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. 2022 Apr 10;79(5):234. doi: 10.1007/s00018-022-04263-9

A53T mutant α-synuclein fibrils formed in macrophage are spread to neurons

Shogo Moriya 1,✉, Michiko Hanazono 1, Takeshi Fukuhara 2,3, Katsuro Iwase 1, Nobutaka Hattori 2,3, Masaki Takiguchi 1
PMCID: PMC11073293  PMID: 35397671

Abstract

Lewy body (LB), which mainly consists of abnormal α-synuclein (αS) aggregates, is a histological hallmark of Parkinson’s disease (PD). αS aggregation and LB inclusions are induced by spreading αS fibrils to neurons; therefore, the formation and transmission of αS fibrils to neurons may play an essential role in initiating LB formation in neurons. αS expressed in neurons is released into the extracellular space and taken up by macrophages and microglia; therefore, we hypothesized that macrophages/microglia play a role in the formation and spread of αS fibrils. In this study, we aimed to investigate the involvement of macrophages/microglia in the formation and spread of αS fibrils using transgenic animals that express human αS in macrophages/microglia. Transgenic zebrafish expressing A53T mutated αS (αS_A53T) in macrophages/microglia revealed αS accumulation in neurons. Transcriptome analysis by RNA-seq of human αS and αS_A53T expressing zebrafish revealed that kinase genes and E3 ubiquitin protein ligase genes were significantly high, and neuronal activity and transport-related Gene Ontology terms were also isolated. Meanwhile, αS_A53T monomers were taken up by A-THP-1 cells; processed to larger molecules, which could be αS fibrils; and released from macrophage cells. Furthermore, the ubiquitin–proteasome system modulated αS fibrils in A-THP-1 cells. αS fibrils suggest being formed from monomers in macrophages and spread to neurons to induce αS aggregates. Therefore, macrophages may play an essential role in the formation of αS aggregates and the pathogenesis of PD.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-022-04263-9.

Keywords: Parkinson’s disease, α-Synuclein, Lewy body, Macrophage, Microglia, Zebrafish

Introduction

Lewy body (LB), which mainly consists of abnormal α-synuclein (αS) aggregates, is one of the histological hallmarks of Parkinson’s disease (PD). Due to the cytotoxicity of αS aggregates [1], it has been suggested that LB formation is involved in the pathogenesis of PD. For studies on LB formation, αS fibrils are applied to cells and animals as seeds of aggregation. Seeded αS fibrils are taken up by cells, inducing αS aggregation through post-translational modifications of αS, such as truncation, phosphorylation, and ubiquitination, followed by LB inclusions, which cause synaptic dysfunction [2]. Injection of αS fibrils into the striatum causes LB-like inclusions in the substantia nigra pars compacta, dopaminergic neuron loss, and motor impairments such as PD [3]. αS fibrils may also be involved in the propagation of αS aggregates. LB-like inclusions caused by the injection of αS fibrils into the striatum have been formed not only in the substantia nigra pars compacta, but also in other brain regions such as the amygdala and cortex [3]. Since peripheral injection of αS fibrils also induces phenotypes of PD, propagation of αS fibrils/aggregates from the peripheral to the central nervous system has been proposed [4]. Therefore, the formation and transmission of αS fibrils to neurons may play an essential role in initiating LB formation in neurons. However, the mechanisms of αS fibril formation in vivo remain to be elucidated.

αS, localized in presynaptic terminals of normal neurons [5], is released into the extracellular space and taken up by recipient cells [6]. The released αS is also taken up and digested in macrophages [7] and microglia [8], which are the resident macrophages in the central nervous system [9]. Meanwhile, αS oligomers are stored in the appendix where immune cells are rich [10, 11]. Since macrophages/microglia take up αS released from neurons, macrophages/microglia can form and release αS oligomers. Furthermore, the released αS oligomers might be transmitted to neurons through interactions between neurons and macrophages/microglia and function as αS fibrils to induce αS aggregation in neurons. Therefore, we hypothesized that macrophages/microglia play a role in the formation and spread of αS fibrils; that is, macrophages/microglia take up αS released from neurons, form αS fibrils, and spread to neurons. In this study, we aimed to investigate the involvement of macrophages/microglia in the formation and spread of αS fibrils using transgenic animals that express human αS in macrophages/microglia. Furthermore, mutants of SNCA gene encoding αS, such as A30P and A53T, are associated with early onset of familial PD [12], form fibrils more rapidly than wild-type in vitro [13]. Therefore, the effect of the αS mutation on the formation and spread of αS fibrils was also investigated.

