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• W2087436387 abstract "•Autonomous CaMKII activity is required not only for LTP, but also for LTD•LTP stimuli (strong but brief) favor traditional substrate site phosphorylation•LTD stimuli (weak but prolonged) instead favor a distinct substrate class•Deciding factor in substrate choice is further Ca2+/CaM stimulation of CaMKII Traditionally, hippocampal long-term potentiation (LTP) of synaptic strength requires Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and other kinases, whereas long-term depression (LTD) requires phosphatases. Here, we found that LTD also requires CaMKII and its phospho-T286-induced “autonomous” (Ca2+-independent) activity. However, whereas LTP is known to induce phosphorylation of the AMPA-type glutamate receptor (AMPAR) subunit GluA1 at S831, LTD instead induced CaMKII-mediated phosphorylation at S567, a site known to reduce synaptic GluA1 localization. GluA1 S831 phosphorylation by “autonomous” CaMKII was further stimulated by Ca2+/CaM, as expected for traditional substrates. By contrast, GluA1 S567 represents a distinct substrate class that is unaffected by such stimulation. This differential regulation caused GluA1 S831 to be favored by LTP-type stimuli (strong but brief), whereas GluA1 S567 was favored by LTD-type stimuli (weak but prolonged). Thus, requirement of autonomous CaMKII in opposing forms of plasticity involves distinct substrate classes that are differentially regulated to enable stimulus-dependent substrate-site preference. Traditionally, hippocampal long-term potentiation (LTP) of synaptic strength requires Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and other kinases, whereas long-term depression (LTD) requires phosphatases. Here, we found that LTD also requires CaMKII and its phospho-T286-induced “autonomous” (Ca2+-independent) activity. However, whereas LTP is known to induce phosphorylation of the AMPA-type glutamate receptor (AMPAR) subunit GluA1 at S831, LTD instead induced CaMKII-mediated phosphorylation at S567, a site known to reduce synaptic GluA1 localization. GluA1 S831 phosphorylation by “autonomous” CaMKII was further stimulated by Ca2+/CaM, as expected for traditional substrates. By contrast, GluA1 S567 represents a distinct substrate class that is unaffected by such stimulation. This differential regulation caused GluA1 S831 to be favored by LTP-type stimuli (strong but brief), whereas GluA1 S567 was favored by LTD-type stimuli (weak but prolonged). Thus, requirement of autonomous CaMKII in opposing forms of plasticity involves distinct substrate classes that are differentially regulated to enable stimulus-dependent substrate-site preference. Long-term potentiation (LTP) and long-term depression (LTD) cause long-term changes of synaptic strength in opposite directions; both are Ca2+ dependent, can occur at the same hippocampal CA3 to CA1 synapses, and are together thought to underlie learning, memory, and cognition (for review, see Collingridge et al., 2010Collingridge G.L. Peineau S. Howland J.G. Wang Y.T. Long-term depression in the CNS.Nat. Rev. Neurosci. 2010; 11: 459-473Crossref PubMed Scopus (678) Google Scholar, Malenka and Bear, 2004Malenka R.C. Bear M.F. LTP and LTD: an embarrassment of riches.Neuron. 2004; 44: 5-21Abstract Full Text Full Text PDF PubMed Scopus (2976) Google Scholar, Martin et al., 2000Martin S.J. Grimwood P.D. Morris R.G. Synaptic plasticity and memory: an evaluation of the hypothesis.Annu. Rev. Neurosci. 2000; 23: 649-711Crossref PubMed Scopus (2105) Google Scholar, Xia and Storm, 2005Xia Z. Storm D.R. The role of calmodulin as a signal integrator for synaptic plasticity.Nat. Rev. Neurosci. 2005; 6: 267-276Crossref PubMed Scopus (325) Google Scholar). Twenty-five years of research has firmly established CaMKII as a major mediator of the postsynaptic mechanisms of LTP (for review, see Colbran and Brown, 2004Colbran R.J. Brown A.M. Calcium/calmodulin-dependent protein kinase II and synaptic plasticity.Curr. Opin. Neurobiol. 2004; 14: 318-327Crossref PubMed Scopus (257) Google Scholar, Coultrap and Bayer, 2012Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35: 607-618Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Lisman et al., 2012Lisman J. Yasuda R. Raghavachari S. Mechanisms of CaMKII action in long-term potentiation.Nat. Rev. Neurosci. 2012; 13: 169-182PubMed Google Scholar). These mechanisms include CaMKII-mediated increase of synaptic AMPA-type glutamate receptor (AMPAR) number (Hayashi et al., 2000Hayashi Y. Shi S.H. Esteban J.A. Piccini A. Poncer J.C. Malinow R. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction.Science. 