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Visual Abstract, The amyloid precursor protein (APP) has been extensively studied as the precursor of the β-amyloid (Aβ) peptide, the major component of the senile plaques found in the brain of Alzheimer’s disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP−/− neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP−/− mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.

Sequential proteolysis of the amyloid precursor protein (APP) and amyloid-β peptide (Aβ) release is an upstream event in Alzheimer’s disease (AD) pathogenesis. The function of APP in neuronal physiology is still, however, poorly understood. Along with its paralog APP-like Proteins 1 and 2 (APLP1-2), APP is involved in neurite formation and synaptic function by mechanisms that are not elucidated. APP is a single-pass transmembrane protein expressed at high levels in the brain that resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell interaction processes and/or activation of intracellular pathways of signal transduction. Along this line, the APP intracellular domain (AICD) was reported to act as a transcriptional factor for targeted gene activation that mediates physiological APP functions. Here, we used an unbiased transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and upon maturation of primary cortical neurons. The transcriptome analysis did not detect any significant differences in expression of previously proposed AICD target genes. The overall transcriptional changes were subtle, but we found that genes clustered in neuronal-activity dependent pathways are dysregulated in the absence of APP. Among these genes, we found the activity-dependent Neuronal PAS domain protein 4 (NPAS4) Immediate Early Gene to be downregulated in the absence of APP. Down-regulation of NPAS4 in APP knock-out (KO) neurons is not related to AICD but to the APP ectodomain. We studied the effect of APP deficiency on GABAergic and glutamatergic transmission, and found an increased production of the inhibitory neurotransmitter GABA in APP KO neurons, along with a reduced expression of the GABA(A) receptors alpha1, suggesting an impaired GABAergic neurotransmission in the absence of APP. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Altogether, our results point out a new role for APP in the regulation of excitatory/inhibitory neurotransmission through the regulation of the activity-dependent NPAS4 gene.

Aβ peptides, the major components of amyloid deposits of Alzheimer’s disease, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have also been reported to contribute to APP processing. However, the respective role of each of these pathways towards APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length APPWTor the β-secretase-derived C99 fragments (β-CTFs) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aβ peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by γ-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 alone is mainly degraded by the proteasome and to a lesser extent by γ-secretase.

The current inability of clinical criteria to accurately identify the “at-risk group” for Amyotrophic Lateral Sclerosis (ALS) development as well as its unknown etiology are fueling the interest in biomarkers aimed at completing clinical approaches for the diagnosis. The Glial cell line-derived neurotrophic factor (GDNF) is a diffusible peptide critically involved in neuronal differentiation and survival. GDNF is largely studied in various neurological and neuromuscular diseases, with a great interest in the peripheral nervous system (PNS). The recent discovery of Amyloid Precursor Protein (APP)-dependent GDNF regulation driving neuro-muscular junctions' formation in APP null transgenic mice, prompts to study whether neurodegeneration relies on loss or gain of APP function and suggests that it could affect peripheral processes. Here, we explored a brand-new aspect of the loss of trophic support in ALS by measuring GDNF, APP, soluble APP fragments and Aβ peptides levels in SOD1WT or SOD1G93A transgenic mouse models of ALS and in human biological fluids [i.e. serum and cerebrospinal fluid (CSF)] from ALS patients and control subjects. Our results show that both GDNF and soluble APP fragments levels are altered at the onset of motor deficits in mice and that their levels are also modified in patient samples. This study indicates that both GDNF and soluble APPα represent possible biomarkers for ALS.

Adenoviral expression of human APP (hAPP), but not of hAPP deleted from its C-terminal intracellular domain, in rat cortical neurons abolishes spontaneous synchronous calcium oscillations. The intracellular domain of APP695 contains several residues that can be phosphorylated. Contrary to non-neuronal cells, a very high phosphorylation of APP on T668 is observed in neurons, which is mediated by JNK, GSK3 and Cdk5 protein kinases. JNK activity, modulated by GSK3, enhances the traffic of phosphorylated APP to nerve terminals, contrary to Cdk5. Here we show that inhibition of GSK3 and JNK restores calcium oscillations in an hAPP expressing neuronal network, whereas inhibition of Cdk5 does not. Expression of mutant hAPPT668A does not inhibit calcium oscillations, and the proportion of hAPPT668A at the plasma membrane is reduced by more than 50%. Altogether, these results indicate that the intracellular domain of APP is needed to inhibit neuronal calcium oscillations because GSK3/JNK phosphorylation of T668 controls APP trafficking at the plasma membrane.

