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 ARCH NEUROL / VOL 69 (NO. 3), MAR 2012 WWW.ARCHNEUROL.COM 385 ©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 neu- rons 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 ap- plied 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 densitomet- ric 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]).