels. We conclude that mitochondrial susceptibility in the putamen plays a role in the development of dyskinesias.

Figure 1. Demographic data for PD samples

Disease duration was significantly longer in dyskinetic-PD patients, a. L-dopa dosage was increased over time, b. Because dyskinetic-PD patients tended to have had PD longer, they received higher annual concentrations of L-dopa, and had had more overall exposure to L-dopa during their lifetime, b, c. However, at similar disease duration timepoints both diagnostic groups of patients had comparable prescription levels of L-dopa. The original cohort consisted of 15 dyskinetic-PD patients, 16 non-dyskinetic-pD patients, and 32 controls, matched for gender, age and PMI (see also table S1).

Figure 2. Decreased mtDNA levels in the dyskinetic-PD putamen, but not cerebellum

MtDNA levels were reduced in dyskinetic-PD samples in the putamen, a, c, but not cerebellum, b. A subgroup of putamen samples, matched for duration of disease and L-dopa exposure, yielded comparable results, c. Insert in panel C shows years of PD diagnosis in the matched groups. Disease duration, d, and average L-dopa p.a., e, were significantly correlated with mtDNA levels in the dyskinetic-PD group, but not in the non-dyskinetic-PD group. a, b, d, e, N=16 control samples, 10 dyskinetic-PD samples, 14 non-dyskinetic-PD samples. c, N=12 control samples, 7 dyskinetic-PD samples, 8 non-dyskinetic-PD samples. Post-hoc t-test ** p<=0.01, * p<=0.05.

Figure 3. Increased number of mtDNA deletions in the dyskinetic-PD putamen

The relative ratio of PCR product in the putamen amplified from the deleted mtDNA region over PCR product from the stable region is reduced in dyskinetic-PD, a. N=20 control samples, 11 dyskinetic-PD samples, 14 non-dyskinetic-PD samples. Increased deletion correlated with decreased mtDNA levels in the PD samples, b. Lower levels of mtDNA were correlated with higher levels of deletion in the dyskinetic-PD group, but not in the non-dyskinetic-PD group. N=16 control samples, 10 dyskinetic-PD samples, 13 non-dyskinetic-PD samples. Note: Less product of the deleted area indicates more mtDNA deletion. Post-hoc t-test ** p<=0.01.

Figure 4. Decreased mRNA levels of genes coding for the mitochondrial electron transport chain in the dyskinetic-PD putamen

A subgroup of samples were matched for disease duration and L-dopa exposure, a. The percentage of probe sets on the Affymetrix gene chip coding for the five complexes of the mitochondrial electron transport chain (mETTs) that were significantly regulated in the various comparisons (p<=0.05, >=80% ‘present’ call in each group) was compared to the percentage of all probe sets regulated in the individual comparisons, b. Positive values signify upregulation, negative values signify downregulation. More probe sets than expected were downregulated, whereas less probe sets than expected were upregulated. N=15 control samples, 7 dyskinetic-PD samples, 7 non-dyskinetic-PD samples. Fisher’s exact test ** p<=0.01, *** p<=0.001. qPCR analysis of a subset of these genes supports the microarray data, c. Gene IDs are shown above each histogram; for gene names see table S3. N=10 control samples, 7 dyskinetic-PD samples, 11 non-dyskinetic-PD samples. T-test * p<=0.05

Figure 5. Effects of DA and its metabolites on striatal neurons in culture

a: DA treatment reduces mtDNA levels in rat primary striatal culture. N= 159 controls, 12 L-dopa, 33 DA, 16 3-MT, 16 DOPAC and 16 HVA. Post-hoc t-test * p<=0.05, *** p<=0.001. b: L-Dopa, DA, their metabolites and metabolizing enzymes. AAAL=aromatic amino acid decarboxylase; COMT= catechol-O-methyl transferase; MAO-B= monoamine oxidase type B. c–f: HPLC analysis of the medium demonstrates that cultures metabolize DA to 3-MT and HVA, c; 3-MT partially to HVA, d; DOPAC fully to HVA, e; do not metabolize HVA, f. 3–24 samples per condition