The dual role of c-src in cell-to-cell transmission of a-synuclein

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The dual role of c-src in cell-to-cell transmission of a-synuclein

Yu Ree Choi

^1,2,3

, Jae-Bong Kim

^1,2,3

, Seo-Jun Kang

^1,2,3

, Hye Rin Noh

^1,2,3

, Ilo Jou

^1,3

, Eun-Hye Joe

^1,2,3

&

Sang Myun Park

^1,2,3,*

Abstract

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons located in the substantia nigra pars compacta and the presence of proteinaceous inclusions called Lewy bodies and Lewy neurites in numerous brain regions. Increasing evidence indicates that Lewy pathology progressively involves additional regions of the nervous system as the disease advances, and the prion-like propagation ofa-synuclein (a-syn) pathology promotes PD progression. Accordingly, the modulation of a-syn transmission may be important for the development of disease-modifying therapies in patients with PD. Here, we demonstrate thata-syn fibrils induce c-src activation in neurons, which depends on the FccRIIb-SHP-1/- 2-c-src pathway and enhances signals for the uptake ofa-syn into neurons. Blockade of c-src activation inhibits the uptake ofa-syn and the formation of Lewy body-like inclusions. Furthermore, the blockade of c-src activation also inhibits the release ofa-syn via activation of autophagy. The brain-permeable c-src inhibitor, saracatinib, efficiently reduces a-syn propagation into neighboring regions in anin vivomodel system. These results suggest a new therapeutic target against progressive PD.

Keywordsc-src; Parkinson’s disease; prion;a-synuclein Subject Categories Molecular Biology of Disease; Neuroscience

DOI10.15252/embr.201948950| Received25July2019| Revised2April2020| Accepted14April2020| Published online5May2020

EMBO Reports (2020)21: e48950

Introduction

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons located in the substantia nigra pars compacta and the presence of proteinaceous inclusions called Lewy bodies (LB) and Lewy neurites (LN) in numerous brain regions [1]. LB and LN are amyloidogenic protein deposits witha-synuclein (a-syn) aggregates as major components. Mutations involvinga-syn have been identified in early-onset familial PD, and genome-wide associated studies (GWAS) revealeda-syngene as a common risk factor for sporadic

PD [2–4]. In addition to PD, protein inclusions with a-syn aggregates have also been observed in other neurodegenerative disorders such as multiple system atrophy and dementia with Lewy body, which are collectively referred to asa-synucleinopathies, suggesting thata-syn plays a key role in their pathogenesis [5].

Lewy pathology has been reported to progressively involve more regions of the nervous system ranging from the olfactory bulb and enteric nervous system to cortical areas as the disease advances [6].

In addition, the presence of LB in grafted neurons [7,8] suggested that a-syn pathology can be propagated between neighboring neurons in a prion-like manner, resulting in the progression of PD.

Substantialin vitroandin vivoexperimental evidence supports the concept of prion-like propagation ofa-syn [9–11], and modulation of a-syn transmission may be important for the development of future disease-modifying therapies in patients witha-synucleinopathies. Nevertheless, the exact mechanism ofa-syn propagation has yet to be investigated.

c-Src is a ubiquitously expressed non-receptor tyrosine kinase, playing a role in proliferation, differentiation, motility, and survival. Especially, it is expressed higher in brain than in most other tissues [12], suggesting that c-src plays an important role in neurons. c-Src plays a key role in growth cone-mediated neurite extension and synaptic plasticity [13] and neuronal differentiation [14]. Src deficiency or blockade of src activity in mice prevents cerebral damage following stroke [15], and PP2, a src family kinase inhibitor, reduces focal ischemic brain injury [16]. Also, the knockdown of c-src protects cells against glutamate-induced loss of viability [17].

Previously, it has been reported that FccRIIb expressed in microglia and neurons acts as a receptor fora-syn fibrils and transmits signals regulating microglial phagocytosis, and a-syn uptake and inclusion body formation in neurons, respectively. Also, SHP-1 in microglia and SHP-1 and SHP-2 in neurons act as a downstream regulator [18,19]. In addition, it has been shown that SHP-1 and SHP-2 dephosphorylate the inhibitory tyrosine 512 of c-src, causing the functional activation of c-src [20,21].

In the present study, we explored the downstream signaling pathway of FccRIIb-SHP-1/-2 in neurons and the role of c-src in cell- to-cell transmission ofa-syn.

1 Department of Pharmacology, Ajou University School of Medicine, Suwon, Korea

2 Center for Convergence Research of Neurological Disorders, Ajou University School of Medicine, Suwon, Korea 3 Department of Biomedical Sciences, BK21Plus Program, Ajou University School of Medicine, Suwon, Korea

*Corresponding author. Tel: +82-31-219-5063; Fax: +82-31-219-5069; E-mail: [email protected]

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Results

a-Syn fibrils enhance c-src activity via FccRIIB-SHP-1/-2pathway in neurons

First, we explored whethera-syn fibrils enhance c-src activity in neurons. We confirmed the state of a-syn fibrils by thioflavin T assay and electron microscopic analysis (Appendix Fig S1A). When SH-SY5Y cells and primary cortical neurons were incubated witha- syn fibrils, the active form of c-src (pY416) was increased, whereas the inactive form of c-src (pY527) was decreased (Fig 1A).

