Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SH-SY5Y cells induced by Aβ25-35

A progressive neurodegenerative disease, Alzheimer’s disease (AD). Studies suggest that highly expressed protein isoaspartate methyltransferase 1 (PCMT1) in brain tissue. In the current study, we explored the effects of neural stem cell-conditioned medium (NSC-CDM) on the PCMT1/MST1 pathway to alleviate Aβ25-35-induced damage in SH-SY5Y cells. Our data suggested that Aβ25-35 markedly inhibited cell viability. NSC-CDM or Neural stem cell-complete medium (NSC-CPM) had a suppression effect on toxicity when treatment with Aβ25-35, with a greater effect observed with NSC-CDM. Aβ25-35 + NSC-CDM group exhibited an increase in PCMT1 expression. sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through the induction of apoptosis and improved p-MST1 expression. Overexpression of PCMT1 reversed the Aβ25-35-induced decrease in cell proliferation and apoptosis. In summary, our findings suggest that NSC-CDM corrects the Aβ25-35induced damage to cells by improving PCMT1 expressions, which in turn reduces phosphorylation of MST1.


Introduction
AD is a multifactorial disease with multiple risk factors. The pathological mechanisms of AD include the deposition of amyloid β (Aβ), astroglia degeneration, hyperphosphorylation and accumulation of the tau protein, neuronal dystrophy, oxidative stress, mitochondrial dysfunction, biological metal homeostasis, and decreased levels of acetylcholine (ACh) (Butterfield et al., 2001;Reddy, 2011). However, the underline molecular mechanism of AD is still unclear, and a real cure for the disease has not yet been found.
Previous studies have shown highly expressed protein isoaspartate methyltransferase 1 (PCMT1) in brain tissue (Zhu et al., 2001). Many studies have shown that PCMT1 acts a critical effect in the regulation of longevity, different types of oxidative stress, heat shock response, and apoptosis (Aleksandra et al., 2014;Jost et al., 2002). Recent research has shown that short-term brain tissue injury (such as subarachnoid hemorrhage (SAH) or traumatic brain injury (TBI)) can up-regulate PCMT1 activity. The up-regulation of PCMT1 is related to the decrease of neurodegenerative factor levels and the anti-apoptotic effect of neurons, thereby inhibiting cerebral edema (Liang et al., 2017;Shi et al., 2017a).
MST1 is a pre-apoptotic protein and the key protein of the Hippo pathway. Activation of MST1 can induce the Bax expression, inhibit the Bcl-2 expression, and cause Caspase 3 activation and apoptosis (Qin et al., 2015). Previous studies reported that, in HEK293 cells, PCMT1 and MST1 colocalized, which inhibited cell apoptosis, and after administration of MST1 activator, apoptosis was stronger than without administration, while PCMT1 expression remained unchanged (Biterge et al., 2014).
Neural stem cells (NSCs) are progenitor cells or sources of differentiation of neurons. NSCs have strong self-renewal ability and an important ultra-clean effect in the process of differentiation. A previous study demonstrated that MVs from human NSCs were able to attenuate neuroinflammation and preserve host neuronal morphology in the irradiated brain (Baulch et al., 2016). In addition, transplantation of NSC-CDM into injured mouse brains not only caused expansion of the NSC population in the subventricular zone but also enhanced the formation of new neurons that migrated to the damaged site (Rhee et al., 2016). NSC-CDM was confirmed to have the capacity of inducing mesenchymal stem cells into neural stem cell-like cells in vitro (Ma et al., 2011), which further suggested that NSC-CDM might enhance endoneurogenesis. Thus, instead of cell transplantation, we use a purified medium containing NSCs paracrine products. Our experiment was to use the protective role of NSC-CDM in cytotoxicity caused by Aβ 25-35 , including the roles of cell viability, cell death, mitochondrial microstructure destruction, and oxidative stress. PCMT1 is stored in mammals and may have a critical effect on the selfhealing process of NSCs. Thus, we hypothesize that NCS-CDM can regulate nerve cell death by regulating the PCMT1/MST1 pathway. These results provide a new target for AD therapy using a single intervention that has multiple effects.

Bioinformatics analysis
Download the gene chip data set GSE5281 from the database GEO (http://www.ncbi.nlm.nih.gov/geo). Download the expression matrix from the GEO database for data processing, and then standardize the data obtained, and finally use the Limma package for differential expression analysis. The hippocampus, medial temporal gyrus, and posterior cingulate tissues of AD patients and normal elderly were analyzed for differential expression, and then the differential genes (|log2FC| ≥ 1, Padj < 0.05) that were up-regulated or down-regulated in the three tissues were screened. 666 differential protein-coding genes were screened. The 666-differential protein-coding genes were screened for GO and KEGG analysis using cluster Profiler. Volcano plots and histograms are completed with ggplot2.

