Age-associated increases of α-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: Is this the target for Parkinson's disease?

www.elsevier.com/locate/ynbdi Neurobiology of Disease 25 (2007) 134 – 149

Age-associated increases of α-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: Is this the target for Parkinson's disease? Yaping Chu and Jeffrey H. Kordower⁎ Department of Neurological Sciences, Rush University Medical Center, 1735 West Harrison Street, Chicago, IL 60612, USA Received 28 August 2006; revised 31 August 2006; accepted 31 August 2006 Available online 18 October 2006 α-Synuclein is a synaptic protein that has been directly linked to both the etiology and pathogenesis of Parkinson's disease. We have previously shown that only nigral neurons in PD expressing αsynuclein inclusions display a loss dopaminergic phenotype. The present study tested the hypothesis that normal aging contributes to this effect. The relative abundance of α-synuclein protein within individual nigral neurons was quantified in eighteen normal humans between the age of 18 and 102 and twenty four rhesus monkeys between the age of 2 and 34. Optical densitometry revealed a robust age-related increase in α-synuclein protein within individual nigral neurons in both species. This effect was specific for nigral α-synuclein as no age-related changes were found in the ventral tegmental area nor were there changes in the nigra for non-pathogenic β-synuclein. The age-related increases in nigral α-synuclein were non-aggregated and strongly associated with age-related decreases in tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine production. In fact, only cells expressing α-synuclein displayed reductions in TH. We hypothesize that age-related increases in α-synuclein result in a subthreshold degeneration of nigrostriatal dopamine which, in PD, becomes symptomatic due to lysosomal failure resulting in protein misfolding and inclusion formation. We further hypothesize that preventing the age-related accumulation of non-aggregated α-synuclein might be a simple and potent therapeutic target for patients with PD. © 2006 Elsevier Inc. All rights reserved. Keywords: Synuclein; Tyrosine hydroxylase; Dopaminergic neuron; Substantia nigra; Aging; Synucleinopathy

Introduction Gene mutations in α-synuclein (Polymeropoulos et al., 1997; Spillantini et al., 1997) provided the first indisputable evidence for the existence of genetic forms of Parkinson's disease (PD). While ⁎ Corresponding author. E-mail address: [email protected] (J.H. Kordower). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2006.08.021

these (Kruger et al., 1998; Singleton et al., 2003; Nishioka et al., 2006) and subsequent (Kitada et al., 1998; Leroy et al., 1998; Bonifati et al., 2002; Le et al., 2003) genetic examples of PD are clearly landmark studies, they remain relatively rare and genetic population studies have indicated that few genes alone contribute to the prevalence of PD (Farrer, 2006; Pankratz and Foroud, 2004). However, interest in α-synuclein remains very high since this protein is a central pathological feature of Lewy bodies and Lewy neurites, pathognomonic features of PD. There is compelling evidence to suggest that overexpression of α-synuclein is toxic to nigral neurons in PD (Singleton et al., 2003; Nishioka et al., 2006; Masliah et al., 2000; Giasson et al., 2002; Shimura et al., 2001). We have demonstrated that, in PD, α-synuclein aggregation in surviving nigral neuron causes a robust down-regulation of dopaminergic markers while adjacent neurons devoid of such aggregates display normal levels of these markers (Chu et al., 2006). A loss of dopaminergic phenotype is one of the first degenerative events that occur in vulnerable nigral neurons, and these data indicate that α-synuclein aggregation may be the trigger for this early pathological cascade. Animal studies support the concept that overexpression of α-synuclein can cause nigrostriatal dysfunction. In Drosophila melanogaster, α-synuclein overexpression results in both the degeneration of dopaminergic (DA) neurons and fly-specific motor deficits (Feany and Bender, 2000). While frank neuronal loss is rarely seen in overexpressing transgenic mice (Masliah et al., 2000; Giasson et al., 2002), these mutants can display a loss of striatal dopamine and a progressive motor syndrome that model specific aspects of the motor deficits seen in PD. More convincing are the gene delivery studies in which viral vectors are used to overexpress α-synuclein within nigral neurons to induce a progressive motor dysfunction that is associated with degeneration of the nigrostriatal system in rats (Kirik et al., 2002; Lo Bianco et al., 2002) and nonhuman primates (Kirik et al., 2003). Aging remains the most compelling and prominent risk factor for the development PD (Baldereschi et al., 2000; Saito et al., 2004; Bender et al., 2006; Lee et al., 1995; Phinney et al., 2006; Andersen et al., 1999; Fearnley and Lees, 1991; Chu et al., 2002).

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An age-related loss of nigrostriatal dopamine is a consistent finding in nonhuman primates and humans, but the magnitude of this loss is insufficient to cause parkinsonian symptoms. It has long been hypothesized that, in sporadic PD, a toxic event may be superimposed upon age-related losses in striatal dopamine and these in combination drive striatal dopamine insufficiency to a level that causes symptoms (Kish et al., 1992; Nieto et al., 2006; Jellinger, 2004; Collier et al., 2005). The role α-synuclein plays in this scenario is just beginning to be understood. Recent data indicate that α-synuclein is upregulated in nigral neurons following administration of MPTP (Nieto et al., 2006; Drolet et al., 2004; Song et al., 2004) and rotenone (Sherer et al., 2003). However, little is known regarding the expression of α-synuclein as a function of normal aging. Jellinger (2004) noted the presence of α-synuclein pathology within the midbrain and limbic cortex of 31% of aged individuals, while Li et al. (2004a,b) used immunoblot data to suggest that αsynuclein was increased in the midbrain region of seven normal aged individuals. The present study examined the expression of αsynuclein within individual nigral neurons across a wide range of normal aging in humans and nonhuman primates and found a profound age-related increase in non-aggregated α-synuclein in both primate species that was associated with age-related decreases in tyrosine hydroxylase (TH). We hypothesize that this age-related increase in α-synuclein is the precursor to α-synuclein inclusions seen in PD. Targeting the age-related changes in αsynuclein might be a valuable therapeutic target to augment dopamine levels and prevent nigrostriatal degeneration in these patients.

