Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway

Microglia are resident immune cells in the brain and are closely associated with central nervous system in�ammation and neurodegenerative diseases. It is known that mTOR pathway plays an important role in the polarization of microglia. Castor1 has been identi�ed as the cytosolic arginine sensor for the mTORC1 pathway, but the role of Castor1 in microglial polarization is still unknown. The purpose of this study was to explore the regulatory effect of Castor1 on microglial polarization and the underlying mechanism. The results demonstrated that Castor1 expression was signi�cantly decreased in LPS and IFN γ treated microglia. Castor1 overexpression inhibited the microglia M1 polarization by reducing the expression of M1 related markers. However, microglia M2 polarization was promoted when Castor1 was overexpressed in IL-4 treated microglia. Mechanistically, Castor1 overexpression inhibited the activation of mTOR signaling pathway. In addition, after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, indicating that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway. We propose that Castor1-mTOR signaling pathway could be considered as a potential target for treatment and intervention of central nervous system-related diseases by regulating microglia polarization.


Introduction
Microglia, the residential cells of the brain, has been proven to be the central nervous system (CNS) sentinels, maintaining normal brain functionality (Colonna and Butovsky, 2017).In brain, microglia presents as different morphologies: radially branched, compact circular and longitudinally branched (Lawson et al., 1990).Microglia participate in two essential functions involving homeostasis and host defense mechanisms (Hickman et al., 2018).The rst function of microglia is screening environmental stimuli and protecting against it, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Glass et al., 2010; Hickman et al., 2013;Stephenson et al., 2018).The second function refers to the physiological house-guarding function, including migrating towards impaired sites, reshaping synapses, and preserving myelin homeostasis (Hickman et al., 2018;Zhan et al., 2014).The microglia functions above are achieved through the heterogeneous activation of microglia in CNS, and the activation can be categorized into M1 and M2 phenotype, which present totally different toxicity on CNS (Tang and Le, 2016).Under the stimulation of LPS and IFN-γ, microglia were activated as M1 phenotype, releasing M1-related markers CD86, iNOS, and M1-related in ammatory cytokines IL-1β, IL-6, TNF-α and MCP-1.In response to the stimulation of IL-4, IL-10 and IL-13, microglia activate to the M2 phenotype, releasing M2-related markers ARG-1 and CD206 as well as m2-related antiin ammatory cytokines TGF-β and IL-10 (Li et al., 2021a).The functional differences between M1 and M2 microglias are closely related to central nervous system diseases, so it is very important to regulate the M1/M2 balance of microglia.Du  enhanced neurogenesis in the hippocampus, and improved functional recovery after traumatic brain injury (Yang et al., 2019).It has also been suggested that chitinase 1 May play a protective role in Alzheimer's disease by promoting the polarization of microglia into the M2 phenotype (Xiao et al., 2017).
Therefore, studying the mechanism of regulating M1 and M2 balance of microglia is of great signi cance for seeking treatment and improving central nervous system injury and related diseases.
Castor1, the GATOR2-interacting cellular arginine sensor of mTORC1, is located on human chromosome 2, which acting in parallel with SLC38A9 to regulate arginine sensing through mTORC1 (Chantranupong et al., 2016).By studying the crystal structure of Castor1, it was found that a ne clipping pocket was carved between the NTD and CTD domains of Castor1 to recognize arginine, revealing the molecular mechanism by which Castor1 senses arginine (Gai et al., 2016).Previous studies have shown that Castor1 is involved in the development of various diseases through the mTOR pathway.Zhou et al. found that Castor1 regulates the progression of lung adenocarcinoma by inhibiting phosphorylation of mTOR and its downstream S6K (Zhou et al., 2018a).Li et al. demonstrated that Castor1 overexpression attenuated mTORC1 activation and thus inhibited cell proliferation of KSHV-transformed cells (Li et al., 2019).However, no studies have been reported in investigating the regulatory effect of Castor1 on microglia polarization.
Mammalian target of rapamycin (mTOR) is an important regulator of cell growth, metabolism and proliferation, and is a catalytic subunit of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Saxton and Sabatini, 2017).The mTOR signaling pathway regulates innate and adaptive immune responses, and one of the main functions of mTORC1 is to regulate downstream p70S6 Kinase(p70S6K) and eukaryotic initiation factor 4E binding protein (4EBPs) to regulate mRNA translation, thus regulating immune responses and microglial in ammatory responses (Keane et al., 2021).Many studies have shown that mTOR signaling pathway is involved in the regulation of microglia polarization, thus affecting the occurrence and development of central nervous system-related diseases.Zhuang et al. found that hydrogen treatment inhibited M1 polarization of microglia and promoted M2 polarization by inhibiting the mTOR pathway, playing a protective role in the brain (Zhuang et al., 2020).Wang et al. 's study showed that Salidroside treatment of microglia reduced the phosphorylation of mTOR and P70S6K, thus participating in the polarization regulation of microglia (Wang et al., 2018).Zhao et al showed that the loss of mTOR in microglia reduced the expression of M1-related in ammatory cytokines, such as TNF-α and IL-1β, and exacerbated the loss of neurons (Zhao et al., 2020).However, the functional role of Castor1 in mTOR mediated microglial polarization remains unclear.
In our study, we found that the expression of Castor1 was reduced in M1-polarized microglia induced by LPS and IFN-γ treatment.In addition, Castor1 overexpression inhibited M1 polarization and promoted M2 polarization of microglia.Further studies demonstrated that Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway.Taken together, this study provides a new method for regulating the polarization characteristics of microglia, and also provides a potential therapeutic target for diseases caused by abnormal polarization of microglia.