2000; 287: 2262-2267Crossref PubMed Scopus (1253) Google Scholar, Opazo et al., 2010Opazo P. Labrecque S. Tigaret C.M. Frouin A. Wiseman P.W. De Koninck P. Choquet D. CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin.Neuron. 2010; 67: 239-252Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and channel conductance, the latter by direct phosphorylation of the GluA1 subunits at S831 (Barria et al., 1997Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation.Science. 1997; 276: 2042-2045Crossref PubMed Scopus (889) Google Scholar, Benke et al., 1998Benke T.A. Lüthi A. Isaac J.T. Collingridge G.L. Modulation of AMPA receptor unitary conductance by synaptic activity.Nature. 1998; 393: 793-797Crossref PubMed Scopus (433) Google Scholar, Derkach et al., 1999Derkach V. Barria A. Soderling T.R. Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 3269-3274Crossref PubMed Scopus (696) Google Scholar, Kristensen et al., 2011Kristensen A.S. Jenkins M.A. Banke T.G. Schousboe A. Makino Y. Johnson R.C. Huganir R. Traynelis S.F. Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating.Nat. Neurosci. 2011; 14: 727-735Crossref PubMed Scopus (202) Google Scholar). LTP stimuli also induce autophosphorylation of CaMKII at T286, which generates Ca2+/CaM-independent “autonomous” activity and is required for LTP induction (Buard et al., 2010Buard I. Coultrap S.J. Freund R.K. Lee Y.S. Dell’Acqua M.L. Silva A.J. Bayer K.U. CaMKII “autonomy” is required for initiating but not for maintaining neuronal long-term information storage.J. Neurosci. 2010; 30: 8214-8220Crossref PubMed Scopus (126) Google Scholar, Coultrap et al., 2012Coultrap S.J. Barcomb K. Bayer K.U. A significant but rather mild contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity.PLoS ONE. 2012; 7: e37176Crossref PubMed Scopus (32) Google Scholar, Giese et al., 1998Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (892) Google Scholar). Notably, “autonomous” CaMKII is by no means fully active, because it can still be ∼5-fold further stimulated by Ca2+/CaM (Coultrap et al., 2010Coultrap S.J. Buard I. Kulbe J.R. Dell’Acqua M.L. Bayer K.U. CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.J. Biol. Chem. 2010; 285: 17930-17937Crossref PubMed Scopus (80) Google Scholar, Miller and Kennedy, 1986Miller S.G. Kennedy M.B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch.Cell. 1986; 44: 861-870Abstract Full Text PDF PubMed Scopus (641) Google Scholar). However, a physiological function for this additional regulation has remained elusive. Although LTP requires NMDA-type glutamate receptor (NMDAR) stimulation, LTD comes in both NMDAR- and metabotropic glutamate receptor (mGluR)-dependent forms (Collingridge et al., 2010Collingridge G.L. Peineau S. Howland J.G. Wang Y.T. Long-term depression in the CNS.Nat. Rev. Neurosci. 2010; 11: 459-473Crossref PubMed Scopus (678) Google Scholar, Malenka and Bear, 2004Malenka R.C. Bear M.F. LTP and LTD: an embarrassment of riches.Neuron. 2004; 44: 5-21Abstract Full Text Full Text PDF PubMed Scopus (2976) Google Scholar). Although LTP requires protein kinase activity and particularly CaMKII, LTD requires protein phosphatase activity (Collingridge et al., 2010Collingridge G.L. Peineau S. Howland J.G. Wang Y.T. Long-term depression in the CNS.Nat. Rev. Neurosci. 2010; 11: 459-473Crossref PubMed Scopus (678) Google Scholar, Malenka and Bear, 2004Malenka R.C. Bear M.F. LTP and LTD: an embarrassment of riches.Neuron. 2004; 44: 5-21Abstract Full Text Full Text PDF PubMed Scopus (2976) Google Scholar, Xia and Storm, 2005Xia Z. Storm D.R. The role of calmodulin as a signal integrator for synaptic plasticity.Nat. Rev. Neurosci. 2005; 6: 267-276Crossref PubMed Scopus (325) Google Scholar) and a potential role of CaMKII is still unclear (Coultrap and Bayer, 2012Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35: 607-618Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). For mGluR-dependent LTD, previous findings were conflicting and indicated either inhibition (Mockett et al., 2011Mockett B.G. Guévremont D. Wutte M. Hulme S.R. Williams J.M. Abraham W.C. Calcium/calmodulin-dependent protein kinase II mediates group I metabotropic glutamate receptor-dependent protein synthesis and long-term depression in rat hippocampus.J. Neurosci. 2011; 31: 7380-7391Crossref PubMed Scopus (82) Google Scholar) or facilitation (Schnabel et al., 1999Schnabel R. Palmer M.J. Kilpatrick I.C. Collingridge G.L. A CaMKII inhibitor, KN-62, facilitates DHPG-induced LTD in the CA1 region of the hippocampus.Neuropharmacology. 1999; 38: 605-608Crossref PubMed Scopus (31) Google Scholar) by CaMKII inhibitors. For NMDAR-dependent LTD, an involvement of CaMKII has been attributed to presynaptic mechanisms (Stanton and Gage, 1996Stanton P.K. Gage A.T. Distinct synaptic loci of Ca2+/calmodulin-dependent protein kinase II necessary for long-term potentiation and depression.J. Neurophysiol. 