Table 1 Subject demographics

Materials and methods

Nonhuman primates

Human cases

Twenty-four rhesus monkeys (8 males, 16 females) ranging in age from 2 to 34 years were analyzed. On the basis of age, the animals were divided into young (n = 4, range 2–12.1 years), middle-aged (n = 10; range, 15–23 years), and aged (n = 10, range 24–34.1) groups. Rhesus monkeys age at a rate of 3:1 as compared to humans (Collier et al., 2005; Andersen et al., 1999). Thus, the groups of monkeys model the equivalent of 6.9–36.3 years, 45–69, and 72–102.3 years of human life, respectively. All monkeys were housed one per cage on a 12-hour on/12-hour off lighting schedule with ad libitum access to food and water. The quality of the animal care exceeded the recommended NIH guidelines. Prior to death, monkeys were pretreated with Ketamine (20 mg/kg, i.m.) and then were deeply anesthetized with sodium pentobarbital (25 mg/kg, i.v.). Prior to perfusion, monkeys were injected with 1 ml of heparin (20,000 IU) into the left ventricle of the heart. Animals were then perfused with normal saline followed by fixation with 4% paraformaldehyde. The brains were then removed from the calvaria and cryoprotected in 30% sucrose in 0.1 M sodium phosphate buffer at 4°C. Serial sections throughout the brain were cut frozen (40 um) on a sliding knife microtome and stored at − 20°C in cryoprotectant (Kompoliti et al., 2004).

To establish the quality of the human tissue, we immunostained sections from each case using a monoclonal tyrosine hydroxylase (TH) with well-known immunostaining patterns. Cases in which brain tissue demonstrated weak TH immunoreactivity were excluded from the study. Tissues from 18 subjects were subsequently analyzed. These subjects (13 males, 6 females; Table 1), all of whom were without neurological or psychiatric illness, ranged in age from 18 to 102 years (average age, 59.65 ± 24.13). On the basis of age, they were divided into three groups: young (n = 6; range, 18–39 years), middle-aged (n = 4; range, 44– 56 years), and aged (n = 8; range, 73–102 years). The average postmortem interval (PMI) was 14.06 ± 7.77 h (range, 3.0– 24.0 h). At autopsy, brains were removed from the calvaria and processed as described previously (Chu et al., 2002, 2006). Briefly, each brain was cut into 1-cm-thick coronal slabs using a Plexiglas brain slice apparatus and then hemisected. The brains were then immersion fixed in 4% paraformaldehyde for 48 h at 4°C and cryoprotected in 0.1 M phosphate buffer saline (PBS; pH 7.4) containing 2% dimethyl sulfoxide (DMSO), 10% glycerol for 24 h followed by 2% DMSO, 20% glycerol in PBS for at least 2 days before sectioning. Slabs containing substantia nigra (SN) were cut perpendicular to the longitudinal axis of the brainstem into 18 adjacent series of 40-μm-thick sections on a freezing sliding microtome. All sections were collected and stored in order in a cryoprotectant solution before processing. The Human Investigation Committee of Rush University Medical Center approved the study.

Case number

Age (years)

Gender

PMI (hours)

Young group 1 2 3 4 5 6 Mean ± SD

18 29 30 35 37 39 31.33 ± 7.6

M F M M M M

10.0 23.0 13.5 n/a 24.0 23.0 19.25 ± 6.01

Middle-aged group 7 8 9 10 Mean ± SD

44 50 52 56 55.16 ± 8.49

M F M M

11.5 20.0 24.0 7.0 20.22 ± 3.54

Aged group 11 12 13 14 15 16 17 18 Mean ± SD

73 80 79 83 84 88 91 102 84.25 ± 9.53

M F F M M F F F

7.3 10.7 5.8 5.5 4.0 3.0 10.7 5.0 6.45 ± 2.80

PMI, postmortem interval; n/a, not available.

Immunohistochemical procedures An immunoperoxidase labeling method was used to visualize α-synuclein labeled profiles. The monoclonal α-synuclein antibody (LB 509, Zymed, CA) recognizes amino acids 115–122 (P37840) of human α-synuclein (Jakes et al., 1999; Spillantini et al., 1997; Sampathu et al., 2003). One series of nigral sections was

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immunostained with α-synuclein (1:5000). After six washes, sections were incubated for 1 h in biotinylated horse anti-mouse IgG (1:200; Vector) followed by the Elite avidin–biotin complex (1:500, ABC kits; Vector). The immunohistochemical reaction was completed with 0.05% DAB and 0.005% H2O2. The distribution and number of α-synuclein immunoreactive (α-synuclein-ir) neurons were evaluated. To control for inter-run variability, all human or monkey cases were stained at the same time.

immune complexes with the antibody and blocking peptide were centrifuged at 10,000 rpm for 20 min. The adsorbed protein/ antibody complex was then used in lieu of the primary antibody. All control experiments resulted in the absence of specific staining. Additionally, a positive control experiment for α-synuclein staining was performed using nigral sections from PD cases.