Extensive research efforts have been conducted over the past decades to understand the processing of the Amyloid Precursor Protein (APP). APP cleavage leads to the production of the beta-amyloid peptide (Abeta), which is the major constituent of the amyloid core of senile plaques found in the brains of patients with Alzheimer's disease (AD). Abeta is produced by the sequential cleavage of APP by beta- and gamma-secretases. Cleavage of APP by gamma-secretase also generates the APP Intracellular C-terminal Domain (AICD) peptide, which might be involved in regulation of gene transcription. Up to now, our understanding of the mechanisms controlling APP processing has been elusive. Recently, APP was found to form homo- or hetero-complexes with the APP-like proteins (APLPs), which belong to the same family and share some important structural properties with receptors having a single membrane spanning domain. Homodimerization of APP is driven by motifs present in the extracellular domain and possibly in the juxtamembrane and transmembrane (JM/TM) domains of the protein. These striking observations raise important questions about APP processing and function: How and where is APP dimerizing? What is the role of dimerization in APP processing and function? Can dimerization be targeted by small molecule therapeutics?

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 gamma-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.

Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimer disease research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including presenilin (PS1 or PS2), the γ-secretase catalytic core. GXXXG and GXXXG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GXXXG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aβ peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AXXXAXXXG motifs. The presence of a mutation causing an inherited form of Alzheimer disease (familial Alzheimer disease) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated, and the impact on complex assembly, activity, and substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggest that AXXXAXXXG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating in the switch between the physiological and pathological functional conformations of the γ-secretase.

One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile plaques in vulnerable regions of CNS. These plaques are formed of aggregated amyloid peptide. Amyloid peptide is released by the cleavage of its precursor (APP). The establishment of cell lines expressing human APP allowed to characterize both amyloidogenic and non-amyloidogneic pathways of APP catabolism and to identify some of the proteins involved in this processing (known as secretases). This led to a better comprehension of amyloid peptide production, which needs to be further characterized since gamma-secretase is as yet not identified; moreover, we still lack a clear overview of the interactions between APP and other proteins promoting Alzheimer's disease (tau, presinilinsellipsis). An important limitation of these cell lines for studying the mechanisms involved in Alzheimer's disease is supported by the observation that human APP expression does not modify transfected cells survival. The infection of primary neuronal cultures with full-length human APP indicates that APP expression induces neuronal apoptosis by itself; this neurotoxicity does not rely on extracellular production of APP derivatives (secreted APP, amyloid peptide). It is now essential to understand, in neuronal models, the production, localization and involvement of amyloid peptide in neurodegenerative processes.

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., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

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., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

The β-amyloid peptide (Aβ) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer's disease (AD). Aβ is produced by the sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Cleavage of APP by γ-secretase also generates the APP Intracellular C-terminal Domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three glycine-xxx-glycine (GxxxG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GxxxG motif led to a drastic reduction of Aβ40 and Aβ42 secretion. β-Cleavage of mutant APP was not inhibited, and reduction of Aβ secretion resulted from inhibition of γ-cleavage. It was anticipated that decreased γ-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GxxxG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM α-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production. These results clearly demonstrate that both orientation and dimerization of the APP TM domain differently affect Aβ and AICD production.

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.

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.

A large ..mu../sup -/ polarization was achieved in muonic Bi atoms with the help of the strong hyperfine field in a polarized nuclear target. Using /sup 209/Bi nuclei polarized to (59 +- 9)% in ferromagnetic BiMn, we observed a ..mu..-e decay asymmetry of (13.1 +- 3.9)%, which gives ..mu../sup -/ polarization per nuclear polarization equal to -1.07 +- 0.35. This value is almost consistent with -0.792 calculated for nuclei with spin I = (9/2) and a positive magnetic moment under the assumption that the hyperfine interaction becomes effective in the lowest muonic states.

O16(,) elastic scattering angular distributions have been measured for incident energies 39.3, 49.5, and 69.5 MeV. These data, and previous measurements at 32.2, 104, and 146 MeV, have been subjected to a global optical model analysis. The deduced global potential has two energy-dependent parameters which are found to vary smoothly with energy and it is uniquely determined by the data. Backward angular distributions measured between 40 and 54 MeV are also presented and shown to be nicely reproduced by the model. The sensitivity of the cross sections to the various regions of the real potential has been investigated as a function of energy using the notch test technique. The low energy behavior of the differential cross sections can be understood in terms of the semiclassical decomposition of Brink and Takigawa. A natural extrapolation of the global potential below 30 MeV is shown to reproduce the wide bump observed in the experimental excitation functions around 20 MeV. This bump is shown to be due to an l=8 shape resonance and is interpreted as the J=8+ member of the K=04+ rotational band of Ne20, in contradiction with the current attribution. Other bound and quasibound states supported by the potential are discussed in the light of orthogonality condition model-type arguments and shown to be consistent with the well-known K=01+ and 0- bands, and with the first three states of the K=04+ band of Ne20. NUCLEAR REACTIONS O16(,), measured (), E=39.3, 49.5, 69.5 MeV; global optical model analysis, E=32-146 MeV; semiclassical decomposition of the scattering amplitude; investigation of the compatibility of the potential description with existing low energy data and comparison with cluster models. © 1983 The American Physical Society.