Involvement of LARP7 in Activation of SIRT1 to Inhibit NF-κB Signaling Protects Microglia from Acrylamide-Induced Neuroinflammation

Acrylamide (AM) is a potent neurotoxin and carcinogen that is mainly formed by the Maillard reaction of asparagine with starch at high temperatures. However, the toxicity mechanism underlying AM has not been investigated from a proteomic perspective, and the regulation of protein expression by AM remains poorly understood. This research was the first to utilize proteomics to explore the mechanism of AM exposure-induced neuroinflammation. Target proteins were obtained by differential protein analysis, functional annotation, and enrichment analysis of proteomics. Then, molecular biology methods, including Western blot, qPCR, and immunofluorescence, were used to verify the results and explore possible mechanisms. We identified 100 key differential metabolites by proteomic analysis, which was involved in the occurrence of various biological functions. Among them, the KEGG pathway enrichment analysis showed that the differential proteins were enriched in the P53 pathway, sulfur metabolism pathway, and ferroptosis. Finally, the differential target protein we locked was LARP7. Molecular biological verification found that AM exposure inhibited the expression of LARP7 and induced the burst of inflammation, while SRT1720 agonist treatment showed no effect on LARP7, but significant changes in inflammatory factors and NF-κB. Taken together, these findings suggested that AM may activate NF-κB to induce neuroinflammation by inhibiting the LARP7-SIRT1 pathway. And our study provided a direction for AM-induced neurotoxicity through proteomics and multiple biological analysis methods.


Introduction
Acrylamide (AM), a vinyl monomer, is a well-recognized potent neurotoxin affecting the nervous system (Song et al. 2017). It can be formed through a heat-induced reaction of asparagine as a source of an amino group and the reducing sugars as a carbonyl group source via the Maillard reaction (Cantrell and McDougal 2021). With the participation of cytochrome p4502e1, AM biotransforms in the liver into glycidamide in the form of epoxide (Hogervorst et al. 2021). AM and glycidamide are neurotoxic (Hogervorst et al. 2021). Acrylamide can damage the brain and spinal cord neurons of zebrafish, causing movement disorders (Komoike and Matsuoka 2019). There are also accumulating reports revealing that AM can inhibit neurotransmitter transmission and thereby inhibit nerve conduction (Kopanska et al. 2018). Given the increasing incidence and prevalence of acrylamide toxicity, elucidating the pathogenesis and identifying the optimal treatment modality for acrylamide-induced peripheral neuropathy is essential.
Neuroinflammation is an important marker of neurotoxicity, and its physiological manifestation is the production of a large number of inflammatory factors under noxious stimuli. Studies have successively found that the levels of inflammatory factors such as interleukin-1β (IL-1β) and interleukin-18  in the cerebral cortex and hippocampus of AM-poisoned animals were significantly higher, and were related to the severity of nerve damage (Santhanasabapathy et al. 2015;Zong et al. 2019). IL-1β is a major regulator of neuroinflammation in the central nervous system (Basu et al. 2004). It can induce other cytokines such as IL-6 and TNFα, further aggravating inflammation (Palomo et al. 2015). IL-1β and IL-6 are involved in the pathogenesis of various neurological diseases (Jin et al. 2020), including viral and bacterial meningitis, multiple sclerosis, Alzheimer's disease, and traumatic brain injury (Campbell et al. 1993).
Sirtuin1 (SIRT1) is a nicotinamide adenosine dinucleotidedependent deacetylase that can regulate DNA expression, apoptosis, and senescence by deacetylating substrate proteins, and participating in physiological or pathological processes (Jiao and Gong 2020). SIRT1 is widely expressed in mouse and human neurons, and studies have shown that SIRT1 is distributed in the hippocampus, prefrontal cortex, and basal ganglia regions (Ramadori et al. 2008;Zakhary et al. 2010). SIRT1 can regulate the inflammatory response of various tissues and cells and is closely related to neuroinflammation (Mendes et al. 2017;Zhang et al. 2020b). SIRT1 can deacetylate the NF-κB subunit p65, and the deacetylation of p65 is suspected to be involved in the inflammatory response, suggesting that the potential regulatory mechanism of SIRT1 in neuroinflammation may involve the NF-κB pathway. As a nuclear transcription factor, NF-κB can regulate the expression of cytokines related to immune, inflammatory, and anti-apoptotic responses. More importantly, NF-κB has been widely regarded as the dominant factor regulating the expression of inflammatory genes. Nuclear translocation of NF-κB in the cytoplasm can induce the production of inflammatory cytokines and trigger the inflammatory response (Guzman et al. 2013), while a large number of inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, can trigger and amplify local inflammation response, which in turn leads to neuroinflammation.
La ribonucleoprotein domain family member 7 (LARP7), a La-related RNA binding protein, interacts with its main target non-coding 7SK RNA and promotes its stability (Uchikawa et al. 2015) (Fig. 1). The evolutionarily conserved LARP7 plays a multifaceted regulatory role in the gene expression of health and disease by integrating into different RNPs. LARP7 regulates the transcription of RNA polymerase 2 (pol II) by banding 7SK snRNA and acts as a cofactor to promote the methylation of the ribose 2′ hydroxyl group methylation (2′-O-methylation) of the spliceosomal U6 snRNA (U6) (Sacks et al. 2018). Decreased expression levels of LARP7 and loss of function caused by gene mutations are related to cancer and Alazami syndrome. It has been reported that LARP7 is associated with degenerative cognitive impairment diseases (Najmabadi et al. 2011). Researchers found enrichment of LARP7 in the nucleoli of rat neurons, and in hippocampal neurons, decreased LARP7 can also reduce the content of perinuclear ribosomes and protein synthesis, while knocking down LARP7 can increase neutrophil inflammation (Hoodless et al. 2016;Maraia et al. 2017). The above studies suggested that LARP7 may have a potential protective effect on tissue damage caused by inflammation. LARP7 is a master regulator controlling the DNA damage response and RNA polymerase II pausing pathways, but its role in the pathogenesis of inflammation is not fully understood (Krueger et al. 2008;Zhang et al. 2020a).
Taken together, in order to reveal the protective effect of LARP7 in neuroinflammation and further explore the neurotoxicity mechanism of AM, our previous study explored the relationship between AM toxicity and inflammation from the perspective of metabolomics (Zhao et al. 2021). The study found that AM exposure inhibited LARP7 expression and altered SIRT1 and NF-κB. Based on a large number of literature reports and previous research results , our group hypothesized that the molecular mechanism of AM-induced neuroinflammation may be the activation of NF-κB by inhibiting the LARP7-SIRT1 pathway ( Fig. 1).

