Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration

Choi, Insup; Zhang, Yuanxi; Seegobin, Steven P.; Pruvost, Mathilde; Wang, Qian; Purtell, Kerry; Zhang, Bin; Yue, Zhenyu

doi:10.1038/s41467-020-15119-w

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Published: 13 March 2020

Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration

Insup Choi¹,
Yuanxi Zhang¹,
Steven P. Seegobin ORCID: orcid.org/0000-0002-4514-9293¹,
Mathilde Pruvost¹,
Qian Wang^1,2,
Kerry Purtell¹,
Bin Zhang² &
…
Zhenyu Yue¹

Nature Communications volume 11, Article number: 1386 (2020) Cite this article

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Abstract

Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. Herein, we report a neuroprotective role for microglia in the clearance of neuron-released α-synuclein. Neuronal α-synuclein activates microglia, which in turn engulf α-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Synucleinphagy requires the presence of microglial Toll-like receptor 4 (TLR4), which induces transcriptional upregulation of p62/SQSTM1 through the NF-κB signaling pathway. Induction of p62, an autophagy receptor, is necessary for the formation of α-synuclein/ubiquitin-positive puncta that are degraded by autophagy. Finally, disruption of microglial autophagy in mice expressing human α-synuclein promotes the accumulation of misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration. Our study thus identifies a neuroprotective function of microglia in the clearance of α-synuclein via TLR4-NF-κB-p62 mediated synucleinphagy.

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Introduction

α-synuclein is produced primarily in neurons and constitutes up to 1% of total cytosolic protein in the brain¹. Although the function of α-synuclein is poorly understood, available evidence suggests its role in synaptic vesicular trafficking^2,3,4. Aggregated α-synuclein is a major component in intraneuronal inclusions known as Lewy bodies (LB) associated with neurodegenerative diseases, such as Parkinson’s disease (PD) and dementia with Lewy body (DLB)^5,6. Increased α-synuclein level due to the multiplication of SNCA alleles is causal to PD^7,8.

Previous evidence suggested “prion-like” cell-to-cell transmission of α-synuclein^9,10. α-synuclein can be secreted by neurons as a result of cellular stress or injury, or as a response to stimulation^11,12,13. Neighboring neurons and glia can engulf and clear extracellular α-synuclein and thus contributing to the regulation of α-synuclein homeostasis in the brain. Interestingly, injection of fibrillar α-synuclein into animal brains causes the spread of LB-like pathology¹⁴, supporting the “Braak hypothesis” of staging in synucleinopathies¹⁵. Therefore, cellular uptake and clearance pathways are key processes to control the deposition and spread of α-synuclein aggregates, thus affecting disease progression.

Although neurons and glia in the brain can ingest and degrade extracellular α-synuclein, microglia show the highest efficiency in vitro¹⁶. Extensive effort has been made toward the identification of cellular pathways of α-synuclein clearance. Several studies reported receptor-mediated internalization of various forms of α-synuclein in neurons and glial cells^17,18,19. However, the exact mechanism for the clearance of internalized α-synuclein remains unclear.

Previous studies suggest that α-synuclein is degraded by macroautophagy, chaperone-mediated autophagy, and the proteasome^20,21,22,23. However, the evidence for macroautophagy (hereafter referred to as autophagy) degradation of α-synuclein is extremely limited^24,25. Autophagy is a bulk degradation pathway responsible for the clearance of protein aggregates and damaged cellular organelles^26,27. Recent evidence demonstrates a strong selectivity for autophagy²⁸. Direct evidence for autophagy in selective degradation of α-synuclein is entirely lacking. Furthermore, since most of the studies of α-synuclein degradation were focused on neurons, whether or not microglia, the prototypical scavenger cell in the brain, take part in the degradation of α-synuclein remains unclear.

Here, we report that microglia ingest and degrade neuron-released α-synuclein through selective autophagy in vitro and in vivo. We document that ingested α-synuclein in microglia is sequestered by autophagosomes for degradation, which is mediated by TLR4-NF-κB signaling through upregulation of the autophagy receptor, p62/SQSTM1. Thus, our results uncover a microglia-specific α-synuclein degradation pathway termed synucleinphagy that regulates α-synuclein homeostasis in the CNS.

Results

Neuron-released α-synuclein is engulfed by microglia in vivo

To understand microglia and α-synuclein interaction in vivo, we employed two mouse models, both of which express wild-type (WT) human α-synuclein (hα-Syn) in a neuron-specific manner. We first verified that mice injected with adeno-associated virus 9 (AAV9) carrying green fluorescent protein (GFP) under the synapsin promoter into the Substantia Nigra pars compacta (SNpc) expressed GFP strictly in neurons (Supplementary Fig. 1a, b). Mice injected with AAV9-hα-Syn in the SNpc showed loss of dopaminergic neurons on the ipsilateral side (Supplementary Fig. 1c), as reported²⁹. In fact, dopamine transporter (DAT) and tyrosine hydroxylase (TH) protein levels were reduced compared with AAV9-GFP-injected mice (Supplementary Fig. 1d, e). Moreover, AAV9-hα-Syn, but not AAV9-GFP injection, caused microglial activation in the striatum as evidenced by the reduced length of processes and terminal branch points as well as increased cell number (Fig. 1a, b).

By RT-qPCR analysis of microglia state-specific gene expression^30,31,32, we found upregulation of several disease-associated signature genes such as ApoE, Axl, and Lpl, inflammatory gene Tnf, and Tgfbr1 at 4-weeks post AAV injection (Fig. 1c; Supplementary Fig. 2). Importantly, we verified the presence of hα-Syn protein in isolated microglia via western blot with hα-Syn-specific antibody (Fig. 1d; Supplementary Fig. 3a). Using Cx3cr1^{CreER-IRES-Eyfp} transgenic mice³³, which express enhanced yellow fluorescent protein (EYFP) under the microglia-specific Cx3cr1 promoter, we detected hα-Syn in Cx3cr1-EYFP-positive microglia (Fig. 1e; Supplementary Fig. 3b).

In transgenic mice overexpressing hα-Syn under the Thy-1 promoter (hα-Syn-Tg), we also found microglial activation in the striatum of 10-month-old mice (Fig. 1f, g). hα-Syn protein was clearly present in microglia through western blot analysis (Fig. 1h) and immunofluorescent staining in Iba-1-positive microglia at the striatum (Fig. 1i; Supplementary Fig. 3c). Noticeably, hα-Syn-Tg mice did not show degeneration of dopaminergic neurons in SNpc up to 12-month-old (Supplementary Fig. 1f). Together, our data from two different mouse models demonstrate that neuron-released hα-Syn induces microglial activation, accompanied by microglial engulfment of extracellular hα-Syn in vivo.

Microglia-engulfed α-synuclein is degraded by autophagy

We next asked if ingested α-synuclein goes through endocytic degradation following phagocytosis or receptor-mediated endocytosis^18,19. We treated cultured primary microglia with recombinant hα-Syn protein and found no evidence of colocalization between hα-Syn and an early endosome marker EEA1 (Supplementary Figs. 3d, 4a). Neither cytochalasin D, a blocker of actin polymerization necessary for phagocytosis, nor Dynasore, a GTPase inhibitor that suppresses both clathrin-dependent and independent endocytosis, inhibited the uptake of hα-Syn protein but rather it increased the uptake (Supplementary Figs. 4b, 5a, 5b).

