Thomas J. Nelson

Oxidation of Cholesterol by Amyloid Precursor Protein and β-Amyloid Peptide

Alzheimers disease (AD) is characterized by accumulation of the neurotoxic peptide β-amyloid, which is produced by proteolysis of amyloid precursor protein (APP). APP is a large membrane-bound copper-binding protein that is essential in maintaining synaptic function and may play a role in synaptogenesis. β-Amyloid has been shown to contribute to the oxidative stress that accompanies AD. Later stages of AD are characterized by neuronal apoptosis. However, the biochemical function of APP and the mechanism of the toxicity of β-amyloid are still unclear. In this study, we show that both β-amyloid and APP can oxidize cholesterol to form 7β-hydroxycholesterol, a proapoptotic oxysterol that was neurotoxic at nanomolar concentrations. 7β-Hydroxycholesterol inhibited secretion of soluble APP from cultured rat hippocampal H19–7/IGF-IR neuronal cells and inhibited tumor necrosis factor-α-converting enzyme α-secretase activity but had no effect on β-site APP-cleaving enzyme 1 activity. 7β-Hydroxycholesterol was also a potent inhibitor of α-protein kinase C, with a Ki of ∼0.2 nm. The rate of reaction between cholesterol and β-amyloid was comparable to the rates of cholesterol-metabolizing enzymes (kcat = 0.211 min-1). The rate of production of 7β-hydroxycholesterol by APP was ∼200 times lower than by β-amyloid. Oxidation of cholesterol was accompanied by stoichiometric production of hydrogen peroxide and required divalent copper. The results suggest that a function of APP may be to produce low levels of 7-hydroxycholesterol. Higher levels produced by β-amyloid could contribute to the oxidative stress and cell loss observed in Alzheimers disease.

Time domains of neuronal Ca2+ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K+ channel cascade

Synaptic changes that underlie associative learning and memory begin with temporally related activity of two or more independent synaptic inputs to common postsynaptic targets. In turn, temporally related molecular events regulate cytosolic Ca2+ during progressively longer-lasting time domains. Associative learning behaviors of living animals have been correlated with changes of neuronal voltage-dependent K+ currents, protein kinase C-mediated phosphorylation and synthesis of the Ca2+ and GTP-binding protein, calexcitin (CE),and increased expression of the Ca2+-releasing ryanodine receptor (type II). These molecular events, some of which have been found to be dysfunctional in Alzheimers disease, provide means of altering dendritic excitability and thus synaptic efficacy during induction, consolidation and storage of associative memory. Apparently, such stages of behavioral learning correspond to sequential differences of Ca2+ signaling that could occur in spatially segregated dendritic compartments distributed across brain structures, such as the hippocampus.

Insulin, PKC signaling pathways and synaptic remodeling during memory storage and neuronal repair

Protein kinase C (PKC) is involved in synaptic remodeling, induction of protein synthesis, and many other processes important in learning and memory. Activation of neuronal protein kinase C correlates with, and may be essential for, all phases of learning, including acquisition, consolidation, and reconsolidation. Protein kinase C activation is closely tied to hydrolysis of membrane lipids. Phospholipases C and A2 produce 1,2-diacylglycerol and arachidonic acid, which are direct activators of protein kinase C. Phospholipase C also produces inositol triphosphate, which releases calcium from internal stores. Protein kinase C interacts with many of the same pathways as insulin; therefore, it should not be surprising that insulin signaling and protein kinase C activation can both have powerful effects on memory storage and synaptic remodeling. However, investigating the possible roles of insulin in memory storage can be challenging, due to the powerful peripheral effects of insulin on glucose and the low concentration of insulin in the brain. Although peripheral for insulin, synthesized in the beta-cells of the pancreas, is primarily involved in regulating glucose, small amounts of insulin are also present in the brain. The functions of this brain insulin are inadequately understood. Protein kinase C may also contribute to insulin resistance by phosphorylating the insulin receptor substrates required for insulin signaling. Insulin is also responsible insulin-long term depression, a type of synaptic plasticity that is also dependent on protein kinase C. However, insulin can also activate PKC signaling pathways via PLC gamma, Erk 1/2 MAP kinase, and src stimulation. Taken together, the available evidence suggests that the major impact of protein kinase C and its interaction with insulin in the mature, fully differentiated nervous system appears to be to induce synaptogenesis, enhance memory, reduce Alzheimers pathophysiology, and stimulate neurorepair.

Poststroke neuronal rescue and synaptogenesis mediated in vivo by protein kinase C in adult brains

Global cerebral ischemia/hypoxia, as can occur during human stroke, damages brain neural networks and synaptic functions. The recently demonstrated protein kinase C (PKC) activation–induced synaptogenesis in rat hippocampus suggested the potential of PKC-mediated antiapoptosis and synaptogenesis during conditions of neurodegeneration. Consequently, we examined the effects of chronic bryostatin-1, a PKC activator, on the cerebral ischemia/hypoxia–induced impairment of synapses and neurotrophic activity in the hippocampal CA1 area and on hippocampus-dependent spatial learning and memory. Postischemic/hypoxic bryostatin-1 treatment effectively rescued ischemia-induced deficits in synaptogenesis, neurotrophic activity, and spatial learning and memory. These results highlight a neuroprotective signaling pathway, as well as a therapeutic strategy with an extended time window for reducing brain damage due to stroke by activating particular PKC isozymes.

