MAPK14-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease


 Background Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), accompanied by chronic neuroinflammation, autophagy dysfunction and SNCA(α-synuclein) accumulation in the form of Lewy bodies. Previous studies show that misfolded SNCA upregulate the inflammatory cascade through NOX activation, which elevated ROS production in microglial cell. However, it’s still not elucidated about the molecular mechanisms between autophagy deficiency and neuroinflammation induced by SNCA accumulation in microglia.Methods We overexpress mice and BV2 cells with SNCAA53T mutant as models of early-onset Parkinson's disease and identified the mechanisms that MAPK14-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease by a series of methods of molecular biology. The results of pathology and animal behavior showed that the inhibition of MAPK14 and NLRP3 had protective effect on Parkinson's disease model.Results Here, we report that MAPK14 (p38α) activates NLRP3 inflammasome via inhibiting TFEB in BV2 cells. Transcription Factor EB (TFEB) signaling negatively regulates NLRP3 inflammasome through increasing LAMP2A expression, which binds to NLRP3 and promotes its degradation via chaperone-mediated autophagy(CMA). Importantly, both MAPK14 inhibitor SB203580 and NLRP3 inhibitor MCC950 not only prevents neurodegeneration in vitro, but also alleviates movement impairment and SNCA/α-synuclein abnormal accumulation in SNCAA53T-tg mice model of Parkinson’s disease.Conclusion Our research reveals an endogenous regulatory mechanism of NLRP3 turnover and microglia-dopaminergic neuron interaction, which may be a potential therapeutic strategy for Parkinson’s disease.


cells. Transcription Factor EB (TFEB) signaling negatively regulates NLRP3 in ammasome through
increasing LAMP2A expression, which binds to NLRP3 and promotes its degradation via chaperonemediated autophagy(CMA). Importantly, both MAPK14 inhibitor SB203580 and NLRP3 inhibitor MCC950 not only prevents neurodegeneration in vitro, but also alleviates movement impairment and SNCA/αsynuclein abnormal accumulation in SNCA A53T -tg mice model of Parkinson's disease.
Conclusion Our research reveals an endogenous regulatory mechanism of NLRP3 turnover and microgliadopaminergic neuron interaction, which may be a potential therapeutic strategy for Parkinson's disease.

Background
Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra, resulting in neuromuscular dysfunction such as rest tremor. The mitochondrial dysfunction, calcium dyshomeostasis, chronic in ammation and oxidative stress are contributed to the molecular pathogenesis of PD. Previous studies shown that neuroin ammation may be the primary factor leading to the process of PD-related pathogenesis and causing the degeneration and loss of dopaminergic neurons. Proin ammatory cytokine such as tumor necrosis factor α and IL-1β were found to be upregulated in the cerebrospinal uid and blood of PD patients 1 . Moreover, activated microglia releases proin ammatory cytokines which lead to neuronal death. Abnormal accumulation of SNCA causes neuroin ammation, in turn, neuroin ammation exacerbates dopaminergic neurons loss. This vicious circle closely associated with the pathological process of PD. The NLRP3 in ammasome was originally de ned by Tschopp that activates in ammatory caspases and release mature IL-1β and IL-18 2 . The NLRP3 in ammation complex contributes to the activation of the innate immune response and causes cell pyrotosis 2,3 . Recent studies demonstrated that NLRP3 in ammasome is involved in the progression of neurodegenerative disease and NLRP3 de ciency plays a protective role in animal models of Alzheimer's disease (AD) and PD 4,5 . Although the mechanisms that NLRP3 in ammasome participates in diverse in ammatory diseases has been extensively investigated, its regulatory networks in microglia are unclear.