We then asked whether autophagy degrades hα-Syn in microglia. We co-stained hα-Syn-treated primary microglia with anti-hα-Syn and anti-ubiquitin or anti-p62/SQSTM1, an autophagy receptor³⁴, and observed colocalization of hα-Syn with ubiquitin or p62 in discrete puncta (middle panels, Fig. 2a; left panels, Fig. 2b). We also treated microglia derived from GFP–LC3 transgenic mice³⁵ with hα-Syn and detected colocalization of the autophagosome marker, GFP–LC3, and hα-Syn (right panels, Fig. 2b). These hα-Syn-associated ubiquitin-, GFP–LC3-, or p62-positive puncta appeared at 6 h and disappeared at 24 h after hα-Syn treatment (arrowheads, Fig. 2a). We also noticed smaller ubiquitin-negative hα-Syn particles that were evident after 6 h, but remained 24 h after treatment (arrows, Fig. 2a). A time-course analysis showed that hα-Syn/ubiquitin-positive puncta emerged at 3 h, and the number of puncta peaked at 6 h, followed by a reduction in number at 24 h post treatment (left panel, Fig. 2c). The puncta number also depended on hα-Syn concentration (right panel, Fig. 2c). In addition, we also observed hα-Syn-associated ubiquitin-, GFP–LC3-positive puncta after treating the medium from AAV9-hα-Syn-infected cortical neuron (arrowheads, Supplementary Fig. 4c), suggesting that hα-Syn-associated LC3 puncta also occurs in microglia in response to physiological form of α-Syn. Furthermore, in GFP–LC3 transgenic mice injected with AAV9-hα-Syn, we found ingested hα-Syn colocalized with GFP–LC3 puncta in Iba-1-positive microglia in the striatum (Fig. 2d). We then performed ultrastructural analysis through electron microscopy (EM) and found that hα-Syn-treated microglia develop large inclusions which are surrounded by double-membrane structures, characteristic of autophagosomes (Fig. 2e).

**Fig. 2: Microglia-engulfed α-synuclein is degraded by autophagy.**

We next treated microglia with SAR405, an inhibitor of VPS34, which is a class III phosphatidylinositol 3-kinase required for autophagosome membrane synthesis³⁶, Bafilomycin A1, or Chloroquine, two different inhibitors of lysosome acidification, after adding hα-Syn. All three drugs prevented the clearance of hα-Syn/ubiquitin-positive puncta (Fig. 2f). By contrast, treatment with Torin1, an inhibitor of mTOR that stimulates autophagy³⁷, facilitated the clearance of puncta (Fig. 2g). We next obtained autophagy-deficient microglia by breeding Cx3cr1^Cre mice, which constitutively express Cre recombinase, with either Atg7^flox/flox or Atg14^flox/flox mice. Atg7 encodes an E1-like enzyme essential in the ubiquitin-like conjugation systems required for autophagy, and Atg14 encodes a positive regulator of VPS34 (refs. ^38,39). The lack of autophagy was confirmed in Atg7-deficient cells or Atg14-deficient cells through the analysis of Atg7, Atg14, p62, and LC3-II (Fig. 2h). In these autophagy-deficient microglia, degradation of hα-Syn/ubiquitin-positive puncta was inhibited (Fig. 2i). We noticed that Atg7 or Atg14 deficiency did not cause microglia death regardless of hα-Syn protein treatment (Supplementary Fig. 4d, e). Thus, we concluded that microglia engulf extracellular α-syn protein, which is sequestered by autophagosomes and degraded by the autophagy–lysosome pathway.

p62 mediates sequestration and degradation of α-synuclein

p62 is known to recognize and interact with ubiquitinated cargo to initiate selective autophagy³⁴. We next investigated the role of p62 in autophagic clearance of hα-Syn. We observed that hα-Syn treatment caused an increase in p62 protein up to 9 h (Fig. 3a). Interestingly, p62 mRNA levels peaked at 3 h, declined at 6 h, and returned to baseline at 9 h post treatment (Fig. 3b), which mirrors the transient increase of hα-Syn/ubiquitin/p62/GFP–LC3-positive puncta (Fig. 2a–c). Actinomycin D, a blocker of mRNA translation, inhibited p62 induction when added before hα-Syn treatment (Supplementary Fig. 4g) and suppressed the formation of hα-Syn/ubiquitin-positive puncta (Supplementary fig. 4h). By contrast, total LC3-II levels changed little after hα-Syn treatment (Supplementary Fig. 4i), suggesting no significant alteration of overall autophagy. Furthermore, other autophagy receptors such as Ndp52, Optineurin, and Nbr1 were not altered (Supplementary Fig. 4i), suggesting that hα-Syn selectively induces p62 expression without affecting other autophagy receptors.

We next treated cultured microglia from p62-KO mice with hα-Syn (Fig. 3c). Remarkably, hα-Syn/ubiquitin-positive puncta were abolished in p62-KO cells (Fig. 3d, e); meanwhile, p62 deficiency did not cause cell death regardless of hα-Syn treatment (Supplementary Fig. 4f), suggesting that p62 is necessary for the formation of hα-Syn/ubiquitin-positive puncta after hα-Syn ingestion, consistent with its role in selective autophagy³⁴. In agreement with the in vitro results (Fig. 3a, b), the number of p62 puncta in microglia was significantly increased in the striatum of AAV9-hα-Syn-injected mice at 6 weeks post injection (Fig. 3f, g) and in 10-month-old hα-Syn-Tg mice compared with controls (Fig. 3h). Furthermore, p62/ubiquitin and hα-Syn puncta colocalized in EYFP-positive (AAV9-hα-Syn-injected Cx3cr1^{CreER-IRES-Eyfp}) or Iba-1-positive (hα-Syn-Tg mice) microglia (Fig. 3i, j). Through co-immunoprecipitation analysis, we found that hα-Syn interacted with specific p62 protein species of high molecular weight in AAV9-hα-Syn-injected brain, hα-Syn-Tg brain, and primary microglia treated with hα-Syn (Fig. 3k–m), supporting that oligomeric p62 directly binds and recruits hα-Syn into autophagosomes. The above data thus demonstrates that hα-Syn-induced p62 upregulation in microglia confers a selective autophagic degradation of ingested hα-Syn.