Proteinase K digested section immunostaining

In order to quantify changes in α-synuclein-ir neuronal number with aging, an optical fractionator unbiased sampling design was used to estimate the total number of α-synuclein-ir neurons within the substantia nigra (Chu et al., 2002, 2006; Gundersen et al., 1988). In each human subject, we evaluated the major portion of the SN that extended from the caudal level of the mammillary bodies to the decussation of the superior cerebellar peduncle. The most rostral portion of the SN was not included in the stereological analysis since it was separated from the brainstem block at autopsy. For each monkey, the whole substantia nigra was evaluated. Approximately seven equispaced sections along the SN were sampled from each human and monkey brain. The section sampling fraction (ssf) was 1/0.055. The distance between sections was approximately 0.72 mm. In cross-section, the SN is located in the ventral midbrain. It has an ellipsoid shape and defined ventrally by the cerebral peduncle and medially by the third cranial nerve rootlets. The substantia nigra was outlined using a 1.25× objective. A systematic sample of the area occupied by the substantia nigra was made from a random starting point (StereoInvestigator 2000 software; Micro-BrightField, Colchester, VT). Counts were made at regular predetermined intervals (x = 313 μm, y = 313 μm), and a counting frame (70 × 70 μm = 4900 μm2) was superimposed on images obtained from tissue sections. The area sampling fraction (asf) was 1/0.05. These sections were then analyzed using a 100× Planapo oil immersion objective with a 1.4 numerical aperture. The section thickness was empirically determined. Briefly, as the top of the section was first brought into focus, the stage was zeroed at the z-axis by software. The stage then stepped through the z-axis until the bottom of the section was in focus. Section thickness averaged 20.21 ± 2.3 μm in the midbrain. The disector height (counting frame thickness) was 11 μm. This method allowed for a 3 μm top guard zones and at least a 3 μm bottom guard zones. The thickness sampling fraction (tsf) was 1/0.54 in the SN. Care was taken to ensure that the top and bottom forbidden planes were never included in the cell counting. α-Synuclein-ir nigral neurons were only counted if the first recognizable labeled profiles came into focus within the counting frame. Using stereological principles, αsynuclein-ir neurons in each case were sampled using a uniform, systematic, and random design. The human midbrain DA neurons contain neuromelanin (NM). NM provides an easily discernible endogenous marker for DA neurons, allowing for an easy assessment of co-localization with α-synuclein in DA neurons. The total number of NM-containing neurons within the human substantia nigra was also estimated stereologically using the same method. The total number of α-synuclein-ir and NM-containing neurons within the SN was calculated using the following formula: N = ∑Q− · 1/ssf · 1/asf · 1/tsf. ∑Q was the total number of raw counts. The coefficients of error (CE) were calculated according to the procedure of Gunderson and colleagues as estimates of precision (West and Gundersen, 1990; Schmitz and Hof, 2000). The values of CE were 0.129 ± 0.05 (range 0.10 to 0.14) in human subjects and 0.125 ± 0.04 (range 0.09 to 0.13) in monkeys.

We used proteinase K (PK) digestion to determine whether the α-synuclein seen in nigral neurons was soluble (non-aggregated) or insoluble (aggregated; Neumann et al., 2004; Miake et al., 2002). Sections containing the substantia nigra were mounted onto gelatin-coated slides and dried for at least 8 h at 55°C. After wetting with TBS-T (10 mM Tris–HCl, pH 7.8; 100 mM NaCl; 0.05% Tween-20), the sections were digested with 50 μg/ml PK (Invitrogen) in TBS-T (10 mM Tris–HCl, pH 7.8; 100 mM NaCl; 0.1% Tween-20) for 3 h at 55°C. The sections were fixed with 4% paraformaldehyde for 10 min. After several washes, the sections were processed for α-synuclein immunostaining as described above. Immunofluorescence A double-label immunofluorescence procedure was employed to determine whether TH expression within nigral neurons was altered in neurons that co-expressed α-synuclein. Sections through the substantia nigra from each brain were incubated in the primary antibody TH (1:500; polyclonal antibody; Chemicon) overnight at room temperature and the goat anti-rabbit antibody coupled to Cy2 (1:200) for 1 h. After six washes in TBS, the sections were blocked again for 1 h in a solution containing 5% goat serum, 2% bovine serum albumin, and 0.3% Triton X-100 in TBS. Sections were then incubated in the second primary α-synuclein (1:2500) monoclonal antibody overnight and the goat anti-mouse antibody coupled to cy5 (1:200) for 1 h. The images were scanned using Olympus Confocal Fluoroview Microscope. A single-label immunofluorescence procedure was also employed to determine the age-related alterations of β-synuclein expression within nigral neurons. One series of nigral sections was incubated in the primary β-synuclein (1:1000) polyclonal antibody (Abcam, MA) overnight and the goat anti-rabbit antibody coupled to cy5 (1:200) for 1 h. The images were analyzed using Olympus Confocal Fluoroview Microscope. Immunohistochemical controls Immunohistochemical control experiments included omission of the primary antibodies or replacement of the primary antibodies with irrelevant IgG matched for protein concentration. Both procedures control for the specificity of the staining procedure and the secondary antibody. The control sections were processed in a manner identical to that described above. An adsorption control experiment for α-synuclein antibody was also performed according to previous methods (Chu et al., 2002). Briefly, the α-synuclein antibody (clone: LB509) was combined with a five-fold volume (by weight) of blocking peptide (peptide sequence: 115–122 of human α-synuclein, P37840; generated by American peptide company, CA) in PBS and incubated overnight at 4°C. The

Stereological quantification of cell number

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Fluorescence intensity measurements We assessed the relative intensity of α-synuclein immunoreactivity within individual nigral neurons as a function of normal aging. All immunofluorescent labeled images were scanned with the Olympus Confocal Fluoroview microscope equipped with argon, krypton lasers, and transparent optics (Olympus America, Inc.). To maintain consistency of the scanned image for each slide, the laser intensity, confocal aperture, PMT voltage, offset, electronic gain, scan speed, image size, filter, and zoom were standardized for consistent background levels using a control section, and these parameters were maintained throughout the entire experiment. Imaging was performed with a 20× objective and a 488-nm or 647-nm excitation. All optical density (OD) measurements were performed using stereological principles of random and systematic sampling. Quantitative OD of α-synuclein immunofluorescence intensity was performed on individual neurons using FLUOVIEW software. Five equispaced sections across the entire rostro-caudal axis of the SN were sampled and evaluated. To account for differences in background staining intensity, five background intensity measurements lacking immunofluorescent profiles were taken from each section. The mean of these five measurements constituted the background intensity. The background intensity was then subtracted from the measured immunofluorescence intensity of each individual neuron to provide a final immunofluorescence intensity value. To control for the specificity of age-related α-synuclein expression, we performed similar analyses evaluating the optical density

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of the non-pathogenic β-synuclein in young, middle-aged, and aged monkeys. Data analysis The neuronal counts and OD measurements were analyzed using a factoral analysis of variance model (ANOVA). When appropriate, post hoc comparisons between groups were performed using the method of Scheffe. Correlations between the dependent measures (neuronal counts or optical density values) with age were performed by using Spearman’s rank correlation. Significance for each analysis was set a priori at 0.05 (two-tailed). Results Specificity of immunostaining To demonstrate the specificity of the immunohistochemical results, we first stained PD nigral sections for α-synuclein-ir. The α-synuclein antibody employed strongly labeled the intracellular inclusions within NM-containing nigral neurons and atrophic neurites in PD brains (Fig. 1A). Intense α-synuclein-ir terminal and perikaryal staining, but no inclusions, were observed in striatum and substantia nigra of all normal aged human (Fig. 1B) and aged monkey (Fig. 1C) cases. In contrast, specific staining was eliminated in sections in which the primary antibody solvent, an irrelevant IgG, or preabsorbed antibody, was substituted for the primary α-synuclein antibody (Fig. 1D).

