The transcription factor EB reduces the intraneuronal accumulation of the beta-secretase-derived APP fragment C99 in cellular and mouse Alzheimer’s disease models

Brains that are affected by Alzheimer’s disease (AD) are characterized by the overload of extracellular amyloid β (Aβ) peptides, but recent data from cellular and animal models propose that Aβ deposition is preceded by intraneuronal accumulation of the direct precursor of Aβ, C99. These studies indicate that C99 accumulation firstly occurs within endosomal and lysosomal compartments and that it contributes to early-stage AD-related endosomal-lysosomal-autophagic defects. Our previous work also suggests that C99 accumulation itself could be a consequence of defective lysosomal-autophagic degradation. Thus, in the present study, we analyzed the influence of the overexpression of the transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, on C99 accumulation occurring in both AD cellular models and in the triple-transgenic mouse model (3xTgAD). In the in vivo experiments, TFEB overexpression was induced via adeno-associated viruses (AAVs), which were injected either into the cerebral ventricles of newborn mice or administrated at later stages (3 months of age) by stereotaxic injection into the subiculum. In both cells and the 3xTgAD mouse model, exogenous TFEB strongly reduced C99 load and concomitantly increased the levels of many lysosomal and autophagic proteins, including cathepsins, key proteases involved in C99 degradation. Our data indicate that TFEB activation is a relevant strategy to prevent the accumulation of this early neurotoxic catabolite

Le complexe g-sécrétase (implications dans la régulation de l'apoptose et la dégradation du peptide amyloïde)

L'activité g-secretase dépendante des présénilines est impliquée dans la production du peptide A-beta et nécessite la formation d'un complexe de haut poids moléculaire contenant au moins 4 protéines partenaires : la préséniline 1 ou 2, la nicastrine, Aph1 et Pen2. Au cours de ma thèse j'ai pu montré une implication de ce complexe gamma-sécrétase dépendant des présénilines dans la régulation de l'apoptose et la dégradation du peptide A-beta. L'implication des présénilines dans les processus de mort cellulaire a déjà été décrite dans la littérature, la présénilines 2 joue un rôle pro-apoptotique alors que la préséniline 1 semble protéger les cellules de l'apoptose. Nos expériences montrent pour la première fois, un phénotype anti-apoptotique de la nicastrine qui est associé à une baisse de l'expression et de l'activité transcriptionnelle de p53. Nous avons également mis en évidence une composante de cette réponse indépendante de p53 puisqu'en absence de p53, la nicastrine présente toujours un phénotype antiapoptotique. Par ailleurs, nous avons établi une fonction du complexe gamma-sécrétase dépendant des présénilines dans la régulation de la dégradation du peptide A-beta. En effet, l'activité gamma -secretase dépendante des présénilines libère en plus du peptide A-beta, sa contrepartie cytosolique, le fragment AICD qui est transloqué au noyau. Nous avons montré pour la première fois, que ce fragment AICD pouvait réguler la transcription de la néprilysine, un enzyme de dégradation du peptide A-beta.NICE-BU Sciences (060882101) / SudocSudocFranceF

γ-Secretase-mediated regulation of neprilysin: influence of cell density and aging and modulation by imatinib.

