Suppression of astrocytic autophagy by αB-crystallin contributes to α-synuclein inclusion formation
Parkinson’s disease (PD) is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions (Lewy bodies) within some remaining neurons in the substantia nigra. Recently, astroglial inclusion body has also been found in some neurodegenerative diseases including PD. However, the underlying molecular mechanisms of how astroglial protein aggregation forms remain largely unknown. Here, we investigated the contribution of αB-crystallin (CRYAB), a small heat shock protein, in α-synuclein inclusion formation in astrocytes.
Small interfering RNA (siRNA)-mediated CRYAB (siCRYAB) knockdown or CRYAB overexpression was performed to investigate the impact of CRYAB on the autophagy in human glioblastoma cell line U251 cells. Co-immunoprecipitation (co-IP) and immunoblotting were used to dissect the interaction among multiple proteins. The clearance of α-synuclein in vitro was evaluated by immunocytochemistry. CRYAB transgenic mice and transgenic mice overexpressing A30P mutant form of human α-synuclein were used to examine the influence of CRYAB to α-synuclein accumulation in vivo.
We found that knockdown of CRYAB in U251 cells or primary cultured astrocytes resulted in a marked augmentation of autophagy activity. In contrast, exogenous CRYAB disrupted the assembly of the BAG3-HSPB8-HSC70 complex via binding with BAG3, thereby suppressing the autophagy activity. Furthermore, CRYAB-regulated autophagy has relevance to PD pathogenesis. Knockdown of CRYAB remarkably promoted cytoplasmic clearance of α-synuclein preformed fibrils (PFFs). Conversely, selective overexpression of CRYAB in astrocytes markedly suppressed autophagy leading to the accumulation of α-synuclein aggregates in the brain of transgenic mice expressing human α-synuclein A30P mutant.
This study reveals a novel function for CRYAB as a natural inhibitor of astrocytic autophagy and shows that knockdown of CYRAB may provide a therapeutic target against proteinopathies such as synucleinopathies.
KeywordsαB-crystallin Astrocytes Parkinson’s disease Autophagy
Amyotrophic lateral sclerosis
Bcl-2-associated athanogene 3
Small heat shock protein
Small interfering RNA (siRNA) targeting CRYAB
Parkinson’s disease (PD) is the most common movement disorder. It is characterized by chronic degeneration of nigral dopaminergic (DA) neurons. There is currently no cure for this devastating neurodegenerative disorder. This is largely due to a limited understanding of its pathogenesis. Over the decades, several hypotheses about PD pathogenesis have been proposed, including mitochondria dysfunction, oxidative stress, abnormal protein aggregation, protein kinase dysfunction, unbalanced calcium homeostasis and neuroinflammation . Abnormal protein aggregation in the remaining nigral DA neurons, known as a Lewy body, is one of the most prominent pathological features in the Parkinsonian brain, in addition to the chronic loss of DA neurons. Over the past few decades, numerous studies have mainly been focused on analyzing protein accumulation and degradation in neuronal cells. This focus is largely attributable to the fact that abnormal aggregates containing disease-causing protein in neuronal cells are the most prominent pathological changes when visualizing the protein aggregates using α-synuclein antibody.
However, accumulating evidence suggests the presence of glial inclusion in PD brain. For example, Braak et al. found the presence of α-synuclein-immunoreactive inclusions in astrocytes in prosencephalic regions . It is hypothesized that astrocytic protein aggregates, such as α-synuclein, are taken up from neurons to be degraded in the lysosome [3, 4]. At advanced disease stages, protein aggregates are accumulated in astrocytes. Indeed, post-mortem brain analysis showed that α-synuclein-immunoreactive inclusions appear frequently in astrocytes as well as neurons in the PD brain [2, 5, 6, 7, 8]. Accumulated protein aggregates are likely to affect astrocyte functions, thereby contributing to neurodegeneration. This notion is supported by several animal studies. For instance, the accumulation of α-synuclein induces astrocytic mitochondrial impairments . Moreover, selective overexpression of an α-synuclein mutant in astrocytes leads to degenerative movement disorder in mice, accompanied by astrogliosis . These data indicate that PD pathogenesis is much more complex than we previously anticipated and glial inclusion may impact on glial function during neurodegeneration thereby contribute to disease progression. It is important to investigate the question of how the glial inclusion is formed during neurodegeneration, a question which has been overlooked in this field for years.
