TDP-43的磷酸化与核丢失在脑出血后继发性脑损伤中的作用及分子机制研究

The autophagy of neuron cell play important roles in the the mechanisms of secondary brain injury after intracerebral hemorrhage (ICH). We have observed the nuclear loss and phosphorylation of TDP-43 might preserve neuron cell autophagy after ICH in our preliminary data. Our hypothesis is that TDP-43 provides protection against secondary brain injury after ICH by stabilizing the autophagy of neuron cell via m-TOR and DCTN1 signaling pathway. The following specific aims at vitro、vivo、mechanism and clinical translation are proposed to address our hypothesis. Firstly, we determine the time course of the nuclear loss and phosphorylation of TDP-43 by western blot, immunofluorescence staining after ICH. Secondly, scanning electron microscope, western blot, and immunofluorescence staining are used to determine the neurovascular protection and its mechanism of the nuclear loss and phosphorylation of TDP-43 by inhibitors and RNA interference. Thirdly, we use the TDP-43 by intranasal application as a clinically relevant translational therapy to protect against early brain injury and provide long term functional improvements after SAH. From multi-level of molecules, cells, tissues and animals, this translationally-focused study is significant due to its potential to yield an effective treatment for ICH patients.

神经元自噬在脑出血(ICH)后继发性脑损伤中起着重要作用。我们预实验结果提示：ICH后神经元的TDP-43发生核丢失与磷酸化，可能引起神经元自噬相关蛋白变化。因此，我们提出假说：ICH后通过其上游的钙调磷酸酶（CN）调控神经元的TDP-43发生核丢失与磷酸化，进而调节其下游m-TOR和DCTN1信号通路调控神经元自噬起到保护作用。我们从体外、活体、机制以及临床验证这一假说。首先，应用Western blot、免疫荧光等检测ICH后不同时间点TDP-43核丢失及其磷酸化在胞核胞浆的表达变化。其次 ，应用扫描电镜、Western blot、免疫荧光，通过慢病毒转染质粒、SiRNA干扰及抑制剂等干预手段检测TDP-43调控神经元自噬的保护作用及分子机制。第三，应用经鼻吸入给药，检测TDP-43产生长期神经功能改善的转化医学作用。最终阐明TDP-43在ICH后脑损伤中的作用，为临床治疗提供新思路。

本研究旨在探讨TAR DNA结合蛋白-43 (TDP-43)在脑出血(ICH)致继发性脑损伤(SBI)中的作用及其机制。脑出血后，TDP-43在细胞核中的表达明显下降，在体内和体外胞浆中的表达增加，表明在脑出血后的SBI过程中，TDP-43从细胞核转到胞浆中。此外，TDP-43的S409/410突变可以抑制其磷酸化，减弱核丢失，并消除皮层下神经元凋亡的增加。抑制TDP-43磷酸化可降低ICH诱导的mTOR活性和dynactin1表达的下调，从而可能缓解ICH诱导的自噬体-溶酶体融合的阻断和自噬体和溶酶体生物发生的增加。然而，TDP-43的下调可能会加重ich诱导的SBI。此外，钙调磷酸酶(CN)可使TDP-43脱磷酸化，氧化血红蛋白可提高CN活性。综上所述，本研究表明，S409/410位点磷酸化的TDP-43功能缺失可能通过抑制mTOR活性和dynactin1表达，从而阻断自噬体-溶酶体融合，诱导LC3II和p62水平升高。这一机制可能在ich诱导的SBI中发挥重要作用，而TDP-43可能是一个潜在的治疗靶点。