Mitogen-activated protein kinases (MAPKs) regulate various cellular processes. The p38 mitogenactivated protein kinase, a prominent member of the MAPKs, not only involved in cell cycle, cell death, development, differentiation, senescence and tumorigenesis, but also functions as a speci c class of serine/threonine kinase regulating the in ammation responses. The important role of MAPK14-mediated in ammatory responses was originally con rmed as the target of the pyridinyl imidazole that inhibited in ammatory cytokines release in LPS-treated monocytes 6 . The MAPK pathways contributed to the increased production of proin ammatory cytokines by microglia that treated with toll-like receptor (TLR) ligands or beta-amyloid (Aβ) 7 . Recently, Mao and colleagues identi ed that MAPK14 inhibits autophagy and promotes microglial in ammatory responses via ULK1 phosphorylation 8 . A few studies have demonstrated that pharmacological inhibition of MAPK14 plays a protective role in several animal models of neurodegenerative disease [9][10][11] . Moreover, studies in vivo have also shown that MAPK14 de ciency plays a protective role in AD via enhancing the level of autophagy 12,13 . Although the protective effect of MAPK14 inhibition and its implication in neurodegenerative diseases are emerging, the mechanisms by which MAPK14 regulates in ammatory responses in microglia remains elusive.
Here, we demonstrate that MAPK14-TFEB pathway regulates NLRP3 in ammasome degradation via CMA. We identify that NLRP3 is a novel substrate for CMA and MAPK14 inhibits TFEB activity via phosphorylation which blocks NLRP3 degradation. Besides, our results reveal that both MAPK14 inhibitor SB203580 and NLRP3 inhibitor MCC950 reduced SNCA-induced microglia activation and dopaminergic neuronal loss. All the results suggest the MAPK14-TFEB pathway may be a potential treatment target for NLRP3 in ammasome-driven neurodegenerative diseases.

Materials And Methods
Animal. The SNCA A53T -tg mice expressing mutant A53T SNCA (the 140 amino acid isoform) under the direction of the mouse prion protein promoter has been described previously 26 . The SNCA A53T -tg mice expressed mutant human A53T SNCA were purchased from Model Animal Research Center of Nanjing University. The structure, location and onset of the inclusions seen in the mutant mice resemble characteristics seen in human neuronal α-synucleinopathies and the transgenic mice are generally used in the study of Parkinson's Disease. Animals were individually housed under light: dark (12:12 h) cycles and provided with food and water.
Balance beam test. Mice were placed on a narrow beam (20 mm/12 mm) suspended 20 cm above soft bedding, and their movement from one end towards the other end was recorded. The number of missteps (paw faults, or slips) was scored during the trip.
Pole test. This test was performed based on the method described by Ogawa 27 used as a measure for bradykinesia. Mice (n = 8 in each group) were placed the top of a pole (55 cm in height, 10 mm in diameter) with a rough surface. Both the time to turn and climb down was recorded.
Administration of the MAPK14 inhibitor SB203580 and MCC950. All animal procedures were approved by the ethics committee of the Southern Medical University. All groups of animals received the inhibitor SB203580 (5 mg/kg) and MCC950 (10 mg/kg) through intraperitoneal injection. In each experimental cohort, male mice were randomly matched for group assignment. SNCA A53T -tg mice were injected with the drug starting from 5 months of age until the end of 9 months. Animals were sacri ced after the nal injection. The midbrain and cortex of half mice were dissected and used for Western blotting analysis.
Meanwhile, the other half mice were perfused with 4% paraformaldehyde and para nized coronal sections were processed for immunohistochemistry assay. Immunohistochemistry. The para n sections of brain tissue were collected for routine immunohistochemistry staining for p-MAPK14 (1:50), TH (1:100), IBA-1 (1:100), IL-1β (1:100), NLRP3(1:100), LAMP2A (1:100) and TFEB (1:100). The area of the midbrain dopaminergic neurons from brain sections according to the anatomical position and the location of TH-positive neurons has been described previously 28 . Immunohistochemistry staining was assessed by two independent pathologists with no prior knowledge of patient characteristics. In order to describe the image where were taken, we provided the model of anatomical position of brain section and the lower power image of TH-positive immunohistochemical brain section. As gure S3 A and S3 B shown that the image of SNpc were taken according to TH positive location and the image of cortex were taken according to their anatomical location. The percentage of positive cells and the expression levels of proteins in the brain tissue were scored according to intensity as previously described. Speci cally, percentages ≤1%, 2%-25%, 26%-50%, 51%-75% and ≥75% were scored as 0, 1, 2, 3 and 4, respectively; non-signi cant brown, light brown, moderate brown and deep brown staining intensities were scored as 0, 1, 2 and 3, respectively. Combined scoring was then calculated and graded as negative (−; 0-1), weak (+; 2-4), moderate (++; 5-8), and strong (+++; 9-12).
