Peripheral Macrophage-derived Exosomes promote repair after Spinal Cord Injury by inducing Local Anti-inflammatory type Microglial Polarization via Increasing Autophagy

Treatment for spinal cord injury (SCI) remains a challenge worldwide, and inflammation is a major cause of secondary injury after SCI. Peripheral macrophages (PMs) have been verified as a key factor that exert anti-inflammatory effects after SCI, but the mechanism is unidentified. As local macrophages, microglia also exert significant effects after SCI, especially polarization. Exosomes show source cell-like biological functions to target cells and have been the subject of much research in recent years. Thus, we hypothesized the PM-derived exosomes (PM-Exos) play an important role in signal transmission with local microglia and can be used therapeutic agents for SCI in a series of in vivo and in vitro studies. For the in vivo experiment, three groups of Sprague-Dawley (SD) rats subjected to spinal cord contusion injury were injected with 200 µg/ml PM-Exos, 20 µg/ml PM-Exos or PBS via the tail vein. Recovery of the rats and of spinal cord function were observed. In vitro, we investigated the potential anti-inflammatory mechanism of PM-Exos and evaluated microglial autophagy, anti-inflammatory type microglia polarization and the upstream signaling pathway. The results showed that spinal cord function and recovery were better in the PM-Exo groups than the control group. In the in vitro study, microglial autophagy levels and the expression of anti-inflammatory type microglia were higher in the experimental groups than the control group. Moreover, the expression of proteins related to the PI3K/AKT/mTOR autophagic signaling pathway was suppressed in the PM-Exo groups. PM-Exos have a beneficial effect in SCI, and activation of microglial autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway, enhancing the polarization of anti-inflammatory type microglia, that may play a major role in the anti-inflammatory process.     

基于PI3K/PTEN/AKT信号途径探讨褪黑素对脊髓损伤大鼠突触可塑性的影响

目的:探讨褪黑素对脊髓损伤大鼠突触可塑性的影响及磷脂酰肌醇3-激酶/张力蛋白同源基因/蛋白激酶B(PI3K/PTEN/AKT)信号途径在其中的作用。方法:选择4月龄SPF级雄性SD大鼠48只,将其随机分为对照组(CON)、模型组(SCI)、褪黑素组(MT)和褪黑素受体拮抗剂组(LUZ),每组12只大鼠。对照组大鼠背部切口后缝合,余下各组大鼠使用改良的Allen's法建立T9水平的脊髓损伤模型。模型建立后,褪黑素组及褪黑素受体拮抗剂组每天腹腔注射褪黑素及褪黑素抑制剂,剂量为12.5 mg·kg~(-1)·d~(-1),对照组和模型组每天注射同体积的生理盐水。治疗后第3、7、14、21、28天进行BBB评分,实验结束处死大鼠取胸椎8-10节段脊髓组织,分别采用免疫组化方法测尼氏小体数量及Western Blot检测PTEN、Synapsin、PSD-95、Gap-43、Akt蛋白的表达。结果:与SCI模型大鼠相比,MT给药干预14 d后的SCI大鼠BBB评分及痛觉压力值均明显降低(P0.05),尼氏小体灰度值提高(P 0.05),PTEN、Synapsin、PSD-95、Gap-43、Akt蛋白的表达均显著上调(P 0.05)。结论:MT可能通过激活PI3K/PTEN/Akt信号途径,上调突触可塑性相关蛋白的表达,促进SCI大鼠突触修复。     

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.     

Aquaporin-4 Mitigates Retrograde Degeneration of Rubrospinal Neurons by Facilitating Edema Clearance and Glial Scar Formation After Spinal Cord Injury in Mice