International audienceProteolytic degradation has emerged as a key pathway involved in controlling levels of the Alzheimer's disease (AD)-associated amyloid-β peptides (Aβ) in the brain. The ectopeptidase, neprilysin (NEP), has been reported as the major Aβ-degrading enzyme in mice and human brains. We have previously shown that NEP expression and activity are regulated by AICD, the intracellular domain of the amyloid-β protein precursor (AβPP) generated by γ-secretase. Thus, NEP transcription, expression, and enzymatic activity are dramatically reduced in fibroblasts devoid of AβPP (the precursor of AICD) or lacking both presenilin (PS) 1 and 2 (two parent proteins contributing to AICD formation). We demonstrate here that NEP expression and activity are influenced by a number of cell passages and density, and we confirm a drastic reduction of NEP expression and activity in AβPP and PS null fibroblasts examined at similar passages and cell densities. Furthermore, Imatinib (Gleevec), a known tyrosine kinase inhibitor was recently shown to elevate AICD in H4 human neuroglioma cells, and this was accompanied by concomitant increases of NEP protein, mRNA levels, and activity. However, the demonstration of a causal link between Imatinib and AICD levels was still lacking. We show here an Imatinib-dependent effect on NEP expression and activity in murine fibroblasts and establish that Imatinib-induced modulation of NEP was abolished by the depletion of AβPP or its homologues APLP1 and APLP2, thereby confirming that Imatinib-mediated control of NEP could indeed be accounted for its effect on AICD

Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy-like behavior, and spatial learning deficits in 3×TgAD and 2×TgAD mice

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TMP21 Transmembrane Domain Regulates γ-Secretase Cleavage*

TMP21 has been shown to be associated with the γ-secretase complex and can specifically regulate γ-cleavage without affecting ϵ-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of γ-secretase activity was investigated independent of ϵ-cleavage using an amyloid-β precursor proteinϵ (APPϵ) construct which lacks the amyloid intracellular domain domain. The APPϵ construct behaves similarly to the full-length precursor protein with respect to α- and β-cleavages and is able to undergo normal γ-processing. Co-expression of APPϵ and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Aβ-positive fragments, consistent with an inhibition of γ-secretase cleavage. The APPϵ system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the γ-secretase complex and could decrease γ-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Aβ production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased γ-cleavage activity

Intraneuronal aggregation of the β-CTF fragment of APP (C99) induces Aβ-independent lysosomal-autophagic pathology

International audienceEndosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers's disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the β-secretase-derived βAPP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was Aβ independent and was even exacerbated when γ-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer's disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with γ-secretase inhibitor-directed therapies

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

International audienceNicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes

β-Amyloid Precursor Protein Intracellular Domain Controls Mitochondrial Function by Modulating Phosphatase and Tensin Homolog–Induced Kinase 1 Transcription in Cells and in Alzheimer Mice Models

International audienceBACKGROUND: Mitophagy and mitochondrial dynamics alterations are two major hallmarks of neurodegenerative diseases. Dysfunctional mitochondria accumulate in Alzheimer's disease-affected brains by yet unexplained mechanisms. METHODS: We combined cell biology, molecular biology, and pharmacological approaches to unravel a novel molecular pathway by which presenilins control phosphatase and tensin homolog-induced kinase 1 (Pink-1) expression and transcription. In vivo approaches were carried out on various transgenic and knockout animals as well as in adeno-associated virus-infected mice. Functional readout and mitochondrial physiology (mitochondrial potential) were assessed by combined procedures including flow cytometry, live imaging analysis, and immunohistochemistry. RESULTS: We show that presenilins 1 and 2 trigger opposite effects on promoter transactivation, messenger RNA, and protein expression of Pink-1. This control is linked to g-secretase activity and b-amyloid precursor protein but is independent of phosphatase and tensin homolog. We show that amyloid precursor protein intracellular domain (AICD) accounts for presenilin-dependent phenotype and upregulates Pink-1 transactivation in cells as well as in vivo in a Forkhead box O3a-dependent manner. Interestingly, the modulation of g-secretase activity or AICD expression affects Pink-1-related control of mitophagy and mitochondrial dynamics. Finally, we show that parkin acts upstream of presenilins to control Pink-1 promoter transactivation and protein expression. CONCLUSIONS: Overall, we delineate a molecular cascade presenilins-AICD-Forkhead box O3a linking parkin to Pink-1. Our study demonstrates AICD-mediated Pink-1-dependent control of mitochondrial physiology by presenilins. Furthermore, it unravels a parkin-Pink-1 feedback loop controlling mitochondrial physiology that could be disrupted in neurodegenerative conditions