Activation of P2X7R inhibits proliferation and promotes the migration and differentiation of Schwann cells

doi:10.21203/rs.3.rs-3451609/v1

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Activation of P2X7R inhibits proliferation and promotes the migration and differentiation of Schwann cells

https://doi.org/10.21203/rs.3.rs-3451609/v1

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published 03 Sep, 2024

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In the vertebrate nervous system, myelination of nerve fibers is crucial for the rapid propagation of action potentials through saltatory conduction. Schwann cells—the main glial cells and myelinating cells of the peripheral nervous system—play a vital role in the process of nerve fiber myelination. A large amount of ATP is secreted in response to injury during the repair of peripheral nerve injuries. This ATP release subsequently the dedifferentiation of the myelinating Schwann cells into repair cells, which is necessary for the regeneration of axons. Subsequently, to restore nerve function, these repair cells redifferentiate into myelinating Schwann cells. Except for P2X4R, purine receptors such as P2X7R also play a significant role in this process. In the current study, decreased expression of P2X7R was observed immediately after sciatic nerve injury, followed by a gradual increase to the normal level of P2X7R expression. In vivo experiments showed that the activation of P2X7R using an agonist injection promoted nerve regeneration, whereas the administration of P2X7R antagonists inhibited nerve regeneration. Further, in vitro experiments supported these findings and demonstrated that P2X7R activation inhibited the proliferation of Schwann cells, but it promoted the migration and differentiation of the Schwann cells. In the current study, it was proposed that the manipulation of P2X7R expression in Schwann cells after nerve injury could be effective in facilitating nerve regrowth and remyelination. This finding would provide novel ideas and strategies for treatment programs in the future aimed at promoting nerve repair and functional recovery.

P2X7 receptor

Schwann cells

Myelination

Peripheral nerve

Regeneration

Abnormal myelination of the nervous system is a common clinical symptom that is caused by various factors such as inflammation, trauma, or poisoning [1]. The peripheral nervous system (PNS), unlike the central nervous system (CNS), has a higher capacity for regeneration that is largely attributable to the plasticity of Schwann cells. Following peripheral nerve injury, the Schwann cells undergo rapid physiological responses to transform into repairing Schwann cells, which create a favorable microenvironment for the growth of axons [2]. Notably, the repairing Schwann cells play a crucial role in several processes as follows: (1) Disintegration of distal axons, including processes such as loss of trophic support, disruption of axonal transport, activation of degenerative signaling pathways, etc. (2) Clearance of axon and myelin debris. The Schwann cells remove debris from the injured site and prepare the environment for the regrowth of axons. (3) Secretion of trophic factors and extracellular matrix. Repairing Schwann cells secrete various factors that support the survival of damaged neurons and promote the regrowth of axons. They also secrete adhesion molecules and components of the extracellular matrix, which aid in axon guidance. (4) Formation of the bands of Büngner. These specialized structures are created by repairing Schwann cells—they serve as a guiding pathway for regrowing axons and help them reach their original targets. (5) Remyelination and axon maturation. The repairing Schwann cells play a crucial role in the remyelination process and ensure proper insulation of regrowing axons, further promoting their maturation [2, 3]. Throughout the aforementioned processes, the Schwann cells undergo various changes, including dedifferentiation, proliferation, migration, redifferentiation, and myelination. Each of these changes is critical for successful regeneration and functional recovery of the peripheral nerves.

After a nerve injury, ATP can be released extracellularly[4, 5]; further, it acts as an injury signal, with a wide range of biological activities. For instance, it plays a role in glia-glia and glia-neuron communication [6, 7]. Furthermore, ATP exerts its effects through the purine receptor P2X, which consists of seven subunits (P2X1R–7R) [8]. These receptors are widely expressed in the CNS, including neurons, microglia, astrocytes, and oligodendrocytes, and are involved in synaptic transmission, neuromodulation, and neuroinflammatory responses [8–10]. High expression levels of P2X4 receptor (P2X4R) and P2X7 receptor (P2X7R) were observed in the Schwann cells [11]. In a rat sciatic nerve crush model, real-time PCR analysis found significant differences in the expression patterns of P2X4R and P2X7R (Fig. 1). Notably, P2X4R expression increased about 3 to 4 times on the first day after the crush injury. Although the upward trend of P2X4R expression decreased after day 1, the expression level remained elevated until 28 days after the injury at approximately twice as high as the baseline. Conversely, P2X7R expression demonstrated a decline of approximately 50% on the first day after the injury. The expression of P2X7R reached a minimum level at day 4 and gradually increased thereafter, returning to the baseline levels by 28 days. The elevated expression of P2X4R was majorly observed in the early stage of the injury; however, P2X7R was predominantly expressed in the middle and late stages (Fig. 1). These findings have been validated in our previous research [11, 12]. The differential expression of P2X4R and P2X7R in the Schwann cells during different stages of nerve injury is suggestive of their involvement in the repair and regeneration processes. Thus, further investigation of the roles and mechanisms of P2X receptors in Schwann cells may provide insights into potential therapeutic targets for nerve injury.

After a sciatic nerve crush injury or transection, both myelinating, as well as non-myelinating Schwann cells, demonstrate the ability to dedifferentiate into immature Schwann cells. Upon axonal regeneration, these cells can differentiate into either myelinating or non-myelinating Schwann cells [13]. The involvement of P2X7R in the process of differentiation and dedifferentiation is of significant interest, and it requires further investigation. The specific role of P2X7R in the regeneration of peripheral nerve injury is yet to be fully elucidated. However, P2X7R activation reportedly promotes the proliferation of Schwann cells in certain contexts [14]. Regarding the migration and differentiation of Schwann cells, the role of P2X7R in Schwann cell differentiation is not well-defined yet. Further research is required to elucidate the specific effects of P2X7R activation on Schwann cell migration, differentiation, and myelination.