In this study, zebrafish (Danio rerio) were used as a model. Macrophage expressed gene 1.1 (mpeg) has been isolated as a macrophage marker gene in zebrafish and mammals [14]. The mpeg promoter-driven transgenes are expressed only in macrophage-lineage cells, including macrophages and microglia in zebrafish [15, 16]. Therefore, the mpeg promoter-driven human SNCA gene expressing zebrafish, and the mpeg promoter-driven A53T-mutated human SNCA gene expressing zebrafish were generated to investigate the effect of αS expression in macrophages/microglia. HuC, which encodes an RNA-binding protein homologous to Drosophila elav, has been isolated as a neuronal marker. Since the HuC promoter-driven transgenes are expressed in neurons [17], the HuC promoter-driven wild-type and A53T-mutated human SNCA-expressing zebrafish were also generated for comparison with the mpeg promoter-driven human SNCA-expressing fish.

Materials and methods

Animals

Zebrafish (Danio rerio) of the AB strain were maintained in aquaria at 28 °C ± 1 °C on a 14-h light, 10-h dark cycle, and fed a standard zebrafish diet twice daily (Meito System, Aichi, Japan). All experimental procedures were performed in accordance with the guideline of the Chiba University Institutional Animal Care and Use Committee.

Generation of transgenic lines

Human SNCA coding region without stop codon amplified with human SNCA full-length cDNA clone (OriGene Technologies, MD, USA) was ligated with Kusabira Orange2 (KO2) coding region amplified with phmKO2-MN1 vector (MBL, Aichi, Japan). 1.86 kb of mpeg promoter region [15] or 3.16 kb of HuC promoter region [17] amplified with zebrafish genome was further ligated to generate the cassette of mpeg:SNCA-KO2 or HuC:SNCA-KO2. The ligated cassette was subcloned into the pT2AL200R150G vector. A53T-mutated SNCA constructs, mpeg:SNCA_A53T-KO2, and HuC:SNCA_A53T-KO2 were generated from pT2AL200R150G-mpeg:SNCA-KO2 and pT2AL200R150G-HuC:SNCA-KO2 by site-directed mutagenesis. mpeg:EGFP and HuC:EGFP constructs were also prepared with amplified EGFP coding region. tol2 transposase mRNA transcribed from the pCS-zT2TP and the construct with phenol red were injected into one-cell stage wild-type embryos, and the embryos were raised at 28.0 °C.

Imaging

Transgenic embryos obtained by incrossing the generated transgenic lines were raised in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) at 28.0 °C and 0.003% 1-phenyl 2-thiourea was added to E3 medium at 1 day post fertilization (dpf). The larvae at five dpf were mounted in 1.2% low melting temperature agarose in E3 medium with 0.008% tricaine. To analyze the localization of KO2 and EGFP, z-stack images were captured with a digital fluorescence microscope under a 4 × or 10 × objective (BZ-X800, KEYENCE, Osaka, Japan) and compressed with standard full focus.

Transcriptome analysis

Total RNAs were isolated from single zebrafish larvae at five dpf of wild type, Tg(mpeg:SNCA-KO2), and Tg(mpeg:SNCA_A53T-KO2) (N = 3 each) with NucleoSpin RNA XS (MACHEREY–NAGEL, Düren, Germany), and submitted to GENEWIZ Japan (Tokyo, Japan) for RNA-seq with the DNBSEQ platform. Sequence reads were aligned to the zebrafish genome (GRCz11.101) with Hisat2 (v2.0.1). The read counts of each gene were normalized with the weighted trimmed mean of M-values scale-normalization method of the edgeR Bioconductor package (v3.32.1) [18]. The likelihood ratio test by the edgeR Bioconductor package was applied for the ANOVA-like test to compare among three groups, followed by comparisons between two groups [19–23]. The genes exhibited a false discovery rate (FDR) lower than 0.01 for the ANOVA-like test, and a more than 2.0-fold change and a FDR lower than 0.01 for a comparison between two groups were isolated.

The number of genes isolated by comparisons among wild type, Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) was analyzed by the DeepVenn diagram (http://www.deepvenn.com/) [24]. Expression profiles of the isolated genes were visualized by heatmap analysis using Clustergrammer (https://maayanlab.cloud/clustergrammer/) [25]. The isolated genes were further processed to Gene Ontology (GO) enrichment analysis based on DAVID [26, 27] with MonaGO (https://monago.erc.monash.edu/) [28]. Biological process was selected in the annotation type, and the significant GO terms with p value less than 0.01 were isolated.

Cell line and culture

A-THP-1 cells were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan, and grown in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37 °C.