Cell Culture and Drug Treatment
The mouse normal brain cell line BV2 was purchased from the Cell Bank of the Chinese Academy of Sciences. All cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin G, and 100 μg/ml streptomycin. The cell incubator was maintained at 37 °C with a humidified 5% CO 2 atmosphere. BV2 cells were treated with 2 mM concentration of AM for 24 h as previously reported (Liu et al. 2015). Subsequently, cells were treated with 5uM concentrations of SRT1720 for 2 h (Cui et al. 2021).

Protein Extraction and Quality Inspection
Cells were collected after 24 h of AM treatment, and the cells were immediately frozen in negative 80 refrigerator for future use, including the control group (without ACR treatment). All samples were taken out under frozen condition and placed on ice; appropriate amount of protein lysate (8 m urea, 1% SDS, including protease inhibitor) was added; after ultrasonic lysis on ice, protein supernatant was extracted by centrifugation; BCA protein was quantified using standard protein provided by Thermo Scientific Pierce BCA kit, and finally SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) electrophoresis was used to evaluate whether the quality of the sample met the requirements of follow-up experiments.

Protein Pretreatment
Reductive alkylation and enzymatic hydrolysis of qualified protein samples were carried out. Triethyl ammonium bicarbonate buffer and tris-(2-carboxyethyl) phosphine were added to the 100 μg protein sample so that the final concentration was 100 mm to react 60 min at 37 ℃, then 40 mM iodoacetamide was added to avoid light for 40 min at room temperature. Then precooled acetone (acetone:sample v/v = 6:1) was added to precipitate 10000 g centrifuge. Trypsin was added and hydrolyzed overnight at 37 ℃, the polypeptides were obtained. The peptides were labeled with TMT reagent (ThermoFisher) added with acetonitrile and hydroxylamine. And a tube of TMT reagent was added to every 100 μg polypeptides. Each group of medium amounts of labeled products was mixed in a tube and drained by a vacuum concentrator.

Separation and Analysis of Peptides
A reversed-phase C18 column was used for high pH liquid phase separation of polypeptide samples redissolved in UPLC sample buffer (2% acetonitrile). The fraction was collected according to peak shape and time, concentrated by vacuum centrifugation, dissolved with mass spectrometry sample buffer (2% acetonitrile and 0.1% formic acid), and analyzed by liquid-phase tandem mass spectrometry (liquid chromatography coupled with tandem mass spectrometry).