The current study aimed to investigate the role of P2X7R in remyelination after sciatic nerve injury by injecting P2X7R agonists or antagonists under the epineurium of the sciatic nerve. The present research could provide valuable insights into P2X7R as a potential therapeutic target in myelinating disorders of the PNS, and contribute to the development of novel treatment strategies.

Animals

Postnatal 1–3 days and adult male Sprague Dawley (SD) rats (weighing 200–250 g) were obtained from the Experimental Animal Center of Nantong University. All the animal experiments were conducted according to the guidelines set by the Committee of Nantong University, and they were approved by the Administration Committee of Experimental Animals, Jiangsu Province, China. All the surgical procedures were performed under anesthesia to minimize the pain and discomfort for the animals.

The sciatic nerve was crushed after a 5 s delay, following the injections. The crush was performed near the bifurcation of the sciatic nerve that is approximately 2 mm from the side of the spinal cord. As previously demonstrated, the crushing was performed twice using a fine hemostat, with each crush lasting for 15 s [15, 16]. P2X7R antagonists (BBG or A438079, Tocris Bioscience) and agonists (BzATP, Sigma) were diluted to a concentration of 1uM, using a phosphate-buffered saline (PBS) at a pH of 7.2 (containing NaCl 8.5 g/L, Na₂HPO₄.12H₂O 29.01g/L, NaH₂PO₄.2H₂O 2.96 g/L). The rats were randomly divided into four groups, and each rat received an injection of 6 µl of the respective solution mentioned above. After the surgery, none of the animals exhibited severe wounds, infections, or contractures. All the animals were housed and fed according to routine procedures, and they were monitored for any change in their general condition.

Cell Culture

Primary cultures of Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old SD rats as per previously established methods [17]. The isolated Schwann cells were cultured in DMEM/10% FBS (Gibico), supplemented with 2 µM Forskolin (Sigma) and 10 ng/mL Human heregulin-β1 (HRG, Proteintech). When the Schwann cell-enriched cultures reached confluence, complement-mediated immunocytolysis was performed to eliminate the residual fibroblasts. The cell proliferation, migration, and differentiation assay was prepared as described previously [16].

For the cell proliferation assay, 50 µM of 5-ethynyl-20-deoxyuridine (EdU, Guangzhou RiboBio) was added to the cell medium. After 24 h, the medium containing EdU was washed and fixed using 4% formaldehyde. EdU labeling was conducted according to the manufacturer’s instructions on the kit, and labeling was performed using the Hoechst 33342 stain (1 µg/ml, Sigma). The images were captured using a DMR fluorescent microscope (Leica Microsystems, Wetzlar, Germany), and the percentage of EdU-positive cells was calculated using 10 random fields in 3 wells.

For the cell differentiation assay, the Schwann cells were plated onto poly-D-lysine (PDL, Sigma) coated coverslips (40000 cells/well) in DMEM containing 10% FBS for 24 hours. Thereafter, they were induced to acquire a differentiated phenotype by exposing to 1 mM 2′-O-Dibutyryladenosine3′,5′-cyclic monophosphate sodium salt (db-Camp, Sigma) and 20 ng/ml HRG for 3 days in a non-proliferating medium (DMEM/F12, 0.5% FBS). Using Western blotting and immunocytochemistry, the expression of markers for both myelinating and non-myelinating Schwann cells as well as changes in cell morphology were analyzed (Fig. S2).

For cell migration assay, the Schwann cells were cultured in a non-proliferating medium (DMEM/F12, 0.5% FBS). Time-lapse videos were captured using an inverted time-lapse Olympus X181 Cell-R microscope, equipped with a Hamamatsu CCD ORCA/AG camera and 960 or 9100 objective lenses. The cells were maintained at 37°C, 5% CO2, and in a humid environment. The movies were processed using the Cell-R software (Olympus).

These experimental assays aimed to investigate the proliferation, migration, and differentiation of Schwann cells in response to various stimuli. The results obtained from these assays would provide valuable insights into the behavior and functional characteristics of Schwann cells, which may further contribute to the understanding of myelinating disorders and the development of potential therapeutic strategies.

Real-time quantitative PCR assay

Total RNAs were extracted using an RNA-Quick Purification Kit (ESscience Biotechnology, Shanghai, China), and cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China). Real-time quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) on a 7300 Real-Time PCR System (Applied Biosystems), following the protocol described in our previous report [18]. The primers used in this experiment were as follows: m-P2X7_F1: 5′-GGTGGGGTGACGAAGTTAGG-3′ m-P2X7_R1: 5′-GACAAAGAATGAGTTCCCCTGC-3′ m-P2X4_F1: 5′-TGTGGCTGTGACCAACACTT-3′ m-P2X4_R1: 5′-TCCTGAGCTGGGACCACATA-3′ The mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that was reference gene. All the reactions were performed in triplicates.