However,a-syn monomers did not induce c-src activity, suggesting that it is species-specific (Fig 1B). In the frontal cortex of A53T transgenic (TG) mice at 8 months of age, which showed Lewy body pathology (Appendix Fig S1B), the active form of c-src (Y416) was increased, whereas the inactive form of c-src (pY527) was decreased (Fig 1C). On the contrary, they were not observed in the frontal cortex of A53T TG mice at 2 months of age (Fig 1C) which did not show any Lewy body pathology (Appendix Fig S1B). These results suggest thata-syn fibrils can enhance c-src activity. Next, FccRIIb knockdown (KD) SH-SY5Y cells were incubated witha-syn fibrils, which did not enhance c-src activity (Fig 1D). SHP-1 or SHP-2 KD SH-SY5Y cells also showed similar results (Fig 1E and F). Addition- ally, treatment with NSC87877 (SHP-1 and SHP-2 inhibitors), PHPS- 1 (SHP-2 inhibitor), or SSG (SHP-1 inhibitor) inhibited the increased c-src activity bya-syn fibrils (Fig 1G), suggesting its dependence on FccRIIb-SHP-1/-2 pathway.

c-Src mediates the uptake ofa-syn into neurons

Inhibition of FccRIIb-SHP-1/-2 pathway decreases the uptake ofa- syn into neurons [18]. To explore whether c-src, as a downstream mediator of FccRIIb-SHP-1/-2 pathway, also regulates the uptake of a-syn, a dual-chamber assay was performed to monitor the cell- derived A53Ta-syn-EGFP uptake into neurons (Fig 2A) [18]. Inhibi- tors of c-src including saracatinib (c-src and Bcr-Abl dual kinase inhibitor) and SKI-1 (c-src inhibitor-1) efficiently inhibited the activation of c-src (Appendix Fig S2A) and decreased the uptake ofa- syn into SH-SY5Y cells (Fig 2B) and primary cortical neurons (Fig 2D). In addition, we generated c-src knockdown (KD) SH-SY5Y stable cell lines (Appendix Fig S2B). Less cell-derived a-syn was absorbed by c-src KD SH-SY5Y cells (Fig 2C) and c-src KD primary cortical neurons (Fig 2E and Appendix Fig S2C). Overexpression of kinase-active c-src (Y529F) [22] enhanced the uptake of a-syn unlike the overexpression of kinase-dead c-src (K297M) [22]

(Fig 2F). Also, overexpression of kinase-active c-src (Y529F) rescued the less uptake of a-syn in c-src KD SH-SY5Y cells (Appendix Fig S2D). Untagged a-syn and A53T a-syn-EGFP were taken up into SH-SY5Y cells and detected similarly (Appendix Fig S2E). After c-src KD SH-SY5Y cells were incubated witha-syn fibrils, the phosphorylation of SHP-1/-2 was not affected (Appendix Fig S2F), further confirming that c-src was a downstream mediator of SHP-1/-2. Furthermore, in a previous study [18], we have demonstrated that FccRIIb does not function as a direct receptor that can be endocytosed witha-syn fibrils into neurons but functions as a receptor transmitting signals to enhance the uptake ofa-syn. The overexpression of kinase-active c-src (Y529F) in FccRIIb KD SH- SY5Y cells also enhanced the uptake ofa-syn (Fig 2G), suggesting

that c-src mediates the uptake of a-syn into neurons, as a downstream mediator of FccRIIb-SHP-1/-2 pathway, and the increase in c-src activity without FccRIIb, a receptor fora-syn fibrils, enhances the uptake ofa-syn into neurons.

c-Src mediatesa-syn aggregates formation in SH-SY5Y cells

Internalized a-syn induces LB-like inclusion body formation with endogenousa-syn [18]. To further confirm the involvement of c-src in the propagation ofa-syn, a coculture assay reported in a previous study (Fig 3A) [18] was performed. This coculture assay can be efficiently used to explore whether cell-deriveda-syn can induce inclusion body formation with endogenous a-syn expressed in neurons by measuring double fluorescence-labeled aggregation puncta [18].

As shown in Fig 3B, saracatinib and SKI-1 efficiently decreased LB- like inclusion body formation in both cells. When c-src KD/A53Ta- syn-EGFP-overexpressing (OE) SH-SY5Y cells were cocultured with A53T a-syn-mCherry OE SH-SY5Y cells, LB-like inclusion body formation in c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells was efficiently suppressed. However, the A53Ta-syn-mCherry OE SH-SY5Y cells were also suppressed (Fig 3C), suggesting that the release ofa- syn from c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells may also be decreased.

c-Src mediatesa-syn release from SH-SY5Y cells by regulating autophagy

To explore whether c-src could mediatea-syn release from neurons, released a-syn into the media was measured using ELISA. a-Syn was less detected in the media after treatment with saracatinib and SKI-1 (Fig 4A).a-Syn was also less detected in the media from c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells compared with that in the media from NT/A53Ta-syn-EGFP OE SH-SY5Y cells (Fig 4B). Next, a dual-chamber assay was performed with c-src KD/A53T a-syn- EGFP OE SH-SY5Y cells as donor cells and control SH-SY5Y cells as recipient cells (Fig 4C). Reduced levels ofa-syn-EGFP were detected in SH-SY5Y cells with c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells than with NT/A53T a-syn-EGFP OE SH-SY5Y cells in the upper chamber (Fig 4D). These results suggested that c-src mediatesa-syn release from SH-SY5Y cells.

To explore whether SHP-1 or SHP-2 as upstream mediators of c- src could also regulate a-syn release, released a-syn in the media was measured from SHP-1 or SHP-2 KD/A53Ta-syn-EGFP OE SH- SY5Y cells. In contrast with c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells, a similar amount of a-syn was detected in the media (Appendix Fig S3A). In dual-chamber assay, a similar amount ofa- syn-EGFP was also detected in SH-SY5Y cells with SHP-1 or SHP-2 KD/A53Ta-syn-EGFP OE as donor cells compared with NT/A53Ta- syn-EGFP OE SH-SY5Y cells as donor cells (Appendix Fig S3B), suggesting that SHP-1 or SHP-2 may not regulate a-syn release in SH-SY5Y cells.