Quantitative real-time PCR
Total RNA was extracted from cells using Trizol reagent (TaKaRa).According to the manufacturer's instructions, Total RNA was reverse transcribed into cDNA using the First-Strand cDNA Synthesis Kit (Applied Biological Materials).SYBR Green Master Mix (Roche) was used for the real-time PCR reactions of the synthesized cDNA in LightCycler 480 system (Roche).The primers used in this study were shown in Supplementary Table 1.Relative gene expression was analyzed by normalization of the cycling threshold (CT) values against the β-actin using the 2 −ΔΔCT method.

Statistical analysis
All experimental data were presented as the mean ± SEM.An unpaired two-sided Student's t test was used to analyze the signi cance between two experimental groups.GraphPad Prism (GraphPad Software) was used to process the data, and P values < 0.05 were considered statistically signi cant.

The Expression of Castor1 was Downregulated in M1 microglia polarization
To investigate the expression and the role of Castor1 in M1 microglia polarization, BV2 cells were treated with 100 ng/mL LPS and 10 ng/mL IFN-γ for 12 h.The expression of M1-related markers and Castor1 was detected by quantitative real-time PCR and western blotting.As expected, compared with the control group, the expression of M1-related markers such as CD86 (P < 0.05), iNOS (P < 0.01), IL-β (P < 0.01), IL-6 (P < 0.01), TNF-α (P < 0.001), MCP-1 (P < 0.001), were signi cantly increased in the LPS/IFN-γ group.And the the expression of Castor1 (P < 0.001) was downregulated in M1 microglia polarization conditions (Fig. 1).These results suggested that Castor1 had a potential role in regulating microglia polarization.

Castor1 overexpression promoted microglia polarization to the M2 phenotype
Microglia will polarize into M1 type or M2 type when confronted with stimulation.These two activated subtypes have different morphology and opposite functions.Since Castor1 overexpression could inhibit M1 polarization, what is the effect of Castor1 overexpression on M2 polarization?To investigate the effect of Castor1 overexpression on M2 polarization, BV2 cells were treated with 20 ng/mL IL-4 for 12 h after they were transfected with pcDNA3.1-vectoror pcDNA3.1-Castor1plasmid for 12 h.Compared with the pcDNA3.1-vecorgroup, the microglia M2 polarization was promoted when Castor1 was overexpressed, as the expression of Arg1 (P < 0.01), CD206 (P < 0.05) and IL-10 (P < 0.05) was signi cantly increased (Fig. 3A, B).And the immuno uorescence staining results further con rmed that Castor1 overexpression promoted the expression of Arg1 and CD206 (Fig. 3C).These results suggested that Castor1 overexpression promoted microglia polarization to the M2 phenotype

