Claudia Marinangeli

Structural features of the KPI domain control APP dimerization, trafficking, and processing.

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz‐type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.—Ben Khalifa, N., Tyteca, D., Marinangeli, C., Depuydt, M., Collet, J.‐F., Courtoy, P. J., Renauld, J.‐C., Constantinescu, S. N., Octave, J.‐N., Kienlen‐Campard, P. Structural features of the KPI domain control APP dimerization, trafficking, and processing. FASEB J. 26, 855–867 (2012). www.fasebj.org

Tauopathy contributes to synaptic and cognitive deficits in a murine model for Alzheimer's disease

Tau alterations are now considered an executor of neuronal demise and cognitive dysfunction in Alzheimers disease (AD). Mouse models combining amyloidosis and tauopathy and their parental counterparts are important tools to further investigate the interplay of abnormal amyloid‐β (Aβ) and Tau species in pathogenesis, synaptic and neuronal dysfunction, and cognitive decline. Here, we crossed APP/PS1 mice with 5 early‐onset familial AD mutations (5xFAD) and TauP301S (PS19) transgenic mice, denoted F+/T+ mice, and phenotypically compared them to their respective parental strains, denoted F+/T– and F–/T+ respectively, as controls. We found dramatically aggravated tauopathy (~10‐fold) in F+/T+ mice compared to the parental F–/T+ mice. In contrast, amyloidosis was unaltered compared to the parental F+/T– mice. Tauopathy was invariably and very robustly aggravated in hippocampal and cortical brain regions. Most important, F+/T+ displayed aggravated cognitive deficits in a hippocampus‐dependent spatial navigation task, compared to the parental F+/T– strain, while parental F–/T+ mice did not display cognitive impairment. Basal synaptic transmission was impaired in F+/T+ mice compared to nontransgenic mice and the parental strains (≥40%). Finally, F+/T+ mice displayed a significant hippocampal atrophy (~20%) compared to nontransgenic mice, in contrast to the parental strains. Our data indicate for the first time that pathological Aβ species (or APP/PS1) induced changes in Tau contribute to cognitive deficits correlating with synaptic deficits and hippocampal atrophy in an AD model. Our data lend support to the amyloid cascade hypothesis with a role of pathological Aβ species as initiator and pathological Tau species as executor.—Stancu, I.‐C., Ris, L., Vasconcelos, B., Marinangeli, C., Goeminne, L., Laporte, V., Haylani, L. E., Couturier, J., Schakman, O., Gailly, P., Pierrot, N., Kienlen‐Campard, P., Octave, J.‐N., Dewachter, I. Tauopathy contributes to synaptic and cognitive deficits in a murine model for Alzheimers disease. FASEB J. 28, 2620–2631 (2014). www.fasebj.org

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

ETHNOPHARMACOLOGICAL RELEVANCE The aqueous decoction of Pterocarpus erinaceus has been traditionally used in Benin against memory troubles. AIM OF THE STUDY New 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). METHODS AND RESULTS We 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. CONCLUSION Pterocarpus erinaceus kino extract appears therefore as a new γ-secretase inhibitor selective towards APP processing.

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

Background: Presenilin TMD8 contains a conserved AXXXAXXXG motif, involved in transmembrane protein interactions. Results: Mutation of PS AXXXAXXXG motifs strongly impacts γ-secretase activity. Conclusion: The geometry and activity of γ-secretase depend on the integrity of the conserved AXXXAXXXG motifs in TMD8. Significance: The PS AXXXAXXXG motif is a key determinant for understanding the structure, assembly, and function 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 Aβ 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.

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.

Assessing Motor Outcome and Functional Recovery Following Nerve Injury

Behavioral tests significantly contribute to our understanding of nerve function after experimental lesions and/or therapeutic intervention. In particular, the rat sciatic nerve has proven to be a valuable animal model to study nerve injury and repair. Here, we describe how to optimally use the commercially available CatWalk system to obtain a detailed and objective analysis of dynamic and static gait parameters.

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.