Recent meta-analysis of genome-wide association studies (GWASs) revealed a set of novel risk loci involved in PD susceptibility, in addition to those that have been previously reported at genome-wide significance levels. These data point to a key role for autophagy and lysosomal biology in PD . Interestingly, among the PD risk loci identified, some of the candidate genes, such as Bcl-2-associated athanogene 3 (BAG3), a member of the BAG family of proteins, and GTP cyclohydrolase I (GCH1), are known to be expressed in both astrocytes and neurons. The expression levels of these genes in astrocytes were preferentially upregulated in the Parkinsonian brain along with protein aggregation  or in response to other pathological stimuli [12, 13], implying that these candidate genes play a role in the glial pathology of PD.
Autophagy is considered as a major system targeting long-lived proteins and dysfunctional organelles for lysosomal degradation. Impaired autophagy activity and significantly increased protein aggregates are found in α-synucleinopathies as well as in tauopathies and amyotrophic lateral sclerosis (ALS) [14, 15, 16, 17, 18]. Moreover, a previous study has shown that a variety of susceptible genes related to degenerative diseases are associated with autophagy-lysosome regulation [19, 20]. Indeed, BAG3 and HSPB8 whose expression was upregulated specifically in astrocytes in the PD brain , have been shown to be able to promote autophagy, indicating that alterd autophagy process in astrocytes is involved in the failure of glial inclusion clearance in PD and may thus contribute to disease onset/progression in neurodegeneration. The persistent existence of glial inclusions in the PD brain raises a question as to whether a yet-unidentified mechanism plays a role in this process, despite the upregulation of BAG3/HSPB8-induced autophagy.
It has been shown that αB-crystallin (CRYAB), a small heat shock protein (sHSP), is mainly expressed in astrocytes and oligodendrocytes in the central nervous system . CRYAB is implicated in various protein aggregation-related neurodegenerative diseases such as Alzheimer’s disease (AD), PD, ALS, tauopathies, Alexander’s disease, and prion disorders [2, 21, 22, 23, 24, 25, 26, 27, 28]. For example, CRYAB and tau are often co-localized in glia with tau pathology . The relationship between increased glial CRYAB expression and abnormal protein accumulation remains unclear. Previously, we found that expression of CRYAB was steadily increased in the ventral midbrain and striatum of an adult mouse brain with aging . Moreover, we demonstrated that the presence of abnormal CRYAB-immunoreactive inclusions in astrocyte-like glial cells in the substantia nigra (SN) of the PD brain is a remarkable pathological feature of the gliosis in PD . We hypothesized therefore that CRYAB may play a role in glial inclusion formation during brain aging and contribute to PD pathogenesis.
In the present study, we tested this hypothesis by investigating the effect of CRYAB on astrocytic autophagy flux in vitro and on the degradation of α-synuclein mutant protein in vivo. We showed that CRYAB functions as a potent inhibitor of astrocytic autophagy induction through interacting with BAG3, which is a key mediator for macroautophagy (hereafter referred to autophagy) induction. By inhibiting the selective process of autophagy, elevated levels of CRYAB leads to the accumulation of aggregated proteins in astrocytes that exacerbates the pathological process of neurodegenerative diseases such as PD.
Adult or neonatal C57BL/6 mice were from Shanghai Laboratory Animal Centre, Chinese Academy of Sciences. CRYAB transgenic mice (FVB-Tg(GFAP-CRYAB)141.6Mes/J) and SNCAA30P (B6.Cg-Tg(THY1-SNCA*A30P)TS2Sud/J) transgenic mice were purchased from the Jackson Laboratory (USA). CRYAB Tg mice in a C57BL/6 (inbred) genetic background generated by 10 backcrosses were used in the entire study. They were maintained on a 12 h light/dark cycle at 23 °C with food and water available ad libitum. All procedures performed were approved by the Institutional Animal Care and Use Committee and were in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Cell culture and transfection
U251 human glioblastoma cell line (Cell Bank, Chinese Academy of Sciences) were maintained in DMEM with 10% fetal bovine serum (FBS) and 100 mM glutamine at 37 °C under 5% CO2. Transient transfection was performed using Lipofectamine 3000 (Invitrogen, L3000–008) according to the manufacturer’s instructions. Knocking down CRYAB was performed as described previously . siRNA duplexes were used (GenePharma, Shanghai, China) and their sequences were as follows: sense: r (GGC CCA AAU UAU CAA GCU A) dTdT; antisense: r (UAG CUU GAU AAU UUG GGC C) dTdG; non-silencing control siRNA: sense: r (UUC UCC GAA CGU GUC ACG U) dTdT; and antisense: r (ACG UGA CAC GUU CGGAGAA) dTdT. At 72 h after transfection, cells were harvested for further analysis.
