High expression of α-synuclein in damaged mitochondria with PLA2G6 dysfunction
© Sumi-Akamaru et al. 2016
Received: 4 February 2016
Accepted: 10 March 2016
Published: 30 March 2016
To clarify the role of α-synuclein (αSyn) in neuronal membrane remodeling, we analyzed the expression of αSyn in neurons with a dysfunction of PLA2G6, which is indispensable for membrane remodeling. αSyn/phosphorylated-αSyn (PαSyn) distribution and neurodegeneration were quantitatively estimated in PLA2G6-knockout (KO) mice, which demonstrate marked mitochondrial membrane degeneration. We also assessed the relationship between αSyn deposits and mitochondria in brain tissue from patients with PLA2G6-associated neurodegeneration (PLAN) and Parkinson’s disease (PD), and quantitatively examined Lewy bodies (LBs) and neurons. The expression level of αSyn was elevated in PLA2G6-knockdown cells and KO mouse neurons. Strong PαSyn expression was observed in neuronal granules in KO mice before onset of motor symptoms. The granules were mitochondrial outer membrane protein (TOM20)-positive. Ultramicroscopy revealed that PαSyn-positive granules were localized to mitochondria with degenerated inner membranes. After symptom onset, TOM20-positive granules were frequently found in ubiquitinated spheroids, where PαSyn expression was low. Axons were atrophic, but the neuronal loss was not evident in KO mice. In PLAN neurons, small PαSyn-positive inclusions with a TOM20-positive edge were frequently observed and clustered into LBs. The surfaces of most LBs were TOM20-positive in PLAN and TOM20-negative in PD brains. The high proportion of LB-bearing neurons and the preserved neuronal number in PLAN suggested long-term survival of LB-bearing neurons. Elevated expression of αSyn/PαSyn in mitochondria appears to be the early response to PLA2G6-deficiency in neurons. The strong affinity of αSyn for damaged mitochondrial membranes may promote membrane stabilization of mitochondria and neuronal survival in neurons.
It is well known that α-synuclein (αSyn) is a pathological marker of Parkinson disease (PD), because αSyn/phosphorylated αSyn (PαSyn) is a main component of Lewy bodies (LBs). The physiological function of αSyn has recently become known. As a pre-synaptic chaperone, αSyn promotes soluble NSF-attachment protein receptor (SNARE)-complex assembly [9, 22]. αSyn also localizes to nuclei and subcellular organelles, including mitochondria and mitochondrion-associated endoplasmic reticulum (ER) membranes [5, 16, 37]. αSyn binds to lipid membranes [42, 43], in particular, to membranes with high curvature, such as synaptic vesicles and mitochondrial inner membranes [5, 25, 52]. The N-terminus of αSyn lies along the surface of the membrane [5, 43], where it senses lipid-packing defects and leads to membrane remodeling and stabilization [7, 29].
Mitochondria comprise an inner and an outer membrane that separate the intermembrane space and the matrix. Mitochondria have various functions, including oxidative phosphorylation, lipid metabolism, endocytosis , apoptosis, and calcium and iron homeostasis . A mitochondrial inner membrane-specific phospholipid, cardiolipin, is crucial for the integrity and function of mitochondria [25, 32]. In the brains of mice lacking αSyn, the mitochondrial lipid composition changes, and complex I/III activity is reduced . Moreover, the N-terminus of αSyn regulates mitochondrial membrane permeability . Together, these findings suggest that αSyn is integral to maintaining mitochondrial function.
Calcium-independent phospholipase A2β, encoded by PLA2G6, has diverse functions, such as releasing lipid mediators, inflammation, vascular relaxation, and secretion, by hydrolyzing the sn-2 ester bond in phospholipids [8, 10, 27, 28, 51]. In PLA2G6-associated neurodegeneration (PLAN) [19, 26], formerly called Seitelberger disease, a variety of neurological deficits are present from infancy, suggesting the importance of PLA2G6 in the brain. PLAN encompasses a number of phenotypes, such as infantile neuroaxonal dystrophy and adult-onset dystonia-parkinsonism (Park14). Although these phenotypes differ in the degree and distribution of neurodegeneration, αSyn/Lewy-related pathology is commonly observed [15, 31, 36].
The pathogenesis of PLA2G6 deficiency is thought to involve dysfunction of mitochondria and membrane remodeling [13, 20]. Pla2g6-knockout (KO) mice show slow progression of motor deficits, and there is a progressive formation of spheroids and tubulovesicular structures [23, 41], similar to that seen in PLAN [11, 17]. Recently, we reported that the spinal cord neurons in Pla2g6-KO mice have ultra-microscopically abnormal mitochondria, with degenerated inner membranes, which are periodic acid Schiff (PAS)-positive, negative for an inner membrane protein (cytochrome c oxidase, CCO), and positive for an outer membrane protein (translocase of the outer mitochondrial membrane, TOM20) on immunohistochemistry [4, 45]. In mass spectrometry, the content of mitochondria-specific phospholipid, cardiolipin, is high in Pla2g6-KO mice. These findings suggest dysregulation of phospholipid metabolism in the mitochondrial inner membrane. We also previously demonstrated low expression of CCO and low production of ATP in PLA2G6-knockdown cells, suggesting mitochondrial dysfunction .