α-synuclein-induced p62 upregulation requires TLR4

We next asked how microglia upregulate p62 transcription in response to extracellular hα-Syn. Previous reports showed α-synuclein can trigger TLR4 or TLR2 activation in cultured glia, which leads to an increase in inflammatory gene transcription^19,40,41. To test TLR4 involvement, we treated microglia from Tlr4-KO mice with hα-Syn and observed no increase in p62 protein and mRNA regardless of the dose (Fig. 4a, b). Also, the number of hα-Syn/ubiquitin-positive puncta present at 6 h after hα-Syn treatment was significantly decreased in Tlr4-KO microglia compared with control cells (Fig. 4c). In addition, we treated cultures with TAK-242, an inhibitor of TLR4 signaling by selectively binding to Cys747 of TLR4 cytoplasmic tail that disrupts its interaction with adaptor molecules TIRAP and TRAM⁴² (Supplementary Fig. 5c). Similar to Tlr4-KO microglia, TAK-242-treated cells showed impaired p62 induction (Fig. 4d) as well as a reduction in the number of hα-Syn/ubiquitin-positive puncta following hα-Syn treatment (Fig. 4e). Furthermore, Tlr4-KO mice injected with AAV-hα-Syn showed reduced p62 puncta number in Iba-1-positive microglia compared with control mice (Fig. 4f, g), indicating that TLR4 is required for microglial p62 induction in response to hα-Syn exposure both in vivo and in vitro.

Lipopolysaccharide (LPS) triggers intracellular signaling via TLR4 binding, leading to the activation of the NF-κB and MAPK pathways. TLR4-LPS binding induces endocytosis of the TLR4-LPS complex and subsequent expression of interferon-beta 1 (Ifnb1) through activation of interferon regulatory factor 3 (IRF3)^43,44. Therefore, we asked if hα-Syn triggers similar TLR4 downstream events. Through RT-qPCR analysis, we found that p62 was also increased by LPS as reported⁴⁵ (Fig. 4h); the major target genes of the NF-κB pathway, Il-1b and Tnf, were upregulated by LPS or hα-Syn treatment. However, LPS has more robust effects in stimulating Il-1b and Tnf expression compared with hα-Syn (Fig. 4h). LPS, but not hα-Syn, induced Ifnb1 expression (Fig. 4h) and increased levels of p-IFR3 (S396), a marker of IRF3 activation (Fig. 4i), indicating the absence of IRF3 signaling in hα-Syn-treated microglia.

To test TLR4 endocytosis, we treated microglia with LPS or hα-Syn protein for 30 min and stained with TLR4 antibody conjugated with phycoerythrin (PE) to monitor the levels of TLR4 at the cell surface. LPS decreased the level of TLR4 on the plasma membrane as a result of endocytosis as reported⁴⁴; by contrast, hα-Syn had little effect on TLR4 levels at the cell surface (Fig. 4j), suggesting that hα-Syn does not induce TLR4 internalization. Lastly, pretreatment with Dynasore did not block p62 induction by hα-Syn (Fig. 4k). The above observations suggest that the hα-Syn-TLR4 interaction stimulates p62 expression without causing TLR4 receptor endocytosis distinctively from LPS-TLR4 signaling.

NF-κB mediates α-synuclein-TLR4 signaling and p62 induction

Upon hα-Syn treatment, microglia activate NF-κB and p38 pathways as indicated by an increase of p-NF-κB (S536), a decrease of IκB level, and an increase of p-p38 (T180/Y182) (Fig. 5a). However, p-ERK1/2 (T202/Y204) and p-JNK (T183/Y185) were not changed (Fig. 5a). We next treated microglia with ML-120B, an inhibitor of IKK-2, which is responsible for IκB phosphorylation and degradation that leads to NF-κB activation, and SB202190 or SB203580, two different inhibitors of p38. While the inhibitory effects of ML-120B, SB202190, and SB203580 were all confirmed in LPS-induced NF-κB activation and gene expression of proinflammatory cytokines (Supplementary Fig. 5d, e), only ML-120B significantly suppressed p62 induction and reduced the number of hα-Syn/ubiquitin-positive puncta after hα-Syn treatment (Fig. 5b, c), suggesting that NF-κB is the primary mediator for TLR4-triggered p62 induction. Supporting this notion, mRNA levels of Il-1b and Tnf were significantly lower in Tlr4-KO than WT cells, despite that both WT and Tlr4-KO microglia show an increase in Il-1b and Tnf expression after hα-Syn treatment (Fig. 5d). By contrast, NRF2 or TFEB pathways were unaltered as shown by the lack of change in the level of NQO-1 and HO-1, two major targets of NRF2, or the intensity of nuclear TFEB in response to hα-Syn^46,47 (Supplementary Fig. 4i, j).

TLR4 is a preferred TLR for α-synuclein to activate NF-κB

Previous studies suggest that in microglia α-synuclein interacts with TLR2 (ref. ¹⁹). We, therefore, tested whether the interaction between hα-Syn-TLR2 activates the NF-κB pathway. We applied a TLR2-blocking antibody (T2.5 clone), which suppressed p-NF-κB in response to the TLR2-specific ligand, Pam₃CSK₄ (Supplementary Fig. 5f). Remarkably, pretreatment with the TLR2-blocking antibody completely suppressed the induction of Il-1b and Tnf in response to hα-Syn in Tlr4-KO microglia (Fig. 5e). Interestingly, the TLR2-blocking antibody showed an enhanced effect of suppression in Tlr4-KO microglia compared with the WT (Fig. 5f), suggesting that TLR2 can partially compensate for TLR4 function in regulating the response to hα-Syn in Tlr4-KO microglia.

To test the specificity of hα-Syn effects on plasma membrane-expressed TLRs⁴⁸, we established the NF-κB-luciferase system expressing NF-κB response element and luciferase reporter gene in HEK293T cells expressing TLR2, TLR2/TLR1, TLR2/TLR6, TLR4/MD2/CD14 (TLR4 complex), or TLR5. First, we validated the specificity of this system by applying specific ligands for each TLR. Application of LPS, Pam₃CSK₄, and recombinant Flagellin peptides selectively increased NF-κB activity in TLR4 complex, TLR2 and TLR2 combinations, and TLR5, respectively (Supplementary Fig. 6). Furthermore, hα-Syn treatment significantly increased NF-κB activity in a dose-dependent manner in TLR4/MD2/CD14-transfected cells compared with YFP transfected cells (Fig. 5g). We also observed that hα-Syn increased NF-κB activity in TLR2, TLR2/1, and TLR2/6-transfected cells comparable with the activity in the TLR4 complex, while no change of the NF-κB activity was observed in YFP or TLR5-transfected cells (Fig. 5h). Furthermore, we applied the TLR2-blocking antibody in cells co-transfected with TLR2 and the TLR4 complex. As expected, pretreatment with TLR2-blocking antibody prevented the increase of NF-κB activity induced by Pam₃CSK₄, but not by LPS (Fig. 5i). Interestingly, application of the TLR2-blocking antibody had little effect on the increase of NF-κB activity induced by hα-Syn (Fig. 5i). These results suggest that hα-Syn prefers TLR4 over TLR2 in regulating NF-κB signaling, while hα-Syn may only act through TLR2 to induce NF-κB signaling in the absence of TLR4.

