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    Knockdown of polypyrimidine tract binding protein facilitates motor function recovery after spinal cord injury
  • Figure 1|Direct reprogramming of primary murine reactive spinal astrocytes into motoneuron-like cells by PTB knockdown in vitro.

    Given the demonstrated direct neuronal conversion of cultured mouse cortical astrocytes after shPTB treatment (Qian et al., 2020), we explored whether PTB silencing led to the conversion of reactive mouse spinal astrocytes into motor neurons in vitro. In a reactive astrocyte model, we treated primary murine spinal astrocytes with lipopolysaccharide (Additional Figure 2A and B). Next, we transduced reactive spinal astrocytes from mice with a lentiviral vector containing a Gfap promoter driving the expression of an shRNA against mouse Ptbp1. When astrocytes were transduced with this vector for 2 days, shPTB significantly downregulated both the protein expression of PTB and mRNA expression of Ptbp1 (Figure 1A–C). At 2 weeks post-reprogramming, shPTB-infected cells showed complex neurite outgrowth, and, by 4 weeks, these cells displayed obvious neuronal morphology, whereas cells transduced with the shCtrl displayed an astrocyte-like flattened and polygonal morphology (Figure 1D). Three weeks after transduction, approximately 41% of shPTB-transduced GFP+ cells demonstrated positive staining for the neuronal marker MAP2 (Figure 1E and F). At 4 weeks post-conversion, the percentage of GFP+/MAP2+ cells was approximately 58%, and approximately 12% of GFP+ cells exhibited expression of the motor neuron marker ChAT (Figure 1G and H). In contrast, cells transduced with the shCtrl lentiviral vector exhibited positive staining only for GFP (Figure 1E and G). These data demonstrate that reactive spinal astrocytes can be reprogrammed into motoneuron-like cells by PTB silencing in vitro.


    Figure 2|Localization of shPTB delivered by AAV in the spinal cord of mice.

    SCI induces the loss of a large number of neurons in the injured area (Anderson et al., 2016). To determine whether PTB silencing replenished neurons, including motor neurons, in an SCI mouse model, we injected AAVs into the oblique opposite sides of the injured spinal area. We used the Gfap promoter to drive shPTB expression. A large number of GFP+ cells were colocalized with GFAP+ astrocytes 1 week after injection (Figure 2A), whereas the GFP+ cells were rarely colocalized with ChAT+ motor neurons (Figure 2B). Quantitatively, approximately 75% of GFP+ cells were colocalized with GFAP+ astrocytes, but no GFP+ cells were colocalized with ChAT+ motoneurons (Figure 2C). 


    Figure 3|PTB knockdown replenished motoneuron-like cells around the injured area after SCI in mice. 

    Moreover, the GFP+ cells did not colocalize with FoxJ1+ neural stem cells (Additional Figure 3A and B). Eleven weeks after AAV-shPTB injection, some GFP+ cells were co-labeled with the neuronal marker NeuN or MAP2 around the injured area (Figure 3A and B). Quantitatively, approximately 30% and 29% of GFP+ cells were co-labeled with NeuN and MAP2, respectively (Figure 3C). Furthermore, approximately 19% of GFP+ cells expressed the motor neuron-specific marker ChAT at 11 weeks after AAV-shPTB injection (Figure 3A–C). At this same time point, neither GFP+ neuron-like cells nor GFP+ motoneuron-like cells were detected around the injured area after AAV-shCtrl injection (Figure 3A–C). Quantitative analysis of neuronal-like cell and motoneuron-like cell populations around the lesion area showed that the number of NeuN+, MAP2+, and ChAT+ cells were more abundant in the AAV-shPTB group compared with that in the AAV-shCtrl group (Figure 3D). In conclusion, these results reveal that shPTB can replenish motoneuron-like cells around the injured area after SCI.


    Figure 6|The PTB-ASO mediated the reprogramming of murine reactive spinal astrocytes to motoneuron-like neurons in vitro. 

