脊髓PSD95介导突触液-液相分离异常参与吗啡耐受形成的机制研究

长期应用吗啡产生的耐受性影响癌痛患者的镇痛治疗。我们的前期研究发现脊髓HSP27参与吗啡耐受的形成，但机制尚不清楚。液-液相分离是新发现的细胞功能调控模式，突触内PSD95棕榈酰化修饰增强可通过改变局部内环境，提高相分离驱动力。我们在预实验中发现吗啡耐受大鼠脊髓神经元突触内微管蛋白、PSD95及其棕榈酰化修饰酶、以及AMPA受体GluR1亚型含量升高。由于HSP27可调控微管运输，进而影响PSD95的突触输送，加之液-液相分离可募集AMPA受体于突触膜，我们据此推测：吗啡所致的HSP27活化，通过微管机制引导PSD95向突触运输，后者通过棕榈酰化修饰导致液-液相分离异常，增强突触膜GluR1募集，导致突触可塑性改变以及吗啡耐受形成。本课题拟应用超微形态学、细胞膜动力学、单粒子跟踪等技术，阐明PSD95介导的液-液相分离在吗啡耐受形成中的作用及调控机制，为探索吗啡耐受精准调控靶点提供新思路。

Repeated morphine administration usually results in the development of morphine tolerance, which seriously affects the analgesic treatment of patients suffering cancer pain. Our previous studies found that heat shock protein (HSP27) at the spinal dorsal horn participated in the development of morphine tolerant, but underlying mechanisms remain elusive. Liquid-liquid phase separation (LLPS) is a novel discovered mechanism regulating cell function. Enhancing palmitoylation of postsynaptic density protein 95 (PSD95) can improve the driving force of phase separation by changing the local internal environment. In our present experiments, we found that the microtubule, PSD95, enzyme of PSD95 palmitoyl modification and AMPA receptor subunit GluR1 expressions were upregulated in the spinal dorsal horn neurons of morphine-tolerance rats. HSP27 can regulate microtubule transport, the latter may influence PSD95 synaptic transmission. Besides, LLPS can recruit synaptic AMPA receptors, we hypothesize that morphine may enhance HSP27 activity, influencing PSD95 transport to synapse by regulating microtubule. PSD95 can cause dysfunction of LLPS by promoting its palmitoylation, which may increase synaptic GluR1 recruitment, and contribute to the changes of synaptic plasticity and facilitate the development of morphine tolerance. In this proposal, multiple methods such as ultra-micromorphology, single particle tracking and dynamics of membrane fluidity will be used to explore the key role and mechanism of PSD95-mediated abnormal liquid-liquid phase separation. The proposed studies may provide novel insights for developing effective regulatory strategies to prevent morphine tolerance development.