Genetic mutation of SNCA  and multiple copies of SNCA  cause PD, suggesting the toxic function due to a genetic abnormality of αSyn [5, 48]. On the other hand, in the brains of elderly people without neuropsychiatric symptoms, where widespread αSyn/Lewy-related pathology is seen , the severity of αSyn/Lewy-related pathology is not associated with the clinical course in PD [14, 18]. In spite of many intensive researches [1, 5, 14, 18, 35, 48], the biological significance of LBs in sporadic PD and other familial PD is not yet fully understood. In this study, we aimed to clarify the reason for αSyn accumulation in neurons, and pathologically analyzed the relationship between αSyn and mitochondrial membranes in PLAN and in Pla2g6-KO mice.
Materials and methods
Generation of Pla2g6-Kd cells
We generated PLA2G6 gene knockdown (Kd) SH-SY5Y human neuroblastoma cells, as described before . Briefly, SH-SY5Y neuroblastoma cell line was obtained from American Tissue Culture Collection (ATCC, Manassas, VA). Cells were grown in Dulbecco’s modified Eagle’s medium high glucose (high-glucose formulation, Nacalai Tesque, Kyoto, Japan) supplemented with 10 % fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cell cultures were all kept at 37 °C. The small interfering RNA (siRNA) targeting human PLA2G6 gene (Life technologies, Carlsbad, CA) and negative control siRNA (Qiagen, Hilden, Germany), were obtained. Subconfluent SH-SY5Y cells were transfected with siRNAs using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA). The targeting sense sequence for human PLA2G6 in SH-SY5Y cells is 5′-GACCAAAGAGCAAGUGACAAAUGUU-3′.
RNA expression analysis
The absence of the PLA2G6 expression was confirmed in Pla2g6-Kd cells, as described before . Briefly, total RNA was extracted from siRNA-transfected SH-SY5Y cells using the RNeasy Kit (Qiagen, Hilden, Germany), and the RNA concentrations were determined spectrophotometrically. cDNA was generated using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) from 100 ng of each RNA sample. RT-PCR was used to confirm reduced expression levels of PLA2G6 gene (data not shown).
Cells were collected after transfection for 48 h. Samples (n = 6 per group) were prepared as described before . Cells, which were transfected with negative control siRNA, were used as control. The protein (10 μg) was separated on 15 % SDS-PAGE, electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, CA, USA), blocked with 5 % nonfat milk and incubated overnight at 4 °C with the primary antibody against GAPDH (1:1000, Millipore) and α synuclein (αSyn, 1:200, IBL, Fujioka, Japan). The bands were visualized with enhanced chemiluminescence’s reagents and exposed to X-ray film.
Cells were fixed in 4 % paraformaldehyde for 30 min. After washing with PBS three times, cells were permeabilized with 0.2 % Triton X-100 for 30 min, and incubated with 10 % skim milk in PBS for 60 min. The antibodies against 20-kDa translocase of the outer mitochondrial membrane (1:100 dilution, TOM20, import receptor, expressed on the mitochondrial outer membrane; Santa Cruz, Dallas, TX) and αSyn (1:100 dilution, LB509, Abcam, Cambridge, England) were used as primary antibodies. Alexa Fluor1488 goat anti-rabbit IgG (H + L) antibody (Life Technologies) and Alexa Fluor1568 goat anti-mouse IgG (H + L) antibody (Life Technologies) were used as the secondary antibodies. Confocal laser-scanned images were obtained using an LSM 510 META (Carl Zeiss, Oberkochen, Germany).
All animals were handled in accordance with the Guidelines for Animal Experimentation of Osaka University (No.26-044-000).
Mice carried a homozygous disruption of Pla2g6 on a C57BL/6 background , aged 15 weeks (n = 3, pre-clinical stage, 1 male and 2 females); 1 year (n = 4, early symptomatic stage, 1 male and 3 females); and 2 years (n = 5, end stage, 2 males and 3 females); and wild-type mice, aged 15 weeks (n = 3, 1 male and 2 females); and 2 years (n = 4, 1 male and 3 females) were obtained. After perfusion of 4 % paraformaldehyde, spinal cords were removed and immersed in the same fixative overnight at 4 °C, after which 4-μm–thick paraffin sections were prepared. Sciatic nerves were also obtained. The sciatic nerves and small pieces of the spinal cord were fixed with 2.5 % glutaraldehyde and processed to epon blocks, as described previously . Epon sections (1-μm–thick) were stained with thionine and PAS.
The research presented in this study has been approved by the University Ethics Committee Osaka University Graduate School of Medicine (No. 10038). Consent for autopsies were obtained from legal representatives for all subjects in accordance with local institutional review board requirements, which was approved by the University Ethics Committee (Osaka University Graduate School of Medicine, Osaka, Japan).
Autopsy samples of one PLAN case (age at death: 20 years; disease duration: 17 years), seven Parkinson’s disease cases (age at death: 73 ± 9 years; disease duration: 13 ± 8 years), and five non-neurodegenerative control cases (age at death: 61 ± 16 years) were obtained. Paraffin-embedded 6-μm–thick sections of the midbrain, at the level of the red nucleus, upper pons, and upper medulla, were prepared and examined, because of the high frequency of LBs in those area in both PLAN [15, 31, 36] and PD.
