Akap

PKA, PP2B

Kinase and phosphatase localization

CaMKII

Signal transduction

Regulate likelihood of LTP induction

This consideration also serves as an important caveat for interpreting results from NMDA receptor knockout mice. This apparently "clean" experimental manipulation, wherein the NMDA receptor is entirely lost, likely results in a large number of secondary effects on molecules normally associated with the NMDA receptor postsynaptically. In fact, experiments using various deletion mutants missing the cytoplasmic anchoring domains of the NMDA receptor have allowed dissection of the role of the NMDA receptor as a scaffolding protein versus its role as a ligand-gated ion channel (50). Deletion of the intracellular domain of the NMDA receptor appears to be sufficient to account for essentially all the physiologic and behavioral deficits observed in NMDA receptor knockout mice—the upshot is that the role of the NMDA receptor as a component of the PSD infrastructure is just as important as its role as a ligand-gated ion channel.

Additional Direct Interactions with the NMDA Receptor

The NMDA receptor NR1 and NR2 sub-units also bind spectrin, the actin-binding protein. This may serve as an additional cytoskeleton anchoring site postsynap-tically. Moreover, this interaction is subject to regulation by phosphorylation— tyrosine phosphorylation of NR2B leads to decreased interactions of spectrin with the receptor, and NR1 interaction with spectrin is modulated by serine/threonine phos-phorylation. However, the role of these effects in synaptic plasticity is not clear at this point.

Finally, as described earlier in the section on direct modulation of NMDA receptors, the scaffolding protein RACK1 promotes formation of a fyn/RACK1/NR2B complex that actually inhibits fyn phosphorylation of the NMDA receptor and diminishes current through the receptor (see Figure 2). Also, PDS-95 modulates src phosphory-lation of NMDARs, and src potentiation of

NMDAR currents appears to require the presence of PSD-95.

Consideration of the complicated structure and regulation of the postsynaptic density complex highlights the importance of thinking of the entire postsynaptic domain as a large functional unit. The NMDA receptor is embedded in a dynamic multiprotein complex that it regulates and in turn that regulates it. While many details of the structural components of the PSD complex are still being worked out and their roles in LTP induction are being actively investigated, it is clear that disrupting one or more of the cogs in this machine can lead to disruption of the proper function of the NMDA receptor.

AMPA Receptors

AMPA receptors, of course, provide the initial depolarization, either locally or distally in the neuron, that ultimately results in NMDA receptor activation. As such, alterations in the AMPA receptor protein or its associated interacting proteins can lead to loss of proper regulation of NMDA receptor activation. However, in this section we will focus on the AMPA receptor as a structural component of the synapse.

AMPA receptors, like NMDA receptor, also reside postsynaptically but are in much more of a state of flux than NMDA receptors. In fact, the average half-life for an AMPA receptor in the postsynaptic membrane is 15 minutes. We will return to some implications of this in the next chapter. Also, as we have discussed in previous chapters, AMPA receptor membrane insertion can be activity-dependent. Thus, the AMPA receptor should probably not be thought of like the NMDA receptor—the NMDA receptor likely serves a frankly structural role in addition to its function as a ligand-gated ion channel while the AMPA receptor is more peripherally associated with the PSD (see reference 51).

The AMPA receptor binds at least two "structural" proteins—Protein Interacting with C Kinase-1 (PICK-1), which binds PKC, and Glutamate Receptor Interacting Protein (GRIP) (see reference 47). GRIP is a multidomain scaffolding protein that likely functions in AMPA receptor trafficking. GRIP also binds to GRIP Associated Protein-1 (GRASP1), a Guanine nucleotide Exchange Factor (GEF) for ras (see references 52 and 53)—the functional role of GRASP1 at the synapse is unclear at present. AMPA receptors can also bind N-Ethyl maleimide Sensitive Factor (NSF), a vesicle-associated protein that may also be involved in receptor membrane insertion is in a fashion reminiscent of its role presynap-tically in neurotransmitter vesicle fusion.

AMPA receptors also bind the A kinase anchoring protein AKAP79, an interaction that appears to be mediated by the PSD-95 homologue SAP-97 (54-56). As the name implies, AKAPs bind and localize PKA by interacting with the regulatory subunits of the kinase. The general role of AKAPs is to help localize PKA near relevant targets such as the AMPA receptor postsynapti-cally. The story is actually more complicated than that, because AKAP79 in the hippocampus also binds and localizes a protein phosphatase, PP2B (aka Calcineurin). As a first approximation, it is useful to think of proteins such as AKAPs as serving a role to increase the signal-to-noise ratio for signal transduction—localizing kinases close to their substrates to increase the efficacy of phosphorylation, but also localizing phosphatases to those same substrates in order to keep their basal phosphory-lation low and to allow for rapid reversal of phosphorylation events once the kinase activation is over (56). AKAP79 may also serve specifically to localize the calcium-sensitive phosphatase PP2B to the AMPA receptor in order to facilitate calcium-dependent AMPA receptor dephosphorylation and down-regulation in LTD (57).

