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α-Synuclein is differentially expressed in mitochondria from different rat brain regions and dose-dependently down-regulates complex I activity
Neuroscience Letters 454 (2009) 187–192
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␣-Synuclein is differentially expressed in mitochondria from different rat brain regions and dose-dependently down-regulates complex I activity Guangwei Liu a , Chunyan Zhang b , Juanjuan Yin a , Xin Li a , Furong Cheng a , Yaohua Li a , Hui Yang b , Kenji Uéda a,d , Piu Chan a , Shun Yu a,c,∗ a Department of Neurobiology and Sino-Japan Joint Laboratory for Neurodegenerative Diseases, Key Laboratory of Neurodegenerative Diseases (Capital Medical University), Ministry of Education, Xuanwu Hospital of China Capital Medical University, 100053 Beijing, China b Beijing Institute for Neuroscience, Capital Medical University of Medical Sciences, 100069 Beijing, China c Institute for Hypoxia Medicine, Xuanwu Hospital of China Capital Medical University, 100053 Beijing, China d Division of Psychobiology, Tokyo Institute of Psychiatry, Tokyo 156-8585, Japan
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Article history: Received 5 August 2008 Received in revised form 5 February 2009 Accepted 25 February 2009 Keywords: Parkinson’s disease ␣-Synuclein Mitochondrion Complex I Brain
a b s t r a c t ␣-Synuclein (␣-Syn) abnormality and mitochondrial deficiency are two major changes in the brain of patients with Parkinson’s disease (PD). A link between ␣-Syn and mitochondria in PD has been demonstrated by a recent study showing that accumulation of ␣-Syn in the mitochondria from the PD-vulnerable brain regions was associated with decreased complex I activity of these mitochondria. In this study, we examined the normal expressions of ␣-Syn in mitochondria from different regions of the rat brain. We showed that ␣-Syn was highly expressed in the mitochondria in olfactory bulb, hippocampus, striatum, and thalamus, where the cytosolic ␣-Syn was also rich. However, the cerebral cortex and cerebellum were two exceptions, which contained rich cytosolic ␣-Syn but very low or even undetectable levels of mitochondrial ␣-Syn. The close quantitative association between mitochondrial and cytosolic ␣-Syn in most brain regions, suggests that the concentration of cytosolic ␣-Syn may determine the amount of ␣-Syn in mitochondria. This is partially supported by the in vitro experiment showing that incubation of ␣-Syn with endogenous ␣-Syn-undetectable cerebellar mitochondria caused a dose-dependent transport of ␣-Syn to the mitochondria. Moreover, we found that the inhibitory effect of ␣-Syn on complex I activity of mitochondrial respiratory chain was also dose-dependent. These results suggest that ␣-Syn in mitochondria is differentially expressed in different brain regions and the background levels of mitochondrial ␣-Syn may be a potential factor affecting mitochondrial function and predisposing some neurons to degeneration. © 2009 Elsevier Ireland Ltd. All rights reserved.
A body of evidence supports that ␣-synuclein (␣-Syn), a neuronal protein originally found in Alzheimer’s disease brains [26], is implicated in the pathogenesis of both autosomal-dominant, familial [19,10,29] and sporadic forms of Parkinson’s disease (PD), as well as in dementia with Lewy bodies [24,1]. However, the mechanism for the role of this protein in PD pathogenesis remains poorly understood. While numerous studies suggest that ␣-Syn aggregates are toxic to dopaminergic neurons [13], there is also report showing that overexpression of this protein can damage dopaminergic neurons even without apparent evidence for the formation of ␣-Syn aggregates [27]. Mitochondrial dysfunction is also shown to implicate in the pathogenesis of PD. For example, complex I deficiency were
∗ Corresponding author at: Department of Neurobiology, Beijing Institute of Geriatrics, Xuanwu Hospital of China Capital Medical University, 45# Changchun Street, Beijing 100053, China. Tel.: +86 10 8319 8890; fax: +86 10 8316 1294. E-mail address: [email protected] (S. Yu). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.02.056
observed in the substantia nigra of PD patients [22]; cybrid cell lines containing a normal nuclear genome but mitochondrial DNA from PD patients caused altered complex I activity, abnormal mitochondrial morphology, and impaired mitochondrial energy-dependent activities [25]. Since ␣-Syn abnormality and mitochondrial dysfunction are both associated with PD, an interaction between ␣-Syn and mitochondria may underlie the PD pathogenesis. Indeed, a few lines of evidence suggest that ␣-Syn and mitochondria interact with each other [30]. On one hand, overexpression of ␣-Syn, which may lead to cytoplasmic accumulation of ␣-Syn, was shown to impair mitochondrial function [7]. On the other hand, an impaired mitochondrial function was shown to induce increased ␣-Syn expression and formation of ␣-Syn inclusions [11]. A direct link between ␣-Syn and mitochondria in PD has been demonstrated by a recent study showing that mitochondria of PD vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of ␣-Syn and decreased complex I activity [2]. This result suggests that in disease conditions the levels of
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mitochondrial ␣-Syn are varied in different brain regions. However, whether in normal conditions mitochondrial ␣-Syn is also differentially expressed in different brain regions remains uninvestigated. The purpose of the present study is to investigate the normal expressions of mitochondrial ␣-Syn in various brain regions and the association of mitochondrial ␣-Syn with cytosolic ␣-Syn. In addition, the effects of ␣-Syn concentration on the complex I activity of mitochondrial respiratory chain were also examined. Five male Wistar rats of 8 weeks purchased from Beijing Vital River Laboratory Animal Co., Ltd. were used to examine the expressions of ␣-Syn in mitochondria and cytoplasm in various brain regions. After deeply anaesthetized, the brains of the rats were removed from the skulls, and the mitochondrial and cytosolic fractions were isolated from the brain tissues according to the procedures described previously with slight modifications [23]. In brief, the tissues dissected from different brain regions were homogenized in the “mitochondria isolation buffer (MIB)” containing 0.32 M sucrose, 1 mM EDTA (K+ salt), and 10 mM Tris–HCl, pH 7.4. The homogenates were centrifuged at 1330 × g for 5 min. The supernatant was preserved, and the pellet was suspended and re-centrifuged as above. Both supernatants were then mixed and centrifuged at 21,250 × g for 10 min. The supernatant was used as cytosolic fraction. The pellet was resuspended in 15% Percoll (Bioshop), and 3 ml of this suspension was laid on two preformed layers consisting of 3.5 ml of 26% Percoll and 3.5 ml of 40% Percoll. The gradient was centrifuged for 5 min at 30,700 × g. The fraction accumulating near the interface of the two lower layers was collected and diluted 1:4 with MIB. The mixture was centrifuged twice at 16,700 × g for 10 min, producing a pellet which contained mitochondria. Protein concentration was determined using BCA Protein Assay Kit (Pierce). Western blot analysis was used to examine the purity of the mitochondrial and cytosolic fractions as well as the expression levels of ␣-Syn. 75 g of proteins were separated by 15% SDS-PAGE, and transferred onto a PVDF membrane, which was then incubated overnight at 4 ◦ C with different primary antibodies (3D5 and 2E3 mouse monoclonal anti-␣-Syn antibodies [28], EQV1, MDV2, and PQE3 rabbit polyclonal anti-␣-Syn antibodies [1], rabbit polyclonal anti-lactate dehydrogenase (LDH) antibody (Rockland Corporation), rabbit polyclonal anti--actin antibody (Santa Cruz), rabbit polyclonal anti-prohibitin antibody (Biomeda), and rabbit polyclonal anti-synaptophysin antibody (Santa Cruz), followed by incubation