Fig. 1. High-power photomicrographs of α-synuclein immunoreactivity within the Lewy bodies (arrows) and neurites (arrowheads) in the substantia nigra of individuals with Parkinson's disease (A). This is contrasted with the non-aggregated staining pattern seen in the neuropil and perikarya (arrows) in the aged human (B) and aged monkey (C) nigra. No α-synuclein immunostaining was observed when the primary antibody was preadsorbed with the specific antigen (D). Neuromelanin appeared as brown granular deposits. Scale bar = 32 μm in panel D (applies to A–D).

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Age-associated increases in α-synuclein immunoreactivity in the human ventral midbrain In normal human cases, α-synuclein immunoreactivity revealed an increase staining intensity as a function of normal aging. In nigral perikarya, α-synuclein-ir was barely detectable in the young group (Figs. 2A, B). With increasing age, intense α-synuclein immunoreactivity was observed within the soma of neurons distributed throughout the substantia nigra pars compacta in middle-aged (Figs. 2C, D) and aged individual (Figs. 2D, F). In this regard, the reaction product indicative of α-synuclein protein often filled the cytoplasm of middle-aged and aged nigral perikarya, a phenomenon rarely seen in young cohorts. In contrast, the intensity and density of α-synuclein-ir neuropil was unaltered across the three age groups attesting to the specificity of changes observed within nigral perikarya. The age-related increases in

nigral α-synuclein stand in contrast to what was observed within the adjacent ventral tegmental area. In this region, α-synuclein immunoreactivity was rarely observed with only an occasional immunoreactive cell seen in the aged group (Fig. 3). Stereological estimates within the nigra revealed a significant age-related increase in the number of the α-synuclein-ir neurons but not the number of melanin-containing profiles (Table 2). A factorial ANOVA revealed a statistically significant difference in the number of α-synuclein-ir neurons within the substantia nigra across groups [F(2,15) = 7.51; P < 0.01]. Post hoc analyses revealed statistically significant differences in the number of α-synuclein-ir neurons between the young and aged groups (P < 0.01), but not between young and middle-aged groups (P > 0.05) or between middle-aged and aged groups (P > 0.05; Fig. 4A). Relative to young individuals, stereological analyses revealed a 269% and a 639% increase in the number of α-synuclein-ir neurons in middle-aged

Fig. 2. Low- (A, C, E) and high-power (B, D, F) photomicrographs of nigral sections from young (A, B), middle-aged (C, D), and aged (E, F) human subjects, illustrating α-synuclein immunoreactivity. α-Synuclein-ir was hardly detectable within the nigral neuron in young subjects (B). As aging advanced, intense α-synuclein-ir neurons (D, F, arrows) were distributed within the substantia nigra pars compacta in middle-aged and aged groups. Scale bar = 32 μm in F (applies to B, D, F), 120 μm in A, C, E.

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Fig. 3. Low- (A, B) and high-power (C, D) photomicrographs of substantia nigra (A, C) and ventral tegmental area (B, D) from an aged human subject, illustrating α-synuclein immunoreactivity. Note that there is only rare α-synuclein immunolabeled neurons in ventral tegmental area in human aging. Scale bar = 32 μm in D (applies to C, D), 120 μm in panels A, B.

and aged individuals respectively (Table 2). Additionally, there was a strong positive correlation between α-synuclein-ir neuronal number and increasing age (r = 0.75; P < 0.001; Fig. 4B). In contrast to α-synuclein-ir neurons, stereological analyses of NMcontaining nigral neurons revealed that the number of neurons making up this population was unaltered as a function of age (F(2,15) = 1.09; P > 0.05; Table 2). In contrast, there were no significant correlations between the number of melanin-containing neurons and age (r = 0.11; P > 0.05). The robust increase in the number of α-synuclein-ir nigral neurons in the face of stable numbers of NM-containing nigral neurons suggests that a phenotypic shift in α-synuclein protein levels from undetectable in the young cases to detectable in the middle and aged cases was responsible for this robust effect. To confirm this hypothesis, we examined the influence of advancing age on relative intracellular α-synuclein protein levels Table 2 Stereological estimates of melanin and α-synuclein neuronal number and optical density in the human substantia nigra Group

Case NM neuronal number number

Young 6 Middle- 4 aged Aged 8 a b

α-Synuclein-ir/ NM number

179,227 ± 53,432 8936 ± 2922 206,435 ± 59,518 32,930 ± 13,814

Optical density of α-synuclein-ir neurons 1572.19 ± 274.94 1725.08 ± 326.41

166,575 ± 56,532 66,012 ± 12,566 a 2469.08 ± 175.34a, b

P < 0.01 compared with young group. P < 0.05 compared with middle-aged group.

Fig. 4. (A) Stereological estimates reveal that the number of α-synuclein-ir nigral neurons significantly increases as a function of age. **P < 0.01 compared with young group. (B) Scatterplots showing the significant correlation between α-synuclein-ir neuronal number and age.

