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.
Understanding the molecular mechanisms of synaptic plasticity underlying learning/memory is of high general significance in its own right, and will ultimately also lead to new therapeutic approaches for conditions that impair brain functions (such as Alzheimer and various forms of intellectual disability). The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is well established as a central regulator of learning/memory and of synaptic potentiation. Recent evidence that implicated CaMKII also in mechanisms of synaptic depression raises a central question: How can CaMKII differentiate between inducing these opposing forms of synaptic plasticity? This proposal will investigate novel mechanisms of CaMKII regulation that can provide a new framework for bi-directional regulation of plasticity by the same mediator. It will determine how these mechanisms regulate synaptic strength in neurons. Their detailed biochemical characterization will provide the basis for further studies to elucidate synaptic physiology and pathology also beyond the scope of this proposal and even the nervous system (as has been the case in the past for other regulatory CaMKII mechanisms). Even on its own, this proposal will provide important insight how neuronal signaling can differentiate between causing opposing effects on synaptic strength, despite activation of the same mediator. Preliminary studies suggest that the 'traditional' tight and exclusive link between CaMKII and synaptic potentiation severely limits our current understanding of synaptic plasticity (and thus also the pathological consequences and possibilities for therapeutic intervention). Thus, the demonstration of CaMKII mechanisms that also regulate synaptic depression will provide a significant advance to the field.
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