Increased oligomerization and phosphorylation of α-synuclein are associated with decreased activity of glucocerebrosidase and protein phosphatase 2A in aging monkey brains

Neurobiology of Aging xxx (2015) 1e11

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Increased oligomerization and phosphorylation of a-synuclein are associated with decreased activity of glucocerebrosidase and protein phosphatase 2A in aging monkey brains Guangwei Liu a, b, Min Chen a, c, Na Mi a, b, Weiwei Yang a, b, Xin Li a, b, Peng Wang a, b, Na Yin a, b, Yaohua Li a, b, Feng Yue a, b, Piu Chan a, b, c, Shun Yu a, b, c, * a b c

Department of Neurobiology, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, Beijing, China Center of Parkinson’s Disease, Beijing Institute for Brain Disorders, Beijing, China Beijing Key Laboratory for Parkinson’s Disease, Beijing, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 June 2014 Received in revised form 2 June 2015 Accepted 3 June 2015

Aging is associated with an increased risk for Parkinson’s disease and dementia with Lewy bodies, in which a-synuclein (a-syn) oligomerization plays key pathogenic roles. Here, we show that oligomeric asyn levels increase with age in the brain of cynomolgus monkeys and are accompanied by a decrease in the expression and activity of glucocerebrosidase (GCase), a lysosomal enzyme whose dysfunction is linked to accumulation of oligomeric a-syn. Besides, levels of a-syn phosphorylated at serine 129 (pS129 a-syn), a modification that promotes a-syn oligomerization also increase with age in the brain and is associated with a reduction in the activity of protein phosphatase 2A (PP2A), an enzyme that facilitates asyn dephosphorylation. The inverse relationship between levels of oligomeric a-syn and pS129 a-syn and activity of GCase and PP2A was more evident in brain regions susceptible to neurodegeneration (i.e., the striatum and hippocampus) than those that are less vulnerable (i.e., cerebellum and occipital cortex). In vitro experiments showed that GCase activity was more potently inhibited by oligomeric than by monomeric a-syn in the lysosome-enriched fractions isolated from brain tissues and cultured neuronal cells. Inhibition of GCase activity induced an elevation of oligomeric a-syn levels, which was shown to increase pS129 a-syn levels and reduce PP2A activity in cultured neuronal cells. The alterations in oligomeric and pS129 a-syns and their association with GCase and PP2A in aging brains may explain the vulnerability of certain brain regions to neurodegeneration in Parkinson’s disease and dementia with Lewy bodies. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: a-Synuclein Parkinson’s disease Dementia with Lewy bodies Glucocerebrosidase Cynomolgus monkey Aging

1. Introduction A major process characterizing aging brains is the formation of fibrous protein inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs), which are associated with age-related neurodegenerative disorders such as Parkinson’s disease (PD), PD dementia (PDD), and dementia with Lewy bodies (DLB) (Jellinger, 2003, 2004; Saito et al., 2004; Vernon et al., 2010). These diseases differ in terms of clinical symptoms and localization of LB pathology (LBP) in the brain, which may be accompanied by other pathological lesions. For example, PD is characterized by impaired motor function with

* Corresponding author at: Department of Neurobiology, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, 45 Changchun Street, Beijing 100053, China. Tel.: þ86 10 8319 8890; fax: þ86 10 8316 1294. E-mail address: [email protected] (S. Yu). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2015.06.004

degeneration of the nigrostriatal dopaminergic system as well as the formation of LBs and LNs in the medullary and olfactory nuclei that spreads gradually to most brain structures, including the dorsal motor nucleus of the vagus nerve, locus coeruleus, substantia nigra, nucleus basalis, hypothalamus, hippocampus, cingulate and temporal gyri, and frontal and parietal cortices (Braak et al., 2003). The formation of extra-nigral LBP is associated with nonmotor complications, with dementia becoming predominant at later stages of PD (Vernon et al., 2010). DLB, which is the second most frequent cause of dementia in the elderly after Alzheimer’s disease, shares clinical and pathological features with PDD: patients with either disease exhibit spontaneous motor parkinsonism and have visual hallucinations and cognitive impairment (Watson et al., 2009), as well as varying degrees of LBP affecting brain stem nuclei and limbic and neocortical areas along with Alzheimer’s diseaseerelated pathological changes such as neurofibrillary tangles and senile plaques (Jellinger, 2003; McKeith et al., 2005).