Castor1 overexpression inhibited mTOR pathway
Previous studies have shown that Castor1, as an upstream regulatory molecule of the mTOR signaling pathway, plays an important role in the pathogenesis of disease.To investigate the effect of Castor1 overexpression on mTOR pathway, BV2 cells were transfected with pcDNA3.1-vecoror pcDNA3.1-Castor1for 12 h and then treated with LPS/IFN-γ for 12 h.And then expression of some key proteins involved in the mTOR pathway was detected, such as mTOR, P70S6K and 4E-BP1 and their respective phosphorylated forms.Results demonstrated that the phosphorylation levels of mTOR (P < 0.05), 4E-BP1 (P < 0.05) and P70S6K (P < 0.05) were signi cantly reduced after overexpression of Castor1 (Fig. 4).The overall results indicated that Castor1 overexpression inhibited the phosphorylation of key proteins involved in the mTOR signaling pathway, thus inhibiting the activity of the mTOR pathway activation.

The regulation of Castor1 on M1 polarization depends on the suppression of the mTOR signaling pathway
Since Castor1 overexpression inhibits the polarization of M1 and also inhibits the activity of the mTOR signaling pathway, we speculated that the regulation of Castor1 on M1 polarization depends on the suppression of the mTOR signaling pathway.To con rm this hypothesis, an experimental group was added on the basis of the previous experiment, namely, microglia were transfected with pcDNA3.1-Castor1for 12 h and treated with mTOR activator (MHY1485, 5 uM) at the same time as being treated with LPS + IFN-γ for 12 h.The results shown that after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, as the expression of M1related markers such as CD86 (P < 0.05), iNOS (P < 0.001), IL-β (P < 0.001), IL-6 (P < 0.05), TNF-α (P < 0.001) and MCP-1 (P < 0.01) were signi cantly increased compared with pcDNA3.1-Castor1 group (Fig. 5).All these results indicated that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway.