Inhibition of the autophagy–lysosomal pathway increases a-syn release from neurons [23–26]. Accordingly, autophagic flux was detected using LC3II and p62 as indicators [27,28]. LC3II, a widely used autophagosome marker, was further detected following treatment with saracatinib and SKI-1. In the presence of bafilomycin A1, LC3II was further increased (Fig 4E). In addition, p62, a widely used marker to monitor autophagic activity, was decreased following

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treatment with saracatinib and SKI (Fig 4E), suggesting an increase in autophagic flux. In c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells, LC3II was also more detected. Bafilomycin A1 further increased LC3II levels (Fig 4F). p62 was also decreased (Fig 4F). c-Src KD primary cortical neurons showed a similar result (Fig 4G), suggesting that autophagy flux was enhanced via downregulation of c-src activity. Conversely, LC3II and p62 levels did not differ in SHP-1/-2 KD cells, compared with NT cells (Appendix Fig S3C). In addition, A53Ta-syn-EGFP was distributed diffusely in the cytosol of A53T a-syn-EGFP OE SH-SY5Y cells. Upon differentiation induced by reti- noic acid (RA) for 5 days, an increase in A53T a-syn-EGFP and spontaneous aggregation of A53T a-syn-EGFP were observed (Appendix Fig S4A, C, and E), consistent with a previous study [29].

The amount of a-syn in Triton X-100 insoluble fraction was also increased by RA (Appendix Fig S4B). However, spontaneous aggregation of a-syn-EGFP was reduced following treatment with saracatinib and SKI-1 (Appendix Fig S4C). The amount of A53T a-syn-EGFP in Triton X-100-insoluble fraction was also less

increased following treatment with saracatinib and SKI-1 (Appendix Fig S4D). In c-src KD/A53T a-syn-EGFP OE SH-SY5Y cells, spontaneous aggregation of A53T a-syn-EGFP was reduced (Appendix Fig S4E). The amount of A53Ta-syn-EGFP in insoluble fraction was also less increased (Appendix Fig S4F). Conversely, levels of spontaneous aggregation of A53T a-syn-EGFP and the amount of A53T a-syn-EGFP in insoluble fraction were similar in SHP-1/-2 KD cells (Appendix Fig S4G and H). In a previous study, Y125 ofa-syn is phosphorylated by src family kinases directly [30], which may affect properties ofa-syn [30]. We generated a SH-SY5Y stable cell line overexpressing A53T/Y125F a-syn-EGFP. Levels of spontaneous aggregation of a-syn-EGFP and the amount ofa-syn- EGFP in insoluble fraction were also similar in A53T/Y125F a-syn cells (Appendix Fig S4I and J), suggesting that the phosphorylation of Y125 a-syn by c-src might not affect the aggregation of a-syn.

These results suggest that the release ofa-syn may reduce via downregulation of c-src activity and enhanced autophagy independent of SHP-1/-2 signaling.

A

D E F G

B C

Figure1. a-Syn fibrils induce activation of c-src in a FccRIIB-SHP-1/-2-dependent manner.

A, B SH-SY5Y cells and primary cortical neurons were treated with1lMa-syn fibrils (A) or monomers (B) for30min. The cells were lysed, and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). ***P<0.001, **P<0.01compared with PBS, unpairedt-test.

C Frontal cortices of A53Ta-syn heterozygous transgenic mice and control mice at the age of9months (old) or2months (young) were lysed, and Western blot was performed with the indicated antibodies (n=3). ***P<0.001compared with WT, unpairedt-test.

D–G FccRIIB KD SH-SY5Y cells (D), SHP-1KD SH-SY5Y cells (E), SHP-2KD SH-SY5Y cells (F), and SH-SY5Y cells with20lM NSC87877,10lM SSG, and1lM PHPS-1(G) were treated with1lMa-syn fibrils for30min. The cells were lysed, and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). ***P<0.001, **P<0.01, and *P<0.05compared with PBS, one-way ANOVA.

Data information: Data are expressed as meanSEM.

Source data are available online for this figure.

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To confirm whether the reduced levels of transferreda-syn in c- src KD cells resulted from limited uptake of transferreda-syn into cells or enhanced degradation of transferreda-syn in recipient cells via autophagy, a dual-chamber assay was performed in the presence of bafilomycin A1. Even in the presence of bafilomycin A1, limited amounts of transferred a-syn were detected in c-src KD SH-SY5Y

cells, although the relative amounts of detected transferred a-syn were increased (Fig 4H), suggesting that the lower detection ofa- syn in c-src KD cells results from the inhibition ofa-syn uptake by the cells. Overall, it was suggested that c-src mediated cell-to-cell transmission of a-syn. We also confirmed it using microfluidic- chamber assay (Appendix Fig S5).

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F G

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Figure2. c-Src expressed in neurons mediates the uptake ofa-syn.

A Schematic diagram showing the dual-chamber assay.

B, D SH-SY5Y cells (B) and primary cortical neurons (D) were cocultured with differentiated A53Ta-syn-EGFP OE SH-SY5Y cells in the presence of5lM saracatinib (sara) or10lM SKI-1for12h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). ***P<0.001against PBS, one-way ANOVA.

C, E NT, c-src KD #1, and c-src KD #2SH-SY5Y cells (C) or primary cortical neurons downregulating c-src (E) were cocultured with differentiated A53Ta-syn-EGFP OE SH-SY5Y cells for12h and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). ***P<0.001 compared with NT cells, one-way ANOVA (C), unpairedt-test (E).

F, G SH-SY5Y cells (F) and FccRIIB KD SH-SY5Y cells (G) were transfected with plasmids of WT c-src, Y529F c-src (kinase-active), or K297M c-src (kinase-dead) tagged with EGFP. After1day, the cells were cocultured with differentiateda-syn OE SH-SY5Y cells for12h and then fixed and immunostained with anti-a-syn antibody.

The intensity was analyzed. Values were derived from three independent experiments (n=3). ***P<0.001, *P<0.05compared with mock-positive cells, one-way ANOVA.

Data information: Data are expressed as meanSEM. Blue indicates DAPI staining. Scale bar indicates20lm.