Preparation of A53T-mutated human α-synuclein (αS_A53T) monomer

Recombinant αS_A53T protein was bacterially expressed and purified as described previously [29] with slight modifications. Briefly, human SNCA_A53T gene in pET16b vector was transformed in Escherichia coli BL21(DE3) strain (Novagen, Birmingham, United Kingdom). Gene expression was induced in LB Miller medium with 10 mM IPTG for 4 h, followed by the freeze-and-thaw process prior to sonication in purification buffer (50 mM Tris–HCl pH7.6, 1 mM EGTA, 1 mM DTT) by Astrason S3000-600 (MISONIX, NY, USA). Cleared lysate was collected after centrifugation at 4 °C, then incubated in boiling water for 5 min. After centrifugation, soluble fraction containing αS proteins was collected and loaded onto the column with Q Sepharose Fast Flow (GE Healthcare, IL, USA) for ion exchange chromatography. αS proteins were captured with column, then washed and eluted in step-wise process in purification buffer with 0.1 and 0.3 M NaCl, respectively. Eluted fraction was subjected for ammonium sulfate precipitation method to concentrate and purify more, followed by the dialysis with 30 mM TrisHCl (pH 7.6) overnight.

αS_A53T monomer treatment

Cells were seeded into 24-well plates at a density of 1.0 × 104 cells/well with 0.5 ml of RPMI medium 1640 supplemented with 10% FBS. 1 μl of appropriately diluted αS_A53T monomers with Opti-MEM (Thermo Fisher Scientific, MA, USA) was applied to each well and incubated for 24 h.

Inhibitor treatment

Cells were seeded into 24-well plates at a density of 1.0 × 104 cells/well with 0.5 ml of RPMI medium 1640 supplemented with 10% FBS. 1 μl of 1 μg/μl αS_A53T monomer, and 1 μl of appropriately diluted l-[5-isoquinolinesulfonyl]-2-methylpiperazine dihydrochloride (H7), a non-selective protein kinase inhibitor [30]; 4[4-(5-nitro-furan-2-ylmethylene)-3, -dioxopyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41), a ubiquitin-activating enzyme E1 inhibitor [31]; or lactacystin, a proteasome inhibitor [32] with Opti-MEM were applied to each well and incubated for 24 h.

Western blotting

Total protein was isolated from single zebrafish larva at five dpf or A-THP-1 cells from a well in 10 μl of tissue lysis buffer (5% SDS, 8 M Urea, 1 mM EDTA, 150 mM NaCl, 50 mM Tris pH 8.0) with sonication for 20 min followed by incubation for 30 min at room temperature. 0.5 μl of the isolated proteins or 1 μl of the medium were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis with 7.5% e-PAGEL (ATTO, Tokyo, Japan), transferred to 0.45 µm nitrocellulose membranes, blocked with EzBlock BSA (ATTO) for 1 h at room temperature, and incubated with primary antibody anti-αS (rabbit monoclonal antibody, 1:1000 dilution; Abcam, Cambridge, UK), anti-phospho-αS (Ser129) (rabbit monoclonal antibody, 1:1000 dilution; Cell Signaling Technology, MA, USA), or anti-β-actin (mouse monoclonal antibody, 1:1000 dilution; Santa Crus Biotechnology, TX, USA) in TBST (20 mM Tris-buffered saline pH 7.5, 0.05% Tween 20) overnight at 4 °C with constant shaking. After wash with TBST, the membranes were incubated for 1 h with the appropriate secondary antibody in TBST. Target proteins were visualized using ImmunoStar LD (FUJIFILM Wako Pure Chemical, Osaka, Japan) and detected with Light-Capture II (ATTO). Bands between 95 and 130 kDa were quantified with CS Analyzer 3.0 (ATTO). Each band intensity was normalized to the β-actin band intensity, and the intensity in the 0 μM group was defined as 1.0. Statistical analysis was performed by one-way ANOVA and Tukey’s post hoc test for multiple comparisons using SPSS 25 (IBM, NY, USA). p < 0.05 was considered as significant.

Gene expression analysis

Total RNA was isolated from cells using TRIsure (Meridian Bioscience, OH, USA) and converted into cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) in 20 μl volume. mRNA levels of the genes were examined by real-time PCR in 10 μl reaction mixtures containing 1 × Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, CA, USA), 0.5 μM each forward and reverse primer, and 1 μl cDNA. The primers used are shown in Table S1. Real-time PCR was performed using a Mx3000P qPCR System (Agilent Technologies) with conditions of 95 °C for 3 min, followed by 40 cycles of 95 °C for 20 s and 65 °C for 20 s followed by a dissociation step. The mRNA levels were normalized to β-actin mRNA with the ddCt method, and the gene expression level in the 0 μM group was defined as 1.0.