Fig. 1
Possible neuroprotective mechanism of LARP7 against acrylamideinduced neurotoxicity. Acrylamide inhibits the expression of SIRT1 by down-regulating the expression of LARP7, which prevents SIRT1 from inhibiting the phosphorylation of NF-KB and triggers cytokine release to induce neuroinflammation

Database Search and Data Statistical Analysis
Submit the original raw file off the MS machine to the Proteome Discoverer server, and check the library using the software version of Proteome Discoverer TM Software 2.4. The protein sequences of specified species in NCBI and Uni-Prot databases or other species databases were selected to search the database. The filtering parameters were Peptide FDR ≤ 0.01. The identification information of proteins and peptides after quality control was counted. According to the expression of protein in different samples, the samples were analyzed by correlation analysis and principal component analysis (PCA). Then, according to the identification of mass spectrometry, all proteins and protein sequences were compared with major databases (Uniprot, NR, GO, KEGG, String) and subcellular localization-related databases, and the annotation information of proteins in each database was obtained. Then the expression abundance of the same protein in different samples was obtained through database search and peak analysis. Through the analysis of the differential expression of proteins among samples (n = 3), the differentially expressed proteins among samples were excavated and screened. Fold change (FC) and p-value were selected as the reference criteria for the screening of differential proteins between groups. Set FC ≥ 1.5 or FC ≤ 0.66, p < 0.05 as the filtering parameter for differential proteins. GO enrichment analysis of the protein concentration was carried out by the software Goatools, and KEGGPATHWAY protein concentration enrichment analysis using Megi database. All the methods were accurate Fisher test. Meiji mainly uses STRING database (http:// string-db. org/) to analyze the protein interaction network, and also refers to the protein interaction network relationship in HPRD (http:// www. hprd. org/), biogrid (http:// thebi ogrid. org), REACTOME (http:// www. react ome. org), and other databases. For species with protein interactions in the database, a protein interaction network was constructed, and then networkX under Python was used to visualize the protein network.

Polymerase Chain Reaction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using the TRIzol reagent (Ther-moFisher Scientific, USA) and quantified using a NanoDrop 2000 Spectrophotometer (Thermo Scientific Fisher). Reverse transcription of 2ug of total RNA was performed using Prime ScriptTM 1st Strand cDNA synthesis Kit (TaKaRa, Japan) according to the manufacturer's instructions, then cDNA was subjected to polymerase chain reaction (PCR) amplification and RT-PCR.
Total RNA was extracted with the RNeasy RNA isolation kit (Qiagen) and reverse transcribed to cDNA with the SuperScript First-strand Synthesis System (Thermo Fisher Scientific). The Real-time PCR reactions were performed with Hieff® qPCR SYBR Green Master Mix (Yeasen biotech) using ABI Prism 7500 system. The primer sequences were documented in Table 1.

Immunofluorescence Assay
Cells were seeded into 12-well plates pre-placed on treated coverslips, and drug treatments were administered 24 h after seeding. Cells were subsequently fixed with 4% paraformaldehyde for 20 min, then permeabilized with ice-cold methanol and blocked in 3% BSA. Cells were then incubated with LARP7 antibody overnight at 4 °C followed by secondary

Statistical Analysis
Statistical analysis was performed using the GraphPad PRISM 8 software, and results from the experiment were expressed as means ± SD. Differences between groups were determined by one-way ANOVA test or Student's t-test. P < 0.05 was considered as statistically significant. Statistical analysis of proteomics has been introduced in Proteomics.

Screening and Visualization of Differential Proteins
To further elucidate the mechanism of acrylamide-induced neurotoxicity, we used proteomics for the first time to study the neural effects of AM exposure. Using TMT quantitative proteomics analysis, through sample correlation analysis and PCA, it is known that the correlation of protein composition between samples within a group is high, and the protein variation between samples between groups is relatively large, which provides information for differential protein analysis (Fig. 2a). By setting the parameters, i.e., upregulated protein FC ≥ 1.5, downregulated protein FC ≤ 0.66, and P ≤ 0.05, the differential expression analysis of the proteins between the samples (N = 3) was carried out. And 47 upregulated proteins and 53 downregulated proteins were mined and screened out, which were finally visualized in the form of volcano plots (Fig. 2b).