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c-Src is involved in lipid raft-dependent endocytosis

To investigate whether c-src signaling in neurons was specific fora- syn uptake, an endocytosis assay was performed using transferrin as a marker for clathrin-dependent endocytosis and lactosylce- ramide (LacCer) as a marker for lipid raft-dependent endocytosis [18,31]. As shown in Fig 5, a-syn fibrils enhanced lipid raft- dependent endocytosis, but not clathrin-dependent endocytosis in SH-SY5Y cells.a-Syn fibrils failed to enhance lipid raft-dependent endocytosis following treatment with saracatinib or SKI-1 (Fig 5A).

In addition, in c-src KD SH-SY5Y cell lines, a-syn fibrils failed to enhance lipid raft-dependent endocytosis (Fig 5B), suggesting that c-src regulates lipid raft-dependent endocytosis and is not specific for the uptake ofa-syn. These results are in agreement with a previous study showing that a-syn fibrils can enhance lipid raft- dependent endocytosis via FccRIIb/SHP-1/-2 signaling [18].

Brain-permeable c-src inhibitor, saracatinib, efficiently inhibits the propagation ofa-syn in A53T TG mice

Previous studies indicated that stereotaxic injection of preformeda- syn fibrils accelerates the formation of intracellular LB or LN-like inclusions in young asymptomatica-syn TG mice, which represent

an useful animal model for the elucidation of prion-like propagation ofa-syn [32–34]. We injecteda-syn fibrils into the unilateral striatum of 8-week-old A53T TG mice. At 30 days post-injection,a-syn lesions were detected in brain bilaterally by immunohistochemistry of pSer129a-syn, a marker of pathological a-syn [35], which was consistent with a previous study [32]. To explore whether the inhibition of c-src diminishes the propagation of a-syn in an in vivo animal model, a brain-permeable c-src inhibitor [36], saracatinib, was administered to 8-week-old A53T mice after injection ofa-syn fibrils for 1 month (Fig 6A). The activated form of c-src (Y416) was increased, whereas the inactive form of c-src (pY527) was decreased after the injection ofa-syn fibrils. Treatment with saracatinib inhibited the increase in the activated form of c-src (Y416) caused by the injection ofa-syn fibrils (Fig 6B). The phosphorylation of SHP-1/-2 was increased after the injection ofa-syn fibrils. On the contrary, the phosphorylation of SHP-1/-2 was not affected by the treatment with saracatinib (Appendix Fig S6). In addition, saracatinib efficiently diminished the propagation ofa-syn lesions (Figs 6C–E, and Appendix Figs S7 and S8). Additionally, to determine the related neuroinflammation, we evaluated astrocytes and microglia by staining for GFAP and Iba-1, respectively. The expression of GFAP, a marker for astrocytic reactivity [37], was increased by the injection ofa-syn fibrils in the cortex and the striatum, whereas treatment A

B

C

Figure3. c-Src mediates Lewy body-like inclusion body formation in SH-SY5Y cells.

A Schematic diagram showing the coculture assay.

B A53Ta-syn-EGFP and A53Ta-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of5lM saracatinib (sara) or10lM SKI-1, respectively, with50lM RA for5days.

C NT or c-src KD #1or c-src KD #2/A53Ta-syn-EGFP and A53Ta-syn-mCherry OE SH-SY5Y cells were cocultured with50lM RA for5days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence-labeled puncta was analyzed. Values were derived from five independent experiments (n=5). ***P<0.001compared with PBS or NT cells, one-way ANOVA.

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with saracatinib slightly diminished GFAP expression (Fig 7A and Appendix Fig S9), suggesting inhibition of astrogliosis. Based on the morphology of microglia, dendritic length, and intensity of Iba-1,

the robust activation of microglia was not accompanied by a-syn propagation (Fig 7B and Appendix Fig S10). However, activated microglia can highly up-regulate MHC class II [38]. The number of A

E

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B C D

Figure4. c-Src mediatesa-syn release from SH-SY5Y cells by regulating autophagy.

A Differentiated A53Ta-syn-EGFP OE SH-SY5Y cells were incubated with5lM saracatinib (sara) or10lM SKI-1for12h.

B Differentiated NT, c-src KD #1, or c-src KD#2/A53Ta-syn-EGFP OE SH-SY5Y cells were cultured12h. Levels ofa-syn in culture media were then measured using ELISA. Values were derived from three independent experiments (n=3). ***P<0.001, **P<0.01compared with control, one-way ANOVA.

C Schematic diagram showing the dual-chamber assay.

D SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, c-src KD #1, or c-src KD #2/A53Ta-syn-EGFP OE SH-SY5Y cells for12h. The cells in the lower chamber were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from five independent experiments (n=5).

***P<0.001compared with NT cells, one-way ANOVA.

E Differentiated A53Ta-syn-EGFP OE SH-SY5Y cells were incubated with5lM saracatinib (sara) or10lM SKI-1in the presence of DMSO or50lM bafilomycin A1 (BafA) for12h.

F, G Differentiated NT and c-src KD/A53Ta-syn-EGFP OE SH-SY5Y cells (F) and NT and c-src KD primary neurons (G) were incubated with50lM BafA for12h. Western blot was performed. Data are representative of three independent experiments (n=3). ***P<0.001, **P<0.01, *P<0.05, one-way ANOVA.

H SH-SY5Y cells were cocultured with differentiated A53Ta-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or50lM BafA for12h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). **P<0.01, one-way ANOVA.

Data information: Data are expressed as meanSEM. Scale bar indicates20lm. Blue indicates DAPI staining.

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MHC class II+microglia is increased in ana-syn fibril-injected rat model [39]. We re-evaluated microglia by staining for MHC class II.

As shown in Fig 7B and Appendix Fig S11, the number of MHC class II+microglia was slightly increased in the cortex and the striatum after the injection ofa-syn fibrils, whereas treatment with saracatinib diminished it. These data suggest that inhibiting c-src activity can efficiently ameliorate the propagation ofa-syn pathology accom- panying micro/astrogliosis.

