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. 2018 Jan;28(1):28-42.
doi: 10.1111/bpa.12461. Epub 2017 Mar 15.

AMBRA1, a novel α-synuclein-binding protein, is implicated in the pathogenesis of multiple system atrophy

Yasuo Miki  1 , Kunikazu Tanji  1 , Fumiaki Mori  1 , Yota Tatara  2 , Jun Utsumi  3 , Hidenao Sasaki  3 , Akiyoshi Kakita  4 , Hitoshi Takahashi  5 , Gian Maria Fimia  6   7 , Koichi Wakabayashi  1
Affiliations

AMBRA1, a novel α-synuclein-binding protein, is implicated in the pathogenesis of multiple system atrophy

Yasuo Miki et al. Brain Pathol. 2018 Jan.

Abstract

The accumulation of abnormal α-synuclein is the major histopathological feature of Lewy body disease and multiple system atrophy (MSA), which are referred to as synucleinopathies. Cytoplasmic degradation systems, such as the autophagy-lysosome and proteasome pathways, are involved in their pathogenesis. Autophagy is tightly regulated by several upstream proteins including UNC-51-like kinase 1/2, beclin1, vacuolar protein sorting-associated protein 34 and autophagy/beclin1 regulator 1 (AMBRA1). Recently, we revealed that both cortical and brainstem-type Lewy bodies were immunopositive for several upstream proteins of autophagy. Therefore, we conducted the present study to elucidate the role of upstream proteins of autophagy in the pathogenesis of MSA. Pathological and biochemical analyses using human brain samples revealed that AMBRA1 is a component of the pathological hallmarks of MSA and upstream proteins of autophagy are impaired in the MSA brain. In vitro and in vivo analyses revealed a ninefold stronger affinity of AMBRA1 with α-synuclein phosphorylated at serine 129 compared with non-phosphorylated α-synuclein. Furthermore, a weak but significant correlation between AMBRA1 overexpression and reduction of abnormal α-synuclein was observed. Silencing AMBRA1 function caused aggregates of α-synuclein in the cytoplasm of mouse primary cultured neurons, which was simulated by the treatment of Bafilomycin, an autophagy inhibitor. Our results demonstrated for the first time that AMBRA1 is a novel hub binding protein of α-synuclein and plays a central role in the pathogenesis of MSA through the degradative dynamics of α-synuclein. These results raise the possibility that molecular modulation targeting AMBRA1 can be a promising candidate for the treatment of synucleinopathies.

Keywords: AMBRA1; autophagy; multiple system atrophy; synucleinopathy; α-synuclein.