with horseradish peroxidase-conjugated goat-antimouse IgG or horseradish peroxidase-conjugated goat-anti-rabbit IgG (Vector Laboratories) (1:5000 in TBST). Immunoreactive bands were revealed by enhanced chemiluminescence (Pierce). Film autoradiograms were analyzed and quantified by computerassisted densitometry using Quantity One (Bio-Rad) system to estimate the relative amount of ␣-Syn. To investigate the effect of ␣-Syn concentration on its mitochondrial translocation, isolated mitochondria were re-suspended in the respiratory buffer (300 mM mannitol, 10 mM KH2 PO4 , 5 mM MgCl2 , pH 7.2 at 37 ◦ C) [20], and were incubated with different concentrations of recombinant human ␣-Syn at 37 ◦ C for different time points. The mitochondria were pelleted by centrifuging at 12,000 × g at 4 ◦ C for 10 min and the reaction was stopped by washing the pellet twice with 500 l of MIB. The mitochondria were re-suspended in an adequate volume (20 l) of MIB for evaluation of the amount of ␣-Syn using Western blot analysis. In some experiments, mitochondria treated by ␣-Syn were fractioned by incubation with digitonin (Sigma) at a concentration of 0.5 mg/mg protein at 4 ◦ C for 15 min [6]. The samples were then diluted using 3 volumes of the respiratory buffer. The inner membrane fraction was obtained as a pellet by centrifugation at 12,500 × g for 15 min, and the supernatant was further centrifuged at 100,000 × g for 60 min to obtain the outer membrane fraction. The purity of the inner membrane fraction
was analyzed using antibodies against an outer membrane protein VDAC1 (Santa Cruz) and an inner membrane protein prohibitin. The presence of ␣-Syn in different membrane fractions was examined using EQV1 anti-␣-Syn antibody. To study the effect of ␣-Syn concentration on complex I activity, the isolated cerebellar mitochondria were incubated with different concentrations of ␣-Syn, fractured by rapid freezing–thawing and measured spectrophometrically for NADH-CoQ oxidoreductase (complex I) activity [16]. The precise localization of ␣-Syn in mitochondria was also examined using pre-embedding immunogold electron microscopy. In this experiment, Wistar rats deeply anesthetized were perfusionfixed with a fixative containing 4% paraformaldehyde and 0.35% glutaraldehyde, and the brains were removed and postfixed with a fixative containing 4% paraformaldehyde, followed by rinse in 20% sucrose. 50 m of brain sections were cut and the procedures for the pre-embedding immunogold electron microscopy were as same as before [28]. All the data are expressed as mean ± SD. Student t-test was used to evaluate the statistical significance between control and each experimental group. Western blot analysis showed that an antibody against a mitochondrial specific protein prohibitin [8], only revealed a 30 kDa band, which is identical to the molecular size of this protein, in the mitochondrial fraction but not in the cytosolic fraction. Conversely, an antibody recognizing the cytoplasmic protein LDH, only detected a 36 kDa protein, which is compatible in molecular size with LDH, in the cytosolic fraction but not in the mitochondrial fraction. Moreover, the mitochondrial fraction reacted very faintly with anti-synaptophysin antibody (Fig. 1A). The presence of ␣-Syn in the mitochondrial faction were examined using five anti-␣-Syn antibodies, which recognize N-terminal (MDV2), NAC domain (EQV1), and C-terminal (PQE3, 3D5, and 2E3) of ␣-Syn [1,28]. Each of the five antibodies revealed a 19 kDa band in the mitochondrial fraction, which was identical in molecular size to the cytosolic ␣-Syn and the recombinant ␣-Syn (Fig. 1B). Pre-absorption of the antibodies with excess recombinant ␣-Syn abolished the bands (data not shown). Pre-embedding immunogold electron microscopic observation using homemade 3D5 mouse monoclonal anti-␣-Syn antibody revealed that mitochondria with positive gold particles for ␣-Syn were localized in the neurons. Photomicrographs with higher magnification showed that the positive gold particles were localized on the outer membrane, the inner membrane, and the intermembrane space of the ␣-Syn-positive mitochondria. Except the membrane of mitochondria, some ␣-Syn-positive gold particles could be also observed in the matrix of the mitochondria (Fig. 1C). The mitochondrial ␣-Syn was differentially expressed in different brain regions. The levels of the mitochondrial ␣-Syn were relatively higher in olfactory bulb, hippocampus, striatum, and thalamus, where the levels of the cytosolic ␣-Syn were also high. In brain stem, the ␣-Syn levels were low in both mitochondrial and cytosolic fractions. However, the cerebral cortex and cerebellum were among the exceptions, which contained relatively rich cytosolic ␣-Syn, but very low or even undetectable levels of mitochondrial ␣-Syn (Fig. 2A and B). Incubation of the cerebellar mitochondria, where there was no detectable endogenous ␣-Syn, with different concentrations of purified recombinant ␣-Syn lead to dose-dependent increase in the amount of ␣-Syn accumulated in the mitochondria during observed 30 min (Fig. 3A), suggesting that ␣-Syn transport onto mitochondria depends on its concentration. The transport was rapid, with the earliest detection of ␣-Syn in the mitochondrial fraction within 5 min. Further extending incubation time did not apparently increase the amount of ␣-Syn in the mitochondrial fraction (Fig. 3B). To rule out the possibility that the ␣-Syn detected in the mitochondrial fraction was because of non-specific binding of the
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Fig. 1. Mitochondrial ␣-Syn detected by Western blot. (A) A mitochondrial protein prohibitin was detected only in the mitochondrial fraction (left), while a cytoplasmic protein LDH was detected only in the cytosolic fraction (middle). In addition, the mitochondrial fraction reacted strongly with anti-prohibitin antibody but very faintly with anti-synaptophysin antibody. Mt: mitochondrial fraction; Cyto: cytosolic fraction; PHB: prohibitin; LDH: lactate dehydrogenase; SYP: synaptophysin. (B) A 19 kDa protein was detected by five anti-␣-Syn-antibodies in the mitochondrial fractions, which share the same molecular size with the cytoplasmic ␣-Syn and the recombinant ␣-Syn; C: mitochondria in the cerebral cortex (A), thalamus (B), and substantia nigra (C). All the mitochondria contain ␣-Syn-positive gold particles, which are different in density. Gold particles can be observed in the mitochondrial matrix (white arrows), outer membrane (black arrow head), inner membrane (white arrow head), and intermembrane space (black arrows) Con: control; Cyto: cytoplasmic fraction; Mt: mitochondrial fraction; M: mitochondria. Bar: 20 nm in (a) and (b), 50 nm in (c).
protein to the surface of the outer membrane, we used digitonin to disrupt the outer membrane of the mitochondria and separate it from the inner membrane. Western blot analysis showed that the inner membrane protein was not identified by an antibody to the outer membrane protein VDAC1 (Fig. 3C) but recognized by an antibody to the inner membrane protein prohibitin (Fig. 3D). The transported ␣-Syn was shown to be mainly localized in the inner membrane fraction, with only small amount present in the outer membrane fraction (Fig. 3D). In fact, a recently study has shown that there is a direct interaction between ␣-Syn and prohibitin [15].
Since ␣-Syn was shown to inhibit complex I activity and the levels of mitochondrial ␣-Syn were different in various brain regions, it is necessary to know the association of ␣-Syn concentration with the activity of the enzyme. For this purpose, isolated cerebellar mitochondria were incubated with different concentrations of ␣-Syn (1 pM, 102 pM, and 104 pM, which is equal to 0.002–20.000 pmol/mg mitochondria). The complex I activity was shown to be inhibited even when ␣-Syn concentration was as low as 1 pM; increasing ␣-Syn concentration caused a further reduction in the activity of complex I (Fig. 4).