1996; 76: 2097-2101Crossref PubMed Scopus (55) Google Scholar, Stevens et al., 1994Stevens C.F. Tonegawa S. Wang Y. The role of calcium-calmodulin kinase II in three forms of synaptic plasticity.Curr. Biol. 1994; 4: 687-693Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and the effect on synaptic strength mediated by postsynaptic AMPARs remains unexplored. However, intriguingly, a recent study identified another CaMKII site on GluA1, S567 (see Figure 2A), which decreases synaptic strength by reducing synaptic localization of AMPARs (Lu et al., 2010Lu W. Isozaki K. Roche K.W. Nicoll R.A. Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1.Proc. Natl. Acad. Sci. USA. 2010; 107: 22266-22271Crossref PubMed Scopus (66) Google Scholar). Here, we manipulated CaMKII by an improved inhibitor, by knockout, and by T286A mutant knockin and demonstrated that NMDAR-dependent LTD requires both CaMKII and its autonomous activity. In hippocampal slices, LTD stimuli induced CaMKII-dependent GluA1 S567 phosphorylation. Biochemical assays with purified protein showed that GluA1 S567 represents a distinct CaMKII substrate class favored by LTD-type stimuli, whereas GluA1 S831 is a traditional substrate favored by LTP-type stimuli. These results demonstrate the regulatory mechanisms that enable autonomous CaMKII to mediate its opposing effects in LTP and LTD. Previous inhibitor studies yielded conflicting results for the involvement of CaMKII in postsynaptic mechanisms of hippocampal LTD (Mockett et al., 2011Mockett B.G. Guévremont D. Wutte M. Hulme S.R. Williams J.M. Abraham W.C. Calcium/calmodulin-dependent protein kinase II mediates group I metabotropic glutamate receptor-dependent protein synthesis and long-term depression in rat hippocampus.J. Neurosci. 2011; 31: 7380-7391Crossref PubMed Scopus (82) Google Scholar, Schnabel et al., 1999Schnabel R. Palmer M.J. Kilpatrick I.C. Collingridge G.L. A CaMKII inhibitor, KN-62, facilitates DHPG-induced LTD in the CA1 region of the hippocampus.Neuropharmacology. 1999; 38: 605-608Crossref PubMed Scopus (31) Google Scholar). Using our more selective CaMKII inhibitor tatCN21 (Buard et al., 2010Buard I. Coultrap S.J. Freund R.K. Lee Y.S. Dell’Acqua M.L. Silva A.J. Bayer K.U. CaMKII “autonomy” is required for initiating but not for maintaining neuronal long-term information storage.J. Neurosci. 2010; 30: 8214-8220Crossref PubMed Scopus (126) Google Scholar, Vest et al., 2007Vest R.S. Davies K.D. O’Leary H. Port J.D. Bayer K.U. Dual mechanism of a natural CaMKII inhibitor.Mol. Biol. Cell. 2007; 18: 5024-5033Crossref PubMed Scopus (142) Google Scholar), we show here CaMKII requirement in an NMDAR-dependent form of LTD that was induced in the hippocampal CA1 area by low-frequency stimulation (LFS; 15 min 1 Hz) (Figures 1A–1C). Synaptic strength was measured by evoked field excitatory postsynaptic potentials (fEPSPs), which were analyzed for slope (Figures 1A–1C) and amplitude (Figure S1). LTD induction was blocked when tatCN21 was added 15 min before LFS and removed immediately after LFS (Figure 1A). Although significant synaptic depression was still apparent immediately after LFS, no LTD developed. Genetic knockout of the CaMKIIα isoform also abolished LTD (Figure 1B), to the same extent as seen after inhibition of all CaMKII isoforms with tatCN21 (Figure 1C). The LFS-induced LTD required NMDAR-dependent Ca2+ influx, because it was blocked by the NMDAR pore blocker MK801 (Figure 1C), consistent with previous findings (Dudek and Bear, 1992Dudek S.M. Bear M.F. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade.Proc. Natl. Acad. Sci. USA. 1992; 89: 4363-4367Crossref PubMed Scopus (1324) Google Scholar, Massey et al., 2004Massey P.V. Johnson B.E. Moult P.R. Auberson Y.P. Brown M.W. Molnar E. Collingridge G.L. Bashir Z.I. Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression.J. Neurosci. 2004; 24: 7821-7828Crossref PubMed Scopus (574) Google Scholar, Mulkey and Malenka, 1992Mulkey R.M. Malenka R.C. Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus.Neuron. 1992; 9: 967-975Abstract Full Text PDF PubMed Scopus (872) Google Scholar, Sanderson et al., 2012Sanderson J.L. Gorski J.A. Gibson E.S. Lam P. Freund R.K. Chick W.S. Dell’Acqua M.L. AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors.J. Neurosci. 2012; 32: 15036-15052Crossref PubMed Scopus (104) Google Scholar). These results show a specific requirement for the CaMKIIα isoform in NMDAR-dependent LTD. The expression of the CaMKIIβ isoform was enhanced in the CaMKIIα knockout mice (Figures 1D and S2), but this did not compensate for loss of CaMKIIα function. Previous studies found elevated CaMKIIβ levels in the postsynaptic densities (PSDs) of CaMKIIα knockout mice (Elgersma et al., 2002Elgersma Y. Fedorov N.B. Ikonen S. Choi E.S. Elgersma M. Carvalho O.M. Giese K.P. Silva A.J. Inhibitory autophosphorylation of CaMKII controls PSD association, plasticity, and learning.Neuron. 