Mitochondrial DNA deletions and neurodegeneration in multiple sclerosis

Abstract

Objective: Cerebral atrophy is a correlate of clinical progression in multiple sclerosis (MS). Mitochondria are now established to play a part in the pathogenesis of MS. Uniquely, mitochondria harbor their own mitochondrial DNA (mtDNA), essential for maintaining a healthy central nervous system. We explored mitochondrial respiratory chain activity and mtDNA deletions in single neurons from secondary progressive MS (SPMS) cases.

Methods: Ninety-eight snap-frozen brain blocks from 13 SPMS cases together with complex IV/complex II histochemistry, immunohistochemistry, laser dissection microscopy, long-range and real-time PCR and sequencing were used to identify and analyze respiratory-deficient neurons devoid of complex IV and with complex II activity.

Results: The density of respiratory-deficient neurons in SPMS was strikingly in excess of aged controls. The majority of respiratory-deficient neurons were located in layer VI and immediate subcortical white matter (WM) irrespective of lesions. Multiple deletions of mtDNA were apparent throughout the gray matter (GM) in MS. The respiratory-deficient neurons harbored high levels of clonally expanded mtDNA deletions at a single-cell level. Furthermore, there were neurons lacking mtDNA-encoded catalytic subunits of complex IV. mtDNA deletions sufficiently explained the biochemical defect in the majority of respiratory-deficient neurons.

Interpretation: These findings provide evidence that neurons in MS are respiratory-deficient due to mtDNA deletions, which are extensive in GM and may be induced by inflammation. We propose induced multiple deletions of mtDNA as an important contributor to neurodegeneration in MS.

Figure 1

Complex IV (COX)/complex II (SDH) histochemical detection of respiratory-deficient neurons in snap frozen MS tissue sections. Complex IV (COX)/complex II (SDH) histochemistry on snap frozen sections from cases with secondary progressive MS showed respiratory-deficient neurons (lacking complex IV and with complex II stained blue, arrowheads) as well as neurons with intact complex IV activity (stained brown, arrow)(A, B) in the GM, (B) predominantly in layer VI, and (C) immediate subcortical WM. Based on morphology both pyramidal and nonpyramidal neurons showed the mitochondrial defect. The majority of respiratory-deficient neurons were located in layer VI (45%) and immediate subcortical white matter (34%). (D, E) Respiratory-deficient cells in GM and WM were confirmed as neurons judged by NeuN immunofluorescent labeling (inset shows respiratory-deficient neuron indicated by arrowhead). (F) The average density of complex IV–deficient neurons, from a median of 7 blocks containing frontal, parietal, temporal, and occipital GM and immediate subcortical WM per case, is shown in relation to age for MS cases (▴) and controls (□). The average density represents respiratory-deficient neurons in whole GM and immediate subcortical WM. The mean density of respiratory-deficient neurons in MS cases was significantly greater (24.97 ± 4.67 neurons/cm², n = 13) than aged control brains (1.59 ± 0.86 neurons/cm², n = 10, p= 0.003). On average 18.54cm² of GM and 2.26cm² of immediate subcortical WM regions were examined per case. The mean age of MS cases (54.9 ± 13.5) was significantly lower than controls (73.8 ± 8.8, p= 0.001). The postmortem delay between MS cases (11.8 ± 4.8 hours) and controls (16.1 ± 7.2 hours) was not significantly different (p= 0.103). Differences between 2 groups were analyzed using the Wilcoxon-Mann-Whitney U-test. Error bars indicate standard error of the mean for each case; ± values indicate standard deviation. COX = cytochrome c oxidase or complex IV; GM = gray matter; MS = multiple sclerosis; SDH = succinate dehydrogenase or complex II; WM = white matter. matter. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

Figure 2

Prevalence of respiratory-deficient neurons in relation to lesions in MS cases. Layer VI neurons in (A) leukocortical (type I) lesions and (C) corresponding NAGM. (B, D) Immediate subcortical WM neurons within WM lesions extending up to the cortex and corresponding NAWM areas [NAWM (SC)]. In A and B, respiratory-deficient neurons, as a percentage of all NeuN-positive cells identified in serial sections, were not significantly different in lesion areas of layer VI (2.01 ± 0.31 out of 1000 neurons in randomly selected type I lesions, n = 22) and lesion areas of immediate subcortical WM (0.23 ± 0.31 out of 1000 neurons, n = 5) compared with corresponding NAGM (4.25 ± 0.41 out of 1000 neurons, n = 22) or WM (1.3 ± 1.9 out of 1000 neurons, n = 5) areas. There was a trend toward more respiratory-deficient neurons in normal-appearing tissue. In C and D, neuronal loss in leukocortical (type I) lesions (in C, 461.49 neurons/mm² ± 97.91) and WM lesions extending up to the cortical margin (in D, 75.76 neurons/mm² ± 36.15) was significant compared to corresponding normal-appearing tissue (GM: 541.93 neurons/mm² ± 91.19; WM: 109.64 neurons/mm² ± 34.84). Neuronal loss in lesion areas was also significant when compared with corresponding areas in control cases (GM: 599.34 neurons/mm²± 93.45; WM: 120.52 neurons/mm² ± 40.67). The density of NeuN-positive cells in normal-appearing tissue was not significantly different compared with corresponding regions in controls. In C and D, parentheses indicate the number of regions quantitated. (E) When the density of respiratory-deficient neurons in GM and immediate subcortical WM in MS were determined with respect to location (frontal, parietal, temporal, and occipital lobes, and plotted in order with frontal blocks to the left and occipital blocks to the right for each case), the density of respiratory-deficient neurons did not significantly differ between the 4 lobes. Differences between 2 groups (in A and B) and more than 2 groups (in C–E) were analyzed using the Wilcoxon-Mann-Whitney U-test and Kruskal-Wallis test, respectively. *p = 0.005, **p = 0.027, and **p = 0.018 for GM and WM, respectively. Error bars and ± indicate standard deviation. GM = gray matter; NAGM = normal appearing gray matter; NAWM (SC)= normal appearing white matter (immediate subcortical region); MS = multiple sclerosis; WM = white matter.