Discussion
Microglia polarize into different phenotypes (mainly M1 type and M2 type) in response to various stimulus and participate in the occurrence and development of diverse central nervous system diseases (Crain et al., 2013;Subhramanyam et al., 2019).It has been found that the mTOR pathway plays an important regulatory role in microglia polarization (Srivastava et al., 2016).However, the role of Castor1 as an upstream regulatory molecule of mTOR signaling pathway in microglia polarization has not been reported.In this experiment, we found that Castor1 overexpression can inhibit M1 polarization and promote M2 polarization, and Castor1's regulation of M1 polarization depends on the inhibition of mTOR signaling pathway.Microglia, as inherent immune cells in the central nervous system, play an important role in the occurrence and development of a series of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease (Zhou et al., 2018b), multiple sclerosis (Benveniste, 1997).In addition, they are also closely related to central nervous system injury, ischemia, in ammation (Wang et al., 2015).Microglia can be divided into in ammatory microglia (M1 type) and anti-in ammatory microglia (M2 type) under different states of activation.M1 microglia mainly express INOS, CD86 and other surface markers, which often induce in ammation and nerve injury (Nguyen et al., 2017).M2 microglia mainly express ARG1, CD206 and other surface markers mainly play the role of nerve repair, induction and anti-in ammatory (Colonna and Butovsky, 2017).Previous studies have shown that M1/M2 balance of microglia plays an important role in maintaining central nervous system homeostasis and preventing neurodegenerative diseases (Song et al., 2016).It has been proved that α -synuclein induced polarization of microglia M1 could amplify neuronal damage and aggravate motor de cits in Parkinson's disease (Du et al., 2018; Tang and Le, 2016).Mirren et al. showed that in the early stage of multiple sclerosis, microglia polarize into the M1 phenotype, leading to neuronal demyelination and nerve damage.In the later stage of multiple sclerosis, microglia polarize into the M2 phenotype, releasing anti-in ammatory factors and promoting myelin regeneration (Miron et al., 2013).Hu X et al. reported that microglia M1 polarization increased during in ammation after ischemic stroke, which expanded the scope of ischemic infarction and further worsened nerve injury (Hu et al., 2015;Hu et al., 2012).Therefore, inhibiting M1 polarization of microglia and promoting M2 polarization of microglia may be a new therapeutic target.Our study induced M1 polarization of microglia using LPS + IFN-γ and found a decrease in Castor1 expression, which has not been previously reported.In order to investigate the effect of Castor1 on microglia polarization, we constructed a pcDNA3.1-Castor1overexpression plasmid vector.After LPS + IFN-γ treatment, Castor1 overexpression inhibited the expression of M1-related markers such as CD86 and INOS.Under The treatment of IL-4, Castor1 overexpression promoted the expression of M2-related markers Arg1 and CD206.This suggested that Castor1 overexpression inhibited M1 polarization and promoted M2 polarization of microglia.
Castor1 is a cytoplasmic arginine sensor consisting of four tandem ACT domains that dissociate from GATOR2 after binding to arginine, thereby activating the mTORC1 signaling pathway and playing an important regulatory role in the occurrence of a variety of diseases (Saxton et al., 2016;Zhuang et al., 2019).Li et al. rst reported that Castor1 could inhibit cell proliferation and transformation and induce cell cycle arrest (Li et al., 2019).Another study have shown that Castor1 has a tumor suppressor function on breast cancer cells, and the activation of Castor1 can be considered as a potential treatment for some cancers (Li et al., 2021b).However, the regulatory effect of Castor1 on microglia has not been reported.mTOR is a serine/threonine protein kinase, belonging to the PI3K-related kinase family, which is regulated by a variety of cell signals and plays a key role in cell proliferation, metabolism, immune response and other processes, and is also closely related to the occurrence of many diseases (Saxton and Sabatini, 2017).It has been reported that mTOR signaling pathway plays a very important role in regulating M1/M2 polarization of microglia.The study of Hu et al. showed that LPS could induce rapid activation of mTOR signaling pathway in microglia.On the contrary, inhibition of the mTOR signaling pathway reduces LPS-induced microglial proin ammatory cytokines (Hu et al., 2020).Li et al. reported that speci c blocking of mTORC1 signaling pathway can reduce LPS-induced M1 Maker expression and increase M2 Maker expression (Li et al., 2016).Therefore, we speculated that the inhibition of M1 polarization in microglia by Castor1 overexpression may depend on its inhibition of mTOR signaling pathway.Thus, the expression of some key proteins involved in the mTOR pathway was detected, such as mTOR, P70S6K and 4E-BP1 and their respective phosphorylated forms.Results demonstrated that the phosphorylation levels of mTOR, 4E-BP1 and P70S6K were signi cantly reduced after overexpression of Castor1.This suggests that Castor1 overexpression inhibits the activity of mTOR signaling pathway in M1 polarization of microglia.In order to verify that Castor1 overexpression inhibits mTOR signaling pathway and thus inhibits M1 polarization of microglia, we treated microglia with mTOR activator MHY1485 and found that after the mTOR signaling pathway was activated, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, as the expression of M1-related markers such as CD86, iNOS, IL-β, IL-6, TNFα, and MCP-1 were signi cantly increased.This indicates that the activation of mTOR signaling pathway antagonizes the inhibitory effect of Castor1 overexpression on microglia M1 polarization, suggesting that the inhibition of Castor1 overexpression on microglia M1 polarization depends on the inhibition of mTOR signaling pathway.

Conclusions
In summary, our study demonstrated that the expression of Castor1 was signi cantly decreased in LPS/IFN-γ treated microglia, and the overexpression of Castor1 inhibited LPS + IFN-γ-induced M1 polarization and promoted IL-4 Induced microglial M2 polarization.The regulatory effect of Castor1 on M1 polarization depends on its inhibition of mTOR signaling pathway and can be antagonized by mTOR signaling pathway activator MHY1485.Therefore, Castor1's regulation of M1/M2 polarization of microglia may make it a potential therapeutic target for neurodegenerative and neuroin ammationrelated diseases.
et al. found that Ki20227 improved neurobehavioral function, reduced cerebral infarction size and played a neuroprotective role by inhibiting M1 polarization of microglia and activating M2 polarization inhibited by NLRP3 pathway (Du et al., 2020).Yang et al. found that Exo-miR-124 treatment promoted M2 polarization of microglia,

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