Statistical analysis was performed by one-way ANOVA and Tukey’s post hoc test for multiple comparisons using SPSS 25 (IBM, NY, USA). p < 0.05 was considered as significant.

Results

Effect of SNCA gene expression in mpeg-expressing cells of zebrafish

Initially, six types of transgenic zebrafish, Tg(mpeg:SNCA- KO2), Tg(mpeg:SNCA_A53T-KO2), Tg(HuC:SNCA-KO2), Tg(HuC:SNCA_A53T-KO2), Tg(mpeg:EGFP), and Tg(HuC:EGFP), were generated using an enhancer trap method [33, 34]. Tg(mpeg:SNCA-KO2) was crossed with Tg(mpeg:EGFP) and localization of αS in larvae at five dpf was examined under a fluorescent microscope (Fig. 1a and b). αS-KO2 was co-localized with EGFP, suggesting that αS is localized in mpeg-expressing cells. Tg(mpeg:SNCA_A53T-KO2) was also crossed with Tg(mpeg:EGFP) (Fig. 1c and d) to evaluate the effect of A53T mutation. In a limited number of cells, EGFP co-localized with αS_A53T-KO2 (Fig. 1c, white arrowheads); however, most of the αS_A53T-KO2 did not co-localize with EGFP. Furthermore, only αS_A53T-KO2 was localized in the fin (Fig. 1c) and in the ventral region of the head (Fig. 1d). Tg(mpeg:SNCA_A53T-KO2) was also crossed with Tg(HuC:EGFP) (Fig. 1e and f). In the lateral view, αS_A53T-KO2 co-localized with EGFP in the fin (Fig. 1e). In the ventral region of the head, αS_A53T-KO2 was co-localized with EGFP (Fig. 1f, white arrowheads). Tg(HuC:SNCA-KO2) was also crossed with Tg(HuC:EGFP) (Fig. 1g), and Tg(HuC:SNCA_A53T-KO2) with Tg(HuC:EGFP) (Fig. 1h). Both αS-KO2 and αS_A53T-KO2 co-localized with EGFP.

Fig. 1.

Fig. 1

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Localizations of transgenes in double transgenic zebrafish larvae at 5 days post fertilization. a, b mpeg-expressing cells are shown in green (mpeg:EGFP). Localization of αS expressed in mpeg-expressing cells is shown in red (mpeg:SNCA-KO2). c, d mpeg-expressing cells are shown in green (mpeg:EGFP). Localization of αS_A53T expressed in mpeg-expressing cells is shown in red (mpeg:SNCA_A53T-KO2). The white arrowheads indicate co-localization of EGFP and SNCA_A53T-KO2. e, f HuC-expressing cells are shown in green (HuC:EGFP). Localization of αS_A53T expressed in mpeg-expressing cells is shown in red (mpeg:SNCA_A53T-KO2). The white arrowheads indicate co-localization of EGFP and SNCA_A53T-KO2. g HuC-expressing cells are shown in green (HuC:EGFP). Localization of αS expressed in HuC-expressing cells is shown in red (HuC:SNCA -KO2). h HuC-expressing cells are shown in green (HuC:EGFP). Localization of αS_A53T expressed in HuC-expressing cells is shown in red (HuC:SNCA_A53T -KO2). (a, c, e, g, h), lateral view; (b, d, f), ventral view. Scale bar: 500 μm (a, c, e, g, h), 100 μm (b, d, f). Insets are for enlargement (c, e)

Because the localization of αS was different between Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2), αS proteins in wild type, Tg(mpeg:SNCA-KO2), Tg(mpeg:SNCA_A53T-KO2), Tg(HuC:SNCA-KO2), and Tg(HuC:SNCA_A53T-KO2) were examined by Western blotting (Fig. 2). Bands at 95 kDa and around 315 kDa were detected in all fish examined, and bands at more than 315 kDa were detected in Tg(mpeg:SNCA-KO2), Tg(HuC:SNCA-KO2), and Tg(HuC:SNCA_A53T-KO2) with anti-αS antibody. A native αS-KO2 band between 43 and 52 kDa was detected in Tg(HuC:SNCA-KO2) and Tg(HuC:SNCA_A53T-KO2); however, no native bands were detected in Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2). Meanwhile, two major bands at 315 kDa and 130 kDa were observed in all the fish examined with anti-phospho-αS antibody in this study.

Fig. 2.