Creation and Analysis of Differential Protein Sets
Then we created a protein set with the screened differential proteins, and divided it into two protein sets. Function annotation of the differentially expressed proteins was performed to understand the function and bioprocess of the differentially expressed proteins. Among them, the GO functional annotation showed that the differential proteins under AM exposure were mainly enriched in cellular process, cellular anatomical entity, and binding process, while the enrichment degree showed slight differences between upregulated and downregulated proteins (Fig. 2c, d). Next, the KEGG pathway enrichment analysis was performed. Our previous metabolomics study showed that AM significantly affected metabolism, and the enrichment pathway results of upregulated differential protein sets, sulfur metabolism, and drug metabolism validated our study again (Fig. 2e,   f). Concordantly, downregulated proteins were enriched in pathways and cellular locations related to the p53 signaling pathway, ferroptosis, cell cycle, and human T cell leukemia.

Effects of AM on LARP7 and Inflammatory Molecules
Current studies have found that LARP7 plays an important role in the human body as a key protective factor. Proteomic differential protein analysis found that AM exposure resulted in a significant downregulation of LARP7 expression in BV2 cells compared with controls. To further understand the function of LARP7 in the pathogenic mechanism of AM, we detected the protein expression of LARP7 at the protein level by Western blot and RT-PCR. The results were consistent with proteomic studies (Fig. 3a, e) that AM inhibited the expression of LARP7. To verify this guess, SIRT1 was detected by immunoblotting, and found that AM inhibition significantly SIRT1 expression (Fig. 3b, d). To determine whether AM modulated expression of cytokines, mRNA expression of IL-1β, IL-6, IL-18, TNF-α, and iNOS were analyzed by RT-PCR. The results illustrated that AM stimulation resulted in a burst of pro-inflammatory cytokines in BV2 microglia compared to untreated cells (Fig. 3f-j). These results are consistent with those of previous studies (Elblehi et al. 2020).

Effects of SRT1720 on LARP7 and Inflammatory in AM Exposure
In view of the differences in protein expression caused by AM exposure and the conjecture verification results about LARP7 protein, we further studied the mechanism of LARP7. SRT1720, an activator of SIRT1, was used to reverse the SIRT1 expression, and observed upregulated SIRT1 expression levels, respectively (Fig. 4a). Then we examined the effect of activation of SIRT1 on the protein expression of LARP7, and the results showed that activation of SIRT1 did not reverse the expression level of LARP7 under AM treatment (Fig. 4b).
Further to the protein expressions of phosphorylated P65 (P-P65) and P65 were investigated by western blot analysis. Quantification of P-P65/total-P65 ratio revealed substantially increased P-P65 levels in AM exposure (Fig. 4c). Further to confirm the expression of LARP7 in AM exposure and determine its subcellular localization, immunofluorescence was performed in BV2 cells. LARP7 protein was mainly localized in the nucleus of BV2 cells, and immunofluorescence staining analysis showed similar results that LARP7 was not regulated by SRT1720 (Fig. 4d). In contrast, SRT1720 reversed the mRNA expression of inflammatory cytokines in cells exposed to AM (Fig. 4e-i).

