Matthias Cacquevel

Rapid purification of active γ‐secretase, an intramembrane protease implicated in Alzheimer’s disease

γ‐Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid‐β precursor protein generates the amyloid‐β protein (Aβ) of Alzheimer’s disease, partially inhibiting γ‐secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ‐secretase, but at low resolution (15 Å). The limited amount of purified γ‐secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X‐ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S‐20) that overexpresses strikingly high levels of all four γ‐secretase components (presenilin, nicastrin, Aph‐1 and Pen‐2). We then used these cells to develop a rapid protocol for the high‐grade purification of proteolytically active γ‐secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.

Imaging brain amyloid deposition using grating-based differential phase contrast tomography

One of the core pathological features of Alzheimers disease (AD) is the accumulation of amyloid plaques in the brain. Current efforts of medical imaging research aim at visualizing amyloid plaques in living patients in order to evaluate the progression of the pathology, but also to facilitate the diagnosis of AD at the prodromal stage. In this study, we evaluated the capabilities of a new experimental imaging setup to image amyloid plaques in the brain of a transgenic mouse model of Alzheimers disease. This imaging setup relies on a grating interferometer at a synchrotron X-ray source to measure the differential phase contrast between brain tissue and amyloid plaques. It provides high-resolution images with a large field of view, making it possible to scan an entire mouse brain. Here, we showed that this setup yields sufficient contrast to detect amyloid plaques and to quantify automatically several important structural parameters, such as their size and their regional density in 3D, on the scale of a whole mouse brain. Whilst future developments are required to apply this technique in vivo, this grating-based setup already gives the possibility to perform powerful studies aiming at quantifying the amyloid pathology in mouse models of AD and might accelerate the evaluation of anti-amyloid compounds. In addition, this technique may also facilitate the development of other amyloid imaging methods such as positron emission tomography (PET) by providing convenient high-resolution 3D data of the plaque distribution for multimodal comparison.

Perineuronal net digestion with chondroitinase restores memory in mice with tau pathology

Alzheimers disease is the most prevalent tauopathy and cause of dementia. We investigate the hypothesis that reactivation of plasticity can restore function in the presence of neuronal damage resulting from tauopathy. We investigated two models with tau hyperphosphorylation, aggregation and neurodegeneration: a transgenic mouse model in which the mutant P301S tau is expressed in neurons (Tg P301S), and a model in which an adeno-associated virus expressing P301S tau (AAV-P301S) was injected in the perirhinal cortex, a region critical for object recognition (OR) memory. Both models show profound loss of OR memory despite only 15% neuronal loss in the Tg P301S and 26% in AAV-P301S-injected mice. Recordings from perirhinal cortex slices of 3 month-old P301S transgenic mice showed a diminution in synaptic transmission following temporal stimulation. Chondroitinase ABC (ChABC) can reactivate plasticity and affect memory through actions on perineuronal nets. ChABC was injected into the perirhinal cortex and animals were tested for OR memory 1 week later, demonstrating restoration of OR memory to normal levels. Synaptic transmission indicated by fEPSP amplitude was restored to control levels following ChABC treatment. ChABC did not affect the progression of neurodegenerative tauopathy. These findings suggest that increasing plasticity by manipulation of perineuronal nets offers a novel therapeutic approach to the treatment of memory loss in neurodegenerative disorders.

FOXO3 determines the accumulation of α-synuclein and controls the fate of dopaminergic neurons in the substantia nigra

Parkinsons disease (PD) is characterized by the selective degeneration of neuronal populations presumably due to pathogenic interactions between aging and predisposing factors such as increased levels of α-synuclein. Here, we genetically modulate the activity of the transcription factor Forkhead box protein O3 (FOXO3) in adult nigral dopaminergic neurons using viral vectors and explore how this determinant of longevity impacts on neuronal fate in normal and diseased conditions. We find that dopaminergic neurons are particularly vulnerable to changes in FOXO3 activity in the substantia nigra. While constitutive activation has proapoptotic effects leading to neuronal loss, inhibition of FOXO-mediated transcription by a dominant-negative competitor causes oxidative damage and is detrimental at high vector dose. To address the role of FOXO3 in PD, we modulate its activity in dopaminergic neurons overexpressing human α-synuclein. In this pathogenic condition, we find that FOXO inhibition has protective effects, suggesting that this transcription factor ultimately contributes to neuronal cell death. Nevertheless, mild FOXO3 activity also protects nigral neurons against the accumulation of human α-synuclein, albeit to a lesser extent. FOXO3 reduces the amount of α-synuclein present in the soluble protein fraction and promotes the coalescence of dense proteinase K-resistant aggregates, with an accumulation of autophagic vacuoles containing lipofuscin. Consistent with these in vivo observations, we find that FOXO3 controls autophagic flux in neuronal cells. Altogether, these results point to FOXO3 as an important determinant of neuronal survival in the substantia nigra, which may oppose α-synuclein accumulation and proteotoxicity.

Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes

Background Mutations linked to early onset, familial forms of Alzheimers disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1–42/Aβ1–40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1–42 production, decreasing Aβ1–40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q. Methodology/Principal Findings We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1–40, Aβ1–42 and APP intracellular domain productions in vitro. Conclusion/Significance Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1–42/Aβ1–40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimers disease.

