A Study of Single Substantia Nigra Neurons

ORIGINAL CONTRIBUTION Relationship Between Mitochondria and ␣-Synuclein A Study of Single Substantia Nigra Neurons

Amy K. Reeve, PhD; Tae-Kyung Park, BSc; Evelyn Jaros, PhD; Graham R. Campbell, PhD; Nichola Z. Lax, PhD; Philippa D. Hepplewhite, BSc; Kim J. Krishnan, PhD; Joanna L. Elson, PhD; Christopher M. Morris, PhD; Ian G. McKeith, MD; Doug M. Turnbull, MD

Objective: To explore the relationship between ␣-sy- pathology and cell loss. Patients with dementia with Lewy nuclein pathology and mitochondrial respiratory chain bodies and idiopathic Parkinson disease fulfilled the clini- protein levels within single substantia nigra neurons. cal and neuropathologic criteria for these diseases.

Design: We examined ␣-synuclein and mitochondrial Results: Our results showed that mitochondrial den- protein expression in substantia nigra neurons of 8 pa- sity is the same in nigral neurons with and without ␣-sy- tients with dementia with Lewy bodies, 5 patients with nuclein pathology. However, there are significantly higher Parkinson disease, and 8 control subjects. Protein ex- levels of the respiratory chain subunits in neurons con- pression was determined using immunocytochemistry fol- taining ␣-synuclein pathology. lowed by densometric analysis. Conclusions: The finding of increased levels of respi- Patients: We examined single substantia nigra neu- ratory chain complex subunits within neurons contain- rons from 5 patients with idiopathic Parkinson disease ing ␣-synuclein does not support a direct association be- (mean age, 81.2 years), 8 patients with dementia with tween mitochondrial respiratory chain dysfunction and Lewy bodies (mean age, 75 years), and 8 neurologically the formation of ␣-synuclein pathology. and pathologically normal control subjects (mean age, 74.5 years). The control cases showed minimal Lewy body Arch Neurol. 2012;69(3):385-393

HE PATHOGENESIS OF IDIO- While mitochondrial dysfunction has pathic Parkinson disease a key role to play in the pathogenesis of (IPD) and dementia with IPD and DLB, its relationship with the Lewy bodies (DLB) re- other key pathogenic factor—the accu- mains unclear. Mitochon- mulation of ␣-synuclein—is less under- drialT involvement has been identified as stood. The accumulation of ␣-synuclein Author Affiliations: Centre for important by several lines of evidence. De- as filamentous inclusions in neurons (Lewy Author Affil Brain Ageing and Vitality, fects of the mitochondrial respiratory bodies [LB]) and in dystrophic neurites is Brain Ageing Institute for Ageing and Health chain, in particular in the activity of com- the pathological hallmark of both IPD and Institute for (Drs Reeve, Krishnan, McKeith, plex I, have been described in homog- DLB. In autosomal recessive parkinson- (Drs Reeve, K and Turnbull), Mitochondrial enized substantia nigra (SN) from pa- ism, homozygous mutations generally re- and Turnbul Research Group, Institute for 1,2 Research Gro Ageing and Health (Ms Park tients with IPD. High levels of deleted sult in nigral degeneration not associated Ageing and H 5 and Drs Campbell, Lax, mitochondrial DNA have been reported in with LB pathology, although LB pathol- and Drs Cam Krishnan, Elson, and Turnbull), the SN of elderly control subjects and pa- ogy has been reported in a compound het- Krishnan, El and Medical Toxicology Centre, tients with Lewy body disease (LBD).3,4 erozygous individual carrying pink1 mu- and Medical Wolfson Unit (Dr Morris), Also, genes encoding proteins that affect tations11 and in individuals carrying Wolfson Uni Newcastle University; UK mitochondrial function have been identi- heterozygous mutations in parkin and Newcastle U National Institute for Health 12-14 National Ins Research Biomedical Research fied in families with autosomal recessive pink1 genes. Research Bio 5 ␣ Centre for Ageing and juvenile parkinsonism including PARK2 Recent studies suggest that -sy- Centre for A Age-related Disease, Newcastle (parkin) (OMIM 602544), PARK6 (pink1) nuclein and mitochondria may have closer Age-related D upon Tyne Hospitals National (OMIM 608309), and PARK7 (DJ-1) interactions than once thought, and their upon Tyne H Health Service Foundation (OMIM 602533). These genes have var- interaction in vitro coincides with mito- Health Servi Trust (Drs Jaros and McKeith ied roles within mitochondria including chondrial dysfunction.15,16 However, much Trust (Drs Ja and Ms Hepplewhite); and the targeting of mitochondria to au- of this data comes from studies examin- and Ms Hepp Neuropathology/Cellular 6,7 Neuropathol Pathology, Royal Victoria tophagy, protection against oxidative ing this relationship in homogenized SN Pathology, R 7,8 17 Infirmary (Dr Jaros), Newcastle stress, and reactive oxygen species sens- tissue or overexpressing cell culture sys- Infirmary (D upon Tyne, England. ing/scavenging.9,10 tems17-19 rather than in single human neu- upon Tyne, E

