Bernadette Allinquant

Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.

Elucidation of O-Glycosylation Structures of the beta-Amyloid Precursor Protein by Liquid Chromatography-Mass Spectrometry Using Electron Transfer Dissociation and Collision Induced Dissociation

Accumulation and deposition of beta-amyloid peptide, a major constituent in neuritic plaques are hallmarks of Alzheimers disease (AD) and AD-related neurodegenerative diseases. beta-Amyloid (Abeta) is derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein present in three major isoforms in brain comprising 695, 751 and 770 amino acids, respectively. Among other post-translational modifications, APP is modified during maturation by N- and O-glycosylation, which are thought to be responsible for its expression and secretion. Unlike N-glycosylation, no sites of O-glycosylation of APP have previously been reported. We report here the identification of three specific O-glycosylation sites of the secreted APP695 (sAPP695) produced in CHO cells, using a combination of high-performance liquid chromatography and electrospray-tandem mass spectrometry. With the use of electron transfer dissociation and collision induced dissociation (ETD and CID), we identified type, composition and structures of the Core 1 type O-linked glycans attached at the residues Thr 291, Thr 292 and Thr 576 of the full-length APP695. The glycosylations comprise multiple short glycans, containing N-acetyl galactosamine (GalNAc), Gal-GalNAc and sialic acid terminated structures. The presence of the glycopeptides in the tryptic mixture was identified using the CID-generated sugar oxonium ions. ETD proved to be valuable for the unambiguous identification of the modified sites as ETD fragmentation occurred along the peptide backbone with little or no cleavage of the glycans. Thus, the combination of the CID and ETD techniques in LC-MS is shown here, as a powerful tool for de novo identification of O-glycosylations at unknown modification sites in proteins.

Activity requires soluble amyloid precursor protein α to promote neurite outgrowth in neural stem cell‐derived neurons via activation of the MAPK pathway

It is known that activity modulates neuronal differentiation in the adult brain but the signalling mechanisms underlying this process remain to be identified. We show here that activity requires soluble amyloid precursor protein (sAPP) to enhance neurite outgrowth of young neurons differentiating from neural stem cells. Inhibition of sAPP secretion and anti‐APP antibodies both abolished the effect of depolarization on neurite outgrowth, whereas exogenous sAPPα, similar to depolarization, induced neurite elongation. Depolarization and sAPPα both required active N‐methyl‐d‐aspartic acid receptor (NMDAR) and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) recruitment to induce neurite outgrowth. However, depolarization and sAPPα played different roles in modulating this signalling cascade. Depolarization induced ERK phosphorylation with fast kinetics via activation of NMDAR. By contrast, acute application of sAPPα did not lead to ERK activation. However, continuous generation of sAPPα was necessary for depolarization‐induced ERK phosphorylation, indicating that sAPPα promotes MAPK/ERK recruitment by an indirect mechanism. In addition, we found that blockade of NMDAR down‐regulated APP expression, whereas depolarization increased sAPPα, suggesting that activity may also act upstream of sAPP signalling by regulating the amount of cellular APP and extracellular sAPPα. Finally, we show that soluble amyloid precursor‐like protein 2 (sAPLP2), but not sAPLP1, is functionally redundant to sAPP in promoting neurite outgrowth and that soluble members of the APP family require membrane‐bound APP to enhance neurite outgrowth. In summary, these experiments indicate a novel role of APP family members in activity‐dependent neuronal differentiation.

Secreted Amyloid Precursor Protein β and Secreted Amyloid Precursor Protein α Induce Axon Outgrowth In Vitro through Egr1 Signaling Pathway

Background sAPPα released after α secretase cleavage of Amyloid Precursor Protein (APP) has several functions including the stimulation of neurite outgrowth although detailed morphometric analysis has not been done. Two domains involved in this function have been described and are present in sAPPβ released at the first step of amyloid peptide cleavage, raising the possibility that sAPPβ could also stimulate neurite outgrowth. We investigated the morphological effects of sAPPα and sAPPβ on primary neurons and identified a key signaling event required for the changes observed. Methodology/Principal Findings Final concentrations of 50 to 150 nM bacterial recombinant sAPPα or sAPPβ added to primary neuronal cultures after 1 day in vitro decreased cell adhesion 24 hours later and primary dendrite length 96 hours later. 150 nM sAPPα and sAPPβ induced a similar increase of axon outgrowth, although this increase was already significant at 100 nM sAPPα. These morphological changes induced by sAPPs were also observed when added to differentiated neurons at 5 days in vitro. Real time PCR and immunocytochemistry showed that sAPPα and sAPPβ stimulated Egr1 expression downstream of MAPK/ERK activation. Furthermore, in primary neurons from Egr1 −/− mice, sAPPs affected dendritic length but did not induce any increase of axon length. Conclusion/Significance sAPPα and sAPPβ decrease cell adhesion and increase axon elongation. These morphological changes are similar to what has been observed in response to heparan sulfate. The sAPPα/sAPPβ stimulated increase in axon growth requires Egr1 signaling. These data suggest that sAPPβ is not deleterious per se. Since sAPPβ and sAPPα are present in the embryonic brain, these two APP metabolites might play a role in axon outgrowth during development and in response to brain damage.