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The major component of LBs and LNs is fibrillated and phosphorylated a-synuclein (a-syn), a small protein normally present in a soluble, monomeric form in neurons (Baba et al., 1998; Gai et al., 1998; Spillantini et al., 1998). The deposition of fibrillated a-syn in LBs and LNs as a basis for diseases such as PD and DLB is supported by the observation that point mutations in and multiplication of the a-syn gene are linked to early-onset familial PD (Chartier-Harlin et al., 2004; Kruger et al., 1998; Polymeropoulos et al., 1997; Singleton et al., 2003; Zarranz et al., 2004). Although a-syn-immunoreactive LBs and LNs are associated with LB diseases, recent studies suggest that soluble asyn aggregates (oligomers or protofibrils) rather than fibrillated a-syn are toxic to neurons (Colla et al., 2012; Malchiodi-Albedi et al., 2011; Roostaee et al., 2013; Volles and Lansbury, 2003). Oligomeric a-syn can induce neuronal death (Dimant et al., 2013; Outeiro et al., 2008; Winner et al., 2011) or cause synaptic dysfunction leading to cognitive impairment in PDD and DLB (Choi et al., 2013; Kramer and Schulz-Schaeffer, 2007; SchulzSchaeffer et al., 2010). Thus, the formation and accumulation of a-syn aggregates in the brain may increase the vulnerability of neurons to degeneration. Mutations in the GBA (glucosidase, beta, acid) gene encoding bglucocerebrosidase (GCase) that cause Gaucher disease (Grabowski, 2008) are a recognized risk factor for PD (Sidransky et al., 2009). GCase is a lysosomal enzyme that hydrolyzes glucosylceramide (GlcCer) into glucose and ceramide (Grabowski, 2008). GBA gene mutations lead to the inhibition of lysosomal function and the accumulation of GlcCer, which promotes a-syn oligomerization by stabilizing soluble oligomeric intermediates (Mazzulli et al., 2011). Conversely, the accumulation of oligomeric a-syn can alter the activity of GCase by modulating its transport from the endoplasmic reticulum to the lysosome (Cooper et al., 2006; Mazzulli et al., 2011; Thayanidhi et al., 2010). This reciprocal interaction between GCase and a-syn may underlie the aggregation of a-syn in the brain of PD patients with GBA mutations; this is supported by findings from autopsies of patients with GBA-associated parkinsonism that revealed elevated levels of oligomeric a-syn and a-syn-immunoreactive LBs and LNs in the cortex and hippocampus (Choi et al., 2011). Although the above evidence suggests an interaction between GCase and a-syn in PD patients with GBA mutations, a recent study has also found a negative correlation between GCase activity and a-syn levels in PD patients without these mutations, suggesting that the interaction is present even in sporadic PD (Murphy et al., 2014). It has been reported that in PD patients serine 129 phosphorylated a-syn (pS129 a-syn) rises from approximately 5 % basal level to 30%e100%, depending on the brain region and pathology severity. This enhancement is directly related to the amount of insoluble protein found in the tissue and dramatically increases with disease progression (Zhou et al., 2011). Studies performed in cell lines associate phosphorylated a-syn with increased formation of soluble oligomers (Arawaka et al., 2006; Kragh et al., 2009), suggesting that a-syn phosphorylation is important for its transformation into pathogenic aggregates. Inversely, aggregated a-syn is shown to reduce the activity of protein phosphotase 2A (PP2A), an enzyme that facilitates a-syn dephosphorylation (Wu et al., 2012). These findings suggest a possibility that the accumulation of a-syn oligomers due to GCase dysfunction could be enhanced by reducing PP2A activity. Aging is the most important risk factor for PD (Zhang et al., 2005). Therefore, investigating the mechanism for a-syn aggregation in the aging brain may explain the age-related increase in PD incidence and the vulnerability of some population of neurons to degeneration in PD. The present study investigated age-dependent alterations in a-syn oligomerization and

phosphorylation and their association with alterations in the activity of GCase and PP2A in various brain regions of cynomolgus monkeys. 2. Methods 2.1. Animals Cynomolgus monkeys (Macaca fascicularis; n ¼ 15) were purchased from a local nonhuman primate breeder (Grandforest Co, Guangxi, China) with detailed individual birth records and quarantine certificates. All animals were healthy and without physical impairments. The animals were acclimated to the laboratory environment for at least 2 months before dissection and were divided into 3 age groups: young (range: 3e4 years; n ¼ 5), middle age (range: 10e12 years; n ¼ 5), and aged (range: 15 years; n ¼ 5). Animals were housed in a primate facility (Wincon TheraCells Biotechnologies Co, Ltd, Nanning, Guangxi, China) accredited by the Association for Assessment and Accreditation of Laboratory Animal Care under a 12:12-hour light-dark cycle with free access to an un interrupted reverse osmosis water supply. Food was available twice daily and supplemented with fresh fruit and vegetables. The experimental protocol was approved by the Institutional Animal Care and Use Committee of WinconTheraCells Biotechnologies (permit no. WD-0312010). For cortical neuronal cultures, newborn Wistar rats were purchased from the Beijing Vital River Laboratory Animal Co Ltd. (Beijing, China) and used within 24 hours of birth. The study was approved by the local animal care and use committee. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996). Necessary euthanization procedures were made for monkeys. The animals were given intramuscular injection of ketamine (10 mg/kg) before transferred to the dissection room. The animals were then deeply anesthetized by intravenous injection of pentobarbital sodium (60 mg/kg) and confirmed complete loss of pain reflex and consciousness before conduction of perfusion and dissection. 2.2. Dissection of monkey brain tissues In deep anesthesia, the thoracic cavity of a monkey was opened, and the heart was exposed. The animal was perfused from the aorta with 2000e3000 mL of 0.01-mM phosphate-buffered saline (PBS) (pH 7.4). The brain was removed from the skull, and the brain tissues were dissected on ice, snap-frozen in liquid nitrogen, and stored at 80  C until use. 2.3. Isolation of mitochondrial and cytosolic fractions Mitochondrial and cytosolic fractions were isolated from brain tissues according to a previously reported procedure with slight modifications (Liu et al., 2009). Briefly, brain tissues from different regions were homogenized in mitochondrial isolation buffer containing 0.32-M sucrose, 1-mM EDTA (Kþ salt), and 10-mM Tris-HCl (pH 7.4). The cytosolic and crude mitochondrial fractions were obtained by differential centrifugation. High purity mitochondria were isolated from the crude mitochondrial fractions by Percoll (Bioshop, Burlington, Ontario, Canada) density gradient centrifugation. Protein concentration was determined using the bicinchoninic acid Protein Assay Kit (Pierce/Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