Plasmids, reagents, and antibodies
The cDNA of BAG3 and CRYAB were amplified using RNA extracted from HEK293 cell, followed by RT-PCR and subcloned into pLenti vector (OBIO, H139).
H2O2 (SinoPharm Chemical Reagent Co., Ltd., China. Cat. #7722-84-1). Rapamycin (Sangon Biotech, China. Cat. #A606203) and chloroquine (Sangon Biotech. Cat. #A506569) were used. The following antibodies were used: anti-LC3 (MBL, Cat. #PM036), anti-β-actin (Sigma, Cat. #A5441), anti-RPS6KB/p70S6 (Cell Signaling, Cat. #2708), anti-phospho-RPS6KB/p70S6 (Cell Signaling, Cat. #9206), anti-mTOR (Cell Signaling, Cat. #2972), anti-phospho-mTOR (Sigma, Cat. #SAB4504043), anti-SQSTM1/p62 (MBL, Cat. #PM045), anti-GAPDH (SAB, Cat. #1336), anti-CRYAB (Santa Cruz Biotech, Cat. #sc-137,143), anti-HSPB8 (Abcam, Cat. #ab4149), anti-HSC70 (Santa Cruz Biotech, Cat. #sc-7298), anti-BAG3 (Abcam, Cat. #ab47124), anti-ubiquitin (DAKO, Cat. #z0458), anti-α-synuclein (Cell Signaling, Cat. #2628), anti-GFP (Santa Cruz Biotech, Cat. #sc-9996).
Western blot analysis and quantification
Western blot was performed as described previously . Briefly, the proteins from lysed cells were denatured and loaded on sodium dodecyl sulfate polyacrylamide gels. Afterwards, the proteins were transferred to PVDF membranes, blocked in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 7.5 and 0.1% Tween 20) containing 5% (w/v) skim milk, and incubated with the corresponding primary and secondary antibodies.
Peroxidase activity was detected with Omni-ECL™ FemtoLight Chemiluminescence Kit (EpiZyme, Cat. #SQ201) and visualized and digitized with ImageQuant (LAS-4000, FujiFilm, Japan). Optical densities of bands were analyzed by using Image J. Protein levels, quantified by computer analysis as the ratio between each immunoreactive band and the levels of β-actin were expressed as a percentage of vehicle-treated control.
To analyze protein interactions in vitro, approximately 5 × 106 U251 cells were harvested after 48 h transfection and lysed in 0.5 ml of RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Pre-cleared lysates were subjected to immunoprecipitation with indicated antibodies directed against the epitope tag as described previously. Precipitated proteins were eluted from the beads by boiling in SDS sample buffer, and separated by SDS-PAGE. Immunoblot assays were performed using the indicated antibodies. The following primary antibodies were used: anti-Flag Affinity gel (Genomics Technology, Cat. #SG4510–10), anti-c-Myc AC (Santa Cruz Biotech, Cat. #sc-40 AC).
Primary astrocytic culture and plasmid transfection
Astrocytes were prepared from the cerebrocortex of wild-type C57BL/6 mice at P0, as described previously . The neonatal cortex was trypsinized and dissociated. Cells were then plated at 75 cm2 flask (Corning, USA) in DMEM containing 10% FBS. Culture media were changed 24 h later and subsequently twice a week. Cultured cells were shaken to remove the top layer of cells sitting over the astroglial monolayer to yield mainly type I astrocytes with a flat morphology between day 5 and 7 in vitro. Before experimental treatments, astrocytic cultures were passaged once. Cells were allowed to reach 90% confluence. Cultures were transfected using lentivirus, which can express CRYAB. The cells transfected with the control lentivirus were included as controls in all experiments.
Immunostaining and confocal microscopy
For immunostaining, cells were fixed in 4% paraformaldehyde followed by 0.1% Triton-X100 in PBS (pH 7.4). After washing twice with PBS, they were incubated in FBS (PBS, pH 7.4, containing 5% FBS) to block nonspecific sites of antibody adsorption. Then, the cells were incubated with corresponding primary antibody followed by incubation of secondary antibody conjugated with either Alex488 or Alex555. The same sample were then incubated with another corresponding primary and secondary antibodies. Each sample was incubated with Hoechst to show the nucleus. The cells were imaged with invert confocal microscope (FV1000; Olympus).