Transmission electron microscopy (TEM). SN4741 cells were washed once with PBS, collected with a cell scraper. The sections of midbrain tissue were collected and washed once with cold PBS. Cell and midbrain tissue pellets were resuspended in the xative containing 2.5% glutaraldehyde in PBS at 4 °C.
Cell and midbrain tissue pellets were xed for 3 h. After rinsing with sodium cacodylate buffer, they were further xed in 1% OsO4 in sodium cacodylate buffer on ice for 1 h and dehydrated with acetone, and then embedded in resin and polymerized at 60 °C for 48 h. Ultrathin sections were obtained on an ultramicrotome and stained with uranyl acetate and lead citrate before observation under Hitachi H-7500 TEM.
BV2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C.Cells were transfected with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.
Western blotting and co-immunoprecipitation. Cytosolic and nuclear fractions were collected using an isolation kit (KeyGEN, Nanjing, China) following the manufacturer's instructions. For Western blotting, cells were collected in RIPA buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 1% Triton X-100 with protease inhibitor cocktail (Roche, Nutley, NJ, USA). The extracts were centrifuged for 10 min at 13,000 rpm at 4 °C, and the supernatants were used for immunoblot analysis. For coimmunoprecipitation, cells were centrifuged at 13,000 g for 10 min at 4 °C, and then the supernatant was transferred into a new tube. And then 5 μg of the corresponding rst antibody was added into the samples and incubating overnight at 4 °C. On the next day, 50 μl agarose beads were added into the samples and incubating 2 h at 4°C. The immunocomplex was collected with centrifugation at 1,500g for 1 min at 4 °C. And washed 3 times with RIPA buffer. Proteins were eluted from beads with 2× SDS loading buffer and subjected to immunoblot analysis. The immunoreactive bands were detected by Odyssey Infrared Imaging System. The band intensity was analyzed with Image J. Antibodies used in Western blotting are diluted by 1000, except TH (1:100) and p-MAPK14(1:100).
Apoptosis and cell death. Cell apoptosis of SN4741 cell was measured using a Cell Apoptosis Kit (Dojindo, Japan) according to the manufacturer's instructions. Percent of cell apoptosis was determined by staining with 2 μM Annexin V and 2 μM Propidium Iodide (PI). The number of cell apoptosis was counted by ow cytometry and images of apoptosis was taken by Laser Scanning Confocal Microscopy. Cell death of primary neuron was detected by staining with PI (1μg/ml) (KeyGEN) according to the manufacturer's instructions. Images were captured by uorescence microscope and analyzed by Image J analysis software.