Autophagy-deficient microglia cause α-synuclein accumulation

We next established microglia-specific Atg7-deficient mice (Cx3cr1^{CreER-IRES-Eyfp}; Atg7^flox/flox) by breeding Cx3cr1^{CreER-IRES-Eyfp} (tamoxifen-inducible Cre) with Atg7^flox/flox mice. Although previous studies showed that deletion of Atg7 specifically in neurons causes neurodegeneration^49,50, we found that Atg7-deficiency had little effect on microglia number and state-specific gene signatures (Supplementary Fig. 7a–g), indicating that loss of autophagy does not cause microglial death or activation for at least up to 7 months following Atg7 deletion.

We then tested the consequence of microglial Atg7-deficiency on α-synuclein homeostasis in the brain by investigating Cx3cr1^{CreER-IRES-Eyfp}; Atg7^flox/flox mice expressing hα-Syn through either AAV injection (Fig. 6a) or transgenic expression (Fig. 6c). After injection of AAV9-hα-Syn, we observed that Atg7-deficient microglia contained more p62 puncta as well as p62/hα-Syn-positive puncta compared with control microglia (Supplementary Fig. 7h, i). Analysis of brain lysate fractions showed that microglial Atg7-deficient brains contained significantly elevated levels of detergent-insoluble hα-Syn at 6 weeks after AAV9-hα-Syn injection, as evidenced by a smear of high-molecular-weight hα-Syn species, compared to littermate controls (Fig. 6b).

**Fig. 6: Autophagy-deficient microglia cause α-synuclein accumulation.**

We also examined microglial Atg7-deficient mice after crossing with hα-Syn-Tg (Cx3cr1^{CreER-IRES-Eyfp}; Atg7^flox/flox; hα-Syn-Tg mice) at 7 months after Atg7 deletion (Fig. 6c). The number of p62 puncta and p62/hα-Syn-positive puncta was significantly higher in Atg7-deficient microglia than control (Fig. 6d, e). Furthermore, microglial Atg7-deficient brains contained elevated levels of detergent-insoluble hα-Syn protein than control mice at 10 months of age (Fig. 6f). Furthermore, microglial Atg7-deficient mice showed increased staining of p-S129 α-synuclein-positive structures, particularly in the lateral SNpc (Fig. 6g, h) and the dorsal striatum (Fig. 6i, j).

Autophagy-deficient microglia promote neurodegeneration

Despite the lack of detectable neurodegeneration in hα-Syn-Tg mice or Cx3cr1^{CreER-IRES-Eyfp}; Atg7^flox/flox mice (Supplementary Fig. 1f; Fig. 7c, d), the compound mice Cx3cr1^{CreER-IRES-Eyfp}; Atg7^flox/flox; hα-Syn-Tg (after the cross of the above two lines of mice) exhibited a decrease in the number of dopaminergic neurons as shown by stereological counting TH-positive neurons and Nissl-positive cells in the SNpc (Fig. 7a, b). These data suggest that disruption of microglial autophagy significantly enhances α-synuclein-mediated neurotoxicity. Taken together, our results demonstrate an important role for microglial autophagy in clearing α-synuclein released from neurons and preventing neurodegeneration.

Discussion

Our study has uncovered a significant role of microglia in clearing extracellular α-synuclein and protecting neurons through selective autophagy, which we term “synucleinphagy”. Here, we show the first evidence that microglia sequester engulfed α-synuclein into autophagosomes for lysosomal degradation, thus solving a long-standing question as to whether wild-type α-synuclein is indeed degraded by autophagy. We identified the mechanism for microglia-specific synucleinphagy, which is mediated through TLR4-NF-κB signaling by transcriptional upregulation of the autophagy receptor, p62/SQSTM1 (Fig. 7e). By contrast, neuron has no change of autophagosomes or p62 protein levels after the treatment with various forms of α-synuclein⁵¹, likely due to lack of the TLR4-NF-κB pathway.

We demonstrate the critical role of TLR4-NF-κB-p62 in mediating synucleinphagy in microglia. TLR4 signaling has been linked to xenophagy, a selective autophagic degradation of infected bacteria, in macrophages. TLR4-NF-κB signaling was previously shown to induce selective autophagy after pathogen invasion^52,53,54. In addition, p62 has a role in the clearance of intra-cytosolic Shigella and Listeria⁵⁵. Thus, synucleinphagy and xenophagy have a shared mechanism through TLR4-mediated signaling. Our study, however, showed that synucleinphagy require specifically p62 as other autophagy receptors, such as Ndp52, Optineurin, or Nbr1, are not upregulated upon TLR4-NF-κB activation. We also noted that during synucleinphagy, LC3-II level was unaltered, despite the increase of p62 levels and colocalization of LC3 puncta with ingested α-synuclein in microglia, suggesting a lack of global upregulation of autophagy but rather a restricted, local selective autophagy. The specific sequestration of α-synuclein into autophagosomes is likely mediated by p62 binding of ubiquitin-linked α-synuclein protein³⁴; future study should identify the specific E3 ligase of α-synuclein in microglia responsible for synucleinphagy. Moreover, synucleinphagy differs from aggrephagy, a selective type of autophagy which degrades intracellular protein aggregates without the involvement of cell surface receptors (e.g., TLR) or NF-κB signaling.

Our result reveals that α-synuclein has a strong preference for TLR4 in signaling NF-κB activity over TLR2 receptor, despite that α-synuclein can act through either receptor, in an agreement with previous reports showing that both receptors interact with α-synuclein (albeit different forms). For example, TLR4 was required for NF-κB activation, and increased inflammation and phagocytic activity in response to various forms of α-synuclein^40,41,56. By contrast, TLR2 also plays a role in neurons in response to α-synuclein in addition to microglia^19,57,58. Future studies should investigate further the significance of other TLRs in mediating α-synuclein degradation as well as inflammation in microglia. Our study also reveals a distinct feature of α-synuclein-triggered TLR4 activation, which does not promote TLR4 endocytosis in contrast to the effect of LPS. The absence of endocytosed TLR4 may prevent Ifnb1 induction or increase of p-IFR3 (S396) level, despite the activation of NF-κB signaling, thus avoiding excessive inflammation and neurotoxicity⁵⁹.

Our report, however, raises many questions with regard to the exact mechanism whereby α-synuclein enters microglia. Our study suggests that microglia ingest and sequester α-synuclein into autophagosomes independent of endocytosis or phagocytosis. Thus, synucleinphagy is a distinct process from LC3-associated phagocytosis (LAP)⁶⁰. A number of studies demonstrated that α-synuclein can enter the cells or penetrate cytoplasmic membrane through endocytosis-independent process^61,62,63. Therefore, our study supports a model that a portion of extracellular α-synuclein interacts with TLR4 (or TLR2), which then triggers NF-κB signaling, while a majority of α-synuclein penetrates cytoplasmic membrane or enters microglia through TLR4 endocytosis-independent manner. The precise process and mechanism for α-synuclein to enter microglia awaits future detailed investigation.

Finally, emerging human genomic studies suggest the autophagy–lysosome system as a converging pathogenic pathway of PD⁶⁴, and many PD-related genes such as PINK1, PARKIN, GBA, and LRRK2, were shown to play a role in autophagy-related pathways in glial cells⁶⁵. Our finding of microglial synucleinphagy via selective autophagy that confers neuroprotection may assist in understanding the role of microglia in preventing synucleinopathies.