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 IMMUNOHISTOCHEMISTRY Table. Clinical and Neuropathologic Features of All DLB and PD Cases Single immunohistochemistry (IHC) for ␣-synuclein (Figure 1A-D) and mitochondrial markers (Figure 1E and F) Cortical Lewy 21 a b has been performed previously. A dual IHC assay was de- Case Disease Body Score SN Cell Loss signed combining staining for ␣-synuclein with the detection 1 DLB 19 UMB = 2/4 of a mitochondrial protein (Figure 1G and Figure 2A and G). LMB = 3/4 We used antibodies to specific subunits of respiratory chain 2 DLB 11 UMB = 2/4 complexes I, II, and IV, and detection of an outer membrane LMB = 3/4 mitochondrial protein, porin, as a marker of mitochondrial 3 DLB 17 UMB = 2/4 density. LMB = 3/4 Immunocytochemistry was performed according to Ma- 4 DLB 10 UMB = 2/4 had et al22 with minor modifications. Antigen retrieval was per- LMB = 3/4 formed in concentrated formic acid followed by high tempera- 5 DLB 16 UMB = 2/4 ture retrieval in 1mM ethylenediaminetetraacetic acid (pH 8.0) LMB = 2/4 ␣ 6 DLB 6, limbic UMB = 2/4 before blocking with 1% normal goat serum. The -synuclein LMB = 4/4 primary antibody (1:30) was then applied for 90 minutes (clone 7 DLB 17, neocortex UMB = 1-2/4 KM51; Novocastra; Leica Biosystems). Biotinylated goat anti- 8 DLB 19 UMB = 2/4 mouse antibodies (Vector Laboratories) were applied, fol- LMB = 3/4 lowed by Vectastain Elite avidin-biotin complex (Vector Labo- 1 PD 13 UMB = 2/4 ratories) and Vectastain Novared substrate (Vector Laboratories). 2 PD 1, brainstem UMB = 1/4 Following this step, sections were stored in TBS overnight at LMB = 1/4 4°C. Immunocytochemistry for mitochondrial proteins (eTable, 3 PD 11 UMB = 3/4 http://www.archneurol.com) was then performed with detec- LMB = 2/4 tion of primary antibodies using the polymer detection system 4 Mixed PD (with 11 UMB = 1/4 (MenaPath kit; Menarini Diagnostics). cerebral amyloid angiopathy) IMAGING AND DENSITOMETRIC ANALYSIS LMB = 4/4 5 PD NA, severe UMB = 2-3/4 Dual IHC–stained sections were analyzed using bright field mi- cortical croscopy and Nuance multispectral imaging (Cri). We took low pathology magnification (ϫ4) images of the SN, defined its boundaries, LMB = 2-3/4 and took 15 random images of neuromelanin-containing neurons within this area. By using single-stain controls for each of Abbreviations: DLB, dementia with Lewy bodies; LMB, lower midbrain; NA, not applicable; PD, Parkinson disease; SN, substantia nigra; UMB, upper the chromogens used in the IHC protocol (Figure 1J-M), in- midbrain. dividual spectra for each color can be created using the Nu- a The cortical Lewy body score for each patient was assessed from the ance software. Owing to the nature of the SN neurons, we gen- whole cortical area samples available from each case. erated spectra for 4 chromogens: blue for hematoxylin, red for b Cell loss scores 0 of 4 (0%-25%), 1 of 4 (25%-50%), 2 of 4 (50%-75%), ␣-synuclein, purple for the mitochondrial proteins, and yellow/ and 4 of 4 (75%-100%). brown for the neuromelanin. These spectra can then be applied to the dual stained sections and the signal from all the rons. To test whether mitochondrial abnormalities in- component chromogens can be extracted.23 The Nuance soft- fluence ␣-synuclein accumulation, we studied individual ware generates black and white images for all the component neurons from the SN in LBD cases. chromogens and a pseudo-colored composite image (Figure 2). The black and white images can then be used for densitometric analysis. METHODS For each pigmented neuron, we measured the intensity of mitochondrial protein staining (per unit area) and assessed the TISSUE type/amount of ␣-synuclein pathology. Mitochondrial protein signals were sampled from a region of the neuron not contain- Formalin-fixed, paraffin-embedded upper midbrain tissue from ing neuromelanin, ␣-synuclein, or the nucleus. Densitometric 5 patients with IPD (mean age, 81.2 years; age range, 75-87 years; analysis is an inverse linear scale ranging from 0 (black) to 250 mean postmortem delay, 52.3 hours; mean tissue fixation, 2.8 (white). Values were inverted by subtraction from 250 so that months), 8 patients with DLB (mean age, 75 years; age range, more intense staining gave a higher value. All densitometric 70-81 years; mean postmortem delay, 39 hours; mean tissue values were normalized to the mean control value for each pro- fixation, 1.9 months), and 8 neurologically and pathologically tein studied, calculated for cells stained in the same batch. Nor- normal control subjects (mean age, 74.5 years; age range, 58-87 malization was performed by expressing each data point from years; mean postmortem delay, 30.6 hours; mean tissue fixa- LBD cases stained at the same time as a percentage of the con- tion, 1.6 months) were included. Control cases showed mini- trol mean for that particular protein. mal Lewy body pathology—however, 1 case showed occa- To minimize variation in intensity of immunostaining, sional LBs, Lewy neurites, and fine granular inclusions—and we performed staining using 1 antibody for all patients and no cell loss (0 of 4 [0%-25% cell loss]). Patients with DLB and control subjects on the same day, where possible. We also IPD fulfilled the clinical and neuropathologic criteria for these compared porin immunostaining in neurons with no pathol- diseases (Table).20 The use of all human tissue had been con- ogy for both control subjects and patients and showed no sented to by the appropriate local research ethics committee difference between the mean values obtained for control and conformed to the UK Medical Research Council guide- subjects and patients (eFigure 1). Therefore, it was reasonable lines on the use of tissue in medical research. to compare all the data from the LBD cases with the control

