Oligodendrocyte Progenitor Cells Increase Chi3l1 Secretion in Exosome to Activate Myh9 and Promote Proliferation through Connexin47 Connected to Astrocytes

Background: Neurodegenerative diseases, caused by the loss of neurons or myelin sheath, are some of the most important neurological diseases that threaten the health of the elderly. In the CNS, oligodendrocytes (OLs) are the only cells that can form myelin. Astrocytes (ASTs) play a generally benecial role in remyelination, including the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) to OLs. However, the specic downstream mechanism is unclear. Methods: This study investigated the proliferation of OPCs in OPCs mono-culture, OPCs culture with ASTs supernatant, and ASTs-OPCs co-culture. Gene Ontology (GO) analysis were used to analyze the differentially expressed genes after transcriptome sequencing of these OPCs. Electron microscope, Nanoparticle Tracking Analysis (NTA), Fluorescence tracing of exosomes and Western blot were used to evaluate the effects of exsomes. Pull-down, co-immunoprecipitation (Co-IP) and mass spectrometry analys were conducted to nd the downstream signal proliferation which is transmitted information into OPCs. Reasults: Direct contact co-culture of ASTs and OPCs promotes the proliferation of OPCs. After Cx47 siRNA interference under ASTs-OPCs co-culture, Chi3l1 secretion in exosome reveals associated decrease, and OPCs proliferation decreased. The cell proliferation induced by Chi3l1 was inhibited after siRNA interfered with Myh9, and the expression of cyclin D1 was also decreased. Conclusions: These results suggest that ASTs transmit information to OPCs by increasing gap junction channel Cx47, thereby promoting the secretion of Chi3l1 in exosome of OPCs. The secretory form of Chi3l1 in exosome might be easier to enter the target cell than in extracellular supernatant, which is benecial to the activation of Myh9 to promote OPCs proliferation. This may be a potential target for drugs rescuing neurodegeneration diseases related to remyelination.


Introduction
The incidence of neurodegenerative disease is gradually increasing, and neurodegenerative disease has become a major disease type that jeopardizes the physical and mental health of the elderly population.Many studies have con rmed that neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis, are associated with damage and destruction of myelin sheath [1][2][3][4]. In the central nervous system (CNS), oligodendrocytes (OLs) are the only cells that can form myelin. The proliferation of OLs depends on the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) [5]. When the body undergoes myelin damage, OPCs that retain proliferative potential proliferate, migrate to the damaged region of the myelin sheath, and differentiate into mature myelin-derived OLs, thereby replacing the myelin sheath that was lost [6,7]. It is very important to regulate OPCs proliferation for the prevention and rehabilitation of the neurodegenerative disease.
Astrocytes (ASTs) are the glial cells with the largest number and volume in the CNS, supporting, protecting and nourishing other nerve cells. There are extensive gap junctions (GJs) between ASTs and OPCs that play a vital role in the process of myelination [8,9]. Among these GJs, the most common is the connexin 47 (Cx47)/Cx43 type [10,11], which, under normal conditions, has an irreplaceable effect on myelin formation [12]. OPCs can connect with ASTs through Cx47 (expressed in OPCs ). Loss of Cx47 causes damage to the myelin sheath and severe demyelinating lesions [13,14].
For example, Chi3l1 increases the malignancy of tumors in gliomas [23,24]. Chi3l1 is also expressed in the brain, as an in ammatory marker in Alzheimer's disease [19,25]. Exosome are small vesicles containing complex protein. Exosome can regulate the biological activity of target cells by carrying proteins, nucleic acids and lipids. There is no report that Chi3l1 exists in exosome.
However, the mechanism that ASTs including the proliferation and differentiation of OPCs is unclear. Studies of GJs Cx47, Chi3l1 in exosome of OPCs are currently lacking, and the relationship between Cx47 and Chi3l1 in exosome has not yet been investigated. Here, transcriptome sequencing, Electron microscope, NTA, pull-down, co-immunoprecipitation (Co-IP) and mass spectrometry results identi ed expression changes about Cx47, Chi3l1, exosome, then explored the mechanism between Cx47 and Chi3l1 in OPCs proliferation.

