脑损伤
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Figure 1｜AAV9P1 has higher affinity for astrocytes and reactive astrocytes (RAs) in vitro.
结果：We used multiple AAV vectors expressing EGFP to transduce primary astrocytes and RAs to evaluate the cell specificity of AAV9P1. To determine the appropriate vector concentration, we used MOIs of 1.0 × 104 and 1.0 × 105. At 2 weeks post-transduction (2 wpt), approximately 0.07% of astrocytes were transfected in the AAV9 P1 group when the MOI was 1.0 × 104. Notably, the percentage of EGFP+ cells was not significantly different compared with the negative control group (Figure 1A). The percentage of AAV9P1-transfected cells in the MOI = 1.0 × 105 group was increased (~0.56%) compared with the MOI = 1.0 × 104 group (Figure 1A and B). Therefore, we chose a MOI of 1.0 × 105 for the subsequent experiments. As shown in Figure 1B, the percentage of EGFP+ primary astrocytes in the AAV9P1, AAV9, and AAV/BBB groups was approximately 0.56%, 0.13%, and 0.16%, respectively. AAV9P1 showed significantly higher transduction efficiency than AAV9 (P < 0.001) and AAV/BBB (P < 0.001). At 4 wpt, approximately 0.53% of AAV9P1-transduced astrocytes expressed EGFP; the percentages of EGFP+ cells were 0.11% and 0.08% in the AAV9 and AAV/BBB groups, respectively (Figure 1C). These results indicate significantly higher AAV9P1-based astrocyte transduction compared with AAV9 (P < 0.001) and AAV/BBB (P < 0.0001). We further studied the affinity of AAV to RAs. Firstly, we acquired RAs using a scratch model. At 3 dps, approximately 23.75% of cells expressed the RA marker nestin. At 7 dps, the percentage of nestin+ cells was approximately 83.02%, indicating that most cells were RAs (Figure 1D and E). These AAV vectors were added to the cell medium at 7 dps. As shown in Figure 1F, the percentage of EGFP+ RAs in the AAV9P1 group was approximately 0.64% at 2 wpt, and approximately 0.11% and 0.079% of cells expressed EGFP in the AAV9 and AAV/BBB groups, respectively. AAV9P1 displayed significantly improved affinity for RAs compared with AAV9 (P < 0.0001) and AAV/BBB (P < 0.0001). At 4 wpt, approximately 0.13%, 0.083%, and 0.047% of RAs expressed EGFP in the AAV9P1, AAV9, and AAV/BBB groups, respectively. Although the percentage of EGFP+ RAs had reduced at 4 wpt, AAV9P1 still displayed a significantly higher transduction efficiency than AAV9 (P < 0.01) and AAV/BBB (P < 0.0001; Figure 1G). These data demonstrate that AAV9P1 has significantly higher transduction efficiency for astrocytes and RAs compared with other AAV vectors in vitro.
Figure 2｜AAV9P1 efficiently targets astrocytes and reactive astrocytes (RAs) in vivo.
结果：To determine if the AAV vectors can target astrocytes and RAs, we developed a CCI model. The process is shown in Figure 2A. Glial scarring was present at 7 dpc and the surrounding area showed significant tissue loss. The expression of nestin, which is typically downregulated in astrocytes, confirmed the formation of RAs (Figure 2B). To compare the specificity of AAV9P1 for astrocytes and neurons, we intravenously injected AAV9P1 at 7 dpc and then stained with GFAP and NeuN (for astrocytes and neurons, respectively) at 2 weeks post-injection (wpi). As shown in Figure 2C, EGFP was mainly detected in GFAP+ astrocytes. Moreover, a large number of transduced cells were positive for the astrocyte nuclear marker, Sox9 (Figure 2D). As shown in Figure 2E, we observed that most AAV9P1-transduced cells express the RA marker, nestin. Approximately 76.82 ± 4.76% of AAV9P1-transduced cells were GFAP+ astrocytes. The percentage of nestin+ RAs and Sox9+ astrocytes was 52.72 ± 8.89% and 42.14 ± 9.50%, respectively. In contrast, we failed to observe EGFP+ neurons (Figure 2F). These data indicate that dual-targeting AAV9P1 can efficiently transduce astrocytes and RAs, not neurons in vivo.
