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Contrasting with its robust expression during embryogenesis, the glial cell line-derived neurotrophic factor (GDNF) is repressed in the adult organism. However, rapid induction of this neuronal growth factor is observed following diverse neuronal insults and it is now widely accepted that the control of its expression could constitute a powerful target in neuropharmacology. We investigated the effects of the neuroprotective drug, riluzole, on the GDNF gene expression in glial cells. Exposure of C6 glioma cells to riluzole (1 microM) significantly increased GDNF protein and mRNA levels. Using luciferase reporter gene constructs encoding fragments of the 5' untranslated region of the rat GDNF gene, we demonstrated that riluzole mediates its effect at the transcription level. Furthermore, luciferase assays revealed the presence of a negative regulatory region within the +343/+587 region of exon 1. This region was shown to contribute to the high sensitivity and specificity of the induction mediated by riluzole in the C6 glioma cell line at pharmacologically relevant concentrations. The effects of riluzole were inhibited by the mitogen-activated protein kinase extracellular signal-related kinase (MEK) inhibitor PD 98059. Together, these results indicated that the induction of GDNF release by riluzole in the C6 glioma cells results from the activation of its corresponding gene promoter through a signalling pathway involving MEK activity. This study suggests that the regulation of GDNF gene transcription in glial cells could contribute to the pharmacological properties of riluzole and possibly other neuroprotective drugs.

Aminoadamantanes are commonly used in the treatment of Parkinson's and Alzheimer's diseases. While these drugs are shown to antagonise ionotropic glutamate receptors on neuronal cells, additional mechanisms could contribute to their neuroprotective properties. The aim of the present study was to investigate the effect of aminoadamantanes on the production of the glial cell line-derived neurotrophic factor (GDNF) in glial cells. For this purpose, we measured the modulation of GDNF release in C6 glioma cell cultures treated for 24 h with amantadine and memantine. Both drugs dose-dependently increased GDNF level in the culture medium with similar potency (submicromolar range) and efficacy (three to four-fold induction). RT-PCR studies revealed that both compounds also increased GDNF mRNA levels and their influence on the GDNF gene transcription was further evidenced using a rat GDNF promoter luciferase reporter assay. Together, these results demonstrate that the neuroprotective effect of amantadine and memantine could involve the regulation of GDNF production by glial cells.

Nucleotide concentrations were normal in adenylosuccinate lyase-deficient fibroblasts, and the succinylpurines were not toxic for cultured neuronal cells.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.

After association with intact Chinese hamster ovary (CHO) cells expressing the rat neurotensin receptor, tritiated neurotensin was rapidly internalized. Internalization was maximal after 30 min and accounted for about 90% of the total associated ligand. Neurotensin internalization was not observed at 0-4 degrees and was inhibited by an excess of unlabelled neurotensin or by the neurotensin non peptide antagonist, SR 48692. Moreover, the incubation of intact cells for 30 min with 10 nM neurotensin resulted in a significant decrease in the number of the cell surface neurotensin receptors. These results indicate that the endocytosis of membrane bound neurotensin in transfected CHO cells resulted from the internalization of the ligand-receptor complex inside the cell, through an agonist-induced process.

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.

The production of amyloid peptide (Abeta) from its precursor (APP) plays a key role in Alzheimer's disease (AD). However, the link between Abeta production and neuronal death remains elusive. We studied the biological effects associated with human APP expression and metabolism in rat cortical neurons. Human APP expressed in neurons is processed to produce Abeta and soluble APP. Moreover, human APP expression triggers neuronal death. Pepstatin A, an inhibitor of aspartyl proteases that reduces Abeta production, protects neurons from APP-induced neurotoxicity. This suggests that Abeta production is likely to be the critical event in the neurodegenerative process of AD.

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.

Parvalbumin mRNA was assayed by Northern blot analysis in muscles from normal and dystropic (mdx) mice. Its content was found to be specifically higher in mdx fast muscles than in control preparations. This suggests an increased expression of the protein in dystrophin-lacking fast fibres. A possible role in calcium homeostasis is discussed.