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Figure 1. Bright field images of single and dual immunohistochemistry (IHC). Different stages of ␣-synuclein accumulation/aggregation in substantia nigra (SN) (A-D). Punctate ␣-synuclein granules (A, arrowheads), irregular inclusion/pre-Lewy body (B, arrowhead), single Lewy body (C, arrow), and multiple Lewy bodies (D, arrow). E, Some SN neurons are deficient for mitochondrial complex I subunits (arrow). In the other neuron, mitochondrial protein staining is very punctate, representing the mitochondria, and it is absent from the nucleus; single IHC for CI20 (purple), hematoxylin (blue), and neuromelanin (yellow/brown). F, All nigral neurons show equal levels of porin; single IHC for porin (control section, purple), hematoxylin (blue), and neuromelanin (yellow/brown). G, SN neurons showing different stages of ␣-synuclein accumulation/aggregation and variable degree of deficiency for mitochondrial COXI subunit; granular ␣-synuclein pathology (red arrow), larger and denser pathology (black arrow), and deficiency for COXI in the neuron lacking any ␣-synuclein pathology (arrowhead). H, SN neuron staining using dual IHC for ␣-synuclein, with the omission of the primary antibody for the mitochondrial proteins. I, SN neuron staining using dual IHC for mitochondrial protein (porin), with the omission of the primary antibody for ␣-synuclein. Single IHC control samples for each of the chromogens used in this study: hematoxylin ( J, blue), ␣-synuclein (K, red), mitochondrial protein (purin) (L, purple), and neuromelanin (M, yellow/brown). Scale bars represent 20 µm.