Immunohistochemistry and immunocytochemistry

The sciatic nerves were fixed in 4% formalin and embedded in an OTC compound (Miles Scientific) for immunohistochemistry, following a previously described protocol [16, 19]. Immunocytochemistry was performed by fixing the cultures and tissues with 4% paraformaldehyde, followed by blocking with Immunol Staining Blocking Buffer (Beyotime, Haimen, Jiangsu Province, China) for 1 h at room temperature (20–25℃). Then, the cultures were incubated overnight at 4°C, with one or two of the following primary antibodies: S-100 protein subunit beta (S100β) antibody (1:500, mouse, Sigma, catalog: AMAB91038), P2X7R antibody (1:200, goat, Abcam, catalog: Ab93354), myelin-associated glycoprotein (MAG) antibody (1:200, rabbit, Invitrogen, catalog: 34-6200), myelin protein-zero (P0) antibody (1:100, chicken, NOVUS, catalog: NB100-1607), and glial fibrillary acidic protein (GFAP) antibody (1:500, mouse, Millipore, catalog: MAB360). After incubating with the primary antibody, the cultures or tissues were incubated with fluorescence-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature (20–25℃). The stained sections were examined using a Zeiss fluorescence microscope, and the images were captured using a CCD Spot camera.

Western blot

Protein samples were prepared as described previously [18]. 20 µg of protein samples were loaded onto a 10% SDS-PAGE gel and separated via electrophoresis. After transferring, the blots were incubated overnight at 4°C with primary antibodies against P2X7R (1:500, rabbit, MILLIPORE, catalog: AB5246), MAG (1:1000), or GAPDH (1:2000, mouse, Proteintech, catalog: 60004-1-Ig). Thereafter, the blots were incubated with HRP-conjugated secondary antibodies and visualized using an ECL Western Blotting Substrate (Pierce). The chemiluminescence signals were detected using the Tanon 5200 Multi system for 1–5 minutes. The bands of interest were analyzed for their intensity using the Image J software (NIH Image; National Institutes of Health).

Electron Microscopic Analysis

Nerve specimens were obtained from three randomly selected rats from each group, and they were examined using electron microscopy. The experimental procedure for electron microscopy was performed as described previously [20]. Briefly, the site of the lesion was removed, fixed, and prepared for quantitative morphometry of the myelinated nerve fibers. To assess the maturity of the fibers, the G-ratio, which represents the ratio of the axon area to the total fiber area, was calculated. The myelinated axon area was measured by digitally tracing the inner and outer layers of the myelinated fiber using the ImageJ software. The G-ratio data were plotted as a scatter plot against the axon area.

Calcium Influx Assay

Schwann cells were seeded into 96-well plates that were pre-treated with poly-D-lysine (PDL), at a density of 5 x 10³ cells per well. After 2 days, the cells were washed using phosphate-buffered saline (PBS) containing sodium chloride (NaCl), sodium phosphate dibasic (Na₂HPO₄.12H₂O), and sodium phosphate monobasic (NaH₂PO₄.2H₂O) at pH 7.2. Next, the cells were incubated with 2 µM fura-4/AM (Sigma) for 20 minutes at 37°C in a balanced air environment with 5% CO₂. After incubation, the cells were washed and maintained in 50 µL of PBS in each well. Fluorescence imaging was performed using a confocal microscope (TCS SP5; Leica Microsystems, Wetzlar, Germany), with excitation at 488 nm. To obtain the measurements, 20 cells were randomly selected from each well, and this process was repeated three times, resulting in a total of 60 cells. Data analysis was conducted using the Metamorph software (Universal Imaging Corp., Downington, PA).

Yo-Pro Uptake Assay

Schwann cells were seeded into 6-well plates that had been treated with PDL, at a density of 3 x 10⁵ cells per well. After 2 days, the cells were randomly divided into five groups. The inhibitors were pre-incubated with the cells for 1 h, followed by the addition of different compounds. Then, the cells were incubated at 37°C for 5 minutes. Next, the cells were digested into single cells, and the cell density was adjusted to 1x10⁶ cells/ml using PBS. To assess the cell viability, 1 µl/ml of Yo-Pro-1 dye was added to the cell suspension, and incubated on ice for 20 to 30 minutes. The fluorescence signal was measured using a fluorescence-activated cell sorting (FACS) Aria cell sorter, and the data were analyzed using FACS Diva software (BD Biosciences, San Jose, CA, USA). The mean fluorescence intensity, standard deviation, and graphs were generated from the data obtained.

Statistical analysis

All the data were expressed as mean ± standard error of the mean (s.e.m). Statistical comparisons between two individual samples were conducted using the Student's t-test, while multiple comparisons were analyzed using one-way ANOVA, followed by Scheffe's post-hoc test. A p-value of less than 0.05 was considered to be statistically significant.

Different expression changes in P2X4 and P2X7 after sciatic nerve injury

Our previous findings demonstrated that P2X4R expression was significantly upregulated following nerve crush injury. Additionally, overexpression of P2X4R in Schwann cells was shown to promote motor and sensory functional recovery and accelerate nerve remyelination following nerve injury via BDNF release [11]. To understand the expression trend that differs from P2X4R, a sciatic nerve crush model was used in this study to investigate P2X7R expression.

Real-time PCR analysis revealed significant differences in the expression trends of P2X4R and P2X7R. Notably, P2X4R expression increased approximately 3–4 times 1 day after the crush injury, followed by a gradual decrease. However, even 28 days after the injury, the expression level of P2X4R remained approximately twice as high as the baseline. Conversely, P2X7R expression demonstrated a decrease of approximately 50% 1 day after the crush injury. It reached its minimum level at 4 days and then gradually increased, returning to the baseline level by 28 days. Notably, the high expression of P2X4R primarily occurred in the early stage of nerve injury, while P2X7R expression was more predominant in the middle and late stages (Fig. 1).

Given the role of P2X4R in promoting nerve regeneration, it is important to understand the specific role of P2X7R in regeneration in peripheral nerve injury.