In a previous study, Hughes et al. [Proc. Natl. Acad. Sci. USA 93 (1996) 2065-2070] demonstrated that the amyloid peptide is able to interact with itself in a two-hybrid system and that interaction is specific. They further supported that the method could be used to define the sequences that might be important in nucleation-dependent aggregation. The sequence of the amyloid peptide can be split into four clusters, two hydrophilic (1-16 and 22-28) and two hydrophobic (17-21 and 29-42). We designed by molecular modeling and tested by the two-hybrid approach, series of mutations spread all over the sequence and changing the distribution of hydrophobicity and/or the spatial hindrance. In the two-hybrid assay, interaction of native Abeta is reproduced. Screening of mutations demonstrates that the C-domain (residues 29-40 (42)), the median domain (residues 17-22) and the N-domain (1-16) are all crucial for interaction. This demonstrates that almost all fragments of the amyloid peptide but a loop (residues 23-28) and the C-term amino acid are important for the native interaction. We support that the folded three-dimensional (3D) structure is the Abeta-Abeta interacting species, that the whole sequence is involved in that 3D fold which has a low secondary structure propensity and a high susceptibility to mutations and thus should have a low stability. The native fold of Abeta could be stabilized in Abeta-Abeta complexes which could in other circumstances facilitate the nucleation event of aggregation that leads to the formation of stable senile plaques.

Neurofibrillary tangles (NFTs) are a characteristic neuropathological lesion of Alzheimer's disease (AD). They are composed of a highly-phosphorylated form of the microtubule-associated protein tau. We are investigating the relationship between NFTs and microtubule stability and how tau phosphorylation and function is affected in transgenic models and by co-expression with beta-amyloid precursor protein and presenilins. In most NFT-bearing neurons, we observed a strong reduction in acetylated alpha-tubulin immunoreactivity (a marker of stable microtubules) and a reduction of the in situ hybridization signal for tubulin mRNA. In transfected cells, mutated tau forms (corresponding to tau mutations identified in familial forms of frontotemporal dementias linked to chromosome 17) were less efficient in their ability to sustain microtubule growth. These observations are consistent with the hypothesis that destabilization of the microtubule network is an important mechanism of cell dysfunction in Alzheimer's disease. The glycogen synthase kinase-3 beta (GSK-3 beta) generates many phosphorylated sites on tau. We performed a neuroanatomical study of GSK-3 beta distribution showing that developmental evolution of GSK-3 beta compartmentalization in neurons paralleled that of phosphorylated tau. Studies on transfected cells and on cultured neurons showed that GSK-3 beta activity controls tau phosphorylation and tau functional interaction with microtubules. Tau phosphorylation was not affected in neurons overexpressing beta-amyloid precursor protein. Transgenic mice expressing a human tau isoform and double transgenic animals for tau and mutated presenilin 1 have been generated; a somatodendritic accumulation of phosphorylated transgenic tau proteins, as observed in the pretangle stage in AD, has been observed but NFTs were not found, suggesting that additional factors might be necessary to induce their formation., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Soluble beta-amyloid protein precursors (beta-APPs) were studied in human brain and cerebrospinal fluid (CSF) after partial purification by ion exchange chromatography. Proteins were analysed in immunoblotting experiments using a monoclonal antibody directed against the N-terminal segment of the beta-APP 770, and by reverse enzymography. In the human brain and CSF, a protein which comigrates with the beta-APP 770 expressed by transfected CHO cells was able to inhibit trypsin.

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.

The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3' untranslated region (3'UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3'UTR containing 8 nucleotides more than the short 3'UTR allows the recovery of an efficiency of translation similar to that of the long 3'UTR. Moreover, the two guanine residues located at the 3' ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3'UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3'UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.

Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells.