Cell incubation and treatment
Neural stem cell complete medium (NSC-CPM), including DMEM/F12, human recombinant epidermal growth factor (EGF; 20 ng/mL) and basic fibroblast growth factor (bFGF; 20 ng/mL), B27 (serum-free medium supplements formulated to provide optimal growth condition for NSC expansion, 1:50), heparin (5 μg/mL), 2 mM L-glutamine, and an antibiotic-antimycotic mixture (1:100, 10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B). Neural stem cell-conditioned medium (NSC-CDM): the cortex region of the E15-18 SD rats was isolated, and the meninges were peeled off on the clean bench. The cortexes were transferred to a 15 mL conical tube containing 3 mL HBSS (Hanks' Balanced Salt Solution) for 5 min, then dissociated into small pieces using a 1 mL-pipette tip. 3 mL of HBSS containing small pieces of cortexes was filtered by 100 nm-filters and centrifuged at 1000 rpm for 10 min at room temperature to get single cells. After that, the cells were resuspended in a completed culture medium. The SH-SY5Y cells culture and the groups were divided as follows: control group; Aβ 25-35 group; Aβ 25-35 + NSC-CPM group; Aβ 25-35 + NSC-CDM group according to our previous study (Jia et al., 2020). Isolating and culturing NSCs and NSC-CDM according to a previous study (Yang et al., 2018).

Apoptosis analysis
Using an in-situ cell death detection kit (Roche, Mannheim, Germany), the cells were grown on coverslips and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled with TUNEL for 15 min at 25°C. After TUNEL labeling, 0.344-mm 2 sections were viewed at 400 × magnification making use of a light microscope (BioTek, VT, USA) to quantify apoptotic cells.

qRT-PCR
The tissues and cell samples RNAs were extracted by RNA Extraction Kit (Promega, Shanghai, China). TB green qPCR master mix (TAKARA BIO INC, Tokyo, Japan) was used for quantitative fluorescence evaluation following with to the manufacturer's requirements. The fluorescence values were expressed by 2 −ΔΔCt and quantified (Livak and Schmittgen, 2001).

Statistical analysis
Data were analyzed by GraphPad 8.0 and presented as mean ± standard deviation (SD). Variance (ANOVA) was used to analyze differences among the different groups, PCMT1 expression in three brain regions between patients with AD and normal elderly were analyzed using the Wilcoxon test. P < 0.05 was regarded as obvious.

Results
The differentially expressed genes of AD patients Differences in expression were analyzed using the limma editing package in three brain regions (hippocampus, medial temporal gyrus, and posterior cingulate) between patients with AD and normal elderly. Volcano map of difference analysis results showed that the PCMT1 gene is downregulated in all three brain regions (Fig. 1A). In addition, compared with normal elderly, in AD patients, the expression of PCMT1 was obviously reduced in the hippocampus, medial temporal gyrus, and posterior cingulate ( Fig. 1B, P < 0.001). This suggests that the low function of PCMT1 is a key factor in the poor prognosis of AD.

Hippocampus
Medial

Role of NSC-CPM or NSC-CDM on PCMT1 expression
To investigate the role of NSC-CPM or NSC-CDM on PCMT1 levels, the PCMT1 in control, Aβ 25-35 (40 μM), Aβ 25-35 (40 μM) + NSC-CPM and Aβ 25-35 (40 μM) + NSC-CDM group was evaluated by western blotting and qRT-PCR. Compared with the control group, western blotting exhibited a reduction in PCMT1 expressions in the Aβ 25-35 group (P < 0.01). The above effects were ameliorated by NSC-CPM or NSC-CDM, with the NSC-CDM group demonstrating a higher PCMT1 expression than the NSC-CPM group ( Fig. 2A). Furthermore, mRNA expression of PCMT1 was like the protein expressions of PCMT1 was found (Fig. 2B).

PCMT1 silencing and overexpression
Compared with the NC group, in the sh-PCMT1 group, the levels of PCMT1 protein and mRNA were markedly reduced (Figs. 2C and 2D). In the sh-PCMT1-2 (48 h) group, the levels of PCMT1 protein and mRNA were minimal. Furthermore, compared with the vector group, the expression Role of PCMT1 on cell proliferation and apoptosis CCK-8 results exhibited that cell proliferation was markedly decreased, and the protective role was suppressed after knockdown of PCMT1 compared with the NC group, between the sh-PCMT1 + NSC-CDM group and sh-PCMT1l group exhibited no statistical difference (Fig. 3A) (P < 0.01 and P > 0.05, respectively). Compared with the vector group, in the PCMT1-OE group, greater cell proliferation was found (P < 0.05), and the inhibition role of Aβ 25-35 (40 μM) on cell proliferation was reversed by PCMT1-OE (Fig. 3B) (P < 0.01). Furthermore, in the sh-PCMT1 group, the TUNEL result exhibited that the apoptotic cell numbers were obviously increased compared with the NC group; there was no statistical difference between the sh-PCMT1 + NSC-CDM group and sh-PCMT1 group; PCMT1 overexpressed reversed the effect of Aβ 25-35induced apoptosis (Figs. 3C and 3D).

