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EVOLUTIONARY HOMOLOGS: Table of contents
PKA and neural facilitation (Short and long term potentiation - a model for learning): Studies in mammals (part 1/2)
Calcineurin is a calcium-sensitive serine/threonine phosphatase that is present at high levels in the hippocampus and enriched at synapses. Once activated, calcineurin can act both directly and indirectly on protein substrates, including CREB. (1) It can dephosphorylate target proteins directly and thereby regulate specific cellular functions. (2) It can modulate an even larger variety of substrates indirectly by its ability to dephosphorylate inhibitor 1 (I-1). I-1, when phosphorylated, inhibits the function of protein phosphatase 1 (PP1). Dephosphorylation of I-1 by calcineurin activates PP1 and leads to the dephosphorylation of a large and independent set of target proteins. One interesting feature of the regulatory actions of calcineurin comes from its interactions with the cAMP cascade. Calcineurin inhibits the action of I-1 by dephosphorylating the site on I-1 phosphorylated by the cAMP-dependent kinase, PKA. Indeed, calcineurin and PKA antagonistically regulate the function of several proteins, including NMDA and GluR6 glutamate receptors (Winder, 1998 and references).
The interactions of PKA and calcineurin are of particular interest in the context of LTP. Based on the requirement for macromolecular synthesis, LTP can be divided into at least two components: an early component (E-LTP) and a late component (L-LTP). Delivery to the Schaffer collateral-CA1 pyramidal cell (SC-CA1) synapse of a single 100 Hz train lasting 1s elicits E-LTP, a relatively short-lived and weak enhancement of synaptic transmission that does not require protein and RNA synthesis and is not dependent on PKA. By contrast, administration of three or four trains of 100 Hz elicits L-LTP, a more robust and stable form of LTP lasting many hours that is dependent on the activation of PKA as well as the synthesis of both RNA and protein. Recent experiments with inhibitors of phosphatases suggest that one role of PKA in LTP in area CA1 may be to suppress the actions of PP1 or PP2A (see Drosophila Twins). In particular, when LTP in area CA1 is induced by strong stimuli it can be blocked by inhibitors of PKA. However, this effect of PKA inhibitors is removed by preincubation of slices with PP1/PP2A inhibitors. This has led to the suggestion that under certain circumstances, PKA may "gate" LTP by suppressing a phosphatase cascade (Winder, 1998 and references).
To investigate the role of phosphatases in synaptic plasticity using genetic approaches, transgenic mice were generated that overexpress a truncated form of calcineurin under the control of the CaMKIIalpha promoter. Mice expressing this transgene show increased calcium-dependent phosphatase activity in the hippocampus. Physiological studies of these mice and parallel pharmacological experiments in wild-type mice reveal a novel, intermediate phase of LTP (I-LTP) in the CA1 region of the hippocampus. This intermediate phase differs from E-LTP by requiring multiple trains for induction and by being dependent on PKA. It differs from L-LTP by not requiring new protein synthesis. These data suggest that calcineurin acts as an inhibitory constraint on I-LTP, one which is relieved by PKA. This inhibitory constraint acts as a gate to regulate the synaptic induction of L-LTP (Winder, 1998 ).
Since phosphatases impose an inhibitory constraint on LTP, these results suggest that PKA is required to suppress phosphatase activity sufficiently to elicit LTP fully. Activation of NMDA receptors increases cAMP levels and PKA activity in CA1 through a calmodulin-dependent process. Therefore, while calcium directly regulates the balance of kinase and phosphatase activity, the generation of cAMP by NMDA-receptor-dependent activation of calcium-sensitive adenylyl cyclases (see Drosophila Rutabaga) can favor kinases further by inducing a PKA-dependent inactivation of the activation of PP1 by calcineurin, through phosphorylation of I-1. It should be noted, however, that although physiological studies suggest the presence of I-1 or an I-1-like protein in CA1, the histological localization of I-1 in CA1 is somewhat controversial. Because I-1 is a member of a family of proteins that modulate phosphatase function, it is possible that another protein from this family mediates the effects reported here (Winder, 1998 and references).