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Figure 2. Separation of signals from individual chromogens on dual immunohistochemistry tissue using multispectral imaging. Bright field images of double immunohistochemistry for ␣-synuclein and mitochondrial proteins. CI20 (A) and porin (B) staining in neurons with ␣-synuclein pathology (black arrowheads) and neurons without ␣-synuclein pathology (red arrowheads). Composite images generated by the Nuance system from the unmixed images of signal from each chromogen (C and D). Note the images are identical to parts A and B, with positive signals from mitochondrial protein (purple) and ␣-synuclein (red) appearing as in the bright field image. Signal from the purple chromogen (E and F; wavelength, 455-500 nm) labeling the mitochondrial protein (E, CI20; F, porin). Note mitochondrial staining is localized in the neuronal cytoplasm and surrounding neuropil but is absent from the nucleus (asterisks). Signal from the red chromogen (wavelength, 500-525 nm) labeling the ␣-synuclein aggregates (G and H). Note that the ␣-synuclein staining is very specific and is only detected in those neurons containing pathology (black arrowheads). The Nuance software does not give a signal for ␣-synuclein in the neuron without pathology (red arrowhead). Signal from the yellow/brown chromogen of neuromelanin (I and J; wavelength, 420-440 nm). Note that the neuromelanin signal is very specific and appears granular in these unmixed images identical to its appearance in the bright field image. Signal from the blue chromogen of hematoxylin (K and L; wavelength, 565-600 nm). Original magnification ϫ40.

cases and to assume that any changes seen were not due to tech- tensity falling below the 10th percentile value for the control nical variation. data for each protein. To determine whether there was a difference between cells with and without pathology, we per- STATISTICS formed a Fisher exact test.

The data sets were not normally distributed and we used a Mann- RESULTS Whitney U test to compare mitochondrial protein staining in neurons with and without pathology. To ascertain whether the staining intensities for mitochondrial proteins changed be- METHOD VALIDATION tween different pathology types, we performed a Bartlett test for equal variances and the Kruskal-Wallis test. To investigate the number of cells that were deficient for certain mitochon- Absolute quantification of protein expression through drial respiratory chain proteins in cases and control subjects the use of densitometric analysis is difficult, but it is using densometric analysis and without assigning arbitrary the only approach that allows us to look at expression ranges, we defined deficient cells as those with a staining in- in neurons within the same section and compare with

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 A Porin and complex II 70-kDa staining intensities B Complex I protein staining intensities in C Complex IV protein staining intensities in in neurons from Lewy body disease cases neurons from Lewy body disease cases neurons from Lewy body disease cases 350 400 200 300

250 300 150 200

150 200 100

100 100 50 50 Staining Intensity per Unit Area Staining Intensity per Unit Area Staining Intensity per Unit Area 0 0 0 Porin Porin C1170 C1170 C120 C120 C119 C119 COXI COXI COXIV COXIV Without With Without With Without With Without With Without With Without With Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology n = 510 n = 210 n = 417 n = 295 n = 586 n = 234 n = 586 n = 269 n = 663 n = 220 n = 752 n = 205

Figure 3. Mitochondrial protein staining is increased in substantia nigra (SN) neurons with ␣-synuclein pathology. There is a significant increase in the levels of several mitochondrial respiratory chain proteins in neurons containing pathology, although the density of mitochondria (porin) and CII70 staining is uniform. A, Staining intensities per unit area for porin (neurons without pathology [NWOP]; 25th percentile, 104.8; median, 120.72; 75th percentile, 166) (neurons with pathology [NWP]; 25th percentile, 98.9; median, 121.044; 75th percentile, 169.4; P = .36) and CII70 in neurons with and without ␣-synuclein pathology (NWOP; 25th percentile, 93.8; median, 121.45; 75th percentile, 179) (NWP; 25th percentile, 105.1; median, 126.96; 75th percentile, 184.7; P = .15). B, Staining intensities for complex I subunits in individual SN neurons with and without pathology (CI19 NWOP; 25th percentile, 74.8; median, 102.58; 75th percentile, 127.7) (NWP; 25th percentile, 103.1; median, 127.03; 75th percentile, 183.3) (CI20 NWOP; 25th percentile, 32.3; median, 72.03; 75th percentile, 120.9) (NWP; 25th percentile, 87.1; median, 115.23; 75th percentile, 136). C, Staining intensities for complex IV subunits in individual SN (COXI NWOP; 25th percentile, 90.5; median, 113.73; 75th percentile, 133.1) (NWP; 25th percentile, 123.2; median, 139.88; 75th percentile, 157.3; (COXIV NWOP; 25th percentile, 77.2; median, 93.89; 75th percentile, 109.2) (NWP; 25th percentile, 91; median, 103.58; 75th percentile, 127.3). *P Ͻ .001. The median of each data set is shown (bar). The data have been normalized to the mean staining intensity for the control data and each data point represents 1 neuron.