Materials And Methods
Experimental animal source.
The study has been approved by the Ethics Committee of Chongqing Medical University. Experimental animals were provided by the Experimental Animal Center of Chongqing Medical University.
Collection of B104 supernatant.
The frozen B104 cell strain was rapidly dissolved in a 37 °C water bath, and the dissolved cell suspension was added to a medium containing 12% fetal bovine serum (FBS, Zhejiang Tianhang Biotechnology, 11011 − 8511). Centrifuged at 1000 rpm for 5 min. The cells were then planted in a petri dish containing 12% FBS medium, which was changed every other day until the cells were 70%-80% con uent. The medium was replaced with a medium containing 1% N-2 supplement (Gibco, A1370701), and cells were incubated for 4 days in the incubator at 37 °C. Then, the supernatant was centrifuged, ltered, collected and stored at -80 °C.
Primary culture OPCs.
The cerebral cells of 1-3-day-old Sprague-Dawley (SD) rats were cultured, and 12% FBS (Gibco, 10099-141) medium was added. After 4-5 days, the medium was replaced with selective medium which containing 20% B104 supernatant and 1% N-2 supplement. Cells were cultivated for 4-6 days. OPCs were separated by 0.01% EDTA digest which cannot digest ASTs because of its weak digestion, and OPCs were seeded in a new medium.
Primary culture ASTs.
The cerebral tissue of newborn SD rats was trypsinized (Gibco, 40127ES60) for 5 min, and the cells were seeded in poly-D-lysine (PDL)-coated dishes and cultured for 5-6 days in a medium containing 12% FBS.
Primary cultured OPCs were digested with 0.01% EDTA and seeded in petri dishes coated with PDL. A medium containing 1% N-2 and 20% B104 supernatant was added, and cells were incubated for 4-6 days.
ASTs and OPCs were digested with 0.25% trypsin and 0.01% EDTA, respectively, and ASTs were seed in the upper chamber of the OPCs. A medium containing 1% N-2 and 20% B104 supernatant was added. Cells were incubated for 4-6 days.
ASTs and OPCs co-culture (group A).
After ASTs were seed in PDL-coated dishes for 1 day, OPCs were then seed on the cell surface of ASTs. A medium containing 1% N-2 and 20% B104 supernatant was added. Cells were incubated for 4-6 days. Digest with 0.01% EDTA when collecting cells.
Interfering agents were added to co-cultured ASTs and OPCs according to the instructions of the siRNA Interference Kit (RiboBio). NC siRNA was a disordered RNA used as a control, and the Cx47 interference sequence was CCGAGAAGACTGTCTTCTT. Digest with 0.01% EDTA when collecting cells.
siRNA interference Cx47 + exosomes ( group Cx47si+) Puri ed OPCs in direct contact with ASTs were cultured in OPC proliferation medium for 24 h. Then, the Cx47 siRNA was transfected and about 9 × 10 10 particles/ml of exosomes were added for 24 h at the same time.
Interfering agents were added to OPCs mono-culture according to the instructions of the siRNA Interference Kit (RiboBio). The interference sequence of Myh9 siRNA1 is: GCCTGTTCTGTGTGGTCAT; siRNA2 is: GCATCGAGTGGAACTTCAT; siRNA3 is: GCGTGACTGGTCTCCTTAA.
Pull-down (group goat and group Ch).
Cell lysate was added to OPCs to extract total cellular protein. The proteins were mixed with goat IgG (group goat) (Beyotime, A7007), Chi3l1 antibodies (group Ch) (Santa Cruz, sc-393484) and incubated overnight at 4 °C with shaking on a shaker. Then, the agarose beads (Santa Cruz, sc-2344) were added to the protein, incubated overnight at 4 °C, and collected. The loading buffer was added to agarose beads and boiled for 10 min, followed by gel electrophoresis. The gel after electrophoresis was subjected to Coomassie blue staining, then the protein bands obtained after the staining were cut out and subjected to mass spectrometry to identify the protein species.