Methods

Animals

B6.B10ScN-Tlr4^lps-del/JthJ mice (Tlr4-KO, #007227), B6J.B6N(Cg)-Cx3cr1^{tm1.1(cre)Jung}/J mice (Cx3cr1^Cre, #025524), and B6.129P2(Cg)-Cx3cr1^{tm2.1(cre/ERT2)Litt}/WganJ mice (Cx3cr1^{CreER-IRES-Eyfp} in this study, #021160) were purchased from Jackson Laboratory (Bar Harbor, ME). Atg7^flox/flox mice and p62-KO mice were kindly gifted from Dr. Masaaki Komatsu (Tokyo, Japan)^38,66. Atg14^flox/flox mice were Dr. Herbert W.Virgin (Washington University School of Medicine, St. Louis, MO)^39,67. GFP–LC3 transgenic mice (C57BL/6J) was described previously³⁵. For generating microglia-specific Atg7-deficient mice, Atg7^flox/flox mice were bred to Cx3cr1^{CreER-IRES-Eyfp} (#021160) mice. Cx3cr1^{CreER-IRES-Eyfp}; Atg7^flox/flox mice and littermate Atg7^flox/flox mice were injected with tamoxifen (intraperitoneal injection, 5 continuous days, 1.5 mg per mouse) to induce Cre recombinase expression. Cx3cr1^{CreER-IRES-Eyfp} mice express Cre recombinase not only in microglia but also in myeloid cell populations in the blood which are replenished by Cx3cr1-negative progenitors, which takes 1 month. However, due to limited turn-over, microglia will continuously express Cre recombinase³³. Therefore, we injected AAV-hα-Syn after 1 month post tamoxifen administration to avoid any possible involvement of Atg7-deficient blood-derived cells in our observation. For obtaining Atg7 or Atg14-deficient cultured cells, Atg7^flox/flox mice or Atg14^flox/flox mice were bred to Cx3cr1^Cre mice (#025524). Mice were maintained under a 12-h light/12-h dark cyclerooms with water accessible at all times. All animal procedures were approved by Icahn School of Medicine at Mount Sinai Animal Care and Use Committee (IACUC-2015-0046).

Generation of Thy-1-SNCA mice (Jax #017682)

The transgenic construct contained a 4.2 kb murine Thy-1 (Gene location on chromosome 9: 43,851,467-43,856,662) promoter fragment containing ~1.6 kb of 5′ flanking sequence plus the 5′ first UTR exon (93 bp), the first intron (2480 bp), and the noncoding region of exon 2 before ATG. This was followed by a 423 bp ORF of human wild-type α-synuclein cDNA (GenBank Acc# NM_000345), and 221 bp of SV40 polyA sequence in a pGL3 vector backbone (Promega). The above strategy was according to the published paper⁶⁸. The microinjection fragment was purified and quantified before the injections. Three injection sessions were performed into C57BL/6Ntac embryos. Founders were identified and mated to wild-type C57BL/6NTac females. Analysis of integration site indicates insertion occurred at Chr11:40456787-40495044 and resulted in a 38.4 Kb deletion⁶⁹. Copy number analysis was also performed on tail clips of the mice using the following primers and probe combination (spanning exons 2 and 3 of hSNCA gene):

Forward primer: 5′-AGGACTTTCAAAGGCCAAGG-3′

Probe (FAM-labeled): 5′-AGTTGTGGCTGCTGCTGAGAAAACC-3′

Reverse primer: 5′-CACTTGCTCTTTGGTCTTCTCAG-3′

The DNA copy number was determined by a standard curve method where for the standard curve the calculated number of copies of (1, 5, 10, 20, 50, and 100 copies) of the 5.1 kb transgene DNA fragment was mixed with 5 ng of DNA prepared from the wild-type C57BL/6NTac mice. The data were analyzed by plotting ΔCt values against the known copy number. A standard curve was run in parallel with the samples prepared from the mice. An average copy number was 1.98 +/− 0.81. Following copy number analysis, the line was sent to The Jackson Laboratory for colony establishment (C57BL/6N-Tg (Thy-1-SNCA)15Mjff/J; Stock No. 017682).

Adeno-associated virus (AAV)

AAV vectors encoding either GFP or human WT α-synuclein were kindly gifted from Dr. Jia-Yi Li (Lund University, Lund, Sweden). AAV9-GFP or AAV9-human WT α-synuclein were packaged using the service provided by Vigene company (Rockville, MD). For establishing an acute PD animal model, 2 µl of 6 × 10¹³ GC/ml of either AAV-GFP or AAV-α-synuclein were injected into the SNpc area of mice (AP: −3.28 mm, ML: −1.5 mm, DV: −4.1 mm) using a stereotaxic apparatus (Kopf Instruments, Tujunga, CA).

Cell culture

Primary microglia were obtained from mixed glia cultured from forebrain of pups from 1- to 3-day-old C57/BL6J WT mice (Jackson Laboratory). Briefly, forebrains were isolated and homogenized into single-cell suspensions by triturating with fire-polished Pasteur pipettes in Dulbecco’s modified Eagle’s medium (DMEM, #11965-092, Gibco^TM, Thermo Fisher Scientific) containing penicillin/streptomycin (#15080-063, Gibco^TM), GlutaMAX^TM (#35050061, Gibco^TM), and heat-inactivated 10% fetal bovine serum (FBS, #S11550, Atlanta Biologicals, Atlanta, GA). Homogenized tissues were plated at 75 cm² T-flask (BD Bioscience, San Jose, CA, USA) and incubated for 2 weeks. Floating microglia were detached from flasks by mild shaking and then filtered through a 70-µm cell strainer to remove cell clumps or debris. Microglia were plated onto culture dishes at an appropriate density. HEK293T cell line was maintained in DMEM supplemented with penicillin/streptomycin and 10% FBS.

Adult microglia isolation

For adult microglia isolation, we adopted the established protocol^32,70. Briefly, whole brains were isolated from mice after whole-body intracardial perfusion with ice-cold D-PBS (Gibco^TM) and homogenized using 15-ml size Dounce homogenizer in 5 ml ice-cold D-PBS with ten strokes. Homogenized tissues were filtered through a 70-µm cell strainer to remove any debris and centrifuged with 1000 × g for 10 min at 4 °C. Cell pellets were resuspended in 5 ml of 70% Percoll^® (P1644, Sigma-Aldrich) made in 10% FBS-containing DMEM and 7 ml of 40% Percoll^® made in D-PBS were laid on the top of it gently in a 15 ml tube (Falcon^TM, CA). After centrifugation with 800 × g speed for 30 min in RT, top layer enriched with myelin was removed and the middle layer (~10 ml) was mixed with ice-cold D-PBS (~40 ml), and centrifuged with 1400 × g for 5 min at 4 °C. This microglia-enriched collection was further used for flow cytometry.