Discussion

During the past decade, prion-like propagation ofa-syn was extensively studied, suggesting that the gradual increase in Lewy pathology, during the progression of PD, may be attributed to prion-like propagation ofa-syn aggregates [9,40,41]. Accordingly, the disrup- tion of intercellular aggregate transmission represents a promising strategy to inhibit disease progression.

Thea-syn fibrils bind to FccRIIb expressed in neurons and acti- vate SHP-1/-2, which accelerates the uptake of a-syn and LB-like inclusion body formation [18]. In the present study, we found that c-src is involved in cell-to-cell transmission of a-syn as a downstream mediator of FccRIIb-SHP-1/-2. The activation of c-src bya- syn fibrils was dependent on FccRIIb-SHP-1/-2 pathway, and the activation of c-src accelerates the uptake of a-syn into neurons.

Furthermore, FccRIIb-SHP-1/-2 signaling enhances the uptake ofa- syn by different modes [18]. The activation of c-src without upstream signaling also accelerated the uptake of a-syn into

neurons, suggesting that c-src could also regulate signals for the uptake ofa-syn as a downstream mediator of FccRIIb-SHP-1/-2.

The expression of c-src has been associated with endocytosis.

Overexpression of cellular src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor [42]. The c-src kinase activity plays a critical role in macropinocytosis and enhances large vesicle formation, clathrin-independent endocytosis of dextran [43]. c-Src-mediated tyrosine phosphorylation is also required for the function of dynamin in ligand-induced signaling receptor internalization [44]. We also observed that the downregulation of c-src activity inhibiteda-syn fibril-induced lipid raft-dependent endocytosis. Accordingly, FccRIIb-SHP-1/-2-c-Src pathway activated by extracellulara-syn fibrils enhanced the mechanism of endocytosis for the uptake ofa-syn.

Interestingly, we also found that the inhibition of c-src activity decreased the release ofa-syn. Although the mechanism of a-syn release is unclear, different secretory pathways have been reported to contribute to its release. a-Syn is released via unconventional and ER-Golgi-independent exocytosis [45], and inhibition of the autophagy–lysosomal pathway increases a-syn release from neurons [23,24,26]. a-Syn is also released in association with exosomes [46] and is increased under lysosomal dysfunction [47].

In addition, a-syn is released by exophagy and the inhibition of autophagosome–lysosome fusion promotes this unconventional secretion [25]. Given thata-syn is degraded by autophagy [48–50], the inhibition ofa-syn degradation induces the accumulation ofa- syn, further releasinga-syn into the extracellular space via different secretory pathways. Activated c-src has been reported to abrogate

A B

Figure5. c-Src is involved in lipid raft-dependent endocytosis.

A, B SH-SY5Y cells in the presence of5lM saracatinib,10lM SKI-1(A) and NT, c-src KD #1, or c-src KD #2SH-SY5Y cells (B) were incubated with1lMa-syn fibrils for 30min, and these cells were further incubated with50nM BODIPY FL C5-LacCer and2.5lg/ml rhodamine-conjugated transferrin for20min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). ***P<0.001compared with control, one-way ANOVA.

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of autophagy in muscle cells [51]. Autophagy is enhanced by the lack of c-src activity via inhibition of mTOR signaling pathway [52].

Src/c-Abl inhibitors promote autophagy and reduce the amount of

misfolded SOD1 protein [53]. We also demonstrated that both genetic inhibition and pharmacological inhibition of c-src activity enhanced autophagy in neurons and blocked the spontaneous

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D

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B

Figure6.

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aggregation of A53Ta-syn in A53Ta-syn OE SH-SY5Y cells following RA treatment. These results suggest that the inhibition of c-src activity also decreases the accumulation ofa-syn aggregation and the further release ofa-syn by enhancing autophagy. In addition, relative amounts of transferred a-syn detected in recipient cells were increased in the presence of bafilomycin A1. It might be due to increaseda-syn release by inhibiting autophagic degradation of a-syn in donor cells and/or increased accumulation of a-syn in recipient cells. We could not decipher which one or both contrib- uted to such increase. Nevertheless, it was noteworthy that the lower detection ofa-syn in c-src KD cells resulted from the inhibition ofa-syn uptake by cells. Accordingly, c-src plays a dual role in cell-to-cell transmission ofa-syn to regulate the uptake ofa-syn via endocytosis and the release and intracellular accumulation ofa-syn via autophagy (Fig 8). However, as an upstream regulator of c-src in cell-to-cell transmission of a-syn, SHP-1 or SHP-2 was not involved in the release ofa-syn. We speculate that it may be due to spatiotemporal differentiation of c-src. c-Src displays dual localization and distribution to endosomal membrane fractions and plasma membrane [54]. Accordingly, c-src in the plasma membranes may be activated by FccRIIb-SHP-1/-2 and in endocytosis, resulting in the enhanced uptake of a-syn, whereas c-src in endosomal membranes may regulate autophagy, resulting in accumulation or release ofa-syn. Further studies are needed to elucidate the precise molecular mechanism of these findings.

We also validated the roles of c-src in cell-to-cell transmission of a-syn in an in vivo model system. We detected pSer129 a-syn immunoreactivity in the contralateral regions after 1 month of stereotaxic injection ofa-syn fibrils in A53T TG mice. The brain of mice exposed to saracatinib during this period showed a dramatic decrease ina-syn pathology compared with control mice, suggesting that inhibition of c-src activity efficiently amelioratesa-syn spread inin vivo. In addition, propagation ofa-syn pathology is known to accompany neuroinflammation [39,55,56]. We also observed that a-syn pathology accompanied an increase in astrocytic reactivity evaluated by GFAP intensity at 1 month after injection. Microglial

activation evaluated by MHC class II expression was also accompanied bya-syn pathology. However, we did not observe any morpho- logical change in microglia evaluated by IBA-1 staining, suggesting that MHC class II expression in microglia might proceed morpholog- ical changes in microglia at early stages ofa-syn pathology-induced neuroinflammation, consistent with a previous study [39].

