PD-1 deficiency aggravates spinal cord injury by regulating the reprogramming of NG2 glia and activating the NgR/RhoA/ROCK signaling pathway.

Spinal cord injury (SCI) is a devastating disorder and a leading cause of disability in adults worldwide. Multiple studies have reported the upregulation of programmed cell death 1 (PD-1) following SCI. However, the underlying mechanism of PD-1 deficiency in SCI is not well established. Therefore, we aimed to investigate the role and potential mechanism of PD-1 in SCI pathogenesis. PD-1 Knockout (KO) SCI mouse model was established, and PD-1 expression was evaluated in tissue samples by western blot assay. We then used a series of function gain-and-loss assays to determine the role of PD-1 in SCI pathogenesis. Moreover, mechanistic assays were performed to explore the association between PD-1, neuron-glia antigen-2 (NG2) glia cells, and miR-23b-5p and then investigated the involved signaling pathway. Results illustrated that PD-1 deficiency enhanced the inflammatory response, neuron loss, and functional impairment induced by SCI. We found that NG2 glia depletion aggravated inflammation, reduced neural survival, and suppressed locomotor recovery in murine SCI model. Further analysis indicated that NG2+ cells were increased in the spinal cord of SCI mice, and PD-1 deficiency increased the number of NG2+ cells by activating the Nogo receptor/ras homolog family member A/Rho kinase (NgR/RhoA/ROCK) signaling. Mechanistically, miR-23b-5p was identified as the negative regulator of PD-1 in NG2 glia. MiR-23b-5p deficiency reduced the expression of inflammatory cytokines, enhanced neural survival, and promoted locomotor recovery in SCI mice, which was counteracted by PD-1 deficiency. In conclusion, PD-1 deficiency exacerbates SCI in vivo by regulating reprogramming of NG2 glia and activating the NgR/RhoA/ROCK signaling.

Lysyl oxidase polymorphisms and susceptibility to osteosarcoma.

Despite the knowledge of many genetic alterations present in osteosarcoma, the complexity of this disease precludes placing its biology into a simple conceptual framework. Lysyl oxidase (LOX) catalyzes the cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. In the current study, we performed genomic sequencing on all seven exons--including the intron-exon splice sites, and the putative promoter region of LOX gene--followed by luciferase reporter assay to analyze the function of newly identified polymorphisms. Associations between LOX polymorphisms and osteosarcoma were then evaluated. Our sequencing data revealed three polymorphisms (-22G/C, 225C/G, and 473G/A) in the exons and promoter region of LOX. The -22G/C polymorphism lies in the downstream core promoter element (DPE) region and caused a decrease in promoter activity of LOX. The prevalence of the -22C allele and 473A allele were significantly increased in osteosarcoma patients compared to controls (odds ratio [OR] = 3.88, 95% confidence interval [CI]= 1.94-7.78, p = 4.18×10(-5), and OR = 1.38, 95%CI = 1.07-1.78, p = 0.013; p 0.0167 was considered significant after Bonferroni correction). Analyzing haplotype showed that the frequency of CCG haplotype (-22, 225, 473) was significantly higher in osteosarcoma cases than in healthy controls after Bonferroni correction (p = 4.46×10(-4)). These results indicate that the -22G/C polymorphism may affect the expression of LOX, and that -22G/C and 473G/A polymorphisms may be new risk factors for osteosarcoma. These findings reveal a potential new pathway by which genetic polymorphisms may affect human diseases.

Lentivirus-siNgR199 promotes axonal regeneration and functional recovery in rats.

BACKGROUND: The limited regenerative capability of central neurons and inhibitory factors are the main causes of axonal regeneration failure after spinal cord injury (SCI). Nogo receptors (NgR) are a family of receptors shared by three factors that inhibit axon outgrowth. In a previous study, siNgR199, an effective lentiviral siRNA vector of NgR1, was constructed and transfected into cortical neurons in vitro, and it effectively promoted axon outgrowth. The present study focused on the therapeutic effect of delivery of a recombinant lentivirus containing siNgR199 in vivo in rats. METHODS: Rat models of traumatic SCI were constructed according to the method developed by Allen. The animals were randomly divided into three groups: group 1, injected with physiological saline; group 2, injected with empty lentivirus vehicle; and group 3, injected with lentivirus carrying siNgR199. The Basso, Beattie, and Bresnahan (BBB) scale was used for assessing hindlimb locomotor function after SCI. The neural tracer biotinylated dextran amine (BDA) and immunohistochemical methods were employed to study axon outgrowth. RESULTS: After injection for 8 weeks, BBB locomotion scores showed that the motor function of the hindlimb recovered better in animals in group 3 (injected with lentivirus carrying siNgR199) than those in groups 1 and 2, which were injected with saline and empty lentivirus vehicle, respectively. In group 3, regenerative nerve fibers were observed at and across the injury site, while very few axonal sprouts were observed in groups 1 and 2. CONCLUSIONS: Injection with lentivirus carrying siNgR199 into the sensorimotor cortex improved axonal regeneration and functional recovery of hindlimb after SCI in rats.

Effect of lentiviral shRNA of Nogo receptor on rat cortex neuron axon outgrowth.

BACKGROUND AND AIMS: Axon growth is crucial for injured neural tissue to recover; however it is difficult to achieve in general. Axon outgrowth is inhibited by the activation of the Nogo receptor (NgR) by one of three different ligands. The present study aimed to suppress the inhibitory effect of the three inhibitory proteins to facilitate axon outgrowth. METHODS: A lentiviral vector, siNgR199 (that has the capacity to interfere with the gene of NgR expression), was constructed for suppressing the gene transcription of NgR. Rat cortex neurons and oligodendrocytes were prepared to observe the effect of siNgR199 on facilitating axon outgrowth. RESULTS: After transfection, the lentiviral siRNA of NgR remained in target neurons for almost two weeks whereas the conventional siRNA of NgR remained in neurons less than five days. Lentivirus-mediated delivery of exogenous small interfering RNA (siNgR199) targeting NgR significantly reduced the expression of this receptor and promoted axon outgrowth. In contrast, provision of naked siRNA targeting NgR (NgRsiRNA) showed less inhibitory effect on NgR protein expression and did not affect axon outgrowth. CONCLUSIONS: Lentiviral siRNA of NgR effectively suppresses the expression of NgR in cultured neurons that facilitates the axon outgrowth. The data implicate that lentiviral siRNA of NgR has therapeutic potential in facilitating the recovery of injured neural tissue.