For quantification of the number of autophagosomes (diameters 0.3–1.0 μm), a total of 50 cells were analyzed using ImageReader software (Fujifilm). Quantification of LC3 and BAG3 fluorescence intensity and analysis of protein co-localization coefficients in 30 cells were performed using ImageReader software (FujiFilm).
Electron microscopic analysis of U251 cells was performed using a standard protocol. In brief, cells were fixed in 2.5% glutaraldehyde in 0.1 M PB (pH 7.4), and post-fixed in aqueous 1% OsO4 followed by 3–4% uranyl acetate. After ethanol and acetone dehydration and embedding in resin, ultrathin (70 nm) sections were then stained with 2% uranyl acetate followed by 0.3% lead citrate. Sections were imaged using transmission electron microscope (Nippon Tekno Co Ltd., model JEOL-1230) at 80 kV. For autophagic vacuole quantification, 20 cells, primary magnification × 20,000, were taken with systematic random sampling from each sample. The cytoplasmic volume fraction of autophagic vacuoles was estimated using ImageReader software (FujiFilm).
Extraction of RIPA-soluble and RIPA-insoluble protein fractions of mouse brain
Extraction of RIPA-soluble and RIPA-insoluble protein fractions from mouse brain tissues was described previously [33, 34, 35]. Previous studies indicated that separation of insoluble fraction was performed on older mice at least 6 months of age [32, 36]. The tissues from one-year-old CRYABTg; SNCAA30P Tg mice were isolated and stored at -80 °C prior to use. Frozen brain tissues were thawed on ice and homogenized in 10 mL/g (v/w) radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% TritonX-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 1 mM EDTA, 50 mM Tris, pH 7.5 with phosphatase and protease inhibitor cocktails). The homogenates were centrifuged at 14,000 g at 4 °C for 30 min and the supernatants were collected as the RIPA-soluble fraction. The pellet was washed with RIPA buffer and centrifuged at 14,000 g at 4 °C for 10 min for three times. The resultant pellet was sonicated in 5 mL/g (v/w) urea buffer (8 M urea, 100 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.0) as urea-soluble fraction.
Alpha-synuclein PFFs exposure
Exposure of U251 cells to α-synuclein PFFs was performed as described previously [4, 37]. Briefly, U251 cells were exposed to 0.5 μM α-synuclein PFFs (a gift from Drs. Lee, Trojanowski and Luk, USA) for 24 h. After the treatment, cells were washed twice with culture medium to remove α-synuclein PFFs. On day 0 and 3 in vitro after exposure, cells were fixed for further analyses.
Statistical analysis was performed using GraphPad software (GraphPad Prism v6.0; GraphPad Software). All the statistical data were presented as mean ± SEM. Statistical significance of the differences was determined using Student t-test. Differences were considered significant at values of P < 0.05.
CRYAB inhibits astrocytic autophagy
We then asked whether CRYAB is capable of influencing autophagy in astrocytes under stress conditions. Hydrogen peroxide (H2O2) has been widely used to induce autophagy in vitro . We evaluated the autophagy activity by measuring p62 levels by using immunoblotting. p62 is known as an autophagy substrate which serves as a reporter of autophagy activity. Promotion of autophagy induced by knockdown of CRYAB resulted in a significant reduction (~ 15%) in p62 levels compared with control (Fig. 1d). Exposure to H2O2 of U251 cells in combination with CRYAB knockdown led to a further reduction in p62 levels (~ 45%), as compared to H2O2 treatment alone. These results suggest that CRYAB also inhibits autophagy in U251 cells under pathological conditions.
To validate whether CRYAB knockdown truly induces autophagy, we performed electron microscopy analysis on U251 cells. It was revealed that knockdown of CRYAB induced robust formation of autophagic vesicles, mature autophagic vesicles and autolysosomes with visible cytoplasmic contents. The total cytoplasmic occupancy of the intracellular autophagic vacuoles was doubled in siCRYAB-treated cells compared with the control (Fig. 1e). Taken together, these data indicate that CRYAB is a crucial autophagic regulator in astrocytes.