Figure 3｜AAV9P1 and AAV/BBB efficiently downregulate PTBP1 in the injury area.
结果：To determine if AAV9P1 and AAV/BBB can efficiently target the injury area and express shRNA and EGFP, we intravenously administrated AAV vectors at 7 dpc (Figure 3A). The IF assay for EGFP expression was performed at 2 wpi. As shown in Figure 3B, EGFP was mainly expressed in the peri-injury cerebral cortex relative to the contralateral cortex, suggesting injury-associated gene expression. This may occur because increased GFAP expression post-TBI facilitates GFAP promoter-based exogenous gene expression. At 4 wpi, WB and qRT-PCR were performed to detect PTBP1 expression in the peri-injury tissue. The results showed that AAV/BBB-shPTBP1 (P < 0.01, compared with AAV/BBB-shCtrl) and AAV9P1-shPTBP1 (P < 0.01, vs. AAV9P1-shCtrl) significantly reduced protein expression of the PTBP1 gene (Figure 3C and D). As shown in Figure 3E, AAV/BBB-shPTBP1 (P < 0.001, compared with AAV/BBB-shCtrl) and AAV9P1-shPTBP1 (P < 0.0001, compared with AAV9P1-shCtrl) significantly reduced mRNA levels of the PTBP1 gene. Taken together, these data confirm AAV-mediated PTBP1 downregulation.
Figure 4｜AAV9P1 and AAV/BBB mediate neuronal reprogramming in vivo.
结果：As shown in Figure 4A, the IF assay was used to determine if AAV9P1-mediated PTBP1 downregulation could efficiently induce neuronal reprogramming at 8 wpi. As shown in Figure 4B and C, approximately 22.71% of EGFP+ cells were neurons after AAV9P1-shPTBP1 administration. In contrast, few EGFP+ neurons were observed after AAV9P1-shCtrl injection. The very small number of EGFP+ neurons may be due to a limitation of the detection method. The percentage of EGFP+ astrocytes was significantly lower after shPTBP1 injection than in the shCtrl group (P < 0.05; Figure 4C). These data indicate that AAV9P1 can induce neuronal reprogramming. We also intravenously injected AAV/BBB and performed the IF assay at 8 wpi. As shown in Figure 4D and E, in both the experimental and control groups, the percentage of NeuN+ cells (55.42 ± 34.57% for shPTBP1, 9.63 ± 8.50% for shCtrl) was increased compared with the AAV9P1 group. Moreover, there were very large inter-group differences. These results suggest that the GFAP promoter may have leaky expression and cause large errors in neuronal reprogramming research. In contrast, AAV9P1 exhibited limited transgene expression and robust astrocyte-to-neuron (AtN) conversion, suggesting that it is a better vehicle for neuronal reprogramming research.
Figure 5｜AAV9P1 mediates conversion of reactive astrocytes (RAs)-to-neurons in vivo.
结果：For further confirmation of neuronal reprogramming, we labeled newly proliferated RAs by intraperitoneal injection of BrdU for one week. Figure 5A shows the schematic of the experimental schedule. As shown in Figure 5B and D, approximately 1.78 ± 0.12% of BrdU+ cells were NeuN+ cells in the AAV9P1-shPTBP1 group. No BrdU+NeuN+ co-labeled cells were detected in the AAV9P1-shCtrl group. These data indicate AAV9P1 mediated conversion of RAs-to-neurons. However, the percentage of GFAP+ cells among the BrdU+ cells in the AAV9P1-shPTBP1 group showed no significant difference compared with the AAV9P1-shCtrl group (Figure 5C and D). These data indicate low conversion efficiency of BrdU-labeled cells. Inhibition of BrdU on neuronal reprogramming may explain the low efficiency of neuronal reprogramming (Wang et al., 2022). These data highlight the importance of good evaluation methods. In summary, we believe that our AAV9P1-based dual-targeting system is a suitable tool to induce neuronal reprogramming.
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