Amyloid precursor protein cytoplasmic domain antagonizes reelin neurite outgrowth inhibition of hippocampal neurons.

The function of the amyloid precursor protein (APP), a key molecule in Alzheimers disease (AD) remains unknown. Among the proteins that interact with the APP cytoplasmic domain in vitro and in heterologous systems is Disabled-1, a signaling molecule of the reelin pathway. The physiological consequence of this interaction is unknown. Here we used an in vitro model of hippocampal neurons grown on a reelin substrate that inhibits neurite outgrowth. Our results show that an excess of APP cytoplasmic domain internalized by a cell permeable peptide, is able to antagonize the neurite outgrowth inhibition of reelin. The APP cytoplasmic domain binds Disabled-1 and retains it in the cytoplasm, preventing it from reaching the plasma membrane and sequesters tyrosine phosphorylated Disabled-1, both of which disrupt reelin signaling. In the context of AD, increased formation of APP cytoplasmic domain in the cytosol released after cleavage of the A beta peptide, could then inhibit reelin signaling pathway in the hippocampus and thus influence synaptic plasticity.

Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors

Brain diseases such as autism and Alzheimers disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biological process. However, current techniques are not sensitive enough to detect minor abnormalities. Here we report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). We show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissociated neurons with a spatial resolution of 12u2005nm and a temporal resolution of 50u2005ms. As proof of principle, we applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concentration (∼30%) found in the brains of patients. In both cases, we show that the FND assay is sufficiently sensitive to detect these changes.

Role of cholesterol metabolism in the pathogenesis of Alzheimer's disease.

Purpose of review Cholesterol has been shown to stimulate the cleavage of amyloid precursor protein (APP) into amyloid peptides involved in Alzheimers disease. However, high level of peripheral cholesterol as a risk factor for Alzheimers disease is still debated. This current review provides an update of the recent literature on cholesterol and APP metabolisms in the brain. Recent findings First, a new relationship between neuronal APP and cholesterol has been shown in which this protein controls cholesterol turnover required for neuronal activity. Second, oxysterols are able to stimulate the synthesis of ATP-binding cassette transporters involved in the exchange of amyloid peptides between the blood and the brain. Third, changes in APP targeting to lipid rafts and/or their composition in cholesterol regulate amyloid peptide production. Summary These recent findings open new areas of investigations to control the neuronal activity and to decrease the amyloid peptide levels in brain, opening on new preventive and therapeutic strategies for Alzheimers disease.

sAβPPα Improves Hippocampal NMDA-Dependent Functional Alterations Linked to Healthy Aging

This study shows a decrease in soluble amyloid-β protein precursor-α (sAβPPα) levels, but no change in sAβPPβ, in the rat hippocampus during healthy aging, associated with the weaker expression of N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) in the CA1 area of hippocampal slices. Exogenous application of recombinant sAβPPα increases NMDAR activation in aged animals and could rescue the age-related LTP deficits described. In contrast, it does not affect basal synaptic transmission or glutamate release. These results indicate that improving synaptic sAβPPα availability at synapses helps in reducing the functional NMDAR-related deregulation of hippocampal networks linked to aging.

Differential Epitope Identification of Antibodies Against Intracellular Domains of Alzheimer's Amyloid Precursor Protein Using High Resolution Affinity-mass Spectrometry

Several polypeptides comprising the carboxy-terminal domain of the 1-amyloid precursor protein (cAPP) were prepared by solid phase peptide synthesis, and employed as antigens for the determination of the epitopes recognised by anti-cAPP antibodies. Selective proteolytic epitope-excision and -extraction on the immobilised immune complexes, in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) were used as major methods for epitope identification. The epitope recognised by a polyclonal anti-cAPP antibody (36-BO) was identified as APP(727-737), a sequence close to the APP transmembrane region. In contrast, the epitope recognised by a monoclonal anti-cAPP antibody (Jonas-mAb) was identified at APP(740-747) to be located more remote from the transmembrane region. The two adjacent, yet distinct epitopes recognised by two different antibodies should provide efficient tools for (i), molecular diagnostic applications, and (ii), the study of intracellular processing pathways of APP relevant to Alzheimers disease, utilising suitable mass spectrometric and molecular imaging approaches.

Changing Paradigms in Down Syndrome: The First International Conference of the Trisomy 21 Research Society.

Down syndrome (DS) is the most common genetic cause of intellectual disability (ID) in humans with an incidence of ∼1:1,000 live births worldwide. It is caused by the presence of an extra copy of all or a segment of the long arm of human chromosome 21 (trisomy 21). People with DS present with a constellation of phenotypic alterations involving most organs and organ systems. ID is present in all people with DS, albeit with variable severity. DS is also the most frequent genetic cause of Alzheimers disease (AD), and ∼50% of those with DS will develop AD-related dementia. In the last few years, significant progress has been made in understanding the crucial genotype-phenotype relationships in DS, in identifying the alterations in molecular pathways leading to the various clinical conditions present in DS, and in preclinical evaluations of potential therapies to improve the overall health and well-being of individuals with DS. In June 2015, 230 scientists, advocates, patients, and family members met in Paris for the 1st International Conference of the Trisomy 21 Research Society. Here, we report some of the most relevant presentations that took place during the meeting.