Saracatinib is a highly selective, orally available, dual-specific c- Src/Abl kinase inhibitor and was developed for the treatment of solid tumors [57]. c-Abl is involved in the pathogenesis of PD via inactiva- tion of parkin [58,59] and/or regulation of a-syn degradation [60].

Additionally, brain-permeable c-Abl inhibitors showed neuroprotective effects in a MPTP mouse model [61–63] and in human A53Ta- syn TG mice [64]. A recent study also reported that brain-permeable c-Abl inhibitor, radotinib, ameliorated a-syn fibril-induced LB/LN- like pathologyin vivo[65], which is similar to our findings. The mean IC50 of saracatinib against c-src is 2.7 nM compared with 30 nM against v-Abl [66], suggesting that saracatinib is more potent in inhibiting c-src than c-Abl. Nevertheless, we could not determine whether saracatinib inhibited c-src alone or both c-src and c-Abl in our experimental model. However, based on ourin vitrodata, it is noteworthy that c-src is also an important target for the development of therapeutic strategies against PD and dual kinase inhibitors may be a more viable option. In addition, saracatinib is under clinical trials against AD due to the inhibitory effect of Fyn kinase [67]. Fyn kinase mediates Aboligomer-induced synaptic dysfunction via PrPC and mGluR5 in AD [68,69]. Similarly,a-syn oligomers also interact with PrPC to induce cognitive impairment via mGluR5 and NMDAR2B [70], although contrasting results were also reported [71].

Until now, several receptors, which bind to a-syn fibrils, have been identified. The a3-subunit of Na⁺/K⁺-ATPase (NKA) was identified as a potential cell surface molecule binding witha-syn fibrils, and this interaction decreased the efficiency of Na⁺extrusion following activation [72]. LAG-3 was identified as a receptor fora- syn fibrils, mediating the transmission of a-syn between neurons and the accumulation of fibrils. In addition, this study also found that neurexin 1b and Abprecursor-like protein 1 (APLP1) acted as

◀

^Figure6. Brain-permeable c-src inhibitor, saracatinib, efficiently inhibits the propagation ofa-syn in A53T TG mice.

Two- to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10lg of recombinanta-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks.

A Schematic diagram showing thein vivoexperiment.

B Fixed brain tissues containing the frontal cortex and the striatum were lysed. Western blot was performed with indicated antibodies. Values were derived from three individual mice (n=3).

C Immunohistochemistry of pSer129a-syn was performed. Arrow indicates pSer129a-syn-positive inclusion. Scale bar indicates50lm.

D Scoring of pSer129immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). ***P<0.001, **P<0.01,

*P<0.05compared with PBS group, unpairedt-test. Data are expressed as meanSEM.

E Schematic representation of the scoring ofa-syn inclusion pathology detected by pSer129staining.

Figure7. Saracatinib efficiently inhibits the micro/astrogliosis in A53T TG mice.^†

A Immunohistochemistry of GFAP was performed. GFAP intensities in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). ***P<0.001, **P<0.01, *P<0.05, one-way ANOVA.

B Immunohistochemistry of IBA-1and MHC class II was performed. Arrow indicates MHC class II-positive cells. Numbers of MHC class II-positive cells in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). ***P<0.001, **P<0.01, one-way ANOVA.

Data information: Data are expressed as meanSEM. Scale bar indicates50lm.

▸

†Correction added on3 July 2020, after first online publication: Figure 7B has been corrected.

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A

B

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putative receptors [73]. Connexin32 was also identified as a receptor for a-syn oligomers to facilitate protein uptake and transfer in neurons and oligodendrocytes [74]. These data suggest thata-syn fibrils may carry diverse receptors similar to Ab.

Recently, different strains ofa-syn fibrils have been extensively studied to explain the diversity of a-synucleinopathy. Current knowledge cannot explain the specificity of the diverse receptors for a-syn fibrils. However, abrogation of cell-to-cell transmission ofa- syn may inhibit the progression of PD, suggesting that the downstream regulators may be more potent than receptors for a-syn.

Thus, targeting c-src, fyn, or c-Abl may be a viable option for the development of therapeutics against AD and PD. Moreover, drugs targeting c-src, fyn, or c-Abl are extensively under development for the treatment of cancer, suggesting that repurposing these drugs facilitates the development of therapeutics against neurodegenerative diseases more efficiently.

In conclusion,a-syn fibrils induced c-src activation in neurons, which is dependent on FccRIIb-SHP-1/-2-c-Src pathway and

enhanced the signals for the uptake ofa-syn into neurons. Inhibition of c-src activation inhibited the uptake of a-syn and LB-like inclusion body formations. Furthermore, abrogation of c-src activation also inhibited the release of a-syn via activation of autophagy.

Treatment with brain-permeable c-src inhibitor, saracatinib, efficiently ameliorateda-syn propagation into neighboring regions in an in vivomodel system. These results suggest a new therapeutic target against the progression of PD.

Materials and Methods

Reagents and antibodies

Antibodies against a-syn (#610786), SHP-1 (#610126), SHP-2 (#610621), and MHC class II (#554926) were purchased from BD Biosciences (Franklin Lakes, NJ). Antibodies against pc-src (Y416) (#6943), pc-src (Y527) (#2105), and total c-src (#2123) were Figure8. Schematic diagram illustrating the molecular mechanism of c-src in cell-to-cell transmission ofa-syn.

a-Syn is released from neurons, taken up into neighboring neurons, and accumulated with endogenously expresseda-syn. In donor cells, c-src impairs autophagy, thus facilitatinga-syn release. In recipient cells, c-src enhances the uptake ofa-syn by regulating endocytosis. Accordingly, c-src plays a dual role in cell-to-cell transmission ofa- syn. It regulates the uptake ofa-syn via endocytosis. It also regulates the release and intracellular accumulation ofa-syn via autophagy.