Proton and Phosphorus Magnetic Resonance Spectroscopy of a Mouse Model of Alzheimer's Disease

The development of new diagnostic criteria for Alzheimers disease (AD) requires new in vivo markers reflecting early pathological changes in the brain of patients. Magnetic resonance (MR) spectroscopy has been shown to provide useful information about the biochemical changes occurring in AD brain in vivo. The development of numerous transgenic mouse models of AD has facilitated the evaluation of early biomarkers, allowing researchers to perform longitudinal studies starting before the onset of the pathology. In addition, the recent development of high-field animal scanners enables the measurement of brain metabolites that cannot be reliably quantified at lower magnetic fields. In this report, we studied a new transgenic mouse model of AD, the 5xFAD model, by in vivo proton and phosphorus MR spectroscopy. This model, which is characterized by an early-onset and a robust amyloid pathology, developed changes in the neurochemical profile, which are typical in the human disease, i.e., an increase in myo-inositol and a decrease in N-acetylaspartate concentrations, as early as in the 40th week of age. In addition, a significant decrease in the γ-aminobutyrate concentration was observed in transgenic mice at this age compared to controls. The pseudo-first-order rate constant of the creatine kinase reaction as well as relative concentrations of phosphorus-containing metabolites were not changed significantly in the 36 and 72-week old transgenic mice. Overall, these results suggest that mitochondrial activity in the 5 × FAD mice is not substantially affected but that the model is relevant for studying early biomarkers of AD.

Motifs in the tau protein that control binding to microtubules and aggregation determine pathological effects

Tau pathology is associated with cognitive decline in Alzheimer’s disease, and missense tau mutations cause frontotemporal dementia. Hyperphosphorylation and misfolding of tau are considered critical steps leading to tauopathies. Here, we determine how motifs controlling conformational changes in the microtubule-binding domain determine tau pathology in vivo. Human tau was overexpressed in the adult mouse forebrain to compare variants carrying residues that modulate tau propensity to acquire a β-sheet conformation. The P301S mutation linked to frontotemporal dementia causes tau aggregation and rapidly progressing motor deficits. By comparison, wild-type tau becomes heavily hyperphosphorylated, and induces behavioral impairments that do not progress over time. However, the behavioral defects caused by wild-type tau can be suppressed when β-sheet breaking proline residues are introduced in the microtubule-binding domain of tau. This modification facilitates tau interaction with microtubules, as shown by lower levels of phosphorylation, and by the enhanced protective effects of mutated tau against the severing of the cytoskeleton in neurons exposed to vinblastine. Altogether, motifs that are critical for tau conformation determine interaction with microtubules and subsequent pathological modifications, including phosphorylation and aggregation.

Focal expression of adeno-associated viral-mutant tau induces widespread impairment in an APP mouse model.

Adeno-associated virus serotype 6 (AAV6) viral vectors encoding mutant and normal tau were used to produce focal tau pathology. Two mutant forms of tau were used; the P301S tau mutation is associated with neurofibrillary tangle formation in humans, and the 3PO mutation leads to rapid tau aggregation and is associated with pathogenic phosphorylation and cytotoxicity in vitro. We show that adeno-associated viral injection into entorhinal cortex of normal and tau knockout animals leads to local overexpression of tau, and the presence of human tau in axons projecting to and emanating from the entorhinal cortex. Starting at 2 months and increasing by 6 months post-injection neurons expressing mutant tau developed hyperphosphorylated tau pathology, in addition to dystrophic neurites. There was neuronal loss in tau-expressing regions, which was similar in normal and in TASTPM mice injected with mutant tau. There was neuroinflammation around plaques, and in regions expressing mutant tau. We saw no evidence that mutant tau had affected amyloid-beta pathology or vice versa. Morris water maze behavioral tests demonstrated mild memory impairment attributable to amyloid-beta pathology at 2 and 4 months, with severe impairment at 6 months in animals receiving adeno-associated viral-3PO. Therefore, TASTPM mice injected with mutant tau displayed many of the main features characteristic of human Alzheimers disease patients and might be used as a model to test new drugs to ameliorate clinical features of Alzheimers disease.

Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimers disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Overexpression of tau in the mouse forebrain using adeno-associated virus (AAV) leads to tau hyperphosphorylation, formation of neurofibrillary tangles and neurodegeneration

Background: Tau hyperphosphorylation is a main pathological hallmark of Alzheimer’s disease and other tauopathies.We have previously shown that hypothermia can induce tau hyperphosphorylation both in vitro and in vivo. In this work, we tested the hypothesis that hypothermic conditions could be used to screen for tau kinase inhibitors. Methods: For this purpose, we used different biological models of gradual complexity such as SH-SY5Y neuronal-like cell cultures, ex vivo metabolically active brain slices, as well as adult non-transgenic mice. Results:Our results show that either direct hypothermia on cells and brain slices,or anesthesia-induced hypothermia in vivo led to tau hyperphosphorylation atmultiple epitopes such asAT270(Thr181),tau-1(Ser195/Ser198/Ser199/Ser202), CP13(Ser202), AT8(Ser202/Thr205), AT180(Thr231), MC-6(Ser235), Ser262and PHF-1(Ser396/Ser404).We further show that LiCl and AR-A014418, two Glycogen Synthase Kinase3(GSK-3) inhibitors, as well as roscovitine a cyclin-dependent kinase 5(Cdk5) inhibitor, can partially or totally prevent hypothermia-induced tau hyperphosphorylation. Importantly, the use of either GSK-3 or Cdk5 inhibitors leads to different pattern of tau phosphorylation reduction, specific of the inhibited kinase, thus allowing us to assess kinase inhibitors specificity. Conclusions: Based on these observations, we propose that hypothermia-induced hyperphosphorylation can be used as a reliable, fast, convenient and inexpensive tool to screen for and characterize tau kinase inhibitors.