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Fig. 3. ␣-Syn transport into mitochondria. Isolated cerebellar mitochondria were incubated with different concentrations of recombinant ␣-Syn. The amount of ␣Syn in mitochondria was increased with the enhancement of ␣-Syn concentration (A). The mitochondrial transport of ␣-Syn was time-independent, with the maximal accumulation of ␣-Syn in the mitochondria found within 5 min (B). An outer membrane mitochondrial protein VDAC1 was detected only in the outer membrane fraction but not in the inner membrane fraction (C). Western blot analysis of the digitonin-disrupted mitochondria showed that ␣-Syn was mainly present in the inner membrane fraction, with only small amount in the outer and intermembrane fraction (D). IM: inner membrane fraction; OM: outer membrane fraction. Fig. 2. Expressions of ␣-Syn in cytosolic and mitochondria in various brain regions. (A) Expressions of cytosolic ␣-Syn in different brain regions. Upper panel: representative immunoblots. Lower panel: bar graphs showing the relative levels of the cytosolic ␣-Syn; (B) expressions of mitochondrial ␣-Syn in different brain regions. Upper panel: representative immunoblots. Lower panel: bar graphs showing the relative levels of the mitochondrial ␣-Syn. Con: recombinant human ␣-Syn; OB: olfactory bulb; HIP: hippocampus; CX: cortex; STR: striatum; Thal: thalamus; CB: cerebellum; BS: brain stem.
The presence of ␣-Syn in mitochondria has been reported in neurons of human [2] and mouse [12] brains as well as in cells overexpressing ␣-Syn [18]. However, the precise localization of this protein in mitochondria remains elusive. Immunoelectron microscopic study on the human brain showed that ␣-Syn-positive gold particles were present on the outer membrane and in the matrix of mitochondria in the neurons [2]. In the mouse brain, however, ␣Syn-positive gold particles were observed only in the mitochondrial membrane of the neurons [12]. In HEK 293 cells overexpressing wild type ␣-Syn, ␣-Syn was shown to locate only to the outer mitochondrial membrane, while in SY5Y cells overexpressing A53T ␣-Syn, ␣-Syn appeared to be localized in both the mitochondrial membrane and matrix [18]. By using pre-embedding immunogold electron microscopic method, which allows higher resolution of the antigen locating sites on well preserved ultrastructures, we also observed that ␣-Syn-immunoreactive mitochondria were localized in the neurons, which is identical to our previous results that ␣-Syn was only expressed in the neurons of the rat brain [28,30]. In the mitochondria, ␣-Syn-positive gold particles were localized in both the membrane and matrix. The membrane bound gold particles could be found on the outer membrane and the inner membrane, or in the intermembrane space of the mitochondria, indicating that ␣-Syn may have different localizations in the mitochondrial membrane. The discrepancy for the precise mitochondrial localization of ␣-Syn reported by different authors is not clear. One possible reason may be due to the difference of the animal species investigated.
Although previous studies on the human and mouse brains showed that ␣-Syn could be found in the mitochondria of both dopaminergic neurons and other neurons, and PD-vulnerable striatum and substantia nigra contained much more mitochondrial ␣-Syn than cerebellum, the relative levels of mitochondrial ␣-Syn in different regions of the normal brain has not yet systemically investigated. In the present study, Percoll density gradient centrifugation [23] was used to isolate mitochondria from rat brains. This method allows preparation of free non-synaptosomal mitochondria with little residual contamination by synaptosomes or myelin. The mitochondrial fraction reacted strongly with the antibody to mitochondrial protein prohibitin but very faintly with the
Fig. 4. Effect of ␣-Syn on complex I activity of mitochondrial respiratory chain. Different concentrations of recombinant ␣-Syn were incubated with the isolated cerebellar mitochondria. After washing out the free ␣-Syn, the mitochondria were fractured by rapid freezing and thawing, and the complex I activity was measured using spectrophotometric analysis of the kinetics of NADH reduction at 340 nm. The complex I activity decreased in a dose-dependent manner (* p < 0.05 vs. control).
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antibody to synaptic protein synaptophysin, suggesting that the mitochondrial fraction isolated with this method mainly contained free mitochondria with only little contamination by synaptosomes. Western blot analysis of the mitochondrial fractions isolated from different brain regions showed that ␣-Syn could be detected in mitochondria from many brain regions, including olfactory bulb, striatum, hippocampus, thalamus, cerebral cortex, and brain stem. However, the levels of mitochondrial ␣-Syn were quite different among the brain regions examined. ␣-Syn were highly expressed in the mitochondria isolated from the olfactory bulb, striatum, hippocampus, and thalamus, where the cytosolic ␣-Syn was also rich. However, in the cerebral cortex and cerebellum, although there was rich cytosolic ␣-Syn, the mitochondrial ␣-Syn was very low or even undetectable. The various levels of mitochondrial ␣-Syn measured in different brain regions may mainly reflect the difference in the ␣-Syn expression in the mitochondria of neurons, since previous immunohistochemical and immunoelectron microscopic studies using the same antibodies showed that ␣-Syn was localized only in the neurons of the rat brain [28,30]. The previous study on ␣-Syn-gene transfected dopaminergic neurons showed that increased cytoplamic ␣-Syn was accompanied by increased mitochondrial ␣-Syn [18]. In the present study, by using isolated rat brain mitochondria to incubate with recombinant ␣-Syn, we observed that the amount of ␣-Syn transported into the mitochondria increased with the enhancement of ␣-Syn concentration. By separating the inner membrane fraction from the outer membrane fraction, we demonstrated that the ␣-Syn transported into mitochondria was mainly accumulated in the inner membrane, ruling out the possibility for the non-specific binding of this protein to the surface of the outer membrane. The dose-dependent ␣-Syn transport to the isolated mitochondria is consistent with the observation on the in vivo brain that the mitochondrial ␣-Syn is quantitatively associated with the cytosolic ␣-Syn in most brain regions except cerebral cortex and cerebellum, indicating that the cytoplasmic concentration of ␣-Syn may determine its amount in the mitochondria in these brain regions. It is not clear why in the cerebral cortex and cerebellum the mitochondrial ␣-Syn are not quantitatively associated with the cytosolic ␣-Syn. One possible reason may be that the mechanism controlling the transport of ␣Syn from cytoplasm to mitochondria in these two brain regions is different from that in other brain regions, which is an issue remains to be addressed. Since ␣-Syn abnormality and mitochondrial complex I deficiency are both implicated in PD pathogenesis, ␣-Syn localization in mitochondria may suggest a functional link between ␣-Syn and complex