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

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


Statistics
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Imaging : Zen 2011 software (v2.6), Zeiss Axioplan2 (v4.9.1) Flow cytometry: FACSDiva (v8.0.1) Data analysis Fiji (ImageJ for image analysis), Stereo Investigator (v 2017, for TH/Nissl counting), Imaris (for representative image generation in vivo), GraphPad Prism v8.3.1 (for quantification and statistical analysis) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
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Field-specific reporting
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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences nature research | reporting summary

October 2018
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Data exclusions We didn't exclude any sample.

Replication
Replication of experiments was successful in three different experiments/cohort and data is accumulated during the analysis process. Results also were replicated in independent experimental setting by different authors.
Randomization All cells analyzed for the quantification were randomly selected. Control mice and their littermate mutant/KO mice were collected from each cohort depending on the genotype and used for the experiment. WT mice injected with either AAV-GFP or AAV-Syn were randomly assigned into different time points.

Blinding
All TH/Nissl counting experiments were confirmed by a blinded test. Also, cell assay experiments including NFkB-luciferase and imaging were performed by more than two people in a blinded test. Protein analyses using Western blotting were not performed blind to load samples by order.

Mycoplasma contamination
All cell lines tested negative for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used.