2002; 36: 493-505Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), an effect also observed here. By contrast, the distribution of the postsynaptic marker PSD-95 was unaltered (Figure S2). A postsynaptic mechanism of the LFS-induced LTD was first indicated by lack of presynaptic changes in paired-pulse facilitation (Figure 1E) and further validated by CaMKII-dependent LTD-specific effects on the phosphorylation state of postsynaptic AMPARs (see below). Like LTP, CaMKII T286 autophosphorylation that generates Ca2+-independent “autonomous” activity is more readily generated by high-frequency stimulation (De Koninck and Schulman, 1998De Koninck P. Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar) and is indeed required for LTP (Giese et al., 1998Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (892) Google Scholar). Thus, we hypothesized that T286-phosphorylated CaMKII promotes LTP, whereas nonphosphorylated CaMKII promotes LTD. However, LTD stimuli also increased CaMKII T286 phosphorylation (Figure 2A). More importantly, CaMKIIα T286A mutant mice were functionally impaired for LTD (Figure 2B). In fact, no significant LFS-induced LTD was observed in these mice at all (Figure 2C), similar to the findings with CaMKIIα knockout mice or with CaMKII inhibition (see Figure 1C). Thus, T286-mediated autonomous CaMKII activity is required not only for LTP, but also for LTD. In contrast to the CaMKIIα knockout mice, the T286A mutant mice showed normal CaMKIIβ expression (Figure S3A). Although CaMKIIα expression appeared slightly reduced, the CaMKIIα to β ratio was not significantly changed (Figure S3A). As previously demonstrated (Giese et al., 1998Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (892) Google Scholar, Silva et al., 1992Silva A.J. Stevens C.F. Tonegawa S. Wang Y. Deficient hippocampal long-term potentiation in α-calcium-calmodulin kinase II mutant mice.Science. 1992; 257: 201-206Crossref PubMed Scopus (1188) Google Scholar), the relationship of stimulus input to fEPSP output before LFS did not differ significantly between the CaMKIIα wild-type and mutant mice (Figure S3B). Thus, the LTD impairments were not due to differences in synaptic efficacy prior to LFS. At first glance, CaMKII dependence of both LTP and LTD appears to indicate that autonomous CaMKII is required for enabling synaptic plasticity, but with other events then determining the direction of plasticity. However, at least for LTP, there is ample previous evidence for a direct role of CaMKII in the synaptic potentiation (Coultrap and Bayer, 2012Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35: 607-618Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Lisman et al., 2012Lisman J. Yasuda R. Raghavachari S. Mechanisms of CaMKII action in long-term potentiation.Nat. Rev. Neurosci. 2012; 13: 169-182PubMed Google Scholar). This includes CaMKII-mediated increase of AMPAR channel conductance by direct phosphorylation of the GluA1 subunits at S831 (Derkach et al., 1999Derkach V. Barria A. Soderling T.R. Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 3269-3274Crossref PubMed Scopus (696) Google Scholar, Kristensen et al., 2011Kristensen A.S. Jenkins M.A. Banke T.G. Schousboe A. Makino Y. Johnson R.C. Huganir R. Traynelis S.F. Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating.Nat. Neurosci. 2011; 14: 727-735Crossref PubMed Scopus (202) Google Scholar). As phosphorylation of GluA1 at another site, S567 (Figure 3A), instead directly reduces the number of synaptic AMPARs (Lu et al., 2010Lu W. Isozaki K. Roche K.W. Nicoll R.A. Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1.Proc. Natl. Acad. Sci. USA. 2010; 107: 22266-22271Crossref PubMed Scopus (66) Google Scholar), this could constitute a direct CaMKII-mediated LTD mechanism. Indeed, LTD stimulation of acute hippocampal slices produced a robust increase in S567 phosphorylation (Figure 3B). This increase was CaMKII dependent, because it was observed only in wild-type mice and not in the CaMKIIα knockout mice (Figure 3B). By contrast, a small LTD-induced increase in S831 phosphorylation immediately after LTD was not statistically significant (Figure 3C), consistent with a previous study (Lee et al., 1998Lee H.K. Kameyama K. Huganir R.L. Bear M.F. NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus.Neuron. 1998; 21: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). As expected after LTD stimuli (Lee et al., 1998Lee H.K. Kameyama K. Huganir R.L. Bear M.F. NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus.Neuron. 