Fig. 2

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αS protein in wild type, Tg(mpeg:SNCA-KO2), Tg(mpeg:SNCA_A53T-KO2), Tg(HuC:SNCA-KO2), and Tg(HuC:SNCA_A53T-KO2) zebrafish

Transcriptome analysis of Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) zebrafish

For further analysis, transcriptome analysis by RNA-seq of wild type, Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2), was performed. After ANOVA-like analysis, the fish were compared to one another. The number of genes isolated by comparisons among wild type, Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) for differential expression, was analyzed using the DeepVenn diagram [24] (Fig. 3a). The number of genes isolated by comparison between Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) was 1008 in total. Expression profiles of the 1008 genes were visualized by heatmap analysis using Clustergrammer [25] (Fig. 3b), and 243 of the 1008 genes were further processed for Gene Ontology (GO) enrichment analysis based on DAVID [26, 27] with MonaGO [28] (Fig. 3c). Four GO terms regulation of cell cycle, response to stress, activation of MAPKKK activity, and oxidation–reduction process were isolated.

Fig. 3.

Fig. 3

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Transcriptome analysis among wild type, Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) zebrafish. a Venn diagram of isolated genes by three comparisons. b Heatmap of genes expressed significantly different between Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) zebrafish. c Gene ontology enrichment analysis with MonaGO of the isolated genes between Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) zebrafish

Meanwhile, the number of genes isolated in both comparisons of wild type vs. Tg(mpeg:SNCA-KO2) and wild type vs. Tg(mpeg:SNCA_A53T-KO2) was 3347 genes. In the isolated genes, expression profiles of post-translational modification-related genes 30 kinase genes and five E3 ubiquitin protein ligase genes were visualized by heatmap analysis (Fig. 4a). Of the 3347 genes, 1448 genes were also processed for GO enrichment analysis using MonaGO (Fig. 4b). The isolated GO terms were related to neuronal activity and transport, suggesting that αS expression does not affect mpeg-expressing cells but affects neuronal cells in both Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2). Since native αS-KO2 was undetected and αS-KO2 was detected at higher molecular weight in Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) (Fig. 2), translated αS-KO2 is suggested to be immediately processed to a higher molecular weight and released from mpeg-expressing cells to stimulate neurons.

Fig. 4.

Fig. 4

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Transcriptome analysis among wild type, Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) zebrafish. a Heatmap of post-translational modification-related genes expressed significantly higher in both Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2). Gene names in black are kinase genes; gene names in red are E3 ubiquitin protein ligase genes. b Gene ontology enrichment analysis with MonaGO of the isolated genes in both Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2)

Processing of αS in macrophage cells

mpeg-expressing cells were suggested to be involved in the processing of αS; therefore, the processing of αS in mpeg-expressing cells was further investigated using A-THP-1 [35], a macrophage cell line derived from THP-1. Initially, A-THP-1 cells were incubated with αS_A53T monomers for 24 h, and the αS protein was examined by Western blotting (Fig. 5a). At all αS concentrations, αS was detected at 95 kDa and below 95 kDa. αS_A53T monomers at 18 kDa were detected at 2 and 20 μg/ml, respectively. Phosphorylated αS was also detected at 130 kDa and between 95 and 130 kDa. αS and phosphorylated αS in the medium after 24-h incubation with αS_A53T monomers were also examined. αS proteins in the medium were detected at the identical molecular weights in cells; however, phosphorylated αS was not detected (Fig. 5b). The αS and phosphorylated αS bands in both cells and the medium were detected at 0 μg/ml. SNCA gene was not expressed in A-THP-1 cells; however, αS was present in the medium without incubation (Fig. 5c). αS concentration in the medium suggests being low since the corresponding band was not detected in Fig. 5b. Therefore, αS can be taken up from the medium and accumulated in cells.

Fig. 5.

Fig. 5

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Investigation of the processing of αS_A53T monomer in macrophage cells. a Effect of αS_A53T monomer treatment on αS protein in A-THP-1 cells. b αS protein in the medium after αS_A53T monomer treatment to A-THP-1 cells. c αS protein in medium. Effect of phosphorylation inhibitor H7 on αS protein (d, e) and E3 ubiquitin protein ligase gene expressions (f–i). Expression or intensity levels of 0 μM H7 were defined as 1.0. All data are presented as the mean ± standard error of the mean (N = 6) and were analyzed by one-way ANOVA and Tukey’s post hoc test for multiple comparisons. **p < 0.01, ***p < 0.001