Hippocampal-dependent memory in mice that express a truncated form of calcineurin was assessed. Mutant mice have normal short-term memory but exhibit a profound and specific defect in long-term memory on both the spatial version of the Barnes maze and on a task requiring the visual recognition of a novel object. To determine whether mutant mice have the capacity for long-term memory, the training protocol was intensified on the spatial version of the Barnes maze by increasing the number of daily training trials. The memory defect is fully reversed, indicating that these mice are capable of forming long-term memory. This rescue experiment suggests that mice overexpressing calcineurin have impaired long-term memory possibly due to a specific defect in the transition between short-term and long-term memory. The memory defect observed was not the result of a developmental abnormality due to the genetic manipulation. In mice in which the expression of the calcineurin transgene is regulated by the tetracycline-controlled transactivator (tTA) system, the spatial memory defect is reversed when the expression of the transgene is repressed by doxycycline. Thus calcineurin has a role in the transition from short- to long-term memory, which correlates with a novel intermediate phase of LTP (Mansuy, 1998).
Two type II regulatory (R) subunits of cAMP-dependent protein kinase (PKA) of 50 and 47 kDa have been identified in Aplysia neurons by several criteria which include phosphorylation by the catalytic subunit of PKA and nanomolar affinity for a peptide fragment of the human thyroid protein Ht 31, properties that in mammals distinguish type II from type I R subunits. The neuronal type II R subunits are differentially localized within cells. For example, the 50-kDa polypeptide is enriched in taxol-stabilized microtubules. In addition, at least seven high molecular mass neuronal RII-binding proteins ranging in mass from 110 to 420 kDa have been demonstrated by a blot overlay technique, which uses 32P-labeled bovine RII alpha as a probe. The RII-binding proteins also exhibit discrete patterns of subcellular localization. For example, the 420 kDa species is enriched in taxol-stabilized microtubules and therefore may serve to anchor the 50-kDa RII subunit. The localization of PKA through the association of RII subunits with the binding proteins may anchor the multifunctional kinase close to key substrates and thereby contribute to the spatial organization required to mediate the orderly phosphorylation events that underly neuronal modulation (Cheley, 1994).
Developing sensory systems are sculpted by an activity-dependent strengthening and weakening of connections. Long-term potentiation (LTP) and depression (LTD) in vitro have been proposed to model this experience-dependent circuit refinement. LTP and LTD induction in vitro was compared with plasticity in vivo in the developing mouse visual cortex of animals mutant for protein kinase A (PKA), a key enzyme implicated in the plasticity of a diverse array of systems. In mice lacking the RIbeta regulatory subunit of PKA, three abnormalities of synaptic plasticity are observed in layer II/III of visual cortex in vitro. These included an absence of (1) extracellularly recorded LTP, (2) depotentiation or LTD, and (3) paired-pulse facilitation. Potentiation is induced, however, by pairing low-frequency stimulation with direct depolarization of individual mutant pyramidal cells. Together these findings suggest that the LTP defect in slices lacking PKA RIbeta lies in the transmission of sufficient net excitation through the cortical circuit. Nonetheless, functional development and plasticity of visual cortical responses in vivo after monocular deprivation does not differ from normal. The loss of all responsiveness in most cortical cells to stimulation of the originally deprived eye can be restored by the reverse suture of eyelids during the critical period in both wild-type and mutant mice. Such an activity-dependent increase in response would seem to require a mechanism like potentiation in vivo. Thus, the RIbeta isoform of PKA is not essential for ocular dominance plasticity, which can proceed despite defects in several common in vitro models of neural plasticity (Hensch, 1998).