Introduction Bien que le metabolisme cellulaire du precurseur du peptide amyloide (APP) ait ete etudie en detail, la fonction precise de la proteine reste inconnue. Le domaine intracellulaire de l’APP (AICD) pourrait controler l’expression de plusieurs genes. Cependant, l’identite de certains genes cibles de l’APP fait l’objet de controverses, et les mecanismes par lesquels l’APP regule leur transcription est inconnue. Le but de notre travail etait de confirmer que l’APP est capable de controler l’expression de certains genes, et d’etudier les mecanismes moleculaires impliques. Methodes Nous avons identifie des genes cibles de l’APP en comparant les profils d’expression de genes dans les fibroblastes embryonnaires de souris (MEFs) exprimant ou non l’APP (APP +/+ et APP -/-). Etant donne que l’AICD est libere de l’APP par une activite γ-secretase preseniline- dependante, nous avons realise des experiences identiques en utilisant des MEFs exprimant ou non les presenilines 1 et 2 (PS +/+ et PS -/-). Resultats Parmi les genes differemment exprimes dans ces deux modeles cellulaires, nous nous sommes particulierement interesses aux genes codant l’aquaporine 1 (AQP1) et CXCL5, dont les expressions sont diminuees dans les MEFs APP-/- et PS -/-. Nous avons montre, par qRT-PCR, immunotransfert et ELISA, que l’expression d’AQP1 et de CXCL5 est fortement diminuee dans les cellules n’exprimant pas l’APP ou PS2. Dans ces cellules, l’expression d’AQP1 et de CXCL5 est retrouvee suite a l’expression stable d’APP ou de PS2 humains. Nous avons ensuite analyse l’activite transcriptionnelle des promoteurs des genes AQP1 et CXCL5, clones en amont du gene rapporteur de la luciferase. L’activite transcriptionnelle des promoteurs des genes AQP1 et CXCL5 etait identique dans les MEFs APP+/+ et APP -/-. De la meme maniere, la stabilite des mRNA AQP1 et CXCL5, mesuree en presence d’actinomycine D, etait identique dans les MEFs APP+/+ et APP-/-. L’utilisation de trichostatine A, un inhibiteur specifique des histones deacetylases (HDACs) de type I et II, a demontre que la regulation de l’expression d’AQP1 et de CXCL5 par l’APP etait sensible a l’acetylation des histones. Nous avons finalement demontre une co-immunoprecipitation d’activite HDAC avec le domaine intracellulaire de l’APP. Conclusions Ce travail demontre que, dans des MEFs, l’expression des genes codant l’AQP1 et CXCL5 est regulee par l’APP et PS2, sans modification de l’activite transcriptionnelle des promoteurs mesuree a l’aide d’un gene rapporteur, ni de la stabilite des mRNAs. Une diminution d’AQP1 renale et de CXCL5 serique, mise en evidence dans des souris APP-/-, suggere un role essentiel de l’APP dans le controle epigenetique de l’expression de ces deux genes.

We have generated transgenic mice expressing the shortest human tau protein, the microtubule-associated protein that composes paired helical filaments in Alzheimer's disease. Transgenic tau transcripts and proteins were strongly expressed in neurons in the developing and adult brain. In contrast to the endogenous tau that progressively disappeared from neuronal cell bodies during development, the human transgenic tau remained abundant in cell bodies and dendrites of a subset of neurons in the adult. This somatodendritic transgenic tau was immunoreactive with antibodies to tau phosphorylated on Thr181 and Thr231 and with the conformation-dependent Alz50 antibody. A few astrocytes expressing the transgenic tau were strongly immunoreactive with antibodies to additional tau phosphorylation sites, ie, at Ser262/ 356 and Ser396/404. All of these phosphorylation sites have been identified in paired helical filaments-tau proteins. In electron microscopy, the transgenic tau was detected into microtubules in axons and in dendrites but not in cell bodies. Neurofibrillary tangles were not detected in transgenic animals examined up to the age of 19 months. These results indicate that transgenic manipulation of tau expression and intracellular targeting is sufficient per se to affect tau compartmentalization, phosphorylation, and conformation partly as it is observed at the pretangle stage in Alzheimer's disease.

alpha 1-tubulin is an isotype of alpha-tubulin, and its mRNA is expressed in the rodent nervous system. A high level of alpha 1-tubulin mRNA in neurones is associated with axonal outgrowth during development as well as with axonal regeneration after axotomy in adult animals. We quantitated alpha 1-tubulin mRNA levels in motor neurone-like NG 108-15 cells using Northern blots in order to determine whether the expression of this neurite outgrowth-associated gene is regulated in NG 108-15 cells during neurite extension and during inhibition of this process by CNS myelin. Here we report that during the acute phase of neurite outgrowth, alpha 1-tubulin mRNA level increases in NG 108-15, a maximal induction of 1.7-fold over the initial level occurring 24 h after neurite outgrowth onset. By contrast, when these cells are plated on CNS myelin alpha 1-tubulin mRNA levels show no such increase. These findings indicate that an increase of the alpha 1-tubulin mRNA level is associated with neurite outgrowth of NG 108-15 cells. More interestingly, this study also demonstrates that the inhibition of neurite outgrowth by CNS myelin may affect the expression of a gene encoding a protein involved in neurite extension.

