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Neural stem cell small extracellular vesicle-based delivery of 14-3-3t reduces apoptosis and neuroinflammation following traumatic spinal cord injury by enhancing autophagy by targeting Beclin-1.

ABSTRACT: Neural stem cell-derived small extracellular vesicles (NSC-sEVs) play an important role in the repair of tissue damage. Our previous in vitro and in vivo studies found that preconditioning with NSC-sEVs promoted the recovery of functional behaviors following spinal cord injury by activating autophagy. However, the underlying mechanisms for such observations remain unclear. In this study, we further explored the mechanisms by which NSC-sEVs repair spinal cord injury via autophagy. We found that NSC-sEVs contain 14-3-3t protein, of which the overexpression or knockdown enhanced and decreased autophagy, respectively. In addition, 14-3-3t overexpression enhanced the anti-apoptotic and anti-inflammatory effects of NSC-sEVs, further promoting functional behavior recovery following spinal cord injury. The overexpression of 14-3-3t was used to further validate the in vivo results through a series of in vitro experiments. Conversely, knockdown of 14-3-3t attenuated the anti-apoptotic and anti-inflammatory effects of NSC-sEVs. Further studies also confirmed that NSC-sEVs increased Beclin-1 expression, with which 14-3-3t interacted and promoted its localization to autophagosome precursors. In this study, we found that NSC-sEVs deliver 14-3-3t, which interacts with Beclin-1 to activate autophagy. Our results indicate that 14-3-3t acts via a newly-discovered mechanism for the activation of autophagy by NSC-sEVs.

SUBMITTER: Rong Y

PROVIDER: S-EPMC6782003 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Neural stem cell small extracellular vesicle-based delivery of 14-3-3t reduces apoptosis and neuroinflammation following traumatic spinal cord injury by enhancing autophagy by targeting Beclin-1.

Rong Yuluo Y Liu Wei W Lv Chengtang C Wang Jiaxing J Luo Yongjun Y Jiang Dongdong D Li Linwei L Zhou Zheng Z Zhou Wei W Li Qingqing Q Yin Guoyong G Yu Lipeng L Fan Jin J Cai Weihua W

Aging 20190928 18

Neural stem cell-derived small extracellular vesicles (NSC-sEVs) play an important role in the repair of tissue damage. Our previous <i>in vitro</i> and <i>in vivo</i> studies found that preconditioning with NSC-sEVs promoted the recovery of functional behaviors following spinal cord injury by activating autophagy. However, the underlying mechanisms for such observations remain unclear. In this study, we further explored the mechanisms by which NSC-sEVs repair spinal cord injury via autophagy. W ...[more]

PMID: 31563124

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Project description:BackgroundSpinal cord injury (SCI) is a severe neurological disorder with many disabling consequences, including persistent neuropathic pain, which develops in about 40 % of SCI patients and is induced and sustained by excessive and uncontrolled spinal neuroinflammation. Here, we have evaluated the effects of lipoxin A4 (LXA4), a member of a unique class of endogenous lipid mediators with both anti-inflammatory and analgesic properties, on spinal neuroinflammation and chronic pain in an experimental model of SCI.MethodsSpinal hemisection at T10 was carried out in adult male CD1 mice and Wistar rats. To test if LXA4 can reduce neuroinflammation and neuropathic pain, each animal received two intrathecal injections of LXA4 (300 pmol) or vehicle at 4 and 24 h after SCI. Sensitivity to mechanical stimulation of the hind paws was evaluated using von Frey monofilaments, and neuroinflammation was tested by measuring the mRNA and/or protein expression levels of glial markers and cytokines in the spinal cord samples after SCI. Also, microglia cultures prepared from murine cortical tissue were used to assess the direct effects of LXA4 on microglial activation and release of pro-inflammatory TNF-α.ResultsLXA4 treatment caused significant reductions in the intensity of mechanical pain hypersensitivity and spinal expression levels of microglial markers and pro-inflammatory cytokines induced by SCI, when compared to rodents receiving control vehicle injections. Notably, the increased expressions of the microglial marker IBA-1 and of the pro-inflammatory cytokine TNF-α were the most affected by the LXA4 treatment. Furthermore, cortical microglial cultures expressed ALX/FPR2 receptors for LXA4 and displayed potentially anti-inflammatory responses upon challenge with LXA4.ConclusionsCollectively, our results suggest that LXA4 can effectively modulate microglial activation and TNF-α release through ALX/FPR2 receptors, ultimately reducing neuropathic pain in rodents after spinal cord hemisection. The dual anti-inflammatory and analgesic properties of LXA4, allied to its endogenous nature and safety profile, may render this lipid mediator as new therapeutic approach for treating various neuroinflammatory disorders and chronic pain with only limited side effects.

