Paul T. Kelly

Postsynaptic injection of CA2+/CaM induces synaptic potentiation requiring CaMKII and PKC activity.

CA2+-regulated protein kinases play critical roles in long-term potentiation (LTP). To understand the role of Ca2+/calmodulin (CaM) signaling pathways in synaptic transmission better, Ca2+/CaM was injected into hippocampal CA1 neurons. Ca2+/CaM induced significant potentiation of excitatory synaptic responses, which was blocked by coinjection of a CaM-binding peptide and was not induced by injections of Ca2+ or CaM alone. Reciprocal experiments demonstrated that Ca2+/CaM-induced synaptic potentiation and tetanus-induced LTP occluded one another. Pseudosubstrate inhibitors or high-affinity substrates of CaMKII or PKC blocked Ca2/CaM-induced potentiation, indicating the requirement of CaMKII and PKC activities in synaptic potentiation. We suggest that postsynaptic levels of free Ca2+/CaM is a rate limiting factor and that functional cross-talk between Ca2+/CaM and PKC pathways occurs during the induction of LTP.

Calmodulin-dependent protein kinase II. Multifunctional roles in neuronal differentiation and synaptic plasticity.

One of the most important mechanisms for regulating neuronal functions is through second messenger cascades that control protein kinases and the subsequent phosphorylation of substrate proteins. Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is the most abundant protein kinase in mammalian brain tissues, and the α-subunit of this kinase is the major protein and enzymatic molecule of synaptic junctions in many brain regions. CaM-kinase II regulates itself through a complex autophosphorylation mechanism whereby it becomes calcium-independent following its initial activation. This property has implicated CaM-kinase II as a potential molecular switch at central nervous system (CNS) synapses. Recent studies have suggested that CaM-kinase II is involved in many diverse phenomena such as epilepsy, sensory deprivation, ischemia, synapse formation, synaptic transmission, long-term potentiation, learning, and memory.During brain development, the expression of CaM-kinase II at both protein and mRNA levels coincides with the active periods of synapse formation and, therefore, factors regulating the genes encoding kinase subunits may play a role in the cell-to-cell recognition events that underlie neuronal differentiation and the establishment of mature synaptic functions. Recent findings have demonstrated that the mRNA encoding the α-subunit of CaM-kinase II is localized in neuronal dendrites. Current speculation suggests that the localized translation of dendritic mRNAs encoding specific synaptic proteins may be responsible for producing synapse-specific changes associated with the processing, storage, and retrieval of information in neural networks.

Developmental Changes in Calmodulin‐Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin‐dependent protein kinase II (CaM‐kinase II). This kinase is comprised of a major 50‐kilodalton (kDa) and a minor 60‐kDa subunit. Experiments examined the developmental properties of CaM kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM‐kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that fore brain CaM‐kinase II undergoes a striking age‐dependent change in subunit composition. In early postnatal forebrain the 60‐kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM‐kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM‐kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.

Changes in the subcellular distribution of calmodulin-kinase II during brain development

Subcellular fractions prepared from rodent forebrain at different postnatal ages were examined for calmodulin-binding proteins using [125I]calmodulin and a gel overlay technique. Synaptic junction (SJ) fractions from newborn brain, which display purity comparable to adult SJ fractions, contain low but detectable amounts of 60 and 50 kdalton calmodulin-binding polypeptides; the latter being the major postsynaptic density protein. These polypeptides have recently been shown to be the calmodulin-binding protein subunits of calmodulin-dependent protein kinase II (CaM-kinase II). CaM-kinase II polypeptides represented the predominent calmodulin-binding proteins in nearly every subcellular fraction examined, regardless of postnatal age. Large increases were observed in the CaM-kinase II content of every subcellular fraction throughout postnatal development. During development, a striking shift in the subcellular distribution of CaM-kinase Ii was observed. Over 4 times as much CaM-kinase II was cytosolic relative to particulate in newborn brain while this ratio was completely reversed in adult brain. Large age-dependent increases in particulate-associated CaM-kinase II were observed in highly purified synaptic plasma membrane (5-fold) and SJ (14-fold) fractions. The CaM-kinase II content of SJ fractions increased approximately 70% between days 24 and 90, a period in development that follows the most active stages of synapse formation in situ. In adult brain, approximately 60% of CaM-kinase II in crude synaptosomal fractions (P2-INT) was recovered in SJ fractions. The CaM-kinase II in SPM fractions from all developmental ages resists solubilization in Triton X-100 and greater than 90% is recovered in SJ fractions. These studies indicate that during brain development the accumulation of SJ-associated CaM-kinase II represents an important process in the molecular and enzymatic maturation of CNS postsynaptic structures.

Developmental changes in morphology and molecular composition of isolated synaptic junctional structures

Synaptic junctional fractions which display subcellular purity that compares favorably to similar fractions prepared from adult have been isolated from immature rat brains. Electron microscopic analysis of immature fractions has revealed age-dependent changes in the morphology of isolated synaptic structures. The recovery of total synaptic junctional protein increased in a linear fashion and was temporally correlated with the appearance of asymmetric synapses in brain. Systematic age-dependent changes were observed in the protein and glycoprotein composition of synaptic membrane and synaptic junction fractions during postnatal development. In isolated synaptic junctions, the major postsynaptic density protein increased approximately 20-fold during postnatal development. Immature synaptic junction fractions contained tubulin and actin in larger relative quantities than are present in synaptic junction fractions isolated from adult brain tissues. Immature synaptic junctions also contained appreciable amounts of postsynaptic membrane glycoproteins that bind concanavalin A (con A).