Co-immunoprecipitation (Co-IP, group goat and group My).
The total cellular protein of OPCs was extracted, mixed with goat IgG and Myh9 antibodies (PRB-440P, Biolegend), incubated overnight at 4 °C with shaking on a shaker. Then, the agarose beads were added to the protein, incubated overnight at 4 °C, and collected. The loading buffer was then added to the beads and boiled for 10 min. Next, the nal obtained protein was analyzed by western blot.
OPCs were digested with 0.01% EDTA and xed with 75% alcohol overnight at 4 °C. The cell cycle was detected by ow cytometry. Immuno uorescence.
The exosomes of groups were resuspended in PBS and dropped onto the copper mesh grid of electron microscopy in the form of water droplets. The droplets were retained on the copper grid for 1 min and xed in 2% uranyl acetate for 1-10 min. Then it were dried naturally at room temperature and observed and photographed under 120 kV biotransmission electron microscope.
The exosome samples were appropriately diluted using 1X PBS buffer to measure the particle size and concentration. The exosomes particle size and concentration using NTA at VivaCell Biosceinces with Zeta View PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software Zeta View 8.04.02. NTA measurement was recorded and analyzed at 11 positions. The Zeta View system was calibrated using 110 nm polystyrene particles. Temperature was maintained around 23 °C -30 °C.
After the total protein of OPCs was extracted, the loading buffer was added and boiled for 10 min. The protein was then subjected to gel electrophoresis, and the strips were cut and electrotransferred. After the strips were blocked with 5% skim milk powder for 2 h, they were incubated with the primary antibody ( EdU. EdU incubation and uorescent staining were performed according to the protocol for the EdU kit (RiboBio, C10310-1, C10310-3). Fluorescence microscopy was used to detect cellular uorescence.
Exosomes of each group were isolated from 50 ml OPC proliferation medium during a 36 h culture period and were suspended in 500 µl PBS. NTA and western blot analysis Alix of exosomal marker protein for exosomes quanti cation.
Cellular uptake of exosomes.
The exosomes were labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, USA). The OPC was coincubated with labeled exosomes at 37 °C for 24 h. Images were detected via confocal microscopy after mounting using Antifade Mounting Medium with DAPI.
Statistical analysis.
One-way analysis of variance was used in this study. All data were processed using SPSS17.

Results
Direct co-culture of ASTs and OPCs promotes the proliferation of OPCs.
OPCs mono-cultured were labeled with PDGFRα ( Fig. 2A green uorescence), and ASTs mono-cultured were labeled with GFAP ( Fig. 2B red uorescence). Cell purity reached more than 95%. OPCs were cultured under three different culture conditions (ASTs-OPCs direct co-culture, OPCs and ASTs secretion co-culture and OPCs mono-culture). EdU staining showed ( Fig. 2B and C) that the proportion of neonatal OPCs (red uorescent-labeled) increased signi cantly under culture in direct contact with ASTs, and ow cytometry ( Fig. 2D and E) revealed that the proportion of OPCs entering the S phase of DNA replication was also signi cantly increased from (6.10 ± 0.27)% in group O to (11.69 ± 3.27)% in group C and (21.46 ± 2.89)% in group A. Therefore, ASTs promote OPCs proliferation through direct contact with OPCs.
A: PDGFRα is speci cally expressed in OPCs,labeled with green uorescence; GFAP is speci cally expressed in ASTs labeled with red uorescence. B: Red indicates the nucleus of the neonucleated cells labeled with EdU and blue is the nucleus labeled with DAPI. C: Statistical analysis of the proportion of newborn cells in each group, **p<0.01 n = 9. D: Statistical analysis of each group cell cycle, **p<0.01 n = 9. E: Detection the cell cycle of OPCs under three different culture conditions by ow cytometry.