Measurement of mitochondrial membrane potential. The mitochondrial membrane potential in cells was assessed using Tetramethylrhodamine methyl ester (TMRE; Thermo Fisher, T669). Cells were washed with 1×PBS and then incubated with TMRE (0.1μM) in medium for 20 min in the dark. The intensity of uorescence was monitored using uorescence microscope. In order to investigate the effects of SNCA accumulation and related toxicity, we used SNCA A53T transgenic mice (SNCA A53T -tg mice). The accumulation of SNCA was identi ed in both the SNpc and the cortex at 9 months of age (Fig.S1 A). To determine the effect of SNCA on NLRP3 in ammasome activation, we measured the expression of core components of this multiprotein complex of the canonical in ammasome in both the SNpc and the cortex of SNCA A53T -tg mice by IHC and Western blotting. In the brain sections of the SNpc and the cortex, NLRP3 in ammasome activation was remarkably intensi ed, as evidenced by increased NLRP3 expression as well as over-production of IL-1β ( Fig.1 A-D). Results of Western blotting revealed that the protein level of NLRP3, ASC, cleaved CASP1 (caspase1) and cleaved IL-1β were increased both in SNpc and cortex of SNCA A53T -tg mice, which further demonstrated that the NLRP3 in ammasome was activated in SNCA A53T -tg mice (Fig.1 E-G). Besides, microglial activation, as assessed morphologically by immunohistochemistry with the classic antibody speci c for Iba-1, microglia was activated in SNCA A53T -tg mice brain both of the SNpc and the cortex (Fig.S1 B). To further con rm this result, PD patients' serum was assayed by ELISA. Consistently, over-production of IL-1β and IL-18 were found in PD patients' serum ( Fig.S1 C-D). Collectively, these data suggest that the NLRP3 in ammasome is activated in the pathological process of PD.

NLRP3 in ammasome activation is suppressed by MAPK14 inhibitor SB203580
To examine whether MAPK14 involved in the NLRP3 in ammasome activation in PD, we rst investigated the levels of MAPK14 phosphorylation in the SNpc and the cortex by IHC. Results showed that compared with wild-type mice, MAPK14 was activated in both the SNpc and the cortex of SNCA A53T -tg mice at 9 months of age ( Fig.2 A, B). Western blotting also identi ed increased phosphorylated MAPK14 and SNCA accumulation in the SNpc and the cortex of SNCA A53T -tg mice (Fig.2 C, D, E).
To assess whether NLRP3 activation is mediated by MAPK14, SNCA A53T -tg mice were injected with MAPK14 inhibitor SB203580 starting at the age of 5 months until the end of 9 months (Fig.S2 A). Then, we analyzed NLRP3 and IL-1β by IHC. The results showed that SB203580 signi cantly reduced the levels of NLRP3 and IL-1β both in SNpc and cortex of SNCA A53T -tg mice (Fig.3A-D). Furthermore, the increased protein levels of NLRP3, ASC, cleaved CASP1, and cleaved IL-1β in SNCA A53T -tg mice (Fig.3 E-G) and BV2-A53T cells were abolished after SB203580 treatment as detected by Western blotting (Fig.S2 B, C) . Also, we have detected the Caspase1 activity as well as mature IL-1β using ELISA in the tissue homogenates and results revealed that SB203580 can decrease the levels of caspase1 activity and mature IL1β in SNCA A53T -tg mice (Fig.S2 D, E). Of note, SB203580 did not affect the transcription of SNCA mRNA (Fig.S2  F). Together, these data suggested that MAPK14 inhibitor SB203580 reduced neuroin ammation caused by SNCA accumulation.

SB203580 induces NLRP3 degradation via chaperone-mediated autophagy
The activation of NLRP3 in ammasome in cells is tightly regulated. Excessive activation of the NLRP3 in ammasome is involved in the pathological process of various diseases including PD. To test whether NLRP3 is degraded by CMA, the interaction between NLRP3 and LAMP2A was examined by immunoprecipitation in brain tissues. The results showed that LAMP2A interacts with NLRP3 in vivo (Fig.4 A). Moreover, we also found that SB203580 enhanced the NLRP3/LAMP2A and NLRP3/ HSPA8 interaction in BV2 cells (Fig.4 B, C).
In CMA, the substrate usually contains a KFERQ-like pentapeptide consensus sequence that is recognized by HSPA8 (HSC70). Inspection of the amino acid sequence of mouse NLRP3, two KFERQ-like motifs, 795QKLVE799 and 989EVLKQ993, were revealed (Fig.S3 A). To determine whether NLRP3 is a bona de substrate for CMA, the rst two amino acids of the mouse NLRP3 KFERQ-like sequence were mutated to alanine (NLRP3AA). The result showed that mutation of the KFERQ-like sequence of NLRP3 abolished its interaction with HSPA8 ( Fig.4 D, E).
