Graph shows quantification of mean±s.e.m. Bafilomycin A1 (BAF) was added for the last 3 h. (J) Cell lysates from BV2 cells cultured with AS fibrils or monomers (1 µM) were collected at different time points and LC3 and β-actin protein levels were examined using western immunoblotting. LC3-positive vesicles in unstimulated or treated BV2 (E) and primary microglial cells (I) were determined using ImageJ particle counting plugin after cell deconvolution ( n=20). (D,H) 3D surface-rendered magnifications of the selected area above. After 12 h cells were immunostained with anti-LAMP1 (red) antibody and primary microglial cells were also stained for LC3. (B–H) BV2 GFP–LC3 cells (B,C) or primary microglial cells (F,G) were left untreated or stimulated with Alexa Fluor 647-labelled AS fibrils (1 µM). Cells were then fixed and stained for LC3. (A) BV2 microglial cells were left untreated or stimulated at different time points with AS monomers (mAS) or fibrils (fAS) at 1 μM. In this scenario, the autophagy pathway plays a critical role as the main mechanism responsible for abnormal protein and organelle degradation ( Osellame and Duchen, 2014).Īlpha-synuclein induces autophagy in microglial cells. In addition, recent reports indicate that AS induces mitochondrial and lysosomal dysfunction and alters vesicular trafficking in PD, which may lead to AS accumulation ( Di Maio et al., 2016 Mazzulli et al., 2016). AS may contribute to PD pathogenesis by distinct mechanisms, but novel evidence suggests that its aberrant fibril conformations are the toxic species that mediate disruption of cellular homeostasis and neuronal death, through effects on various intracellular targets including synaptic function ( Peelaerts et al., 2015). Neuronal loss in the substantia nigra, which causes striatal dopamine deficiency, and Lewy bodies, intracellular inclusions containing aggregates of alpha-synuclein (SNCA, AS hereafter), are the neuropathological hallmarks of PD. Parkinson's disease (PD) is a late-onset neurodegenerative disorder that mainly affects the motor system. However, the mechanistic basis underlying the specific interaction between OPTN and TBK1 in these disorders is still elusive.
Interestingly, mutations of OPTN and TBK1 are both associated with neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Creutzfeld–Jacob disease and Pick's disease ( Korac et al., 2013 Li et al., 2016). Inhibition and depletion of TBK1 or OPTN blocks the efficient turnover of depolarised mitochondria. As an upstream binding partner for the autophagy receptor, TBK1 phosphorylates OPTN on damaged mitochondria, leading to the formation of a TBK1–OPTN complex. Recently, it has also been shown that the TBK1–OPTN axis targets damaged mitochondria for degradation via PINK1/parkin-mediated mitophagy ( Moore and Holzbaur, 2016). They also showed that this process was regulated by the activation of TANK-binding kinase 1 (TBK1), which binds and phosphorylates OPTN on Ser177, leading to enhanced binding to Atg8 proteins such as LC3 ( Wild et al., 2011). (2011) characterised an autophagic adaptor, optineurin (OPTN), as a key component of pathogen-induced autophagy. Numerous autophagic receptors have been reported to control the delivery of specific cargoes to the lysosomes through autophagy. This article has an associated First Person interview with the first author of the paper. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.
Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells.
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