Soluble beta-amyloid induction of Alzheimer's phenotype for human fibroblast K+ channels.

Although beta-amyloid is the main constituent of neurite plaques and may play a role in the pathophysiology of Alzheimer's disease, mechanisms by which soluble beta-amyloid might produce early symptoms such as memory loss before diffuse plaque deposition have not been implicated. Treatment of fibroblasts with beta-amyloid (10 nM) induced the same potassium channel dysfunction previously shown to occur specifically in fibroblasts from patients with Alzheimer's disease--namely, the absence of a 113-picosiemen potassium channel. A tetraethylammonium-induced increase of intracellular concentrations of calcium, [Ca2+]i, a response that depends on functional 113-picosiemen potassium channels, was also eliminated or markedly reduced by 10 nM beta-amyloid. Increased [Ca2+]i induced by high concentrations of extracellular potassium and 166-picosiemen potassium channels were unaffected by 10 nM beta-amyloid. In Alzheimer's disease, then, beta-amyloid might alter potassium channels and thus impair neuronal function to produce symptoms such as memory loss by a means other than plaque formation.

Calcium responses in human fibroblasts: a diagnostic molecular profile for Alzheimer's disease.

We have previously identified alterations of K+ channel function, IP3-mediated calcium release, and Cp20 (a memory-associated GTP binding protein) in fibroblasts from AD patients vs. controls. In the present study we introduce a scoring system based on these response alterations that integrates two or more alterations (and their degree) in AD vs. control fibroblasts. This scoring system generates an index that distinguishes AD patients from controls with both high specificity and sensitivity. We also show that low doses of bradykinin elicit intracellular calcium release almost exclusively in AD cell lines in an all or none fashion that provide a clear measurement of enhanced IP3-mediated function in AD vs. controls.

Ionic and signal transduction alterations in Alzheimer's disease: relevance of studies on peripheral cells.

Several lines of evidence indicate that Alzheimer's disease (AD) has systemic expression. Systemic changes are manifested as alterations in a number of molecular and cellular processes. Although, these alterations appear to have little or no consequence in peripheral systems, their parallel expression in the central nervous system (CNS) could account for the principal clinical manifestations of the disease. Recent research seems to indicate that alterations in ion channels, calcium homeostasis, and protein kinase C (PKC) can be linked and thereby constitute a model of pathophysiological relevance. Considering the difficulties of studying dynamic pathophysiological processes in the disease-ridden postmortem AD brain, peripheral tissues such as fibroblasts provide a suitable model to study molecular and cellular aspects of the disease.

Enhanced BK-induced calcium responsiveness in PC12 cells expressing the C100 fragment of the amyloid precursor protein.

Several lines of evidence have implicated the amyloid precursor protein (APP) and its metabolic products as key players in Alzheimer's disease (AD) pathophysiology. The approximately 100 amino acid C-terminal fragment (C100) of APP has been shown to accumulate intracellularly in neurons expressing familial AD (FAD) mutants of APP and to cause neurodegeneration when expressed in transfected neuronal cells. Transgenic animals expressing this fragment in the brain also exhibit some neuropathological and behavioral AD-like deficits. Here, we present evidence that PC12 cells expressing the C100 fragment either via stable transfections or herpes simplex virus-mediated infections show alterations in calcium handling that are similar to those previously shown in fibroblasts from AD patients. This alteration in calcium homeostasis may contribute to the deleterious effects of C100 in PC12 cells. Our data also lend support for a pathophysiological role for C100 since it induces an alteration thought to play an important role in AD pathology.

Endoplasmic reticulum as a source of Ca2+ in neurotransmitter secretion.

Depolarization of the synaptosomal membrane by a rapid elevation of [K+]0 induces secretion of adenosine-5'-triphosphate (ATP) as well as the specific neurotransmitters. In addition to the classical [Ca2+]0-dependent mode, we have found that ATP secretion also occurred in the absence of extracellular calcium [( Ca2+]0 less than 1 microM). The extent of both modalities of secretion depended on membrane potential, and the [Ca2+]0-independent secretion proceeded at a rate that was substantially smaller than that of the [Ca2+]0-dependent mode at all membrane potentials examined. We propose that intracellular stores may provide the Ca2+ required for exocytosis in the [Ca2+]0-independent mode of ATP secretion. To test this hypothesis, we searched for the presence of Ca(2+)-release channels gated by intracellular messengers in our synaptosomal preparation. We fused membrane vesicles from lysed synaptosomes with acidic phospholipid bilayers formed at the tip of a patch pipette and found that these membranes contained a Ca(2+)-selective channel. The properties of this channel resemble those of the Ca(2+)-release channel reconstituted from sarcoplasmic reticulum membrane vesicles. These include size of the single open-channel conductance (75 pS Cs+ as the main current carrier), activation by adenine nucleotides (ATP), ryanodine and caffeine, and inhibition by ruthenium red.