Regulation of αS processing in macrophage cells

Kinase genes and E3 ubiquitin protein ligase genes were isolated by transcriptome analysis; therefore, the involvement of phosphorylation and ubiquitination in the processing of αS was investigated. The association between αS and phosphorylated αS in A-THP-1 cells was evaluated using H7, a non-selective protein kinase inhibitor [30]. Phosphorylation of αS was significantly suppressed with 102 μM H7 (Fig. 5d, e). An αS greater than 95 kDa was not detected and a band between 95 and 130 kDa was significantly low with 102 μM H7 (Fig. 5d, e). The effect of protein kinase inhibition by H7 on ubiquitination-related gene expression was also evaluated. Expression of HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 and 2 (HERC1 and 2), HECT domain E3 ubiquitin protein ligase 4 (HECTD4), and HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1), which are E3 ubiquitin protein ligase genes expressed in A-THP-1 cells, were examined. HERC1 (Fig. 5f) and HUWE1 (Fig. 5g) expression was significantly lower, and HERC2 (Fig. 5h) and HECTD4 (Fig. 5i) expression was significantly higher at 102 μM H7.

PYR-41, which is a ubiquitin-activating enzyme E1 inhibitor [31], was used to evaluate the effect of ubiquitination on kinase gene expression. From Fig. 4a, tau tubulin kinases (TTBKs), protein kinase C (PRKC), and microtubule associated serine/threonine kinase (MAST) were selected, and polo-like kinase 2 (PLK2) is known to phosphorylate αS at Ser129 [36]. In this study, the expression of TTBK2, MAST1, MAST2, PLK1, PRKCB, and PRKCZ, which are members of these families expressed in A-THP-1 cells, was examined. αS and phosphorylated αS were significantly lower with 101 μM PYR-41 (Fig. 6a, b). Expression of MAST2 (Fig. 6c), TTBK2 (Fig. 6d), PLK1 (Fig. 6e), and PRKCB (Fig. 6f) was significantly lower with 101 μM PYR-41. The expression of MAST1 and PRKCZ (Fig. 6g and h) were unaffected by PYR-41 treatment. These results suggest that ubiquitination affects the expression of protein kinase genes, but phosphorylation does not directly affect the expression of E3 ubiquitin protein ligase genes.

Fig. 6.

Fig. 6

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Effect of ubiquitination inhibitor PYR-41in the processing of αS_A53T monomer in macrophage cells. Effect of ubiquitination inhibitor PYR-41 on αS protein (a, b), kinase gene expressions (c–h). Expression or intensity levels of 0 μM PYR-41 were defined as 1.0. All data are presented as the mean ± standard error of the mean (N = 6) and were analyzed by one-way ANOVA and Tukey’s post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Since ubiquitination affected kinase gene expression, the ubiquitin–proteasome system may be involved in this regulation. Lactacystin, which is a proteasome inhibitor [32], was used to evaluate its association with the ubiquitin–proteasome system. αS significantly decreased with 101 μM lactacystin (Fig. 7a, b). Expression levels of MAST1 (Fig. 7c, MAST2 (Fig. 7d), TTBK2 (Fig. 7e), and PRKCB (Fig. 7f) were significantly higher at 101 μM lactacystin, PLK1 expression was not significantly different (Fig. 7g), and PRKCZ expression was significantly lower at 101 μM (Fig. 7h). Meanwhile, the expression of HERC1 (Fig. 7i) and HECTD4 (Fig. 7j) was significantly higher with 101 μM H7 treatment, and HERC2 (Fig. 7k) and HUWE1 (Fig. 7l) expression was significantly higher at 100 and 101 μM H7.

Fig. 7.

Fig. 7

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Involvement of the proteasome system in the processing of αS_A53T monomer in macrophage cells. Effect of proteasome inhibitor lactacystin on αS protein (a, b), kinase gene expressions (c–h), and E3 ubiquitin protein ligase gene expressions (i–l). Expression or intensity levels of 0 μM lactacystin were defined as 1.0. All data are presented as the mean ± standard error of the mean (N = 6) and were analyzed by one-way ANOVA and Tukey’s post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Discussion

In this study, the involvement of macrophages/microglia in the formation and spread of αS fibrils and the effects of αS fibrils on neuronal cells were investigated using human SNCA-expressing zebrafish, Tg(mpeg:SNCA-KO2), and Tg(mpeg:SNCA_A53T-KO2). αS suggests being processed to larger molecules in mpeg-expressing cells and stimulating neuronal cells in both Tg(mpeg:SNCA -KO2) and Tg(mpeg:SNCA_A53T-KO2). Furthermore, αS_A53T monomers suggest being taken up by A-THP-1 cells and processed into larger molecules. Unphosphorylated αS was detected in the medium, although phosphorylated αS was not detected. Therefore, unphosphorylated αS suggests being released after processing of larger αS molecules.

