The transcription factor Stat-1 is essential for Schwann cell differentiation, myelination and myelin sheath regeneration.

BACKGROUND: Myelin sheath is a crucial accessory to the functional nerve-fiber unit, its disruption or loss can lead to axonal degeneration and subsequent neurodegenerative diseases (NDs). Notwithstanding of substantial progress in possible molecular mechanisms underlying myelination, there is no therapeutics that prevent demyelination in NDs. Therefore, it is crucial to seek for potential intervention targets. Here, we focused on the transcriptional factor, signal transducer and activator of transcription 1 (Stat1), to explore its effects on myelination and its potential as a drug target. METHODS: By analyzing the transcriptome data obtained from Schwann cells (SCs) at different stages of myelination, it was found that Stat1 might be involved in myelination. To test this, we used the following experiments: (1) In vivo, the effect of Stat1 on remyelination was observed in an in vivo myelination mode with Stat1 knockdown in sciatic nerves or specific knockdown in SCs. (2) In vitro, the RNA interference combined with cell proliferation assay, scratch assay, SC aggregate sphere migration assay, and a SC differentiation model, were used to assess the effects of Stat1 on SC proliferation, migration and differentiation. Chromatin immunoprecipitation sequencing (ChIP-Seq), RNA-Seq, ChIP-qPCR and luciferase activity reporter assay were performed to investigate the possible mechanisms of Stat1 regulating myelination. RESULTS: Stat1 is important for myelination. Stat1 knockdown in nerve or in SCs reduces the axonal remyelination in the injured sciatic nerve of rats. Deletion of Stat1 in SCs blocks SC differentiation thereby inhibiting the myelination program. Stat1 interacts with the promoter of Rab11-family interacting protein 1 (Rab11fip1) to initiate SC differentiation. CONCLUSION: Our findings demonstrate that Stat1 regulates SC differentiation to control myelinogenic programs and repair, uncover a novel function of Stat1, providing a candidate molecule for clinical intervention in demyelinating diseases.

The Schwann cell-specific G-protein Gαo (Gnao1) is a cell-intrinsic controller contributing to the regulation of myelination in peripheral nerve system.

Myelin sheath abnormality is the cause of various neurodegenerative diseases (NDDs). G-proteins and their coupled receptors (GPCRs) play the important roles in myelination. Gnao1, encoding the major Gα protein (Gαo) in mammalian nerve system, is required for normal motor function. Here, we show that Gnao1 restricted to Schwann cell (SCs) lineage, but not neurons, negatively regulate SC differentiation, myelination, as well as re-myelination in peripheral nervous system (PNS). Mice lacking Gnao1 expression in SCs exhibit faster re-myelination and motor function recovery after nerve injury. Conversely, mice with Gnao1 overexpression in SCs display the insufficient myelinating capacity and delayed re-myelination. In vitro, Gnao1 deletion in SCs promotes SC differentiation. We found that Gnao1 knockdown in SCs resulting in the elevation of cAMP content and the activation of PI3K/AKT pathway, both associated with SC differentiation. The analysis of RNA sequencing data further evidenced that Gnao1 deletion cause the increased expression of myelin-related molecules and activation of regulatory pathways. Taken together, our data indicate that Gnao1 negatively regulated SC differentiation by reducing cAMP level and inhibiting PI3K-AKT cascade activation, identifying a novel drug target for the treatment of demyelinating diseases.

The Etv1/Er81 transcription factor coordinates myelination-related genes to regulate Schwann cell differentiation and myelination.