In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding sites, influence KATP channels and sulfonylurea receptors properties in insulinoma and cardiac cells. In this study, a negative allosteric action of fluorescein derivatives on glibenclamide binding sites has been shown in rat cortical neurons. This supports the hypothesis of interactions between nucleotide- and sulfonylurea-binding sites within the sulfonylurea receptor.

Using a rat C6 brain tumor model, we studied the antitumor effects of Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene transfer followed by ganciclovir treatment. C6 glioma cells were transfected in vitro with the HSV-tk gene, and tested for their sensitivity to ganciclovir. Although there was no surviving cell at a 30 microM ganciclovir concentration, unmodified C6 cells were not affected by the drug. For in vivo experiments, intracerebral tumors were induced in rats by stereotactic injection of 10(4) HSV-tk-modified C6 cells. Ten days later, the animals were treated with intraperitoneal injections of ganciclovir for 21 days. The tumors evolution was evaluated by high resolution magnetic resonance imaging. In 33% of the rats, the signal intensity of the tumors became heterogeneous, with development of highly hyperintense areas, and a complete tumor regression was subsequently noted. Histological examination of successfully treated tumors revealed progressive necrosis with formation of cysts. The survival time of the HSV-tk/ganciclovir treated animals was consistently increased, all rats surviving more than 30 days and 33% of them being still alive after 80 days.

Alzheimer's disease, the most frequent cause of dementia, is characterized by the formation in the brain of neurofibrillary tangles and senile plaques. Neurofibrillary tangles are composed of bundles of paired helical filaments containing the microtubule-associated protein tau. In autopsy-derived brain samples from patients with Alzheimer's disease, tau is hyperphosphorylated and constitutes a promising disease marker. Senile plaques contain a small amyloid peptide derived from the amyloid precursor protein. Mutations of the amyloid precursor protein gene have been identified in rare cases of familial Alzheimer's disease, suggesting a causal role for amyloid peptide deposition in the disease. However, Alzheimer's disease has been demonstrated to be characterized by an important genetic heterogeneity. The identification of pathogenic DNA mutations, different from those of the amyloid precursor protein gene, will reveal whether the corresponding genes are involved in either an increased production of the amyloid peptide or a decrease of its removal, or in the fibrillogenic properties of the peptide, which seem to be related to its toxicity. Several mammalian cells are able to produce the amyloid peptide from its precursor. Understanding the cellular mechanisms that determine how cleavages occur in cells could help to identify new strategies for modulating amyloid peptide production. In attempts to produce animal models of Alzheimer's disease, investigators have used transgenic strategies. To date, these efforts have not been very successful. However, the expression in transgenic mice of both mutated amyloid peptide precursor and amyloid associated proteins should prove useful for examining the importance of putative etiological factors, and for testing novel therapies including anti-amyloidogenic strategies.

When neuronal adhesion is reduced by heparin, cultured neurons detach from the substratum and form clusters connected by neuritic fascicles. 24 h after treatment, Northern blots analysis revealed an increase in the expression of the 9-kb MAP2 mRNA in the heparin-treated neuronal cultures. MAP2 being associated with the cross-bridges between microtubules, this increased expression could be an attempt of the cells to rigidify their cytoskeleton to compensate for the reduced attachment. However, the MAP2 protein content was not increased after a 24-h heparin treatment. We discuss possible explanations for this observation and their implications on MAP2 metabolism.

A new mouse monoclonal antibody was raised to a bacterial fusion protein between beta-galactosidase and the extracellular domain of the human amyloid protein precursor (APP). In immunoblotting experiments, this monoclonal antibody labelled the bacterial fusion protein used as an immunogen, the human brain APP, and different full-length APP isoforms expressed by transfected cells. In immunocytochemistry, the monoclonal antibody stained the dystrophic neurites of abundant senile plaques found in the cerebral cortex of patients with Alzheimer's disease. Using bacterial expression of several cDNA fragments, the epitope was mapped to an amino acid sequence of APP not investigated before., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Total RNA was purified from rat forebrain at different postnatal ages and analyzed by Northern blot using a specific neurotensin receptor RNA probe. The rat neurotensin receptor mRNA was present in high amount during the first 10 days of life. Thereafter, it rapidly decreased and was undetected after 20 days. [3H]neurotensin binding experiments performed on the same tissues indicated that the total amount of neurotensin receptors increased during the first week and was maximal between day 7 and day 10. This plateau was followed by an important loss (70%) of neurotensin receptors. These results indicate that an important reduction in the genetic expression of the neurotensin receptor after day 10 may probably account for the [3H]neurotensin binding profile observed in rat forebrain during the postnatal ontogeny.

