Info

Maint/Expr

No (119, 121)

?

No (119)

in synaptic plasticity. However, those PKC inhibitors reported to be isoform-selective do not generally differ greatly in their potency for inhibiting the various isoforms of PKC, limiting their effectiveness for hippocampal slice physiology studies. Obviously, transgenic mouse knockout technologies have been used to good effect to selectively eliminate specific protein kinase isoforms. Thus, at the present time, utilization of knockout technology to evaluate the roles of PKC isoforms in LTP and learning is a very appealing prospect.

The first isoform-specific investigation of a role for PKC in LTP involved characterization of a knockout of the brain-specific gamma isoform of PKC (41). Indeed, this was one of the very first knockout animal studies ever published. In these studies, very modest effects on hippocampus-dependent memory were observed with the loss of PKCy. However, the PKCy knockout animal has a very pronounced but idiosyncratic LTP deficit. Tetanus-induced LTP in area CA1 is completely lost in gamma knockout animals (see Figure 8). However, LTP can be recovered in PKCy-deficient mice by delivery of an LTD-inducing stimulus prior to LTP-inducing tetanic stimulation (41).

FIGURE 7 Domain structures of isoforms of PKC. The regulatory and catalytic domains of various PKC isoforms illustrated regions of structural conservation. Autophosphorylation sites in the conventional isoforms are indicated by red dots. Figure courtesy of Coleen Atkins.

These data suggest that the role of PKCy is limited to the induction of LTP and that the gamma isoform of PKC is not necessary for LTP maintenance. (One alternative possibility is that LTD-inducing stimulation recruits the capacity of another PKC isoform to compensate for the lack of PKCy.) The intriguing model has been proposed that a loss of phosphorylation of the postsynaptic PKC substrate neurogranin contributes to this phenotype, via the neuro-granin gate mechanism that we discussed in the last chapter. Overall, these studies suggest the hypothesis that while PKCy is involved in regulating LTP induction, other isoforms of PKC are involved in LTP maintenance.

Along these lines, Ed Weeber in my lab, in collaboration with Michael Leitges and others, evaluated a PKCP knockout mouse model (42). The PKCP knockout phenotype was in some ways the mirror image of the PKCy knockout. Deletion of the PKCP gene resulted in pronounced memory defects: strong attenuation of cued and contextual fear conditioning. However, the beta knockout animal had no discernable LTP phenotype in area CA1 of hippocampus3 (see Figure 8). Based on these studies it seems clear that the beta isoforms of PKC are not necessary for tetanus-induced, NMDA receptor-dependent LTP induction or early maintenance in area CA1 of hippocampus.

These observations do not, of course, preclude the involvement of PKCP in other forms of LTP in area CA1 and in synaptic plasticity in other brain regions. The data specifically suggest an important role for the beta isoforms of PKC in the synaptic plasticity underlying amygdala-dependent associative learning because of the loss of amygdala-dependent fear conditioning in these animals. There is, moreover, a potential role for PKCP in synaptic plasticity in area CA1, as we observed an attenuation of phorbol ester-induced potentiation of synaptic transmission in area CA1. Interestingly, this finding contrasts with mice deficient in the gamma isoform of PKC, which have no loss of phorbol ester-induced synaptic facilitation (43). These several observations, when taken

3 This also is an example of a result that dissociates hippocampal LTP from learning behavior. We will return to this issue in detail in Chapter 9.

FIGURE 8 Hippocampal LTP in PKC isoform-specific knockout mice. Hippocampal slices obtained from PKC beta (upper)-, and gamma (middle)-, (lower)-deficient mice or wild-type mice (+/+ in upper panel) were given an LTP-inducing stimulus (arrows) delivered after stable baseline responses were recorded for 20 minutes. Each set of tetani consisted of a single train of 100-Hz stimulation for 1 second, while maintaining slices at 25°C. Reproduced with permission from Weeber et al. (42) and Abeliovich, et al. (41).

FIGURE 8 Hippocampal LTP in PKC isoform-specific knockout mice. Hippocampal slices obtained from PKC beta (upper)-, and gamma (middle)-, (lower)-deficient mice or wild-type mice (+/+ in upper panel) were given an LTP-inducing stimulus (arrows) delivered after stable baseline responses were recorded for 20 minutes. Each set of tetani consisted of a single train of 100-Hz stimulation for 1 second, while maintaining slices at 25°C. Reproduced with permission from Weeber et al. (42) and Abeliovich, et al. (41).

altogether, suggest the possible specific involvement of the beta isoforms of PKC in neuromodulation in area CA1.