Deparaffinized sections were incubated for 30 min with 0.3 % H2O2 to quench endogenous peroxidase activity, and were then washed with phosphate-buffered saline (PBS). The primary antibodies used were rabbit polyclonal antibodies against α-Syn (1:2000 dilution for mouse, 1:5000 dilution for human; Sigma − Aldrich, St. Louis, MO), TOM20 (1:40 dilution for mouse, 1:100 dilution for human, Santa Cruz), cathepsin D (lysosomal enzyme, 1:400 dilution, Dako, Glostrup, Denmark), ubiquitin (1:2000 dilution, DAKO), and mouse monoclonal antibodies against PαSyn (specific for Ser129-phosphortlated αSyn, 1:800 dilution for mouse, 1:2000 dilution for human, pSyn#64; Wako, Osaka, Japan) and cytochrome c oxidase subunit IV (CCO, respiratory complex IV, expressed on the mitochondrial inner membrane; 1:300 dilution for mouse, 1: 1000 dilution for human; Invitrogen) and KDEL (Lys-Asp-Glu-Leu, 1:500 dilution, Enzo Life Sciences, Farmingdale, NY). Goat anti-rabbit and anti-mouse immunoglobulins conjugated to peroxidase-labeled dextran polymer (Dako Envision+, Dako) were used as secondary antibodies. Reaction products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA), and hematoxylin was used to counterstain the cell nuclei. The immunostaining patterns were compared in serial sections. Some sections were additionally stained with Luxol Fast Blue (LFB) or PAS.
For double immunohistochemistry, two primary antibodies were combined, including antibodies for αSyn (αSyn or PαSyn), mitochondrial membrane markers (CCO or TOM20), ubiquitin, and tyrosine hydroxylase (TH). The VECTASTAIN ABC-AP kit (Vector Laboratories) and ALKALINE PHOSPHATASE SUBSTRATE KIT IV BCIP/NBT (Vector Laboratories) were used for the secondary antibody and visualization of reaction products, respectively.
Quantitative pathological analysis of anterior horn cells and sciatic nerves of mice
We estimated the number of neurons filled with PαSyn-positive granules and the number of motor neurons in the anterior part of mouse cervical spinal cord and myelinated fibers in sciatic nerves. The neurons with clear nucleoli and cell bodies with a diameter greater than 25 μm, presumed to be alpha motor neurons, were counted . To this end, video images were obtained for each 4-μm–thick Nissl-stained paraffin section and each 1-μm–thick toluidine blue-stained epon section, using a digital camera (KEYENCE VB-7010, KEYENCE, Osaka, Japan) attached to a light microscope (ECLIPSEE800, Nikon, Tokyo, Japan). The diameters of motoneurons showing clear nucleoli and cell bodies, and the myelinated fibers in the sciatic nerves were measured using image analysis software (VH-H1A5, KEYENCE). Four sections of cervical cords were examined for each mouse. For the sciatic nerves, three fields (100× magnification) per mouse were examined. The number of motoneurons, large myelinated fibers (diameter, >10 μm), and total myelinated fibers in wild-type mice (2-years-old) and Pla2g6-KO mice (1-year-old and 2-years-old) was counted. The density of myelinated fibers (per mm2) was calculated for each mouse. The differences in the number of motoneurons and the fiber density between wild-type mice and Pla2g6-KO mice were statistically analyzed using the Wilcoxon rank sum test.
Ultrathin sections of the spinal cord from Pla2G6-KO mice aged 15 weeks, 56 weeks, or 100 weeks were prepared and stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope (H-7650; Hitachi High-Technologies Co., Tokyo, Japan).
Quantitative pathological analysis of TOM20-expression on the surface of human LBs
Double immunohistochemistry was performed for PαSyn (in brown) and for TOM20 (in blue) in the substantia nigra, in the locus coeruleus, or in the dorsal motor nuclei of the vagus nerve. Neuronal round or spherical PαSyn-positive inclusions with a clear contour were defined as LBs. In double immunohistochemistry, PαSyn-positive LBs (brown) were separated into three types, according to the expression pattern of TOM20 (blue) on the surface: TOM20-negative, partially TOM20-positive, and completely TOM20-positive (Fig. 7). Where less than half of the LB surface was positive for TOM20, the LB was classified as “partially TOM20-positive” and where more than half of the LB surface was positive for TOM20, the LB was classified as “completely TOM20-positive”. We counted the number of each type of LB in the substantia nigra, the locus coeruleus, and dorsal motor nuclei of the vagus in each patient. Slides were examined with a × 40 objective, and the microscope images were viewed on video-screen. The contours of the substantia nigra and the locus coeruleus, and dorsal motor nuclei of the vagus were drawn on the slide. The number of LBs with various surface expression levels of TOM20 was compared between PLAN and PD sections.
