The recent demonstration of K+ channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca2+-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphatemediated release of intracellular Ca2+.A number of cellular changes have been observed in fibroblasts from patients with Alzheimer disease (AD). These include abnormality of glucose and energy-related metabolism (1), defective release of a cholinergic factor (2), abnormal f8-amyloid expression and processing (3), changes in Ca2+ metabolism (30-34), and altered p-adrenergic-induced cAMP formation (4). The recent demonstration of K+ channel dysfunction in AD fibroblasts (5, 6) and past observations of Ca2+-mediated K+ channel modulation during memory storage (7) suggested that AD, which is characterized by memory loss and other cognitive deficits (8, 9), might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin (10-12), an agent that activates phospholipase C (PLC) to generate inositol 1,4,5-trisphosphate (1P3) (13)(14)(15) different for AD and control fibroblasts. f-Amyloid protein (23-25) itself, while causing the previously observed inactivation of K+ channels in AD fibroblasts, had no effect on the bombesin-elicited Ca2+ signals. These and other findings, together with measurements of bombesin receptor number, suggest that PLC/G-protein coupling and/or IP3 receptors are responsible for differences in Ca2+ responses between AD and non-AD fibroblasts. METHODSCell Lines. Human skin fibroblasts (Table 1) were purchased from the Coriell Cell Repositories (Camden, NJ). Cells were seeded and maintained as described (5). The number of passages was not significantly different between groups [AD, 10.9 ± 1.3 (mean ± SEM), n = 10; AC, 11.5 + 0.8, n = 8;...
Alzheimer's disease (AD) characteristically presents with early memory loss. Regulation of K ؉ channels, calcium homeostasis, and protein kinase C (PKC) activation are molecular events that have been implicated during associative memory which are also altered or defective in AD. PKC is also involved in the processing of the amyloid precursor protein (APP), a central element in AD pathophysiology. In previous studies, we demonstrated that benzolactam (BL), a novel PKC activator, reversed K ؉ channels defects and enhanced secretion of APP␣ in AD cells. In this study we present data showing that another PKC activator, bryostatin 1, at subnanomolar concentrations dramatically enhances the secretion of the ␣-secretase product sAPP␣ in fibroblasts from AD patients. We also show that BL significantly increased the amount of sAPP␣ and reduced A40 in the brains of APP[V717I] transgenic mice. In a more recently developed AD double-transgenic mouse, bryostatin was effective in reducing both brain A40 and A42. In addition, bryostatin ameliorated the rate of premature death and improved behavioral outcomes. Collectively, these data corroborate PKC and its activation as a potentially important means of ameliorating AD pathophysiology and perhaps cognitive impairment, thus offering a promising target for drug development. Because bryostatin 1 is devoid of tumor-promoting activity and is undergoing numerous clinical studies for cancer treatment in humans, it might be readily tested in patients as a potential therapeutic agent for Alzheimer's disease.
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.
Alterations in amyloid precursor protein (APP) metabolism, calcium regulation, oxidative metabolism, and transduction systems have been implicated in Alzheimer's disease (AD). Limitations to the use of postmortem brain for examining molecular mechanisms underscore the need to develop a human tissue model representative of the pathophysiological processes that characterize AD. The use of peripheral tissues, particularly of cultured skin fibroblasts derived from AD patients, could complement studies of autopsy samples and provide a useful tool with which to investigate such dynamic processes as signal transduction systems, ionic homeostasis, oxidative metabolism, and APP processing. Peripheral cells as well as body fluids (i.e., plasma and CSF) could also provide peripheral biological markers for the diagnosis of AD. The criteria required for a definite diagnosis of AD presently include clinical criteria in association with histopathologic evidence obtained from biopsy or autopsy. Thus, the use of peripheral markers as a diagnostic tool, either to predict or at least to confirm a diagnosis, may be of great importance.
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.
Pigment epithelium‐derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 µM glutamate, glutamate‐induced neuronal death was significantly reduced. The protective effect of rPEDF was dose‐dependent in the range from 0.023 to 7.0 nM (1–500 ng/ml), with a half‐maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate‐induced neurotoxicity, possibly via a calcium‐related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.
Since memory loss is characteristic of Alzheimer disease (AD), and since K+ channels change during acquisition of memory in both molluscs and mammals, we investigated K+ channel function as a possible site of AD pathology and, therefore, as a possible diagnostic index as well. A 113-pS tetraethylammonium (TEA)-sensitive K+ channel was consistently absent from AD fibroblasts, while it was often present in young and aged control fibroblasts. A second (166-pS) K+ (28). Membrane potential was measured both prior to obtaining outside-out patches and by using the "perforated-patch" technique (28,37). Before recording, culture medium was replaced with the following solution (mM): NaCl 150, KCI 5, CaCl2 2, MgCl2 1, Hepes (NaOH) 10, pH = 7.4. Pipettes were made from Blue Tip capillary tubes (i.d. 1.1-1.2 mm) by using a BB-CH Mecanex puller and then filled with a high-K+ solution (mM): KCI 140, CaCl2 2, MgCl2 1, Hepes (NaOH) 10, pH = 7.4. Pipette resistances were -6 MQl. Records were obtained by using an Axopatch-1C amplifier (dc to 10 kHz), stored on tape (Toshiba PCM video recorder), and later transferred to a personal computer using an Axolab interface. Only recordings lasting for at least 3 min were considered for final analysis. The pClamp suite of programs was used for singlechannel data acquisition and analysis. Amplifier, interface, and software were obtained from Axon Instruments (Foster
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