To further assess whether CMA is involved in the regulation of NLRP3, we treated BV2 cells with serum deprivation or AR7 (10 µM, 24 h) or QX77 (10 µM, 24 h), the latter two chemicals are well-known CMA agonists. The results showed that both serum deprivation and CMA agonists (AR7, QX77) increased the levels of LAMP2A and reduced the levels of NLRP3 (Fig.4 F-I). Moreover, the level of LAMP2A in the SNpc of mice was analyzed. Treatment with MAPK14 inhibitor SB203580 elevated the level of LAMP2A, a key receptor for CMA ( Fig.4 J, K).

SB203580 activates TFEB-mediated autophagy
Our data in Figure 2 and 3 demonstrate that MAPK14 mediates the NLRP3 in ammasome activation in BV2 cells and in SNCA A53T -tg mice, as evidenced by increased levels of NLRP3, cleaved CASP1, and IL-1β. Furthermore, in Figure 4, we showed that MAPK14 regulates NLRP3 turn over through CMA. All these prompt us to examine the role of MAPK14 in all over autophagy regulation, which may subsequently contribute to the NLRP3 in ammasome activation 14 . TFEB is a master protein for lysosomal biogenesis.
In SNpc sections from SNCA A53T -tg mice, the total levels of TFEB and LAMP1 were decreased, while the SQSTM1/p62 level was increased (Fig.5 A, B). To determine whether SNCA aggregation affects the TFEB nuclear translocation, subcellular localization of TFEB was examined by sub-cellular fractionation. The result showed that the nuclear TFEB level decreased in SNpc sections of SNCA A53T -tg mice (Fig.S4 A, B).
In order to examine whether MAPK14 activation is involved in TFEB-mediated autophagy in the PD model, we treated SNCA-A53T overexpressing BV2 cells with MAPK14 inhibitor SB203580 (10 μM, 24 h). The results showed that MAPK14 inhibitor SB203580 signi cantly elevates TFEB-mediated autophagy, as evidenced by the increased LAMP1, MAP1LC3/LC3 and the decrease of SQSTM1 (Fig.5 C, D). Furthermore, both Western blotting and immuno uorescence revealed that SB203580 prompted TFEB nuclear translocation from the cytoplasm (Fig.5 E-H). Meanwhile, SB203580 enhanced the biogenesis of lysosomes (Fig. S4 C, D). Besides, SB203580 also increased the level of TFEB in SNpc of mice as detected by IHC (Fig.5 I, J). All these data suggested that SB203580 promotes TFEB-mediated autophagy.
5. MAPK14 interacts with and phosphorylates TFEB at serine 211 Next, we aimed to elucidate the mechanism by which MAPK14 regulates TFEB-mediated autophagy. To evaluate whether TFEB could be a substrate of MAPK14, we rst detected the interaction between MAPK14 and TFEB by co-immunoprecipitation. As expected, immunoprecipitation analysis showed that MAPK14 interacted with TFEB both in BV2 cells and mice brain (Fig.6 A-E). Of note, SB203580 reduced the MAPK14/TFEB interaction that enhanced by SNCA A53T aggregation (Fig.6 B-E).