ASTs promote OPCs proliferation by inducing Cx47 expression in OPCs.
The OPCs collected under the above three conditions were subjected to transcriptome sequencing, and differentially expressed genes were screened by Gene Ontology (GO) analysis. Genes enriched in channel activity (please see Supplementary Material 1) are displayed in a heat map (Fig. 3A). Among these genes, the difference in Cx47 expression was signi cant among the three groups. To examine the relationship between Cx47 and OPCs proliferative capacity, the expression level of Cx47 after Cx47 siRNA interference was tested under ASTs-OPCs co-culture conditions. Western blot and immuno uorescence con rmed that Cx47 siRNA successfully reduced the expression of Cx47 (Fig. 3B, C, D and E). Then, we examined the proliferative capacity and cell cycle of OPCs after Cx47 siRNA interference. The relative number of red uorescent-labeled neonatal cells was signi cantly reduced after decreasing Cx47 expression ( Fig. 3F and G), and ow cytometry revealed that the ability of OPCs to enter the S phase of DNA replication was inhibited with decreased Cx47 expression ( Fig. 3H and I). Thus, the ability of ASTs to promote the proliferation of OPCs is closely related to Cx47. Cx47 regulates the expression of chi3l1 in OPCs under ASTs-OPCs co-culture conditions.
Cx47 promotes the proliferation of OPCs. GO analysis of the transcriptome sequencing in OPCs under Cx47 presence (group A) or absence (group Cx47si) conditions revealed that these proliferation-related genes are mainly concentrated in extracellular exosomes (Fig. 4A). A secondary analysis of these genes (please see Supplementary Material 2) by volcano map suggested that Chi3l1 may be a downstream signal for the Cx47-induced proliferation of OPCs (Fig. 4B). Therefore, we examined the expression of Chi3l1 after Cx47 siRNA interference under ASTs-OPCs co-culture conditions. The western blot results showed that the gray value of the Chi3l1 band decreased from (1.01 ± 0.05) in group A and (1.04 ± 0. 22) in group NCsi1 to (0.48 ± 0.15) in group Cx47si (Fig. 4C and D). The immuno uorescence results also con rmed that the expression of Chi3l1 in OPCs decreased after Cx47 siRNA interference ( Fig. 4E and F).
A: GO analysis was performed on the differentially expressed genes of group A and group Cx47si, and combined with the bubble plot and barplot results, the differentially expressed genes of the extracellular exosome were selected for further analysis, |log2 (FC)|≥ 1.5. B: The volcano map analysis of the differential genes enriched in extracellular exosomes. The red and green dots denote the upregulated and downregulated differentially expressed genes, respectively. C: Western blot detected the expression of Chi3l1 after Cx47 siRNA interference. D: Statistical analysis of the expression of Chi3l1 by western blot, **p<0.01, n = 5. E: Statistical analysis of the expression of Chi3l1 by immuno uorescence, **p<0.01, n = 9. F: Detection of the uorescence intensity of Chi3l1 after Cx47 siRNA interference.
Cx47 regulates the expression of Chi3l1 secretion in exosome to promotes OPCs proliferation.
Cx47 promoting the secretion of exosomes in OPCs. NTA revealed that the exosomal concentration increased from 9 × 10 9 particles/ml in the CO-OPC group to 8 × 10 10 particles/ml in the ASTs-OPCs group. The exosomal concentration was signi cantly reduced in the CX47 siRNA group (8.8 ± 0.4 × 10 9 particles/ml) compared with those in the ASTs-OPCs (1.5 ± 0.1 × 10 10 particles/ml) ( Fig. 5A and B). The 98.6% particles of exosome were approximately 135 nm ( Fig. 5A and C). Exosome of the culture were detected by TEM and western blot analyses. TEM demonstrated vesicles of approximately 100 nm in diameter (Fig. 5C). Western blot results con rmed the special markers of exosome Alix and CD63 proteins and expression of Chi3l1 in OPCs secreted exosome (Fig. 5D).