Atrophy of upper motor neurons hampers axonal regeneration and functional recovery following spinal cord injury (SCI). Apart from the severity of primary injury, a series of secondary pathological damages including spinal cord edema and glial scar formation affect the fate of injured upper motor neurons. The aquaporin-4 (AQP4) water channel plays a critical role in water homeostasis and migration of astrocytes in the central nervous system, probably offering a new therapeutic target for protecting against upper motor neuron degeneration after SCI. To test this hypothesis, we examined the effect of AQP4 deficiency on atrophy of rubrospinal neurons after unilateral rubrospinal tract transection at the fourth cervical level in mice. AQP4 gene knockout (AQP4^?/?) mice exhibited high extent of spinal cord edema at 72 h after lesion compared with wild-type littermates. AQP4^?/? mice showed impairments in astrocyte migration toward the transected site with a greater lesion volume at 1 week after surgery and glial scar formation with a larger cyst volume at 6 weeks. More severe atrophy and loss of axotomized rubrospinal neurons as well as axonal degeneration in the rubrospinal tract rostral to the lesion were observed in AQP4^?/? mice at 6 weeks after SCI. AQP4 expression was downregulated at the lesioned spinal segment at 3 days and 1 week after injury, but upregulated at 6 weeks. These results demonstrated that AQP4 not only mitigates spinal cord damage but also ameliorates retrograde degeneration of rubrospinal neurons by promoting edema clearance and glial scar formation after laceration SCI. This finding supports the notion that AQP4 may be a promising therapeutic target for SCI.     

Antioxidant effect of quercetin against acute spinal cord injury in rats and its correlation with the p38MAPK/iNOS signaling pathway

AimsThe present study aimed to investigate the correlation between quercetin (Que) and the p38 mitogen-activated protein kinase (p38MAPK)/inducible nitric oxide synthase (iNOS) signaling pathway and to explore its regulating effect on secondary oxidative stress following acute spinal cord injury (SCI), so as to elucidate the protective effects and mechanism associated with Que treatment during acute SCI.Main methodsSprague–Dawley rats were randomly divided into sham-surgery, SCI, Que, methylprednisolone (MP), and specific p38MAPK inhibitor SB203580 treatment groups. Acute SCI models were established in rats by a modified Allen's method. Real-time PCR analysis, western blot assay and immunohistochemistry for molecular changes in the p38MAPK/iNOS signaling pathway, determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, reflecting the levels of secondary oxidative stress, and functional or behavioral data, reflecting changes induced by Que and control treatments post-SCI were performed.Key findingsQue significantly increased Basso, Beattie and Bresnahan scores and inclined plane test scores in SCI rats similar to the positive control drug, MP. Que significantly inhibited increases in phosphorylated-p38MAPK (p-p38MAPK) and iNOS expression and reduced the rate of iNOS-positive cells in rats with SCI, similar to the effects of SB203580. In addition, both Que and SB203580 reduced MDA content and enhanced SOD activity in SCI rats, with Que effects being stronger.SignificanceThese experimental findings indicate that in SCI rats, Que has protective effects on the spinal cord by the potential mechanism of inhibiting the activation of p38MAPK/iNOS signaling pathway and thus regulating secondary oxidative stress.     

bFGF通过抑制Nogo-A信号减少脊髓损伤后胶质瘢痕形成

脊髓损伤后胶质瘢痕的形成是阻碍神经恢复的关键原因之一。碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）具有良好的神经保护及促进脊髓损伤的修复作用,然而其对于胶质瘢痕的影响及其机制仍不清楚。本研究通过采用血管动脉夹（30 g）夹闭雌性SD大鼠脊髓2 min造成急性脊髓损伤模型并予以每天皮下注射bFGF（80 μg/kg）,探讨bFGF促进脊髓损伤的恢复作用是否涉及到胶质瘢痕调控和Nogo-A/NgR信号的相关机制。通过检测损伤后28 d,各组BBB评分和斜板试验,发现bFGF显著促进脊髓损伤后大鼠运动功能的恢复。HE及尼氏染色显示,bFGF处理组相对于生理盐水处理组,其神经元明显增多,空洞面积减少。同时,星形胶质细胞标记物GFAP免疫荧光结果表明,bFGF减少胶质瘢痕形成,抑制星形胶质细胞过度激活。同样,通过Western 印迹检测发现,bFGF处理后,胶质瘢痕相关蛋白（如GFAP, neurocan）以及神经突生长抑制蛋白（Nogo-A）信号通路相关蛋白质表达量下降。上述结果表明,bFGF可能通过抑制Nogo-A信号蛋白的表达,从而抑制胶质瘢痕的形成,促进脊髓损伤的恢复。此机制研究为脊髓损伤的治疗和恢复提供全新的思路和药物靶点。     

Thrombin Enhances NGF-Mediated Neurite Extension via Increased and Sustained Activation of p44/42 MAPK and p38 MAPK

Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling.     