To identify the regulatory function of MAPK14 on TFEB, we examined the role of MAPK14 on the interaction between TFEB and14-3-3 proteins. As phosphorylation of TFEB at serine 211 by mTORC1 has been proved to promote TFEB binding to 14-3-3, we detected the level of TFEB phosphorylation with a phospho-Ser211 speci c antibody. It showed that SB203580 reduced the TFEB phosphorylation at serine 211 (Fig.6 F, G). To explore the mechanism by which MAPK14 regulates TFEB's function, we analyzed the effect of overexpression kinase-dead MAPK14 mutant on TFEB's nuclear translocation. Compared with MAPK14 WT, kinase-dead MAPK14 overexpression increased the nuclear translocation of TFEB (Fig.6 H, I) and enhanced the biogenesis of lysosome (Fig.6 J, K). Meanwhile, kinase-dead MAPK14 overexpression declined the levels of NLRP3, ASC, C-CASP1 detected by WB (Fig.6 L, M). To further con rm the effects from p38alpha-MAPK (MAPK14), we analyzed the effect of MAPK14 siRNA. As gure S 5 shown, MAPK14 siRNA increased the biogenesis of lysosome (Fig. S 5A-B). Also, MAPK14 siRNA decreased the levels of NLRP3, C-CASP1 detected by WB. (Fig. S 5 C, D). These results supported that MAPK14 activation suppresses TFEB function via phosphorylating TFEB at serine 211 and inhibiting TFEB nuclear translocation.
These nding including the interaction between MAPK14 and TFEB , the inhibitory effects of MAPK14 inhibitor and the activation of NLRP3 in ammasome regulated by autophagy, raise the possibility that the MAPK14 may curb TFEB to increase NLRP3 in ammasome activation. To test the hypothesis, we rstly overexpressed TFEB in SNCA-A53T BV2 cells. The results showed that TFEB overexpression reduced NLRP3 and cleaved CASP1 as detected by Western blotting (Fig. S 6 A, B). To examine whether MAPK14 activates NLRP3 via regulating TFEB, we compared the effects of SB203580 treatment, TFEB knockdown, and both SB203580 treatment and TFEB knockdown in SNCA-A53T BV2 cells. Remarkably, the overactivation of NLRP3 in ammasome and cleaved CASP1 resulted from SNCA-A53T was almost completely reversed by SB203580, while TFEB knockdown eliminated the effect of SB203580 (Fig. S 6 C,  D). Similarly, the effect of SB203580 in IL-1β, IL-18 were abrogated in SNCA-A53T BV2 cells with TFEB knockdown (Fig. S 6 E-F). To further test whether the autophagy/lysosome pathway is responsible for MAPK14 inhibition induced NLRP3 degradation, we treated SNCA-A53T BV2 cells with autophagy/lysosome inhibitor CQ (10 µM, 4 h). The results showed that autophagy/lysosome inhibitor CQ blocked SB203580-induced NLRP3 degradation (Fig. S 6 G, H). These results supported that MAPK14 activates NLRP3 in ammasome via disturbing TFEB-mediated autophagy.

SNCA-induced NLRP3 activation in microglia promoted dopaminergic neuronal loss
To examine whether microglial activation is responsible for the death of dopaminergic neurons, SN4741 cells were treated with conditioned medium form microglia. After appropriate treatments, Cell death in SN4741 cells were determined by ow cytometry. As gure 7 A-B shown, conditioned medium form A53T SNCA microglia caused dopaminergic neuronal loss, while NLRP3 inhibitor MCC950 (10 µM, 12h) and MAPK14 inhibitor SB203580 can effectively eliminate the effect.
To address whether NLRP3 activation is involved in dopaminergic neuronal loss in vivo, SNCA A53T -tg mice were fed with NLRP3 inhibitor MCC950 starting at the age of 5 months until the end of 9 months (Fig.S7 A). Interestingly, MCC950 signi cantly inhibited the activation of microglia in SNCA A53T -tg mice, which suggests that NLRP3 plays a key role in the activation of microgliain PD (Fig7 C-D) . Then, we analyzed the levels of TH in SNpc by WB and IHC. The results showed that MCC950 signi cantly reduced the levels of dopaminergic neuronal loss in SNpc of SNCA A53T -tg mice (Fig. 7 E-H). Furthermore, Balance beam test and pole test showed that MCC950 treatment signi cantly improved the motor activity in SNCA A53T -tg mice (Fig. S 7 B, C).