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within nigral neurons with computer-assisted fluorescence intensity measurements. Using the Fluoroview software coupled to an Olympus confocal microscope, we found that only a few of nigral neurons in young and middle-aged subjects displayed detectable α-synuclein immunofluorescence (Figs. 5A1, B1). Those nigral neurons with detectable α-synuclein-ir displayed low optical density measures indicative of low levels of protein. As aging progressed, individual neurons in aged cases displayed intense α-synuclein-ir immunofluorescence within nigral perikarya (Fig. 5C1). To confirm these qualitative observations, we performed quantitative fluorescence intensity measurement on individual α-synuclein-ir neurons from all 18 human subjects. An analysis of variance revealed that OD of α-synuclein immunofluorescence intensity was significantly different between the young, middle-aged, and aged group [F(2,15) 8.02; P < 0.005]. Post hoc analyses revealed statistically significant differences in αsynuclein fluorescence intensity between the young and aged groups (P < 0.01), between the middle-aged and aged groups (P < 0.05), but not between young and middle-aged groups (P > 0.05; Fig. 6A). Relative to young group, the OD of α-synuclein-ir fluorescence intensity was increased 9.7% in

middle-aged individuals and 56.6% in the aged individuals (Table 2). The rise in α-synuclein fluorescence intensity was significantly correlated with increasing age (r = 0.69, P < 0.002; Fig. 6B). We wanted to establish whether the increase of α-synuclein protein was associated with the decrease of TH expression in the nigral neurons (Figs. 5A2, B2, C2). Thus, we analyzed TH immunoreactive (TH-ir) fluorescence intensity separately in nigral neurons that did or did not contain α-synuclein immunofluorescence profiles. We found that, as a function of age, the expression of TH did not change in neurons with undetectable expression of α-synuclein [F(2,15) = 0.89; P > 0.05]. When evaluated independent of age, TH-ir intensity was significantly decreased in the neurons with α-synuclein profiles (OD = 2479.72 ± 369.24) when compared with the neurons without α-synuclein-ir profiles (OD = 3355.27 ± 404.87; P < 0.01). Evaluating TH-ir as a function of age and α-synuclein expression, an analysis of variance revealed a difference in the expression of TH-ir within α-synuclein containing profiles [F(2,15) = 7.67; P < 0.001]. Post hoc analyses of TH-ir OD within α-synuclein-ir cytoplasm revealed that, relative to both young (P < 0.01) and middle-aged (P < 0.05) individuals, there was a statistically significant decrease in TH-ir in aged nigral

Fig. 5. Laser confocal microscopic images of substantia nigra from young (A1–A3), middle-aged (B1–B3) and aged (C1–C3) subjects, illustrating the intensity of α-synuclein (A1, B1, C1), TH (A2, B2, C2) immunofluorescence and the co-localization of both proteins (merged pictures; A3, B3, C3). Note that TH (C2, C3; arrows) immunofluorescent intensity was diminished in the neurons with α-synuclein (B1, C1, arrows) accumulation as compared with the neurons without α-synuclein accumulation (C2, C3; arrowheads). No α-synuclein profile was seen in the nigral neuron in young group. Scale bar = 72 μm in C3 (applies to A1–C3).

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Fig. 6. (A) Histogram showing that the optical density (OD) of α-synucleinir nigral neurons was significantly increased with aging processes. **P < 0.01 compared with young group, ~~P < 0.05 compared with middleaged group. (B) Scatterplots illustrate the correlation between OD of α-synuclein-ir neurons and age.

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not between middle-aged and aged groups (P > 0.05). Relative to young monkeys, the number of α-synuclein-ir nigral neurons increased 169% and 215% in the middle-aged and aged groups respectively (Fig. 10A). Correlational analyses demonstrated a strong association between the number of α-synuclein-ir neurons and advancing age (r = 0.68; P < 0.001; Fig. 10B). Confocal microscopic analyses further verified that the αsynuclein immunofluorescent profiles were seldom detected in the nigral neurons of young monkeys (Fig. 11A1). In contrast, with advancing age, numerous intensely immunofluorescent α-synuclein labeled nigral neurons were seen in middle-aged (Fig. 11B1) and aged monkeys (Fig. 11C1). These qualitative observations were supported by quantitative assessments (Table 3). An analysis of variance demonstrated a statistically significant difference in the OD of α-synuclein-ir neurons across the three age groups (F(2,21) = 6.07; P < 0.01). Post hoc analyses revealed a significant difference in the OD of α-synuclein-ir neurons between young and aged groups (P < 0.01), but not between young and middleaged groups (P > 0.05) or middle-aged and aged groups (P > 0.05, Fig. 12A). In comparison to young monkeys, the OD of αsynuclein-ir neurons was increased 46.47% in middle-aged and 83.08% in aged groups. A highly significant positive correlation was also found between the increasing OD of α-synuclein-ir neurons and increasing age (r = 0.68, P < 0.0002; Fig. 12B). Confocal microscopy was used to assess the degree of colocalization and the relationship of staining intensities between TH and α-synuclein. TH-ir fluorescence intensity was significantly lower in the neurons with α-synuclein-ir cytoplasm than

neurons (Fig. 7A). Correlative analysis verified that the age-related expression of TH-ir OD was inversely correlated with α-synuclein-ir OD (r = − 0.55; P < 0.001; Fig. 7B). Age-associated increases in α-synuclein immunoreactivity in the nonhuman primate ventral midbrain In young monkeys, a few light α-synuclein-ir neurons were detected within the substantia nigra (Fig. 8B). As aging progressed, numerous intense α-synuclein-ir neurons were seen within the nigra of middle-aged and aged monkeys (Figs. 8D, F). The intensity of α-synuclein-ir neurons appeared greatest in the aged monkey nigra (Fig. 8F) relative to the middle-aged or young monkey nigra (Fig. 8D). In contrast, there was a similar density and intensity of α-synuclein-ir terminals in the striatum and substantia nigra across groups (Figs. 8A, C, E), again attesting to the specificity of the changes seen within nigral perikarya. In VTA, α-synuclein-ir neurons were not detected in the young and middleaged monkeys. Similar to what was seen in the human cases, an occasional lightly stained α-synuclein-immunoreactive cell was observed in the aged monkey VTA (Fig. 9). Stereological estimates demonstrated a significant increase in the number of α-synuclein-ir perikarya in the substantia nigra of the middle-aged and aged monkeys relative to young monkeys (Table 3). A factorial ANOVA confirmed a statistically significant difference in the number of α-synuclein-ir neurons within the substantia nigra across the age groups (F(2,21) = 6.91; P < 0.005). Post hoc analyses revealed a significant difference in the number of α-synuclein-ir neurons between the young and middle-aged groups (P < 0.05) and between the young and aged groups (P < 0.01) but

Fig. 7. (A) Histogram illustrating the optical density (OD) of TH-ir nigral neurons was significantly decreased in the neurons with α-synuclein-ir as compared with the neurons without detectable α-synuclein-ir. Assessing TH-ir as a function of age and α-synuclein expression, the TH-ir OD was significantly decreased in aged group. **P < 0.01 compared with young group, – –P < 0.05 compared with middle-aged group. (B) Scatterplots illustrate a negative correlation between TH-ir OD and α-synuclein-ir OD.