    Given the demonstrated capability of ASOs to degrade their target mRNAs (Bennett et al., 2019; Qian et al., 2020; Maimon et al., 2021), downregulation of PTB by PTB-ASO suggests a clinically feasible strategy. As shown in Figure 6A–C, compared with the control ASO, PTB-ASO transfection markedly reduced PTB protein and Ptbp1 mRNA expression in astrocytes after 2 days. At 5 weeks post-transfection, we evaluated the morphology of reprogrammed cells and the expression of the mature neuronal marker MAP2 as well as the motor neuron marker ChAT via immunocytochemistry (Figure 6D). Quantitative analysis of MAP2+ and ChAT+ cells among total Hoechst+ cells showed that approximately 64% of the reprogrammed cells exhibited neurite outgrowth and expressed MAP2, and approximately 14% of the reprogrammed cells exhibited positive ChAT staining (Figure 6D and E). These results suggest that PTB-ASO significantly induced the conversion of reactive spinal astrocytes into motoneuron-like cells in vitro.


    Figure 7|The PTB-ASO reduces the density of the glial scar and replenishes motoneuron-like cells around the spinal cord lesion.

    As previously described, the glial scar has a dual role in SCI repair. Some studies have suggested that moderate reduction in the glial scar can play a positive role in functional recovery after SCI (Rodriguez et al., 2014; Hesp et al., 2018; Yang et al., 2020b). We determined the effects of PTB ASOs on glial scar density by GFAP staining of the injured spinal cord. As shown in Figure 7A, at 12 weeks after SCI, control ASO-injected mice showed typical glial scar morphology with interdigitating astrocytes that displayed a considerable number of parallel processes. PTB-ASO-injected mice also exhibited integrated glial scar structures, which were different from the exhaustive ablation of glial scars in some previous reports. In PTB ASO-injected mice, glial scar-derived astrocytes were loosely distributed with fewer parallel processes (Figure 7A). Quantitative analysis of the immunofluorescent intensity of GFAP around the lesion area revealed a moderate decrease in the density of glial scars in PTB ASO-injected mice compared with that in control ASO-injected mice (Figure 7B). 


    SCI results in irreversible loss of neurons in the injured area, and the regenerative capacity of the damaged neurons is limited (Sofroniew, 2018). To explore the potential beneficial function of the PTB-ASO in SCI mice, we determined whether injection of the PTB-ASO into the injured spinal cord replenished neurons, including motor neurons. This strategy may be helpful for promoting functional recovery after massive SCI-associated loss of neurons in the lesion area. We found that control ASO-injected mice exhibited an obvious decrease in NeuN+ and ChAT+ cells in the region proximal to the lesion area compared with the distal region at 12 weeks after SCI (Figure 7A). In contrast, PTB-ASO-injected mice retained many NeuN+ and ChAT+ cells in the region proximal to the lesion at a level similar to that observed in the distal site (Figure 7A). Quantitatively, the number of NeuN+ and ChAT+ cells in the region proximal to the lesion area was greater in PTB-ASO-injected mice compared with that in control ASO-injected mice (Figure 7C). 


    Figure 8|The PTB-ASO prevented apoptosis of injured spinal cord cells. 

    TUNEL staining was utilized to detect cell apoptosis around the lesion area at 12 weeks post-SCI. We found that the number of TUNEL+ cells around the lesion area was lower in PTB-ASO-injected mice than in control ASO-injected mice (Figure 8A and B). Furthermore, quantitative RT-PCR analysis of mRNA from injured spinal tissue exhibited lower expression levels of the apoptotic markers caspase-3 and caspase-9 in PTB-ASO-injected mice compared with those in control ASO-injected mice (Figure 8C). These findings revealed that the PTB-ASO reduced glial scar density without disrupting its overall structure, replenished motoneuron-like cells around the SCI lesion area, and decreased apoptotic cell death in the injured spinal cord.


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  • 发布日期: 2022-07-20  浏览: 151
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