Yanling Zhu

Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration

The migration of olfactory ensheathing cells (OECs) is essential for pioneering the olfactory nerve pathway during development and for promoting axonal regeneration when implanted into the injured central nervous system (CNS). In the present study, recombinant Nogo-66 enhanced the adhesion of OECs and inhibited their migration. Using immunocytochemistry and western blot, we showed that the Nogo-66 receptor (NgR) was expressed on OECs. When NgR was released from the cell surface with phosphatidylinositol-specific phospholipase C or neutralized by NgR antibody, the effect of Nogo-66 on OEC adhesion and migration was markedly attenuated. Nogo-66 was found to promote the formation of focal adhesion in OECs and inhibited their membrane protrusion through the activation of RhoA. Furthermore, the co-culture migration assay demonstrated that OEC motility was significantly restricted by Nogo-A expressed on Cos7 cell membranes or oligodendrocytes. Moreover, treatment with anti-NgR antibody facilitated migration of implanted OECs in a spinal cord hemisection injury model. Taken together, we demonstrate, for the first time, that Nogo, a myelin-associated inhibitor of axon regeneration in the CNS, enhances the adhesion and inhibits the migration of OECs via NgR regulation of RhoA.

Glial cell line-derived neurotrophic factor promotes olfactory ensheathing cells migration.

Olfactory ensheathing cells (OECs) are a unique type of macroglia with axonal growth‐promoting properties. The migrating ability of OECs in CNS is essential for neural regeneration. However, little is known about the extracellular and intracellular factors that regulate OEC migration. In the present study, we examined the effects of glial cell line‐derived neurotrophic factor (GDNF) on OECs migration. Initially, the “scratch” migration assay, Boyden chamber assay, and explant migration assay showed that GDNF could promote OECs migration in vitro. Treatment of OECs with GDNF also induced cytoskeleton reorganization and up‐regulated expression of cytoskeleton proteins. GDNF‐induced OECs migration was demonstrated depending on GFRα‐1 and Ret receptor, and activation of JNK and Src signaling cascades. Furthermore, GDNF was found to promote implanted OECs migration in a spinal cord hemisection injury model. Together, we report, to our knowledge for the first time, that GDNF stimulate OECs migration in vitro and in vivo.

Olfactory ensheathing cells promote migration of Schwann cells by secreted nerve growth factor

Transplantation of Schwann cells (SCs) and olfactory ensheathing cells (OECs) have emerged as very promising therapies for spinal cord repair. The important features of interaction between SCs and OECs are beginning to be appreciated, although the underlying mechanism remains unclear. In the present study, we tested the effects of OECs on SCs migration using a range of in vitro migration assays. We found that SCs migrated abundantly upon OECs monolayer, and the migration‐promoting effects were identified to be due to the secreted diffusible factors in OEC‐derived conditioned medium (OEC‐CM). Furthermore, neutralizing nerve growth factor (NGF) in OEC‐CM with NGF antibody could block this effect. Moreover, we found that NGF promotes SCs migration even on astrocyte monolayer. Taken together, these findings provide the first evidence that OECs can promote SCs migration in astrocytic environment by secreted NGF.

Reactive astrocytes inhibit the survival and differentiation of oligodendrocyte precursor cells by secreted TNF-α.

Axonal demyelination is a consistent pathological characteristic of spinal cord injury (SCI). Although an increased number of oligodendrocyte progenitor cells (OPCs) is observed in the injured spinal cord, they fail to convert into mature oligodendrocytes. However, little is known about the underlying mechanism. In our study, we identified a link between inhibition of OPC survival and differentiation and reactive astrocytes in glial scar that was mediated by tumor necrosis factor-α (TNF-α). Initially, both glial scar tissue and reactive astrocyte-conditioned medium were shown to inhibit OPC differentiation. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunochemistry revealed that OPCs expressed type 1 TNF-α receptor (TNF-R1). When TNF-α or TNF-R1 was neutralized with antibody, the effect of reactive astrocyte-conditioned medium or recombinant TNF-α protein on OPC differentiation was markedly attenuated. In addition, reactive astrocyte-conditioned medium was also shown to induce OPC apoptosis. All these findings provide the first evidence that reactive astrocytes release TNF-α to inhibit OPC survival and prevent them from differentiating into mature oligodendrocytes, suggesting a mechanism for the failure of remyelination after SCI.

Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

Olfactory ensheathing cells (OECs) are a unique type of glial cells that have axonal growth-promoting properties. OEC transplantation has emerged as a promising experimental therapy of axonal injuries and demyelinating diseases. However, some fundamental cellular properties of OECs remain unclear. In this study, we found that the distinct OEC subpopulations exhibited different migratory properties based on time-lapse imaging of single isolated cells, possibly due to their different cytoskeletal organizations. Moreover, OEC subpopulations displayed different attractive migratory responses to a gradient of lysophosphatidic acid (LPA) in single-cell migration assays. Finally, we found that OEC subpopulations transformed into each other spontaneously. Together, these results demonstrate, for the first time to our knowledge, that distinct OEC subpopulations display different migratory properties in vitro and provide new evidence to support the notion of OECs as a single cell type with malleable functional phenotypes.

