DYNAMICS OF SIGNAL TRANSDUCTION IN NEURONS

描述（由申请人提供）：本提案首先旨在改进，开发和测试强大的新分子技术，以监测和操纵神经元中的动态信号转导。基于光诱导电子转移的电压指示器的目标包括更高的灵敏度、基因靶向性和更长的波长。由藻胆蛋白工程而成的新型远红色荧光蛋白是很有前途的细胞周期状态、Ca2+和蛋白酶活性的体内指标的构建块。测量和控制神经元活动的一种新的替代方法是设计一种人工转录因子，通过同时高[Ca2+]和光照激活，大大改善内源性活性报告如c-fos。基因编码的“快照报告者”将在触发照明精确定义的时间内捕获整个大神经元集合的活动模式，然后驱动效应基因的表达来标记这些细胞，并允许选择性兴奋，抑制或消融来测试它们的功能重要性。可被红光激活的嵌合红视通道允许通过完整颅骨的深部神经元的光遗传激发，但其峰值波长应进一步增加，其对蓝光的剩余灵敏度应受到抑制。通过更有效的单线态产氧蛋白，以及引入单线态氧传感GFP来绘制抑制的空间范围，可以改善光遗传学对突触释放的抑制。这两种光遗传学工具都将用于解剖恐惧条件反射中的杏仁核回路。产生氧气的单线态蛋白质现在可以分裂成两个互补的片段，只有在嵌合伴侣结合后才具有光活性。这种互补系统可以让蛋白质之间的相互作用和激酶和蛋白酶活性被捕获，以便随后通过电子显微镜进行可视化。一种标记在药理学上确定的时间内产生的蛋白质的基因编码标签可能适用于完整大脑中蛋白质的图像合成和降解，这要归功于纳米颗粒的发展，纳米颗粒可以通过血脑屏障输送小分子药物。这种纳米颗粒也可以帮助临床药物输送到大脑。这些技术将被用来测试一个新的假设，即非常长期的记忆，如恐惧条件反射，是以神经周围网络（PNN）中孔的模式存储的，PNN是一种特殊的细胞外基质，包裹着成熟的神经元，限制突触的形成。PNN与突触的三维交织将通过连续切片电子显微镜成像。通过小鼠的脉冲追踪15N标记和人类尸体大脑的14C含量来比较PNN和突触内成分的寿命。基因编码的指标和抗新表位抗体可以提高蛋白酶在体内活性的空间和时间分辨率

DESCRIPTION (provided by applicant): This proposal first aims to improve, develop, and test powerful new molecular techniques to monitor and manipulate dynamic signal transduction in neurons. Goals for voltage indicators based on photoinduced electron transfer include greater sensitivity, genetic targeting, and longer wavelengths. New far-red fluorescent proteins engineered from phycobiliproteins are promising building blocks for in vivo indicators of cell cycle status, Ca2+, and protease activity. A novel alternative approach to measuring and manipulating neuronal activity is to engineer an artificial transcription factor activated by simultaneous high [Ca2+] and illumination, greatly improving on endogenous activity reporters such as c-fos. The genetically encoded "snapshot reporter" will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, then drive expression of effector genes to mark those cells and allow selective excitation, inhibition, or ablation to test their functional importance. A chimeric channelrhodopsi activatable by red light permits optogenetic excitation of deep neurons through the intact skull, but its peak wavelength should be further increased and its residual sensitivity to blue light suppressed. Optogenetic inhibition of synaptic release will be improved by a more efficient singlet-oxygen generating protein, and introducing a singlet- oxygen-sensing GFP to map the spatial extent of inhibition. Both optogenetic tools will be applied to dissect amygdalar circuits n fear conditioning. The singlet-oxygen generating protein can now be split into two complementary fragments that only become photoactive after being brought together by chimeric partners. This complementation system may allow protein-protein interactions and kinase and protease activity to be captured for subsequent visualization by electron microscopy. A genetically encoded tag that marks proteins made during a pharmacologically defined period may become applicable to image synthesis and degradation of proteins in intact brain, thanks to development of nanoparticles that deliver small molecule drugs across the blood-brain barrier. Such nanoparticles may also aid clinical drug delivery to the brain. Such techniques will be used to test a new hypothesis that very long-term memories such as fear conditioning are stored as the pattern of holes in the perineuronal net (PNN), a specialized extracellular matrix that envelops mature neurons and restricts synapse formation. The 3-D intertwining of PNN and synapses will be imaged by serial-section electron microscopy. Lifetimes of PNN vs. intrasynaptic components will be compared by pulse-chase 15N labeling in mice and 14C content in human cadaver brains. Genetically encoded indicators and anti-neoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. New techniques including genetic knockouts, better pharmacological inhibitors, and the snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Biosynthesis of PNN components and proteases will be imaged.