研究背景：

多聚嘧啶束结合蛋白1（Polypyrimidine tract binding protein 1，Ptbp1）是一种重要的RNA结合蛋白，在神经元分化和成熟相关的可变剪接调控过程中发挥着关键作用。通过敲低 Ptbp1基因的表达，直接在原位诱导星形胶质细胞向神经元 (astrocyte-to-neuron，AtN) 转分化以替代受损的神经元，已被报道在多种神经退行性疾病模型中具有一定的治疗效果。与神经干细胞移植相比，原位转分化避免了潜在的致瘤性、感染及免疫排斥等副作用，并且相较于化学小分子重编程操作更加简便易行。如果Ptbp1敲降能够成功实现星形胶质细胞到神经元的转分化，将标志着神经再生领域的一项重大进步。然而，一些研究在成功敲降Ptbp1后未观察到预期的AtN转分化现象，此外严格的谱系示踪技术也未能验证转分化神经元确实来源于星形胶质细胞，同时发现存在“神经元泄漏”现象导致原有神经元被错误标记，Ptbp1介导的转分化过程仍存在较多争议，许多细节尚未得到系统性研究。最新研究表明，Smoothened激动剂（Smoothened agonist，SAG）能显著增强小分子诱导的重编程，然而SAG在Ptbp1诱导的AtN转分化中的作用尚未被探索，研究其影响可能为优化转分化策略提供新的思路。

研究目的：

    系统研究Ptbp1敲降在生理及不同炎性反应状态下诱导大鼠脊髓星形胶质细胞向神经元转分化的过程，从新的角度为当前转分化实验的争议提供可能的解释，并探索SAG对转分化过程的影响及其潜在调控机制。

研究方法：

通过多次差速贴壁法纯化并扩增原代大鼠脊髓星形胶质细胞，免疫荧光染色及定量PCR鉴定细胞纯度。分别使用两条短发夹RNA（short hairpin RNA，shRNA）和小干扰RNA（small interfering RNA，siRNA）转染细胞，定量PCR和Western Blot检测Ptbp1的敲降效率；活细胞长时程监测系统观察转分化细胞形态变化及突起生长；转录组测序技术分析基因表达变化；免疫荧光染色每周检测Tuj1或MAP2（神经元标志物），以及GFAP（星形胶质细胞标志物）或Ptbp1的表达情况；膜片钳记录转分化第4周细胞的动作电流及动作电位，评估电生理特性。在原代星形胶质细胞中添加脂多糖（lipopolysaccharide，LPS）诱导炎性活化，构建神经炎症环境下的反应性星形胶质细胞模型；再加入地塞米松逆转炎性活化，构建逆转活化细胞模型，通过定量PCR验证反应性星形胶质细胞相关因子的表达变化，随后在活化及逆转活化细胞中敲降Ptbp1，通过免疫荧光染色检测Tuj1的表达情况。最后，在转分化体系中加入SAG，通过多电极阵列（multi-electrode array，MEA）在第1周和第2周分别检测转分化细胞的神经电信号，评估电生理功能变化；定量PCR检测Shh信号通路中关键转录因子Gli1和Gli2的表达；定量PCR和Western Blot检测不同时间点Ptbp2的表达变化；免疫荧光染色检测Ptbp1、Ptbp2、Tuj1或MAP2的表达。

研究结果：

    shRNA和siRNA均成功敲低大鼠脊髓星形胶质细胞Ptbp1的表达，转分化细胞的形态和性质开始向神经元方向转变，随时间推移逐渐增强并最终趋于稳定。转录组测序结果显示涉及神经分化、轴突生长及突触形成的相关基因表达发生显著差异。膜片钳检测到转分化细胞产生动作电流，但未能生成动作电位，免疫荧光染色进一步表明转分化细胞为发育早期的未成熟神经元。LPS诱导的反应性星形胶质细胞在敲降Ptbp1后的神经元转分化能力显著下降，但通过地塞米松逆转炎性活化状态可部分恢复其转分化潜能。在敲降Ptbp1后的转分化体系中加入SAG后成功激活了Shh信号通路，MEA检测到逐渐增强的神经电信号，转分化细胞中原本维持高表达的Ptbp2出现明显下调，并开始表达成熟神经元标志物。

研究结论：

敲降Ptbp1可诱导大鼠脊髓星形胶质细胞转分化为早期未成熟神经元，且不同炎性状态的星形胶质细胞转分化能力存在显著差异。此外，SAG通过激活Shh信号通路并诱导转分化神经元Ptbp2的下调，促进其进一步发育为具备电生理功能的成熟神经元，最终促进神经再生。

Background:

