Serena Stanga

Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing

Alzheimers disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β‐amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full‐length APP dimerizes more than its α and β C‐terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ‐cleavage site. We show that both non‐familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C‐terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non‐amyloidogenic processing.

Characterization of Pterocarpus erinaceus kino extract and its gamma-secretase inhibitory properties

ETHNOPHARMACOLOGICAL RELEVANCEnThe aqueous decoction of Pterocarpus erinaceus has been traditionally used in Benin against memory troubles.nnnAIM OF THE STUDYnNew strategies are needed against Alzheimer׳s disease (AD), for, to date, AD treatment is symptomatic and consists in drugs treating the cognitive decline. An interesting target is the β-amyloid peptide (Aβ), whose accumulation and progressive deposition into amyloid plaques are key events in AD aetiology. Identifying new and more selective γ-secretase inhibitors or modulators (none of the existing has proven so far to be selective or fully efficient) appears in this respect of particular interest. We studied the activity and mechanisms of action of Pterocarpus erinaceus kino aqueous extract, after the removal of catechic tannins (KAST).nnnMETHODS AND RESULTSnWe tested KAST at non-toxic concentrations on cells expressing the human Amyloid Precursor Protein (APP695), as well as on primary neurons. Pterocarpus erinaceus extract was found to inhibit Aβ release in both models. We further showed that KAST inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that KAST is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit the cleavage of Notch, another γ-secretase substrate responsible for major detrimental side effects observed with γ-secretase inhibitors. Epicatechin was further identified in KAST by HPLC-MS.nnnCONCLUSIONnPterocarpus erinaceus kino extract appears therefore as a new γ-secretase inhibitor selective towards APP processing.

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

Besides its crucial role in the pathogenesis of Alzheimers 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. FASEB J. 30, 1696–1711 (2016). www.fasebj.org

Gamma-Secretase Inhibitor Activity of a Pterocarpus erinaceus Extract

Background: Accumulation of β-amyloid peptides (Aβ) and its progressive deposition into amyloid plaques are key events in the aetiology of Alzheimers disease (AD). To date, AD treatment is symptomatic and consists of drugs treating the cognitive decline. Objective: Identifying molecules specifically targeting Aβ production or aggregation represents a huge challenge in the development of specific AD treatments. Several molecules reported as γ-secretase inhibitors or modulators have been evaluated, but so far none of them have proven to be selective or fully efficient. We have previously investigated the potential interest of plant extracts and we reported that Pterocarpus erinaceus stem-bark extract was active on Aβ release. Our aim here was to characterize the mechanisms by which this extract reduces Aβ levels. Methods: We tested P. erinaceus extract at non-toxic concentrations on cells expressing the human amyloid precursor protein (APP695) or its amyloidogenic β-cleaved C-terminal fragment (C99), as well as on neuronal cell lines. P. erinaceus extract was found to inhibit Aβ release. We further showed that this extract inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that P. erinaceus extract is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit γ-secretase-dependent Notch intracellular domain release. Conclusion: P. erinaceus extract appears as a new potent γ-secretase inhibitor selective towards APP processing.

Glycines from the APP GXXXG/GXXXA Transmembrane Motifs Promote Formation of Pathogenic Aβ Oligomers in Cells

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G29XXXG33 and G38XXXA42 motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.

Presenilin 2-Dependent Maintenance of Mitochondrial Oxidative Capacity and Morphology

Mitochondrial dysfunction plays a pivotal role in the progression of Alzheimers disease (AD), and yet the mechanisms underlying the impairment of mitochondrial function in AD remain elusive. Recent evidence suggested a role for Presenilins (PS1 or PS2) in mitochondrial function. Mutations of PSs, the catalytic subunits of the γ-secretase complex, are responsible for the majority of inherited AD cases (FAD). PSs were shown to be present in mitochondria and particularly enriched in mitochondria-associated membranes (MAM), where PS2 is involved in the calcium shuttling between mitochondria and the endoplasmic reticulum (ER). We investigated the precise contribution of PS1 and PS2 to the bioenergetics of the cell and to mitochondrial morphology in cell lines derived from wild type (PS+/+), PS1/2 double knock-out (PSdKO), PS2KO and PS1KO embryos. Our results showed a significant impairment in the respiratory capacity of PSdKO and PS2KO cells with reduction of basal oxygen consumption, oxygen utilization dedicated to ATP production and spare respiratory capacity. In line with these functional defects, we found a decrease in the expression of subunits responsible for mitochondrial oxidative phosphorylation (OXPHOS) associated with an altered morphology of the mitochondrial cristae. This OXPHOS disruption was accompanied by a reduction of the NAD+/NADH ratio. Still, neither ADP/ATP ratio nor mitochondrial membrane potential (ΔΨ) were affected, suggesting the existence of a compensatory mechanism for energetic balance. We observed indeed an increase in glycolytic flux in PSdKO and PS2KO cells. All these effects were truly dependent on PS2 since its stable re-expression in a PS2KO background led to a complete restoration of the parameters impaired in the absence of PS2. Our data clearly demonstrate here the crucial role of PS2 in mitochondrial function and cellular bioenergetics, pointing toward its peculiar role in the formation and integrity of the electron transport chain.

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 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.

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 Alzheimers 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.