7. SB203580 plays a protective role in SNCA A53T -tg mice The above data showed that MAPK14-TFEB pathway is involved in NLRP3 in ammasome activation, which prompts us to test whether MAPK14 inhibitor SB203580 has a protective role in SNCA A53T -tg mice.
Wild type and SNCA A53T -tg mice were treated with or without the MAPK14 inhibitor SB203580. Balance beam test and Pole test showed that SB203580 treatment signi cantly improved the motor activity in SNCA A53T -tg mice (Fig.8 A, B). In addition, abnormal accumulation of SNCA-A53T could be alleviated by MAPK14 inhibition (Fig.8 C-F). Synapse loss correlates with cognitive impairment in neurodegenerative diseases including AD and PD 15,16 . To examine the protective effect of MAPK14 inhibition, we detected the presynaptic proteins synapsin-1 (SYN1) in SNCA A53T -tg mice. The results showed that SB203580 rescues the reduced level of SYN1 in SNCA A53T -tg mice (Fig.8 G, H). Under TEM, SNCA A53T -tg mice have fewer synaptic vesicles, while SB203580 treatment partially restores synaptic vesicle loss (Fig.8 I, J).
Taken together, these data indicated that MAPK14 inhibition provides protection in SNCA A53T -tg mice.

Discussion
Parkinson Disease (PD) is a common, age-related neurodegenerative disease a icting more than 2-3% of people over the 65 and more than 4% of the population by the age of 85. The progressive loss of dopaminergic neurons in the substantia nigra (SN) eventually results in motor alterations of PD. The precise mechanism underlying the pathogenesis of PD is not yet been fully elucidated. Previous studies shown the defect of autophagy-lysosomal pathway (ALP) and neuroin ammation are contributor in the PD pathophysiology. Accumulating evidence suggests that immune signaling cascades are regulated by autophagy, while the mechanisms that modulate the in ammasome activation process is still unclear. In our study, NLRP3 was identi ed as a novel chaperone-mediated autophagy (CMA) substrate and activation of CMA promotes NLRP3 degradation and thereby inhibits overproduction of proin ammatory cytokines in microglia. Moreover, we demonstrated that MAPK14 activation caused by SNCA A53T inhibits TFEB-mediated CMA, which promotes NLRP3 in ammasome activation. Furthermore, we found that both MAPK14 and NLRP3 inhibitor can reduce SNCA-induced microglia activation and dopaminergic neuronal loss. Therefore, MAPK14-TFEB pathways regulates NLRP3 degradation via CMA in SNCAinduced microglia activation, which promotes dopaminergic neuronal loss.
The chronic neuroin ammation in PD has been intensively investigated in the past decade, but the precise origin of the CNS in ammation response remains unclear. Evidences found that neuroin ammatory response is a key factor in the pathological process of PD 17 . Activated microglia and increased levels of pro-in ammatory factors were identi ed in postmortem analysis and PD models.
SNCA abnormal aggregation can appears in neuron and microglia, which not only disrupts neuronal function, also causes activation of microglia that increase phagocytic activity and pro-in ammatory cytokines production 18 . It is still controversial whether SNCA can trigger microglia responses and pathological reaction. Our results not only identi ed that NLRP3 was a novel CMA substrate, but also revealed that MAPK14 activation caused by SNCA inhibits TFEB activity that blocked CMA, and then decreased NLRP3 degradation in microglia. Studies found that SNCA abnormal aggregation activated microglia, which led to death of dopaminergic neurons in the substantia nigra of the midbrain 19 . Toll-like receptor 2 (TLR2) of microglia was identi ed as the receptor for secreted SNCA, which transport SNCA into microglial cytoplasm 20 . Overexpression of α-synuclein increases microglial activation in vivo.
However, SNCA internalization still appears when TLR2 is de cient, suggesting α-synuclein uptake relies on multiple receptor systems 21 .