P2X7R expressed on Schwann cells

To confirm the expression of P2X7R about peripheral nerve regeneration, the sciatic nerve crush model was used to conduct experiments in the current study. Immunohistochemical analysis revealed that P2X7R expression started to decline 2 days after the injury, and reached its lowest point on the seventh day. Subsequently, it gradually increased and returned to the baseline level approximately 4 weeks after the injury (Fig. 2A). The expression of myelin-associated glycoprotein (MAG), a protein involved in myelin formation, was also examined, and it was found that MAG followed a similar trend as P2X7R (Fig. 2B). Western blot analysis further corroborated these findings, and demonstrated that P2X7R expression decreased—starting from 0.5 h after injury and reaching its minimum level at 6 h (Fig. 2C). These results suggested that P2X7R plays a crucial role in the early stages of nerve injury.

Since the sciatic nerve contains various cell types that include fibroblasts and macrophages, the expression and function of P2X7R in Schwann cells were investigated, which are known to contribute to myelination in the PNS. To explore P2X7R expression in the Schwann cells, we utilized a Schwann cell purification culture technique and performed immunofluorescence and Western blot analysis. Consistent with a previous report [14, 21], it was observed that Schwann cells predominantly expressed P2X7R. Double immunohistochemistry for S100β (a marker for Schwann cells) and P2X7R revealed their co-localization in vitro, primarily in the cell membrane and cytoplasm (Fig. S1B). Similar results were also observed in vivo (Fig. S1A). These findings suggested that P2X7R was indeed expressed on Schwann cells, and may play a crucial role in Schwann cells during peripheral nerve regeneration.

Activation of P2X7R helps in myelin sheath regeneration after sciatic nerve injury

To investigate the effects of P2X7R on myelin regeneration in the PNS, a study was conducted using a rat crush model of sciatic nerve injury. In this model, agonists or antagonists (dissolved in 6µl of normal saline) were administered into the epineurium of the sciatic nerve on the first day and seventh day after the injury. Two weeks later, we collected injury points from each group and examined the expression of myelin proteins MAG and P0 using frozen section immunohistochemistry and Western Blot techniques.

The results of the current study demonstrated that the activation of P2X7R significantly increased MAG and P0 expression, while the inhibition of P2X7R led to a significant decrease in their expression (Fig. 3A and B). Additionally, transmission electron microscopy was used to analyze the myelin structure, and it was observed that the G-ratio value, which indicates the thickness of the myelin sheath, was significantly lower in the group that was treated with the P2X7R agonist BzATP as compared to the control group. Conversely, the G-ratio values were higher in the groups treated with the P2X7R antagonists BBG and A438079, as compared to the control group (Fig. 3C).

Based on these findings, it was concluded that during the process of sciatic nerve injury and repair, P2X7R activation plays a beneficial role in the promotion of myelin regeneration.

P2X7R affects the release of Ca²⁺ and the permeability of cell membranes in Schwann cells

P2X7R is a trimeric ion channel that is activated by extracellular adenosine 5'-triphosphate (ATP). Previous studies have shown that P2X7R is highly expressed in Schwann cells[14, 21]. To better understand the functional properties of P2X7R in Schwann cells, intracellular calcium (Ca²⁺) concentration was monitored using a fluorescent-labeled probe, namely Fluo-4-AM. In this study, various P2X7R antagonists (200 nM BBG, 100 µM A438079, or 100 µM oATP) and agonists (1M ATP or 100 µM BzATP) were used to induce changes in intracellular Ca²⁺ levels.

It was observed that the application of ATP caused a rapid increase in cytosolic Ca²⁺ levels, which was significantly reduced when BBG, A438079, or oATP were used (Fig. 4A-a and A-c). Among these antagonists, A438079 had the most pronounced effect. Similar results were observed with BzATP (Fig. 4A-a and A-c). The above findings indicated that P2X7R activation promoted the release of Ca²⁺ in Schwann cells.

Based on previous reports, it is known that when the P2X7R ligand-gated ion channel is open, substances with molecular weight less than 900 Da can enter the cell [22]. Therefore, Yo-Pro-1, which has a molecular weight of 629 Da, was selected to evaluate the effect of P2X7R agonists or antagonists on cell permeability. The results demonstrated that the P2X7R agonist BzATP significantly (P < 0.01) increased the permeability of Schwann cells, while the P2X7R antagonist BBG effectively antagonized the increase in permeability induced by BzATP (Fig. 4A-d). These findings further support the notion that P2X7R is as a functional receptor in Schwann cells.

Overall, this study provides evidence that P2X7R activation in Schwann cells leads to an increase in intracellular Ca²⁺ levels and promotes changes in cell permeability. These findings contribute to our understanding of the functional role of P2X7R in Schwann cells and its potential implication in cellular processes.

P2X7R affects the proliferation, migration, and differentiation of Schwann cells

During the myelination process, Schwann cells undergo proliferation, migration, and differentiation to form the myelin sheath. As the Schwann cells differentiate, they reduce their division activity, establish a 1:1 relationship with axons, and upregulate the expression of myelin proteins such as MAG and P0 [23, 24].

In the current study, the effect of P2X7R agonists and antagonists on the proliferation, migration, and differentiation of Schwann cells was investigated. To assess the proliferation, EdU labeling was utilized in primary cultured Schwann cells treated with P2X7R agonists and antagonists. The findings of the study revealed that P2X7R antagonists, including BBG, A438079, and oxATP, promoted Schwann cell proliferation to varying degrees, with BBG exhibiting the most pronounced effect. Conversely, the P2X7R agonist BzATP significantly inhibited the proliferation of Schwann cells (Fig. 4A-b&B). These results suggested that P2X7R activation inhibited the proliferation of Schwann cells.