CRYAB inhibits autophagy in a mTOR-independent manner
Moreover, we determined whether CRYAB suppresses autophagy through the inhibition of autophagosome-lysosome fusion. We found that in the presence of chloroquine (CQ), an inhibitor for the autophagosome-lysosome fusion, CRYAB knockdown further augmented the accumulation of autophagosomes compared with CQ treatment alone (Fig. 2h), suggesting that CRYAB does not regulate autophagy through the inhibition of autophagolysosome degradation. Taken together, these results suggest that CRYAB may regulate autophagy in the upstream of mTOR pathway.
CRYAB inhibits BAG3-dependent autophagy via disruption of the assembly of chaperon complex BAG3-HSPB8-HSC70
Moreover, previous studies have shown that BAG3-mediated autophagy is required by its interaction with HSC70 and HSPB8 [42, 43]. It was observed that BAG3 did bind to HSC70 and HSPB8 in U251 cells (Fig. 3c). Thus, we speculated that CRYAB may bind to BAG3 and competitively inhibit the formation of BAG3-HSPB8-HSC70 complex. To test this, we performed BAG3 co-IP assays using U251 cell lines stably overexpressing CRYAB-Flag or empty vector as control. It was found that HSC70, HSPB8 and CRYAB could be detected in BAG3 immunoprecipitates and the levels of HSC70 were decreased in the immunoprecipitates from cells overexpressing CRYAB (Fig. 3d-e). These results suggest that elevation of CRYAB levels can disrupt the assembly of the protein complex BAG3-HSPB8-HSC70.
CRYAB inhibits the degradation of aggregated proteins in astrocytes by regulating autophagy
To determine whether the impact of CRYAB knockdown on α-synuclein degradation is attributable to enhanced expression of BAG3 which is known to promote autophagy, we assessed the expression levels of BAG3 after CRYAB knockdown in U251 cells. We found that the expression levels of BAG3 were not significantly altered after CRYAB knockdown (Fig. 5h), suggesting that CRYAB knockdown-induced augmentation of α-synuclein aggregate clearance result from elevated BAG3 activity but not its expression.
Astrocytic CRYAB promotes the accumulation of α-synuclein in vivo
Previous studies suggest that CRYAB does not regulate autophagy in neonatal rat ventricular myocytes . However, we found CRYAB to be required for the regulation of autophagy in astrocytes; as in an adult mouse brain CRYAB is enriched in astrocytes and oligodendrocytes, providing a molecular basis for the CRYAB-dependent inhibition of astrocytic autophagy. These data demonstrate the intriguing specialization of CRYAB function in the brain. CRYAB participates in the regulation of astrocytic autophagy of the brain, representing a mechanistically distinct molecular machinery utilized in the regulation of autophagy in astrocytes.
Numerous studies have shown that sHSPs, including CRYAB, are often considered to be molecular chaperones, that participate in the ubiquitin-proteasome degradation process. sHSPs help hydrolyse misfolded proteins by recognizing and combining with them, to prevent cellular toxicity under certain stress conditions [49, 50]. CRYAB also plays a role in inhibiting inflammation in the process of degenerative diseases [29, 51, 52, 53]. In the present study, we demonstrated a novel function for CRYAB in astrocytic autophagy regulation. The discrepancy between our findings in the present study and others suggests multifaceted functions of CRYAB. CRYAB plays distinct functions depending on circumstances related to the nature of insults, site of injury and disease staging. We speculate that, under normal conditions or at an early stage of PD, CRYAB functions as a molecular chaperone, participating in the ubiquitin proteasome pathway for misfolded protein degradation. Additionally, it inhibits inflammation to maintain cellular homeostasis under stress conditions. While at the advanced disease stages, despite of the increased levels of CRYAB, CRYAB is not able to prevent the progression of neuroinflammation. Meanwhile, as the protein aggregations become a more severe burden, they are released into the extracellular space and taken up by glial cells, such as astrocytes and microglia [2, 4]. Elevated levels of CRYAB in astrocytes prevent aggregated proteins from degradation by inhibiting autophagy, which in turn accelerates the degeneration of both neuronal and glial cells.
How the function of CRYAB switches is currently unclear. It may be attributable to a particular state of CRYAB determined by its phosphorylation at different sites. This is supported by recent studies showing that CRYAB in different phosphorylation forms plays distinct roles. Levels of CRYAB ser59 are increased significantly under pathological conditions and it plays a role in accelerating inflammation . Therefore, it is necessary to distinguish the different phosphorylation forms of CRYAB in the various context of neurodegenerative diseases.