Background: Axonal myelination is critical for the functioning of vertebrate nervous system. Myelin sheath malformation or degeneration can cause a variety of neurological diseases. Our previous study identified multiple potential myelination-related transcriptional factors (TFs), including expressed sequence tag (ETS) variant transcription factor 1 (Etv1)/Er81, via gene microarray analysis of Schwann cells (SCs) at various myelination stages. Etv1 is known to be involved in the regulation of neuronal specialization, muscle spindle differentiation, and sensorimotor connectivity. However, to our knowledge, to date, there are no relevant studies that Etv1 regulates SC myelination. Methods: To investigate the roles of Etv1 in SC re-myelination, an in vivo mouse myelination model was used, in which the sciatic nerve is crushed. Etv1 in nerves was knocked down via in situ injection of cholesterol-modified Etv1-small interfering (si)RNA. The expression of myelin-associated glycoprotein (MAG) was evaluated by Western blotting (WB) and immunohistochemistry (IHC). Myelination was assessed by transmission electron microscopy (TEM). The effects of Etv1 on SC proliferation, migration, and differentiation were assessed in vitro using the EdU cell proliferation kit, a culture-insert scratch assay, a SC aggregate sphere migration assay on the axons of dorsal root ganglions (DRGs), and a SC differentiation model. Chromatin immunoprecipitation (ChIP) united with quantitative real-time PCR (qPCR), known as ChIP-qPCR, and luciferase activity reporter assays were performed to explore the possible mechanisms by which Etv1 controls SC differentiation and myelination. Results: The results demonstrated that Etv1 promoted myelination by facilitating SC proliferation, migration, and differentiation. Etv1 expression in SCs was upregulated during re-myelination, and knocking down Etv1 expression dramatically abrogated SC re-myelination in the crushed sciatic nerves. Moreover, silencing of Etv1 by siRNA in SCs in vitro inhibited its migration, proliferation, and differentiation. The results of ChIP-qPCR and luciferase reporter assay showed that Etv1 may regulate SC differentiation and myelination by binding to the promoters of myelination-related genes, such as MAG and Runx2, to initiate their transcription. Conclusions: Taken together, these findings demonstrated a previously unknown role of Etv1 in SC differentiation and myelination, providing a candidate molecular target for clinical interventions in demyelinating diseases.

Decellularization alters the unfavorable regenerative adverse microenvironment of the injured spinal cord to support neurite outgrowth.

Background: Acellular tissue has been transplanted into the injury site as an external microenvironment to intervene with imbalance microenvironment that occurs after spinal cord injury (SCI) and stimulating axonal regeneration, although the mechanism is unclear. Given decellularization is the key means to obtain acellular tissues, we speculated changes in the internal components of tissue caused by decellularization may be the key reason why acellular tissues affect remodeling of the microenvironment. Methods: Complete spinal cord crush in a mouse model was established, and the dynamic of extracellular matrix (ECM) expression and distribution during SCI was studied with immunohistochemistry (IHC). Normal spinal cord (NSC) and 14-day injury spinal cord (ISC) were obtained to prepare the decellularized NSC (DNSC) and decellularized ISC (DISC) through a well-designed decellularization method, and the decellularization effects were evaluated by residual DNA content determination, hematoxylin and eosin staining (H&E), and IHC. Rat dorsal root ganglia (DRG) were co-cultured with NSC, ISC, DNSC, and DISC to evaluate their effect on neurite outgrowth. Furthermore, the mechanisms by which decellularized tissue promotes axonal growth were explored with proteomics analysis of the protein components and function of 14-day ISC and DISC. Results: We found the expression of the four main ECM components (collagen type I and IV, fibronectin, and laminin) gradually increased with the progression of SCI compared to NSC, peaking at 14 days of injury then slightly decreasing at 21 days, and the distribution of the four ECM proteins in the ISC also changed dynamically. H&E staining, residual DNA content determination, and IHC showed decellularization removed cellular components and preserved an intact ECM. The results of co-cultured DRG with NSCs, ISCs, DNSCs, and DISCs showed DNSCs and DISCs had a stronger ability in supporting neurite outgrowth than NSC and ISC. We found through proteomics that decellularization could remove proteins associated with inflammatory responses, scarring, and other pathological factors, while completely retaining the ECM proteins. Conclusions: Taken together, our findings demonstrate decellularization can optimize the imbalanced microenvironment after SCI by removing components that inhibit spinal cord regeneration, providing a theoretical basis for clinical application of acellular tissue transplantation to repair SCI.