Polypyrimidine tract binding protein 1 (Ptbp1) is an important RNA-binding protein that plays a crucial role in regulating alternative splicing events related to neuron differentiation and maturation. Direct in situ astrocyte-to-neuron (AtN) conversion to replace damaged neurons via Ptbp1 knockdown has demonstrated therapeutic effects in several neurodegenerative disease models. Compared to the transplantation of neural stem cells, in situ AtN conversion avoids the potential risks of tumorigenicity, infection and immune rejection. Additionally, it is also simpler and more practical than small molecule reprogramming. If Ptbp1 knockdown can successfully induce trans-differentiation from astrocytes to neurons, it will mark a breakthrough in the field of neural regeneration. However, some studies failed to observe the expected AtN conversion after successful Ptbp1 knockdown. Moreover, rigorous lineage tracing techniques cannot confirm that the converted neurons are indeed derived from astrocytes, and the phenomenon of “neuronal leakage” led to the mislabeling of pre-existing neurons. As a result, the Ptbp1-mediated AtN conversion process remains controversial, with many details still lacking systematic investigation. Recent studies have shown that Smoothened agonist (SAG) can significantly enhance small molecule-induced reprogramming, whereas its role in Ptbp1-induced AtN conversion has not been explored. Investigating its effects may provide new insights for optimizing Ptbp1-mediated AtN conversion strategies.

Objective:

To systematically study the process of Ptbp1 knockdown-induced astrocyte-to-neuron conversion in rat spinal cord astrocytes under both physiological and different inflammatory states, aiming to provide possible explanations from a new perspective for the current controversies surrounding trans-differentiation experiments, as well as to explore the effects of SAG on this conversion process and its underlying mechanisms.

Methods:

Primary rat spinal cord astrocytes were purified and amplified by multiple times of differential adherence method, and their purity was further verified through immunofluorescence staining and RT-qPCR. Short hairpin RNA (shRNA) and small interfering RNA (siRNA) were used to transduce the cells, and RT-qPCR and Western blot were used to assess the knockdown efficiency of Ptbp1. A live-cell long-term monitoring system was used to observe morphological changes and processes growth during conversion. Transcriptome sequencing technology was performed to analyze gene expression changes. Immunofluorescence staining was conducted weekly to monitor the expression of Tuj1 or MAP2 (neuronal markers), and GFAP (astrocyte marker) or Ptbp1. Patch-clamp recordings were performed at the fourth week of conversion to measure action currents and action potentials, evaluating the electrophysiological properties of converted cells. Lipopolysaccharide (LPS) was used to induce inflammatory activation in primary astrocytes, establishing a reactive astrocyte model under neuroinflammatory conditions. Dexamethasone (DEX) was further added to reverse the inflammatory activation, establishing a reverse-reactive astrocyte model. RT-qPCR was used to verify changes in the expression of reactive astrocyte-related factors. Ptbp1 was then knocked down in both reactive and reverse-reactive astrocytes, and Tuj1 expression was analyzed by immunofluorescence staining respectively. Finally, SAG was introduced to the neural conversion system, and the electrical activity of converted cells was detected using multi-electrode arrays (MEA) recordings at week 1 and week 2 respectively, to compare the electrophysiological changes with or without SAG treatment. RT-qPCR was used to detect the expression of key transcription factors Gli1 and Gli2 in the sonic hedgehog (Shh) signaling pathway. Moreover, Ptbp2 expression was measured at different time points by RT-qPCR and Western blot, and the expression of Ptbp1, Ptbp2, Tuj1, or MAP2 was further evaluated by immunofluorescence staining.

Results：

Both shRNA and siRNA successfully knocked down the expression of Ptbp1 in rat spinal cord astrocytes, and the morphology and properties of the converted cells began to shift toward a neuronal phenotype, gradually enhancing and eventually stabilizing over time. The results of transcriptomic sequencing showed significant changes in the expression of genes related to neurodifferentiation, axon growth, and synapse formation. Patch-clamp recordings detected action currents in the converted cells, but no action potentials were generated. Immunofluorescence staining further indicated that the converted cells were immature neurons that had not yet matured. Furthermore, the neuronal conversion ability of LPS-induced reactive astrocytes was significantly reduced after Ptbp1 knockdown, but the capacity for neural conversion was partially restored by dexamethasone treatment after the reversal of inflammatory activation. Moreover, in the Ptbp1 knockdown-induced conversion system, the addition of SAG successfully activated the Shh signaling pathway of converted cells. MEA recordings detected progressively increasing neural electrical signals. Ptbp2, which had been maintained at a high level, was significantly downregulated, and the converted then began to express mature neuronal markers.

Conclusion：

Knocking down Ptbp1 can induce the conversion of rat spinal cord astrocytes into early immature neurons, with the conversion potential of astrocytes varying significantly under different inflammatory states. Additionally, SAG activates the Shh signaling pathway and induces the downregulation of Ptbp2 in the converted neurons, promoting their further development into mature neurons and acquisition of electrophysiological functions, ultimately facilitating neural regeneration.