The beta A4 peptide is the major constituent of the amyloid core of abundant senile plaques found in the cerebral cortex of patients with Alzheimer's disease. This amyloid peptide is synthesized as part of a large transmembrane amyloid protein precursor or APP. In addition to the highly expressed transmembrane APP isoforms, an mRNA encoding a secreted APP lacking the transmembrane domain has been identified. A cleavage of the transmembrane protein also yields an extracellular soluble APP fragment. The effect of phorbol esters on the release of the extracellular APP was studied in transfected Chinese hamster ovary cells which stably express either a transmembrane or a secreted APP isoform. The activation of protein kinase C by phorbol-12,13-dibutyrate increased the extracellular release of the transmembrane APP resulting from its proteolytic cleavage, while 4-beta-phorbol, which does not activate protein kinase C, did not significantly affect the recovery of the soluble APP. On the contrary, the recovery of APP secreted in the culture medium without proteolytic cleavage was not increased by protein kinase C-mediated phosphorylation.

In the present study, we describe the effects of RP 67580, a substance P non-peptide antagonist, in binding and second messenger experiments performed using transfected Chinese hamster ovary cells expressing the rat NK1 receptor. The cDNA sequence encoding the rat brain substance P receptor was transfected in Chinese hamster ovary cells, and cellular clones which stably express the corresponding protein were isolated. [3H]Substance P binding was performed in homogenates of these transfected cells and revealed the presence of NK1 receptors in displacement experiments, using peptide analogs of three mammalian tachykinins (substance P, neurokinin A, neurokinin B). Scatchard analysis indicated a KD value of 0.33 +/- 0.13 nM and a Bmax value of 5.83 +/- 1.16 pmol/mg of protein. RP 67580, a selective NK1-receptor antagonist was found to displace the specific binding of [3H]substance P. When [3H]RP 67580 was used as a ligand, it displayed a high affinity (KD value: 1.22 +/- 0.27 nM) in transfected cell homogenates and only competed with NK1 receptor ligands. Substance P stimulated the hydrolysis of phosphoinositide in a time- and concentration-dependent manner and this effect was mimicked by selective agonists of the NK1 receptor ([Pro9]SP and septide). RP 67580 did not induce any accumulation of inositol phosphates, but was found to inhibit the inositol phosphate increase mediated by substance P, without affecting the maximal response. From these results, one may conclude that the receptor expressed by the transfected Chinese hamster ovary cells revealed similar binding characteristics as the NK1 receptor present in the rat brain and also confirmed the high affinity and the antagonist properties of RP 67580.

Myoclonus epilepsy and ragged-red-fiber syndrome (MERRF) is caused by a point mutation at nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA. We analyzed leukocyte DNA from nine members of a large MERRF family using a new technique, solid-phase minisequencing. Quantitative analysis of the tRNA(8344Lys) mutation showed that the mutated mtDNA comprised from 9 to 72% of the total mtDNA in the leukocytes of these individuals. The minisequencing method is a promising tool for the diagnosis of MERRF. In addition to the identification of the tRNA(8344Lys) mutation, the relative amount of mutated mtDNA can be simultaneously determined in the same assay from one blood sample.

Processing of amyloid precursor protein (APP) by γ-secretase is the last step in the formation of the Aβ peptides associated Alzheimer's disease. MAS solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP in membrane bilayers that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618-660), we target the glycines within the TM domain, as well as residues at the γ- and e-cleavage sites. We find that GxxxG motifs involving Gly625, Gly629 and Gly633 mediate TM helix homodimerization, and that the TM helix breaks at the transition point near the e-cut site. We show that the insertion of three consecutive leucines at the transition point in APP695 inhibits e-cleavage leading to a low production of Aβ peptides and an accumulation of the α- and β- C-terminal fragments. The leucine insertion extends the TM domain by one helical turn, whereas an insertion of three glycines does not, demonstrating that the helix-to-coil transition is required for γ-secretase processing. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM α-helix.

