Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

Alpha‐synuclein (αS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of αS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased αS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side‐chains eliminates electrostatic repulsion in the C‐terminal tail of αS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C‐terminal tail and the NAC (non‐amyloid‐β component) region are maintained and may lead to the formation of a globular domain. Transient long‐range contacts between the C‐terminus of the protein and regions N‐terminal to the NAC region are also preserved. Thus, the release of long‐range contacts does not play a role in the increased aggregation of αS at low pH, which we instead attribute to the increased hydrophobicity of the protein.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Helix periodicity, topology, and dynamics of membrane-associated alpha-synuclein

The protein alpha-Synuclein (aS) is a synaptic vesicle-associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinson's disease. The normal function of aS depends on a membrane-associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane-binding domain of aS is helical and suggested that it may exhibit an unusual structural periodicity. Here we present a study of the periodicity, topology, and dynamics of detergent micelle-bound aS using paramagnetic spin labels embedded in the micelle or attached to the protein. We show that the helical region of aS completes three full turns every 11 residues, demonstrating the proposed 11/3 periodicity. We also find that the membrane-binding domain is partially buried in the micelle surface and bends toward the hydrophobic interior, but does not traverse the micelle. Deeper submersion of certain regions within the micelle, including the unique lysine-free sixth 11-residue repeat, is observed and may be functionally important. There are no long-range tertiary contacts within this domain, indicating a highly extended configuration. The backbone dynamics of the micelle-bound region are relatively uniform with a slight decrease in flexibility observed toward the C-terminal end. These results clarify the topological features of aS bound to membrane-mimicking detergent micelles, with implications for aS function and pathology.     

Effect of amyloids on the vesicular machinery: implications for somatic neurotransmission

Certain neurodegenerative diseases are thought to be initiated by the aggregation of amyloidogenic proteins. However, the mechanism underlying toxicity remains obscure. Most of the suggested mechanisms are generic in nature and do not directly explain the neuron-type specific lesions observed in many of these diseases. Some recent reports suggest that the toxic aggregates impair the synaptic vesicular machinery. This may lead to an understanding of the neuron-type specificity observed in these diseases. A disruption of the vesicular machinery can also be deleterious for extra-synaptic, especially somatic, neurotransmission (common in serotonergic and dopaminergic systems which are specifically affected in Alzheimer''s disease (AD) and Parkinson''s disease (PD), respectively), though this relationship has remained unexplored. In this review, we discuss amyloid-induced damage to the neurotransmitter vesicular machinery, with an eye on the possible implications for somatic exocytosis. We argue that the larger size of the system, and the availability of multi-photon microscopy techniques for directly visualizing monoamines, make the somatic exocytosis machinery a more tractable model for understanding the effect of amyloids on all types of vesicular neurotransmission. Indeed, exploring this neglected connection may not just be important, it may be a more fruitful route for understanding AD and PD.     

The Parkinson's disease protein LRRK2 impairs proteasome substrate clearance without affecting proteasome catalytic activity

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common known cause of Parkinson''s disease (PD). The clinical features of LRRK2 PD are indistinguishable from idiopathic PD, with accumulation of α-synuclein and/or tau and/or ubiquitin in intraneuronal aggregates. This suggests that LRRK2 is a key to understanding the aetiology of the disorder. Although loss-of-function does not appear to be the mechanism causing PD in LRRK2 patients, it is not clear how this protein mediates toxicity. In this study, we report that LRRK2 overexpression in cells and in vivo impairs the activity of the ubiquitin-proteasome pathway, and that this accounts for the accumulation of diverse substrates with LRRK2 overexpression. We show that this is not mediated by large LRRK2 aggregates or sequestration of ubiquitin to the aggregates. Importantly, such abnormalities are not seen with overexpression of the related protein LRRK1. Our data suggest that LRRK2 inhibits the clearance of proteasome substrates upstream of proteasome catalytic activity, favouring the accumulation of proteins and aggregate formation. Thus, we provide a molecular link between LRRK2, the most common known cause of PD, and its previously described phenotype of protein accumulation.     