Triptolide promotes spinal cord repair by inhibiting astrogliosis and inflammation

Spinal cord injury (SCI) is a cause of major neurological disability, and no satisfactory treatment is currently available. Traumatic SCI directly damages the cell bodies and/or processes of neurons and triggers a series of endogenous processes, including neuroinflammatory response and reactive astrogliosis. In this study, we found that triptolide, one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, inhibited astrogliosis and inflammation and promoted spinal cord repair. Triptolide was shown to prevent astrocytes from reactive activation by blocking the JAK2/STAT3 pathway in vitro and in vivo. Furthermore, astrocytic gliosis and glial scar were greatly reduced in injured spinal cord treated with triptolide. Triptolide treatment was also shown to decrease the ED‐1 or CD11b‐positive inflammatory cells at the lesion site. Using neurofilament staining and anterograde tracing, a significantly greater number of regenerative axons were observed in the triptolide‐treated rats. Importantly, behavioral tests revealed that injured rats receiving triptolide had improved functional recovery as assessed by the Basso, Beattie, and Bresnahan open‐field scoring, grid‐walk, and foot‐print analysis. These results suggested that triptolide promoted axon regeneration and locomotor recovery by attenuating glial scaring and inflammation, and shed light on the potential therapeutic benefit for SCI.

Olfactory ensheathing cells: The primary innate immunocytes in the olfactory pathway to engulf apoptotic olfactory nerve debris

The olfactory system is an unusual tissue in which olfactory receptor neurons (ORNs) are continuously replaced throughout the life of mammals. Clearance of the apoptotic ORNs corpses is a fundamental process serving important functions in the regulation of olfactory nerve turnover and regeneration. However, little is known about the underlying mechanisms. Olfactory ensheathing cells (OECs) are a unique type of glial cells that wrap olfactory axons and support their continual regeneration from the olfactory epithelium to the bulb. In the present study, OECs were identified to exist in two different states, resting and reactive, in which resting OECs could be activated by LPS stimulation and functioned as phagocytes for cleaning apoptotic ORNs corpses. Confocal analysis revealed that dead ORNs debris were engulfed by OECs and co‐localized with lysosome associated membrane protein 1. Moreover, phosphatidylserine (PS) receptor was identified to express on OECs, which allowed OECs to recognize apoptotic ORNs by binding to PS. Importantly, engulfment of olfactory nerve debris by OECs was found in olfactory mucosa under normal turnover and was significantly increased in the animal model of olfactory bulbectomy, while little phagocytosis by Iba‐1‐positive microglia/macrophages was observed. Together, these results implicate OEC as a primary innate immunocyte in the olfactory pathway, and suggest a cellular and molecular mechanism by which ORNs corpses are removed during olfactory nerve turnover and regeneration.

Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-α in Spinal Cord Lesion of Rat

Background After spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site. Methods and Findings In the present study, we identified a link between OECs migration and reactive astrocytes in glial scar that was mediated by the tumor necrosis factor-α (TNF-α). Initially, the Boyden chamber migration assay showed that both glial scar tissue and reactive astrocyte-conditioned medium promoted OECs migration in vitro. Reactive astrocyte-derived TNF-α and its type 1 receptor TNFR1 expressed on OECs were identified to be responsible for the promoting effect on OECs migration. TNF-α-induced OECs migration was demonstrated depending on activation of the extracellular signal-regulated kinase (ERK) signaling cascades. Furthermore, TNF-α secreted by reactive astrocytes in glial scar was also showed to attract OECs migration in a spinal cord hemisection injury model of rat. Conclusions These findings showed that TNF-α was released by reactive astrocytes in glial scar and attracted OECs migration by interacting with TNFR1 expressed on OECs via regulation of ERK signaling. This migration-attracting effect of reactive astrocytes on OECs may suggest a mechanism for guiding OECs migration into glial scar, which is crucial for OECs-mediated axons regrowth beyond the spinal cord lesion site.

Olfactory ensheathing cells: attractant of neural progenitor migration to olfactory bulb.

Olfactory ensheathing cells (OECs) are the glial cells that derive from the olfactory placode, envelop olfactory axons in the course of migration from the olfactory epithelium to the olfactory bulb and reside primarily in the olfactory nerve layer. OECs transplantation as a promising experimental therapy for axonal injuries has been intensively studied; however, little is known about their roles in olfactory bulb development. In this study, we examined the effects of OECs on the migration of neural progenitors in rostral migratory stream (RMS). Initially, the neurosphere migration assay showed that OEC‐conditioned medium promoted progenitors to migrate from RMS neurospheres in a concentration dependent manner. Moreover, co‐culturing OECs nearby the RMS explants led to asymmetric migration of explants in different developing stages. However, OECs could influence the migration in a distance not further than 1.5 mm. Finally, slice assay that mimic the circumstance in vivo revealed that OECs had a chemoattractive activity on RMS neural progenitors. Together, these results demonstrate that OECs attract neural progenitors in RMS through the release of diffusible factors and it is likely that OECs mainly influence radial migration in the olfactory bulb but not tangential migration of the RMS invivo during development. This suggests a previously unknown function for OECs in olfactory development and a novel mechanism underlying the targeting of RMS cells.

Ethyl pyruvate promotes spinal cord repair by ameliorating the glial microenvironment

BACKGROUND AND PURPOSE Spinal cord injury (SCI) triggers a series of endogenous processes, including neuroinflammation and reactive astrogliosis, which may contribute to the failure of neural regeneration and functional recovery. In the present study, the effect of ethyl pyruvate on spinal cord repair was explored.