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.

A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10). The inserts of the immunoreactive clones were short (0.9-1.1 kbp). One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx. 1.9 kbp) and widely overlapping cDNA inserts. They did not contain the first two nucleotide residues of the initiator codon, nor the 5′-end untranslated portion of the mRNA. These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5′-end of the already known cDNA sequence. The nucleotide sequence consists of 48 bp of 5′-end non-coding region, 1695 bp of coding region and 212 bp of 3′-end non-coding region including a 20 bp poly(A) tail. The signal peptide and the mature protein subunit are 18 and 547 residues long respectively. Tyr is confirmed as N-terminal residue. The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing [Korza & Ozols (1988) J. Biol. Chem. 263, 3486-3495; Ozols (1989) J. Biol. Chem. 264, 12533-12545]. The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus. The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells [Munro & Pelham (1987) Cell 48, 899-907]; -HDEL in yeast [Pelham, Hardwick & Lewis (1988) EMBO J. 7, 1757-1762]). We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum. In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.

cDNA probes specific for the A4 amyloid peptide precursor hybridize with a 3.2–3.4 kb mRNA doublet which can be attributed to the use of two polyadenylation sites. Different chimeric mRNAs were synthesized by in vitro transcription of the coding region of the chicken lysozyme or the chloramphenicol acetyl transferase followed by two 3′ untranslated regions of the A4 amyloid peptide precursor mRNA using the two possible polyadenylation sites. In vivo translation of these mRNA constructs in Xenopus oocytes indicates that the long mRNAs using the second polyadenylation site produce higher amounts of proteins as compared to the short mRNAs. This effect on the translation is not related to a higher stability of the long mRNA in oocytes.

Characterization of a Monoclonal-antibody Raised Against the Extracellular Domain of the Amyloid Precursor Protein Which Recognizes Tau-proteins

Neurofibrillary tangles and senile plaques, two neuropathological markers of Alzheimer's disease, may both contain peptide fragments derived from the β amyloid protein. Human β amyloid peptide precursor cDNAs have been isolated from normal foetal and adult brain libraries. In peripheral tissue and cultured cells, a novel precursor containing a protease inhibitor domain has been cloned. A cDNA library from the cerebral cortex of a patient with sporadic Alzheimer's disease was constructed and several clones coding for the β amyloid peptide precursor were isolated cDNAs containing two types of insertion coding for a serine protease inhibitor domain were identified. The use of another polyadenylation site available in the 3′-untranslated region of the mRNA was observed. These results indicate that, in one patient with Alzheimer's disease, different RNA species coding for the β amyloid peptide precursor arise by alternative splicing of a single transcriptional unit, and use different polyadenylation sites.

cDNA clones for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were isolated from rat liver expression libraries in λgt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full-length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.

There is a high affinity binding of [3H]PN 200-110 (Kd = 0.21 nM) to slow calcium channels in cultured neurones. Several calcium antagonists, which recognize the [3H]PN 200-110 binding site, did not affect the K+-induced calcium uptake. The calcium channel activator BAY K 8644 increased the calcium uptake in depolarizing conditions and this effect was antagonized by pharmacological concentrations of calcium entry blockers. We conclude that the dihydropyridine binding site is involved in the modulation of calcium entry through the voltage-sensitive channel in depolarized cultured neurones.

The reticuloendothelial system is responsible for removing old and damaged erythrocytes from the circulation, allowing iron to return to bone marrow for hemoglobin synthesis. Cultured bone marrow macrophages were loaded with 59Fe-labelled erythroblasts and iron mobilization was studied. After erythroblast digestion, iron taken up by macrophages was found in ferritin as well as in a low-molecular-weight fraction. The analysis of iron mobilization from macrophages shows: (1) the iron was mobilized as ferritin. (2) A higher mobilization was observed when apotransferrin was present in the culture medium. (3) In the presence of apotransferrin in the culture medium, part of the iron was found as transferrin iron. (4) Iron transfer from ferritin to apotransferrin was observed in a cell-free culture medium and this process was temperature independent. The results indicate that after phagocytosis of 59Fe-labelled erythroblasts by macrophages, iron is mobilized as ferritin. In the plasma, this iron can be transferred to apotransferrin.