I. In human dopaminergic neurons, ␣-Syn accumulation in the mitochondria was shown to cause reduced mitochondrial complex I activity [2]. Our study on the isolated cerebellar mitochondria confirmed this, indicating that the inhibition of complex I activity by ␣-Syn was dose-dependent, with the minimal effective concentration being as low as 1 pM. The effect of ␣-Syn on complex I activity of mitochondria may have special physiological and pathophysiological meaning. It is known that oxidative phosphorylation on mitochondria produces ATP for cell functioning, which leads to large amounts of generation of free radicals detrimental to mitochondria and other cellular components, which are thought to be the main reason for aging [4]. We speculate that proper suppression of complex I activity by ␣-Syn is beneficial to reducing free radical production and mitochondrial damage. However, in abnormal conditions, abnormal accumulation of ␣-Syn in mitochondria may inhibit complex I activity to a greater extent, leading to reduced ATP production, increased ROS generation, and accelerated neurodegeneration [2]. To support this, both PD and amyotrophic lateral sclerosis (ALS) are characterized by impairments of mitochondria and increased accumulation of ␣-Syn in the neurons of some vulnerable regions
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in the brain or the spinal cord [14,21,3]. Some experiments on animals and cells provide additional evidence for the deleterious role of ␣-Syn in mitochondrial function in abnormal conditions. For example, ␣-Syn overexpression made mitochondria more vulnerable to complex I inhibitor [17]; interaction of ␣-Syn with mitochondria caused release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components [18]; mice lacking ␣-Syn are resistant to mitochondrial toxins [9]; the prolonged mitochondrial complex I inhibition due to continuous MPTP infusion is alleviated when ␣-Syn is removed [5]. Taken together, the present results that mitochondrial ␣-Syn is differentially expressed in different brain neurons and dosedependently down-regulates the complex I activity suggests that the background levels of mitochondrial ␣-Syn may be a potential factor affecting mitochondrial function and predisposing some neurons to degeneration. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30570646 and 30430280), Ministry of Science and Technology of China (2006CB500701, 2006AA02A408), Natural Science Foundation of Beijing (7022011), and Japan Society for the Promotion of Science (JSPS) (C11680774 and B 14380363) and Funding Project for Academic Human Resources Development in Institutions of Higher Learning under Jurisdiction of Beijing Municipality PHR (IHLB). References [1] K. Arima, K. Uéda, N. Sunohara, S. Hirai, Y. Izumiyama, H. Tonozuka-Uehara, M. Kawai, Immunoelectron microscopic demonstration of NACP/alpha-synucleinepitopes on the filamentous component of Lewy bodies in Parkinson’s disease and in dementia with Lewy bodies, Brain Res. 808 (1998) 93–100. [2] L. Devi, V. Raghavendran, B.M. Prabhu, N.G. Avadhani, H.K. Anandatheerthavarada, Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain, J. Biol. Chem. 283 (2008) 9089–9100. [3] M.J. Doherty, T.D. Bird, J.B. Leverenz, Alpha-synuclein in motor neuron disease: an immunohistologic study, Acta. Neuropathol. 107 (2004) 169–175. [4] E. Dufour, N.G. Larsson, Understanding aging: revealing order out of chaos, Biochim. Biophys. Acta 1658 (2004) 122–132. [5] F. Fornai, O.M. Schlüter, P. Lenzi, M. Gesi, R. Ruffoli, M. Ferrucci, G. Lazzeri, C.L. Busceti, F. Pontarelli, G. Battaglia, A. Pellegrini, F. Nicoletti, S. Ruggieri, A. Paparelli, T.C. Südhof, Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alphasynuclein, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3413–3418. [6] T. Gotow, M. Shibata, S. Kanamori, O. Tokuno, Y. Ohsawa, N. Sato, K. Sahara, Y. Yayoi, T. Watanabe, J.F. Leterrier, M. Linden, E. Kominami, Y. Uchiyama, Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells, Cell Death Differ. 7 (2000) 666–674. [7] L.J. Hsu, Y. Sagara, A. Arroyo, E. Rockenstein, A. Sisk, M. Mallory, J. Wong, T. Takenouchi, M. Hashimoto, E. Masliah, Alpha-synuclein promotes mitochondrial deficit and oxidative stress, Am. J. Patho. 157 (2000) 401–410. [8] E. Ikonen, K. Fiedler, R.G. Parton, K. Simons, Prohibitin, an antiproliferative protein, is localized to mitochondria, FEBS Lett. 358 (1995) 273–277. [9] P. Klivenyi, D. Siwek, G. Gardian, L. Yang, A. Starkov, C. Cleren, R.J. Ferrante, N.W. Kowall, A. Abeliovich, MF. Beal, Mice lacking alpha-synuclein are resistant to mitochondrial toxins, Neurobiol. Dis. 21 (2006) 541–548. [10] R. Krüger, W. Kuhn, T. Müller, D. Woitalla, M. Graeber, S. Kösel, H. Przuntek, J.T. Epplen, L. Schöls, O. Riess, Ala30Pro mutation in the gene encoding alphasynuclein in Parkinson’s disease, Nat. Genet. 18 (1998) 106–108. [11] H.J. Lee, S.Y. Shin, C. Choi, Y.H. Lee, S.J. Lee, Formation and removal of alphasynuclein aggregates in cells exposed to mitochondrial inhibitors, J. Biol. Chem. 277 (2002) 5411–5417. [12] W.W. Li, R. Yang, J.C. Guo, H.M. Ren, X.L. Zha, J.S. Cheng, D.F. Cai, Localization of alpha-synuclein to mitochondria within midbrain of mice, Neuroreport 18 (2007) 1543–1546. [13] Q.L. Ma, P. Chan, M. Yoshii, K. Uéda, ␣-Synuclein aggregation and neurodegenerative diseases, J. Alzheimers Dis. 5 (2003) 139–148. [14] L.J. Martin, Transgenic mice with human mutant genes causing Parkinson’s disease and amyotrophic lateral sclerosis provide common insight into mechanisms of motor neuron selective vulnerability to degeneration, Rev. Neurosci. 18 (2007) 115–136. [15] M.A. McFarland, C.E. Ellis, S.P. Markey, R.L. Nussbaum, Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions, Mol. Cell Proteomics 7 (2008) 2123–2137.
192
G. Liu et al. / Neuroscience Letters 454 (2009) 187–192
[16] V. Mildaziene, Z. Nauciene, R. Baniene, J. Grigiene, Multiple effects of 2,2 ,5,5 tetrachlorobiphenyl on oxidative phosphorylation in rat liver mitochondria, Toxicol. Sci. 65 (2002) 220–227. [17] M. Orth, S.J. Tabrizi, A.H. Schapira, J.M. Cooper, Alpha-synuclein expression in HEK293 cells enhances the mitochondrial sensitivity to rotenone, Neurosci. Lett. 351 (2003) 29–32. [18] M.S. Parihar, A. Parihar, M. Fujita, M. Hashimoto, P. Ghafourifar, Mitochondrial association of alpha-synuclein causes oxidative stress, Cell Mol. Life Sci. 65 (2008) 1272–1284. [19] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, I.G. Di, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045–2047. [20] Z. Roland, M. Christophe, B. Aldo, A.E.B. Alberto, T. Jean-Paul, Resveratrolinduced limitation of dysfunction of mitochondria isolated from rat brain in an anoxia-reoxygenation model, Life Sci. 71 (2002) 3091–3108. [21] S. Sasaki, Y. Horie, M. Iwata, Mitochondrial alterations in dorsal root ganglion cells in sporadic amyotrophic lateral sclerosis, Acta. Neuropathol. 114 (2007) 633–639. [22] A.H. Schapira, J.M. Cooper, D. Dexter, J.B. Clark, P. Jenner, C.D. Marsden, Mitochondrial complex I deficiency in Parkinson’s disease, J. Neurochem. 54 (1990) 823–827. [23] N.R. Sims, Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation, J. Neurochem. 55 (1990) 698–707.
