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

蛋白磷酸酶 1 抑制剂 2 在突触缩放中的作用

• 批准号: 1457336
• 负责人: Houhui Xia
• 金额: $ 39.5万
• 依托单位: Louisiana State University Health Sciences Center
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1457336&HistoricalAwards=false
• 关键词: role; protein; phosphatase; inhibitor; synaptic

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.

大脑神经元可以感觉到神经元网络的活动水平。如果活动水平太低或太高，神经元可以调整神经元之间的通信水平。实现这一点的方式还没有完全被理解。在之前的工作中，研究人员发现了一种有趣的方式，涉及一种名为蛋白质磷酸酶-1(PP1)的蛋白质，该蛋白质受另一种名为抑制物-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方面的作用。这项工作将为未来的动物研究奠定基础，以了解大脑如何适应视觉体验或剥夺。对环境光线的适应对日常生活很重要，因此这些研究将对理解包括人类在内的其他动物的视觉产生广泛影响。该项目将为博士后研究员、本科生和高中生，包括代表不足的少数民族和妇女，提供在这个EPSCoR州接受研究培训的机会。研究将集中在一个丰富的酶，蛋白磷酸酶-1(PP1)，及其调节因子，抑制物-2(I-2)，在双向突触伸缩中的功能。这项研究还将确定I-2如何调节PP1在突触后密度蛋白95千克道尔顿(PSD95)上丝氨酸295(Ser295)的去磷酸化过程中的功能，PSD95是突触结构和神经元通讯的关键分子。肌球蛋白轻链激酶(MLCK)通过丝氨酸43(Ser43)的I-2磷酸化调节I-2的影响将在PP1对Ser295的PSD95去磷酸化和突触伸缩的背景下确定。以原代培养的大鼠皮质神经元为模型系统，PI应用荷包牡丹碱增强神经元活性或河豚毒素(TTX)降低神经元活性。通过表达I-2短发夹状RNA片段RNA(ShRNA)来抑制I-2的表达，并用免疫印迹法检测其对Ser295处PSD95磷酸化的影响。通过电生理记录观察I-2kD对突触传递的影响。通过Western blotting检测不同MLCK亚型的shRNAs对I-2pS43的影响，将确定负责Ser43上I-2磷酸化的MLCK亚型的同源性。MLCK亚型对双向突触伸缩的影响将通过表达相应shRNA的神经元的电生理记录来确定。