To evaluate the effects of P2X7R on the migration and differentiation of Schwann cells, we employed a Schwann cell differentiation model. The Schwann cells were treated with BBG, A438079, oxATP, and BzATP, and their movement trajectories were observed using a live cell imaging workstation (Fig. 4C-E). After 3 days of culture, the expression of myelin proteins MAG and P0 was assessed through immunofluorescence and Western blot analysis (Fig. 5A, D, and F). The results demonstrated that the addition of BBG and oxATP significantly inhibited cell movement (Fig. 4C-E). Additionally, BzATP treatment significantly increased MAG expression, while oxATP and A438079 decreased MAG and P0 expression (Fig. 5A, D, and F). Therefore, P2X7R activation promoted Schwann cell migration and differentiation.

These findings provided valuable insights into the role of P2X7R in Schwann cell proliferation, migration, and differentiation, thereby contributing to the understanding of mechanisms underlying myelin sheath formation.

The purine receptor P2X7R, an ATP receptor, is involved in the regulation of various physiological and pathological processes after nerve injury [5, 8, 21, 25]. Our previous studies showed that among its seven subtypes (P2X1R-P2X7R), P2X4R-7Rs are highly expressed in Schwann cells [11]. In the current study, it was found that P2X7R is expressed in myelinating Schwann cells as well as in non-myelinating Schwann cells in vivo (Fig. S1 A). In vitro experiments using primary purified Schwann cells or differentiated Schwann cells confirmed the co-expression of P2X7R with S100β protein (Fig. 5B and S1 B). These findings were consistent with previous studies by Song et al. (2015) and Luo et al. (2013), which showed that Schwann cells predominantly expressed P2X7R [14, 21]. Early immunohistochemical analyses conducted by Faroni et al. (2014) demonstrated that in the wild-type peripheral nerves, P2X7R expression was primarily observed in myelinating Schwann cells, and only a few non-myelinating Schwann cells showed immunopositivity for P2X7R [25].

After a peripheral nerve injury, a cascade of pathological and physiological events occurs at the site of the injury that leads to Wallerian degeneration in the distal stump and regional axonal degeneration in the proximal stump [26, 27]. Moreover, Schwann cells play a vital role in the repair process by undergoing rapid physiological changes, including dedifferentiation, formation of Büngner bands, and production of neurotrophic factors and extracellular matrix (ECM) molecules. These repairing Schwann cells stimulate axonal regeneration and proliferation, leading to the redifferentiation of Schwann cells that wrap around the regenerating axons to form new myelin sheaths. Notably, this process continues, until the regenerating axons extend to their target organs and enable functional nerve regeneration [3]. Therefore, Schwann cells, which are the myelinating cells of the peripheral nerve system, play a crucial role in this intricate process. In the current study, a rat crush model of sciatic nerve injury was utilized to investigate the role of P2X7R in peripheral nerve regeneration. We employed a timed, fixed-point, and quantitative administration method to inject P2X7R inhibitors and agonists into the epineurium of the sciatic nerve. Two weeks after the injury, we evaluated the expression of myelin proteins and recovery of the myelin sheath using Western Blot analysis and transmission electron microscopy (Fig. 3). The results indicated that the activation of P2X7R was beneficial for regeneration, as evidenced by improved expression of myelin proteins and myelin sheath recovery. Based on these findings, it was proposed that P2X7R may promote peripheral nerve remyelination by inhibiting Schwann cell proliferation and enhancing their migration and differentiation. In the experiments in the current study that used the sciatic nerve crush model, a decrease in P2X7R expression was observed starting from 0.5 h after the injury and reaching the minimum level at 6 hours (Fig. 2C). Additionally, experiments were conducted using purified Schwann cells, where P2X7R inhibitors and agonists were added. It was found that the activation of P2X7R significantly stimulated the release of calcium ions (Ca²⁺) and increased the cell membrane permeability (Fig. 4A-a, A-c, and A-d). Conversely, the addition of P2X7R inhibitors led to the opposite outcome (Fig. 4A-a, A-c, and A-d). These findings suggested that P2X7R may serve as an important functional receptor during the early stages of nerve injury. Overall, the study provided valuable insights into the role of P2X7R in peripheral nerve regeneration and suggested its potential as a target for therapeutic interventions.

In the early stage of sciatic nerve injury, highly differentiated Schwann cells undergo rapid dedifferentiation and enter a proliferative cycle [28]. The current study found that the protein level of P2X7R started declining 0.5 hours after the sciatic nerve injury, as observed using Western blot analysis. This corresponded to the experiment where the addition of a P2X7R inhibitor to purified Schwann cells significantly promoted their proliferation. However, the expression of P2X7R reached its lowest level at 6 hours, and then increased gradually (Fig. 2B). This suggested that the upregulation of P2X7R may be involved in the preparation for the redifferentiation of Schwann cells and remyelination of newly formed axons. In conclusion, the response of cells varies at different time points, indicating a dynamic regulation of P2X7R expression during the process of nerve injury and repair. In the current study, the effect of P2X7R inhibitors and agonists on the proliferation, migration, and differentiation of Schwann cells was primarily investigated. Various experimental techniques such as EdU labeling, live cell workstations, and Schwann cell differentiation models were employed to assess these effects (Fig. 4D-E and Fig. 5). The results demonstrated that the P2X7R activation inhibited Schwann cell proliferation while promoting their migration and differentiation. These findings were consistent with our in vivo experiments, wherein a P2X7R agonist injection into the sciatic nerve epithelium facilitated regeneration. Collectively, these studies suggested that targeting Schwann cell P2X7R could be a promising approach for treating disorders associated with PNS myelination.

