The role of protein phosphatase-1 inhibitor-2 in synaptic scaling

大脑神经元可以感觉到神经元网络的活动水平。如果活动水平过低或过高，神经元可以调节神经元之间的交流水平。这是如何完成的还没有完全理解。在之前的工作中，研究人员发现了一种有趣的方法，它涉及一种名为蛋白磷酸酶-1 （PP1）的蛋白质，这种蛋白质受另一种名为抑制剂-2 （I-2）的蛋白质的调节。在这个项目中，研究人员将研究这两种蛋白质在调节神经元通讯中的作用。这项工作将为未来在动物身上的研究奠定基础，以了解大脑如何适应视觉体验或视觉剥夺。适应环境光对日常生活非常重要，因此这些研究将对包括人类在内的其他动物的视觉理解产生广泛的影响。该项目将为博士后、本科生和高中生提供机会，包括代表性不足的少数民族和妇女，在这个EPSCoR州接受研究培训。研究将集中于一种丰富的酶，蛋白磷酸酶-1 （PP1）及其调节因子，抑制剂-2 （I-2）在双向突触缩放中的功能。该研究还将确定I-2如何调节PP1在突触后密度蛋白95千道尔顿（PSD95）丝氨酸295 （Ser295）去磷酸化中的功能，PSD95是突触结构和神经元通信的关键分子。肌球蛋白轻链激酶（MLCK）通过I-2丝氨酸43位点（Ser43）磷酸化对I-2调控的影响将在PP1作用于PSD95丝氨酸295位点去磷酸化和突触缩放的背景下确定。以大鼠皮层初级神经元为模型系统，PI将使用双管碱来增强神经元活性或使用河豚毒素（TTX）来降低神经元活性。I-2将通过表达I-2短发夹RNA片段RNA （ShRNA）被敲低（KD），并通过western blotting检测其对PSD95 Ser295磷酸化的影响。i - 2kd对突触传递的影响将通过电生理记录来检测。通过western blotting检测不同MLCK亚型shrna对I-2pS43的影响，可以确定负责I-2 Ser43磷酸化的MLCK亚型的身份。MLCK同种异构体对双向突触缩放的影响将通过表达相应ShRNA的神经元的电生理记录来确定。

Brain neurons can sense the activity level of neuron networks. If the activity levels are too low or too high, the neurons can adjust the communication level between the neurons. The way that this is accomplished is not completely understood. In previous work, the researcher found one interesting way that involves a protein called protein phosphatase-1 (PP1) that is regulated by another protein called inhibitor-2 (I-2). In this project, the researcher will investigate the role of these two proteins in regulating neuron communications. This work will lay the foundation for future studies in animals to understand how the brain adapts to visual experience or deprivation. Adaptation to environmental light is important for daily life, so these studies will have broad impact on understanding vision in other animals, including humans. The project will provide opportunities for a postdoctoral fellow, undergraduate, and high school students, including underrepresented minorities and women, to be trained in research in this EPSCoR state. Studies will focus on the function of an abundant enzyme, protein phosphatase-1 (PP1), and its regulator, inhibitor-2 (I-2), in bi-directional synaptic scaling. The study will also determine how I-2 regulates PP1 function in the dephosphorylation of serine 295 (Ser295) on postsynaptic density protein 95 kilo Dalton (PSD95), a molecule critical for synapse architecture and neuronal communication. The impact of I-2 regulation by myosin light chain kinases (MLCK) via I-2 phosphorylation at serine 43 (Ser43) will be determined in the context of both PP1 function on PSD95 dephosphorylation at Ser295 and synaptic scaling. Using primary rat cortical neurons as a model system, the PI will apply bicuculline to enhance neuronal activity or tetrodotoxin (TTX) to decrease neuronal activity. I-2 will be knocked-down (KD) by expression of I-2 short hairpin RNA fragment RNA (ShRNA), and its effect on PSD95 phosphorylation at Ser295 will be examined by western blotting. The effect of I-2 KD on synaptic transmission will be examined by electrophysiological recording. The identity of the MLCK isoform responsible for I-2 phosphorylation at Ser43 will be determined by examining the effect of ShRNAs of various MLCK isoforms on I-2pS43 by western blotting. MLCK isoform effects on bi-directional synaptic scaling will be determined using electrophysiological recordings of neurons expressing the corresponding ShRNA.