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2.4. Preparation of lysosome-enriched fractions The lysosome-enriched fraction of brain tissue was prepared as previously described (Murphy et al., 2014). Briefly, freshly-frozen tissue was homogenized in homogenization medium composed of 0.32-M sucrose, 1-mM EDTA, 10-mM Tris-HCl (pH 7.4), and a protease inhibitor cocktail (complete, EDTA-free; Roche Diagnostics, Mannheim, Germany). The total homogenate was centrifuged to sediment the nuclear pellet and cellular debris. The pellet was washed twice, and the resultant supernatant was centrifuged to obtain a lysosome-enriched pellet, which was resuspended in homogenization medium. Protein concentration was measured with the bicinchoninic acid assay. 2.5. Preparation of a-syn oligomers Recombinant human a-syn was solubilized in sterile PBS (pH 7.0) to a final concentration of 100 mM. An Eppendorf tube containing 100 mM of a-syn solution was sealed with parafilm and incubated at 37  C for 7 days with continuous shaking (650 rpm) on an Eppendorf Thermomixer Comfort (Eppendorf AG 22331, Hamburg, Germany). The incubated solution was further subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to isolate the a-syn oligomers from the dimers and monomers. The a-syn oligomers were purified from the gel using a Micro Protein Recovery Kit (Sangon, Biotech, Shanghai, China) according to the protocol provided, aliquoted, and stored at 80  C until use. 2.6. Preparation of phosphorylated a-syn pS129 a-syn was prepared from recombinant human a-syn as previously described (Sasakawa et al., 2007). Purified a-syn was first incubated with casein kinase II (New England Biolabs, Ipswich, MA, USA), and the resultant pS129 a-syn was purified by anionexchange chromatography and verified by immunoblotting with an antibody against the pS129 a-syn (Epitomics, Burlingame, CA, USA) combined with mass spectrometry. The pS129 a-syn was concentrated by ammonium sulfate precipitation. 2.7. ELISAs for oligomeric and pS129 a-syns The oligomeric a-syn levels in brain tissues and cell lysates were measured by the enzyme-linked immunosorbent assay (ELISA) (ElAgnaf et al., 2006) using nonbiotinylated and biotinylated 3D5 mouse monoclonal antibodies (Yu et al., 2007) for capture and detection, respectively. After completion of the immunoreaction, the contents of each well of the ELISA plate were incubated with 100 mL of ExtrAvidin Alkaline Phosphatase (E-2636; Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20,000 in blocking buffer and then reacted with the enzyme substrate p-nitrophenyl phosphate (N1891; Sigma-Aldrich). The reaction was allowed to proceed for 30 minutes at room temperature, after which the absorbance was read at 405 nm using a Multiskan MK3 microplate reader (Thermo Scientific, UT, USA). To detect pS129 a-syn, 0.1 mg/mL of anti-pS129 a-syn polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in coating buffer was used for capture. The remaining steps were the same as those for the detection of oligomeric a-syn by ELISA. 2.8. PP2A activity assay PP2A activity in brain homogenates was measured as previously described (Qi et al., 2011) using a PP2A Colorimetric Assay Kit (GenMed Scientifics Inc, Arlington, MA, USA). The protein

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concentration in the supernatant was determined by the Bradford assay (GMS 30030.1; GenMed Scientifics Inc) and was normalized to 5 mg/mL. 2.9. Western blot analysis Western blot analysis was performed as previously described (Alim et al., 2002). Briefly, protein samples (30 mg protein/lane) were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidenedifluoride membrane, and then incubated with rabbit monoclonal anti-GCase antibody (1:500; Epitomics, Burlingame, CA, USA) overnight at 4  C, followed by incubation with horseradish peroxidaseeconjugated goat anti-rabbit IgG (1:5000; Vector Laboratories, Burlingame, CA, USA) for 1 hour at 4  C. Immunoreactivity was detected using the enhanced chemiluminescence reagent (Promega, Madison, WI, USA). b-tubulin was used as a loading and internal control to enable sample normalization. 2.10. GCase activity assay GCase activity was determined using the QuantiChrom bGlucosidase Assay Kit (DBGD-100; BioAssay Systems Inc, Hayward, CA, USA). Distilled water (20 mL) was added to 2 wells of a clearbottom 96-well plate; 200 mL of either distilled water or calibrator was then added to the wells for a total volume of 220 mL. Samples (20 mL) were loaded in the other wells, and 200 mL of working reagent was added to each sample for a final reaction volume of 220 mL. The solutions were mixed by briefly tapping the plate, and optical density at 405 nm was measured immediately (t ¼ 0) and again after 20 minutes (t ¼ 20 minutes) on a plate reader and used to calculate GCase activity of the sample (U/L) based on the hydrolysis of 1-mM substrate per minute by 1 unit of enzyme at pH 7.0. 2.11. Incubation of the lysosome-enriched fraction with a-syn Isolated lysosomes were resuspended in phosphate buffer (0.1 mol/l Na2HPO4,12H2O, 0.1 mol/l NaH2PO4,2H2O, pH 7.0) and incubated at 37  C for 60 minutes with different concentrations (0.1, 1, or 10 mM) of a-syn monomer and oligomer before GCase activity was measured. 2.12. Primary cultures of rat cortical neurons Newborn rats were decapitated. The brains were removed and bilateral cortices were dissected. Primary rat cortical neurons were dissociated as described previously (Yu et al., 2008) and plated at a density of 1  105 cells/cm2 in poly-L-lysine-coated 35-mm diameter dishes (Nunc, Roskilde, Denmark) or flasks (Corning, NY, USA) in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) containing 10% horse serum (HyClone Laboratories, South Logan, UT, USA), 10% fetal calf serum (HyClone Laboratories), 100 U/mL penicillin, and 100 mg/mL streptomycin. 2.13. MES 23.5 dopaminergic cell culture Mesencephalon  neuroblastoma N18TG2 hybrid cells (MES 23.5 dopaminergic cells, a gift from Dr. Weidong Le at Baylor College of Medicine) (Crawford et al., 1992) were cultured in Dulbecco’s Modified Eagle’s Medium and/or F12 medium (Gibco, Grand Island, NY, USA) supplemented with 5% fetal calf serum and Sato’s ingredients as previously described (Yu et al., 2004). The cells were transferred to 35-mm dishes for immunocytochemistry and 25-cm2 flasks for the GCase activity assay and oligomeric a-syn detection.