250 Staining intensity for complex I 19kDa subunit in neurons with and without pathology for all LBD cases No pathology With pathology

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150

100 Staining Intensity per Unit Area

0 LBD1LBD2 LBD3 LBD4 LBD5 LBD6 LBD7 LBD8 LBD9 LBD10 LBD11 LBD12 Cases

Figure 4. Mitochondrial complex I 19-kDa subunit staining is increased in neurons containing ␣-synuclein pathology in all cases. The mean mitochondrial CI19 staining increases in neurons containing ␣-synuclein pathology compared with those without pathology for 11 of 12 cases. For LBD6, there is a very small decrease in staining intensity in cells with pathology compared with those without pathology, but this may be explained by the small sample size when considering individual cases. LBD indicates Lewy body disease.

control cases. Several publications have shown that expression in neurons that correlate with the bio- antibodies to several respiratory chain complexes (in- chemical defect seen (eFigure 2). cluding those used in this article) can detect changes that correlate with a biochemical defect.22,24,25 In addi- MITOCHONDRIAL PROTEIN LEVELS IN SN tion, we used the same method to explore protein NEURONS OF NORMAL CONTROL SUBJECTS expression in patients with neurodegeneration second- ary to primary defects of the mitochondrial genome Using brain sections from normal control subjects, we mea- and we were able to show clear defects of protein sured the normal expression of different mitochondrial pro-

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 teins in 5298 single pigmented SN neurons (eFigure 3). There was minimal evidence of LBs and ␣-synuclein ag- A Complex I 19-kDa subunit staining ∗ ∗∗ gregates in these subjects (6.5% of cells contained pathol- 250 ∗ ogy), which was much lower than in IPD and DLB cases

(29% of cells contained pathology [1433 of 4947 neu- 200 rons]). There was wide variation of staining intensities in normal control individuals for all 6 mitochondrial proteins studied in this investigation. 150

100

MITOCHONDRIAL PROTEIN LEVELS IN SN Staining Intensity

NEURONS WITH AND WITHOUT PATHOLOGIC 50 ␣-SYNUCLEIN AGGREGATES

0 +/– + ++ +++ The data obtained from both IPD and DLB cases showed n = 278 n = 81 n = 97 n = 52 the same results and because IPD and DLB are postu- B 20 lated to be on a clinical spectrum, we pooled all the data 250 Complex IV subunit I staining from these cases to ensure robust statistical signifi- cance. From this point forward, LBD refers to any data 200 from both IPD and DLB cases. We analyzed a total of 4947 single SN neurons from LBD cases. Porin IHC showed that the median and range of the 150 relative mitochondrial density were equal in neurons with and without ␣-synuclein pathology in LBD cases (P = .36) 100