Transforming growth factor beta induces a beta-responsive calcium fluxes in neurons.

The beta-amyloid (a beta) peptide is a neurotoxic peptide that accumulates in the brains of Alzheimer patients, but is also present in body fluids at subnanomolar levels. The potential effects of these low levels of a beta are unclear. We have recently shown that physiologic levels of a beta increase tyrosine phosphorylation and induce increases in cytosolic calcium. The basement membrane mixture, Matrigel, is required for observation of the a beta-induced calcium response. We now show that transforming growth factor beta (TGF beta) is the active component in Matrigel eliciting the a beta/calcium response. The response to the type of TGF beta varies depending on the cell type with TGF beta 1 eliciting a beta responsiveness in olfactory neuroblasts, and TGF beta 2 eliciting a beta responsiveness in PC12 cells.

Benzolactam (BL) enhances sAPP secretion in fibroblasts and in PC12 cells.

Activation of protein kinase C is known to favor the alpha-secretase processing of the Alzheimer's disease (AD) amyloid precursor protein (APP), resulting in the generation of non-amyloidogenic soluble APP (sAPP). Consequently, the relative secretion of amyloidogenic Abeta1-40 and Abeta1-42(3) is reduced. This is particularly relevant since fibroblasts and other cells expressing APP and presenilin AD mutations secrete increased amounts of total Abeta and/or increased ratios of Abeta1-42(3)/Abeta1-40. Interestingly, PKC defects have been found in AD brain alpha and beta isoforms) and in fibroblasts (alpha isoform) from AD patients. Here, we use a novel PKC activator (benzolactam, BL) with improved selectivity for the alpha, beta and gamma isoforms to enhance sAPP secretion in fibroblasts from AD patients and in PC12 cells. Incubation (2 h) of AD fibroblasts with BL (1 and 10 microM) resulted in significant increases of sAPP secretion over basal levels. sAPP secretion in BL-treated AD cells was also slightly higher compared to control BL-treated fibroblasts, which only showed significant increases of sAPP secretion after treatment with 10 microM BL. Staurosporine (a PKC inhibitor) eliminated the effects of BL in both control and AD fibroblasts. BL and a related compound (LQ12) also caused an approximately 3-fold sAPP secretion in PC12 cells. The use of a novel and possibly non-tumorigenic PKC activator may prove useful to favor non-amyloidogenic APP processing and is, therefore, of potential therapeutic value.

Outgrowths from Hermissenda photoreceptor somata are associated with activation of protein kinase C.

We have found changes in the morphology of photoreceptor somata from the mollusc Hermissenda that are produced by application of 12,13-phorbol dibutyrate (PDBU), an activator of PKC, in combination with elevated intracellular Ca2+ levels. The changes in morphology were expressed as rapid and repetitive outgrowths and additionally as more general changes in shape of the soma. Application of 4 alpha-PMA, a phorbol ester which does not activate PKC, did not produce these changes. The functional integrity of the photoreceptors in these dissociated eye preparations was maintained throughout the period of incubation with PDBU according to standard electrophysiological criteria. It has previously been shown that classical conditioning produced a reduction of dendritic volume in the type B photoreceptor of Hermissenda, a specific locus for associative memory storage. These changes in dendritic morphology were correlated with increased resistance across the cell membrane caused by learning-induced reductions of outward somatic K+ currents. Such conditioning-specific reductions of somatic K+ currents appear to depend on the phosphorylation of a 20-kDa G-protein (CP20) mediated by the Ca2+ and phospholipid-dependent kinase, protein kinase C (PKC). Thus PKC activity may be important in structural changes of the synaptic region of specific neurons involved in associative memory. The results of the present study suggest that the effects of PKC activation may also include structural changes in the soma of these same neurons.

Physiological levels of beta-amyloid increase tyrosine phosphorylation and cytosolic calcium.

The a beta peptide is a neurotoxic peptide that accumulates in the brains of Alzheimer patients, but is also present in body fluids at subnanomolar levels. The potential effects of these low levels of a beta are unclear. We now show that one such action is to increase tyrosine phosphorylation in PC12 cells and olfactory neuroblasts. Application of a beta 25-35 or a beta 1-40 induces a dose-dependent increase in the tyrosine phosphorylation in both whole cells and in vitro. The increase in tyrosine phosphorylation is both rapid and sensitive, being stimulated by picomolar doses of a beta and occurring within 1 min of application. Calcium imaging experiments provide further support for the role of tyrosine phosphorylation in the action of a beta. While a beta does not alter calcium metabolism under basal conditions, the addition of a beta induces a rapid increase in cytoplasmic calcium in olfactory neuroblasts that have been treated with the tyrosine phosphatase inhibitor, sodium orthovanadate or in PC12 cells treated with nerve growth factor. These responses could be blocked by the tyrosine kinase inhibitor, herbimycin. These calcium responses displayed an obligate requirement for the presence of matrix proteins. The identification of a rapid, sensitive assay for the action of a beta may facilitate investigations of its mechanism of action.