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Culture dishes and flasks were precoated with poly-L-lysine as previously described (Yu et al., 2004).

3. Results 3.1. Characterization of the ELISAs for oligomeric and pS129 a-syns

2.14. Immunocytochemistry Different concentrations of a-syn monomer and oligomer were added to the medium of primary neuronal and MES 23.5 dopaminergic cells at final concentrations in the range of 0.2e5.0 mM. After 48 hours, the cells were washed with PBS several times, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.3% Triton X100. The cells were incubated overnight at 4  C with the 3D5 monoclonal antibody (1:1000), followed by a 2-hour incubation with Alexa Fluor 594 goat anti-mouse IgG (1:2000; Invitrogen, Carlsbad, CA, USA) at room temperature and a 5-minute incubation with DAPI (Sigma-Aldrich). Cells were mounted and observed by confocal laser microscopy (MRC 1024; Bio-Rad, Hercules, CA, USA). 2.15. Statistical analysis Data are expressed as mean  standard deviation. One-way analysis of variance followed by Dunnett’s multiple comparisons test was used to evaluate differences between effects of different concentrations of GCase inhibitor on GCase activity or a-syn oligomer levels. Other data were analyzed by 2-way analysis of variance followed by Tukey’s multiple comparisons test to evaluate differences between groups. p < 0.05 was considered statistically significant.

Freshly prepared a-syn sample and incubated a-syn sample were examined by Western blot. A single band of around 18 kDa was detected in the freshly prepared sample by an antibody (3D5) recognizing wild-type human a-syn (Yu et al., 2007), which was identical to the molecular size of a-syn monomer. By contrast, this antibody revealed several bands in the incubated sample, which corresponded to dimers and various sizes of oligomers including trimers, tetramers, and pentamers. The a-syn oligomers were isolated from the monomers and dimers and were used as the protein standard for the ELISA for oligomeric a-syn (Fig. 1A). Both the freshly prepared sample (containing only monomeric a-syn) and the purified mixture of oligomeric a-syn were analyzed by the ELISA method. A linear relationship between absorbance and a-syn oligomer concentration was observed, with an R2 value of 0.9898. There was no signal detected for the freshly prepared a-syn (Fig. 1B). These results confirmed that oligomeric a-syn was specifically detected by the ELISA. To detect pS129 a-syn, a commercial antibody recognizing pS129 a-syn and biotinylated 3D5 were used for capture and detection, respectively. Synthesized pS129 a-syn was used as the standard to assess the specificity of the ELISA for pS129 a-syn. After confirming the purity by Western blot (Fig. 1C), the synthesized pS129 a-syn was analyzed by the ELISA method. A linear

Fig. 1. Detection of oligomeric and phosphorylated a-syn by enzyme-linked immunosorbent assay (ELISA). (A) Western blot analysis of freshly prepared a-syn samples (lane 1), unpurified incubated a-syn samples (lane 2), and purified incubated a-syn samples, using an antibody specific for human wild-type (WT) a-syn. (B) ELISA for a-syn monomers and oligomers. A linear relationship was observed between absorbance and a-syn oligomer concentration (R2 ¼ 0.9898). No signal was detected for a-syn monomers. (C) Western blot analysis of WT a-syn (lane 1) and a-syn phosphorylated at serine 129 (pS129 a-syn) (lane 2 and 3) using antibodies against WT a-syn (lane 1 and 2) and pS129 a-syn (lane 3). pS129 a-syn was only recognized by an antibody recognizing phosphorylated a-syn, which aggregated into oligomers. (D) ELISA for WT and pS129 a-syns. A linear relationship was observed between absorbance and pS129 a-syn concentration (R2 ¼ 0.9671). There was no concentration-dependent change in absorbance for unmodified a-syn. Abbreviations: a-syn, a-synuclein; p-a-syn, phosphorylated a-synuclein; wt-a-syn, wild-type a-synuclein.