Figure 3

mtDNA deletions are present throughout the GM in MS cases. A cortical MS lesion (arrowheads) is identified by (A) lack of PLP and (B) presence of LCA staining. (C) Complex IV (COX)/complex II (SDH) histochemistry was performed in adjacent sections mounted on membrane slides (Leica). In C, approximately 250 × 250-μm² regions from lesions (*) and NAGM were laser-microdissected. Scale bar: 250μm. Long-range PCR using DNA extracted from GM regions from (D) MS cases (shows findings in MS03, aged 42 years) and (E) controls (shows findings in CON01 and CON09, aged 60 and 84 years, respectively) show evidence of multiple deletions of mtDNA (amplified fragments smaller than 11kb). In D, MS cases, mtDNA deletions were extensive in cortical lesions (lanes 1–4) and in NAGM (lanes 5–8). In E, aged controls, mtDNA deletions were less apparent in cortical GM (all lanes) and only the wild-type bands (faint band at 11kb) were observed in a proportion of sampled regions (lanes 2 and 4). In E, the mtDNA deletions in controls also vary in size consistent with multiple deletions of mtDNA as evident in the lanes of cases CON1 and CON09. + indicates wild-type 11-kb band amplified from control DNA. Scale bar: 500μm. COX = cytochrome c oxidase or complex IV; GM = gray matter; LCA = leukocyte common antigen; NAGM = normal appearing gray matter; MS = muscular sclerosis; PLP = proteolipid protein; SDH = succinate dehydrogenase or complex II; WM = white matter. matter. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

Figure 4

mtDNA deletions are present within neurons in MS. (A) Following complex IV (COX)/complex II (SDH) histochemistry and immunofluorescent labeling of NeuN, single respiratory-deficient neurons (i, arrowhead) and neurons with complex IV activity (brown) were isolated from membrane slides using laser microdissection (ii). Scale bar: 20 μm. (B) Long-range PCR identified multiple deletions of mtDNA in respiratory efficient neurons (brown) when DNA was extracted from 5 pooled, closely located neurons (shown in each lane). mtDNA deletions were evident within neurons throughout the MS GM (MS03 shown with each lane representing different regions sampled from frontal, parietal, temporal, and occipital lobes). When (C) 5 and (D) 2 respiratory-deficient neurons from MS cases were pooled there was also evidence of mtDNA deletions. In D, the minimum number of neurons needed to extract adequate quantity of mtDNA for long-range PCR was 2. In C and D, the number of mtDNA deletions detected by long-range PCR rarely exceeded the number of respiratory-deficient neurons pooled, consistent with clonal expansion. As previously reported, the full-length product is not always apparent, particularly when mtDNA deletions are present possibly due to bias of PCR., (E, F) Sequencing of the deleted mtDNA extracted from long-range PCR gels identified the breakpoints of deletions, as shown in the electropherogram, confirming both long-range and real-time PCR findings. (G) The large-scale deletions encompassed much of the major arc of the mitochondrial genome including genes of complex I (green), complex III (red), complex IV (orange), complex V (blue), and tRNAs (black). Scale bar: 10μm. + indicates wild-type 11kb band amplified from control DNA. COX = cytochrome c oxidase or complex IV; GM = gray matter; MS = muscular sclerosis; SDH = succinate dehydrogenase or complex II. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

Figure 5

High heteroplasmy levels of mtDNA deletions in single respiratory-deficient neurons. (A) The heteroplasmy level of mtDNA deletions at a single-cell level was determined using real-time PCR and DNA extracted from single neurons; respiratory-deficient (blue) neurons contained significantly greater levels of mtDNA deletions (41.22%± 22.64), judged by ND1 and ND4 assay, compared with those with intact complex IV activity (brown) in MS cases (16.43%± 10.72, **p < 0.001) and controls (20.31%± 13.71, *p= 0.001). Based on 95% confidence intervals of heteroplasmy level in respiratory-efficient neurons (brown) from MS cases (n = 6) and controls (n = 5), 55% of respiratory-deficient neurons in MS cases had the increased heteroplasmy levels. We did not find a correlation between heteroplasmy level and density of HLA-positive cells. Both GM and WM neurons were analyzed (53% and 47%, respectively of all neurons analyzed). In total, 34, 56, and 80 single neurons were included for control COX+, MS COX+, and MS COX− neurons. (B) High heteroplasmy levels at a single neuronal level were detected in respiratory-deficient neurons in MS cases, independent of age. Immunohistochemical labeling of (C) COX-I and (D) COX-II in adjacent sections using DAB (brown), both of which are mtDNA-encoded catalytic subunits of complex IV, and NeuN (Vector® SG, blue) identify neurons lacking mtDNA-encoded subunits (arrowheads). Scale bar: 16 μm. Kruskal-Wallis test was used to compare difference between groups. COX+= respiratory-efficient neurons with intact complex IV activity (brown histochemical stain); COX−= respiratory-deficient neurons devoid of complex IV and with complex II activity; GM = gray matter; HLA = human leukocyte antigen; MS = multiple sclerosis; WM = white matter. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]