An interesting finding in the current research was the differential expression trend of P2X4R and P2X7R in the co-culture model of Schwann cells, as observed in the microarray data analysis. P2X4R showed high expression levels in the early stages, while P2X7R was primarily expressed in the middle and late stages [11, 12]. This pattern was consistent with the regeneration of sciatic nerve after injury, as shown in the current study (Fig. 2B) and previous research using Western blot analysis [11]. The role of P2X7R in peripheral nerve myelination has been previously investigated by Faroni et al. (2014) using a P2X7R knockout mice model. They observed that P2X7R knockout resulted in a significant decrease in the expression of myelin proteins in the sciatic nerve. Additionally, the number of myelinated axons was also reduced in the knockout mice as compared to that in the control group [25]. The importance of P2X7R in peripheral nerve remyelination was examined at the morphological and molecular levels, and this phenomenon was also strongly confirmed in the data obtained in the current study. Overall, the data of this study strongly supported the notion that P2X7R plays a crucial role in the function of Schwann cells and peripheral nerve myelination. Further, the differential expression patterns of P2X4R and P2X7R suggested distinct roles for these receptors at different stages of nerve injury and repair. Further research is required to elucidate the underlying mechanisms and explore the therapeutic potential of targeting P2X4R and P2X7R in peripheral nerve disorders.

P2X7R is a membrane protein that functions as a ligand-gated ion channel receptor, and it is implicated in various human ion channel diseases. Upon activation, P2X7R allows charged sodium and calcium particles to enter the cell, leading to the activation of various signaling pathways that are involved in immune responses, neurotransmitter release, oxidative stress, cell proliferation, and apoptosis [29, 30]. A unique feature of P2X7R is its ability to undergo complete desensitization upon activation, thereby allowing a continuous influx of charged particles and triggering inflammatory signaling pathways [31–33]. Additionally, P2X7R can interact with other proteins by altering its conformation, leading to the modulation of the related pathways. For instance, in macrophages, the presence of ATP induces a structural change in P2X7R, thereby enabling its binding to the channel protein Panx1. This interaction triggers the NFκB signaling pathway, resulting in the generation of pro-IL-1β through Toll-like receptor (TLR) activation. Subsequently, caspase-1 is activated, leading to the release of IL-1β. These findings highlight the multifaceted role of P2X7R in cellular processes and its involvement in various disease mechanisms [34]. Understanding the intricate signaling pathways associated with P2X7R activation can provide valuable insights for the development of therapeutic strategies targeting P2X7R in human diseases. In October 2019, the Mansoor team utilized single-particle cryoelectron microscopy to investigate the structure of full-length P2X7R of rats in both its apo- and ATP-bound states. This study aimed to unravel the distinct functional characteristics of P2X7R and elucidate the structure of its large and enigmatic cytoplasmic domain. The findings provided valuable insights about the low affinity of P2X7R for ATP, thereby elucidating the role of palmitoylation in preventing receptor desensitization. Further, the findings revealed a novel folding pattern of the cytoplasmic domain and the presence of a binuclear zinc ion complex and a high-affinity guanosine nucleotide binding site within the cytoplasmic domain of P2X7R. However, the specific functions of these elements remain unknown [31]. Nevertheless, the aforementioned discoveries, along with other related research, would establish the groundwork for future experiments that aim to unravel the unique signaling mechanisms of P2X7R [31, 35].

The impact of Schwann cell P2X7R on axon myelination has not been investigated extensively yet. In the current study, it was demonstrated that the activation of P2X7R inhibits proliferation, promotes migration, and enhances the differentiation of Schwann cells, which play a crucial role in the proper development and regeneration of the peripheral nerves. It is hypothesized that P2X7R signaling may influence myelination by regulating the expression of genes related to myelin formation and maintenance, thereby promoting the formation and stability of myelin sheaths around the axons. However, further research is required to comprehensively understand the precise mechanisms by which P2X7R affects axon myelination, and further explore its potential therapeutic implications in peripheral nerve disorders.

Acknowledgments

We thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.

Authors contributions

Study conception and design, manuscript draft: W.S, G.C; experiment implementation: W.S, X.H, Z.L, J.X, J.S, Z.W, and Y.Z; data analysis: Y.G, L.X, and W.S. All the authors have read and approved the final version of the manuscript.

Funding

We acknowledge the support from the National Natural Science Foundation of China (82001296, 32070998, 32271054, 82272169), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics Approval

The animal experiments conducted in this study were carried out in compliance with the institutional animal care and use guidelines of Nantong University, and they were authorized by the Animal Ethics Committee of Nantong University, China (approval No. IACUC20201010-001).

Ethics Approval

All the animal experiments were conducted in accordance with the Institutional Animal Care and Use Guidelines of Nantong University and were ethically approved by the Administration Committee of Experimental Animals, Jiangsu Province, China.

Consent to Participate

Informed consent was obtained from all the individual participants included in the study.

Consent for Publication

All the co-authors approved the final version of the manuscript and agreed to submit it to Molecular Neurobiology.

Competing Interests The authors declare no conflict of interest.