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Fig. 2. Age-related changes in levels of cytosolic and mitochondrial a-syn oligomers. (A) Age-related alterations in levels of cytosolic a-syn oligomers. Levels of oligomeric a-syn were higher in the striatum and hippocampus than in the cerebellum and occipital cortex and increased with age in the striatum and hippocampus. (B) Age-related alterations in levels of mitochondrial a-syn oligomers. Levels of oligomeric a-syn were higher in the striatum than in other brain regions. Unlike in the cytosol, an age-related increase in mitochondrial a-syn was observed in all brain regions. Tukey’s multiple comparisons test after 2-way analysis of variance, *p < 0.05 and **p < 0.01 versus young age group; #p < 0.05 and ##p < 0.01 versus middle age group (n ¼ 5). Abbreviations: a-syn, a-synuclein; CB, cerebellum; HIP, hippocampus; OC, occipital cortex; STR, striatum.

relationship was observed between absorbance and pS129 a-syn concentration, with an R2 value of 0.9671. There were no concentration-dependent changes in absorbance for wild-type asyn (Fig. 1D). 3.2. Age-related increase in a-syn oligomerization Age-related changes in a-syn oligomerization in the brain were examined. Levels of oligomeric a-syn in cytosolic fractions differed across brain regions, with the higher levels detected in the striatum and hippocampus than in the cerebellum and occipital cortex. Compared with the young age group, significant age-related elevation of cytosolic a-syn oligomers was detected in middle age and aged groups in the striatum and hippocampus and not in the cerebellum and occipital cortex (Fig. 2A). Compared with cytosolic fractions, mitochondrial fractions contained higher levels of oligomeric a-syn in each brain region, particularly in the striatum and hippocampus, where an age-dependent increase in cytosolic a-syn oligomers was evident. Different from cytosolic fractions, the levels of mitochondrial a-syn oligomers increased significantly with age in all brain regions (Fig. 2B).

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Fig. 3. Age-related changes in GCase protein expression and enzymatic activity. (A) A representative result of Western blot analysis of GCase expression in different brain regions. (B) Levels of GCase expression were lower in the striatum and hippocampus than in the cerebellum and occipital cortex, particularly in the aged group. (C) GCase activity decreased with age in all brain regions, corresponding to the age-dependent decrease in GCase expression. Tukey’s multiple comparisons test after 2-way analysis of variance, *p < 0.05 and **p < 0.01 versus young age group; #p < 0.05 and ##p < 0.01 versus middle age group (n ¼ 5). Abbreviations: CB, cerebellum; GCase, glucocerebrosidase; HIP, hippocampus; OC, occipital cortex; STR, striatum.

3.3. Age-related decrease in GCase expression and activity In contrast to oligomeric a-syn, whose levels were higher in the striatum and hippocampus than in the cerebellum and occipital cortex and increased with age, GCase expression and activity were lower in the striatum and hippocampus than in the cerebellum and occipital cortex and decreased with age (Fig. 3AeC). The inverse relationship between GCase expression and activity and a-syn oligomerization was especially evident in the striatum and hippocampus, indicating a potential link between the 2 proteins. 3.4. Age-related increase in a-syn phosphorylation and decrease in PP2A activity It has been shown that a-syn phosphorylation facilitates its oligomerization (Arawaka et al., 2006; Kragh et al., 2009; Zhou et al., 2011), and aggregated a-syn reduces the activity of PP2A, an enzyme that dephosphorylates a-syn (Wu et al., 2012). Therefore, it is possible that the age-related increase in a-syn

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oligomeric a-syns dose-dependently reduced the activity of GCase, with a more potent effect observed for the oligomers (Fig. 5). 3.6. Inhibition of GCase activity by a-syn in cultured neuronal cells To verify the discrepancy between effects of monomeric and oligomeric a-syns on GCase activity in living cells, different concentrations of monomeric and oligomeric a-syns were added to the culture medium of primary cortical neurons and MES 23.5 dopaminergic cells. The cells were then fixed and immunofluorescently labeled with an antibody against human a-syn to visualize the intracellular translocation of the exogenous a-syn. Primary neurons (Fig. 6A) and MES 23.5 cells (Fig. 6B) treated with a-syn monomer showed stronger diffused a-syn-immunoreactive signal than untreated (PBS) cells, which intensified with the elevation of a-syn concentration. In both types of cells, the signal for a-syn was stronger in the nucleus than in the cytoplasm. Very few, if any, of granular structuresdlikely a-syn aggregatesdwere observed. In contrast to monomeric a-syn-treated cells, cells treated with a-syn oligomers had numerous a-syn-immunoreactive aggregates in the cytoplasm, which also increased with the increase of a-syn concentration (Fig. 6A and B). These results indicate that exogenously added monomeric and oligomeric a-syns can enter into the primary neurons and MES 23.5 dopaminergic cells. On this basis, we studied the effect of a-syn on GCase activity in the cultured cells. The activity of GCase was measured 24 hours after a-syn was added to the medium. Both monomeric and oligomeric a-syns reduced the activity of GCase as a function of a-syn concentration, with a more potent effect observed for the oligomers (Fig. 7A and B).

