Rene Etcheberrigaray

Lithium Decreases Membrane-Associated Protein Kinase C in Hippocampus: Selectivity for the α 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 α, in the absence of any changes in the labeling of either the β(I/II) or γ 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, lithiums 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.

Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity.

Abstract: 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.

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.

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.

Use of cultured fibroblasts in elucidating the pathophysiology and diagnosis of Alzheimer's disease

Altered signal transduction systems in Alzheimer’s disease (AD) are likely to be of pathophysiological importance, because these processes are critical to cellular and brain functions. In addition to their classic role of converting “environmental” stimuli into intracellular signals, these transduction systems modulate multiple aspects of cellular function, including posttranslational modification and processing of molecules such as amyloid precursor protein‘,2 and Alterations in those two proteins in particular are generally considered to play a critical role in the pathophysiology of aging and AD. Impaired oxidative metabolism is also known to accompany AD, and oxidative deficits can alter signal transduction system^.^,^ One important difficulty in defining the relation of changes in signal transduction or oxidation to the causation of AD lies in determining if the abnormality is of pathophysiological importance or if it is an epiphenomenon occurring secondary to neurodegeneration. Thus, whether primary defects in oxidative metabolism or in a particular signal transduction system might cause the characteristic structural changes remains unclear. An understanding

Molecular mechanisms of memory and the pathophysiology of Alzheimer's disease.

Research on molecular and biophysical mechanisms of associative learning and memory storage identified a number of key elements that are phylogenetically conserved. In both vertebrates and invertebrates, K+ channels, PKC, Cp20, and intracellular Ca2+ regulation play a fundamental role in memory mechanisms. Because memory loss is the hallmark and perhaps the earliest sign of Alzheimers disease, we hypothesized that these normal memory mechanisms might be altered in AD. With the use of a variety of experimental methodologies, our results revealed that one of the critical elements in memory storage, K+ channels, are dysfunctional in AD fibroblasts. Moreover, beta-amyloid induced the same K+ dysfunction in normal cells. Intracellular Ca2+ release, also associated with molecular memory mechanisms, was found altered in fibroblasts from patients with AD. The results therefore strongly suggest that biophysical and molecular mechanisms of associative learning could be altered in AD and that they may contribute to the memory loss observed early in the disease.

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.

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 Alzheimers 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.

Soluble β-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 Alzheimers 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.

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 Alzheimers 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.