Jean-Noël Octave

Calcium-mediated transient phosphorylation of tau and amyloid precursor protein followed by intraneuronal amyloid-beta accumulation.

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-β peptide (Aβ) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Aβ accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Aβ. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Amyloid precursor protein controls cholesterol turnover needed for neuronal activity.

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full‐length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG‐CoA reductase (HMGCR)‐mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co‐immunoprecipitated and co‐localized in the Golgi. This interaction prevented Site‐2 protease‐mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24‐hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity.

Epigenetic control of aquaporin 1 expression by the amyloid precursor protein

Cellular processing of the amyloid precursor protein (APP) has been extensively studied, but its precise function remains elusive. The intracellular domain of APP has been proposed to regulate expression of several genes by mechanisms that are largely unknown. We report that APP regulates expression of the aquaporin 1 (AQP1) gene in mouse embryonic fibroblasts and in transgenic mice. AQP1 mRNA and protein were down‐regulated in fibroblasts lacking APP or presenilin 2 in which AQP1 expression was restored by stable expression of full‐length APP or presenilin 2 but not by APP deleted from its carboxy‐terminal domain. The transcriptional activity of the AQP1 gene promoter and the stability of AQP1 mRNA were identical in fibroblasts expressing or not expressing APP. Control of AQP1 expression by APP was sensitive to trichostatin A, an histone deacetylase inhibitor, and histone deacetylase activity coimmunoprecipitated with APP. Altogether, these data show that a presenilin‐2‐ dependent γ‐secretase activity releases the intracellular domain of APP involved in the epigenetic control of AQP1 expression. Since AQP1 is found in astrocytes surrounding senile plaques, this epigenetic control of AQP1 expression could have important implications in Alzheimer disease.—Huysseune, S., Kienlen‐Campard, P., Hébert, S., Tasiaux, B., Leroy, K., Devuyst, O., Brion, J.‐P., De Strooper, B., Octave, J.‐N. Epigenetic control of aquaporin 1 expression by the amyloid precursor protein. FASEB J. 23, 4158‐4167 (2009). www.fasebj.org

Iron mobilization from cultured hepatocytes: effect of desferrioxamine B.

When cultured rat hepatocytes prelabelled for different times at 37 degrees with 59Fe are reincubated for 1 hr in a fresh medium, radiolabelled iron is released in the washout medium as a function of the prelabelling time, and behaves like low molecular weight material on isokinetic centrifugation in sucrose gradients. When apotransferrin or desferrioxamine B are present in the reincubation medium, the kinetics of iron release are similar but the absolute amounts of radiolabelled iron found in the culture medium are much greater. In the presence of apotransferrin, most of the 59Fe released from the cells distributes as transferrin whereas with desferrioxamine B, almost all the 59Fe is extracted by benzyl alcohol indicating its chelation by the drug. Cell fractionation data indicate that iron accumulated by hepatocytes is rapidly incorporated into cytosol ferritin, and this seems to be a preferred source of iron for the chelator.

Iron mobilization from cultured rat bone marrow macrophages.

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.

Iron mobilization from cultured rat fibroblasts and hepatocytes. Effect of various drugs.

Ramon RAMA*, Jean-Noel OCTAVE, YvesJacques SCHNEIDER+, Jean-Claude SIBILLE, Joseph N. LIMET, Jean-Claude MARESCHAL, Andre TROUET and Robert R. CRICHTON Universitt Catholique de Louvain (Laboratoire de Chimie Physiologique and Unit& de Biochirnie) and International institute of Ceitular and Molecular Pathology, place L. Pasteur, B-1348 Lolivain-La-~el~ye and 75, avenue Hippocrate, 3-l 200 B~xe~ies~ Bel~.uFn

Down-regulation of 3H-lofentanil binding to opiate receptors in different cultured neuronal cells.