Finding an experimental system to examine the relationship between LTP, an experimental paradigm for long term memory, and the behaviorial aspects of long term memory has been difficult because LTP is a cellular response to stimulation while memory is tested behaviorly. To explore the role of protein kinase A in the late phase of long-term potentiation (L-LTP) and memory, transgenic mice were generated that express R(AB), an inhibitory form of the regulatory subunit of PKA, only in the hippocampus and other forebrain regions. In these R(AB) transgenic mice, hippocampal PKA activity is reduced, and L-LTP is significantly decreased in area CA1, without affecting basal synaptic transmission or the early phase of LTP. Moreover, the L-LTP deficit is paralleled by behavioral deficits in spatial memory (as tested in the hidden platform version of the Morris water maze task. This is a hippocampus-dependent task that relies on the ability of the animal to learn and remember the relationships between multiple distal cues and the platform) and in long-term but not short-term memory for contextual fear conditioning (as measured by associating a neutral conditioned stimulus such as a tone with an aversive unconditioned stimulus, such as foot shock). These deficits in long-term memory are similar to those produced by protein synthesis inhibition. Thus, PKA plays a critical role in the consolidation of long-term memory. This correlation between deficits in L-LTP and impaired behavioral long-term memory is not absolute and may be overridden by other factors. Nevertheless, it is possible to propose a molecular model for the late phase of LTP and explicit forms of long-term memory storage. Whereas short term memory involves Calmodulin sensitive activation of Nitric oxide synthase and activation of kinases such as CaM Kinase II, long term memory involves the Calmodulin activation of adenylyl cyclase and the consequent cAMP dependent activation of PKA; in turn, this targets transcription factors such as CREB (Abel, 1997).
The effects of mutations in protein kinase A (PKA) were assessed on long-term potentiation (LTP) in the mossy fiber pathway. Tests were made of this pathway's relationship to spatial and contextual learning. Ablation by gene targeting of the C beta 1 or the RI beta isoform of PKA produces a selective defect in mossy fiber LTP, providing genetic evidence for the role of these isoforms in the mossy fiber pathway. Despite the elimination of mossy fiber LTP, the behavioral responses to novelty, spatial learning, and conditioning to context are unaffected. Thus, contrary to current theories about hippocampal function, mossy fiber LTP does not appear to be required for spatial or contextual learning. In the absence of mossy fiber LTP, adequate spatial and contextual information might reach the CA1 region via other pathways from the entorhinal cortex (Huang, 1995).
The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). PKA activity is required for both homosynaptic LTD and depotentiation. A specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, these data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits (Brandon, 1995).
The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. Homozygous mutants in the Cbeta1-subunit isoform showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants are unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that is only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but fail to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway (Qui, 1996).
Gene expression regulated by the cAMP response element (CRE) has been implicated in synaptic plasticity and long-term memory. It has been proposed that CRE-mediated gene expression is stimulated by signals that induce long-term potentiation (LTP). To test this hypothesis, Mice were prepared that were transgenic for a CRE-regulated reporter construct. Long-lasting long-term potentiation (L-LTP) in the hippocampus was studied, because it depends on cAMP-dependent protein kinase activity (PKA) and de novo gene expression. CRE-mediated gene expression was markedly increased after L-LTP, but not after decremental UP (D-LTP). Furthermore, inhibitors of PKA blocked L-LTP and associated increases in CRE-mediated gene expression. These data demonstrate that the signaling required for the generation of L-LTP but not D-LTP is sufficient to stimulate CRE-mediated transcription in the hippocampus (Impey, 1996).
Memory storage consists of a short-term phase that is independent of new protein synthesis and a long-term phase that requires the synthesis of new proteins and RNA. A cellular representation of these two phases has been demonstrated recently for long-term potentiation (LTP) in both the Schaffer collateral and the mossy fibers of the hippocampus, a structure widely thought to contribute to memory consolidation. By contrast, much less information is available about the medial perforant pathway (MPP), one of the major inputs to the hippocampus. Both a short-lasting and a long-lasting potentiation (L-LTP) can be induced in the MPP of rat hippocampal slices by applying repeated tetanization in reduced levels of magnesium. This potentiation is dependent on the activation of NMDA receptors. The early, transient phase of LTP in the MPP does not require either protein or RNA synthesis, and it is independent of protein kinase A activation. By contrast, L-LTP required the synthesis of proteins and RNA, and is selectively blocked by inhibitors of cAMP-dependent protein kinase (PKA). Forskolin, an adenylate cyclase (See Drosophila Rutabaga) activator, also induced a L-LTP that was attenuated by inhibition of transcription. These results demonstrate that, like LTP in the Schaffer collateral and mossy fiber pathways, MPP LTP also consists of a late phase that is dependent on protein and RNA synthesis and PKA activity. Thus, cAMP-mediated transcription appears to be a common mechanism for the late form of LTP in all three pathways within the hippocampus (Nguyen, 1996).
Continued: PKA and neural facilitation (Short and long term potentiation - a model for learning): Studies in mammals part 2/2

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