Aijun Huang

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.

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.

Glial cell line-derived neurotrophic factor promotes survival and induces differentiation through the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathway respectively in PC12 cells.

PC12-GFRalpha1 cells, a clonal cell line engineered to express glial cell line-derived neurotrophic factor receptor alpha1 were constructed. Given glial cell line-derived neurotrophic factor could induce the differentiation and promote the survival of PC12-GFRalpha1 cells at low concentrations, the cells provide an unlimited source of monoclonal cells for studies on the signal transduction pathway of glial cell line-derived neurotrophic factor. To characterize the involvement of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in the biological effect of glial cell line-derived neurotrophic factor, we used the mitogen-activated protein kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor LY294002. PD98059 blocked glial cell line-derived neurotrophic factor-induced PC12-GFRalpha1 cells neurite formation in a dose-dependent manner, without significantly altering cell viability. LY294002 reversed the survival-promoting effect of glial cell line-derived neurotrophic factor on the PC12-GFRalpha1 cells in serum-deprived medium. The present study demonstrates that phosphatidylinositol 3-kinase pathway seems to mediate the survival-promoting effect of glial cell line-derived neurotrophic factor on PC12-GFRalpha1 cells, while the activation of mitogen-activated protein kinase pathway could be an important step in mediating PC12-GFRalpha1 cells differentiation induced by glial cell line-derived neurotrophic factor. Therefore, it is inferred that similar intracellular signaling components are used by distinct growth factors toward a common biological effect.

The effects of ciliary neurotrophic factor on neurological function and glial activity following contusive spinal cord injury in the rats

Ciliary neurotrophic factor (CNTF) has been implicated in the pathophysiology of injury to the central nervous system. The rapid increase in CNTF production following spinal cord injury (SCI) in rats is thought to serve a role in the neuronal survival and functional recovery. In this study, 40 SD rats were divided into four groups: sham-operated group, saline-treated group, 5- and 10-microg CNTF group. Saline and CNTF were given through lumbar intrathecal catheter for 10 days after T10 segment of spinal cord were injured by modified Allen contusion method. Animals were behaviorally tested for 6 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and inclined plane test. At the end of 6 week, rubrospinal neurons of five rats in each group were labeled by retrograde transport of the horseradish peroxidase (HRP) from the lesion site, and then the labeled red nucleus neuron (RN) numbers were counted. Additional rats were histologically assessed for tissue sparing and neuronal loss and reactive gliosis at the injury site and adjacent areas. Rats treated with CNTF regained greater improvements in hindlimb function than controls. The amount of spared tissue was significantly higher in CNTF-treated animals than in controls. After CNTF treatment, the number of HRP-labeled RN neurons were significantly increased. Astrocytes and microglia reactivity was more pronounced in CNTF-treated animals than in controls. These results indicate that intrathecal infusion of exogenous CNTF following SCI may significantly reduce tissue damage and protect the rubrospinal descending tracks and enhances functional recovery, and may also induce more gliosis.

MSX3 Switches Microglia Polarization and Protects from Inflammation-Induced Demyelination

The major challenge for progressive multiple sclerosis therapy is the promotion of remyelination from inflammation-induced demyelination. A switch from an M1- to an M2-dominant polarization of microglia is critical in these repair processes. In this study, we identified the homeobox gene msh-like homeobox-3 (Msx3) as a new pivotal regulator for microglial polarization. MSX3 was induced during microglia M2 polarization and repressed in M1 cells. The expression of MSX3 in microglia was dynamically regulated during experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. The overexpression of MSX3 in microglia promoted M2 but impeded M1 polarization. Interrupting MSX3 expression in microglia accelerated inflammation-induced demyelination and neurodegeneration. The conditioned medium from MSX3-transduced microglia promoted oligodendrocyte progenitor survival, differentiation, and neurite outgrowth. The adoptive transfer of MSX3-transduced microglia suppressed EAE and facilitated remyelination within the murine CNS in EAE and the LPC model. Mechanically, chromatin immunoprecipitation assays also indicated that MSX3 directly regulated three key genes associated with microglia M2 polarization, including Pparg, Stat6, and Jak3. Importantly, we found that overexpression of MSX3 in human-derived microglia represents the M2 phenotype and ameliorated EAE after intraventricular injection. Our findings suggest a new homeobox protein-dependent mechanism for driving microglia M2 polarization and identify MSX3 as an attractive therapeutic approach for preventing inflammation-induced demyelination and promoting remyelination.

Rap1GAP interacts with RET and suppresses GDNF-induced neurite outgrowth.

Glial cell line-derived neurotrophic factor (GDNF) was originally recognized for its ability to promote survival of midbrain dopaminergic neurons, but it has since been demonstrated to be crucial for the survival and differentiation of many neuronal subpopulations, including motor neurons, sympathetic neurons, sensory neurons and enteric neurons. To identify possible effectors or regulators of GDNF signaling, we performed a yeast two-hybrid screen using the intracellular domain of RET, the common signaling receptor of the GDNF family, as bait. Using this approach, we identified Rap1GAP, a GTPase-activating protein (GAP) for Rap1, as a novel RET-binding protein. Endogenous Rap1GAP co-immunoprecipitated with RET in neural tissues, and RET and Rap1GAP were co-expressed in dopaminergic neurons of the mesencephalon. In addition, overexpression of Rap1GAP attenuated GDNF-induced neurite outgrowth, whereas suppressing the expression of endogenous Rap1GAP by RNAi enhanced neurite outgrowth. Furthermore, using co-immunoprecipitation analyses, we found that the interaction between RET and Rap1GAP was enhanced following GDNF treatment. Mutagenesis analysis revealed that Tyr981 in the intracellular domain of RET was crucial for the interaction with Rap1GAP. Moreover, we found that Rap1GAP negatively regulated GNDF-induced ERK activation and neurite outgrowth. Taken together, our results suggest the involvement of a novel interaction of RET with Rap1GAP in the regulation of GDNF-mediated neurite outgrowth.

LncRNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination

The regulation of inflammation is pivotal for preventing the development or reoccurrence of multiple sclerosis (MS). A biased ratio of high‐M1 versus low‐M2 polarized microglia is a major pathological feature of MS. Here, using microarray screening, we identify the long noncoding RNA (lncRNA) GAS5 as an epigenetic regulator of microglial polarization. Gain‐ and loss‐of‐function studies reveal that GAS5 suppresses microglial M2 polarization. Interference with GAS5 in transplanted microglia attenuates the progression of experimental autoimmune encephalomyelitis (EAE) and promotes remyelination in a lysolecithin‐induced demyelination model. In agreement, higher levels of GAS5 are found in amoeboid‐shaped microglia in MS patients. Further, functional studies demonstrate that GAS5 suppresses transcription of TRF4, a key factor controlling M2 macrophage polarization, by recruiting the polycomb repressive complex 2 (PRC2), thereby inhibiting M2 polarization. Thus, GAS5 may be a promising target for the treatment of demyelinating diseases.

Neuroprotective effects of nitidine against traumatic CNS injury via inhibiting microglia activation

Glial cell response to injury has been well documented in the pathogenesis after traumatic brain injury (TBI) and spinal cord injury (SCI). Although microglia, the resident macrophages in the central nervous system (CNS), are responsible for clearing debris and toxic substances, excessive activation of these cells will lead to exacerbated secondary damage by releasing a variety of inflammatory and cytotoxic mediators and ultimately influence the subsequent repair after CNS injury. In fact, inhibition of microgliosis represents a therapeutic strategy for CNS trauma. We here showed that nitidine, a benzophenanthridine alkaloid, restricted reactive microgliosis and promoted CNS repair after traumatic injury. Nitidine was shown to prevent cultured microglia from LPS-induced reactive activation by regulation of ERK and NF-κB signaling pathway. Furthermore, the nitidine-mediated inhibition of microgliosis was also shown in injured brain and spinal cord, which significantly increased neuronal survival and decreased neural tissue damage after injury. Importantly, behavioral analysis revealed that nitidine-treated mice with SCI had improved functional recovery as assessed by Basso Mouse Scale and swimming test. Together, these findings indicated that nitidine increased CNS tissue sparing and improved functional recovery by attenuating reactive microgliosis, suggestive of the potential therapeutic benefit for CNS injury.

Disulfiram and Diphenhydramine Hydrochloride Upregulate miR-30a to Suppress IL-17-Associated Autoimmune Inflammation

T-helper 17 (Th17) cells play an important role in the pathogenesis of multiple sclerosis (MS), an autoimmune demyelinating disease that affects the CNS. In the present study, MicroRNA sequencing (miRNA-seq) was performed in mouse Th0 and Th17 cells to determine the critical miRNAs that are related to Th17 differentiation. We found that miR-30a was significantly downregulated during mouse Th17 differentiation. In addition, the level of miR-30a in CD4+ T cells from peripheral blood of MS patients and experimental autoimmune encephalomyelitis (EAE) animal models was also decreased and inversely correlated with the expression of interleukin 17a, the canonical cytokine of Th17 cells. Moreover, overexpression of miR-30a inhibited Th17 differentiation and prevented the full development of EAE, whereas interference of miR-30a promoted Th17 differentiation. Mechanism studies showed that miR-30a reduced IRF4 expression by specifically binding with the 3′-untranslated region. Through screening of 640 different Food and Drug Administration (FDA)-approved drugs, we found that disulfiram and diphenhydramine hydrochloride were effective candidates for inhibiting Th17 differentiation and ameliorating EAE development through upregulating miR-30a. To our knowledge, the present work is not only the first miRNA-seq study focusing on Th17 differentiation, but also the first chemical screening for FDA-approved drugs that inhibit Th17 differentiation through regulating miRNA expression. SIGNIFICANCE STATEMENT The present work is the first miRNA sequencing (miRNA-seq) study focusing on T-helper 17 (Th17) differentiation. By miRNA deep sequencing, we found that miR-30a was downregulated during Th17 differentiation. miR-30a was also decreased in CD4+ T cells from multiple sclerosis patients and experimental autoimmune encephalomyelitis (EAE) mice. miR-30a reduced IRF4 expression by specific binding with the 3′-untranslated region and thus suppressed Th17 differentiation and prevented the full development of EAE. Interestingly, by performing a chemical screen with Food and Drug Administration-approved small-molecule drugs, we found that disulfiram and diphenhydramine upregulated miR-30a and suppressed Th17-associated autoimmune demyelination.