Estimation of dopaminergic neuron density and the proportion of LB-bearing neurons in controls and PD and PLAN patients
Double immunohistochemistry was performed against PαSyn (brown) and TH (blue) in human brainstem sections. In double immunohistochemistry, we counted the number of TH-positive (dopaminergic) neurons bearing LBs and total TH-positive neurons in the substantia nigra and the locus coeruleus . Slides were examined with a × 40 objective, and the microscope image was viewed on a video-screen. The contours of the substantia nigra and the locus coeruleus were drawn on the slide. The neuron density over three fields was calculated for each patient. The density (per mm2) of TH-positive neurons bearing LBs and total TH-positive neurons were compared between PLAN, PD (n = 7), and control (n = 5) individuals.
Estimation of the influence of LBs on TH expression levels in dopaminergic neurons
Double immunohistochemistry was performed for PαSyn (brown) and TH (blue). The immunostaining of TH was compared between dopaminergic neurons with LBs and those without LBs, in the substantia nigra and locus coeruleus. The brainstem was compared between PLAN and PD (n = 7) individuals.
All statistical analyses of histopathological data in mice were performed using SPSS Statistics software for Windows version 20.0 (SPSS, Chicago, IL). Data are expressed as the mean (SD). Mann–Whitney U test was used to analyze the ratio of αSyn/GADPH in cultured cells, the number of neurons and the density of large myelinated fibers and total myelinated fibers in mice. P values of less than 0.05 were considered as statistically significant.
High expression of αSyn in Pla2g6-knockdown cells
Strong expression of αSyn in the spinal cord of Pla2g6-KO mice at the preclinical-stage
Strong expression of PαSyn in CCO-negative, TOM20-positive granules in young Pla2g6-KO mice
The time course of Pα-syn expression in neurons and the number of motor neurons and myelinated fibers of Pla2g6-knockout (KO) mice
2y (n = 4)
15w (n = 3)
1y (n = 4)
2y (n = 5)
Number of neurons filled with PαSyn (+) granules
Number of AHCs
Sciatic nerve (distal axon)
Density of myelinated fibers
Sciatic nerve (distal axon)
Density of large myelinated fibers
Morphological changes of PαSyn-positive granules in Pla2g6-KO mice with age
At the end stage (2 years), some of the PαSyn-positive granules showed degenerated membranes (Fig. 3d, e). Using double immunohistochemistry, PαSyn was shown to co-localize with TOM20 in the granules and the degenerated membrane of the granules (Fig. 3f).
Semi-thin and ultra-microscopic image of small PαSyn-positive granules in Pla2g6-KO mice
To define the structure of small PαSyn-positive granules in Pla2g6-KO mouse neurons, we compared the the structures observed on semi-thin or ultra-microscopic pictures with the small PαSyn-positive granules seen on paraffin sections. In thionine-stained semi-thin sections, we observed dark colored granules in the cytoplasm and proximal axons of neurons in young KO mice (Fig. 3g). The granules were clearly PAS-positive (Fig. 3h), and showed a similar size and distribution as PαSyn-positive granules. Ultra-microscopically, round mitochondria could be seen in which the cristae were partially replaced by small dense granules both in the perinuclear space of neurons (Fig. 3i) and in axons (Fig. 3j). These abnormal mitochondria and round bodies were of a similar size and had a similar distribution as the PAS-positive granules in semi-thin sections, suggesting that PAS-positive granules were indicative of PαSyn-loading abnormal mitochondria.
In the aged KO mice, fewer round mitochondria could be seen in which the cristae were partially replaced by small dense granules in the perinuclear space of neurons (Fig. 3k). In the axons, mitochondria with branching and tubular cristae were frequently seen, and were clustered together (Fig. 3l). In the spheroid, highly degenerated mitochondria, tubulovesicular structures and degenerated membranes were combined to form an irregular structure (Fig. 3m), suggesting severe degeneration of mitochondrial membranes.
Different distribution of ubiquitin and PαSyn in Pla2g6-KO mice
Significant decrease in the density of large myelinated fibers, but not neuron number, in the spinal cord
To clarify the degree of neurodegeneration in axons and neurons that contained PαSyn-positive granules, from young mice, we estimated the number of axons and neurons in Pla2g6-KO mice after symptom onset. Axonal degeneration progressed as reported previously  and myelinated fibers became atrophic with age. Quantitative analysis (Table 1) revealed a significant decrease in large myelinated fibers (>10 μm in diameter) with age (KO mice 1y, n = 4, p =0.057; KO mice 2 y, n = 5, p <0.05). The density of the total myelinated fibers in Pla2g6-KO mice after onset was similar to that in the wild-type mice. The number of anterior horn cells in Pla2g6-KO mice at the early symptomatic stage was similar to that of wild-type mice. Even at the end stage, the number of neurons was not significantly different between Pla2g6-KO and wild-type mice (p = 0.067).
Prominent formation of Lewy Bodies (LBs) in a PLAN patient with a mutation in PLA2G6
PαSyn -positive small inclusions colocalized with the mitochondrial membrane protein in PLAN
Clustering of PαSyn-positive small inclusions to form a larger neuronal inclusion in PLAN
To clarify the relationship between PαSyn-positive small inclusions and LBs, we analyzed the distribution of PαSyn-positive small inclusions in neurons affected in PLAN. In PLAN neurons, small PαSyn-positive inclusions with a TOM20-positive edge appeared to cluster, and formed a larger narrow (PLAN: Fig. 6f) or round (PLAN: Fig. 6g) inclusion. In some LBs, small PαSyn-positive inclusions with a TOM20-positive edge appeared to cover the surface (PLAN: Fig. 6h), whereas the immunoreactivity of PαSyn was reduced in the center. PαSyn and TOM20-positive membranous structures also appeared to cover the surface of LBs (Fig. 6i).