Soluble beta-amyloid induces Alzheimer's disease features in human fibroblasts and in neuronal tissues.

It has been shown that K+ channels, Cp20 (a 20kD GTP-binding protein), and intracellular calcium release, play a key role in associative memory storage. These same elements have been shown to be altered in fibroblasts from Alzheimer's Disease (AD) patients. In addition, it has been shown that PKC, also implicated in memory storage and closely related to the above mentioned components, is also altered in AD fibroblasts. Moreover, beta-amyloid was capable of inducing an AD-like phenotype for K+ channels and Cp20 in otherwise normal fibroblasts, providing additional evidence for the potential involvement of these components in AD and suggesting a possible pathological consequence of soluble beta-amyloid elevation in AD. Preliminary evidence shows that comparable changes in potassium channel function are also present in human olfactory neuroblasts from AD patients. These results indicate that the observed changes not only occur in peripheral tissues such as fibroblasts, but also in neural tissue, the primary site of AD pathology.

[Topography and reactivity of the visual evoked potential]. / Topografía y reactividad del potencial evocado visual.

Augmenting/reducing (A/R) of visual evoked potentials (VEP) has been repeatedly observed in central derivations, with some subjects increasing, and others decreasing, VEP amplitude with increasing intensity of stimulation. Central derivations also exhibit hemispheric lateralization regarding A/R. This paper explores central and occipital VEP in the same population of 16 healthy, right-handed male subjects, stimulated with binocular light flashes 10 usec in duration delivered by à Grass PS2 stimulator at a rate of 1/sec and at the intensities of 0.36, 0.72 and 1.44 joules at source. Amplitudes and latencies of components P1, N1 and P2 agreed with those reported in the literature. On the basis of amplitude/intensity slope functions of "peak to peak" amplitudes (P1N1 and NIP2) at Cz, 10 augmenters (slope greater than 0) and 6 reducers (slope smaller than 0) were found. Compared to central leads (C3 and C4) occipital ones (O1 and O2) did not exhibit significant interhemispheric differences. Vertex augmenters for N1P2 were occipital reducers and vice-versa. The different characteristics of A/R at occipital and central leads are interpreted in terms of stages of visual information processing in primary and association areas and functional significance of VEP components.

[Ionic channels and second messenger alterations in Alzheimer's disease. Relevance of studies in nonneuronal cells]. / Alteraciones de canales iónicos y segundos mensajeros en la enfermedad de Alzheimer. Relevancia de estudios en células extraneurales.

INTRODUCTION: Numerous observations indicate that, while the predominant clinical expression arises from brain pathology, Alzheimer s disease (AD) has systemic expression at the cellular and molecular levels. Although these alterations seem to be inconsequential outside the central nervous system, their parallel expression in the brain could be considered a plausible pathophysiological model and explain part of the clinical manifestations; in particular those related to memory loss. DEVELOPMENT: Recent research has provided experimental evidence of a direct or indirect linkage between alteration in ion channels, PKC, calcium homeostasis and amyloid processing in peripheral tissues. Some evidence also indicates similar phenomena in the brain, attesting to the relevance of the changes in non CNS cells. CONCLUSION: Considering the difficulties of using post mortem material to study dynamic and/or early event in mostly end stage, disease ridden tissues, peripheral cells such as fibroblasts offer a model to study cellular aspects of AD pathophysiology.

Calcium alterations in Alzheimer's disease: pathophysiology, models and therapeutic opportunities.

Calcium plays a pivotal role in mediating many important biological functions. The intracellular calcium concentration is tightly regulated by a variety of systems and mechanisms. Calcium is sequestered by various organelles such as mitochondria and/or endoplasmic reticulum and extruded across the plasma membrane by energy-dependent transport systems. Different Ca2+-binding proteins are also involved in these processes. Alterations in calcium homeostasis might be critically implicated in brain aging and in the neuropathology of Alzheimer's disease (AD). In fact, one of the postulated mechanisms of beta-amyloid toxicity seems to involve a Ca2+ dysregulation accompanied with enhanced vulnerability to excitotoxic stimuli. Although brain characteristic lesions-plaques and tangles-constitute the hallmarks of AD, accumulated evidence suggests the systemic feature of this disease. Therefore peripheral cell lines may represent a useful approach to explore the cellular pathophysiology of AD, including calcium alterations and associated phenomena.

Multiple potassium and chloride channels in the human colon carcinoma cell line SW1116.