After treatment with PYR-41, inhibition of ubiquitination was associated with decreases in MAST2, TTBK2, PLK1, and PRKCB gene expression. However, inhibition of phosphorylation by H7 was not associated with E3 ubiquitin protein ligase gene expression. Therefore, ubiquitination can regulate protein phosphorylation by modulating the expression of protein kinase genes, such as MAST2, TTBK2, and PRKCB in macrophage cells. Meanwhile, increases in HERC1, HERC2, HECTD4, and HUWE1 gene expression were associated with increases in MAST1, MAST2, TTBK2, and PRKCB gene expression after lactacystin treatment. The proteasome system suggests being associated with E3 ubiquitin protein ligase gene expression, followed by protein kinase gene expression. Since macrophage cells take up αS monomers to degrade through lysosomal and proteasomal pathways [37], this association could be involved in the degradation of αS through the ubiquitin–proteasome system. Phosphorylated αS was not released from A-THP-1 cells. Therefore, phosphorylation of αS may be required to degrade αS through the ubiquitin–proteasome system. Meanwhile, unphosphorylated αS was affected by H7, PYR-41, and lactacystin in macrophage cells. Unphosphorylated αS suggests being released from macrophage cells; therefore, the ubiquitin–proteasome system may be involved in the release of unphosphorylated αS as well. In this study, only macrophages were examined. However, microglia are the resident macrophages in the central nervous system [9] and also take up αS to degrade [7]; therefore, microglia could play an identical role in the brain.

In this study, 0 μg/ml αS_A53T treatment exhibited no significant difference from the other αS_A53T monomer concentrations. Since A-THP-1 cells suggest taking up αS from the medium, wild-type αS suggests being processed like αS_A53T as well. Furthermore, no dose-dependent response suggests that A-THP-1 cells hold limited amount of αS within cells. In the transcriptome analysis, expression of neuronal activity-related genes was isolated in both Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2), although the immune system-related genes were not isolated. Therefore, macrophages suggest stimulating neurons by releasing unphosphorylated αS in both Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2). On the other hand, four GO terms in the biological process —regulation of cell cycle, response to stress, activation of MAPKKK activity, and oxidation–reduction process —were isolated when Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) were compared. The four isolated GO terms mainly consisted of growth arrest and DNA damage-inducible (gadd) 45 family genes, which were significantly lower in Tg(mpeg:SNCA_A53T-KO2). Since the Gadd45 family is involved in neurodevelopment [38], αS_A53T accumulation in neurons could inhibit neuronal development of Tg(mpeg:SNCA_A53T-KO2). The expression differences between Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2) could be due to the accumulation of αS.

αS_A53T-KO2 accumulated not only in neurons, but also throughout the body of Tg(mpeg:SNCA_A53T-KO2). In this study, both wild-type and A53T-mutated αS suggest being released from mpeg-expressing cells and stimulating neurons. Transcriptome analysis revealed that transport-related genes were isolated in both Tg(mpeg:SNCA-KO2) and Tg(mpeg:SNCA_A53T-KO2); therefore, both released wild-type and A53T-mutated αS could be taken up by neurons. However, wild-type αS did not accumulate in Tg(mpeg:SNCA-KO2). In αS fibril-seeded neuronal cells, autophagosome-like vesicles are formed, and αS is degraded by autophagy and the proteasome [2, 39]; therefore, wild-type αS taken up to neurons in Tg(mpeg:SNCA-KO2) suggests being degraded. On the other hand, αS_A53T accumulated in Tg(mpeg:SNCA_A53T-KO2). αS_A53T causes a high aggregation propensity [40, 41]; therefore, aggregation of αS_A53T may inhibit degradation. Larger αS_A53T molecules more than 315 kDa were not detected in Tg(mpeg:SNCA_A53T-KO2) by Western blotting unlike the other transgenic zebrafish. αS_A53T aggregates may be insoluble owing to aggregation propensity. Therefore, αS_A53T released from mpeg-expressing cells could be aggregated and accumulated throughout the body, including neurons. Meanwhile, αS_A53T was also expressed in Tg(HuC:SNCA_A53T-KO2). αS proteins, especially those with higher molecular weights detected by Western blotting, were different from Tg(mpeg:SNCA_A53T-KO2) and similar to Tg(mpeg:SNCA-KO2) and Tg(HuC:SNCA -KO2), suggesting that αS_A53T does not aggregate in neurons of Tg(HuC:SNCA_A53T-KO2). Therefore, αS released in the form of fibrils from mpeg-expressing cells including macrophages/microglia, could act as a seed for the accumulation of αS throughout the body including the brain. Furthermore, macrophages/microglia are involved in the maintenance of neurons [42]; therefore, αS aggregates may be propagated by spreading αS fibrils from macrophages/microglia. In this study, several major bands were detected with anti-αS antibody and anti-phospho-αS antibody in wild-type zebrafish. Since wild type zebrafish does not express αS, bands detected in wild type suggest not being αS bands. Therefore, phosphorylated αS was not detected in transgenic zebrafish. Phosphorylated αS in mpeg-expressing cells may be undetected due to sensitivity since total protein from a whole body was examined.