Project description:BACKGROUND:The prognosis of spinal cord injury (SCI) is closely related to secondary injury, which is dominated by neuroinflammation. There is evidence that ?-synuclein aggregates after SCI and that inhibition of ?-synuclein aggregation can improve the survival of neurons after SCI, but the mechanism is still unclear. This study was designed to investigate the effects of ?-synuclein on neuroinflammation after SCI and to determine the underlying mechanisms. METHOD:A T3 spinal cord contusion model was established in adult male Sprague-Dawley rats. An SNCA-shRNA-carrying lentivirus (LV-SNCA-shRNA) was injected into the injury site to block the expression of ?-synuclein (forming the SCI+KD group), and the SCI and sham groups were injected with an empty vector. Basso-Beattie-Bresnahan (BBB) behavioural scores and footprint analysis were used to detect motor function. Inflammatory infiltration and myelin loss were measured in the spinal cord tissues of each group by haematoxylin-eosin (HE) and Luxol Fast Blue (LFB) staining, respectively. Immunohistochemistry, Western blot analysis, and RT-qPCR were used to analyse protein expression and transcription levels in the tissues. Immunofluorescence was used to determine the morphology and function of glial cells and the expression of matrix metalloproteinase-9 in the central canal of the spinal cord. Finally, peripheral serum cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS:Compared with the SCI group, the SCI+KD group exhibited reduced inflammatory infiltration, preserved myelin, and functional recovery. Specifically, the early arrest of ?-synuclein inhibited the pro-inflammatory factors IL-1?, TNF-?, and IL-2 and increased the expression of the anti-inflammatory factors IL-10, TGF-?, and IL-4. The neuroinflammatory response was regulated by reduced proliferation of Iba1+ microglia/macrophages and promotion of the shift of M1-polarized Iba1+/iNOS+ microglia/macrophages to M2-polarized Iba1+/Arg1+ microglia/macrophages after injury. In addition, compared with the SCI group, the SCI+KD group also exhibited a smaller microglia/astrocyte (Iba1/GFAP) immunostaining area in the central canal, lower MMP-9 expression, and improved cerebrospinal barrier function. CONCLUSION:Lentivirus-mediated downregulation of ?-synuclein reduces neuroinflammation, improves blood-cerebrospinal barrier function, promotes functional recovery, reduces microglial activation, and promotes the polarization of M1 microglia/macrophages to an M2 phenotype to confer a neuroprotective immune microenvironment in rats with SCI.

Project description:Spinal cord injury (SCI) can cause severe irreversible motor dysfunction and even death. Neural stem cell (NSC) transplantation can promote functional recovery after acute SCI in experimental animals, but numerous issues, including low-transplanted cell survival rate, cell de-differentiation, and tumor formation need to be resolved before routine clinical application is feasible. Recent studies have shown that transplanted stem cells facilitate regeneration through release of paracrine factors. Small extracellular vesicles (sEVs), the smallest known membrane-bound nanovesicles, are involved in complex intercellular communication systems and are an important vehicle for paracrine delivery of therapeutic agents. However, the application of NSC-derived small extracellular vesicles (NSC-sEVs) to SCI treatment has not been reported. We demonstrate that NSC-sEVs can significantly reduce the extent of SCI, improve functional recovery, and reduce neuronal apoptosis, microglia activation, and neuroinflammation in rats. Furthermore, our study suggests that NSC-sEVs can regulate apoptosis and inflammatory processes by inducing autophagy. In brief, NSC-sEVs increased the expression of the autophagy marker proteins LC3B and beclin-1, and promoted autophagosome formation. Following NSC-sEV infusion, the SCI area was significantly reduced, and the expression levels of the proapoptotic protein Bax, the apoptosis effector cleaved caspase-3, and the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly reduced, whereas the expression level of the anti-apoptotic protein Bcl-2 was upregulated. In the presence of the autophagy inhibitor 3MA, however, these inhibitory effects of NSC-sEVs on apoptosis and neuroinflammation were significantly reversed. Our results show for the first time that NSC-sEV treatment has the potential to reduce neuronal apoptosis, inhibit neuroinflammation, and promote functional recovery in SCI model rats at an early stage by promoting autophagy.

Dataset Information

Neural stem cell small extracellular vesicle-based delivery of 14-3-3t reduces apoptosis and neuroinflammation following traumatic spinal cord injury by enhancing autophagy by targeting Beclin-1.

Publications

Neural stem cell small extracellular vesicle-based delivery of 14-3-3t reduces apoptosis and neuroinflammation following traumatic spinal cord injury by enhancing autophagy by targeting Beclin-1.

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