[24] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowsky, R. Jakes, M. Goedert, Alpha-synuclein in Lewy bodies, Nature 388 (1997) 839–840. [25] R.H. Swerdlow, J.K. Parks, J.N. Davis 2nd, D.S. Cassarino, P.A. Trimmer, L.J. Currie, J. Dougherty, W.S. Bridges, J.P. Bennett Jr., G.F. Wooten, W.D. Parker, Matrilineal inheritance of complex I dysfunction in a multigenerational Parkinson’s disease family, Ann. Neurol. 44 (1998) 873–881. [26] K. Uéda, H. Fukushima, E. Masliah, Y. Xia, A. Iwai, M. Yoshimoto, D.A. Otero, J. Kondo, Y. Ihara, T. Saitoh, Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 11282–11286. [27] J. Xu, S.Y. Kao, F.J.S. Lee, W. Song, L.W. Jin, B.A. Yankner, Dopamine-dependent neurotoxicity of ␣-synuclein: A mechanism for selective neurodegeneration in Parkinson disease, Nat. Med. 8 (2002) 600–606. [28] S. Yu, X. Li, G. Liu, J. Han, C. Zhang, Y. Li, S. Xu, C. Liu, Y. Gao, H. Yang, K. Uéda, P. Chan, Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody, Neuroscience 145 (2007) 539–555. [29] J.J. Zarranz, J. Alegre, J.C. Gomez-Esteban, E. Lezcano, R. Ros, L.Vidal AmpueroI, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E. GomezTortosa, T. del Ser, D.G. Munoz, J.G. de Yebenes, The new mutation, E46K, of alphasynuclein causes Parkinson and Lewy body dementia, Ann. Neurol. 55 (2004) 164–173. [30] L. Zhang, C. Zhang, Y. Zhu, Q. Cai, P. Chan, K. Uéda, S. Yu, H. Yang, Semiquantitative analysis of alpha-synuclein in subcellular pools of rat brain neurons: an immunogold electron microscopic study using a C-terminal specific monoclonal antibody, Brain Res. 1244 (2008) 40–52.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
␣-Synuclein is differentially expressed in mitochondria from different rat brain regions and dose-dependently down-regulates complex I activity Guangwei Liu a , Chunyan Zhang b , Juanjuan Yin a , Xin Li a , Furong Cheng a , Yaohua Li a , Hui Yang b , Kenji Uéda a,d , Piu Chan a , Shun Yu a,c,∗ a Department of Neurobiology and Sino-Japan Joint Laboratory for Neurodegenerative Diseases, Key Laboratory of Neurodegenerative Diseases (Capital Medical University), Ministry of Education, Xuanwu Hospital of China Capital Medical University, 100053 Beijing, China b Beijing Institute for Neuroscience, Capital Medical University of Medical Sciences, 100069 Beijing, China c Institute for Hypoxia Medicine, Xuanwu Hospital of China Capital Medical University, 100053 Beijing, China d Division of Psychobiology, Tokyo Institute of Psychiatry, Tokyo 156-8585, Japan
a r t i c l e
i n f o
Article history: Received 5 August 2008 Received in revised form 5 February 2009 Accepted 25 February 2009 Keywords: Parkinson’s disease ␣-Synuclein Mitochondrion Complex I Brain
a b s t r a c t ␣-Synuclein (␣-Syn) abnormality and mitochondrial deficiency are two major changes in the brain of patients with Parkinson’s disease (PD). A link between ␣-Syn and mitochondria in PD has been demonstrated by a recent study showing that accumulation of ␣-Syn in the mitochondria from the PD-vulnerable brain regions was associated with decreased complex I activity of these mitochondria. In this study, we examined the normal expressions of ␣-Syn in mitochondria from different regions of the rat brain. We showed that ␣-Syn was highly expressed in the mitochondria in olfactory bulb, hippocampus, striatum, and thalamus, where the cytosolic ␣-Syn was also rich. However, the cerebral cortex and cerebellum were two exceptions, which contained rich cytosolic ␣-Syn but very low or even undetectable levels of mitochondrial ␣-Syn. The close quantitative association between mitochondrial and cytosolic ␣-Syn in most brain regions, suggests that the concentration of cytosolic ␣-Syn may determine the amount of ␣-Syn in mitochondria. This is partially supported by the in vitro experiment showing that incubation of ␣-Syn with endogenous ␣-Syn-undetectable cerebellar mitochondria caused a dose-dependent transport of ␣-Syn to the mitochondria. Moreover, we found that the inhibitory effect of ␣-Syn on complex I activity of mitochondrial respiratory chain was also dose-dependent. These results suggest that ␣-Syn in mitochondria is differentially expressed in different brain regions and the background levels of mitochondrial ␣-Syn may be a potential factor affecting mitochondrial function and predisposing some neurons to degeneration. © 2009 Elsevier Ireland Ltd. All rights reserved.
A body of evidence supports that ␣-synuclein (␣-Syn), a neuronal protein originally found in Alzheimer’s disease brains [26], is implicated in the pathogenesis of both autosomal-dominant, familial [19,10,29] and sporadic forms of Parkinson’s disease (PD), as well as in dementia with Lewy bodies [24,1]. However, the mechanism for the role of this protein in PD pathogenesis remains poorly understood. While numerous studies suggest that ␣-Syn aggregates are toxic to dopaminergic neurons [13], there is also report showing that overexpression of this protein can damage dopaminergic neurons even without apparent evidence for the formation of ␣-Syn aggregates [27]. Mitochondrial dysfunction is also shown to implicate in the pathogenesis of PD. For example, complex I deficiency were
∗ Corresponding author at: Department of Neurobiology, Beijing Institute of Geriatrics, Xuanwu Hospital of China Capital Medical University, 45# Changchun Street, Beijing 100053, China. Tel.: +86 10 8319 8890; fax: +86 10 8316 1294. E-mail address: [email protected] (S. Yu). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.02.056
observed in the substantia nigra of PD patients [22]; cybrid cell lines containing a normal nuclear genome but mitochondrial DNA from PD patients caused altered complex I activity, abnormal mitochondrial morphology, and impaired mitochondrial energy-dependent activities [25]. Since ␣-Syn abnormality and mitochondrial dysfunction are both associated with PD, an interaction between ␣-Syn and mitochondria may underlie the PD pathogenesis. Indeed, a few lines of evidence suggest that ␣-Syn and mitochondria interact with each other [30]. On one hand, overexpression of ␣-Syn, which may lead to cytoplasmic accumulation of ␣-Syn, was shown to impair mitochondrial function [7]. On the other hand, an impaired mitochondrial function was shown to induce increased ␣-Syn expression and formation of ␣-Syn inclusions [11]. A direct link between ␣-Syn and mitochondria in PD has been demonstrated by a recent study showing that mitochondria of PD vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of ␣-Syn and decreased complex I activity [2]. This result suggests that in disease conditions the levels of
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mitochondrial ␣-Syn are varied in different brain regions. However, whether in normal conditions mitochondrial ␣-Syn is also differentially expressed in different brain regions remains uninvestigated. The purpose of the present study is to investigate the normal expressions of mitochondrial ␣-Syn in various brain regions and the association of mitochondrial ␣-Syn with cytosolic ␣-Syn. In addition, the effects of ␣-Syn concentration on the complex I activity of mitochondrial respiratory chain were also examined. Five male Wistar rats of 8 weeks purchased from Beijing Vital River Laboratory Animal Co., Ltd. were used to examine the expressions of ␣-Syn in mitochondria and cytoplasm in various brain regions. After deeply anaesthetized, the brains of the rats were removed from the skulls, and the mitochondrial and cytosolic fractions were isolated from the brain tissues according to the procedures described previously with slight modifications [23]. In brief, the tissues dissected from different brain regions were homogenized in the “mitochondria isolation buffer (MIB)” containing 0.32 M sucrose, 1 mM EDTA (K+ salt), and 10 mM Tris–HCl, pH 7.4. The homogenates were centrifuged at 1330 × g for 5 min. The supernatant was preserved, and the pellet was suspended and re-centrifuged as above. Both supernatants were then mixed and centrifuged at 21,250 × g for 10 min. The supernatant was used as cytosolic fraction. The pellet was resuspended in 15% Percoll (Bioshop), and 3 ml of this suspension was laid on two preformed layers consisting of 3.5 ml of 26% Percoll and 3.5 ml of 40% Percoll. The gradient was centrifuged for 5 min at 30,700 × g. The fraction accumulating near the interface of the two lower layers was collected and diluted 1:4 with MIB. The mixture was centrifuged twice at 16,700 × g for 10 min, producing a pellet which contained mitochondria. Protein concentration was determined using BCA Protein Assay Kit (Pierce). Western blot analysis was used