Fig. 4. Age-related alterations in a-syn phosphorylation and PP2A activity. (A) Levels of pS129 a-synclein were measured by enzyme-linked immunosorbent assay and were found to increase with age in different brain regions. (B) PP2A enzymatic activity declined with age in different brain regions. Tukey’s multiple comparisons test after 2way analysis of variance, *p < 0.05 and **p < 0.01 versus young age group; #p < 0.05 and ##p < 0.01 versus middle age group (n ¼ 5). Abbreviations: CB, cerebellum; HIP, hippocampus; OC, occipital cortex; p-a-syn, phosphorylated a-syn; PP2A, phosphatase 2A; STR, striatum.

3.7. GCase inhibition increases a-syn oligomerization in neuronal cells Loss-of-function mutations in GBA are associated with increased levels of Triton X-100-soluble, high molecular weight a-syn species, which are likely toxic a-syn oligomers (Mazzulli et al., 2011). Application of conduritol-b-epoxide (CbE), a specific inhibitor of GCase activity, to a human dopaminergic cell line induced a-syn monomer accumulation (Cleeter et al., 2013). However, the

oligomerization is also associated with its phosphorylation and dephosphorylation enzyme. To demonstrate this possibility, the age-related changes in pS129 a-syn levels and PP2A activity were examined. Similar to the trends observed for levels of oligomeric asyn, the levels of pS129 a-syn were higher in the striatum and hippocampus than in the cerebellum and occipital cortex and increased as a function of age (Fig. 4A). In contrast, the PP2A activity was relatively lower in the striatum and hippocampus than in the cerebellum and occipital cortex and decreased with age (Fig. 4B). These results indicate an association between a-syn phosphorylation and PP2A activity over the course of aging. 3.5. Inhibition of GCase activity by a-syn in isolated brain lysosomes

a-Syn and GCase has been shown to interact under lysosomal solution conditions (pH 5.5) (Yap et al., 2011), indicating that a-syn may directly modulate GCase activity. To determine this possibility, lysosomes were isolated from the occipital cortex of young monkeys, where the expression and activity of GCase are relatively higher among the 4 brain regions investigated (Fig. 3). Various concentrations of monomeric and oligomeric a-syn were incubated with the lysosomal extracts under lysosomal solution conditions before GCase activity was measured. Both monomeric and

Fig. 5. a-Synclein (a-syn)-induced decrease in GCase activity. Lysosome-enriched fractions were incubated for 60 minutes with indicated concentrations of a-syn monomers and oligomers. GCase activity was measured with an enzymatic activity assay. Both forms of a-syn induced a dose-dependent decrease in GCase activity, with a more potent effect found for the oligomers. Tukey’s multiple comparisons test after 2way analysis of variance, *p < 0.05 and **p < 0.01 versus lysozyme-treated group; # p < 0.05 versus a-syn monomer-treated group (n ¼ 5). Abbreviation: GCase, glucocerebrosidase.

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Fig. 6. Intracellular translocation of a-syn monomers and oligomers in primary neurons and MES 23.5 dopaminergic cells. (A) Monomeric and oligomeric a-syns were added to the culture medium of primary cortical neurons and intracellular translocation of the exogenous a-syn proteins was examined by immunocytochemistry using an anti-a-syn antibody. In PBS-treated control neurons, a-syn immunoreactivity was faintly observed in the nucleus but not in the cytoplasm. In neurons treated with a-syn monomer, diffused immunoreactive signals (red) were observed mainly in the nucleus (DAPI, blue) and to a lesser degree in the cytoplasm (arrows), which increased with the enhancement of a-syn concentration. In neurons treated with a-syn oligomer, a-syn-positive granules (red, arrow heads) were observed in the cytoplasm and increased in number with increasing a-syn concentration. (B) Intracellular translocation of a-syn in MES 23.5 dopaminergic cells. Cells treated with a-syn monomer showed diffuse a-syn-immunoreactive signals (red) in the cytoplasm (arrows) and nucleus (DAPI, blue), which intensified with increasing a-syn concentration. In contrast, cells treated with a-syn oligomer had a-syn-immunoreactive granules (arrow heads) in the cytoplasm, with some also present in the nucleus. Scale bar ¼ 25 mm. Abbreviations: a-syn, a-synclein; PBS, phosphate-buffered saline. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

opposite result has also been reported, demonstrating that pharmacological inhibition of GCase activity does not affect a-syn levels in these cells (Dermentzaki et al., 2013). In the present study, the effect of reduced GCase activity on a-syn oligomerization was investigated in cultured primary neurons and MES 23.5 dopaminergic cells. Different concentrations of CbE were added to the culture medium, followed by the addition of 5-mM monomeric asyn 2 hours later; after 24 hours, cytosolic levels of oligomeric a-syn were measured by the ELISA. With the increase in CbE concentration, the activity of GCase decreased, whereas the levels of oligomeric a-syn increased (Fig. 8A and B), indicating that loss of GCase activity leads to increased a-syn oligomerization.