Bryostatin Enhancement of Memory in Hermissenda

Bryostatin, a potent agonist of protein kinase C (PKC), when administered to Hermissenda was found to affect acquisition of an associative learning paradigm. Low bryostatin concentrations (0.1 to 0.5 ng/ml) enhanced memory acquisition, while concentrations higher than 1.0 ng/ml down-regulated the pathway and no recall of the associative training was exhibited. The extent of enhancement depended upon the conditioning regime used and the memory stage normally fostered by that regime. The effects of two training events (TEs) with paired conditioned and unconditioned stimuli, which standardly evoked only short-term memory (STM) lasting 7 min, were—when bryostatin was added concurrently—enhanced to a long-term memory (LTM) that lasted about 20 h. The effects of both 4- and 6-paired TEs (which by themselves did not generate LTM), were also enhanced by bryostatin to induce a consolidated memory (CM) that lasted at least 5 days. The standard positive 9-TE regime typically produced a CM lasting at least 6 days. Low concentrations of bryostatin (<0.5 ng/ml) elicited no demonstrable enhancement of CM from 9-TEs. However, animals exposed to bryostatin concentrations higher than 1.0 ng/ml exhibited no behavioral learning. Sharp-electrode intracellular recordings of type-B photoreceptors in the eyes from animals conditioned in vivo with bryostatin revealed changes in input resistance and an enhanced long-lasting depolarization (LLD) in response to light. Likewise, quantitative immunocytochemical measurements using an antibody specific for the PKC-activated Ca2+/GTP-binding protein calexcitin showed enhanced antibody labeling with bryostatin. Animals exposed to the PKC inhibitor bisindolylmaleimide-XI (Ro-32-0432) administered by immersion prior to 9-TE conditioning showed no training-induced changes with or without bryostatin exposure. However, if animals received bryostatin before Ro-32, the enhanced acquisition and demonstrated recall still occurred. Therefore, pathways responsible for the enhancement effects induced by bryostatin were putatively mediated by PKC. Overall, the data indicated that PKC activation occurred and calexcitin levels were raised during the acquisition phases of associative conditioning and memory initiation, and subsequently returned to baseline levels within 24 and 48 h, respectively. Therefore, the protracted recall measured by the testing regime used was probably due to bryostatin-induced changes during the acquisition and facilitated storage of memory, and not necessarily to enhanced recall of the stored memory when tested many days after training.

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

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new ϵ-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKCϵ 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/PS1δ, which produce large quantities of β-amyloid peptide (Aβ), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of Aβ by 60–70%. In contrast to the marked activation of α-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of α-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the Aβ-lowering ability of these PKCϵ activators is caused by increasing the rate of Aβ degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.

Reduction of beta-amyloid levels by novel PKCepsilon activators

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new ϵ-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKCϵ 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/PS1δ, which produce large quantities of β-amyloid peptide (Aβ), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of Aβ by 60–70%. In contrast to the marked activation of α-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of α-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the Aβ-lowering ability of these PKCϵ activators is caused by increasing the rate of Aβ degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.

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

Background: ApoE4 is a genetic risk factor for sporadic AD. PKC is involved in synaptogenesis and shows abnormalities in aging and AD. Results: ApoE3 (not apoE4), acting through LRP1, protects synapses against ASPD by inducing PKCϵ. Conclusion: ApoE3 stimulates synaptogenesis and protection against ASPD by increasing PKCϵ synthesis. Significance: ApoE3 may reduce the risk for AD by stimulating PKCϵ synthesis. Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCϵ, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCϵ inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCϵ mRNA and expression of the PKCϵ protein. Amyloid β specifically blocked the expression of PKCϵ but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCϵ pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.

GABA-mediated synaptic interaction between the visual and vestibular pathways of Hermissenda.

Abstract: The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual‐vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CMS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 μM), the cholinergic antagonist atropine (250 μM), and the serotonergic antagonist imipramine (50 μM). In contrast, the GABAA antagonist bicuculline (250 μM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 μM)M) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 μM) onto the terminal branches of the B cell resulted in a concentration‐dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABAB agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from −70 to −80 mV and were consistent with mixed Cl‐ and K+ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual‐vestibular interactions in this animal, and suggest a possible role of GABA in visual‐vestibular associative learning.

Protection against β-Amyloid-induced Apoptosis by Peptides Interacting with β-Amyloid

β-Amyloid peptide produces apoptosis in neurons at micromolar concentrations, but the mechanism by which β-amyloid exerts its toxic effect is unknown. The normal biological function of β-amyloid is also unknown. We used phage display, co-precipitation, and mass spectrometry to examine the protein-protein interactions of β-amyloid in normal rabbit brain in order to identify the biochemical receptors for β-amyloid. β-Amyloid was found to bind primarily to proteins involved in low density lipoprotein and cholesterol transport and metabolism, including sortilin, endoplasmic reticulum-Golgi intermediate compartment 2 (ERGIC2), ERGIC-53, steroid 5α-reductase, and apolipoprotein B. β-Amyloid also bound to the C-reactive protein precursor, a protein involved in inflammation, and to 14-3-3, a protein that regulates glycogen synthase kinase-3β, the kinase involved in tau phosphorylation. Of eight synthetic peptides identified as targets of β-amyloid, three were found to be effective blockers of the toxic effect of β-amyloid on cultured neuronal cells. These peptides bound to the hydrophobic region (residues 17–21) or to the nearby protein kinase C pseudo-phosphorylation site (residues 26–30) of β-amyloid, suggesting that these may be the most critical regions for β-amyloid effector action and for aggregation. Peptides or other small molecules that bind to this region may protect against β-amyloid toxic effect by competitively blocking its ability to bind β-amyloid effector proteins such as sortilin and 14-3-3.