The PI3K/AKT Pathway and Renal Cell Carcinoma

The phosphatidylinositol 3 kinase(PI3K)/AKT pathway is genetically targeted in more pathway components and in more tumor types than any other growth factor signaling pathway,and thus is frequently activated as a cancer driver.More importantly,the PI3K/AKT pathway is composed of multiple bifurcating and converging kinase cascades,providing many potential targets for cancer therapy.Renal cell carcinoma(RCC) is a high-risk and high-mortality cancer that is notoriously resistant to traditional chemotherapies or radiotherapies.The PI3K/AKT pathway is modestly mutated but highly activated in RCC,representing a promising drug target.Indeed,PI3 K pathway inhibitors of the rapalog family are approved for use in RCC.Recent large-scale integrated analyses of a large number of patients have provided a molecular basis for RCC,reiterating the critical role of the PI3K/AKT pathway in this cancer.In this review,we summarize the genetic alterations of the PI3K/AKT pathway in RCC as indicated in the latest large-scale genome sequencing data,as well as treatments for RCC that target the aberrant activated PI3K/AKT pathway.     

Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice

Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n = 22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.     

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord

Background aimsIn this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI).MethodsShort hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale.ResultsAnimals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment.ConclusionsThe improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.     

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells

Background

Acute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury.

Results

Enhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H₂O₂ toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS.

Conclusions

Our data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.     

Characteristics of the PI3K/AKT and MAPK/ERK pathways involved in the maintenance of self-renewal in lung cancer stem-like cells

Lung cancer is the leading cause of cancer-related mortality worldwide due to its early asymptomatic and late metastasis. While cancer stem cells (CSCs) may play a vital role in oncogenesis and development of lung cancer, mechanisms underlying CSCs self‐renewal remain less clear. In the present study, we constructed a clinically relevant CSCs enrichment recognition model and evaluated the potential functions of phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT) and mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathways in lung cancer via bioinformatic analysis, providing the basis for in depth mechanistic inquisition. Experimentally, we confirmed that PI3K/AKT pathway predominantly promotes proliferation through anti-apoptosis in lung adenocarcinoma cells, while MAPK/ERK pathway has an overwhelming superiority in regulating the proliferation in lung CSCs. Further, utilizing stemness score model, LLC-Symmetric Division (LLC-SD) cells and mouse orthotopic lung transplantation model, we elucidated an intricate cross-talk between the oncogenic pathway and the stem cell reprograming pathway that impact stem cell characteristics as well as cancer biology features of lung CSCs both in vitro and in vivo. In summary, our findings uncovered a new insight that PI3K/AKT and MAPK/ERK pathways as oncogenic signaling pathway and/or stem cell signaling pathway act distinctively and synergistically to regulate lung CSCs self-renewal.     

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown.Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations.Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro.Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.     

miRNAs在脊髓损伤中作用的研究进展

脊髓损伤是一个重要的公共卫生难题,脊髓损伤可划分为三个病理生理阶段:原发性损伤期、继发性损伤期和慢性损伤期。基因表达的改变在脊髓损伤中起到了重要作用,miRNAs可以调控转录后所有基因的表达,所以miRNAs是脊髓损伤中一个很具有研究价值的研究对象。miRNAs是20-25碱基组成的非编码RNA,通过与靶mRNAs 3‘UTR结合下调其表达实现的对mRNA翻译进程的调控。miRNAs与中枢神经系统的发育、功能和疾病有密切关系。脊髓损伤后miRNAs通过调节中性粒细胞和炎性反应通路在炎性应答中起到了重要作用;miRNAs在细胞凋亡中表现出了复杂的功能,其表达的改变可能同时刺激和抑制凋亡;miRNAs可通过增强星形胶质细胞肥大和调节胶质瘢痕的进程;miRNAs的下调可能通过促进轴突靶向作用、神经元存活和轴突生长来促进损伤脊髓部位再生进程。目前脊髓损伤仍是现代医学的难题,对神经系统疾病中miRNAs作用的研究,为脊髓损伤治疗提供了一种新的治疗方案,也是将来研究中的热点。     

Regulation of Autophagy and Ubiquitinated Protein Accumulation by bFGF Promotes Functional Recovery and Neural Protection in a Rat Model of Spinal Cord Injury

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.     

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.