Katona I, Weis J (2017) Diseases of the peripheral nerves. Handb Clin Neurol 145:453-474. doi:10.1016/B978-0-12-802395-2.00031-6
Nocera G, Jacob C (2020) Mechanisms of Schwann cell plasticity involved in peripheral nerve repair after injury. Cell Mol Life Sci 77 (20):3977-3989. doi:10.1007/s00018-020-03516-9
Gu X, Ding F, Williams DF (2014) Neural tissue engineering options for peripheral nerve regeneration. Biomaterials 35 (24):6143-6156. doi:10.1016/j.biomaterials.2014.04.064
Abbracchio MP, Burnstock G, Verkhratsky A, Zimmermann H (2009) Purinergic signalling in the nervous system: an overview. Trends Neurosci 32 (1):19-29. doi:10.1016/j.tins.2008.10.001
Cheng RD, Ren W, Luo BY, Ye XM (2023) The role of purinergic receptors in neural repair and regeneration after spinal cord injury. Neural Regeneration Research 18 (8):1684-1690. doi:10.4103/1673-5374.363186
Fields RD, Burnstock G (2006) Purinergic signalling in neuron-glia interactions. Nat Rev Neurosci 7 (6):423-436. doi:10.1038/nrn1928
Rodrigues RJ, Tome AR, Cunha RA (2015) ATP as a multi-target danger signal in the brain. Front Neurosci 9:148. doi:10.3389/fnins.2015.00148
Illes P, Muller CE, Jacobson KA, Grutter T, Nicke A, Fountain SJ, Kennedy C, Schmalzing G, Jarvis MF, Stojilkovic SS, King BF, Di Virgilio F (2021) Update of P2X receptor properties and their pharmacology: IUPHAR Review 30. Brit J Pharmacol 178 (3):489-514. doi:10.1111/bph.15299
Burnstock G (2016) An introduction to the roles of purinergic signalling in neurodegeneration, neuroprotection and neuroregeneration. Neuropharmacology 104:4-17. doi:10.1016/j.neuropharm.2015.05.031
Tang Y, Illes P (2017) Regulation of adult neural progenitor cell functions by purinergic signaling. Glia 65 (2):213-230. doi:10.1002/glia.23056
Su WF, Wu F, Jin ZH, Gu Y, Chen YT, Fei Y, Chen H, Wang YX, Xing LY, Zhao YY, Yuan Y, Tang X, Chen G (2019) Overexpression of P2X4 receptor in Schwann cells promotes motor and sensory functional recovery and remyelination via BDNF secretion after nerve injury. Glia 67 (1):78-90. doi:10.1002/glia.23527
Zhang B, Su W, Hu J, Xu J, Askar P, Bao S, Zhou S, Chen G, Gu Y (2022) Transcriptome Analysis of Schwann Cells at Various Stages of Myelination Implicates Chromatin Regulator Sin3A in Control of Myelination Identity. Neurosci Bull. doi:10.1007/s12264-022-00850-9
Hall S (2005) The response to injury in the peripheral nervous system. J Bone Joint Surg Br 87b (10):1309-1319. doi:10.1302/0301-620x.87b10.16700
Song XM, Xu XH, Zhu J, Guo Z, Li J, He C, Burnstock G, Yuan H, Xiang Z (2015) Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury. Purinergic Signal 11 (2):203-213. doi:10.1007/s11302-015-9445-8
Chen G, Kim YH, Li H, Luo H, Liu DL, Zhang ZJ, Lay M, Chang W, Zhang YQ, Ji RR (2017) PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1. Nat Neurosci 20 (7):917-926. doi:10.1038/nn.4571
Gu Y, Wu Y, Su W, Xing L, Shen Y, He X, Li L, Yuan Y, Tang X, Chen G (2018) 17beta-Estradiol Enhances Schwann Cell Differentiation via the ERbeta-ERK1/2 Signaling Pathway and Promotes Remyelination in Injured Sciatic Nerves. Front Pharmacol 9:1026. doi:10.3389/fphar.2018.01026
Su WF, Gu Y, Wei ZY, Shen YT, Jin ZH, Yuan Y, Gu XS, Chen G (2016) Rab27a/Slp2-a complex is involved in Schwann cell myelination. Neural Regen Res 11 (11):1830-1838. doi:10.4103/1673-5374.194755
Chen G, Park CK, Xie RG, Ji RR (2015) Intrathecal bone marrow stromal cells inhibit neuropathic pain via TGF-beta secretion. J Clin Invest 125 (8):3226-3240. doi:10.1172/JCI80883
Shen YT, Gu Y, Su WF, Zhong JF, Jin ZH, Gu XS, Chen G (2016) Rab27b is Involved in Lysosomal Exocytosis and Proteolipid Protein Trafficking in Oligodendrocytes. Neurosci Bull 32 (4):331-340. doi:10.1007/s12264-016-0045-6
Chen G, Zhang Z, Wei Z, Cheng Q, Li X, Li W, Duan S, Gu X (2012) Lysosomal exocytosis in Schwann cells contributes to axon remyelination. Glia 60 (2):295-305. doi:10.1002/glia.21263
Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X (2013) P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord. Cell Death Dis 4:e829. doi:10.1038/cddis.2013.343
Volonte C, Apolloni S, Skaper SD, Burnstock G (2012) P2X7 receptors: channels, pores and more. CNS Neurol Disord Drug Targets 11 (6):705-721. doi:10.2174/187152712803581137
Askar P, Xu JH, Hu JX, Shangguan JH, Sun HL, Zhou SL, Yang XM, Chen G, Su WF, Gu Y (2022) The Etv1/Er81 transcription factor coordinates myelination-related genes to regulate Schwann cell differentiation and myelination. Ann Transl Med 10 (16). doi:10.21037/atm-22-3489
Muppirala AN, Limbach LE, Bradford EF, Petersen SC (2021) Schwann cell development: From neural crest to myelin sheath. Wires Dev Biol 10 (5). doi:10.1002/wdev.398
Faroni A, Smith RJ, Procacci P, Castelnovo LF, Puccianti E, Reid AJ, Magnaghi V, Verkhratsky A (2014) Purinergic signaling mediated by P2X7 receptors controls myelination in sciatic nerves. J Neurosci Res 92 (10):1259-1269. doi:10.1002/jnr.23417
Conforti L, Gilley J, Coleman MP (2014) Wallerian degeneration: an emerging axon death pathway linking injury and disease. Nature Reviews Neuroscience 15 (6):394-409. doi:10.1038/nrn3680
Zhang K, Jiang MS, Fang YS (2021) The Drama of Wallerian Degeneration: The Cast, Crew, and Script. Annu Rev Genet 55:93-113. doi:10.1146/annurev-genet-071819-103917
Wang H, Zhang P, Yu J, Zhang F, Dai W, Yi S (2019) Matrix metalloproteinase 7 promoted Schwann cell migration and myelination after rat sciatic nerve injury. Mol Brain 12 (1):101. doi:10.1186/s13041-019-0516-6
Di Virgilio F, Sarti AC, Grassi F (2018) Modulation of innate and adaptive immunity by P2X ion channels. Curr Opin Immunol 52:51-59. doi:10.1016/j.coi.2018.03.026
Zou J, Vetreno RP, Crews FT (2012) ATP-P2X7 receptor signaling controls basal and TNFalpha-stimulated glial cell proliferation. Glia 60 (4):661-673. doi:10.1002/glia.22302
McCarthy AE, Yoshioka C, Mansoor SE (2019) Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization. Cell 179 (3):659-670 e613. doi:10.1016/j.cell.2019.09.017
Koshimizu T, Koshimizu M, Stojilkovic SS (1999) Contributions of the C-terminal domain to the control of P2X receptor desensitization. J Biol Chem 274 (53):37651-37657. doi:10.1074/jbc.274.53.37651
Yan Z, Khadra A, Li S, Tomic M, Sherman A, Stojilkovic SS (2010) Experimental characterization and mathematical modeling of P2X7 receptor channel gating. J Neurosci 30 (42):14213-14224. doi:10.1523/JNEUROSCI.2390-10.2010
Pelegrin P, Surprenant A (2009) The P2X(7) receptor-pannexin connection to dye uptake and IL-1beta release. Purinergic Signal 5 (2):129-137. doi:10.1007/s11302-009-9141-7
Costa-Junior HM, Sarmento Vieira F, Coutinho-Silva R (2011) C terminus of the P2X7 receptor: treasure hunting. Purinergic Signal 7 (1):7-19. doi:10.1007/s11302-011-9215-1