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Fig. 8. Low- (A, C, E) and high-power (B, D, F) photomicrographs through the mid-substantia nigra from young (A, B), middle-aged (C, D), and aged (E, F) rhesus monkeys illustrating the distribution of α-synuclein immunoreactivity. Note the intense α-synuclein-ir neurons in middle-aged (D; arrows) and aged (F; arrows) animals. The intensity of α-synuclein-ir neurons was higher in aged monkeys (F) relative to the middle-aged monkeys. Scale bar = 32 μm in panel F (applies to B, D, F), 600 μm in A, C, E.

the neurons without detectable α-synuclein-ir profiles (Fig. 11). Quantitative TH-ir fluorescence intensity measurements confirmed that the OD of TH-ir fluorescence intensity was significantly decreased in the neurons with α-synuclein-ir profiles (1391.60 ± 350.92) as compared with the neurons without α-synuclein-ir profiles (1716.56 ± 424.64; P < 0.01) regardless of age. When age was included in the analyses (Fig. 13), the OD of TH-ir fluorescence was significantly different in the neurons with α-synuclein-ir profiles (F(2,21) = 3.77, P < 0.05) but not in the neurons without α-synuclein-ir [F(2,21) = 0.62; P > 0.05]. Post hoc analyses of the OD of TH-ir fluorescence intensity in the

neurons with α-synuclein-ir cytoplasm revealed statistically significant differences between young and middle-aged groups (P < 0.05) and between young and aged groups (P < 0.05) but not between middle-aged and aged groups (P > 0.05). The correlative analysis revealed that the linear relationship between TH-ir and α-synuclein-ir fluorescence intensity was not significant (r = 0.23, P > 0.05). To establish the specificity of these age-related changes to α-synuclein, control experiments were performed in monkeys comparing the distribution and staining intensity of β-synuclein immunoreactivity, a close, but non-pathogenic, α-synuclein homo-

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Fig. 9. Low- (A, B) and high-power (C, D) photomicrographs through the substantia nigra (A, C) and ventral tegmental area (B, D) from an aged monkey. Note that while intense α-synuclein was seen in the substantia nigra, only rare VTA neurons displaying light α-synuclein immunostaining neurons were seen in the ventral tegmental area. Scale bar = 32 μm in panel D (applies to C, D), 600 μm in panels A, B.

log. Abundant β-synuclein immunoreactive profiles were observed in the neuropil. Moderate β-synuclein immunolabeling was detected in nigral neuronal perikarya. When compared to the young group, there were no qualitative differences in the patterns or intensity of β-synuclein immunofluorescence between found in young, middle, and aged groups. Quantitative β-synuclein-ir OD measurements revealed a stable expression of this protein across the three age groups (P > 0.05, Fig. 14). Alterations of α-synuclein immunoreactivity with PK treatment PK digestion was used to determine whether the age-related expression of α-synuclein was soluble (non-aggregated) or insoluble (aggregated). In this study, PK digested soluble α-synuclein completely. Without PK treatment, nigral sections from aged humans and monkeys stained robustly for α-synuclein labeling neuropil and perikarya (Figs. 15A, C). In contrast, αsynuclein immunoreactivity was virtually eliminated in sections

Table 3 The number and optic density of α-synuclein-ir nigral neurons in monkey

Fig. 10. (A) Histogram illustrating that the number of α-synuclein-ir nigral neurons significantly increases in monkeys as a function of age. *P < 0.05, **P < 0.01 compared with young group. (B) Scatterplots showing the correlation between the number of α-synuclein-ir neurons and age.

Group

Case number

Total neuronal number

Optic density

Young Middle-aged Aged

4 10 10

17,656 ± 4411 47,604 ± 22,592* 55,683 ± 13,736**

359.07 ± 88.370 525.94 ± 163.03 657.38 ± 148.64**

*P < 0.05, **P < 0.05 compared with young group.

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Fig. 11. Laser confocal microscopic images of substantia nigra from young (A1–A3), middle-aged (B1–B3), and aged (C1–C3) rhesus monkeys, illustrating the intensity of α-synuclein (A1, B1, C1) and TH (A2, B2, C2) immunofluorescence and the co-localization α-synuclein and TH (merged pictures; A3, B3, C3). Note that TH (B2, C2; arrows) immunofluorescent intensity is diminished in neurons with detectable α-synuclein (B1, C1, B3, C3; arrows) relative to nigral neurons without detectable α-synuclein (arrowhead). No α-synuclein profiles were seen in the nigral neuron in young group. Scale bar = 72 μm in C2 (applies to A1–C3).

treated with PK (Figs. 15B, D). This indicates that the α-synuclein that accumulates in nigral perikarya as a function of age is soluble and non-aggregated. As a positive control, we treated PD brains with PK. In the PD brains, α-synuclein stained inclusions and atrophic neurites and this staining pattern was unaltered by proteinase K digestion (Fig. 15F). This indicates that the accumulation of α-synuclein in PD is distinct from the accumulation seen in normal aging. Discussion The present study demonstrates the robust association between α-synuclein and aging within nigral neurons in both humans and nonhuman primates. In this regard, α-synuclein was almost undetectable within nigral perikarya in young monkeys and humans as α-synuclein immunoreactivity in these cases was restricted to fibers and terminals in keeping with this protein’s well-established role in synaptic processes (Totterdell and Meredith, 2005; Yavich et al., 2004). The effects of aging on α-synuclein expression within nigral perikarya were quite impressive. In humans, there was a 269% and 639% increase