AKAP79 also serves an additional scaffolding protein function. It binds to cyclase-coupled receptors such as the beta-adrenergic receptor, localizing recep tor, effector, kinase, substrate, and phos-phatase all together in a supramolecular complex. In the context of the hippocampal pyramidal neuron synapse, this might allow for enhanced beta adrenergic receptor modulation of AMPA receptor function, enhancing AMPA receptor function via PKA-dependent phosphory-lation. This might serve in the induction of LTP as a mechanism whereby cyclase-coupled receptors can augment AMPA receptor-mediated membrane depolarization and indirectly augment NMDA receptor activation.

AKAP79 also binds to PKC, again localizing this kinase near its substrate, the AMPA receptor. By analogy to the scenario outlined earlier for AKAP/PKA, this scaffolding activity might facilitate PKC-coupled receptor augmentation of AMPA receptor function during the induction of LTP as well. As we will discuss in the next chapter, PKC phosphorylation of AMPA receptors also contributes to E-LTP expression, and of course AKAP79 localization of PKC near AMPA receptors would help facilitate this mechanism as well.

CaMKII

CaMKII is highly enriched at the post-synaptic density complex. This enrichment in part occurs through CaMKII binding to the actin cytoskeleton, and the anchor for the cytoskeleton is the NMDA receptor as we have discussed extensively. Thus, one purpose of the NMDA receptor/PSD-95/ cytoskeleton complex is to help localize CaMKII to the PSD domain. This keeps a critical effector of the NMDA receptor, CaMKII, tightly bound and localized for effective responsiveness to NMDA receptor activation. Interaction of CaMKII with the PSD also can be regulated by CaMKII autophosphorylation—John Lisman has proposed this as a mechanism contributing to the maintenance of E-LTP, a model that we will return to in the next chapter.

While the various scaffolding proteins— PSD-95 and the like—that we discussed ealier are involved upstream of the NMDA receptor, regulating its function, CaMKII is downstream of the NMDA receptor. However, I list it as a component of the synaptic infrastructure necessary for proper NMDA receptor function because it is such an important and direct target of the NMDA receptor—in essence loss of CaMKII function may functionally translate as equivalent to loss of NMDA receptor function. In addition, CaMKII binding to the PSD complex may play a structural role in concert with the actin cytoskeleton to serve as part of the infrastructure necessary for the NMDA receptor to function appropriately.

V. LTP INDUCTION COMPONENT 4—FEED-FORWARD AND FEEDBACK MECHANISMS THAT REGULATE THE LEVEL OF CALCIUM ATTAINED

There is a clear consensus that elevation of postsynaptic calcium is necessary for LTP induction, so clearly any process that modulates the postsynaptic calcium level has the capacity to affect LTP induction. Unfortunately that's about where the clarity ends. In this section, we will deal with some of the known processes whereby calcium levels in the postsynaptic spine are regulated. (See table for summary.)

In one sense, dendritic spines are specialized calcium-handling compartments (see Sabatini, Oertner, and Svoboda

(58) for a very nice treatment of this idea). They contain many molecules dedicated to calcium handling that affect the kinetics of calcium elevation, kinetics which are a critical determinant for (a) whether synaptic strength is changed and (b) whether LTP or LTD is induced. A few generalizations can be made in this context based on published work in this area. One, calcium elevation in a spine is largely compartmentalized to that spine—the narrow spine neck greatly limits calcium diffusion out of the spine in response to NMDA receptor activation, for example. Two, the level of calcium attained determines whether synaptic strength goes up or down—modest levels yield synaptic depression (LTD or depotentiation) and higher levels yield LTP. Three, the kinetics of calcium entry secondary to NMDA receptor activation versus back-propagating action potential/VDCC-dependent calcium influx are different—NMDA receptor activation gives a much longer-lasting elevation of spine calcium than does activation of VDCCs. Four, a seminal finding from Rob Malenka's lab made clear that a relatively prolonged (> 2 seconds) elevation of postsynaptic calcium is necessary for LTP induction in CA1 pyramidal neurons. The basic idea is that this prolonged calcium elevation is necessary to trigger the biochemical processes subserving LTP maintenance (which we will discuss in the next chapter). With these four principles in mind it becomes quite clear that regulating the kinetics of calcium handling and the steady-state level of

Table 4 Calcium Feedback and Feed-Forward Mechanisms

Molecule/Organelle Role Modulator/Regulator

VDCCs Augment NMDA receptor-dependent PKA

Ca influx Ca influx due to bpAPs Regulate ERK activation

Endoplasmic reticulum Ca efflux from ER, limit LTP? PLC-coupled receptors

(Ca-ATPase/IP3R/RyR)

Presynaptic mitochondria Regulate presynaptic Ca levels Unknown calcium achieved locally in the dendritic spine is going to be a critical component of LTP induction. What is not clear are the exact molecular processes that impinge upon these variables.

In this section, I will briefly overview two postsynaptic processes and one presy-naptic process that are involved in synaptic calcium handling and describe some of the available literature investigating the role of these processes in LTP induction. The specific systems I will describe are postsy-naptic Voltage-dependent Calcium Channels (VDCCs), the postsynaptic endoplasmic reticulum (ER), and presynaptic mitochondria. The precise roles of these three systems/processes in LTP induction are quite murky at present, and many more years of work are likely to be necessary before a clear picture emerges concerning exactly what is happening with these molecules and organelles during LTP induction. However, a number of studies using inhibitors and knockouts of various components of these systems have been published, and a brief review of these studies is appropriate in order to set the stage for thinking about this category of molecular mechanisms.

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