Quantitative analysis of LBs with TOM20 present on their surface
Proportion of LB-bearing dopaminergic neurons and neuronal density
The dopaminergic neuron density and the proportion of Lewy body-bearing neurons
Density of dopaminergic neurons
(n = 5)
(n = 7)
(n = 1)
LB harbouring neuron
LB harbouring neuron
Preserved expression of TH in dopaminergic neurons, despite the presence of LBs
The expression of tyrosine hydroxylase in dopaminergic neurons bearing Lewy bodies (LBs)
(n = 7)
(n = 1)
(n = 7)
(n = 1)
Number of LB bearing neurons with the variety of tyrosine hydroxylase expression
Discussion and conclusions
In this study, we demonstrated elevated expression of αSyn both in cultured cells and in mice with PLA2G6 deficiency. Prominent accumulation of PαSyn in damaged mitochondria was shown in Pla2g6-KO mice, which constitute a model of PLA2G6-associated neurodegeneration (PLAN). In PLAN neurons, small PαSyn-positive granules and Lewy Bodies (LBs) were covered with mitochondrial membrane component on the surface. These findings suggest that αSyn/PαSyn associates with damaged mitochondria in PLAN.
Strong expression of αSyn was evident in Pla2g6-Kd neuroblastoma cells and KO mouse neurons, suggesting that endogenous αSyn was induced in neurons due to PLA2G6-deficiency. In Pla2g6-KO mice, surprisingly high levels of PαSyn expression were observed in neurons; this was never seen in the wild-type mice. PαSyn was detected on the surface of tiny granules, which had a staining pattern that was apparently different from that of the toxic strains of αSyn, including fibrils and ribbons [6, 33]. Immunohistochemical and ultra-microscopic data suggest that PαSyn-positive and PAS-positive granules were abnormal mitochondria with degenerated inner membranes in KO mice, but not lysosomes or endoplasmic reticulum, as has been shown previously [4, 45].
Injury of the inner mitochondrial membrane leads to permeabilization of mitochondria [2, 32, 50]. In KO mice from young, many neuronal mitochondria with degenerated inner membrane were present, but neither autophagic degradation (mitophagy) nor ubiquitination of mitochondria were observed. Mitochondrial membrane permeabilization also induces apoptosis [32, 49]. More than 8 months after the appearance of mitochondria with degenerated inner membranes, due to PLA2G6 deficiency, neuronal loss was not significant in mice, suggesting that apoptosis did not occur to neurons. TOM20-positive granules were ubiquitin-negative in neurons and ubiquitin-positive in spheroids, suggesting ubiquitination of mitochondria occurs in degenerated axons, but not in neurons.
In aged KO mice, PαSyn-positive granules became more degenerated morphologically, but the PαSyn expression level was maintained. αSyn/PαSyn seems to have a strong affinity for the mitochondrial membrane [37, 52]. However, considering the different localization of PαSyn and ubiquitin in axons, PαSyn seems to disappear from mitochondria before mitochondrial ubiquitination, following membrane permeabilization. The elevated expression of αSyn/PαSyn in mitochondria may be related to membrane stabilization .
In the substantia nigra of 26S proteasome depleted mice, neurodegeneration is accompanied by neuronal inclusions similar to Pale bodies , the precursors of LB . The inclusions contain mainly abnormal mitochondria , suggesting that Pale body or LB might be associated with abnormal mitochondria with bad quality control owing to dysfunction of ubiquitin proteasome system. As previously reported, LB formation is prominent in PLAN [15, 31, 36]. Considering the severity of mitochondrial degeneration in Pla2g6-KO mice, the association of Pale bodies or LBs with mitochondria would be strongly expected in PLAN.
Our double immunohistochemical data in PLAN revealed that the surface of PαSyn-positive small inclusions were TOM20-positive and CCO-negative, which is the same expression pattern of granules seen in Pla2g6-KO mouse neurons. αSyn/PαSyn appears to have a strong affinity for mitochondrial membrane components both in Pla2g6-KO mice and in the PLAN case investigated here. Moreover, the small inclusions appeared to cluster into a large inclusion, possibly due to the strong affinity of αSyn for the mitochondrial membrane.
LB extracts from PD brains have been reported to trigger αSyn pathology and motor deficits in mice and monkeys, which suggests that LBs contain neurotoxic materials . However, the presence of LBs in dopaminergic neurons was not associated with the low cytoplasmic expression of TH, either in the autopsied PLAN or PD brains studied here, although this occurs early in PD . This suggests that the presence of LB would not necessarily convey negative effects on the micro-environment of neurons, in spite of the neurotoxic materials inside the LB.
