Reduction of β-Amyloid Levels by Novel Protein Kinase Cϵ Activators

Abstract

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new ⑀-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKC⑀ in a dose-dependent manner. Unlike PKC activators that bind to the 1,2-diacylglycerol-binding site, such as bryostatin and phorbol esters, which produce prolonged down-regulation, the new activators produced sustained activation of PKC. When applied to cells expressing human APPSwe/PS1␦, which produce large quantities of ␤-amyloid peptide (A␤), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of A␤ by 60 -70%. In contrast to the marked activation of ␣-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of ␣-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the A␤-lowering ability of these PKC⑀ activators is caused by increasing the rate of A␤ degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.Alzheimer disease is characterized by the accumulation of aggregated ␤-amyloid (A␤), 2 which is a 4-kDa peptide produced by the proteolytic cleavage of amyloid precursor protein (APP) by ␤-and ␥-secretases. Oligomers of A␤ are the most toxic, whereas fibrillar A␤ is largely inert. Monomeric A␤ is found in normal patients and has an as-yet undetermined function. The earliest consistent cytopathological change in Alzheimer disease is loss of synapses (1, 2). Finding a way to protect against the loss of synapses is a major therapeutic goal. Protein kinase C (PKC) activators have demonstrated neuroprotective activity in animal models of Alzheimer disease (3), depression (4), and stroke (5). Bryostatin, a potent PKC activator, also increases the rate of learning in rodents, rabbits, and invertebrates (4, 6, 7). This effect is accompanied by increases in levels of synaptic proteins spinophilin and synaptophysin and structural changes in synaptic morphology (8). PKC activators also can reduce the levels of A␤ and prolong the survival of Alzheimer disease transgenic mice (3). Evidence suggests that PKC␣ and ⑀ are the most important PKC isoforms in eliciting these changes. Antisense inhibition of PKC␣ blocks secretion of sAPP␣, whereas indirect activators of PKC, such as carbachol, increase sAPP␣ secretion (9). Experiments with specific PKC isozyme inhibitors also point to PKC⑀ as the isozyme that most effectively suppresses A␤ production (10). Thus, isoform-specific PKC activators are highly desirable as potential anti-Alzheimer drugs. Specific activators are preferable to compounds such as bryostatin that show less specificity among conventional and novel forms of PKC because nonspecific activation of PKC␦ or ␤ could produce undesirable side effects.One compound known to activate PKC⑀ specifically is DCP-LA...

Bryostatin-1 Restores Hippocampal Synapses and Spatial Learning and Memory in Adult Fragile X Mice

“…The hyperabundance of immature-looking lengthened dendritic spines could be the results of a failed/delayed maturation (Cruz-Martin et al, 2010;Harlow et al, 2010) and activity-dependent synaptic elimination (Pfeiffer et al, 2010). In short, bryostatin-1-like agents (Sun and Alkon, 2005;Nelson et al, 2009;DeChristopher et al, 2012) …”

“…One problem with inhibition of the mGluR signaling, for instance, is that the inhibition exaggerates spine immaturity in the fragile X mice (Cruz-Martin et al, 2010), an effect opposite to the intended therapeutic outcomes. Here, we show that bryostatin-1 (Nelson et al, 2009;DeChristopher et al, 2012), a highly potent and relatively specific protein kinase C (PKC) « activator (also of PKCa), with pharmacologic profiles of rapid mGluR desensitization, synaptogenesis, and synaptic maturation/repairing (Hongpaisan and Alkon, 2007;Sun et al, 2008;Hongpaisan et al, 2011), rescues synaptic and memory functions and other phenotypic features in adult fragile X mice. Bryostatin-1 has a much lower ED 50 for inducing PKC« translocation than its ED 50 s for PKCa or PKCd (Szallasi et al, 1994).…”

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95

“…DCPLA-ME was synthesized in our laboratory following the method described earlier (34,70) and shown to be specific for PKC⑀. Primary antibodies (rabbit polyclonal anti-PKC⑀ (sc-214), rabbit polyclonal anti-PKC␣ (sc-208), rabbit polyclonal anti-PKC␦ (sc-213), mouse monoclonal anti-synaptophysin (sc-17750), and mouse monoclonal anti-␤-actin (sc-47778)) were obtained from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA).…”

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

“…Bryostatin 1 was purchased from Biomol International. DCPLA and DCPLA-ME were synthesized in our laboratory following the method described earlier (28). Primary antibodies (PKC-⑀, ␤-actin, RACK1, synaptophysin, MAP-2, and PSD-95) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).…”

“…The PKC activator DCPLA methyl ester (DCPLA-ME) (Fig. 1E) is a derivative of DCPLA, which associates with the PKC phosphatidylserine-binding site and specifically activates PKC⑀ (27,28). Unlike diacylglycerol-binding PKC activators, phosphatidylserine-binding activators produce little or no down-regulation of PKC (29).…”