in the number of perikarya displaying detectable α-synuclein-ir in middle and aged individuals relative to young cohorts. The fluorescence intensity of α-synuclein-ir on a per neurons basis was increased 56.6% in aged individuals as well. In rhesus monkeys, the number of detectable α-synuclein-ir neurons increased 169% and 215% in middle aged and aged monkeys relative to young. As seen with humans, the fluorescence intensity of individual nigral neurons was increased in middle and aged monkeys by 46% and 83% respectively. These agerelated increases in α-synuclein are supported by the immunoblot data reported by Li et al. (2004a,b) examining frozen midbrain samples. The importance of these findings is enhanced by the specificity of these changes. First, the expression of β-synuclein, a non-pathogenic analogue of α-synuclein (Ohtake et al., 2004; Lincoln et al., 1999), is unchanged as a function of aging, suggesting that the age-related expression in α-synuclein is a specific pathological event. Second, the age-related expression of α-synuclein within the adjacent VTA of monkeys and humans was a relatively rare event, indicating that the age-related accumulation of α-synuclein is specific for the vulnerable region of PD and is rarely expressed within an adjacent dopaminergic

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Fig. 14. Histogram illustrating that the optical density (OD) of β-synucleinir nigral neurons was not altered as a function of advancing age.

Fig. 12. (A) Histogram illustrating that the optical density (OD) of α-synuclein-ir nigral neurons is significantly increased in monkey nigral neurons as a function of age. **P < 0.01 compared with young group. (B) Scatterplots showing the correlation between the OD of α-synuclein-ir neurons and age.

region that is resistant to degeneration. Thirdly, the obvious increases in α-synuclein were preferentially observed within perikarya. Although we cannot rule out the presence of subtle changes in axons or terminals since the expression patterns in these cellular compartments were not quantified, there were no apparent alterations in monkeys or humans as a function of age. Lastly, it should to be noted that the age-related expression of αsynuclein expression seen in humans and monkeys is not in the form of inclusions as is seen in PD (e.g. Chu et al., 2006). The cytoplasmic appearance of the reaction product may be analogous to the microaggregates described by Meredith and coworkers at the ultrastructural level (Meredith et al., 2004). However, our data clearly indicate that the increases in α-synuclein are distinct from

Fig. 13. Histogram illustrating that, in monkeys, the optical density (OD) of TH-ir nigral neurons was significantly decreased in nigral neurons with detectable α-synuclein-ir when compared with the OD of TH-ir nigral neurons without detectable α-synuclein-ir profiles. *P < 0.05 compared with young group.

the inclusions seen in PD as the former was soluble and digested by proteinase K while the latter was insoluble and impervious to proteinase K digestion. It is well established that, in all species examined, including monkeys (McCormack et al., 2004) and humans (Muthane et al., 1998; Chu et al., 2002), the number of nigral neurons does not change as a function of age. However, there can be a profound phenotypic down-regulation of dopaminergic markers (Chu et al., 2002; McCormack et al., 2004; Dawson et al., 1999; Ma et al., 1999; Emborg et al., 1998). Indeed, we have previously demonstrated decreases in TH and dopamine transporter optical density in aged monkeys and humans to such a degree that many nigral neurons become undetectable using these immunohistochemical markers. It is theoretically possible that the increased number of cells expressing α-synuclein within nigral neurons is due to neurogenesis. However, the stability of the number of nigral neurons as a function of age, the lack of neurogenesis within the nigra under any experimental condition (Frielingsdorf et al., 2004) and the robust increase of α-synuclein optical density on a per neuron basis all support the concept that the increased number of α-synuclein positive nigral neurons reflects an increase in detectable protein. A question that arises from these data is why does such an agerelated increase occur? One possibility is that there is enhanced synthesis of α-synuclein within nigral neurons that leads to enhanced detectability as a function of age. Existing studies using northern blot analysis indicate that this is not the case (Le et al., 2002). However, additional studies employing in situ hybridization techniques on individual neurons are currently underway to further test this hypothesis. More likely, however, is that α-synuclein accumulates within the cell due to impaired clearance of the protein. Indeed, impaired clearance results in protein misfolding, and this has been hypothesized to be a main culprit in cellular dysfunction and death in neurodegenerative diseases (Snyder and Wolozin, 2004; Dawson and Dawson, 2003; Li et al., 2004a,b). While initially thought to be cleared by the proteosome, it is becoming more clear that α-synuclein is selectively translocated into lysosomes for degradation by chaperone-mediated autophagy (Cuervo et al., 2004; Lee et al., 2004; Rockenstein et al., 2005), perhaps in collaboration with the proteosome (Mandel et al., 2005). For example, Lee et al. (2004) used COS-7 cells to demonstrate that α-synuclein is degraded using a lysosomal pathway and blockade of this pathway induces the formation of α-synuclein aggregates. This degradation of α-synuclein occurs through the lysosome via chaperones such as heat shock protein-70, which when overexpressed protects dopamine neurons from degeneration

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Fig. 15. High-power photomicrographs of nigral sections from normal aged subject (A, B), rhesus monkey (C, D), and Parkinson's disease (E, F), illustrating the patterns of α-synuclein immunoreactivity with (B, D, F) and without (A, C, E) proteinase K treatment. α-Synuclein immunolabeling (A, C, E; arrows) observed on the sections without proteinase K treatment was virtually eliminated following proteinase K treated sections (B, D, F). In aged monkeys and humans indicating that the α-synuclein accumulation seen in normal aging is soluble. In contrast, immunoreactive inclusions (arrowheads) and neurites were detected on both sections of PD with and without proteinase K treatment, indicating that the inclusions are insoluble. Scale bar = 32 μm in F (applies to A–F).