1998; 21: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar), GluA1 phosphorylation at the PKA site S845 (Roche et al., 1996Roche K.W. O’Brien R.J. Mammen A.L. Bernhardt J. Huganir R.L. Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit.Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar) decreased (Figure S4). But how can CaMKII differentiate between GluA1 phosphorylation at S567 versus S831 after LTD versus LTP? In order to address this question, the regulation of the GluA1 phosphorylation sites by CaMKII was compared in biochemical assays with purified protein. CaMKII was made “autonomous” by prephosphorylation at T286, and subsequent GluA1 phosphorylation was tested in the presence or absence of an additional Ca2+/CaM stimulus (Figure 3D). GluA1 S831 phosphorylation was significantly further stimulated by Ca2+/CaM, with Ca2+-independent autonomous activity less than 20% of maximal stimulated activity (Figure 3D), as expected for traditional CaMKII substrates (Coultrap et al., 2010Coultrap S.J. Buard I. Kulbe J.R. Dell’Acqua M.L. Bayer K.U. CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.J. Biol. Chem. 2010; 285: 17930-17937Crossref PubMed Scopus (80) Google Scholar). By contrast, GluA1 S567 phosphorylation was not further enhanced by additional Ca2+/CaM stimulation, resulting in autonomy of 100% or more (Figure 3D). As an important consequence of this differential regulation, GluA1 S831 was phosphorylated much more readily than S567 under the maximally Ca2+/CaM-stimulated conditions, whereas S567 was phosphorylated much more readily than S831 by autonomous CaMKII in absence of further stimulation (see Figure 3D). Thus, LTP-type stimuli (strong but brief) indeed favor S831 phosphorylation, whereas LTD-type stimuli (weak but prolonged) instead favor S567 phosphorylation (Figure 3D, right panels). Elevated CaMKII autonomy was previously observed for T-site binding T substrates (Bayer et al., 2001Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Interaction with the NMDA receptor locks CaMKII in an active conformation.Nature. 2001; 411: 801-805Crossref PubMed Scopus (578) Google Scholar, Coultrap et al., 2010Coultrap S.J. Buard I. Kulbe J.R. Dell’Acqua M.L. Bayer K.U. CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.J. Biol. Chem. 2010; 285: 17930-17937Crossref PubMed Scopus (80) Google Scholar), but this elevation was more modest and still allowed for some positive regulation by Ca2+/CaM (Coultrap et al., 2010Coultrap S.J. Buard I. Kulbe J.R. Dell’Acqua M.L. Bayer K.U. CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.J. Biol. Chem. 2010; 285: 17930-17937Crossref PubMed Scopus (80) Google Scholar). Furthermore, the GluA1 loop 1 lacks sequence homology with T substrates (Figure 4A). More importantly, GluA1 loop 1 showed no T-site binding: whereas the T-substrate peptide AC3 effectively competed with T-site-mediated CaMKII binding to GluN2B in vitro, a peptide derived from GluA1 loop 1 did not (Figure 4B). Thus, GluA1 S567 represents a class of CaMKII substrate sites that is distinct from both traditional substrates (such as GluA1 S831) and T substrates (such as GluN2B S1303). The decision between Ca2+-dependent induction of LTP versus LTD is thought to be determined by differential activation of protein kinase versus phosphatase. Indeed, whereas LTP requires CaMKII and its autonomous activity (for review, see Coultrap and Bayer, 2012Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35: 607-618Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Lisman et al., 2012Lisman J. Yasuda R. Raghavachari S. Mechanisms of CaMKII action in long-term potentiation.Nat. Rev. Neurosci. 2012; 13: 169-182PubMed Google Scholar), LTD requires calcineurin, a Ca2+-activated phosphatase (for review, see Collingridge et al., 2010Collingridge G.L. Peineau S. Howland J.G. Wang Y.T. Long-term depression in the CNS.Nat. Rev. Neurosci. 2010; 11: 459-473Crossref PubMed Scopus (678) Google Scholar, Malenka and Bear, 2004Malenka R.C. Bear M.F. LTP and LTD: an embarrassment of riches.Neuron. 2004; 44: 5-21Abstract Full Text Full Text PDF PubMed Scopus (2976) Google Scholar, Xia and Storm, 2005Xia Z. Storm D.R. The role of calmodulin as a signal integrator for synaptic plasticity.Nat. Rev. Neurosci. 2005; 6: 267-276Crossref PubMed Scopus (325) Google Scholar). However, this study provides evidence that LTD also requires autonomous CaMKII activity. Thus, autonomous CaMKII mediates both LTP and LTD. Importantly, our results also provide evidence for a mechanism by which CaMKII can indeed induce both of these two opposing forms of synaptic plasticity (as illustrated in Figure 4C). LFS-induced LTD is most robust prior to 3 weeks of age (Collingridge et al., 2010Collingridge G.L. Peineau S. Howland J.G. Wang Y.T. Long-term depression in the CNS.Nat. Rev. Neurosci. 