Kentaro Murakami

Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2

Unsaturated fatty acids (oleic acid and arachidonic acid) activate purified protein kinase C independently of phospholipid and Ca2+. Oleic acid activation of protein kinase C is as effective as phosphatidylserine and Ca2+. K a, values for oleic acid and arachidonic acid are 50 and 53 μM, respectively. In contrast to the cis fatty acids, a trans form (elaidic acid) or a saturated fatty acid (stearic acid) has little or no effect on protein kinase C activation. If cis fatty acid liberation is physiologically important, this suggests that another mechanism may exist for protein kinase C activation, in addition to phospholipase C/phosphatidylinositol turnover signaling, possibly via the liberation of cis fatty acids by the Ca2+‐dependent phospholipase A2 system.

Protein kinase C inhibitors eliminate hippocampal long-term potentiation

Recent findings suggest that protein kinase C (PKC) regulates the persistence of long-term potentiation (LTP). To test the hypothesis that PKC inhibition would decrease persistence of potentiation we applied PKC inhibitors (mellitin, polymyxin B, H-7) by micropressure ejection to the intact hippocampus either before or after LTP induction. When inhibitor was given 15 min before LTP, initial potentiation was unaffected, yet responses decayed to baseline levels by 60 min after the onset of potentiation. PKC inhibitor treatment 10 min after LTP onset induced decay of responses to pre-LTP baseline levels within 50 min of ejection. Inhibitor applied 60 min after LTP onset induced substantial decay but not to baseline levels. Potentiation was unaffected by inhibitor treatment 4 h after the induction of LTP. Measurement of PKC subcellular distribution revealed that inhibitor significantly reduced the proportion of PKC associated with the membrane. These findings represent the first demonstration that PKC inhibitors prevent persistence of potentiation. They also suggest that PKC regulates the persistence of synaptic enhancement beginning after its onset, and that PKCs role decreases with time after the induction of enhancement.

Phorbol ester promotes growth of synaptic plasticity

Iontophoretic application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to the intact rat hippocampus enhances potentiation produced by subsequent high frequency stimulation of the perforant path. The decay of the enhanced population spike amplitude recorded in the hilar dentate gyrus was prevented in animals receiving ejections of TPA, as compared to controls which decayed to baseline values within 2 h following high frequency stimulation. In fact, growth of the potentiated response was observed beginning at 45 min. Similar results were observed with the population excitatory postsynaptic potential slope, a measure of the synaptic response. Since tumour-promoting phorbol esters are known to translocate and activate protein kinase C (PKC) and PKC is translocated to the membrane following hippocampal potentiation, a role for membrane-associated PKC in the regulation of synaptic plasticity is suggested.

A newly discovered protein kinase C activator (oleic acid) enhances long-term potentiation in the intact hippocampus

Protein kinase C can be activated by oleate, an unsaturated fatty acid. Since protein kinase C is activated by long-term potentiation, we wished to determine whether iontophoretic ejection of oleate into the intact hippocampal dentate gyrus of urethane-anesthetized rats would cause an enhancement of the response potentiated by high frequency stimulation of the perforant path. Oleate ejection did significantly enhance the persistence of the potentiated response. Moreover, a growth of the response beyond the initial potentiation was seen. Arachidonate, which stimulates protein kinase C to a lesser degree, had a significant preservation effect, but no effect on growth of the response. After vehicle and elaidate (trans-stereoisomer of oleate) ejections, the potentiated response decayed to baseline values. In addition, the persistence of the potentiated response observed two hours after its induction was positively correlated with the ability of an unsaturated fatty acid to activate protein kinase C in vitro. The present results support the proposal that protein kinase C activation enhances synaptic strength. It is suggested that one mechanism for this activation may be PLA2-mediated release of oleate.

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1s possible role in age-related decline of hippocampal synaptic plasticity.

Dietary cis-fatty acids that increase protein F1 phosphorylation enhance spatial memory.

Activation of protein kinase C (PKC) facilitates long-term potentiation (LTP), a model of memory, and increases its substrate protein F1 (aka GAP43) phosphorylation in direct relation to synaptic enhancement. Unsaturated fatty acids (c-FAs) which activate purified PKC, when injected into hippocampus, enhance LTP. To determine if dietary c-FAs could alter memory itself as well as brain PKC substrate (F1) metabolism, rats were maintained for 10 weeks on fatty acid diets enriched in mono-unsaturated oleic acid (OA; 20% olive oil, w/w), or a mono- and di-unsaturated mixture of oleate/linoleate (O/L; 20% corn oil), or a saturated fatty acid diet of laurate/myristate (L/M; 20% hydrogenated coconut oil). The O/L diet group was superior to the OA and L/M groups in spatial memory performance after the first two weeks of acquisition and in later achievement of criterion performance. The O/L diet had a significantly higher hippocampal protein F1 in vitro phosphorylation than in both the OA and L/M in trained and non-trained animals. Significantly, animals that made fewer errors showed higher F1 phosphorylation (r = -0.70). Diet both increases brain PKC substrate phosphorylation and enhances maze learning, confirming the feasibility of enhancing learning and memory by dietary regimens derived from basic neurochemical studies of synaptic plasticity.