Structure and topology of the non-amyloid-beta component fragment of human alpha-synuclein bound to micelles: implications for the aggregation process

Human alpha-synuclein is a small soluble protein abundantly expressed in neurons. It represents the principal constituent of Lewy bodies, the main neuropathological characteristic of Parkinson's disease. The fragment corresponding to the region 61-95 of the protein, originally termed NAC (non-amyloid-beta component), has been found in amyloid plaques associated with Alzheimer's disease, and several reports suggest that this region represents the critical determinant of the fibrillation process of alpha-synuclein. To better understand the aggregation process of alpha-synuclein and the role exerted by the biological membranes, we studied the structure and the topology of the NAC region in the presence of SDS micelles, as membrane-mimetic environment. To overcome the low solubility of this fragment, we analyzed a recombinant polypeptide corresponding to the sequence 57-102 of alpha-synuclein, which includes some charged amino acids flanking the NAC region. Three distinct helices are present, separated by two flexible stretches. The first two helices are located closer to the micelle surface, whereas the last one seems to penetrate more deeply into the micelle. On the basis of the structural and topological results presented, a possible pathway for the aggregation process is suggested. The structural information described in this work may help to identify the appropriate target to reduce the formation of pathological alpha-synuclein aggregation.     

Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of nine sterol intermediates. We found that lanosterol is significantly (～50%) and specifically reduced in the nigrostriatal regions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis. Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic reticulum to mitochondria in dopaminergic neurons exposed to MPP+, suggesting that lanosterol might exert its survival effect by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.     

Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated-namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)-are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.     

Structural and mechanistic basis of Parl activity and regulation

The mitochondrial rhomboid protease Parl governs apoptosis, morphology, metabolism and might be implicated in Parkinson's disease, but the structural basis of its activity and complex regulation remain unknown. We report the discovery of γ-cleavage, a proteolytic event on the loop connecting the first transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the protease. This cleavage disrupts the '1+6' structure that defines every mitochondrial rhomboid and generates a new form of Parl, PROD (Parl-rhomboid-domain). Structure-function analysis of Parl suggests that γ-cleavage could be implicated in eliminating Parl proteolytic activity, and structural modeling of PROD reveals structural conservation with the bacterial rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based mechanism of catalysis, Parl appears to use a conserved mitochondrial rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis. This work provides unexpected insights into the structural determinants regulating Parl stability and activity in vivo, and reveals a complex cascade of proteolytic events controlling the function of the protease in the mitochondrion.     

Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α‐synuclein

The discoveries of mutations in SNCA were seminal findings that resulted in the knowledge that α‐synuclein (αS) is the major component of Parkinson's disease‐associated Lewy bodies. Since the pathologic roles of these protein inclusions and SNCA mutations are not completely established, we characterized the aggregation properties of the recently identified SNCA mutations, H50Q and G51D, to provide novel insights. The properties of recombinant H50Q, G51D, and wild‐type αS to polymerize and aggregate into amyloid were studied using (trans,trans)‐1‐bromo‐2,5‐bis‐(4‐hydroxy)styrylbenzene fluorometry, sedimentation analyses, electron microscopy, and atomic force microscopy. These studies showed that the H50Q mutation increases the rate of αS aggregation, whereas the G51D mutation has the opposite effect. However, H50Q and G51D αS could still be similarly induced to form intracellular aggregates from the exposure to exogenous amyloidogenic seeds under conditions that promote their cellular entry. Both mutant αS proteins, but especially G51D, promoted cellular toxicity under cellular stress conditions. These findings reveal that the novel pathogenic SNCA mutations, H50Q and G51D, have divergent effects on aggregation properties relative to the wild‐type protein, with G51D αS demonstrating reduced aggregation despite presenting with earlier disease onset, suggesting that these mutants promote different mechanisms of αS pathogenesis.