SW1116 cells have a profound capacity for secreting mucin molecules bearing the Lewisa epitope. Mucin molecules with the same epitope have been found to be elevated in the serum of patients with cystic fibrosis, a disease with defective ion channels. We therefore decided to study ion channels in this cell line. In the present work, we report the presence of two K(+)-channels and two Cl(-)-channels in the apical membrane of SW1116 cells. One of the K(+)-channels has a large conductance (approximately 278 pS), anomalous rectifying properties, and is inactivated rapidly. The second type exhibited a linear I/V curve (19 pS), was voltage insensitive and inactivation was not observed. In cell-attached patches, spontaneous openings of chloride channels were seen with higher frequency than previously reported in other colon carcinoma cell lines or airway epithelial cells. Inside-out experiments allowed identification of two different Cl(-)-channels (Cl(-)-1 and Cl(-)-2). Both exhibited rectification, but in opposite directions, and both were insensitive to NIPAB.

Restoration of TEA-induced calcium responses in fibroblasts from Alzheimer's disease patients by a PKC activator.

Several alterations in fibroblasts of Alzheimer's disease (AD) patients have been described, including alterations in calcium regulation, protein kinase C (PKC), and potassium (K+) channels. Studies have also found reduced levels of the alpha isoform of PKC in brains and fibroblasts of AD patients. Since PKC is known to regulate ion channels, we studied K+ channel activity in fibroblasts from AD patients in the presence of (2S, 5S)-8-(1-decynyl)benzolactam (BL), a novel activator of PKC with improved selectivity for the alpha, beta, and gamma isoforms. We present evidence for restoration of normal K+ channel function, as measured by TEA-induced [Ca2+]i elevations, due to activation of PKC by BL. Representative patch-clamp data further substantiate the effect of BL on restoration of 113pS K+ channel activity. Immunoblotting analyses using an alpha-isozyme-specific PKC antibody confirm that BL-treated fibroblasts of AD patients show increased PKC activation. The present study suggests that PKC activator-based restoration of K+ channels may offer another approach to the investigation of AD pathophysiology, which in turn could lead to the development of a useful model for AD therapeutics.

Lithium decreases membrane-associated protein kinase C in hippocampus: selectivity for the alpha isozyme.

We investigated the effects of lithium on alterations in the amount and distribution of protein kinase C (PKC) in discrete areas of rat brain by using [3H]phorbol 12,13-dibutyrate quantitative autoradiography as well as western blotting. Chronic administration of lithium resulted in a significant decrease in membrane-associated PKC in several hippocampal structures, most notably the subiculum and the CA1 region. In contrast, only modest changes in [3H]phorbol 12,13-dibutyrate binding were observed in the various other cortical and subcortical structures examined. Immunoblotting using monoclonal anti-PKC antibodies revealed an isozyme-specific 30% decrease in hippocampal membrane-associated PKC alpha, in the absence of any changes in the labeling of either the beta (I/II) or gamma isozymes. These changes were observed only after chronic (4 week) treatment with lithium, and not after acute (5 days) treatment, suggesting potential clinical relevance. Given the critical role of PKC in regulating neuronal signal transduction, lithium's effects on PKC in the limbic system represent an attractive molecular mechanism for its efficacy in treating both poles of manic-depressive illness. In addition, the decreased hippocampal membrane-associated PKC observed in the present study offers a possible explanation for lithium-induced memory impairment.

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo.

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.

Classical conditioning and protein kinase C activation regulate the same single potassium channel in Hermissenda crassicornis photoreceptors.

The patch-clamp technique was used to study the effects of classical conditioning and protein kinase C (PKC) activation on K+ channels of identified neurons in the snail Hermissenda crassicornis. Here we present evidence that classical conditioning and PKC activation similarly modify the same K+ channel. K+ channels were recorded in cells from animals with different training experience. The 64-pS K+ channel appeared with significantly lower frequency in the conditioned group compared to the frequencies in control animals (naive and unpaired). In addition, when present, the 64-pS channel exhibited a lower percentage of open time and an increased interval between opening bursts in cells from conditioned animals. The 42-pS K+ channel was observed with about the same frequency in all three groups, and its percentage of open time was invariant, regardless of the animal's experience. Incubation of the photoreceptor with the PKC activator phorbol 12,13-dibutyrate (PDBu) led to a profound decrease in the percentage of open time of the 64-pS K+ channel, from 35.7% in the control group to 2.5% in the PDBu-treated group. The inactive phorbol 4 alpha-phorbol 12-myristate 13-acetate had no effect. The use of the PKC inhibitor H-7 significantly blocked the phorbol effect. Inside-out patches obtained from phorbol preincubated cells likewise showed the same effect of PDBu on K+ channels, but the effect was not observed when phorbol was added after the cell-free patches were obtained from nontreated cells. By contrast, the percentage of open time of the 42-pS K+ channel remained unchanged after phorbol treatment.