A certain proportion of LB-bearing neurons is reported to occur in the substantia nigra (3.6 % on average), and this does not correlate with the symptoms or disease duration [14, 18]. In our case of PLAN, a high proportion of dopaminergic neurons harbor LBs, suggesting the long-term survival of these neurons. In particular, the neurons were preserved well in the locus coeruleus, despite the presence of multiple LBs. This suggests that LBs in PLAN have a low toxicity. As αSyn/PαSyn-loading mitochondria with degenerated inner membrane did not lead to neuronal death in mice, high concentrations of mitochondrial membrane components on the surface of LBs may be associated with the low toxicity of LBs in PLAN through the stabilization of the mitochondrial lipid membrane [7, 29].
In conclusion, αSyn/PαSyn accumulation in damaged mitochondria of Pla2g6-KO mice and LBs in PLAN indicates a common pathological mechanism, which includes the strong affinity of αSyn for damaged mitochondrial membranes. PLAN patients with late onset tend to show prominent αSyn accumulation in neurons . Endogenous αSyn could be neuroprotective by stabilizing mitochondrial membranes in neurodegenerative diseases, such as PLAN and possibly even in PD, which may indicate a new target for the treatment of α-synucleinopathy.
This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science, JSPS (H.SA. G.B.), Scientific Research on Innovative Areas from MEXT (H.M.), Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) (H.M.), Practical Research Project for Rare/Intractable Disease from Japan Agency for Medical Research and Development, AMED (S.K., H.M.), and a Budget for Promoting Priority Measures in Discretionary Funds of the President in Tottori University (S.K.). The authors would like to thank H. Hayakawa for his expert assistance with biological analysis, K. Inoue for her expert assistance with pathological analysis, A. Sone for technical assistance, and R. Yoshida for fantastic illustrations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Araki K, Yagi N, Ikemoto Y, Choong CJ, Hayakawa H, Beck G, Sumi H, Fujimura H, Moriwaki T, Nagai Y, Goto Y, Mochizuki H. Synchrotron FTIR micro-spectroscopy for structural analysis of Lewy bodies in the brain of Parkinson’s disease patients. Sci Rep. 2015;5:17625.View ArticlePubMedPubMed CentralGoogle Scholar
- Ashrafi G, Schwarz TL. The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ. 2013;20:31–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Beck G, Shinzawa K, Hayakawa H, Baba K, Yasuda T, Sumi-Akamaru H, Tsujimoto Y, Mochizuki H. Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1. PLoS One. 2015;10(10), e0141629.View ArticlePubMedPubMed CentralGoogle Scholar
- Beck G, Sugiura Y, Shinzawa K, Kato S, Setou M, Tsujimoto Y, Sakoda S, Sumi-Akamaru H. Neuroaxonal dystrophy in calcium-independent phospholipase A2β deficiency results from insufficient remodeling and degeneration of mitochondrial and presynaptic membranes. J Neurosci. 2011;31:11411–20.View ArticlePubMedGoogle Scholar
- Bendor JT, Logan TP, Edwards RH. The Function of α-Synuclein. Neuron. 2013;79:1044–66.View ArticlePubMedGoogle Scholar
- Bousset L, Pieri L, Ruiz-Arlandis G, Gath J, Jensen PH, Habenstein B, Madiona K, Olieric V, Böckmann A, Meier BH, Melki R. Structural and functional characterization of two alpha-synuclein strains. Nat Commun. 2013;4:2575.View ArticlePubMedPubMed CentralGoogle Scholar
- Braun AR, Lacy MM, Ducas VC, Rhoades E, Sachs JN. α-Synuclein-induced membrane remodeling is driven by binding affinity, partition depth, and interleaflet order asymmetry. J Am Chem Soc. 2014;136:9962–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Burke JE, Dennis EA. Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Res. 2009;50(Suppl):S237–42.PubMedPubMed CentralGoogle Scholar
- Burré J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Südhof TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science. 2010;329:1663–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Cheon Y, Kim HW, Igarashi M, Modi HR, Chang L, Ma K, Greenstein D, Wohltmann M, Turk J, Rapoport SI, Taha AY. Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice. Biochim Biophys Acta. 2012;1821:1278–86.View ArticlePubMedPubMed CentralGoogle Scholar
- Cowen D, Olmstead EV. Infantile neuroaxonal dystrophy. J Neuropathol Exp Neurol. 1963;22:175–236.View ArticlePubMedGoogle Scholar
- Ellis CE, Murphy EJ, Mitchell DC, Golovko MY, Scaglia F, Barceló-Coblijn GC, Nussbaum RL. Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein. Mol Cell Biol. 2005;25:10190–201.View ArticlePubMedPubMed CentralGoogle Scholar