     

Vitamin D-binding protein interacts with Aβ and suppresses Aβ-mediated pathology

The level of vitamin D-binding protein (DBP) is increased in the cerebrospinal fluid of patients with Alzheimer''s disease (AD), suggesting a relationship with its pathogenesis. In this study, we investigated whether and how DBP is related to AD using several different approaches. A pull-down assay and a surface plasmon resonance binding assay indicated direct interactions between purified DBP and amyloid beta (Aβ), which was confirmed in the brain of AD patients and transgenic AD model mice by immunoprecipitation assay and immunohistochemical double-staining method. Moreover, atomic force microscopic examination revealed that DBP reduced Aβ aggregation in vitro. DBP also prevented Aβ-mediated death in cultured mouse hippocampal HT22 cell line. Finally, DBP decreased Aβ-induced synaptic loss in the hippocampus and rescued memory deficits in mice after injection of Aβ into the lateral ventricle. These results provide converging evidence that DBP attenuates the harmful effects of Aβ by a direct interaction, and suggest that DBP is a promising therapeutic agent for the treatment of AD.     

A simple algorithm locates beta-strands in the amyloid fibril core of alpha-synuclein, Abeta, and tau using the amino acid sequence alone

Fibrillar inclusions are a characteristic feature of the neuropathology found in the alpha-synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Familial forms of alpha-synucleinopathies have also been linked with missense mutations or gene multiplications that result in higher protein expression levels. In order to form these fibrils, the protein, alpha-synuclein (alpha-syn), must undergo a process of self-assembly in which its native state is converted from a disordered conformer into a beta-sheet-dominated form. Here, we have developed a novel polypeptide property calculator to locate and quantify relative propensities for beta-strand structure in the sequence of alpha-syn. The output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of the beta-sheet core in alpha-syn fibrils. In particular, the plot features three peaks, the largest of which is completely absent for the nonfibrillogenic protein, beta-syn. We also report similar significant correlations for the Alzheimer's disease-related proteins, Abeta and tau. A substantial region of alpha-syn is capable [corrected] of converting from its disordered conformation into a long [corrected] alpha-helical protein. We have developed the aforementioned algorithm to locate and quantify the alpha-helical hydrophobic moment in the amino acid sequence of alpha-syn. As before, the output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of alpha-helical structure in membrane bilayer-associated alpha-syn.     

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.     

Autophagy and human diseases

Autophagy is a major intracellular degradative process that delivers cytoplasmic materials to the lysosome for degradation. Since the discovery of autophagy-related (Atg) genes in the 1990s, there has been a proliferation of studies on the physiological and pathological roles of autophagy in a variety of autophagy knockout models. However, direct evidence of the connections between ATG gene dysfunction and human diseases has emerged only recently. There are an increasing number of reports showing that mutations in the ATG genes were identified in various human diseases such as neurodegenerative diseases, infectious diseases, and cancers. Here, we review the major advances in identification of mutations or polymorphisms of the ATG genes in human diseases. Current autophagy-modulating compounds in clinical trials are also summarized.     

帕金森病患者血浆alpha-synuclein、Abeta及tau蛋白变化特点

目的：探索帕金森病（Parkinson''s disease,PD）患者血浆中alpha- 突触核蛋白、Abeta及tau 蛋白变化情况。方法：募集2014 年4 月至 2015 年4 月来我院就诊的PD 患者62 例,正常对照人群59 例,采集两组人群的基本临床信息,测定血浆中琢- 突触核蛋白、 Abeta40、Abeta42、pT181-tau 蛋白、pT231-tau 蛋白和总tau 蛋白浓度,比较两组之间的差异,同时进行相关性分析。结果：PD患者血浆 alpha- 突触核蛋白和pT181-tau 蛋白浓度显著高于对照组(P 值分别为0.001,0.019),而两组间Abeta40、Abeta42、pT231-tau 蛋白和总tau 蛋白浓度无明显差异（P>0.05）。相关性分析提示PD 患者血浆alpha-突触核蛋白和pT181-tau 蛋白浓度与患者年龄、性别、教育程度、 病程、高血压、糖尿病、Hoehn/ Yahr 分级及Schwab &England 评分无相关性（P>0.05）。结论：虽然PD患者血浆琢- 突触核蛋白和 pT181-tau 蛋白高于正常对照组,但尚不适宜作为PD 的生物标志物。     

Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild‐type rats

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation‐prone proteins such as αSN, which is central in the development of Parkinson''s disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT‐positive aggregates that contain higher‐order β‐sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross‐seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.     