Flow cytometry

Microglia-enriched pellets were resuspended in 500 µl of FACS buffer (1% FBS in ice-cold D-PBS), and incubated with 10 µg/ml of anti-mouse CD16/CD32 (#14-0161-82, Invitrogen) for 15 min on the ice to block Fc-receptor on microglia followed by 5 µg/ml of CD45 monoclonal antibody (I3/2.3) conjugated with APC-Cyanine7 (#A15395, Invitrogen) and 2 µg/ml of CD11b monoclonal antibody (M1/70) conjugated with PerCP-Cyanine5.5 (#45-0112-82, Invitrogen) for 30 min on the ice. After washed, the microglia population (CD11b^High/CD45^Intermediate) was gated and collected as shown in Supplementary Fig. 2 using FACSDiva v8.0.1 (BD Biosciences). In an initial validation step, microglia from Cx3cr1^{CreER-IRES-Eyfp} were stained with CD45 and CD11b antibodies and the ratio of CD11b^High/CD45^Intermediate cell population among EYFP-positive cells, and vice versa was checked by setting each other’s cell population as a parent gate as shown in Supplementary Fig. 2.

Quantitative RT–PCR

cDNA was synthesized from the total RNA with AffinityScript™ Multi-Temp RT (Stratagene) with oligo dT18 as primer. For real-time PCR, PlatinumTaq DNA polymerase (Invitrogen) and a SYBR green (Molecular Probes) containing buffer were used. The real-time PCRs were performed using a thermocycler (ABI7900HT; Applied Biosystems). The PCR conditions used were: 95 °C for 2 min, 40 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s using the following primer pairs (Eurofins MWG Operon):

Actin, 5′-AGGTGACAGCATTGCTTCTC-3′ (sense), 5′-GCTGCCTCAACACCTCAAC-3′ (antisense);

Axl, 5′-AGCACAGTCTGCAAACTCC-3′ (sense), 5′-CTACCTCTAGCTCCGTAGGTT-3′ (antisense);

ApoE, 5′-AACCGCTTCTGGGATTACCTG-3′ (sense), 5′-CTCTCCCTCGGCTAGGCAT-3′ (antisense);

Cd9, 5′-TAACTTCATCTTCTGGCTCGCT-3′ (sense), 5′-AAACCAACCAGCATCATGAGG-3′ (antisense);

Clec7a, 5′-ACTTCAGCACTCAAGACATCC-3′ (sense), 5′-TGGCTTCCTTTCTCTGATCC-3′ (antisense);

Csf1r, 5′-CCTCAAACGTGGAGACACCAA-3′ (sense), 5′-CGTGTGCCAACATCATTGCT-3′ (antisense);

Cst7, 5′-TTCAACAACTGCACAAATGACA-3′ (sense), 5′-GGCCTTTCACCACCTGTACCA-3′ (antisense);

Cx3cr1, 5′-CAACCCCTTTATCTACGCCTT-3′ (sense), 5′-GACCCATCTCCCTCGCTTG-3′ (antisense);

HexB, 5′-TACAAGAACCAGTAGCCGTCCT-3′ (sense), 5′-CTCTAAACCTCGTAACGCTCC-3′ (antisense);

H2-Ab1, 5′-GCCCTCAACCACCACAACAC-3′ (sense), 5′-AGTCCCCATTCCTAATAAGCTGT-3′ (antisense);

Il-1b, 5′-CCTCTGATGGGCAACCACTT-3′ (sense), 5′-TTCATCCCCCACACGTTGAC-3′ (antisense);

Il-6, 5′-ACAGAAGGAGTGGCTAAGGA-3′ (sense), 5′-CGCACTAGGTTTGCCGAGTA-3′ (antisense);

Ifnb1, 5′-AGATGTCCTCAACTGCTCTC-3′ (sense), 5′-AGATTCACTACCAGTCCCAG-3′ (antisense);

Ifng, 5′-TGGCAGGAGATGTCTACACT-3′ (sense), 5′-GAAGCACCAGGTGTCAAGTC-3′ (antisense);

Itgax, 5′-CTGCTGCCACCAACCCTTC-3′ (sense), 5′-AGCCATCAATCAGGAACACGA-3′ (antisense);

Lpl, 5′-ACAAGTTTTAGAGCAGGACCAT-3′ (sense), 5′-TTGCACAGCAGTTTACAAGCATC-3′ (antisense);

Olfml3, 5′- GACACAGAACCCAGCTTTGC-3′ (sense), 5′- GCTACAGTCCGTCACCATATCGT-3′ (antisense);

p62/Sqstm1, 5′-GAAGCTGCCCTATACCCACA-3′ (sense), 5′-CCCGATGTCGTAATTCTTGGTC-3′ (antisense);

P2ry12, 5′-TTTGCTGGGCTCATCACGAAC-3′ (sense), 5′-ACTGAAGTAACTTGGCACACC-3′ (antisense);

Tgfbr1, 5′-GATCCATCACTAGATCGCCCTT-3′ (sense), 5′-CCGACCTTTGCCAATGCTT-3′ (antisense);

Tmem119, 5′-CTGACATTCTGGCTGCTACC-3′ (sense), 5′-CACCCTTCACAGGCTTTGCTC-3′ (antisense);

Tnf, 5′-TCACTGGAGCCTCGAATGTC-3′ (sense), 5′-GTGAGGAAGGCTGTGCATTG-3′ (antisense);

Trem2, 5′-GTCCCAAGCCCTCAACACC-3′ (sense), 5′-TCCTCACCCAGCTGCCGACA-3′ (antisense).

The cycle threshold (Ct) for the gene transcript was normalized to the average Ct for transcripts of the housekeeping gene, Actin, amplified in each reaction. Relative quantitation of normalized transcript abundance was determined using the comparative Ct method (∆∆Ct).

Immunostaining

For brain tissue staining, mice were anesthetized and transcardially perfused with ice-cold PBS, and brains were obtained. Then, brains were fixed overnight at 4 °C in 4% paraformaldehyde. Fixed brains were stored at 4 °C in a 30% sucrose solution until they sank. A series of coronal sections (30 µm) were obtained with a cryostat (Leica, Wetzlar, Germany). Coronal sections were incubated with blocking/permeabilization buffer (5% goat serum and 0.25% Triton X-100 in PBS) at RT for 30 min and incubated with primary antibodies against Aquaporin 4 (AB3594, Millipore, 1:500), GFAP (#130300, Thermofisher Scientific, 1:500), GFP/YFP (MA5-15256, Thermofisher Scientific, 1:500), Iba-1 (019-19741, Wako, Japan, 1:500), NeuN (MAB377, Chemicon, 1:500), p62 (GP62-C, Progen, Germany, 1:250), α-synuclein (clone MJFR1, ab138501, Abcam, 1:500; clone syn211 conjugated with Alexa594 fluorescein, sc-12767 AF594, Santa Cruz Biotechnology, 1:200), or ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology, 1:500) at 4 °C overnight followed by secondary antibodies conjugated with Alexa-fluorescein. For 3,3′-diaminobenzidine (DAB) staining, serial sections were rinsed three times with PBS, treated with 3% H₂O₂ for 3 min, and rinsed with PBS containing 0.1% Triton X-100 (PBST). After incubating in blocking solution (10% Goat serum and 0.25% Triton X-100 in PBS), sections were incubated overnight at room temperature with primary antibodies against Tyrosine hydroxylase (AB152, Millipore, 1:1000) and p-S129 α-synuclein (ab51253, Abcam, 1:1000). After rinsing in PBST, sections were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 1 h and the avidin/biotin system (Vector Laboratories) for 30 min and visualized using a DAB solution (Vector Laboratories). Sections were then mounted on gelatin-coated slides and examined under a bright-field microscope (Olympus Optical, BX51, Tokyo, Japan). For cultured primary microglia, cells were fixed with 4% paraformaldehyde at 4 °C for 15 min and incubated with microwave-boiled antigen retrieval solution (#CTS013, R&D System) for 5 min. After washing with PBS, cells were incubated with blocking/permeabilization buffer (1% BSA and 0.1%Triton X-100 in PBS) at RT for 30 min and incubated with primary antibodies including EEA1 (610457, BD Bioscience, 1:100), α-synuclein (clone MJFR1, ab138501, Abcam, 1:500; clone 2f12, MABN1817, Millipore; clone 42, 610787 BD Bioscience, 1:100; clone 204, #2647, Cell Signaling, 1:100), p62 (GP62-C, Progen, Germany, 1:250), ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology, 1:100), and TFEB (A303-673A, Bethyl Laboratories Inc, TX, 1:200) at 4 °C overnight followed by secondary antibodies. Images were obtained using a confocal microscope with Zen 2011 software (Zeiss LSM 780, Carl Zeiss, Jena, Germany).