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Figures

Figure 1
Figure 1
Immunoreactivity of phosphorylated α‐synuclein (p‐α‐syn) and AMBRA1 in the brains of patients with multiple system atrophy (MSA). Immunoreactivity for p‐α‐syn (A, C, E) and AMBRA1 (B, D, F) in glial cytoplasmic inclusions (GCIs) (A, B), neuronal cytoplasmic inclusions (NCIs) (C, D) and threads (E, F). Bars = 10 μm.
Figure 2
Figure 2
Double‐labeling immunofluorescence demonstrating co‐localization of AMBRA1 and p‐α‐syn in GCIs (A–C), NCIs (D–F) and threads (white arrows) (G–I). AMBRA1 appears green, and p‐α‐syn appears red. Bars = 20 μm.
Figure 3
Figure 3
Western blot analysis of total brain lysates from MSA patients and normal controls. Raw data for the levels of ULK1, ULK2, AMBRA1, TRAF6, beclin1, VPS34, p62, LC3 and actin in the cerebellar white matter of patients with MSA (n = 5) and normal controls (n = 5) (A). In patient with MSA, there were significantly increased expression levels of ULK1 (B), ULK2 (C) and AMBRA1 (D). On the other hand, TFAF6 was significantly decreased in patients with MSA (E). No significant difference in the levels of beclin1 (F), VPS34 (G), p62 (H) and LC3‐II relative to LC3‐I (I) between MSA patients and normal controls. *P < 0.05; **P < 0.01.
Figure 4
Figure 4
Interaction of AMBRA1 with wild type (WT) α‐syn in mammalian cells. (A) Myc‐tagged AMBRA1 constructs are illustrated. FL, full length; F1–3, fragments 1–3. (B) Characterization of the site of AMBRA1 responsible for α‐syn binding. HEK293 cells were co‐transfected with Myc‐tagged AMBRA1 and RH‐tagged WT α‐syn. Protein extracts (input) were subjected to TALON purification under native conditions, in which cobalt attracts RH‐tagged protein. Purified WT α‐syn bound to the F3 portion of AMBRA1. (C–E) Proximity ligation assay (PLA) in mammalian cells. (C) No signals are recognized in the absence of the primary antibodies. (D) With the use of anti‐α‐syn (syn211) and AMBRA1 antibodies as primary antibodies for PLA, binding of endogenous α‐syn and AMBRA1 are visualized as red signals (white arrowheads). (E) Co‐transfection of WT α‐syn and AMBRA1 showed intense signals (white arrows). Bars = 20 μm.
Figure 5
Figure 5
Interaction and affinity of AMBRA1 with phospho‐mimic S129E α‐syn in mammalian cells. HEK293 cells were co‐transfected with a combination of Flag‐tagged AMBRA1 and WT α‐syn, or phospho‐mimic S129E α‐syn. Inputs were subjected to immunoprecipitation (IP) using an anti‐Flag antibody. (A) Purified complexes were then analyzed by immunoblotting with an anti‐Flag antibody, showing bands of 150 kDa corresponding to the molecular weight of AMBRA1 in AMBRA1‐transfected cells (arrowhead, top). In cells transfected with AMBRA1 and WT α‐syn, an anti‐α‐syn (RH and 4D6) antibodies detected a band of 16 kDa in input and IP (arrow). A high molecular weight smear appeared in cells transfected with AMBRA1 and S129E α‐syn (asterisk, bottom). (B, C) Sensorgrams of the binding of WT α‐syn and S129E mutant to immobilized AMBRA1. Various concentrations of WT α‐syn and S129E mutant were injected onto the AMBRA1‐immobilized sensor chip. RU, resonance units.
Figure 6
Figure 6
Protein‐protein interactions of AMBRA1 in the brains of MSA patients. Protein extracts from the cerebral cortex of normal controls and MSA patients, and cerebellar white matter of normal controls and MSA patients were immunoprecipitated using an anti‐AMBRA1 antibody. Representative cases of each brain region are shown in this figure. (A) An anti‐AMBRA1 antibody was utilized for western blot analysis of purified proteins, showing a more intense band of AMBRA1 in the cerebral cortex (lanes 2 and 6) and cerebellar white matter of MSA patients (lanes 4 and 8) than normal controls (the cerebral cortex, lanes 1 and 5; the cerebral cortex, lanes 3 and 7). A band with molecular mass of 52 kDa was observed in the cerebral cortex of MSA (arrow). (B) An anti‐α‐syn antibody (4D6) detected intense bands with a molecular weight of 16 kDa corresponding to native α‐syn in comparison with the same region between MSA patients and normal controls (white arrowhead) (right box; long time exposure). (C) For western blot analysis of immunoprecipitated proteins, a rabbit anti‐p‐α‐syn antibody was utilized. In inputs of MSA, a rabbit anti‐p‐α‐syn antibody detected a 16 kDa band. In an IP sample of the cerebral cortex of MSA, a rabbit anti‐p‐α‐syn antibody detected a band of 52 kDa, corresponding to the band detected by the anti‐AMBRA1 antibody (arrow). (D–F) PLA in the brain of MSA patients. (D) No signals were observed in the absence of primary antibodies for AMBRA1 and p‐α‐syn. Signals in GCIs (white arrows) (E) and NCIs (F). Bars = 20 μm.
Figure 7
Figure 7
Overexpression of AMBRA1 in the degradation of S129E α‐syn in mammalian cells. (A) Increased expression of Bip, an endoplasmic reticulum stress marker, after transfection with more than 0.5 μg AMBRA1. (B) Expression levels of AMBRA1, p62, LC3, S129E α‐syn and actin in cells treated with 0.25 μg S129E α‐syn with HaloTag and 0.25 μg AMBRA1 or its empty vector. Significant decrease in the LC3‐II/LC3‐I ratio (C) and p62 in AMBRA1 overexpressing cells (D) along with a reduction of S129E α‐syn (E). The molecular weight of S129E α‐syn with HaloTag is 50 kDa. Values are expressed as means and standard deviation of independent three experiments. *P < 0.05.
Figure 8
Figure 8
Silencing AMBRA1 function in mouse primary cultured neurons. (A) Decreased expression level of AMBRA1 by AMBRA1 siRNA (arrowhead). (B–E) Immunofluorescence analysis demonstrating transfection of AMBRA1 siRNA caused dot‐like structures of α‐syn in the cytoplasm, compared with that of control siRNA. (F) Semi‐quantitative analysis showing significantly increased number of dot‐like structures of α‐syn in the cytoplasm. LC3 appears green, and α‐syn appears red. Bars = 10 μm. **P < 0.01.
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