Fig.S1.tif
Fig. S1 P2X7R expression on Schwann cells. (A) Immunohistochemistry staining for S100β (green), a marker for Schwann cells, and P2X7R (red) in vivo. The images show co-localization of S100β and P2X7R, indicating the expression of P2X7R on Schwann cells. Scale bar, 75 μm. (B) Immunohistochemistry staining for S100β (red) and P2X7R (green) in vitro. The images demonstrate co-localization of S100β and P2X7R, confirming the expression of P2X7R on Schwann cells. Scale bar, 25 μm.
Fig.S2.tif
Fig. S2 Schwann cell differentiation model. (A) Immunofluorescence images showing the expression of myelin proteins MAG (green) and P0 (red) in Schwann cells after 3 days of differentiation. Cells were cultured under differentiation conditions. Scale bar, 50 μm. (B) Western blot analysis of myelin protein MAG and P0 expression in Schwann cells after 3 days of differentiation. The results are presented as fold-change in comparison with the control group, with GAPDH as the protein loading control. *P < 0.05, compared with the control group, one-way ANOVA, n = 3 rats/group.

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You are reading this latest preprint version

Activation of P2X7R inhibits proliferation and promotes the migration and differentiation of Schwann cells

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Cell Culture

Real-time quantitative PCR assay

Immunohistochemistry and immunocytochemistry

Western blot

Electron Microscopic Analysis

Calcium Influx Assay

Yo-Pro Uptake Assay

Statistical analysis

Results

Different expression changes in P2X4 and P2X7 after sciatic nerve injury

P2X7R expressed on Schwann cells

Activation of P2X7R helps in myelin sheath regeneration after sciatic nerve injury

P2X7R affects the release of Ca²⁺ and the permeability of cell membranes in Schwann cells

P2X7R affects the proliferation, migration, and differentiation of Schwann cells

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Activation of P2X7R inhibits proliferation and promotes the migration and differentiation of Schwann cells

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Cell Culture

Real-time quantitative PCR assay

Immunohistochemistry and immunocytochemistry

Western blot

Electron Microscopic Analysis

Calcium Influx Assay

Yo-Pro Uptake Assay

Statistical analysis

Results

Different expression changes in P2X4 and P2X7 after sciatic nerve injury

P2X7R expressed on Schwann cells

Activation of P2X7R helps in myelin sheath regeneration after sciatic nerve injury

P2X7R affects the release of Ca2+ and the permeability of cell membranes in Schwann cells

P2X7R affects the proliferation, migration, and differentiation of Schwann cells

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

P2X7R affects the release of Ca²⁺ and the permeability of cell membranes in Schwann cells