Discussion
NSCs have the common characteristics of self-renewing stem cells and the characteristics of directed neural differentiation (Hwang et al., 2014;Lewis, 2017). NSC-CDM has been shown to exert antiapoptotic effects in vitro and in vivo (Liang et al., 2014). PCMT1 is highly expressed in brain tissue, including the substantia nigra, blue plaque, and paraventricular nucleus (Zhu et al., 2001). PCMT1 is highly active and pleiotropic, so even small differences in overall PCMT1 activity may have a profound impact on the longterm function of the central nervous system (CNS) (Zhu et al., 2006). Bioinformatics analysis found that PCMT1 activity in the brain tissue of AD patients was significantly decreased (Johnson et al., 1991). Therefore, we speculate that PCMT1, a conservative gene with aging protein repair, has a key effect in the self-renewal process of NSCs, and the paracrine effect of NSCs can up-regulate PCMT1 expression.
In this study, we cultured SH-SY5Y cells induced by Aβ 25-35 with NSC-CPM and NSC-CDM. The results exhibited that the expression of PCMT1 protein and mRNA increased markedly in NSC-CPM and NSC-CDM, and NSC-CDM had a stronger effect than NSC-CPM (P < 0.05). PCMT1 has been associated with apoptosis. Sambri et al. (2011) found that microRNA 15a/16-1 regulates apoptosis in hepatocellular tumor cells through PCMT1, and Liang et al. (2017) showed that CGP3466B plays a neuroprotective role by regulating apoptosis through the PCMT1/Mst1 pathway in rats with traumatic brain injury. MST1 is also involved in tumor occurrence and overgrowth of cells (Perumal et al., 2017), as MST1 deficiency can cause tumor formation that is associated with apoptosis of multiple cell types (Shi et al., 2017b). Overexpression of PCMT1 has been shown to prevent apoptosis induced by Bax in neurons and COS-1 cells. Overexpression of PCMT1 has also been shown to block H 2 O 2 -induced apoptosis through regulation of the methylation of various anti-apoptotic proteins, including Hsp90, Hsp70, and Bcl-xL, in vascular endothelial cells (Bidinosti et al., 2010;Huebscher et al., 1999;Loforese et al., 2017;Maroto and Perez-Polo, 2002). Increased PCMT1 activity is also associated with neuroprotection due to the prevention of neuronal apoptosis and cerebral edema (Liang et al., 2017;Shi et al., 2017a).
The present study demonstrates that Aβ 25-35 inhibits cell viability and induces apoptosis in SH-SY5Y cells, which was reversed by treatment with NSC-CDM. In the present study, a decrease in PCMT1 expression levels was observed in the Aβ 25-35 -treated group. However, these effects were ameliorated by treatment with NSC-CDM. CCK-8 analysis showed that after knockdown of PCMT1 using sh-PCMT1, there was significantly reduced cell viability. Overexpression of PCMT1 resulted in increased cell viability and reversal of the effects of Aβ 25-35 treatment. In addition, using a TUNEL assay, we found that the number of apoptotic cells was markedly higher in SH-SY5Y cells with sh-PCMT1 treatment, whereas overexpression of PCMT1 reversed the effect of Aβ 25-35 induced apoptosis. Western blotting showed that sh-PCMT1 and Aβ 25-35 treatment significantly reduced PCMT1 expression, reduced the ratio of Bcl 2/Bax, increased the ratio of cleaved caspase 3/caspase 3, and increased the ratio of p-MST1/T-MST1. sh-PCMT1 inhibited the protective effects of NSC-CDM, while overexpression of PCMT1 reversed these effects.

Conclusion
Taken together, the NSC-CDM up-regulates the PCMT1 expression, acts on the classic Hippo apoptosis pathway by inhibiting the activation of MST1, and reduces the apoptosis of Aβ 25-35 -induced SH-SY5Y cells.
Availability of Data and Materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' Contribution: WXW, Article writing and revision, data analysis; JGY, study conceptions procurement of samples, paper drafting; YHN, DZY, procurement of samples and results analysis; LY, data collecting and analysis; SCC, study conception and paper drafting. All authors read and approved the final version of the present research manuscript and agreed to be accountable for all aspects of the work.
Ethical Approval: All samples for this study were collected in accordance with the Helsinki Declaration and were approved by the local Ethical Committee.
Funding Statement: The authors received no specific funding for this study.
Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.