Cx47 regulates the expression of Chi3l1 secretion in exosome The exosomal secretion was signi cantly increase in the ASTs-OPCs group compared with that in the CO-OPC group. The expression of Chi3l1 in exosome of ASTs-OPCs group(10.29 ± 1.71) is signi cantly higher than the exosome of supernatant of the CO-OPC group(2.87 ± 0.31) (Fig. 5C&D). SiRNA CX47 signi cantly reduce the expression of Chi3l1 in exosome of the siRNA CX47 group (2.09 ± 0.58) compared with in the ASTs-OPCs group (Fig. 5E&G).
SiRNA CX47 signi cantly decreased the proliferative capacity of OPCs. The results showed a lower percentage of S phase OPCs in group CX47si(17.59 ± 0.64%) than those in groups ASTs-OPCs (26.78 ± 0.78%). The ability of siRNA CX47 to inhibit proliferation OPCs was alleviated by exosomes supplementation. Exosomes labeled with PKH26 were coincubated in the ASTs-OPCs group for 24 h to evaluate whether they could be internalized by cells. PKH26 was observed to transfer into cells (Fig. 5I).
The proportion of OPCs in S phase increased from 17.38 ± 0.32% in CX47si group to 22.07 ± 0.15% in the CX47si + group (Fig. 5J and K). These results indicate that exosome play a vital role in the proliferation of OPCs.
Many studies have reported that Chi3l1 can promote tumor cell growth during tumor progression [22,26], but the role of OPCs has not been reported. To test whether Chi3l1 also promotes cell proliferation in OPCs, 5 ng/ml and 10 ng/ml exogenous Chi3l1 were added to OPCs under mono-culture conditions.
Exogenous Chi3l1 increased the proportion of green uorescent-labeled neonatal OPCs ( Fig. 6A and B) and the proportion of OPCs entering the S phase of the cell cycle ( Fig. 6C and D), con rming that Chi3l1 also promotes cell proliferation in OPCs.
A: After addition of exogenous Chi3l1 at 5 ng/ml and 10 ng/ml, respectively, EdU detected the proliferative capacity of OPCs, and the neonatal OPCs were labeled with green uorescence. B: Statistical analysis of the proportion of new cells detection by EdU, **p<0.01, n = 9. C: Statistical analysis of each group cell cycle, **p<0.01, *p<0.05, n = 5. D: Flow cytometry detected the proportion of each cell cycle after adding exogenous Chi3l1.
Chi3l1 may bind Myh9 to transmit information to OPCs.
Chi311 is an exocrine glycoprotein with a molecular weight of 40 kDa. Its role in promoting cell proliferation must depend on the receptor, but Chi311 has no speci c receptor. A pull-down experiment was performed on the protein homogenate of OPCs using agarose beads combined with a Chi3l1 antibody, and the protein bound to the agarose beads was electrophoresed. The gel was then stained with Coomassie blue, and the bands were cut for mass spectrometry for identi cation of the protein species (Fig. 7A). A total of 111 proteins (please see Supplementary Material 3) were detected by mass spectrometry, of which Myh9 had the highest degree of peptide matching (Fig. 7B). Then, we used the Myh9 antibody for Co-IP experiments and performed western blot on the pulled-down proteins, revealing that the protein pulled down by the Myh9 antibody contained Chi3l1 (Fig. 7C). The experiment proves that Chi3l1 is able to bind Myh9.
A: The electrophoresis gel of the pull-down protein was stained with Coomassie brilliant blue. Myh9 was identi ed in the strip labeled "a". B: The map of Myh9 analyzed by mass spectrometry with the amino acid sequence shown above the map. C: Western blot of the protein pulled down by the Myh9 antibody with goat IgG used as a control.
The addition of 10 ng/ml exogenous Chi3l1 was shown to promote the proliferation of OPCs. On this basis, Myh9 siRNA interference was performed to observe whether OPCs proliferation was inhibited. The three siRNA interference sequences in the siRNA kit were screened and veri ed to select the siRNA sequence with the best interference effect. The western blot results ( Fig. 8A and C) showed that the M1 interference sequence was most effective, which was con rmed by immuno uorescence (Fig. 8D and E).