(Figure 3A). Therefore, any differences in intensity of Staining Intensity

respiratory chain subunit staining between neurons with 50 and without pathology were unlikely due to changes in overall mitochondrial mass. Staining for complex II 70 0 kDa showed a slight increase in cells with pathology +/– + ++ +++ (P = .05), but this was less significant than for other pro- n = 233 n = 84 n = 89 n = 26 teins studied. This is relevant because complex II is the only complex of the electron transport chain that con- Figure 5. The increase in mitochondrial protein staining is not related to the tains no subunits encoded by mitochondrial DNA. The extent of ␣-synuclein pathology present within single neurons. The level of mitochondrial protein varies little between neurons containing different staining intensity for this subunit is not affected by de- amounts of ␣-synuclein pathology. A, Results from neurons stained for fects of mitochondrial DNA and in fact, forms the basis CI19. *P Ͻ .001, **P Ͻ .05. B; Results for COXI. The median of each data of our cytochrome C oxidase (COX)/succinate dehydro- set is shown (bar); each data point represents 1 neuron. genase activity assay.26 For all other mitochondrial respiratory chain proteins studied (CI19, CI20, COXI, and COXIV), the median intensity of staining was significantly greater in those cells containing ␣-synuclein pathology than in those with- RELATIONSHIP OF ␣-SYNUCLEIN PATHOLOGY out (P Ͻ .001) (Figure 3). Results for both complex I sub- TYPE TO EXPRESSION OF units show that a large number of the cells without pa- MITOCHONDRIAL PROTEINS thology have very low expression of complex I subunits, entirely compatible with previous reports of reduced ac- To examine the relationship between the expression of tivity of complex I in aging SN27 (Figure 3B). This dif- mitochondrial proteins and the extent of ␣-synuclein pa- ference in staining intensities between neurons with and thology, we graded the density of ␣-synuclein staining without pathology was also seen for complex IV. The re- as few granules (ϩ/−), granules or a small pre-Lewy body sult for COXI is particularly relevant because IHC for this (ϩ), larger pre-Lewy body (ϩϩ), and very dense pre- respiratory chain subunit has been shown to correlate with Lewy or large Lewy body (ϩϩϩ).28,29 The only statisti- complex IV activity.22 These results suggest that respi- cal differences between data sets occurred when consid- ratory dysfunction is not directly involved in the forma- ering the staining for CI19 (Figure 5A) and porin tion of ␣-synuclein aggregates (Figure 3C). (between ϩ vs ϩϩϩ and ϩϩ vs ϩϩϩ [P Ͻ .05]). For This pattern of staining is similar for all patients all other mitochondrial proteins, there was no statistical (Figure 4). Most patients (with the exception of those difference (P Ͼ .05) (Figure 5B; eg, COXI). From these with LBD6) showed an increase of staining for mitochon- results, it appears that in individual SN neurons, in- drial complex I 19-kDa subunit in neurons containing creased levels of expression of the majority of mitochon- ␣-synuclein pathology compared with those devoid of drial proteins is not related to the progression of ␣-sy- pathology. This further validates our observation be- nuclein aggregates from granules to Lewy bodies and is cause the staining intensities of neurons of a single case certainly not as dramatic as observed between cells with are the same as when we consider the whole group. and without pathology.

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15 Total Cells, % Total

0 0-10 10-2020-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 Percentiles of Control Staining Intensities

B 35 Complex I 19-kDa staining

15 Total Cells, % Total

0 0-10 10-2020-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 Percentiles of Control Staining Intensities

C D E

Figure 6. Distributions of staining intensity of complex I in substantia nigra neurons of control and Lewy body disease cases. Percentage of substantia nigra neurons, which fall into each of the 10% staining intensity brackets, for Lewy body disease case neurons compared with control cases and only those with pathology. Note that neurons with a staining intensity in the bottom 10% of this range were defined as deficient. A, Complex I 20-kDa subunit. B, ComplexI 19-kDa subunit. C-E, Examples of neurons stained with different intensities throughout the range of complex I 20-kDa: intense staining (C), moderate staining (D), and weak (deficient) staining (E). Scale bars represent 20 µm.

COMPLEX I DEFICIENCY IN PIGMENTED LBD SN that were deficient for complex I subunits (Figure 6). In normal aged SN, 9.94% of neurons (36 of 362) are Since previous data suggest a role for complex I defi- deficient for CI20, while 9.85% are deficient for CI19. ciency in PD, we determined the number of neurons In LBD, we determined that 18.0% of neurons contain-