Discussion
Neuroinflammation is pathologically associated with many neurodegenerative disorders (Leng and Edison 2021). However, elucidation of the specific mechanism of acrylamideinduced neurotoxicity has been hampered. In biomedical studies, proteomics profiling techniques have proven to be powerful new tools for uncovering complex biological processes, which aid in the exploration of novel mechanisms of disease pathogenesis and project future approaches to personalized medicine (Aslam et al. 2017). In this study, we performed biomolecular studies of AM-treated BV2 cells using proteomics and screened for LARP7 that interacts with RNA polymerase II (Pol II) transcribed noncoding RNA (Hasler et al. 2021). As a nuclear protein, LARP7 has been reported to be involved in various diseases, including cardiac damage and Alazami syndrome. LARP7 function in neuroinflammation has not been previously studied. We initially unveiled in this study that AM exposure resulted in the inhibition of LARP7 expression, accompanied by the These results suggested that neuroinflammation may be the main cause of AM-induced brain injury, and the inhibition of LARP7 may be a key to triggering neuroinflammation.
According to the KEGG enrichment analysis and differential gene expression analysis of proteomics, we learned that AM exposure caused abnormal sulfur metabolism and drug metabolism, and found that downregulated proteins were mainly enriched in the p53 signaling pathway, ferroptosis, cell cycle, and human T cell leukemia virus 1 infection. Previous studies also showed that AM exposure leads to changes in inflammation-related metabolites and metabolic pathways in brain tissue (Zhao et al. 2021). These are the results of differential protein pathway enrichment studies by proteomics. Sulfur metabolism is involved in multiple facets of cellular metabolism related to several responses to stress (Miller and Schmidt 2020). There are multiple lines of evidence for an impaired sulfur amino acid metabolism in autism spectrum disorder and inflammatory diseases. And bisulfite, an end product of sulfur metabolism, abrogated ozone-induced AHR and attenuated ozone-induced neutrophil inflammation in mice (Kasahara et al. 2019). The p53 tumor suppressor gene was among the most frequently mutated genes in human cancer, which suppresses cancer formation through its role in regulating the cell cycle and apoptosis (Huang et al. 2018;Tong et al. 2020). It has been shown that exposure to AM induces development of colon cancer by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis (Zhang 2009). This is one of the main reasons why acrylamide is listed as a carcinogen. One of the other pathways for differential protein enrichment that has to be highlighted is ferroptosis. Most organ damage and degenerative diseases are caused by ferroptosis, and the central nervous system is the most active part of the body's oxygen metabolism, and it is easy to become the main target organ of oxidative damage. Current studies have confirmed that ferroptosis is related to oxidative metabolism (Jiang et al. 2021), and that acrylamide causes nerve damage by inducing and affecting oxidative metabolism (Yilmaz et al. 2017). Neuroinflammation and iron appear to be tightly regulated, as the inflammatory milieu is associated with iron accumulation, which is associated with NDD and neuroinflammation (Fernández-Mendívil et al. 2021). Specifically, acrylamide may cause neuroinflammation by inducing ferroptosis, contributing to triggering neuronal apoptosis. Sulfur metabolism and ferroptosis are directly or indirectly related to the occurrence of inflammation, revealing the mechanism of AM-induced inflammation from a new perspective. The functions of sirt1 are diverse and complex. It mainly interacts with different substrates such as FOXO, PGC-1α, P53, and NF-κB; participates in glucose and lipid metabolism, cardioprotection, and neuroprotection; and exerts its regulatory function on genes (Imperatore et al. 2017). Inflammatory stimuli can induce sirt1 inhibition and transcriptional activation of P53 and P65, leading to apoptosis and neurodegeneration. Although relatively fewer studies of LARP7, several reports have pointed out the important role of LARP7 in the life process.
Through the verification experiments, we can know that the neuroinflammation caused by AM exposure may be related to the inhibition of LARP7 expression. By exploring the expression of P-P65 when LARP7 is inhibited, we can conclude that the lower expression of LARP7 activates the NF-κB inflammatory pathway. SRT1720 can activate sirt1 for neuroprotection (Khader et al. 2017). As we all know, if the activation of sirt1 has no effect on LARP7, it means that sirt1 is not an upstream regulatory protein of LARP7, and LARP7 may regulate the expression of sirt1. This conclusion has now been confirmed (Yu et al. 2021). Cardiomyocyte-specific knockout of LARP7 reduces SIRT1 activity, impairs mitochondrial biogenesis and oxidative respiration, and causes myocardial dysfunction Yu et al. 2021). The treatment results of SRT1720 also reiterated that LARP7 can regulate the protein expression of sirt1. The detection results of mRNA expression levels of cytokines also confirmed that LARP7 mediates the expression of sirt1 to guide the changes of cytokines. Therefore, we speculate that acrylamide can further influence neuroinflammation by downregulating LARP7 and reducing Sirt1 to activate the NF-κB pathway.

Conclusion
Collectively, the present study evaluated the response of LARP7 to AM-induced neuroinflammation, showing that LARP7 regulates the SIRT1/NF-κB signaling pathway to affect neuroinflammation. On the other hand, our data provided a potential mechanism to explore the AM neurotoxicity. We will also further explore the regulatory role of LARP7 in Sirt1/NF-κB, so as to fully understand the molecular mechanism of AM-induced neuroinflammation.
Author Contribution Material preparation, data collection, and analysis were performed by Jinxiu Guo, Hongjia Xue, Wenxue Sun, Shiyuan Zhao, and Junjun Meng. The first draft of the manuscript was written by Jinxiu Guo. Haitao Zhong, and Pei Jiang supervised the study. Fig. 4 Effects of SRT1720 on LARP7 and inflammatory in AM exposure. a SRT1720 reverse the SIRT1 expression after AM treatment. b The effect of SRT1720 on the expression activity of LARP7 after AM treatment. c Administration of SRT1720 after AM treatment affected the relative expression of P-p65. d Immunofluorescence staining was used to detect the expression and localization of LARP7 and the regulation of LARP7 by SRT1720 treatment. e-i SRT1720 reversed the mRNA expression of inflammatory cytokines ◂