As shown in Fig. 6, we found that CRYAB derived from astrocytes enhanced the accumulation of insoluble α-synuclein in SNCAA30P Tg mice driven by Thy1 promoter. Given that Thy1 promoter-driven expression of Cre recombinase is largely restricted to neurons , it raises the question as to why astrocytic CRYAB promotes α-synuclein accumulation in neuronal cells. Interestingly, a study, using luciferase reporter mice produced by Thy1-Cre mice, showed that Thy1 promoter-driven expression of luciferase reporter gene can be detected in both neurons and astrocytes , suggesting that Thy1 promoter is not exclusive for targeting gene expression in neurons, as previously expected. This is supported by a direct evidence showing that in SNCAA30P Tg mice, accumulated α-synuclein could be detected in both astrocytes and neurons on the midbrain and brainstem sections . Thus, an alternative explanation for the results shown in Fig. 6 is that astrocytic CRYAB promotes the accumulation of insoluble α-synuclein in astrocytes of SNCAA30P Tg mice in a cell-autonomous fashion.
We showed in the present study that CRYAB inhibits BAG3-mediated selective autophagy which is an adaptive process to maintain protein homeostasis under stress [57, 58]. The BAG3-HSPB8-HSP70 complex promotes thedegradation of aggregated proteins which is vital for age-related neurodegenerative diseases. During aging, theexpression of BAG3 is induced under oxidative stress and plays a neuroprotective role . Through inhibition of proteasome system, BAG3 can also be upregulated along with aggresome provoking . Through inhibition of proteasome system, BAG3 can also be upregulated along with aggresome provoking . The overexpression of BAG3 in primary cultured neurons significantly promotes Tau degradation In an ALS model, neuronal BAG3 expression is higher in the spinal cords of mutant SOD transgenic mice . A misfolded proteins, such as mutant SOD can also be targeted by BAG3/HSC70, following autophagic degradation . Thus, BAG3-mediated macroautophagy has been considered as a potential therapeutic target for neurodegenerative diseases. Interestingly, during aging or in degenerative diseases, BAG3-HSPB8 expression was more significantly increased in astrocytes than neurons, suggesting that astrocytic autophagy regulation is involved in neurodegeneration and actively responds to extracellular aggregates . Our results showed that the selective overexpression of CRYAB in astrocytes resulted in the heavier accumulation of α-synuclein in aged mice, indicating that suppression of astrocytic autophagy can lead to the aggregation of α- synuclein. Our findings provide new insight into the role of astrocytic autophagy in the degradation of abnormal protein aggregates.
In summary, our study demonstrates that CRYAB is a potent inhibitor of astrocytic autophagy via interacting with BAG3, demonstrating a new signalling pathway that negatively regulates the degradation of α-synuclein aggregates. The present study suggests that the balance between CRYAB-BAG3 and BAG3-HSPB8-HSC70 complexes in astrocytes is an integral components of neuropathology in neurodegeneration. Aberrant increases in the levels of CRYAB inhibit astrocytic autophagy, leading to more severe protein aggregation. The findings that knockdown of astrocytic CRYAB promotes the degradation of aggregated proteins suggest that CRYAB may be a new therapeutic target for future intervention in PD.
We thank Ms. YD Li and JC Hou for excellent technical assistance in genotyping; Dr. Q Hu and his colleagues at the Optical Imaging Center of ION for technical support in confocal microscopy; Dr. Y Kong and her colleagues at the EM core-facility of ION for technical support in electron microscopy; Drs. VM Lee, J Trojanowski and KC Luk for providing α-synuclein PFFs.
This work was supported by grants from the Natural Science Foundation of China (31430036, 91742116, U1801681), National Key Basic Research Program of China (2015CB553500), Key Research Program of Frontier Sciences (QYZDJ-SSW-SMC002), Strategic Priority Research Program of Chinese Academy of Science (XDB32020100), Shanghai Municipal Science and Technology Major Project (2018SHZDZX05), and the Shanghai Municipal Science and Technology Commission (17ZR1435300 to SZZ).
Availability of data and materials
The data supporting the conclusions of this article are available from the corresponding author upon request.
SZL and YSG conducted most of the in vivo and in vitro experiments and the data analysis. PZL contributed to pilot experiments. SZZ and SY contributed to in vivo experiments. YQY contributed to cell cultures. XMW, FD and XSG contributed to discussion. JWZ supervised the project and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All procedures performed were approved by the Institutional Animal Care and Use Committee and were in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Consent for publication
The authors declare that they have no competing interests.
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