Specificity of presenilin-1- and presenilin-2-dependent γ-secretases towards substrate processing.

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 ß-amyloid peptide (Aß) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aß 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.

APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

Presenilin transmembrane domain 8 conserved AXXXAXXXG motifs are required for the activity of the γ-secretase complex.

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 Aß 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.

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 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.

Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release.

The amyloid precursor protein (APP) has been extensively studied as the precursor of the ß-amyloid (Aß) 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.

Contribution of the Endosomal-Lysosomal and Proteasomal Systems in Amyloid-ß Precursor Protein Derived Fragments Processing.

Aß peptides, the major components of Alzheimer's disease (AD) amyloid deposits, 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 been reported to contribute to APP processing. However, the respective role of each of these pathways toward APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length wild type APP or the ß-secretase-derived C99 fragment (ß-CTF) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aß 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 is mainly degraded by the proteasome and to a lesser extent by γ-secretase.

A Role for GDNF and Soluble APP as Biomarkers of Amyotrophic Lateral Sclerosis Pathophysiology.

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 Aß 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.

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.

Epigenetic regulations of immediate early genes expression involved in memory formation by the amyloid precursor protein of Alzheimer disease.

We previously demonstrated that APP epigenetically regulates Egr1 expression both in cultured neurons and in vivo. Since Egr1 is an immediate early gene involved in memory formation, we wondered whether other early genes involved in memory were regulated by APP and we studied molecular mechanisms involved. By comparing prefrontal (PF) cortex from wild type (APP+/+) and APP knockout mice (APP-/-), we observed that APP down regulates expression of four immediate early genes, Egr1, c-Fos, Bdnf and Arc. Down regulation of Egr1, c-Fos and Bdnf transcription resulted from a decreased enrichment of acetylated histone H4 on the corresponding gene promoter. Further characterization of H4 acetylation at Egr1 and c-Fos promoters revealed increased acetylation of H4K5 and H4K12 residues in APP-/- mice. Whereas APP affected Egr1 promoter activity by reducing access of the CREB transcription factor, its effect on c-Fos appeared to depend on increased recruitment of HDAC2 histone deacetylase to the gene promoter. The physiological relevance of the epigenetic regulation of Egr1 and c-Fos gene transcription by APP was further analyzed following exposure of mice to novelty. Although transcription of Egr1 and c-Fos was increased following exposure of APP+/+ mice to novelty, such an induction was not possible in APP-/- mice with a high basal level of expression of these immediate early genes. Altogether, these results demonstrate that APP-mediated regulation of c-Fos and Egr1 by different epigenetic mechanisms is needed for their induction during exposure to novelty.

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.

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.

Inhibition of neuronal calcium oscillations by cell surface APP phosphorylated on T668.

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.

What is the role of amyloid precursor protein dimerization?

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 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?

Amyloidogenic processing but not amyloid precursor protein (APP) intracellular C-terminal domain production requires a precisely oriented APP dimer assembled by transmembrane GXXXG motifs.

The beta-amyloid peptide (Abeta) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer disease. Abeta is produced by the sequential cleavage of the amyloid precursor protein (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. APP contains three Gly-XXX-Gly (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 Abeta40 and Abeta42 secretion. beta-Cleavage of mutant APP was not inhibited, and reduction of Abeta secretion resulted from inhibition of gamma-cleavage. It was anticipated that decreased gamma-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 alpha-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production.

Phosphorylation of APP695 at Thr668 decreases gamma-cleavage and extracellular Abeta.

Phosphorylation of human APP695 at Thr668 seems to be specific to neuronal tissue and could affect Abeta production. Metabolism of APP mutated at Thr668 residue was analyzed in CHO cell line and primary cultures of rat cortical neurons. By site-directed mutagenesis, T668A or T668D substitutions were introduced in wild-type APP695. In CHO cells, wild-type APP695 was very slightly phosphorylated at Thr668 and produced similar levels of extracellular Abeta40 as compared to APPT668A. On the contrary, APPT668D was more efficiently cleaved by beta-secretase. However, accumulated betaCTF were less cleaved by gamma-secretase and less extracellular Abeta40 was produced. Decreased susceptibility to cleavage by gamma-secretase was confirmed upon expression of C99T668D. In neurons, part of APP695 was phosphorylated at Thr668. Following neuronal expression of APPT668A, extracellular Abeta40 production was increased. In conclusion, phosphorylation of human APP695 at Thr668 increases APP beta-cleavage but decreases its gamma-cleavage and extracellular Abeta40 production.

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis.

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.