The adipocyte differentiation protein APMAP is an endogenous suppressor of Aβ production in the brain

The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic syste

Calcium-dependent dynamics of cadherin interactions at cell–cell junctions

Cadherins play a key role in the dynamics of cell–cell contact formation and remodeling of junctions and tissues. Cadherin–cadherin interactions are gated by extracellular Ca^(2+), which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (τ = 1.17 ± 0.06 s^(−1)) upon chelation of extracellular Ca^(2+). A cadherin mutant with reduced adhesive activity (W2A) exhibited a faster, more substantial loss of homophilic interactions (τ = 0.86 ± 0.02 s^(−1)), suggesting two types of native cadherin interactions—one that is rapidly modulated by changes in extracellular Ca^(2+) and another with relatively stable adhesive activity that is Ca^(2+) independent. The Ca^(2+)-sensitive dynamics of cadherin interactions were transmitted to the cell interior where β-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction

The adipocyte differentiation protein APMAP is an endogenous suppressor of Aβ production in the brain

The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system

Alzheimer's disease mutations in APP but not γ-secretase modulators affect epsilon-cleavage-dependent AICD production

Pathological amino-acid substitutions in the amyloid precursor protein (APP) and chemical gamma-secretase modulators affect the processing of APP by the gamma-secretase complex and the production of the amyloid-beta peptide A beta 42, the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early-onset familial Alzheimer's disease affect both gamma- and epsilon-cleavage sites, by raising the A beta 42/40 ratio and inhibiting the production of AICD50-99, one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to gamma- secretase modulators, which shift A beta 42 production towards the shorter A beta 38, but unequivocally spare the epsilon-site and APP- and Notch-ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its gamma- site, modifying at the same time, the solvation of the epsilon-site

Anti-nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells

The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic

Mercury is a direct and potent γ-secretase inhibitor affecting Notch processing and development in Drosophila

Prenatal exposure to mercury causes neurodevelopmental disorders and neurological pathologies in infants, such as microcephaly and mental retardation. Despite critical importance, the molecular interactions leading to mercury toxicity are yet to be elucidated. We first used a cell-free assay to investigate mercury effects on purified γ-secretase activity. Next, we treated adult Drosophila melanogaster with mercury and collected control and mercury-treated embryos, which were subjected to mild hypotonic protein extraction, or immunostained to reveal nervous system morphology. Embryos left to develop into adults were examined for wing phenotypes. Relative to control metals, we found that mercury strongly inhibits in vitro γ-secretase processing of both amyloid-β precursor protein (APP) and Notch. Mercury inhibited APP and Notch cleavage in a dose-dependent manner, with IC(50) values of 50-125 nM, and is therefore comparable in potency to benchmark γ-secretase inhibitors. Immunoblot analysis of embryonic protein extracts showed that mercury inhibits Notch cleavage by γ-secretase in vivo. This is accompanied by severe neurodevelopmental abnormalities in embryos and adult wing-notching phenotypes. Our findings provide first evidence that mercury is a direct and potent γ-secretase inhibitor and suggest that inhibition of γ-secretase and disruption of the Notch developmental pathway potentially contribute to mercury-induced toxicity in the nervous system