The PKC beta knockout studies indicate that, in and of itself, loss of the function of the PKCP isoforms does not lead to a loss of E-LTP maintenance. Similarly, loss of PKCy also does not lead to an inability to maintain E-LTP. That leaves us with the final calcium-sensitive isoform of PKC,

PKCa. Recent studies from my lab, again by Ed Weeber in collaboration with Michael Leitges and others, support the hypothesis that the alpha isoform of PKC is involved in E-LTP maintenance. Knockout of PKCa leads to a loss of tetanus-induced LTP in area CA1 in the absence of effects on baseline synaptic transmission or hippocampal morphology. While these studies are at a very early stage, hopefully future work will clarify the mechanisms involved in the regulation of PKCa activity in E-LTP, and the role of this PKC isoform in E-LTP maintenance.

Persistent Activation of PKC in E-LTP

How is it that PKC is utilized in generating a persisting signal in E-LTP? In 1991 Eric Klann and his colleagues published the first direct demonstration of persistent protein kinase activation in LTP, and they specifically identified PKC as one of the kinases involved in this process (29). Since that time Eric, and independently Todd Sacktor, have done an extensive series of studies to define the mechanisms for persistent activation of PKC in E-LTP. No aspect of this work has been straightforward. It is safe to say that none of the mechanisms that we all initially thought were the most likely to be involved in persistent PKC activation in LTP have subsequently turned out to be involved. Specifically, the two main hypotheses in the early days were membrane insertion and calpain-mediated proteolysis. Neither of these turned out to be involved in maintaining NMDA receptor-dependent LTP in area CA1, although they likely are involved in other forms of LTP (36).

In the next sections, I will describe some of the mechanisms that have turned out to be involved in persistent PKC activation in E-LTP in area CA1: oxidation, autophospho-rylation, and increased synthesis. These all serve as unique and interesting examples of now neurons can solve the biochemical problem of generating a lasting signal capable of affecting synaptic function.

Oxidation of PKC

Eric Klann and his colleagues have discovered a quite novel route for persistent PKC activation in LTP, and indeed a novel signal transduction mechanism in its own right. In oxidative activation of PKC (and other proteins regulated by oxidation), a reactive oxygen species directly reacts chemically with its target. Thus, instead of binding reversibly to an allosteric site, the second messenger, in this case, causes a direct and persistent modification of an amino acid side chain of its effector enzyme. This reaction is probably not readily reversible—thus, the modification has a built-in persistence, lasting until the PKC molecule is broken down completely.

In the case of oxidative PKC activation in LTP, the reactive species is superoxide or a superoxide-derived reactive oxygen species such as peroxynitrite or hydrogen peroxide (see Figure 9 and and 44). PKC is activated by reactive oxygen species in a complex manner. Hydrogen peroxide and superoxide increase both autonomous (i.e., calcium-independent) and calcium/phospholipid-dependent PKC activity. The a, piI, e, and Z isoforms of PKC are autonomously activated by reactive oxygen species through thiol side-chain oxidation and release of zinc from the cysteine-rich "zinc finger" regions of PKC (see Figure 9). In a biochemical tour de force, Lauren Knapp and Eric Klann showed that the generation of this persistently activated form of PKC occurs concomitantly with induction of NMDA receptor-dependent E-LTP (45).

Several lines of evidence show that the source of the reactive oxygen species in LTP is secondary to NMDA receptor activation (44). For example, NMDA receptor activation in area CA1 of hippocampal slices results in superoxide free-radical production, providing a source of reactive oxygen species. This finding is nicely complemented by the observation that superoxide

FIGURE 9 A model for oxidative activation of PKC in LTP. Calcium influx through the NMDA receptor triggers production of reactive oxygen species (NO, superoxide, and peroxynitrite), which directly act on cysteine side-chains in PKC. This oxidation results in Zn release and autonomously active PKC. Reactive oxygen species can also cross the synapse as retrograde messengers to activate PKC presynaptically.

FIGURE 9 A model for oxidative activation of PKC in LTP. Calcium influx through the NMDA receptor triggers production of reactive oxygen species (NO, superoxide, and peroxynitrite), which directly act on cysteine side-chains in PKC. This oxidation results in Zn release and autonomously active PKC. Reactive oxygen species can also cross the synapse as retrograde messengers to activate PKC presynaptically.

scavengers inhibit E-LTP induction in area CA1. Finally, NMDA receptor blockade blocks the generation of the persistently activated oxidized form of PKC in E-LTP. However, the precise source of the superoxide-derived messenger is not known at this time, and this is an area of active investigation. Possibilities include NOS (which can produce superoxide as well as nitric oxide), NADPH oxidases, mitochondrial electron transport, and lipid peroxidases.