to examine the purity of the mitochondrial and cytosolic fractions as well as the expression levels of ␣-Syn. 75 g of proteins were separated by 15% SDS-PAGE, and transferred onto a PVDF membrane, which was then incubated overnight at 4 ◦ C with different primary antibodies (3D5 and 2E3 mouse monoclonal anti-␣-Syn antibodies [28], EQV1, MDV2, and PQE3 rabbit polyclonal anti-␣-Syn antibodies [1], rabbit polyclonal anti-lactate dehydrogenase (LDH) antibody (Rockland Corporation), rabbit polyclonal anti--actin antibody (Santa Cruz), rabbit polyclonal anti-prohibitin antibody (Biomeda), and rabbit polyclonal anti-synaptophysin antibody (Santa Cruz), followed by incubation with horseradish peroxidase-conjugated goat-antimouse IgG or horseradish peroxidase-conjugated goat-anti-rabbit IgG (Vector Laboratories) (1:5000 in TBST). Immunoreactive bands were revealed by enhanced chemiluminescence (Pierce). Film autoradiograms were analyzed and quantified by computerassisted densitometry using Quantity One (Bio-Rad) system to estimate the relative amount of ␣-Syn. To investigate the effect of ␣-Syn concentration on its mitochondrial translocation, isolated mitochondria were re-suspended in the respiratory buffer (300 mM mannitol, 10 mM KH2 PO4 , 5 mM MgCl2 , pH 7.2 at 37 ◦ C) [20], and were incubated with different concentrations of recombinant human ␣-Syn at 37 ◦ C for different time points. The mitochondria were pelleted by centrifuging at 12,000 × g at 4 ◦ C for 10 min and the reaction was stopped by washing the pellet twice with 500 l of MIB. The mitochondria were re-suspended in an adequate volume (20 l) of MIB for evaluation of the amount of ␣-Syn using Western blot analysis. In some experiments, mitochondria treated by ␣-Syn were fractioned by incubation with digitonin (Sigma) at a concentration of 0.5 mg/mg protein at 4 ◦ C for 15 min [6]. The samples were then diluted using 3 volumes of the respiratory buffer. The inner membrane fraction was obtained as a pellet by centrifugation at 12,500 × g for 15 min, and the supernatant was further centrifuged at 100,000 × g for 60 min to obtain the outer membrane fraction. The purity of the inner membrane fraction
was analyzed using antibodies against an outer membrane protein VDAC1 (Santa Cruz) and an inner membrane protein prohibitin. The presence of ␣-Syn in different membrane fractions was examined using EQV1 anti-␣-Syn antibody. To study the effect of ␣-Syn concentration on complex I activity, the isolated cerebellar mitochondria were incubated with different concentrations of ␣-Syn, fractured by rapid freezing–thawing and measured spectrophometrically for NADH-CoQ oxidoreductase (complex I) activity [16]. The precise localization of ␣-Syn in mitochondria was also examined using pre-embedding immunogold electron microscopy. In this experiment, Wistar rats deeply anesthetized were perfusionfixed with a fixative containing 4% paraformaldehyde and 0.35% glutaraldehyde, and the brains were removed and postfixed with a fixative containing 4% paraformaldehyde, followed by rinse in 20% sucrose. 50 m of brain sections were cut and the procedures for the pre-embedding immunogold electron microscopy were as same as before [28]. All the data are expressed as mean ± SD. Student t-test was used to evaluate the statistical significance between control and each experimental group. Western blot analysis showed that an antibody against a mitochondrial specific protein prohibitin [8], only revealed a 30 kDa band, which is identical to the molecular size of this protein, in the mitochondrial fraction but not in the cytosolic fraction. Conversely, an antibody recognizing the cytoplasmic protein LDH, only detected a 36 kDa protein, which is compatible in molecular size with LDH, in the cytosolic fraction but not in the mitochondrial fraction. Moreover, the mitochondrial fraction reacted very faintly with anti-synaptophysin antibody (Fig. 1A). The presence of ␣-Syn in the mitochondrial faction were examined using five anti-␣-Syn antibodies, which recognize N-terminal (MDV2), NAC domain (EQV1), and C-terminal (PQE3, 3D5, and 2E3) of ␣-Syn [1,28]. Each of the five antibodies revealed a 19 kDa band in the mitochondrial fraction, which was identical in molecular size to the cytosolic ␣-Syn and the recombinant ␣-Syn (Fig. 1B). Pre-absorption of the antibodies with excess recombinant ␣-Syn abolished the bands (data not shown). Pre-embedding immunogold electron microscopic observation using homemade 3D5 mouse monoclonal anti-␣-Syn antibody revealed that mitochondria with positive gold particles for ␣-Syn were localized in the neurons. Photomicrographs with higher magnification showed that the positive gold particles were localized on the outer membrane, the inner membrane, and the intermembrane space of the ␣-Syn-positive mitochondria. Except the membrane of mitochondria, some ␣-Syn-positive gold particles could be also observed in the matrix of the mitochondria (Fig. 1C). The mitochondrial ␣-Syn was differentially expressed in different brain regions. The levels of the mitochondrial ␣-Syn were relatively higher in olfactory bulb, hippocampus, striatum, and thalamus, where the levels of the cytosolic ␣-Syn were also high. In brain stem, the ␣-Syn levels were low in both mitochondrial and cytosolic fractions. However, the cerebral cortex and cerebellum were among the exceptions, which contained relatively rich cytosolic ␣-Syn, but very low or even undetectable levels of mitochondrial ␣-Syn (Fig. 2A and B). Incubation of the cerebellar mitochondria, where there was no detectable endogenous ␣-Syn, with different concentrations of purified recombinant ␣-Syn lead to dose-dependent increase in the amount of ␣-Syn accumulated in the mitochondria during observed 30 min (Fig. 3A), suggesting that ␣-Syn transport onto mitochondria depends on its concentration. The transport was rapid, with the earliest detection of ␣-Syn in the mitochondrial fraction within 5 min. Further extending incubation time did not apparently increase the amount of ␣-Syn in the mitochondrial fraction (Fig. 3B). To rule out the possibility that the ␣-Syn detected in the mitochondrial fraction was because of non-specific binding of the
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Fig. 1. Mitochondrial ␣-Syn detected by Western blot. (A) A mitochondrial protein prohibitin was detected only in the mitochondrial fraction (left), while a cytoplasmic protein LDH was detected only in the cytosolic fraction (middle). In addition, the mitochondrial fraction reacted strongly with anti-prohibitin antibody but very faintly with anti-synaptophysin antibody. Mt: mitochondrial fraction; Cyto: cytosolic fraction; PHB: prohibitin; LDH: lactate dehydrogenase; SYP: synaptophysin. (B) A 19 kDa protein was detected by five anti-␣-Syn-antibodies in the mitochondrial fractions, which share the same molecular size with the cytoplasmic ␣-Syn and the recombinant ␣-Syn; C: mitochondria in the cerebral cortex (A), thalamus (B), and substantia nigra (C). All the mitochondria contain ␣-Syn-positive gold particles, which are different in density. Gold particles can be observed in the mitochondrial matrix (white arrows), outer membrane (black arrow head), inner membrane (white arrow head), and intermembrane space (black arrows) Con: control; Cyto: cytoplasmic fraction; Mt: mitochondrial fraction; M: mitochondria. Bar: 20 nm in (a) and (b), 50 nm in (c).
protein to the surface of the outer membrane, we used digitonin to disrupt the outer membrane of the mitochondria and separate it from the inner membrane. Western blot analysis showed that the inner membrane protein was not identified by an antibody to the outer membrane protein VDAC1 (Fig. 3C) but recognized by an antibody to the inner membrane protein prohibitin (Fig. 3D). The transported ␣-Syn was shown to be mainly localized in the inner membrane fraction, with only small amount present in the outer membrane fraction (Fig. 3D). In fact, a recently study has shown that there is a direct interaction between ␣-Syn and prohibitin [15].
Since ␣-Syn was shown to inhibit complex I activity and the levels of mitochondrial ␣-Syn were different in various brain regions, it is necessary to know the association of ␣-Syn concentration with the activity of the enzyme. For this purpose, isolated cerebellar mitochondria were incubated with different concentrations of ␣-Syn (1 pM, 102 pM, and 104 pM, which is equal to 0.002–20.000 pmol/mg mitochondria). The complex I activity was shown to be inhibited even when ␣-Syn concentration was as low as 1 pM; increasing ␣-Syn concentration caused a further reduction in the activity of complex I (Fig. 4).