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supplied by Cell Signaling Technology (Danvers, MA). Antibody against pSer129a-syn (#ab51253) was ordered from Abcam (Cam- bridge, UK) for Western blot analysis. Antibody against IBA-1 (#019-19741) and pSer 129a-syn (#015-25191) was purchased from Wako (Richmond, VA). Antibody against GFAP (#RA22101) was purchased from Neuromics (Montreal, QC). Saracatinib for cellular studies was procured from Santa Cruz Biotechnology (Santa Cruz, CA). Saracatinib for mouse experiments was purchased from Cayman Chemical Company (Ann Arbor, MI). SKI-1 (c-src inhibitor 1), RA, and bafilomycin A1 were obtained from Sigma (St. Louis, MO). Rhodamine-conjugated transferrin and boron-dipyrromethene (BODIPY) FL C5-LacCer were purchased from Molecular Probes (Leiden, the Netherlands).a-Syn was prepared as described previously [19]. To preparea-syn fibrils, 5 mg/ml monomerica-syn was incubated at 37°C with continuous agitation at 250 rpm for 2 weeks.

a-Syn fibrils were sonicated on ice for 3–5 s at 3 W using an ultra- sonic processor VC 505 (Sonics & Materials, Inc., Newtown, CT) and stored at80°C until use asa-syn fibrils. The status ofa-syn fibrils was determined by thioflavin T binding assay and electron microscopy (Appendix Fig S1A).

Plasmids and transfection

The cDNAs for WT c-src, c-src K297M (dominant-negative c-src), and c-src Y592F (constitutively active c-src) were gifted by Prof.

Park K-S at Kyung Hee University School of Medicine, Seoul, Korea [22]. They were subcloned into pEGFP using PCR for expressing WT, K297M, and Y592F c-src fused with EGFP in the C-terminal. All plasmids were confirmed by sequencing. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Cell culture

SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO2and 95% air. Primary cortical neurons were cultured from Sprague-Dawley rat embryos on embryonic day 18 and maintained in neurobasal medium (Invit- rogen) with GlutaMAX^TM-I (Thermo, Waltham, MA), and B-27 supplement (Invitrogen). All animal procedures were conducted according to the guidelines established by the Ajou University School of Medicine Ethics Review Committee (IACUC No. 2016- 0047).

Generation of stable cell lines

FccRIIb, SHP-1, and SHP-2 knockdown (KD) SH-SY5Y cells were prepared as described previously [18]. c-Src KD SH-SY5Y cells were prepared using lentiviral constructs expressing shRNA for human c- src (KD #1: TRCN 0000038150, KD #2: TRCN 0000195339) (Sigma, St. Louis, MO). Lentiviruses were generated by transfecting HEK293TN cells with a mixture of pVSV-G, pGAG-pol, and pLKO.1- puro containing shRNAs against c-src gene using Lipofectamine 2000. Supernatants containing these lentiviruses were collected at 48 h after transfection. Samples were centrifuged at 250gfor 5 min followed by filtering with a 0.45-lm syringe filter and added into SH-SY5Y cells. Stable cell lines were then selected using puromycin.

A53Ta-syn-EGFP and A53Ta-syn-mCherry OE SH-SY5Y cells were prepared as described previously [18]. c-Src KD/A53Ta-syn-EGFP OE SH-SY5Y cells were developed using lentiviral transfection of A53T a-syn-EGFP in c-src KD SH-SY5Y cells and selected using a FACSAria III.

Dual-chamber and coculture assays

Dual-chamber and coculture assays were performed as described previously [18]. For dual-chamber assay, A53Ta-syn-EGFP OE SH- SY5Y cells or WTa-syn OE SH-SY5Y cells were used as the donor cells and differentiated by treatment with 50lM RA for 5 days. The SH-SY5Y cells, or primary cortical neurons cultured on the coverslip in a 12-well plate as recipient cells, were cocultured with differentiated A53Ta-syn-EGFP OE SH-SY5Y cells or WTa-syn OE SH-SY5Y cells cultured on the insert for 12 h. The recipient cells were prepared for staining. To measure the amount of internalizeda-syn, five random fields were selected and intensities of more than 100 cells were analyzed using a MetaMorph software (Molecular Devices). For the coculture assay, A53T a-syn-EGFP OE SH-SY5Y cells and A53Ta-syn-mCherry OE SH-SY5Y cells were cocultured on the coverslip in a 12-well plate (1:1 ratio) for the indicated times in the presence of 50lM RA. The cells were prepared for confocal microscopic analysis. To measure the inclusion body formation of a-syn fibrils, five random fields were selected and more than 100 cells were analyzed. Cells containing double fluorescence-labeled puncta were counted manually. The number of A53Ta-syn-EGFP OE SH-SY5Y cells containing double fluorescence-labeled puncta and the number of A53Ta-syn-mCherry OE SH-SY5Y cells containing double fluorescence-labeled puncta were counted separately and expressed as percentages of total cells analyzed.

Western blot

Cells and mouse brain were lysed in an ice-cold RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxy- cholate, 150 mM NaCl) containing a protease inhibitor cocktail (Cal- biochem, Germany) and a phosphatase inhibitor cocktail (GenDEPOT, Baker, TX). For Western blot using fixed brain tissue, the samples were prepared as described previously [75]. Briefly, fixed brain tissues were lysed with lysis buffer (300 mM Tris–HCl, pH 8.0, 2% SDS) containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail. The samples were incubated for 30 min on ice and then for 60 min at 100°C followed by 2 h at 60°C.

The lysates were centrifuged at 22,000gfor 30 min at 4°C, and the supernatant was collected. The protein concentrations were determined with a BCA protein assay kit (Bio-Rad, Hercules, CA).

Proteins were resolved by SDS–PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the indicated antibodies. They were then visualized using an enhanced chemiluminescence (ECL) system (Thermo, Waltham, MA).