Here, 10 ng/ml exogenous Chi3l1 protein was added to mono-culture OPCs, and M1 siRNA was used to knockdown expression of Myh9. EdU staining (Fig. 8F and G) showed that M1 siRNA decreased the number of green uorescent-labeled neonatal cells, and ow cytometry ( Fig. 8I and J) revealed that M1 siRNA suppressed the ability of OPCs to enter the S phase. Western blot (Fig. 8B and H)

Discussion
Neurodegenerative diseases, caused by the loss myelin sheath, are some of the most important neurological diseases that threaten the health of the elderly. In the CNS, OLs are the only cells that can form myelin [27]. The proliferation of OLs depends on the proliferation and differentiation of OPCs. It is very important to regulate OPCs proliferation for the prevention and rehabilitation of the neurodegenerative disease. The interaction between glial cells exerts a regulatory function on the growth of glial cells themselves. As the most abundant glial cells, ASTs have an important in uence on the proliferation of OPCs under both pathological and physiological conditions [28][29][30][31]. Under normal conditions, ASTs provide nutrients and necessary cytokines for the growth and survival of OPCs, such as lipids, tumor growth factor β1 (TGFβ1), chemokine stromal-derived factor 1 (CXCL12), and broblast growth factor receptor 3 (FGFR3) [9,28,[32][33][34]. Moreover, the effect of ASTs on OLs may be bidirectional regulation. For example, in MS, ASTs can promote in ammation to strengthen myelin damage on the one hand, and on the other hand protect OLs and axons from in ammatory damage [35]. It can be seen that when the body undergoes demyelination, ASTs can regulate the growth of OLs to compensate for the loss of myelin [31,[36][37][38][39][40].
A similar phenomenon was found in this study, which examined the proliferation ability of OPCs under conditions of OPCs mono-culture, ASTs supernatant culture, and ASTs-OPCs co-culture. The OPCs in direct contact with ASTs were found to proliferate vigorously, indicating that the chemical channels produced by the direct contact between ASTs and OPCs can inevitably transmit information that promotes OPC proliferation and cannot be replaced by AST supernatant.
GJs are information channels formed by hemichannel protein subunits or hexamer linkers between adjacent cells, allowing small molecules and ions to pass through [41,42]. Their molecular weights range from 25-62 kDa and are used to name different gap junction proteins [43]. There are 21 GJ proteins expressed in the human CNS [44], of which OLs mainly express three types, including Cx47, Cx32 and Cx29, while ASTs mainly express Cx43, Cx30 and Cx26 [45,46]. In addition to the GJs formed between OLs and their own cells, Cx47/Cx43 and Cx32/Cx30 are mainly formed between OLs and ASTs, whereas Cx29 does not participate in the formation of GJs with ASTs [47]. Cx47 is expressed in the oligodendrocyte cell line and is a hemichannel that can be linked to Cx43 expressed by ASTs in the brain [48]. GO analysis of differentially expressed genes in OPCs under three culture conditions, screened 47 differentially expressed genes related to channel activity, which Cx47 expression was signi cantly different between the three groups and highly expressed in group A. The stable expression of Cx47 depends on the presence of ASTs [10,49]. Speci cally, Cx47 siRNA interference in ASTs-OPCs co-culture conditions inhibited the proliferative capacity of OPCs, thus con rming that Cx47 could transmit proliferation information to OPCs.
Biological information analysis of the transcriptome sequencing results revealed that ASTs can increased secretion of Chi3l1 in exosome and exosome of OPCs by Cx47. It suggesting that these increased Chi3l1 in exosome and exosome might be responsible for OPCs proliferation..