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Fig. 3. ␣-Syn transport into mitochondria. Isolated cerebellar mitochondria were incubated with different concentrations of recombinant ␣-Syn. The amount of ␣Syn in mitochondria was increased with the enhancement of ␣-Syn concentration (A). The mitochondrial transport of ␣-Syn was time-independent, with the maximal accumulation of ␣-Syn in the mitochondria found within 5 min (B). An outer membrane mitochondrial protein VDAC1 was detected only in the outer membrane fraction but not in the inner membrane fraction (C). Western blot analysis of the digitonin-disrupted mitochondria showed that ␣-Syn was mainly present in the inner membrane fraction, with only small amount in the outer and intermembrane fraction (D). IM: inner membrane fraction; OM: outer membrane fraction. Fig. 2. Expressions of ␣-Syn in cytosolic and mitochondria in various brain regions. (A) Expressions of cytosolic ␣-Syn in different brain regions. Upper panel: representative immunoblots. Lower panel: bar graphs showing the relative levels of the cytosolic ␣-Syn; (B) expressions of mitochondrial ␣-Syn in different brain regions. Upper panel: representative immunoblots. Lower panel: bar graphs showing the relative levels of the mitochondrial ␣-Syn. Con: recombinant human ␣-Syn; OB: olfactory bulb; HIP: hippocampus; CX: cortex; STR: striatum; Thal: thalamus; CB: cerebellum; BS: brain stem.
The presence of ␣-Syn in mitochondria has been reported in neurons of human [2] and mouse [12] brains as well as in cells overexpressing ␣-Syn [18]. However, the precise localization of this protein in mitochondria remains elusive. Immunoelectron microscopic study on the human brain showed that ␣-Syn-positive gold particles were present on the outer membrane and in the matrix of mitochondria in the neurons [2]. In the mouse brain, however, ␣Syn-positive gold particles were observed only in the mitochondrial membrane of the neurons [12]. In HEK 293 cells overexpressing wild type ␣-Syn, ␣-Syn was shown to locate only to the outer mitochondrial membrane, while in SY5Y cells overexpressing A53T ␣-Syn, ␣-Syn appeared to be localized in both the mitochondrial membrane and matrix [18]. By using pre-embedding immunogold electron microscopic method, which allows higher resolution of the antigen locating sites on well preserved ultrastructures, we also observed that ␣-Syn-immunoreactive mitochondria were localized in the neurons, which is identical to our previous results that ␣-Syn was only expressed in the neurons of the rat brain [28,30]. In the mitochondria, ␣-Syn-positive gold particles were localized in both the membrane and matrix. The membrane bound gold particles could be found on the outer membrane and the inner membrane, or in the intermembrane space of the mitochondria, indicating that ␣-Syn may have different localizations in the mitochondrial membrane. The discrepancy for the precise mitochondrial localization of ␣-Syn reported by different authors is not clear. One possible reason may be due to the difference of the animal species investigated.
Although previous studies on the human and mouse brains showed that ␣-Syn could be found in the mitochondria of both dopaminergic neurons and other neurons, and PD-vulnerable striatum and substantia nigra contained much more mitochondrial ␣-Syn than cerebellum, the relative levels of mitochondrial ␣-Syn in different regions of the normal brain has not yet systemically investigated. In the present study, Percoll density gradient centrifugation [23] was used to isolate mitochondria from rat brains. This method allows preparation of free non-synaptosomal mitochondria with little residual contamination by synaptosomes or myelin. The mitochondrial fraction reacted strongly with the antibody to mitochondrial protein prohibitin but very faintly with the
Fig. 4. Effect of ␣-Syn on complex I activity of mitochondrial respiratory chain. Different concentrations of recombinant ␣-Syn were incubated with the isolated cerebellar mitochondria. After washing out the free ␣-Syn, the mitochondria were fractured by rapid freezing and thawing, and the complex I activity was measured using spectrophotometric analysis of the kinetics of NADH reduction at 340 nm. The complex I activity decreased in a dose-dependent manner (* p < 0.05 vs. control).
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antibody to synaptic protein synaptophysin, suggesting that the mitochondrial fraction isolated with this method mainly contained free mitochondria with only little contamination by synaptosomes. Western blot analysis of the mitochondrial fractions isolated from different brain regions showed that ␣-Syn could be detected in mitochondria from many brain regions, including olfactory bulb, striatum, hippocampus, thalamus, cerebral cortex, and brain stem. However, the levels of mitochondrial ␣-Syn were quite different among the brain regions examined. ␣-Syn were highly expressed in the mitochondria isolated from the olfactory bulb, striatum, hippocampus, and thalamus, where the cytosolic ␣-Syn was also rich. However, in the cerebral cortex and cerebellum, although there was rich cytosolic ␣-Syn, the mitochondrial ␣-Syn was very low or even undetectable. The various levels of mitochondrial ␣-Syn measured in different brain regions may mainly reflect the difference in the ␣-Syn expression in the mitochondria of neurons, since previous immunohistochemical and immunoelectron microscopic studies using the same antibodies showed that ␣-Syn was localized only in the neurons of the rat brain [28,30]. The previous study on ␣-Syn-gene transfected dopaminergic neurons showed that increased cytoplamic ␣-Syn was accompanied by increased mitochondrial ␣-Syn [18]. In the present study, by using isolated rat brain mitochondria to incubate with recombinant ␣-Syn, we observed that the amount of ␣-Syn transported into the mitochondria increased with the enhancement of ␣-Syn concentration. By separating the inner membrane fraction from the outer membrane fraction, we demonstrated that the ␣-Syn transported into mitochondria was mainly accumulated in the inner membrane, ruling out the possibility for the non-specific binding of this protein to the surface of the outer membrane. The dose-dependent ␣-Syn transport to the isolated mitochondria is consistent with the observation on the in vivo brain that the mitochondrial ␣-Syn is quantitatively associated with the cytosolic ␣-Syn in most brain regions except cerebral cortex and cerebellum, indicating that the cytoplasmic concentration of ␣-Syn may determine its amount in the mitochondria in these brain regions. It is not clear why in the cerebral cortex and cerebellum the mitochondrial ␣-Syn are not quantitatively associated with the cytosolic ␣-Syn. One possible reason may be that the mechanism controlling the transport of ␣Syn from cytoplasm to mitochondria in these two brain regions is different from that in other brain regions, which is an issue remains to be addressed. Since ␣-Syn abnormality and mitochondrial complex I deficiency are both implicated in PD pathogenesis, ␣-Syn localization in mitochondria may suggest a functional link between ␣-Syn and complex I. In human dopaminergic neurons, ␣-Syn accumulation in the mitochondria was shown to cause reduced mitochondrial complex I activity [2]. Our study on the isolated cerebellar mitochondria confirmed this, indicating that the inhibition of complex I activity by ␣-Syn was dose-dependent, with the minimal effective concentration being as low as 1 pM. The effect of ␣-Syn on complex I activity of mitochondria may have special physiological and pathophysiological meaning. It is known that oxidative phosphorylation on mitochondria produces ATP for cell functioning, which leads to large amounts of generation of free radicals detrimental to mitochondria and other cellular components, which are thought to be the main reason for aging [4]. We speculate that proper suppression of complex I activity by ␣-Syn is beneficial to reducing free radical production and mitochondrial damage. However, in abnormal conditions, abnormal accumulation of ␣-Syn in mitochondria may inhibit complex I activity to a greater extent, leading to reduced ATP production, increased ROS generation, and accelerated neurodegeneration [2]. To support this, both PD and amyotrophic lateral sclerosis (ALS) are characterized by impairments of mitochondria and increased accumulation of ␣-Syn in the neurons of some vulnerable regions