2010; 11: 459-473Crossref PubMed Scopus (678) Google Scholar, Dudek and Bear, 1993Dudek S.M. Bear M.F. Bidirectional long-term modification of synaptic effectiveness in the adult and immature hippocampus.J. Neurosci. 1993; 13: 2910-2918Crossref PubMed Google Scholar), and our recordings were done at 2–2.5 weeks of age. Although LTP undergoes a developmental switch from PKA to CaMKII dependence between week 1 and 4 in mice (Yasuda et al., 2003Yasuda H. Barth A.L. Stellwagen D. Malenka R.C. A developmental switch in the signaling cascades for LTP induction.Nat. Neurosci. 2003; 6: 15-16Crossref PubMed Scopus (258) Google Scholar), CaMKII mediates LTP already in 2-week-old mice (Hinds et al., 1998Hinds H.L. Tonegawa S. Malinow R. CA1 long-term potentiation is diminished but present in hippocampal slices from alpha-CaMKII mutant mice.Learn. Mem. 1998; 5: 344-354PubMed Google Scholar) and rats (Wikström et al., 2003Wikström M.A. Matthews P. Roberts D. Collingridge G.L. Bortolotto Z.A. Parallel kinase cascades are involved in the induction of LTP at hippocampal CA1 synapses.Neuropharmacology. 2003; 45: 828-836Crossref PubMed Scopus (44) Google Scholar). Thus, our results do not reflect a developmental switch from CaMKII-dependence of LTD to LTP. Instead, the CaMKIIα isoform and its phospho-T286-induced autonomous activity mediate both LTD and LTP mechanisms at the same developmental stage. CaMKII T286 autophosphorylation is more readily induced by high-stimulation frequencies (Bayer et al., 2002Bayer K.U. De Koninck P. Schulman H. Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations.EMBO J. 2002; 21: 3590-3597Crossref PubMed Scopus (88) Google Scholar, Chao et al., 2011Chao L.H. Stratton M.M. Lee I.H. Rosenberg O.S. Levitz J. Mandell D.J. Kortemme T. Groves J.T. Schulman H. Kuriyan J. A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin- dependent kinase II holoenzyme.Cell. 2011; 146: 732-745Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, De Koninck and Schulman, 1998De Koninck P. Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar), which has been proposed to be important in the signal processing during LTP induction (Coultrap and Bayer, 2012Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35: 607-618Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Then, why is T286 phosphorylation also induced and required in LTD? Although T286 autophosphorylation acts as a spike frequency detector, it also acts as a detector of spike number (as well as spike duration and amplitude), thereby processing all of these input parameters (De Koninck and Schulman, 1998De Koninck P. Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar). Consequently, efficient T286 autophosphorylation should indeed be expected from both LTP stimuli (strong but brief, such as two 1 s trains of 100 pulses at 100 Hz) and LTD stimuli (weak but prolonged, such as 15 min of 900 pulses at 1 Hz). In any case, because T286 phosphorylation is required for both CaMKII-dependent LTD and LTP, it is not the factor differentiating between the opposing CaMKII effects on synaptic strength. Then what does determine whether the autonomous activity of T286-phosphorylated CaMKII induces LTD or LTP? Our results provide evidence that this involves differential substrate site selection, with phosphorylation of traditional CaMKII substrates promoting LTP, and phosphorylation of a distinct and differentially regulated substrate class instead promoting LTD. LTP stimuli are well established to induce CaMKII-mediated phosphorylation of GluA1 at S831, which, in turn, results in the increase in AMPAR conductance observed after LTP (Barria et al., 1997Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation.Science. 1997; 276: 2042-2045Crossref PubMed Scopus (889) Google Scholar, Benke et al., 1998Benke T.A. Lüthi A. Isaac J.T. Collingridge G.L. Modulation of AMPA receptor unitary conductance by synaptic activity.Nature. 1998; 393: 793-797Crossref PubMed Scopus (433) Google Scholar, Derkach et al., 1999Derkach V. Barria A. Soderling T.R. Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 3269-3274Crossref PubMed Scopus (696) Google Scholar, Kristensen et al., 2011Kristensen A.S. Jenkins M.A. Banke T.G. Schousboe A. Makino Y. Johnson R.C. Huganir R. Traynelis S.F. Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating.Nat. Neurosci. 2011; 14: 727-735Crossref PubMed Scopus (202) Google Scholar). With GluA1 S567, we here identify an LTD-induced CaMKII substrate site. Notably, S567 phosphorylation has been shown to decrease synaptic AMPAR localization, and thereby currents (Lu et al., 2010Lu W. Isozaki K. Roche K.W. Nicoll R.A. Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1.Proc. Natl. Acad. Sci. USA. 2010; 107: 22266-22271Crossref PubMed Scopus (66) Google Scholar). Thus, differential CaMKII-mediated phosphorylation of two distinct sites, S831 and S567, on the same protein, GluA1, can indeed promote either synaptic depression or potentiation. But what controls the differential substrate site selection by autonomous CaMKII in LTD versus LTP? One difference lies in the strength of additional stimulation by Ca2+/CaM, which is much stronger during LTP compared to LTD, with higher Ca2+ concentrations that lasts throughout the entire induction period for LTP, compared to lower Ca2+ concentrations that repeatedly fall back to basal levels during the induction of LTD (Helmchen, 2002Helmchen F. Raising the speed limit—fast Ca(2+) handling in dendritic spines.Trends Neurosci. 2002; 25 (discussion 441): 438-441Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, Ismailov et al., 2004Ismailov I. Kalikulov D. Inoue T. Friedlander M.J. The kinetic profile of intracellular calcium predicts long-term potentiation and long-term depression.J. Neurosci. 2004; 24: 9847-9861Crossref PubMed Scopus (82) Google Scholar, Zucker, 1999Zucker R.S. Calcium- and activity-dependent synaptic plasticity.Curr. Opin. Neurobiol. 1999; 9: 305-313Crossref PubMed Scopus (507) Google Scholar). Such additional stimulation further increases activity even of “autonomous” CaMKII, at least toward traditional substrates (Coultrap et al., 2010Coultrap S.J. Buard I. Kulbe J.R. Dell’Acqua M.L. Bayer K.U. CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.J. Biol. Chem. 2010; 285: 17930-17937Crossref PubMed Scopus (80) Google Scholar, Miller and Kennedy, 1986Miller S.G. Kennedy M.B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch.Cell. 1986; 44: 861-870Abstract Full Text PDF PubMed Scopus (641) Google Scholar). In our biochemical assays, such further Ca2+/CaM stimulation of “autonomous” CaMKII indeed significantly increased its activity for GluA1 S831 (by >5-fold, as expected), but, remarkably, not for GluA1 S567. The effect of this regulation on GluA1 phosphorylation was that biochemical LTP-type stimuli (brief but strong) primarily targeted S831, whereas LTD-type stimuli (weak but prolonged) primarily targeted S567. Thus, this regulation readily explains the substrate site selection after different stimuli in hippocampal slices. Synaptic GluA1 removal by S567 phosphorylation is clearly a regulatory step that would promote LTD. However, determining the necessity and sufficiency of this phosphorylation for LTD induction and expression will require further detailed investigations. Notably, in LTP, CaMKII is required for orchestrating multiple signaling events, but with individual events (such as GluA1 S831 phosphorylation) neither necessary nor sufficient, even though each clearly promotes and/or facilitates LTP (for review, see Coultrap and Bayer, 2012Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35: 607-618Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Lisman et al., 2012Lisman J. Yasuda R. Raghavachari S. Mechanisms of CaMKII action in long-term potentiation.Nat. Rev. Neurosci. 2012; 13: 169-182PubMed Google Scholar). Thus, a similarly complex situation may be expected for the CaMKII-mediated signaling that is required for LTD. Given this complexity and redundancy of plasticity pathways, it is even more remarkable that the “autonomous” form of CaMKII is essential, not only in LTP but also in LTD. Taken together, CaMKII and its autonomous activity mediate two opposing forms of NMDAR-dependent synaptic plasticity. “Autonomous” CaMKII requires additionally further stimulation by Ca2+/CaM for maximal phosphorylation of traditional CaMKII substrates. By contrast, GluA1 S567 represent examples of a distinct class of LTD-specific CaMKII substrates for which “autonomous” phosphorylation is not further stimulated by Ca2+/CaM. Thus, we propose that phosphorylation of traditional CaMKII substrates promotes LTP, whereas phosphorylation of the substrate class identified here instead promotes LTD (as illustrated in Figure 4C). The T286A mice were described previously (Giese et al., 1998Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (892) Google Scholar); the CaMKIIα knockout mice are a newly generated strain (see Figure S2). All animal treatments and housing were in accordance with the University of Colorado Denver Institutional Animal Care and Use Committee. Electrophysiological recordings, western analysis, in vitro phosphorylation assays, immunocytochemistry, and protein-protein binding assays as well as preparation of hippocampal slices, extracts, and purified proteins were done essentially as described previously (Buard et al., 2010Buard I. Coultrap S.J. Freund R.K. Lee Y.S. Dell’Acqua M.L. Silva A.J. Bayer K.U. CaMKII “autonomy” is required for initiating but not for maintaining neuronal long-term information storage.J. Neurosci. 