Autophosphorylation of calmodulin-kinase II in synaptic junctions modulates endogenous kinase activity

Previous studies have purified from bfain a Ca2+/calmodulin‐dependent protein kinase II (designated CaM‐kinase II) that phosphorylates synapsin I, a synaptic vesicle‐associated phosphoprotein. CaM‐kinase II is composed of a major Mr 50K polypeptide and a minor Mr60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin‐dependent manner. Recent studies have demonstrated that the 50K component of CaM‐kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM‐kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [α‐32P]8‐azido‐ATP was used to demonstrate that the 60K and 50K polypeptides of SJ‐associated CaM‐kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin‐dependent manner. Experiments using 32P‐labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I‐calmoduhn binding sites. These results suggested that the autophosphorylation of CaM‐kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I‐calmodulin overlay techniques and sodium dodecyl Solfate‐polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM‐kinase II polypeptides bound more calmodulin (50–70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM‐kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaMkinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM‐kinase II. The CaM‐kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300–1,000 nM). These findings show that the degree of autophosphorylation of CaM‐kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinases activity at CNS synapses in situ.

Cellular and molecular bases of memory: synaptic and neuronal plasticity.

Discoveries made during the past decade have greatly improved our understanding of how the nervous system functions. This review article examines the relation between memory and the cellular mechanisms of neuronal and synaptic plasticity in the central nervous system. Evidence indicating that activity-dependent short- and long-term changes in strength of synaptic transmission are important for memory processes is examined. Focus is placed on one model of synaptic plasticity called long-term potentiation, and its similarities with memory processes are illustrated. Recent studies show that the regulation of synaptic strength is bidirectional (e.g., synaptic potentiation or depression). Mechanisms involving intracellular signaling pathways that regulate synaptic strength are described, and the specific roles of calcium, protein kinases, protein phosphatases, and retrograde messengers are emphasized. Evidence suggests that changes in synaptic ultrastructure, dendritic ultrastructure, and neuronal gene expression may also contribute to mechanisms of synaptic plasticity. Also discussed are recent findings about postsynaptic mechanisms that regulate short-term synaptic facilitation and neuronal burst-pattern activity, as well as evidence about the subcellular location (presynaptic or postsynaptic) of mechanisms involved in long-term synaptic plasticity.

Evidence that the 40,000 Mr phosphoprotein influenced by high frequency synaptic stimulation is the alpha subunit of pyruvate dehydrogenase.

We have previously shown that brief periods of high frequency synaptic stimulation of the rat hippocampus influence the endogenous phosphorylation of a 40,000 Mr brain protein (Browning et al.). The results of the present study demonstrate that this brain phosphoprotein is enriched in a purified mitochondrial fraction and co-migrates with the alpha-subunit of pyruvate dehydrogenase in sodium dodecyl sulfate polyacrylamide gels. Comparisons of total and partial proteolytic fingerprints indicate that the two proteins are essentially identical. In addition, the phosphorylation of the 40,000 Mr brain protein is sensitive to both dichloroacetate and magnesium as has been reported for pyruvate dehydrogenase. Taken together these data provide persuasive evidence that the brain protein is the alpha-subunit of pyruvate dehydrogenase and thereby raise the possibility that even very short periods of synaptic activity influence an enzyme of particular importance to mitochondrial metabolism in brain.

Calcium/calmodulin-dependent protein kinase II regulates hippocampal synaptic transmission

Extracellular application of protein kinase inhibitors was used to examine the role of calcium/calmodulin-dependent protein kinase II (CaM-KII) in synaptic transmission in the CA1 region of rat hippocampus. Bath application of the broad spectrum, membrane permeable kinase inhibitor H7 (250 microM) decreased excitatory synaptic responses elicited in hippocampal slices. Whereas H7 inhibits several protein kinases and has non-specific effects, several synthetic peptides have been developed as specific inhibitors of CaM-KII. Using in situ phosphorylation in hippocampal slices, we demonstrate that extracellular application of synthetic peptide inhibitors of CaM-KII preferentially suppresses the phosphorylation of synapsin I at the CaM-KII specific site. This suppression was not reversed by the application of a calcium ionophore indicating the decrease in phosphorylation does not result only from blockade of presynaptic calcium influx. Thus, it appears the peptides gain access to intracellular compartments and retain their inhibitory properties. Further, we found that extracellular application of these peptide inhibitors decreased excitatory synaptic responses elicited in the CA1 region of hippocampal slices with relative potencies consistent with their ability to block CaM-KII activity in vitro. Peptide application did not alter the input resistance of postsynaptic cells nor responses elicited by glutamate iontophoresis. These results suggest that CaM-KII activity, possibly through phosphorylation of presynaptic synapsin I, is required for sustained synaptic transmission at mammalian synapses.

Glutamate iontophoresis induces long-term potentiation in the absence of evoked presynaptic activity

Protocols that induce long-term potentiation (LTP) typically involve afferent stimulation. We tested the hypothesis that LTP induction does not require presynaptic activity. The significance of this hypothesis is underscored by results suggesting that LTP expression may involve activity-dependent presynaptic changes. An induction protocol using glutamate iontophoresis was developed that reliably induced LTP in hippocampal slices without afferent stimulation. Iontophoresis LTP was Ca2+ dependent, was blocked by MK-801, and occluded tetanus-induced LTP. Iontophoresis LTP was induced when excitatory postsynaptic potentials were completely blocked by adenosine plus tetrodotoxin. Our results suggest constraints on the involvement of presynaptic mechanisms and putative retrograde messengers in LTP induction and expression; namely, these processes must function without many forms of activity-dependent presynaptic processes.