Confocal microscopy

Cells cultured on the coverslips were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The fixed cells were washed several times with PBS and incubated in the absence or presence of the permeabilization buffer (PBS

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containing 0.1% Triton X-100) for 5 min at room temperature. After washing several times with PBS, the cells were blocked with block- ing buffer (PBS containing 5% bovine serum albumin) for 30 min at room temperature and then incubated with the indicated antibodies overnight at 4°C. The samples were incubated with Alexa 488- or Alexa 594-conjugated secondary antibodies (Jackson ImmunoRe- search, West Grove, PA) for 2 h and then with Hoechst for 10 min.

They were mounted and observed under a confocal microscope (Zeiss, Germany).

Enzyme-linked immunosorbent assay (ELISA)

SH-SY5Y cells were differentiated by treatment with 50lM RA for 5 days. Culture media were replaced by fresh media. Cells were then further incubated for 12 h in the presence of 50lM RA. The culture supernatant was collected. Samples were centrifuged at 1,500 rpm for 5 min to remove dead cells and debris. Supernatants were filtered through 0.45-lm syringe filters (Millipore, MA, USA) and stored at20°C. Samples were thawed on ice right before use and analyzed using a human a-synuclein ELISA kit (Elabscience, TX, USA) according to the manufacturer’s instructions.

Animals

We used M83 mice-overexpressing A53T-mutated human a-syn under the control of mouse prion protein promoter (B6;C3-Tg(Prnp- SNCA*A53T)83Vle/J, The Jackson Laboratory) and C57BL/6 mice (Orient Bio). M83 mice were obtained by crossing hemizygous M83 mice, produced by crossing male homozygous M83 mice and female C57BL/6 mice. All animal procedures were conducted according to the guidelines established by the Ajou University School of Medicine Ethics Review Committee (IACUC No. 2016-0047).

Intracerebral injection and drug treatment

Two- to three-month-old hemizygous M83 mice were anesthetized with intraperitoneal injection of 2,2,2-tribromoethanol (250 mg/kg) and injected into the striatum with sterile PBS or 10lg of recombi- nant human WT a-syn fibrils (5 mg/ml) (coordinates: anterior–

posterior, +1.0 mm; medial–lateral, 1.8 mm; dorsal–ventral, 3.2 mm from the bregma) using a 10-ll Hamilton syringe with single needle (33 gauge) at an injection rate of 0.4ll/min, and the needle was placed at the injection site for 10 min. After recovery from surgery, mice were fed with saracatinib 10 mg/kg/day by oral gavage for 4 weeks. Each vehicle and saracatinib were dissolved in 0.5% hydroxypropyl methylcellulose (HPMC) and 0.1% Tween-80 in 250ll sterile water.

Immunohistochemistry

The brain was removed after perfusion with PBS and post-fixed in 4% paraformaldehyde for 24 h, and cryoprotected in 30% sucrose in PBS for 4 days. Fixed brains were cut on a vibratome (Leica, Germany) at 35lm thickness. The free-floating sections were washed three times for 10 min in PBS and treated with 3% H2O2

(Sigma, St. Louis, MO) in PBS for 5 min to inactivate endogenous peroxidase following by blockade with 1% BSA/0.2% Triton X-100 in PBS for 1 h. Primary antibodies were added to the sections and

incubated overnight at 4°C. After incubation with the biotinylated secondary antibody (Vector Laboratories Burlingame, CA) for 1 h at room temperature, sections were washed with PBS and incubated with ABC reagent (Vector Laboratories Burlingame, CA) for 1 h.

Sections were stained using 3⁰,3⁰-diaminobenzidine (DAB) (Sigma, St. Louis, MO) at room temperature and mounted on slides. Sections were dehydrated with ethanol and xylene and mounted with permount solution and coverslipped. Sections were analyzed using the BX51(Olympus, Japan) and the pictureframe software (MBF Bioscience Inc., Williston, VT, USA).

Pathology scoring

The pathology was scored as described previously [76]. Briefly, we counted the pSer129-stained neurons and set a score ranging from 0 to 5 specific for each single brain region [0: absence; 1: sparse (1 soma); 2: mild (neurites, 2–10 soma); 3: moderate (11–20 soma and many neurites); and 4: severe]. Next, we determined the average score for each brain region per animal and calculated the average score per group. Counting was performed using the BX51 and a stereoinvestigator software (MBF Bioscience Inc., Williston, VT, USA).

GFAP intensity analysis

All images were obtained using an EDF active mode (Z-stack align- ment module) of an Axio Scan Z1 Slide Scanner (Carl Zeiss). To measure GFAP intensity, 5–6 fields were measured for each region (three sections per mice). Background intensity was taken from area adjacent to the measured area. After background intensity was subtracted, a ratio of the intensity was calibrated as % using the ImageJ software.

Quantification of MHC class II-positive cells

Quantification of MHC class II-positive cells was performed as described previously [38] with some modifications. Briefly, for counting MHC class II-positive cells, three sections were used, including the cortex and the striatum region per mice. Counting was performed using×20 objective connecting the BX51 and a stereoinvestigator software. Numbers represent the raw total number of MHC class II-positive cells per mice multiplied by 12 to extrapolate the population estimate.

Statistical analysis

All values are expressed as meansSEM. Statistical significance was evaluated using the unpaired t-test or one-way ANOVA (GraphPad Prism software, San Diego, CA, USA).

Expanded Viewfor this article is available online.

Acknowledgements

We thank Prof. Park K-S at Kyung Hee University School of Medicine, Seoul, Korea, for providing c-src plasmids. This research was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (Ministry of Science and ICT) (Grants NRF-2017R1E1A1A01073713 and NRF-2019R1A5A2026045).

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Author contributions

YRC and SMP designed the study. YRC, J-BK, HRN, and S-JK performed the experiments. YRC, J-BK, HRN, S-JK, IJ, E-HJ, and SMP analyzed and discussed the data. YRC, J-BK, and SMP wrote and edited the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

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