Acidic nanoparticles protect against α‐synuclein‐induced neurodegeneration through the restoration of lysosomal function

Parkinson''s disease (PD) is an age‐related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, associated with the accumulation of misfolded α‐synuclein and lysosomal impairment, two events deemed interconnected. Protein aggregation is linked to defects in degradation systems such as the autophagy‐lysosomal pathway, while lysosomal dysfunction is partly related to compromised acidification. We have recently proven that acidic nanoparticles (aNPs) can re‐acidify lysosomes and ameliorate neurotoxin‐mediated dopaminergic neurodegeneration in mice. However, no lysosome‐targeted approach has yet been tested in synucleinopathy models in vivo. Here, we show that aNPs increase α‐synuclein degradation through enhancing lysosomal activity in vitro. We further demonstrate in vivo that aNPs protect nigral dopaminergic neurons from cell death, ameliorate α‐synuclein pathology, and restore lysosomal function in mice injected with PD patient‐derived Lewy body extracts carrying toxic α‐synuclein aggregates. Our results support lysosomal re‐acidification as a disease‐modifying strategy for the treatment of PD and other age‐related proteinopathies.     

Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent molecular lesions so far found in Parkinson's disease (PD), an age-dependent neurodegenerative disorder affecting dopaminergic (DA) neuron. The molecular mechanisms by which mutations in LRRK2 cause DA degeneration in PD are not understood. Here, we show that both human LRRK2 and the Drosophila orthologue of LRRK2 phosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP), a negative regulator of eIF4E-mediated protein translation and a key mediator of various stress responses. Although modulation of the eIF4E/4E-BP pathway by LRRK2 stimulates eIF4E-mediated protein translation both in vivo and in vitro, it attenuates resistance to oxidative stress and survival of DA neuron in Drosophila. Our results suggest that chronic inactivation of 4E-BP by LRRK2 with pathogenic mutations deregulates protein translation, eventually resulting in age-dependent loss of DA neurons.     

α‐synuclein suppresses microglial autophagy and promotes neurodegeneration in a mouse model of Parkinson’s disease

The cell‐to‐cell transfer of α‐synuclein (α‐Syn) greatly contributes to Parkinson''s disease (PD) pathogenesis and underlies the spread of α‐Syn pathology. During this process, extracellular α‐Syn can activate microglia and neuroinflammation, which plays an important role in PD. However, the effect of extracellular α‐Syn on microglia autophagy is poorly understood. In the present study, we reported that extracellular α‐Syn inhibited the autophagy initiation, as indicated by LC3‐II reduction and p62 protein elevation in BV2 and cultured primary microglia. The in vitro findings were verified in microglia‐enriched population isolated from α‐Syn‐overexpressing mice induced by adeno‐associated virus (AAV2/9)‐encoded wildtype human α‐Syn injection into the substantia nigra (SN). Mechanistically, α‐Syn led to microglial autophagic impairment through activating toll‐like receptor 4 (Tlr4) and its downstream p38 and Akt‐mTOR signaling because Tlr4 knockout and inhibition of p38, Akt as well as mTOR prevented α‐Syn‐induced autophagy inhibition. Moreover, inhibition of Akt reversed the mTOR activation but failed to affect p38 phosphorylation triggered by α‐Syn. Functionally, the in vivo evidence showed that lysozyme 2 Cre (Lyz2 ^cre)‐mediated depletion of autophagy‐related gene 5 (Atg5) in microglia aggravated the neuroinflammation and dopaminergic neuron losses in the SN and exacerbated the locomotor deficit in α‐Syn‐overexpressing mice. Taken together, the results suggest that extracellular α‐Syn, via Tlr4‐dependent p38 and Akt‐mTOR signaling cascades, disrupts microglial autophagy activity which synergistically contributes to neuroinflammation and PD development.