Microglia cell number and morphology analysis

From two brain slices from each animal, z-stack pictures were obtained with 20× magnification at multiple areas of the striatum using a confocal microscope after staining with Iba-1 antibody (Zeiss LSM 780, Carl Zeiss, Jena, Germany). Then, microglial cell number was counted from z-stack projected pictures using “cell counter” plug-in in Fiji software (National Institutes of Health, MD). For morphology analysis, the length of processes and terminal points of branches were measured by a semi-automated way using the “filament” plug-in in Imaris 3D interactive visualization software (Bitplane, Zurich, Switzerland). To show the colocalization of two interest proteins or the location of proteins in microglia, any immunostaining signals outside of Iba-1-staining area were masked using “surface” and “mask” plug-in in Imaris software, and each protein was made into the surface according to each staining signal. Then, final 3D rendering surface images were made by combining each protein surface to make a representative image.

Brain fractionation

Frozen brains (one hemisphere each animal) were homogenized with Dounce homogenizer in 2 ml of sucrose buffer (0.32 M Sucrose in 50 mM Tris-HCl, pH 7.4) added with Halt™ Protease and Phosphatase Inhibitor Cocktail (#78440, Thermofisher Scientific), and spun-down for 10 min at 1000 × g 4 °C. The supernatant (~1.3 ml) was collected and 0.9 ml of lysates were mixed with 0.1 ml of 10% Triton X-100 to make 1%Triton X-100 in sucrose buffer. After spin-down for 10 min at 16,000 × g 4 °C, the supernatant was collected into the new tube (detergent-soluble fraction), and the pellet was washed with 1% Triton X-100 two times to remove any soluble proteins. Finally, the washed pellet was resuspended in 0.1 ml of 1% SDS/1% Triton X-100/sucrose buffer and sonicated with the probe-tip sonicator in level ‘2’ for 10 s (Fisher Scientific 550 Sonic Dismembrator). After spin-down for 5 min at 16,000 × g RT, the supernatant was collected into the new tube (detergent-insoluble fraction). Protein concentration was determined by Pierce BCA Protein Assay Kit (#23225, Thermofisher Scientific).

Immunoprecipitation

Brain lysate from 1% Triton X-100/sucrose buffer fractions or cultured microglia lysed in 1% Triton X-100 PBS were incubated with α-synuclein antibody (clone 2f12, MABN1817, Millipore) in 4 °C overnight followed by Dynabeads™ Protein G (#10009D, Thermofisher Scientific). After washing with 1% Triton X-100 PBS three times, proteins that bound to beads were released by boiling in LDS sample buffer (Thermofisher Scientific) at 95 °C for 10 min.

Western blot analysis

Cultured cells were lysed on ice in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 1 mM NaF, 0.25% Na-deoxycholate, 1 mM Na₃VO₄ and 150 mM NaCl) containing Halt™ protease and phosphatase inhibitor cocktail (#78440, Thermofisher Scientific). For FACS-sorted microglia, cells were lysed in 1% Triton/8 M Urea/PBS buffer to maximize the yield, sonicated with the probe-tip sonicator in level “2” for 10 s (Fisher Scientific 550 Sonic Dismembrator). For AAV model, total ~30,000 microglia sorted from FACS were used and, for transgenic mice, ~120,000 cells were used for each group. Lysates were centrifuged, and proteins in the supernatant were separated by NuPAGE^® Precast Gel System (Thermofisher Scientific). Membranes were incubated with primary antibodies, including actin (#3700 S, Cell Signaling, 1:50000), ATG7 (MAB6608, R&D, 1:1000), ATG14 (PD026, MBL, 1:1000), dopamine transporter (MAB369, Millipore, 1:1000), ERK1/2 (#9107S, Cell Signaling, 1:1000), p-ERK1/2 (Thr202/Tyr204, #4370P, Cell Signaling, 1:1000), GFP/YFP (MA5-15256, Thermofisher Scientific, 1:2000), IκB (#4814, Cell Signaling, 1:1000), p-IRF3 (S396, #29047, Cell Signaling, 1:1000), IRF3 (#4302, Cell Signaling, 1:1000), JNK (#9252S, Cell Signaling, 1:1000), p-JNK (Thr183/Tyr185, #4671S, Cell Signaling, 1:1000), LC3B (#2775S, Cell Signaling, 1:1000), NBR1 (16004-1-AP, Proteintech, 1:1000), NDP52 (12229-1-AP, Proteintech, 1:1000), NQO-1 (11451-1-AP, Proteintech, 1:1000), HO-1 (sc-10789, Santa Cruz, 1:1000) p-NF-κB (S536, #3033S, Cell Signaling, 1:1000), Optineurin (10837-1-AP, Proteintech, 1:1000), p38 (#9212S, Cell Signaling, 1:1000), p-p38 (Thr180/Tyr182, #4511S, Cell Signaling, 1:1000), p62 (PM066, MBL, 1:50000), p62 (18420-1-AP, Proteintech, 1:1000), α-synuclein (clone MJFR1, ab138501, Abcam, 1:2000; clone 2f12, MABN1817, Millipore, 1:2000; clone 42, 610787 BD Bioscience, 1:1000; clone D37A6, # 4179, Cell Signaling, 1:1000), or Tyrosine hydroxylase (T2928, Sigma, 1:2000) at 4 °C overnight, washed with Tris-buffered saline containing 0.1% Tween 20, incubated with secondary antibodies, and visualized with SuperSignal™ West Pico Chemiluminescent Substrate (Thermofisher Scientific). Uncropped scan images for the main figures are presented in Supplementary Fig. 8.