in Drosophila (Auluck et al., 2002) and prevents α-synuclein accumulation in transgenic mice (Klucken et al., 2004). The carboxyl terminus of the HSP70 interacting protein may be a determining factor as to whether α-synuclein degradation takes a lysosomal or proteosomal path (Shin et al., 2005). It is well established that lysosomal function declines with cellular aging (Keller et al., 2004), and thus the molecular events associated with age-related lysosomal dysfunction form the basis for the hypothesis that age-related increases in α-synuclein are the result of this defect. This hypothesis is supported by studies in transgenic mice (Meredith et al., 2002), and future studies confirming this hypothesis in primates are warranted. The fact that there are age-related increases in α-synuclein is interesting, but becomes even more relevant within the context of cellular dysfunction and neurological disease. The critical aspect of

the present series of experiments is not just the demonstrated accumulation of α-synuclein within aging nigral neurons but its strong association with a loss of dopamine phenotype, one of the earliest cellular manifestations seen within the substantia nigra in Parkinson's disease (Chu et al., 2002; Fearnley and Lees, 1991; Kastner et al., 1993; Ross et al., 2004). In both humans and monkeys, accumulation of α-synuclein within nigral perikarya was associated with decreased TH expression within those same neurons. Indeed, for both species, regardless of age, decreased TH was only observed within nigral neurons displaying detectable α-synuclein-ir and, regardless of age, adjacent neurons without detectable synuclein expression displayed stable levels of TH. These age-related decreases in nigral TH likely mediate the loss of striatal dopamine that has been demonstrated by others (McCormack et al., 2004; Kawamura et al., 2003). These data are

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Fig. 16. Hypothetical process by which α-synuclein influences nigrostriatal function during normal aging and PD. (top) During normal aging, soluble α-synuclein accumulates within nigral perikarya and this causes a phenotypic down-regulation of dopamine. The level of dopamine insufficiency is not severe enough to induce the cardinal symptoms of PD. (bottom) In PD, the same process occurs, but at some point, lysosomal function is overwhelmed, the progressive loss of dopaminergic function becomes more severe, and the magnitude of dopamine dysfunction is sufficient for the cardinal symptoms of PD to occur.

complementary to what we had previously observed in patients with Parkinson's disease? (Chu et al., 2006). In PD patients, cells without α-synuclein-ir displayed the greatest expression of TH. Cells with non-aggregated α-synuclein displayed diminished, but detectable TH. Lastly, the expression of TH was virtually undetectable in nigral neurons with α-synuclein-ir inclusions. How do the age-related increases in non-aggregated αsynuclein potentially relate to the symptoms observed in patients with Parkinson's disease? Based upon our present and previous (Chu et al., 2006) findings, we hypothesize that age-related decreases in nigrostriatal dopamine are mediated by increases in non-aggregated α-synuclein (Fig. 16, top). This accumulation may be due to a suboptimal, but still functioning, lysosome. The increase in α-synuclein is never of a sufficient magnitude to drive dopamine levels past a threshold that would engender the cardinal signs and symptoms of PD. In PD, for reasons that still remain to be determined, we hypothesize that the lysosomal burden of the nigral neuron is overwhelmed, the age-related accumulation of αsynuclein becomes further intensified and misfolded to form an inclusion. These events cause the cell to completely lose its dopaminergic phenotype, dopamine levels pass a critical threshold, and cause symptoms to emerge (Fig. 16, bottom). One should note that all of the data presently discussed regarding α-synuclein expression and nigrostriatal degeneration focus on the dopaminegic phenotype and not frank neuronal

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degeneration. Indeed, the link between α-synuclein and neuronal survival always involves supraphysiological overexpression in transgenic Drosophila (Feany and Bender, 2000; Auluck et al., 2002) or rats and monkeys receiving viral vector injections to the midbrain (Kirik et al., 2002, 2003). Under the physiological conditions of normal aging, there is a loss of phenotype without cell death. Under the physiological conditions of PD, there is a clear link between α-synuclein expression and dopamine phenotype (Chu et al., 2006). The best association between α-synuclein, PD and cell death is the postmortem cases from patients with the αsynuclein mutation (Polymeropoulos et al., 1997; Singleton et al., 2003). However, these cases still cannot determine whether the physiological overexpression of α-synuclein resulted in a phenotypic down-regulation of nigrostriatal dopamine and other factors related to other alterations in the genome were responsible for the cell death. Dissociating α-synuclein's effects on phenotype versus neuronal viability is a fruitful area for study. Finally, are the present data relevant for PD therapy? If one accepts the hypothesis that α-synuclein overexpression is associated with down-regulation of the dopamine phenotype, then suppressing its expression might reverse the striatal dopamine insufficiency that causes cardinal symptoms. One possibility is to disaggregate already formed inclusion bodies, and there is some evidence to support this approach (e.g. Li et al., 2004a,b; Hayashita-Kinoh et al., 2006). However, a more parsimonious approach might be to never allow the α-synuclein to accumulate to levels that would allow protein misfolding and the formation of inclusions. Techniques such as siRNA (Sapru et al., 2006) or the development of small molecules (Iwata et al., 2003; HayashitaKinoh et al., 2006) that reduce non-aggregated α-synuclein expression could be delivered to the PD patient early in the disease process and prevent or reverse the loss of dopamine phenotype that causes the PD cardinal symptoms. These events could lessen the lysosomal burden and potentially prevent the neuronal degeneration that ultimately occurs within the nigra of these patients. In summary, the present data illustrate a robust increase in αsynuclein protein as a function of human aging and this change is strongly associated with decreases in nigrostriatal activity. Similar findings were observed in young and aged nonhuman primates, and these animals can be employed as a natural animal model for this pathology. We believe that the age-related increase αsynuclein puts an added burden on an already challenged lysosome, ultimately causing inclusion bodies to form in PD nigral neurons and drive dopamine levels past a symptomatic threshold. Based upon these data and hypotheses, we propose that reducing age-related α-synuclein expression might prevent inclusion formation and may serve as a powerful target for therapeutic intervention. References Andersen, A.H., Zhang, Z., Zhang, M., Gash, D.M., Avison, M.J., 1999. Age-associated changes in rhesus CNS composition identified by MRI. Brain Res. 829, 90–98. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M., 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295, 865–868. Baldereschi, M., Di Carlo, A., Rocca, W.A., Vanni, P., Maggi, S., Perissinotto, E., Grigoletto, F., Amaducci, L., Inzitari, D., 2000. Parkinson's disease and parkinsonism in a longitudinal study: two-fold

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