- Glynn P. Neuronal phospholipid deacylation is essential for axonal and synaptic integrity. Biochim Biophys Acta. 2013;1831:633–41.View ArticlePubMedGoogle Scholar
- Greffard S, Verny M, Bonnet AM, Seilhean D, Hauw JJ, Duyckaerts C. A stable proportion of Lewy body bearing neurons in the substantia nigra suggests a model in which the Lewy body causes neuronal death. Neurobiol Aging. 2010;31:99–103.View ArticlePubMedGoogle Scholar
- Gregory A, Westaway SK, Holm IE, Kotzbauer PT, Hogarth P, Sonek S, et al. Neurodegeneration associated with genetic defects in phspholipase A2. Neurology. 2008;71:1402–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Guardia-Laguarta C, Area-Gomez E, Rüb C, Liu Y, Magrané J, Becker D, Voos W, Schon EA, Przedborski S. α-Synuclein is localized to mitochondria-associated ER membranes. J Neurosci. 2014;34:249–59.View ArticlePubMedPubMed CentralGoogle Scholar
- Hedley-Whyte ET, Gilles FH, Uzman BG. Infantile neuroaxonal dystrophy. A disease characterized by altered terminal axons and synaptic endings. Neurology. 1968;18:891–6.View ArticlePubMedGoogle Scholar
- Jellinger KA. Neuropathology of sporadic Parkinson’s disease: Evaluation and changes of concepts. Mov Disord. 2012;27:8–30.View ArticlePubMedGoogle Scholar
- Khateeb S, Flusser H, Ofir R, Shelef I, Narkis G, Vardi G, Shorer Z, Levy R, Galil A, Elbedour K, Birk OS. PLA2G6 mutation underlies infantile neuroaxonal dystrophy. Am J Hum Genet. 2006;79:942–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Kinghorn KJ, Castillo-Quan JI, Bartolome F, Angelova PR, Li L, Pope S, Cochemé HM, Khan S, Asghari S, Bhatia KP, Hardy J, Abramov AY, Partridge L. Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction. Brain. 2015;138(Pt 7):1801–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Kordower JH, Olanow CW, Dodiya HB, Chu Y, Beach TG, Adler CH, Halliday GM, Bartus RT. Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease. Brain. 2013;136(Pt 8):2419–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Lai Y, Kim S, Varkey J, Lou X, Song JK, Diao J, Langen R, Shin YK. Nonaggregated α-synuclein influences SNARE-dependent vesicle docking via membrane binding. Biochemistry. 2014;53:3889–96.View ArticlePubMedPubMed CentralGoogle Scholar
- Malik I, Turk J, Mancuso DJ, Montier L, Wohltmann M, Wozniak DF, Schmidt RE, Gross RW, Kotzbauer PT. Disrupted membrane homeostasis and accumulation of ubiquitinated proteins in a mouse model of infantile neuroaxonal dystrophy caused by PLA2G6 mutations. Am J Pathol. 2008;172:406–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Marland JR, Hasel P, Bonnycastle K, Cousin MA. Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals. J Biol Chem. 2016;291:2080–6.PubMedPubMed CentralGoogle Scholar
- Monteiro JP, Oliveira PJ, Jurado AS. Mitochondrial membrane lipid remodeling in pathophysiology: a new target for diet and therapeutic interventions. Prog Lipid Res. 2013;52:513–28.View ArticlePubMedGoogle Scholar
- Morgan NV, Westaway SK, Morton JE, Gregory A, Gissen P, Sonek S, et al. PLA2G6, encoding a phospholipase A2, is mutated in neurodegenerative disorders with high brain iron. Nat Genet. 2006;38:752–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Morrison K, Witte K, Mayers JR, Schuh AL, Audhya A. Roles of acidic phospholipids and nucleotides in regulating membrane binding and activity of a calcium-independent phospholipase A2 isoform. J Biol Chem. 2012;287:38824–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Murakami M, Taketomi Y, Miki Y, Sato H, Hirabayashi T, Yamamoto K. Recent progress in phospholipase A2 research: from cells to animals to humans. Prog Lipid Res. 2011;50:152–92.View ArticlePubMedGoogle Scholar
- Ouberai MM, Wang J, Swann MJ, Galvagnion C, Guilliams T, Dobson CM, Welland ME. α-Synuclein senses lipid packing defects and induces lateral expansion of lipids leading to membrane remodeling. J Biol Chem. 2013;288:20883–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Paine SM, Anderson G, Bedford K, Lawler K, Mayer RJ, Lowe J, Bedford L. Pale body-like inclusion formation and neurodegeneration following depletion of 26S proteasomes in mouse brain neurones are independent of α-synuclein. PLoS One. 2013;8, e54711.View ArticlePubMedPubMed CentralGoogle Scholar
- Paisán-Ruiz C, Li A, Schneider SA, Holton JL, Johnson R, Kidd D, Chataway J, Bhatia KP, Lees AJ, Hardy J, Revesz T, Houlden H. Widespread Lewy body and tau accumulation in childhood and adult onset dystonia-parkinsonism cases with PLA2G6 mutations. Neurobiol Aging. 2012;33:814–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Paradies G, Paradies V, De Benedictis V, Ruggiero FM, Petrosillo G. Functional role of cardiolipin in mitochondrial bioenergetics. Biochim Biophys Acta. 2014;1837:408–17.View ArticlePubMedGoogle Scholar