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 ing no pathology (66 of 366) are deficient for CI19, phosphate and thus, it can only occur in cells with nor- while only 0.9% of neurons containing ␣-synuclein mal mitochondrial function. A situation may then arise pathology (1 of 107) are deficient (P Ͻ .001). For whereby ␣-synuclein clearance cannot cope with the in- CI20, 10.4% of all LBD cells containing no pathology creased levels of aggregating protein and the Lewy body (31 of 298) are deficient compared with only 1.5% of forms. In this context, the observation of little or no ␣-sy- ␣-synuclein–containing neurons (2 of 126) nuclein pathology in the respiratory chain deficient SN (P Ͻ .001). Thus, very few neurons containing neurons would seem entirely fitting. ␣-synuclein pathology are deficient for complex I sub- A final consideration is the type of ␣-synuclein being units and that deficiency occurs predominantly in investigated. There still remains some debate as to which neurons that do not contain pathology. of the ␣-synuclein forms is potentially toxic. ␣-Sy- nuclein can exist in many forms, some of which have the COMMENT ability to interact with lipid membranes and cause the formation of pores within them. We have only examined insoluble forms of ␣-synuclein, and it is possible that Our data shows that within individual nigral neurons, it is these forms that are less harmful. In neurons, dam- the presence of pathologic ␣-synuclein aggregates is as- aged ␣-synuclein forms soluble oligomers and protofi- sociated with significantly higher expression of respira- brils, which have been shown to damage membranes and tory chain subunits. ␣-Synuclein pathology and deficien- have other detrimental effects. Neurons that show mi- cies of mitochondrial respiratory chain subunits, which tochondrial dysfunction may be unable to aggregate these are 2 factors thought to play important roles in the patho- toxic forms of ␣-synuclein. Again, it could potentially be genesis of synucleinopathies, appear to affect different the same double-hit hypothesis, as previously men- populations of nigral neurons. tioned, of increased toxic ␣-synuclein and mitochon- Although we only studied protein expression levels drial dysfunction leading to cell death. Neurons with nor- in fixed tissues, the expression of a number of the se- mal mitochondrial function may be better equipped to lected proteins reflects the activity of respiratory chain respond to the toxic ␣-synuclein species and bring them complexes within mitochondria.22 We had expected that together into a LB, thus protecting the cell from the dam- mitochondrial dysfunction would lead directly to ␣-sy- aging effects of soluble forms of this protein. ␣-Sy- nuclein pathology. A deficiency of mitochondrial respi- nuclein is believed to have detrimental influences on mi- ratory chain subunits could lead to adenosine triphos- tochondrial function but again, the form of ␣-synuclein phate depletion, which would impair a number of cellular responsible remains unclear.18,36 processes including protein degradation through both While many previous studies have found an associa- chaperone-mediated autophagy and the ubiquitin pro- tion between mitochondrial dysfunction and the accu- teasome system.30 Defects of mitochondrial respiratory mulation of ␣-synuclein in homogenized tissue, our data chain subunits can cause increased free radical produc- shows that at the single cell level, these 2 pathologies seem tion in cell culture31-34 and potentially lead to damage of not to occur in the same cells. The data presented in this intracellular proteins and increased load on the protea- article shows that while mitochondrial dysfunction and some. ␣-synuclein pathology are important in LBD, mitochon- There are a number of potential explanations for why drial dysfunction appears not to be the catalyst for ␣-sy- cells containing ␣-synuclein pathology appear to have in- nuclein accumulation at the single cell level. creased expression of mitochondrial respiratory chain proteins, with no increase in overall mitochondrial density; Accepted for Publication: October 19, 2011. we discuss 3 of them. First, it is feasible that mitochon- Correspondence: Doug M. Turnbull, MD, Mitochondrial drial dysfunction and ␣-synuclein accumulation pro- Research Group, Institute of Ageing and Health, The Medi- vide a toxic double hit for neurons. Those cells showing calSchool,NewcastleUniversity,NewcastleuponTyne,NE2 both severe mitochondrial dysfunction and ␣-synuclein 4HH, England ([email protected]). accumulation are lost thus, explaining why it is ex- Author Contributions: Study concept and design: Reeve, tremely rare to find cells with both low levels of mito- Campbell, Krishnan, McKeith, and Turnbull. Acquisition chondrial protein expression and ␣-synuclein pathol- of data: Reeve, Park, Hepplewhite, Morris, and McKeith. ogy. However, data from this study examining the Analysis and interpretation of data: Reeve, Park, Jaros, Lax, relationship between different stages of ␣-synuclein ac- Elson, and Turnbull. Drafting of the manuscript: Reeve, Mor- cumulation and mitochondrial dysfunction would not ris, and Turnbull. Critical revision of the manuscript for im- support this. portant intellectual content: Reeve, Park, Jaros, Campbell, Second, there is a possibility that normal mitochon- Lax, Hepplewhite, Krishnan, Elson, Morris, McKeith, and drial function is required for the accumulation of ␣-sy- Turnbull. Statistical analysis: Reeve and Elson. Obtained fund- nuclein into Lewy bodies. Aggregating protein needs to ing: McKeith and Turnbull. Administrative, technical, and be transported along axons/neurites to the cell body, where material support: Morris. Study supervision: Park, Jaros, it is proposed that it accumulates into aggresomes, which McKeith, and Turnbull. eventually form Lewy bodies. Recruitment of mitochon- Financial Disclosure: None reported. dria and ubiquitin proteasome system proteins then oc- Funding/Support: Tissue for this study was provided by curs, facilitating the clearance and degradation of aggre- the Newcastle Brain Tissue Resource, which is funded gating protein.35 The transport and clearance of by grants from the UK Medical Research Council ␣-synuclein will require mitochondrial adenosine tri- (G0400074) and the Alzheimer’s Society and Alzhei-

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