CaMKII substrate-selection in opposing forms of synaptic plasticity

描述(申请人提供)：长时程增强(LTP)和抑制(LTD)是两种截然相反的形式的钙依赖突触可塑性，分别由高频或低频刺激诱导，并被认为是学习和记忆的基础。超过22年的研究已经将钙/钙调蛋白依赖的蛋白激酶II(CaMKII)与LTP调节紧密联系在一起。然而，最近的证据(包括这项提案的初步结果)表明，有限公司也有CAMKII要求。值得注意的是，LTP-和LTD-机制都涉及T286的自动磷酸化，它产生钙离子非依赖的CaMKII的“自主”活性。那么，在LTP与LTD中，是什么控制了相反的下游响应？这一提议将检验这样一种假设，即LTP和LTD刺激不同地调节“自主”CaMKII的底物选择性，从而对突触强度造成相反的影响。最近的研究结果表明，“自主的”CaMKII并不是完全活跃的，而是可以被钙离子/钙调素(至少对于常规的“R-底物”)显著地进一步刺激。初步研究表明，这种“自主的”CaMKII的进一步刺激确实是增强突触强度所必需的。更多的初步研究表明，某些特定底物(现在被称为CaM保护的“C-底物”)的磷酸化可以被CaM/CaM显著抑制。因此，“自主的”CaMKII的活性可以由钙/钙调素以底物依赖的方式双向调节。具体的假设是，R底物的磷酸化是由LTP刺激诱导的，然后促进突触增强，而C底物的磷酸化是由LTD刺激诱导的，然后促进突触抑制。最近的研究结果表明，(与传统观点相反)LTP后产生的CaMKII自主性很快被逆转，而LTP后产生的自主性更持久(尽管产生速度较慢)。在LTP过程中，CaMKII的自主性仅在LTP后显著超过钙/CaM刺激，在LTP过程中确实应该促进R-底物和抑制C-底物，在LTP过程中抑制R-底物和促进C-底物。事实上，初步研究将两种不同的C底物蛋白与CaMKII在突触后LTD机制中的新功能联系起来。有趣的是，其中一种蛋白质还含有一个额外的R底物位点，它与LTP相连。该建议将：(1)通过直接比较确定LTP与LTD后CaMKII自主的时间进程，并确定LTP与LTD诱导与维护对CaMKII自主的要求。(在LTP中，CaMKII自主最近被证明是诱导所必需的，但不是维持所必需的)。(AIMS 2+3)两种不同的C-底物：确定它们在LTP和LTP过程中磷酸化的生化机制和细胞功能。这将包括确定在LTP刺激期间干扰抑制(通过破坏CaM保护)如何在功能上影响突触增强(期望LTP刺激现在额外地参与由C底物介导的特定LTD机制)。这一建议的结果将建立CaMKII与突触后LTD的牢固联系，阐明新的CaMKII在体外和神经元中的调节机制，并为“自主的”CaMKII如何介导两种截然不同的突触可塑性提供机制解释。

DESCRIPTION (provided by applicant): Long-term potentiation (LTP) and depression (LTD) are two opposing forms of Ca2+-dependent synaptic plasticity that are induced by high or low frequency stimulation, respectively, and thought to underlie learning and memory. Over 22 years of research have firmly linked the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) to LTP regulation. However, recent evidence (including by preliminary results of this proposal) indicates CaMKII requirement also in LTD. Notably, both LTP- and LTD-mechanisms involve T286 auto-phosphorylation, which generates Ca2+ -independent "autonomous" activity of CaMKII. What, then, controls the opposing downstream response in LTP versus LTD? This proposal will test the hypothesis that LTP- and LTD-stimuli differentially regulate substrate-selectivity of "autonomous" CaMKII, thereby causing opposite effects on synaptic strength. Recent results showed that "autonomous" CaMKII is not fully active, but can instead be significantly further stimulated by Ca2+/CaM (at least for regular "R-substrates"). Preliminary studies showed that such further stimulation of "autonomous" CaMKII is indeed required for enhancing synaptic strength. Additional preliminary studies showed that phosphorylation of some specific substrates (now termed CaM-protected "C-substrates") can instead be significantly inhibited by Ca2+/CaM. Thus, activity of "autonomous" CaMKII can be bi-directionally regulated by Ca2+/CaM, in a substrate-dependent manner. The specific hypothesis is that phosphorylation of R-substrates is induced by LTP-stimuli and then promotes synaptic potentiation, while phosphorylation of C- substrates is induced by LTD-stimuli and then promotes synaptic depression. Recent results suggested that (-contrary to traditional view-) CaMKII autonomy generated after LTP is quickly reversed, while autonomy after LTD is much more persistent (though generated more slowly). This timecourse of regulation, with CaMKII autonomy significantly outlasting the Ca2+/CaM- stimulus only after LTD but not after LTP, should indeed promote R-substrates and suppress C- substrates during LTP, and suppress R-substrates and promote C-substrates during LTD. Indeed, preliminary studies linked two different C-substrate proteins to the novel functions of CaMKII in postsynaptic LTD mechanisms. Interestingly, one of these proteins also contains an additional R-substrate site, which was instead linked to LTP. This proposal will: (Aim 1) Determine the timecourse of CaMKII autonomy after LTP versus LTD in direct comparison, and determine the requirement of CaMKII autonomy in LTD induction versus maintenance. (In LTP, CaMKII autonomy was recently shown to be required for induction but not maintenance). (Aims 2+3) Two different C-substrates: Determine the biochemical mechanisms and the cellular functions of their phosphorylation during LTD versus suppression during LTP. This will include determining how disrupting the suppression during LTP-stimuli (by disrupting the CaM- protection) functionally affects synaptic potentiation (with the expectation that LTP-stimuli now additionally engage specific LTD-mechanisms mediated by the C-substrates). The results of this proposal will establish a firm link of CaMKII to postsynaptic LTD, elucidate novel CaMKII regulation mechanisms in vitro and in neurons, and provide a mechanistic explanation how "autonomous" CaMKII can mediate two distinct and opposing forms of synaptic plasticity.