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in the brain or the spinal cord [14,21,3]. Some experiments on animals and cells provide additional evidence for the deleterious role of ␣-Syn in mitochondrial function in abnormal conditions. For example, ␣-Syn overexpression made mitochondria more vulnerable to complex I inhibitor [17]; interaction of ␣-Syn with mitochondria caused release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components [18]; mice lacking ␣-Syn are resistant to mitochondrial toxins [9]; the prolonged mitochondrial complex I inhibition due to continuous MPTP infusion is alleviated when ␣-Syn is removed [5]. Taken together, the present results that mitochondrial ␣-Syn is differentially expressed in different brain neurons and dosedependently down-regulates the complex I activity suggests that the background levels of mitochondrial ␣-Syn may be a potential factor affecting mitochondrial function and predisposing some neurons to degeneration. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30570646 and 30430280), Ministry of Science and Technology of China (2006CB500701, 2006AA02A408), Natural Science Foundation of Beijing (7022011), and Japan Society for the Promotion of Science (JSPS) (C11680774 and B 14380363) and Funding Project for Academic Human Resources Development in Institutions of Higher Learning under Jurisdiction of Beijing Municipality PHR (IHLB). References [1] K. Arima, K. Uéda, N. Sunohara, S. Hirai, Y. Izumiyama, H. Tonozuka-Uehara, M. Kawai, Immunoelectron microscopic demonstration of NACP/alpha-synucleinepitopes on the filamentous component of Lewy bodies in Parkinson’s disease and in dementia with Lewy bodies, Brain Res. 808 (1998) 93–100. [2] L. Devi, V. Raghavendran, B.M. Prabhu, N.G. Avadhani, H.K. Anandatheerthavarada, Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain, J. Biol. Chem. 283 (2008) 9089–9100. [3] M.J. Doherty, T.D. Bird, J.B. Leverenz, Alpha-synuclein in motor neuron disease: an immunohistologic study, Acta. Neuropathol. 107 (2004) 169–175. [4] E. Dufour, N.G. Larsson, Understanding aging: revealing order out of chaos, Biochim. Biophys. Acta 1658 (2004) 122–132. [5] F. Fornai, O.M. Schlüter, P. Lenzi, M. Gesi, R. Ruffoli, M. Ferrucci, G. Lazzeri, C.L. Busceti, F. Pontarelli, G. Battaglia, A. Pellegrini, F. Nicoletti, S. Ruggieri, A. Paparelli, T.C. Südhof, Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alphasynuclein, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3413–3418. [6] T. Gotow, M. Shibata, S. Kanamori, O. Tokuno, Y. Ohsawa, N. Sato, K. Sahara, Y. Yayoi, T. Watanabe, J.F. Leterrier, M. Linden, E. Kominami, Y. Uchiyama, Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells, Cell Death Differ. 7 (2000) 666–674. [7] L.J. Hsu, Y. Sagara, A. Arroyo, E. Rockenstein, A. Sisk, M. Mallory, J. Wong, T. Takenouchi, M. Hashimoto, E. Masliah, Alpha-synuclein promotes mitochondrial deficit and oxidative stress, Am. J. Patho. 157 (2000) 401–410. [8] E. Ikonen, K. Fiedler, R.G. Parton, K. Simons, Prohibitin, an antiproliferative protein, is localized to mitochondria, FEBS Lett. 358 (1995) 273–277. [9] P. Klivenyi, D. Siwek, G. Gardian, L. Yang, A. Starkov, C. Cleren, R.J. Ferrante, N.W. Kowall, A. Abeliovich, MF. Beal, Mice lacking alpha-synuclein are resistant to mitochondrial toxins, Neurobiol. Dis. 21 (2006) 541–548. [10] R. Krüger, W. Kuhn, T. Müller, D. Woitalla, M. Graeber, S. Kösel, H. Przuntek, J.T. Epplen, L. Schöls, O. Riess, Ala30Pro mutation in the gene encoding alphasynuclein in Parkinson’s disease, Nat. Genet. 18 (1998) 106–108. [11] H.J. Lee, S.Y. Shin, C. Choi, Y.H. Lee, S.J. Lee, Formation and removal of alphasynuclein aggregates in cells exposed to mitochondrial inhibitors, J. Biol. Chem. 277 (2002) 5411–5417. [12] W.W. Li, R. Yang, J.C. Guo, H.M. Ren, X.L. Zha, J.S. Cheng, D.F. Cai, Localization of alpha-synuclein to mitochondria within midbrain of mice, Neuroreport 18 (2007) 1543–1546. [13] Q.L. Ma, P. Chan, M. Yoshii, K. Uéda, ␣-Synuclein aggregation and neurodegenerative diseases, J. Alzheimers Dis. 5 (2003) 139–148. [14] L.J. Martin, Transgenic mice with human mutant genes causing Parkinson’s disease and amyotrophic lateral sclerosis provide common insight into mechanisms of motor neuron selective vulnerability to degeneration, Rev. Neurosci. 18 (2007) 115–136. [15] M.A. McFarland, C.E. Ellis, S.P. Markey, R.L. Nussbaum, Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions, Mol. Cell Proteomics 7 (2008) 2123–2137.
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G. Liu et al. / Neuroscience Letters 454 (2009) 187–192
[16] V. Mildaziene, Z. Nauciene, R. Baniene, J. Grigiene, Multiple effects of 2,2 ,5,5 tetrachlorobiphenyl on oxidative phosphorylation in rat liver mitochondria, Toxicol. Sci. 65 (2002) 220–227. [17] M. Orth, S.J. Tabrizi, A.H. Schapira, J.M. Cooper, Alpha-synuclein expression in HEK293 cells enhances the mitochondrial sensitivity to rotenone, Neurosci. Lett. 351 (2003) 29–32. [18] M.S. Parihar, A. Parihar, M. Fujita, M. Hashimoto, P. Ghafourifar, Mitochondrial association of alpha-synuclein causes oxidative stress, Cell Mol. Life Sci. 65 (2008) 1272–1284. [19] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, I.G. Di, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045–2047. [20] Z. Roland, M. Christophe, B. Aldo, A.E.B. Alberto, T. Jean-Paul, Resveratrolinduced limitation of dysfunction of mitochondria isolated from rat brain in an anoxia-reoxygenation model, Life Sci. 71 (2002) 3091–3108. [21] S. Sasaki, Y. Horie, M. Iwata, Mitochondrial alterations in dorsal root ganglion cells in sporadic amyotrophic lateral sclerosis, Acta. Neuropathol. 114 (2007) 633–639. [22] A.H. Schapira, J.M. Cooper, D. Dexter, J.B. Clark, P. Jenner, C.D. Marsden, Mitochondrial complex I deficiency in Parkinson’s disease, J. Neurochem. 54 (1990) 823–827. [23] N.R. Sims, Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation, J. Neurochem. 55 (1990) 698–707.
[24] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowsky, R. Jakes, M. Goedert, Alpha-synuclein in Lewy bodies, Nature 388 (1997) 839–840. [25] R.H. Swerdlow, J.K. Parks, J.N. Davis 2nd, D.S. Cassarino, P.A. Trimmer, L.J. Currie, J. Dougherty, W.S. Bridges, J.P. Bennett Jr., G.F. Wooten, W.D. Parker, Matrilineal inheritance of complex I dysfunction in a multigenerational Parkinson’s disease family, Ann. Neurol. 44 (1998) 873–881. [26] K. Uéda, H. Fukushima, E. Masliah, Y. Xia, A. Iwai, M. Yoshimoto, D.A. Otero, J. Kondo, Y. Ihara, T. Saitoh, Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 11282–11286. [27] J. Xu, S.Y. Kao, F.J.S. Lee, W. Song, L.W. Jin, B.A. Yankner, Dopamine-dependent neurotoxicity of ␣-synuclein: A mechanism for selective neurodegeneration in Parkinson disease, Nat. Med. 8 (2002) 600–606. [28] S. Yu, X. Li, G. Liu, J. Han, C. Zhang, Y. Li, S. Xu, C. Liu, Y. Gao, H. Yang, K. Uéda, P. Chan, Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody, Neuroscience 145 (2007) 539–555. [29] J.J. Zarranz, J. Alegre, J.C. Gomez-Esteban, E. Lezcano, R. Ros, L.Vidal AmpueroI, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E. GomezTortosa, T. del Ser, D.G. Munoz, J.G. de Yebenes, The new mutation, E46K, of alphasynuclein causes Parkinson and Lewy body dementia, Ann. Neurol. 55 (2004) 164–173. [30] L. Zhang, C. Zhang, Y. Zhu, Q. Cai, P. Chan, K. Uéda, S. Yu, H. Yang, Semiquantitative analysis of alpha-synuclein in subcellular pools of rat brain neurons: an immunogold electron microscopic study using a C-terminal specific monoclonal antibody, Brain Res. 1244 (2008) 40–52.