HEK293T-NF-κB-luciferase assay system

HEK293T cells were seeded into a 96-well plate at a density of 2 × 10⁴ cells per well overnight. Cells were transfected using Lipofectamine^® 2000 (Invitrogen) with pcDNA3 vectors encoding either TLR1 (Addgene #13014), TLR2 (Addgene #13015), TLR4 (Addgene #13018), MD2 (Addgene #13028), CD14 (Addgene #13645), TLR5 (Addgene #13019), or TLR6 (Addgene #13020) with pGL4.32[luc2P/NF-κB-RE/Hygro] vector containing five copies of an NF-κB response element (NF-κB-RE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis) (#N111A, Promega, Madison, WI). Vectors were gifts from the Doug Golenbock Lab through Addgene. After 24 h, cells were treated with either EndoClear^TM human recombinant α-synuclein (AS5555, Anaspec, CA), lipopolysaccharide (LPS, #L7770, Sigma-Aldrich), Pam₃CSK₄ (#tlrl-pms, Invivogen, CA), or recombinant Flagellin (Rec FLA-ST, #tlrl-flic-10, Invivogen, CA). Following 24 h of stimulation by the ligand, the luciferase activity was measured using the ONE-Glo^TM Luciferase assay system (#E6110, Promega, Madison, WI).

Electron microscope

Cell cultures grown on Permanox slides (Electron Microscopy Sciences (EMS), Hatfield, PA) were taken from incubation, and directly placed in 2% glutaraldehyde (EMS) and 2% paraformaldehyde (EMS)/0.1 M sodium cacodylate buffer (EMS) for a minimum of 2 h at 4 °C. Cells were washed, fixed in 1% aqueous osmium tetroxide at RT for 1 h, washed, and transferred to 2% aqueous uranyl acetate at RT for 1 h. Slides were washed with distilled water, dehydrated in an ascending aqueous ethanol series, and then embedded in Epon resin (EMS). Inverted BEEM capsules (#3, EMS) were placed directly over regions of interest, filled with fresh resin, and transferred to a vacuum oven for heat polymerization at 60 °C for 12–24 h. To separate the cells from the slides, a hot plate was heated to 60 °C, and the slides placed directly on a pre-heated hot plate for exactly 3 min and 30 s. The capsules were removed from the hot plate, and carefully dislodged free from the slide using a plier. Ultrathin (85 nm) sections were collected onto 300 mesh copper grids (EMS) using a Leica UC7 ultramicrotome (Leica Biosystems Inc., Buffalo Grove, IL), contrast stained with uranyl acetate and lead citrate, and imaged on a Hitachi 7700 transmission electron microscope (Hitachi High Technologies America, Inc., Dallas, TX) equipped with an AMT 2 K x 2 K digital camera (Advanced Microscopy Techniques, Corp., Woburn, MA).

Image analysis and quantification

For counting p62-positive or p62/α-synuclein-positive puncta in microglia in vivo, fixed brain slices were stained with primary antibodies against human α-synuclein, p62, and Iba-1 and z-stack pictures were obtained with ×63 magnification (1024 × 1024 pixels) at multiple areas of the striatum using a confocal microscope (Zeiss LSM 780, Carl Zeiss). Puncta were counted inside of Iba-1-positive area in every single focal plane throughout the whole z-stack manually. In cultured cells treated with EndoClear^TM human α-synuclein (AS5555, Anaspec, CA), pictures were taken with ×20 magnification from multiple areas (up to 15 areas per coverslip), and ubiquitin/α-synuclein-positive puncta were counted by a plug-in “Cell counter” in Fiji software (National Institutes of Health, MD). Then, the number of puncta each picture was divided by cell number and normalized to each control group. For quantification of p-S129 α-synuclein DAB staining, pictures were taken with ×20 magnification, and positive signals were counted by a semi-automated way by using “Find Maxima” in Fiji software (National Institutes of Health, MD) after setting the threshold. Values were normalized to the average values from the control group in each cohort. For stereology counting of TH-positive or Nissl-stained cell bodies in the SNpc, one in every six sections was selected with a random start, and a total of five brain slices on average were used for each mouse for IHC labeling for TH, including DAB enhancement, followed by cresyl violet staining to reveal all neurons. A Zeiss Axioplan2 was used for tissue slice imaging with a ×20 objective, and Stereo Investigator was used to estimating the total number of neurons in the region of interest using the following parameters: frame sizes, 150 × 150 μm; grid sizes, 250 × 250 μm; top guard zone height, 2 μm; and optical dissector height, 8 μm. These parameters yielded a coefficient of error <10% throughout the analysis.

Statistics and reproducibility

The statistical significance of differences between two groups was determined using the unpaired two-tailed Student’s t test or Mann–Whitney U test based on the normality test. For multiple-means comparisons, statistical significance was determined by one-way analysis of variance followed by Newman–Keuls post hoc test or two-way analysis of variance with Bonferroni post hoc test using GraphPad Prism 5 (GraphPad Software, CA, USA). All values are reported as mean ± SEM. Data are representative of at least three independent experiments unless indicated. The source data underlying Figs. 1b, c, g, 2c, f, g, i, 3a, b, e, g, h, 4a–e, g, h, j, k, 5a–i, 6e, f, h, j, 7b, d, and Supplementary Fig. 1e, f, 4b, d–j, 5b, e, 6a–c, 7b, d, e, g, i are provided as a Source Data file.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files.

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Acknowledgements

This work was supported by an NIH grant (RO1, NS060123, and P50NS094733). We thank the assistance of members in the core facilities for Flow Cytometry, Microscopy, and qPCR at the Icahn School of Medicine at Mount Sinai. We thank NeuroScience Associates (Michael J. Fox Foundation) for the Thy-1-SNCA animal information and experiment. We are grateful to all members of Yue laboratory at the Icahn School of Medicine at Mount Sinai.

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Authors and Affiliations

Departments of Neurology and Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
Insup Choi, Yuanxi Zhang, Steven P. Seegobin, Mathilde Pruvost, Qian Wang, Kerry Purtell & Zhenyu Yue
Department of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
Qian Wang & Bin Zhang

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Insup Choi
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Yuanxi Zhang
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Steven P. Seegobin
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Mathilde Pruvost
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Qian Wang
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Kerry Purtell
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Bin Zhang
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Zhenyu Yue
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Contributions

Z.Y. conceived the project, supervised the entire study, and wrote the paper; I.C. designed, performed, and analyzed most of the experiments and wrote the paper; Y.Z. helped for the stereotaxic injection and stereological counting; S.S. performed NF-κB-luciferase experiments; M.P. helped in vitro results and analysis; Q.W. and B.Z. helped for analyzing the data; K.P. helped for preparing mice and writing the paper.

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Correspondence to Zhenyu Yue.

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Choi, I., Zhang, Y., Seegobin, S.P. et al. Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration. Nat Commun 11, 1386 (2020). https://doi.org/10.1038/s41467-020-15119-w

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Received: 16 January 2020
Accepted: 17 February 2020
Published: 13 March 2020
DOI: https://doi.org/10.1038/s41467-020-15119-w

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