3.8. Increase in a-syn phosphorylation and decrease in PP2A activity in a-syn-treated cells As shown in brain tissues, increased a-syn oligomerization was associated with increased a-syn phosphorylation and decreased PP2A activity. To verify this association in living cells, we added different concentrations of monomeric or oligomeric a-syn to the medium of primary neurons and MES 23.5 cells and measured alterations in pS129 a-syn levels and PP2A activity. In both types of cells, monomeric and oligomeric a-syns led to an increase in pS129 a-syn levels (Fig. 9A and C) and a decrease in PP2A activity (Fig. 9B and D). Compared with monomeric a-syn, oligomeric a-syn

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Fig. 7. a-Syn-induced reduction in GCase activity in cultured cells. Primary neurons (A) and MES 23.5 dopaminergic cells (B) treated with a-syn monomer and oligomer showed dose-dependent decreases in GCase activity, with the oligomers having a greater effect. Tukey’s multiple comparisons test after 2-way analysis of variance, *p < 0.05 and **p < 0.01 versus control; #p < 0.05 and ##p < 0.01 versus monomer groups (n ¼ 10). Abbreviations: a-syn, a-synclein; GCase, glucocerebrosidase.

showed more potent effect on a-syn phosphorylation and PP2A activity. 4. Discussion In the present study, age-dependent alterations in a-syn oligomerization and phosphorylation and their association with GCase and PP2A in the brain were investigated in cynomolgus monkeys of different ages (3e4, 10e12, and 15 years). Instead of using quantitative Western blot analysis, ELISA methods specific for oligomeric and pS129 a-syns were applied to insure accurate measurements of oligomeric and pS129 a-syn levels in brain tissues. The data obtained showed that the levels of oligomeric and pS129 a-syns were higher and increased more evidently with age in the hippocampus and striatum than in the cerebellum and occipital cortex, which were inversely associated with age-dependent reductions in the expression and activity of GCase and the activity of PP2A, indicating a potential link among the 4 proteins. Previous studies have revealed a bidirectional positive feedback loop between a-syn and GCase (Mazzulli et al., 2011). On one hand, loss of GCase activity leads to the accumulation of GlcCer, which stabilizes a-syn oligomers; on the other hand, a-syn accumulation blocks the transport of GCase from the endoplasmic reticulum to the lysosome, resulting in GCase depletion from the

lysosome. This reciprocal interaction between GCase and a-syn can explain partially the inverse relationship between increased levels of oligomeric a-syn and decreased expression and activity of GCase in aging monkey brains. In addition to the indirect interaction between a-syn and GCase, our results obtained in the lysosomal extracts isolated from brain tissues suggest that a-syn may directly inhibit the lysosomal GCase activity, with the oligomers being more potent than the monomers. These results are in accordance with a previous study that a-syn can interact with GCase (Yap et al., 2011). However, it remains unknown how a-syn inhibits GCase activity and why a-syn oligomers inhibit GCase activity more strongly than the monomers. One possibility is that a-syn oligomers have a higher binding affinity with GCase and can therefore modulate its enzymatic activity to a greater extent in isolated lysosomes. The colocalization of a-syn and GCase in LBs indicates that aggregated a-syn can tightly bind to GCase (GokerAlpan et al., 2010).The inhibitory effect of a-syn on GCase activity was not only observed in isolated lysosomes but also in the cultured neuronal cells. In the latter condition, the oligomeric a-syn was still more potent than the monomeric a-syn in reducing GCase activity. Different from conditions in isolated lysosomes, in cultured neuronal cells, in addition to directly inhibiting GCase, a-syn oligomers may indirectly reduce GCase activity by blocking its transport from the endoplasmic reticulum to the lysosome (Mazzulli et al., 2011; Thayanidhi et al., 2010). How is reduced GCase activity linked to age-dependent increase in a-syn oligomerization? One possible mechanism is that reduced GCase activity may result in GlcCer accumulation, which promotes a-syn oligomerization by stabilizing soluble oligomeric intermediates (Mazzulli et al., 2011). Another potential mechanism is that reduced GCase activity may increase a-syn phosphorylation at serine 129, a modification that has been shown to promote a-syn aggregation (Ferrer et al., 2011; Walker et al., 2013). Because of the function that GCase can catalyze the breakdown of GlcCer into glucose and ceramide (Motabar et al., 2012), a reduction in GCase activity will decrease the production of ceramide, an activator of PP2A (Chalfant et al., 1999; Dobrowsky et al., 1993), which functions as a dephosphorylation enzyme for a-syn. This will reduce PP2A activity, leading to an increase in a-syn phosphorylation and further aggregation (Fujiwara et al., 2002; Lee et al., 2011). In the striatum and hippocampus, where a-syn levels were higher, there were increased levels of pS129 a-syn and decreased activity of PP2A. In contrast, in the cerebellum and occipital cortex, where levels of asyn oligomers were lower, there were lower levels of pS129 a-syn and higher activity of PP2A. In addition, in all brain regions investigated, the levels of pS129 a-syn increased with age, which was accompanied by age-dependent decrease in PP2A activity. This inverse relationship was also observed in cultured neuronal cells, where increased levels of pS129 a-syn were associated with decreased PP2A activity. As additional evidence to support the above mechanism, a study on autopsy brain samples from subjects with sporadic PD revealed that GCase protein levels and enzyme activity were selectively reduced in the early stages of PD in regions with increased a-syn levels and decreased ceramide concentration (Murphy et al., 2014). The above findings suggest that the accumulation of oligomeric a-syn by GCase dysfunction could be enhanced by reducing PP2A activity. The present results do not exclude other mechanisms that may promote age-dependent increase in a-syn phosphorylation. For example, oxidative stress levels are generally higher in the striatum and hippocampus (Crivello et al., 2005) and high intrinsic oxidative stress in susceptible neurons has been shown to inactivate PP2A (Foley et al., 2007), which in turn increases a-syn phosphorylation. Conversely, increased a-syn oligomers can directly inhibit PP2A activity (Wu et al., 2012) in addition to indirect inhibition of GCase