One additional interesting aspect of this model is that superoxide and other reactive oxygen species, like NO, can cross cell membranes by mechanisms that are still under investigation. Thus, oxidative activation of PKC may not be limited to the postsynaptic compartment (see Figure 9). This mechanism presents an intriguing possibility for a retrograde signaling mechanism in E-LTP, especially given that presynaptic PKC activation apparently is sufficient to give increased neuro-transmitter release.

Finally, I should note that not just PKC but several protein kinases and phos-phatases are regulated by reactive oxygen species, as are a variety of transcription factors. Typically, protein kinases are activated by reactive oxygen species, whereas protein phosphatases are inhibited, potentially enabling a concerted modulation of protein phosphorylation levels within the cell similar to what we have already talked about with the cAMP gate. Persistent phosphatase inhibition in E-LTP is also a potential mechanism contributing to E-LTP maintenance (see reference 6; reviewed in reference 44).

PKC Autophosphorylation in LTP

PKC autophosphorylation is also a mechanism for generating a persisting signal in E-LTP. As part of his early studies into the mechanisms of persistent PKC activation in LTP, Eric Klann also observed that E-LTP is associated with a phosphatase-reversible alteration in PKC

immunoreactivity (30). This finding suggested that increased phosphorylation of PKC might contribute to its autonomous activation in LTP. In a follow-up series of studies, we tested the hypothesis that PKC phosphorylation is increased during E-LTP expression, utilizing an antibody we generated that is selective for autophospho-rylated PKC (46).

PKC is known to autophosphorylate at sites in three domains in vitro: an amino-terminal pair of sites near the autoinhibitory domain, a pair of sites in the hinge region, and a carboxy-terminal pair of sites (47; see Figure 7). Additional studies have revealed that the enzyme is phospho-rylated at two other sites; a transphosphory-lation on the activation loop (T500 in PKC |II) and an autophosphorylation at an additional C-terminal site (S660 in PKC |3II) (48). Three of the known phosphorylation sites (T500, T641, and S660) are likely to be phosphorylations occurring concomitant with maturation of the kinase (49, 50).

We studied autophosphorylation at the carboxy-terminal pair of autophospho-rylation sites (S634/T641) for several reasons. First, there is a high degree of sequence conservation among the classical PKC isoforms in this domain (see Figure 7). Second, site-directed mutagenesis studies demonstrated that autophosphorylation in this domain has important functional consequences (51, 52), including protection of the enzyme from down-regulation and causing the enzyme to associate with the actin cytoskeleton, a potential localization mechanism.

In our studies of PKC autophospho-rylation in LTP, we found that PKC has its C-terminal autophosphorylation persistently increased in LTP (46; see Figure 10). Thus, one persisting signal in E-LTP is an elevated level of autophosphorylated PKC. While this may sound reminiscent of the preceeding story with CaMKII autophos-phorylation, it is known that PKC autophosphorylation occurs by an intramolecular reaction (49, 53). This means that,

FIGURE 10 PKC autophosphorylation in LTP. (A) The three pairs of PKC autophosphorylation sites in the classical isoforms of PKC (red dots). Panels B through D show increased PKC phosphorylation in LTP. (B) Representative physiologic recording data for the three conditions used to investigate PKC phosphorylation in LTP. The Y-axis is the normalized initial slope of the field EPSP. Note that the high-frequency stimulation (HFS) and HFS + APV (50 pM D, L-APV) samples each received three pairs of tetanic stimulation trains (marked with arrows). All HFS and HFS + APV samples were taken 45 minutes to1 hour after delivery of the third period of tetanic stimulation. (C) Representative Western blots of individual area CA1 subregions for each of the 3 conditions employed in these studies. Each is paired with an appropriate control slice (CTL) from the same hippocampus. In the upper panels, blotting is with antiserum 96160 (phospho-PKC). In the lower panels, anti-p44 MAP kinase blot is used to control for protein loading (Erk1-CT). (D) Mean normalized phospho-PKC immunoreactivity. Error bars are SEM: for LTP n = 9, for test stim (LFS) n = 3, for HFS + APV, n = 4. HFS is significantly different from control, LFS, and HFS + APV (p < .05, one-way ANOVA). Adapted from Sweatt et al. (46).

unlike CaMKII, which can self-perpetuate its autophosphorylation by transphos-phorylation of adjacent subunits, PKC autophosphorylation at these sites cannot be self-perpetuating by having one PKC molecule phosphorylate another.

This led us to wonder how the increased autophosphorylation of PKC was preserved in the presence of ongoing phosphatase activity in the neuron. To explain our observation, we developed the "protected site" model for PKC phosphorylation in LTP. In this model, PKC phosphorylation occurs at sites protected sterically by adjacent portions of the PKC molecule, limiting accessibility of phosphatase and preserving the enzyme in a phosphorylated state. Atomic resolution modeling and in vitro experiments supported key predictions of this model (see Box 2).

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