SummaryThere was stereospecific binding of 3H-lofentanil (KD value = 1.53 nM) to membranes of neuroblastoma-glioma NG 108-15 cells which are known to bear high affinity binding sites for enkephalin derivatives (δ-opiate receptor subtype). There was no high affinity specific binding of the μ-opiate specific ligand 3H-sufentanil. The specific binding of 3H-lofentanil to δ-opiate receptor subtype was down-regulated (decrease in Bmax value without change in the KD value) after prolonged incubation of the cells in the presence of leu- and met- enkephalin (0.1 μM). There was no down-regulation of the opiate receptors (3H-lofentanil and 3H-d-ala-d-leu-enkephalin specific binding) after incubation of NG 108-15 cells with drugs from the fentanyl series (alfentanil or sufentanil).In cultured neurones from rat forebrain (15 day old embryos), the 3H-lofentanil binding was specific with high affinity (KD: 0.048 nM) and a slow dissociation rate similar to that in adult rat cortex. Drugs of the fentanyl series (4-anilino-piperidines) were potent displacers whereas agonists of the δ- (enkephalin derivatives), σ (phencyclidine, haloperidol, 3-hydroxyphenyl-propylpiperidine) or K- (U 50488) opiate sites had a low affinity (Ki > 0.5 μM) for 3H-lofentanil specific binding sites. Since there was also specific binding of 3H-sufentanil, the opiate receptors in cultured neurones seem to be mainly of the μ-subtype and this is consistent with the ontogeny of opiate receptors subtypes. These receptors were down-regulated after incubation in the presence of etorphine, sufentanil and alfentanil but not enkephalin derivatives.These results strongly suggest specific binding of 3H-sufentanil and 3H-lofentanil mainly to the so-called μ-opiate receptors in cultured neurones and a specific binding of 3H-lofentanil to lower affinity δ-opiate receptors in neuroblastoma-glioma cells. The down-regulation of the μ-opiate binding sites in cultured neurones and that of the δ-site in neuroblastoma × glioma hybrid cells were dose-and temperature-dependent, induced by the corresponding high affinity agonists and prevented by naloxone. Morphine did not induce down-regulation of μ or δ receptor sites, possibly because of a partial antagonist effect on both receptor subtypes.

Epigenetic Induction of EGR-1 Expression by the Amyloid Precursor Protein during Exposure to Novelty.

Following transcriptome comparison of primary cultures isolated from brain of mice expressing or not the amyloid precursor protein APP, we found transcription of the EGR-1 gene to be regulated by APP. In primary cultures of cortical neurons, APP significantly down regulated EGR-1 expression at both mRNA and protein levels in a γ-secretase independent manner. The intracellular domain of APP did not interact with EGR-1 gene promoter, but enrichment of acetylated histone H4 at the EGR-1 promoter region was measured in APP-/- neurons, as well as in brain of APP-/- mice, in which increase in EGR-1 expression was also measured. These results argue for an important function of APP in the epigenetic regulation of EGR-1 gene transcription both in vitro and in vivo. In APP-/- mice, constitutive overexpression of EGR-1 in brain impaired epigenetic induction of this early transcriptional regulator during exposure to novelty. Altogether, these results indicate an important function of APP in the epigenetic regulation of the transcription of EGR-1, known to be important for memory formation.

Cortical cells reveal APP as a new player in the regulation of GABAergic neurotransmission.

The amyloid precursor protein (APP) modulates synaptic activity, resulting from the fine tuning of excitatory and inhibitory neurotransmission. GABAergic inhibitory neurotransmission is affected by modifications in intracellular chloride concentrations regulated by Na+-K+-2Cl− cotransporter 1 (NKCC1) and neuronal K+-Cl− cotransporter 2 (KCC2), allowing entrance and efflux of chloride, respectively. Modifications in NKCC1 and KCC2 expression during maturation of cortical cells induce a shift in GABAergic signaling. Here, we demonstrated that APP affects this GABA shift. Expression of APP in cortical cells decreased the expression of KCC2, without modifying NKCC1, eliciting a less inhibitory GABA response. Downregulation of KCC2 expression by APP was independent of the APP intracellular domain, but correlated with decreased expression of upstream stimulating factor 1 (USF1), a potent regulator of Slc12a5 gene expression (encoding KCC2). KCC2 was also downregulated in vivo following APP expression in neonatal mouse brain. These results argue for a key role of APP in the regulation of GABAergic neurotransmission.

Amyloid precursor protein reduction enhances the formation of neurofibrillary tangles in a mutant tau transgenic mouse model

Alzheimers disease is characterized by the presence of 2 neuropathological lesions: neurofibrillary tangles, composed of tau proteins which are highly phosphorylated and phosphorylated on uncommon sites, and amyloid plaques, containing the Aß peptides generated from the amyloid precursor protein (APP). Reduction of some APP proteolytic derivatives in Alzheimers disease such as sAPPα fragment has been reported and sAPPα has been shown to affect tau phosphorylation. To investigate inxa0vivo the effect of absence of APP protein and its fragments on tau phosphorylation and the formation of neurofibrillary tangles, we have generated mice deleted for APP gene and overexpressing a human mutant tau protein and developing neurofibrillary tangles (APPKOTg30 mice). These APPKOTg30 mice showed more severe motor and cognitive deficits, increased tau phosphorylation, increased load of neurofibrillary tangles, and increased p25/35 ratio in the brain, compared with Tg30 mice. These data suggest that APP and/or its proteolytic derivatives interfere with the formation of neurofibrillary tangles in a transgenic mouse model that will be useful for investigating the relationship between APP and tau.