- Peelaerts W, Bousset L, Van der Perren A, Moskalyuk A, Pulizzi R, Giugliano M, Van den Haute C, Melki R, Baekelandt V. α-Synuclein strains cause distinct synucleinopathies after local and systemic administration. Nature. 2015;522(7556):340–4.View ArticlePubMedGoogle Scholar
- Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science. 1997;276:2045–7.View ArticlePubMedGoogle Scholar
- Recasens A, Dehay B, Bové J, Carballo-Carbajal I, Dovero S, Pérez-Villalba A, Fernagut PO, Blesa J, Parent A, Perier C, Fariñas I, Obeso JA, Bezard E, Vila M. Lewy body extracts from Parkinson disease brains trigger a-synuclein pathology and neurodegeneration in mice and monkeys. Ann Neurol. 2014;75:351–62.View ArticlePubMedGoogle Scholar
- Riku Y, Ikeuchi T, Yoshino H, Mimuro M, Mano K, Goto Y, Hattori N, Sobue G, Yoshida M. Extensive aggregation of α-synuclein and tau in juvenile-onset neuroaxonal dystrophy: an autopsied individual with a novel mutation in the PLA2G6 gene-splicing site. Acta Neuropathol Commun. 2013;1:12.View ArticlePubMedPubMed CentralGoogle Scholar
- Robotta M, Gerding HR, Vogel A, Hauser K, Schildknecht S, Karreman C, Leist M, Subramaniam V, Drescher M. Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation. Chembiochem. 2014;15:2499–502.View ArticlePubMedGoogle Scholar
- Ross OA, Braithwaite AT, Skipper LM, Kachergus J, Hulihan MM, Middleton FA, et al. Genomic investigation of alpha-synuclein multiplication and parkinsonism. Ann Neurol. 2008;63:743–50.View ArticlePubMedGoogle Scholar
- Sengoku R, Saito Y, Ikemura M, Hatsuta H, Sakiyama Y, Kanemaru K, Arai T, Sawabe M, Tanaka N, Mochizuki H, Inoue K, Murayama S. Incidence and extent of Lewy body-related alpha-synucleinopathy in aging human olfactory bulb. J Neuropathol Exp Neurol. 2008;67:1072–83.View ArticlePubMedGoogle Scholar
- Shen J, Du T, Wang X, Duan C, Gao G, Zhang J, Lu L, Yang H. α-Synuclein amino terminus regulates mitochondrial membrane permeability. Brain Res. 2014;1591:14–26.View ArticlePubMedGoogle Scholar
- Shinzawa K, Sumi H, Ikawa M, Matsuoka Y, Okabe M, Sakoda S, Tsujimoto Y. Neuroaxonal dystrophy caused by group VIA phospholipase A2 deficiency in mice: a model of human neurodegenerative disease. J Neurosci. 2008;28:2212–20.View ArticlePubMedGoogle Scholar
- Shvadchak VV, Yushchenko DA, Pievo R, Jovin TM. The mode of α-synuclein binding to membranes depends on lipid composition and lipid to protein ratio. FEBS Lett. 2011;585:3513–9.View ArticlePubMedGoogle Scholar
- Snead D, Eliezer D. Alpha-synuclein function and dysfunction on cellular membranes. Exp Neurobiol. 2014;23:292–313.View ArticlePubMedPubMed CentralGoogle Scholar
- Sumi H, Nagano S, Fujimura H, Kato S, Sakoda S. Inverse correlation between the formation of mitochondria-derived vacuoles and Lewy-body-like hyaline inclusions in G93A superoxide-dismutase-transgenic mice. Acta Neuropathol. 2006;112:52–63.View ArticlePubMedGoogle Scholar
- Sumi-Akamaru H, Beck G, Kato S, Mochizuki H. Neuroaxonal dystrophy in PLA2G6 knockout mice. Symposium: Neurodegeneration with Brain Iron Accumulation. Neuropathology. 2015;35:289–302.View ArticlePubMedGoogle Scholar
- Wakabayashi K, Tanji K, Odagiri S, Miki Y, Mori F, Takahashi H. The Lewy body in Parkinson’s disease and related neurodegenerative disorders. Mol Neurobiol. 2013;47:495–508.View ArticlePubMedGoogle Scholar
- Wilson RS, Nag S, Boyle PA, Hizel LP, Yu L, Buchman AS, Shah RC, Schneider JA, Arnold SE, Bennett DA. Brainstem aminergic nuclei and late-life depressive symptoms. JAMA Psychiatry. 2013;70:1320–8.View ArticlePubMedGoogle Scholar
- Yamada M, Iwatsubo T, Mizuno Y, Mochizuki H. Overexpression of a-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphorylation of a-synuclein and activation of caspase-9: resemblance to pathogenetic changes in Parkinson’s disease. J Neurochem. 2004;91:451–61.View ArticlePubMedGoogle Scholar
- Yin H, Zhu M. Free radical oxidation of cardiolipin: chemical mechanisms, detection and implication in apoptosis, mitochondrial dysfunction and human diseases. Free Radic Res. 2012;46:959–74.View ArticlePubMedGoogle Scholar
- Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao Z, Zhang X, Zhao C, Choi J, Shi J, Song K, Turk J, Ma ZA. Protection of pancreatic beta-cells by group VIA phospholipase A (2)-mediated repair of mitochondrial membrane peroxidation. Endocrinology. 2010;151:3038–48.View ArticlePubMedPubMed CentralGoogle Scholar
- Zigoneanu IG, Yang YJ, Krois AS, Haque E, Pielak GJ. Interaction of α-synuclein with vesicles that mimic mitochondrial membranes. Biochim Biophys Acta. 2012;1818:512–9.View ArticlePubMedPubMed CentralGoogle Scholar