死亡相关蛋白激酶1磷酸化Tau介导微管解聚引起脊髓损伤后轴突变性的机制研究

The spinal cord injury can cause neuronal axon degeneration. It is known that Tau phosphorylation leads to axonal degeneration via microtubule depolymerization. Previous our studies demonstrated that death associated protein kinase 1 (DAPK1), which was activated in the ischemia, phosphorylated Tau mediating neuronal death. In our further studies, the axons are shortened after over-expression of Tau phosphorylation in neuron cells, whereas, the axons are growth after over-expression of Tau non-phosphorylation when compared to wild type. There are the increasing expresions of DAPK1 and Tau phosphorylation in the model of spinal cord ischemic injury in mice. Either genetic deletion of DAPK1 kinase domain (KD) in mice (DAPK1-KD/) or blocking DAPK1 by systematic application of a membrane permeable peptide improves neurological functions against spinal cord injury. Hereby, we assume that after spinal cord ischemic injury, DAPK1 is activated and phosphorylated Tau, and then induces axonal degeneration via microtubule depolymerization. This study intends to further elucidate the DAPK1-Tau interaction and its cellular and molecular mechanism of neuron death via detecting microtubule morphology, axon associated protein expression and axonal transmission function in vitro cultured neurons and in-vivo DAPK1KD knockout mice in spinal cord injury model. Our study will provide substantial evidence and possible molecular target for the treatment of spinal cord injury.

脊髓损伤后可导致神经元轴突变性。已知Tau磷酸化介导微管解聚引起轴突变性。我们前期研究证实：缺血性卒中小鼠模型中，激活的死亡相关蛋白激酶1（DAPK1）可磷酸化Tau介导神经元死亡。进一步实验发现：对比野生型Tau，过表达磷酸化Tau可引起神经元轴突缩短, 而过表达非磷酸化Tau促进轴突形成；脊髓缺血性损伤小鼠模型中，DAPK1被激活，且磷酸化Tau的表达上调；运用DAPK1激酶域敲除的转基因小鼠或外源性多肽阻断DAPK1-Tau，小鼠脊髓损伤程度减轻。据此我们假设：脊髓损伤后激活DAPK1，通过磷酸化Tau介导微管解聚，促进轴突变性。本研究拟采用原代神经元和转基因小鼠，通过氧糖剥夺与光化学诱导脊髓缺血模型，观察DAPK1磷酸化Tau及其对微管形态、轴突相关蛋白表达以及轴突传递功能的影响，以深入阐明DAPK1-Tau作用引起脊髓损伤后轴突变性的机制，为抗脊髓损伤治疗提供理论依据与分子靶标。

为进一步探讨DAPK1磷酸化Tau的作用，明确脊髓损伤（SCI）后轴突变性的机制，我们进行深入研究。首先检测DAPK1KD-/-小鼠和同窝生野生型小鼠DAPK1KDloxp/loxp SCI后DAPK1的活性、Tau的磷酸化以及轴突相关蛋白knesin和synapsin1的表达。结果显示DAPK1KDloxp/loxp SCI后DAPK1的活性以及Tau pS262上调，knesin和synapsin1下降，而DAPK1KD-/-小鼠Tau pS262较DAPK1KDloxp/loxp 相比下调，knesin和synapsin1下调减轻。细胞层面，原代神经元转染TauS262D慢病毒组突起长度缩短；knesin和Synapsin 1的蛋白水平较TauS262A组下调；而mEPSC较TauS262A组下降，提示TauS262受DAPK1调节；敲除DAPK1激酶域可以保护SCI后的轴突的完整性。以上研究证实SCI中DAPK1与Tau的相互作用规律，为后续SCI的防治提供相关策略。另一方面，我们发现一个在神经损伤中有抑制DAPK1活性作用的突触前分子Caytaxin。结果显示, 1质谱发现Caytaxin与DAPK1结合,Caytaxin与DAPK1均在突触前定位；2缺血性卒中(MCAO)6h后，DAPK1的活性则较假手术组明显增加，Caytaxin 的表达水平在缺血早期（6h）之前侧较对侧有明显的增加，随后表达下降，而神经元凋亡也在MCAO后期（6h以后）出现，12h后明显增强；3DAPK1与Caytaxin共定位，二者相互作用在MCAO后明显增强；4细胞实验发现Caytaxin可以抑制DAPK1活性；5过表达或者抑制Caytaxin可以缓解或者加重MCAO后的缺血损伤。综上, 我们首次发现针对DAPK1活性的内源性抑制分子Caytaxin,在缺血性卒中早期DAPK1与Caytaxin作用，抑制DAPK1活性，保护神经元。因此，针对DAPK1和Caytaxin的相互作用设计小分子多肽进行治疗，很可能为缺血性卒中的治疗提供新的治疗靶点与策略。我们已经发现SCI中DAPK1的活性对疾病转归和恶化有着直接影响，我们会进一步检测SCI中过表达Caytaxin是否也可以抑制DAPK1的活性，缓解SCI，为SCI后减轻神经元死亡探索可能的干预方法。