Protein Kinase C

Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C, or PKC) is a ubiquitous enzyme, highly enriched in brain, where it plays a major role in regulating both pre- and postsynaptic aspects of neurotransmission (Nishizuka 1992; Stabel and Parker 1991). PKC is one of the major intracellular mediators of signals generated upon external stimulation of cells via a variety of neurotransmitter receptors (including muscarinic [M1, M3, M5], noradrenergic [tti], and serotonergic [5-HT2] receptors) that induce the hydrolysis of various membrane phospholipids.

Activation of PKC by DAG appears to involve the binding of the lipid to a specific regulatory site on the enzyme, resulting in an increase in the Ca2+ affinity, and thus its stimulation at physiological ionic concentrations. Ca2+ is also believed to contribute to PKC activation by facilitating the interaction of the enzyme with the lipid bilayer and hence with acidic phospholipids and DAG. PKC is now known to exist as a family of closely related subspecies, has a heterogeneous distribution in brain (with particularly high levels in presynaptic nerve terminals), and, together with other kinases, appears to play a crucial role in the regulation of synaptic plasticity and various forms of learning and memory. The multiple closely related PKC isoforms are all activated by endoplasmic reticulum (which serves as a vast web and framework for Ca2+-binding proteins to capture and sequester Ca ). Ca buffering/triggering proteins are nonuniformly distributed, so there is considerable subcellular variation of Ca concentrations (e.g., near a Ca channel). The primary mechanism for Ca calcium exit phospholipids and DAG, albeit with slightly different kinetics. The isoforms can be subclassified according to Ca2+ dependence: the "conventional" PKCs {",

&II, if) are dependent on Ca2+ for activity, whereas several others, termed "novel" ■?, 9) and "atypical" are calcium independent

(Nishizuka 1992). The conventional, novel, and atypical isozymes all share activation by phospholipids or DAG and an autoinhibitory pseudosubstrate region, which maintains the enzyme in an inactive state until activated. However, the subgroups are activated by different activators. The conventional PKCs require calcium, acidic phospholipids, and DAG for activation; the novel PKCs do not require calcium, and the atypical PKCs do not require calcium or DAG. PKC isozymes that do not share the pseudosubstrate region (i'/PKD and «■) have been described, which suggests a possible different mode of action.

The differential tissue distribution of PKC isozymes, as well as the fact that several isoforms are expressed within a single cell type, suggests that each isozyme may exert distinct cellular functions. At present, it is unclear whether such putative functional specificity arises from differential in vivo activation, differential substrate specificity, or a combination thereof. PKC has many growth-regulating properties in immature cells and has additional cell-specific responses in individual mature cells (Kanashiro and Khalil 1998). One protein whose activity is modulated by PKC is myristoylated alanine-rich C kinase substrate (MARCKS). This protein functions as a regulated crossbridge between actin and the plasma membrane, contributing to the cytoskeleton of the cell and subsequently to neuronal plasticity (Aderem 1992) (see Figure 1-13). PKC is also an important activator of phospholipase A2, thus linking the phosphoinositide cycle with arachidonic acid pathways (see Figure 1-13). Arachidonic acid functions as an important mediator of second-messenger pathways within the brain and is regulated by chronic lithium (Axelrod et al. 1988; Rapoport 2001). The activation of phospholipase A2 by PKC (and other pathways) results in arachidonic acid release from membrane phospholipids (Axelrod 1995). This release of arachidonic acid from cellular membrane allows for the subsequent formation of a number of eicosanoid metabolites such as prostaglandins and thromboxanes. These metabolites mediate numerous subsequent intracellular responses and, because of their lipid permeable nature, transsynaptic responses.

PKC also has been demonstrated to be active in many other cellular processes, including stimulation of transmembrane glucose transport, secretion, exocytosis, smooth muscle contraction, gene expression, modulation of ion conductance, cell proliferation, and desensitization of extracellular receptors (Nishizuka 1992). One of the best-characterized effects of PKC activation in the CNS is the facilitation of neurotransmitter release. Studies have suggested that PKC activation may facilitate neurotransmitter release via a variety of mechanisms, including modulating several ionic conductances regulating Ca2+ influx, upstream steps regulating release of Ca2+ from intracellular stores, recruitment of vesicles to at least two distinct vesicle pools, and the Ca2+ sensitivity of the release process itself (discussed in Bown et al. 2002). Abundant data also suggest that the PKC signaling pathway may play an important role in the pathophysiology and treatment of bipolar disorder (Manji and Lenox 1999). Thus, elevations in PKC isozymes have been reported in postmortem brain and platelets in bipolar patients; more importantly, in animal and cell-based models, lithium and valproate exert strikingly similar effects on PKC isozymes and substrates in a time frame mimicking their therapeutic actions.

A recent whole-genome association study of bipolar disorder has further implicated this pathway. Of the risk genes identified, the one demonstrating by far the strongest association with bipolar disorder was diacylglycerol kinase, an immediate regulator of PKC (Baum et al. 2008). In animal models of mania, several studies have demonstrated that both acute and chronic amphetamine exposure produces an alteration in PKC activity and its relative cytosol-to-membrane distribution, as well as the phosphorylation of a major PKC substrate, GAP-43, which has been implicated in long-term alterations of neurotransmitter release. Increased hedonistic drive and increased tendency to abuse drugs are well-known facets of manic behavior; notably, PKC inhibitors attenuate these important aspects of the manic-like syndrome in rodents (Einat and Manji 2006; Einat et al. 2007). Recent preclinical studies have specifically investigated the antimanic effects of tamoxifen (since this is the only CNS-penetrant PKC inhibitor available for humans). These studies showed that tamoxifen significantly reduced amphetamine-induced hyperactivity and risk-taking behavior (Einat et al. 2007). Finally, with respect to cognitive dysfunction associated with mania, Birnbaum et al. (2004) demonstrated that excessive activation of PKC dramatically impaired the cognitive functions of the prefrontal cortex and that inhibition of PKC protected cognitive function. In summary, preclinical biochemical and behavioral data support the notion that PKC activation may result in manic-like behaviors, whereas PKC inhibition may be antimanic. These preclinical data, along with animal studies discussed above, have prompted clinical studies of PKC inhibitors and mood dysregulation. A number of small studies have found that tamoxifen, a nonsteroidal antiestrogen and a PKC inhibitor at high concentrations, possesses antimanic efficacy (Bebchuket al. 2000; Kulkarni et al. 2006). Most recently, a double-blind, placebo-controlled trial of tamoxifen in the treatment of acute mania was undertaken (Zarate et al. 2007). Subjects showed significant improvement in mania on tamoxifen compared with placebo as early as 5 days, and the effect size for the drug difference was very large after 3

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