The activation of NLRP3 in ammasome usually requires upregulation of the transcription of NLRP3 and pro-IL-1β by NF-κB. However, recent studies report that NLRP3 in ammasome can be regulated independently of transcription. For instance, lys-63-speci c deubiquitinase BRCC36 promotes NLRP3 deubiquitination and facilitates NLRP3 activation. Previous in vitro study showed that NLRP3 in ammasome is activated by pathologic SNCA. In this work, we revealed that the degeneration of NLRP3 through CMA is an essential event for the regulation of NLRP3 in ammasome. Our study demonstrated that MAPK14-TFEB pathway regulates NLRP3 in ammasome in microglia which is involved in the pathological process of PD. Our data are in line with the previous study demonstrating the protective effect of MAPK14 inhibition and TFEB in PD models 11,22 . Several studies revealed that MAPK14 activation is closely connected to autophagy dysfunction. Mao and colleagues identi ed that MAPK14 regulates macroautophagy and CMA via phosphorylating ULK and LAMP2A respectively 23,24 . Chen et al.
reported that MAPK14 inhibition promotes mitophagy 11 . And recent studies have shown that autophagy plays a key role in in ammasome regulation 25 . Notably, autophagy dysfunction disrupts cell homeostasis thus induces excessive activation of in ammasomes. This study provided evidence showing that MAPK14/TFEB-mediated CMA regulating the NLRP3 in ammasome. Although we identi ed the role of MAPK14 in activation of NLRP3 in ammasome in SNCA A53T mice, the mechanism may be applicable in other neurodegenerative diseases.

Conclusion
In conclusion, we uncovered a novel NLRP3 degradation pathway by CMA in microglia, which is negative regulation by MAPK14 through direct phosphorylating TFEB at ser211, inhibits the transcription of autophagy gene. Moreover, MAPK14 inhibitor SB203580 provided protection via enhancing TFEBmediated CMA and reducing NLRP3 in ammasome activation both in vitro and vivo. Given that neuroin ammatory response is deeply involved in neurodegenerative diseases, the mechanisms we identi ed here shed new light on the neurodegenerative process. In addition, the MAPK14-TFEB mediated CMA may be a potential therapeutic target for PD.

Consent for publication
All authors have approved of the contents of this manuscript and provided consent for publication.

Availability of data and materials
All data in this study are included in this published article and its supplementary information les. SNCAA53T-tg and wild type mice. *p<0.05 (Student's t-test). (E-G) Cell lysates from the cortex and SNpc of 9 months old SNCAA53T-tg or wild type mice were immunoblotted. The protein levels of NLRP3, ASC, cleaved CASP1, IL-1β were statistically analyzed in F and G. Mean ± SEM, n = 6, *p < 0.05(Student's ttest).  NLRP3 is degraded through CMA (A) Brain lysates from mice were used for IP using anti-NLRP3 or anti-LAMP2A antibody. (B, C) Cell lysates from BV2 cells were used for IP with NLRP3. SB203580 increased the NLRP3/LAMP2A and NLRP3/ HSPA8 interaction and showed in C. (D, E) Cell lysates from BV2 cells were used for IP with anti-Flag antibody. Wild type NLRP3-Flag but not NLRP3AA-Flag was coimmunoprecipitated with HSPA8. (F, G) Cell lysates from BV2 cells were immunoblotted to determine the levels of LAMP2A and NLRP3 after starvation. The protein levels of LAMP2A and NLRP3 were statistically analyzed in I and J. Mean ± SEM, n = 3, *p < 0.05. (H, I) Cell lysates from BV2 cells were immunoblotted to determine the levels of LAMP2A and NLRP3 after treatment with AR7 and QX77. The protein levels of LAMP2A and NLRP3 were statistically analyzed in L. Mean ± SEM, n = 3, *p < 0.05. (J, K) IHC demonstrating decreased LAMP2A protein levels were rescued by SB203580 in the SNpc of SNCAA53T-tg mice. Statistical analysis of the scores of LAMP2A staining were shown in N. *p < 0.05.