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Fig. 8. Inhibition of GCase activity promotes a-syn oligomerization. Primary neurons (A and B) and MES 23.5 dopaminergic cells (C and D) were treated with indicated concentrations of the GCase inhibitor conduritol-b-epoxide (CbE). Cells were lysed 24 hours later and a-syn oligomer levels were measured by enzyme-linked immunosorbent assay. In both cell types, with increasing CbE concentration, GCase activity decreased (A and C) while a-syn oligomer levels increased (B and D). Dunnett’s multiple comparisons test after 1 way analysis of variance, *p < 0.05 and **p < 0.01 versus control (n ¼ 6). Abbreviations: a-syn, a-synclein; GCase, glucocerebrosidase.

Fig. 9. Increase in a-syn phosphorylation and decrease in PP2A activity in a-syn-treated cells. Primary neurons (A and B) and MES 23.5 dopaminergic cells (C and D) were treated with either a-syn monomer and oligomer. Twenty-four hours later, pS129 a-syn levels and PP2A activity were measured. With the increase in a-syn concentration, increased pS129 a-syn levels (A and C) were found in primary neurons and MES 23.5 dopaminergic cells, which were accompanied by reduced PP2A activity (B and D). Compared with a-syn monomers, a-syn oligomers had more potent effects on pS129 a-syn levels and PP2A activity. Tukey’s multiple comparisons test after 2-way analysis of variance, *p < 0.05 and **p < 0.01 versus control; #p < 0.05 and ##p < 0.01 versus monomer groups (n ¼ 5). Abbreviations: a-syn , a-synuclein; p-a-syn, phosphorylated a-syn; PP2A, phosphatase 2A.

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activity. Moreover, it is also possible that age-dependent increase in a-syn phosphorylation may be also affected by some kinases. For example, polo-like kinase 2 has been reported to increase in the brain of PD patients and is able to phosphorylate a-syn at serine 129 (Mbefo et al., 2010). In addition to differences in cytosolic levels of oligomeric a-syn across brain regions, a-syn oligomerization also varied among mitochondrial fractions isolated from different brain regions and increased with age. Mitochondrial fractions from the striatum had the highest levels of oligomeric a-syn, followed by those from the hippocampus, cerebellum, and occipital cortex. Previous studies have shown that striatal mitochondria contain higher levels of monomeric a-syn as compared with those in the cerebellum (Devi et al., 2008; Liu et al., 2009). We report here for the first time that oligomeric a-syn levels are also higher in mitochondria of the striatum than those of other brain regions. This may be due to the increased oxidative stress in the striatum, which is known to promote a-syn translocation to mitochondria (Cole et al., 2008; Crivello et al., 2005). In addition, our previous study found that increased levels of a-syn in the cytosol may contribute to their accumulation in mitochondria (Liu et al., 2009). The accumulation of oligomeric a-syn with age can increase the risk of mitochondrial dysfunction by disrupting the mitochondrial membrane (Hashimoto et al., 2003) or by inhibiting complex I activity (Chinta et al., 2010; Liu et al., 2009). The results of the present study can partially explain the vulnerability of some brain regions to LBP. For example, the striatum and hippocampus are vulnerable to LBP in PD and DLB patients, especially those with GBA mutations (Braak et al., 2003; Sidransky and Lopez, 2012). In contrast, the cerebellum and occipital cortex are relatively insensitive to LBP (Braak et al., 2003; Sidransky and Lopez, 2012). We propose that an age-related increase in a-syn oligomerization will predispose some regions to LBP, and when combined with genetic and environmental risk factors, the levels of a-syn oligomers in these regions will exceed the pathogenic threshold, leading to the formation of LBP and neurodegeneration. This may be what has happened to the brain with diseases such as PD and DLB. Disclosure statement The authors declare that they have no competing interests. Acknowledgements This work was supported by grants from the National Basic Research Program (“973” Program) of China (2011CB504101), Natural Science Foundation of China (81071014 and 81371200), National Science and Technology Support Program (2012BAI10B03), Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality (PHR200907113), National High Technology Research and Development Program (“863” Program) of China (2006AA02A408), and the Natural Science Foundation of Beijing (7122035). References Alim, M.A., Hossain, M.S., Arima, K., Takeda, K., Izumiyama, Y., Nakamura, M., Kaji, H., Shinoda, T., Hisanaga, S., Uéda, K., 2002. Tubulin seeds alpha-synuclein fibril formation. J. Biol. Chem. 277, 2112e2117. Arawaka, S., Wada, M., Goto, S., Karube, H., Sakamoto, M., Ren, C.H., Koyama, S., Nagasawa, H., Kimura, H., Kawanami, T., Kurita, K., Tajima, K., Daimon, M., Baba, M., Kido, T., Saino, S., Goto, K., Asao, H., Kitanaka, C., Takashita, E., Hongo, S., Nakamura, T., Kayama, T., Suzuki, Y., Kobayashi, K., Katagiri, T., Kurokawa, K., Kurimura, M., Toyoshima, I., Niizato, K., Tsuchiya, K., Iwatsubo, T., Muramatsu, M., Matsumine, H., Kato, T., 2006. The role of G-protein-coupled

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