2010; 30: 8214-8220Crossref PubMed Scopus (126) Google Scholar, Coultrap et al., 2010Coultrap S.J. Buard I. Kulbe J.R. Dell’Acqua M.L. Bayer K.U. CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.J. Biol. Chem. 2010; 285: 17930-17937Crossref PubMed Scopus (80) Google Scholar, Coultrap et al., 2012Coultrap S.J. Barcomb K. Bayer K.U. A significant but rather mild contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity.PLoS ONE. 2012; 7: e37176Crossref PubMed Scopus (32) Google Scholar, Sanderson et al., 2012Sanderson J.L. Gorski J.A. Gibson E.S. Lam P. Freund R.K. Chick W.S. Dell’Acqua M.L. AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors.J. Neurosci. 2012; 32: 15036-15052Crossref PubMed Scopus (104) Google Scholar, Vest et al., 2007Vest R.S. Davies K.D. O’Leary H. Port J.D. Bayer K.U. Dual mechanism of a natural CaMKII inhibitor.Mol. Biol. Cell. 2007; 18: 5024-5033Crossref PubMed Scopus (142) Google Scholar) and detailed in the Supplemental Experimental Procedures. The in vitro GluA1 phosphorylation assays were done at 30°C with 10 nM (kinase subunits) of T286-phosphorylated CaMKII and 2 μM GST fusion proteins of the cytoplasmic GluA1 C-tail (containing S831) or loop1 (containing S567), in presence of either Ca2+/CaM or EGTA. Phospho-site detection utilized specific antibodies (Lu et al., 2010Lu W. Isozaki K. Roche K.W. Nicoll R.A. Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1.Proc. Natl. Acad. Sci. USA. 2010; 107: 22266-22271Crossref PubMed Scopus (66) Google Scholar). Immunodetection values after western analysis were quantified as described (Buard et al., 2010Buard I. Coultrap S.J. Freund R.K. Lee Y.S. Dell’Acqua M.L. Silva A.J. Bayer K.U. CaMKII “autonomy” is required for initiating but not for maintaining neuronal long-term information storage.J. Neurosci. 2010; 30: 8214-8220Crossref PubMed Scopus (126) Google Scholar, Coultrap et al., 2012Coultrap S.J. Barcomb K. Bayer K.U. A significant but rather mild contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity.PLoS ONE. 2012; 7: e37176Crossref PubMed Scopus (32) Google Scholar, Vest et al., 2007Vest R.S. Davies K.D. O’Leary H. Port J.D. Bayer K.U. Dual mechanism of a natural CaMKII inhibitor.Mol. Biol. Cell. 2007; 18: 5024-5033Crossref PubMed Scopus (142) Google Scholar). NMDAR-dependent LTD was induced in aCSF (in mM: 126 NaCl, 3.0 KCl, 1.5 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 11 D-glucose, and 25.9 NaHCO3) by 15 min 1 Hz stimulation for electrophysiology (at test-stimulus intensity, which was set to elicit 50%–60% of maximal response before LTD induction) or by 3 min 20 μM NMDA for biochemistry (Sanderson et al., 2012Sanderson J.L. Gorski J.A. Gibson E.S. Lam P. Freund R.K. Chick W.S. Dell’Acqua M.L. AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors.J. Neurosci. 2012; 32: 15036-15052Crossref PubMed Scopus (104) Google Scholar). In contrast to chemically induced LTP, this chemically induced LTD is well established and utilizes the same mechanisms as NMDAR-dependent electrically induced LTD (Lee et al., 1998Lee H.K. Kameyama K. Huganir R.L. Bear M.F. NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus.Neuron. 1998; 21: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Slices from 21- to 24-day-old mice were used for biochemistry. Statistical analysis (∗∗p < 0.01; ∗p < 0.05; ns: not significant) for all multiple comparisons was done by ANOVA with Newman-Keuls post hoc analysis. Significance of LTD was analyzed by paired t test compared to baseline before LFS. We thank Drs. Matt Kennedy, Paco Herson, and Guiying Deng (U. Colorado) for helpful discussions and critical reading of the manuscript. We thank Drs. Giese, Silva, and Yasuda for the T286A mutant mice. We thank Dr. Wallace Chick for generating the gene targeting vector. The research was supported by National Institutes of Health grants NS048154 (Rocky Mountain Neurological Disorders Center grant), NS040701 (to M.L.D.), NS080851, and NS081248 (to K.U.B.). The University of Colorado is currently seeking patent protection for tatCN21, its derivatives, and its uses. Download .pdf (1.08 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figures S1–S4" @default.
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• W2087436387 title "Autonomous CaMKII Mediates Both LTP and LTD Using a Mechanism for Differential Substrate Site Selection" @default.
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