Exosome are carriers of information exchange and material transfer between cells. Exosome can participate in cell immune response, migration, differentiation, proliferation and other functions [50]. The results of electron microscopy, immunoblotting and nanoparticle tracking analysis showed that astrocytes adhesion increased the secretion of almost 10 times exosome of OPCs. At the same time, The concentration of Chi3l1 in exosome was also much higher than that Chi3l1 in OPCs. It is suggested that Chi3l1 of exosome is a high concentration transport form. In other words, ASTs can increased autocrine and paracrine of Chi3l1 in exosomes and exosomes of OPCs by Cx47.
Chi3l1 is a member of the glycoprotein hydrolase 18 family and has the ability to bind chitin but has no enzymatic activity [51][52]. Chi3l1 is a tumor-associated factor that is highly expressed in various tumor cells by activating the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT and nuclear factor (NF)-κB pathways to promote tumor growth, invasion and metastasis [26,[53][54][55]. It is also an in ammatory factor that activates the in ammatory response [19,[56][57][58]. This study found that Chi3l1 also promotes cell proliferation in OPCs.
Chi3l1 is a secreted protein that inevitably plays a biological role by binding to the corresponding receptor. Because Chi3l1 has no speci c receptors in reports, many studies have focused on nding receptors for Chi3l1. Interleukin-13 receptor (IL-13R), CRTH2 and CD44 can interact with Chi3l1 to act as a receptor [20,[59][60][61].
In this study, we used pull-down,mass spectrometry and Co-IP to nd the speci c combination of Myh9 and Chi3l1. These experiments showed that Myh9 is the downstream signal that Chi3l1 transmits proliferation information into OPCs.
Myh9, also known as nonmuscle myosin heavy chain IIa (NMMHC-IIA), is located on chromosome 22q12.3 [62] and is expressed in many cell types. Myh9 is a cytoskeletal component composed of complex multimeric protein [63]. It is essential for biological processes such as cell migration, division and signal transduction [62,[64][65][66]. It also has angiogenesis, vascular remodeling abilities in vascular endothelial cells [66] and promotes tumor invasion, metastasis in some tumors [64,67]. Furthermore, Myh9 promotes tumor invasion and metastasis in some tumors [65,[68][69][70]. In addition, some studies have shown that Myh9 also contributes to cell proliferation, cell contraction, adhesion and cytokinesis. In this study, Myh9 siRNA interference on OPCs under the added exogenous Chi3l1 condition revealed that Myh9 knockdown blocked the proliferation of OPCs induced by Chi3l1. The expression of cyclin D1 also decreased with Myh9 siRNA interference.
Our research and other studies have shown that exogenous Chi3l1 can promote the proliferation of OPCs. However, the study also showed that Chi3l1 was secreted in the form of exosome. We speculated that the secretory form of Chi3l1 in exosome might be easier to enter the target cell than in extracellular supernatant, which is bene cial to the activation of Myh9.
In summary, ASTs activate the expression of Chi3l1 in OPCs via gap junction Cx47. Subsequently, the expression and secretion of Chi3l1 in exosome of OPCs is increased. Then, Chi3l1 binds Myh9 of OPCs and transmits proliferation information into the cells to stimulate expression of cyclin D1, thereby promoting OPCs proliferation (Fig. 9). It provides a new understanding for the treatment of neurodegenerative diseases by promoting myelin sheath regeneration.

Conclusions
ASTs play a generally bene cial role in the proliferation and differentiation of OPCs. However, the speci c mechanism is unclear. This study shown that direct contact and secretory exosomes between ASTs and OPCs plays an important role in the proliferation of OPCs. These results suggest that ASTs transmit information to OPCs by increasing gap junction Cx47. More interestingly, ASTs also regulates the expression of Chi3l1 in exosomes of OPCs via gap junction. The exosomes may be the special transport form of Chi3l1 in the cells. It is bene cial to the transmembrane transport of Chi3l1 and the proliferation of OPCs. The Chi3l1 binds Myh9 of OPCs and stimulate expression of cyclin D1. It provides a new understanding for proliferation of OPCs. This may be a potential help for drugs rescuing neurodegeneration diseases related to remyelination.