αS expressed in macrophages/microglia [43, 44] and/or taken up from neurons can give rise to the formation of αS fibrils and phosphorylated. Phosphorylated αS can be degraded under the control of the ubiquitin–proteasome system. Unphosphorylated αS fibrils could also be released. Since neuronal cells take up αS fibrils via cell-surface receptors [45], αS fibrils released from macrophages/microglia can be taken up by neurons and degraded by autophagy and the proteasome [39]. When αS fibrils cannot be degraded in neurons, αS fibrils are aggregated and cause LB formation (Fig. 8). This mechanism can be involved in the neuron-to-neuron propagation of αS aggregates. αS is propagated neurons in Tg(mpeg:SNCA_A53T-KO2), although αS is not expressed in neurons. Since microglia, intestinal macrophages, and macrophages are involved in the maintenance of neurons, the proposed mechanism can be involved in the propagation of αS from the peripheral to the central nervous system. This mechanism can be applied to sporadic PD, that is, when the activity of macrophages/microglia and/or neurons declines, undegraded αS fibrils accumulate in neurons, which causes αS aggregation and LB formation. Alterations of the innate immune system in PD have been reported, such as precedence of gastrointestinal dysfunction over motor symptoms in PD [46], an association of gut inflammation with the onset of PD [47], and activation of macrophages and microglia in PD [48, 49]. During aging, which is a major risk factor for PD, neuronal activity declines [50], and macrophages and microglia also decrease phagocytosis and immune reactions [51, 52]. These alterations may affect the mechanisms proposed in this study and cause PD by causing LB formation.

Fig. 8.

Fig. 8

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Proposed mechanisms αS aggregate formation. αS expressed in macrophage and/or taken up from neurons is processed to αS fibrils and phosphorylated. Phosphorylated αS is degraded by the ubiquitin–proteasome system. Unphosphorylated αS fibrils are also released, taken up to neurons and degraded. When αS fibrils are unable to be degraded in neurons, αS fibrils are aggregated

Conclusions

In this study, the involvement of macrophages/microglia in the formation and spread of αS fibrils and the regulatory mechanisms of αS in macrophages were investigated. αS was processed to larger molecules, which could be αS fibrils, and phosphorylated in macrophages. Phosphorylated αS can be degraded under the control of the ubiquitin–proteasome system. Unphosphorylated αS fibrils were released from macrophages, and taken up by neurons. αS_A53T fibrils were accumulated in neurons. Therefore, αS fibrils suggest being formed from monomers in macrophages/microglia and spread to neurons to induce αS aggregates. Macrophages/microglia may play an essential role in the formation of αS aggregates and the pathogenesis of PD.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 182 KB) (181.9KB, pdf)
Supplementary file2 (TXT 11908 KB) (11.6MB, txt)

Acknowledgements

We thank Dr. Motoyuki Ito and Dr. Takamasa Mizoguchi for their technical supports, Dr. Koichi Kawakami and Dr. Yoshiko Takahashi for providing pT2AL200R150G vector and pCS-zT2TP vector, and Ms. Emiko Arita and Ms. Keiko Tamai for their administrative supports. We also thank GENEWIZ Japan to assist our RNA-seq experiment, KEYENCE for their technical supports, and Editage (www.editage.com) for English language editing.

Author contributions

SM conceptualized the study. SM, KI and MT acquired funding and SM and MT supervised the project. SM, MH, TF and NH conducted experiments and analyzed the data. SM drafted and edited the manuscript. SM, TF and MT reviewed the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by JSPS KAKENHI 19K07838 (SM), JSPS KAKENHI 16H03257 (MT, KI, SM), and Grants from Chiba Foundation for Health Promotion Disease Prevention (SM).

Availability of data and materials

All data are available in the main text and supplementary materials.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval and consent to participate

All experimental procedures were performed in accordance with the guideline of the Chiba University Institutional Animal Care and Use Committee (Approval No. 2-402 and 3-384).

Consent for publication

Not applicable.

Author information

Not applicable.

Footnotes

Publisher's Note

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