PROJECTS SUPPORTED FOR 2014

Ecole Pratique des Hautes Etudes, Bordeaux – Bordeaux

Brain Topology of Alzheimer disease presymptomatic phase

SUMMARY OF PROPOSED RESEARCH

It is now admitted that AD is a long run disease, with a long presymptomatic phase. The application of disease-modifying therapy at this very late phase of the disease could partly explain its inefficiency. That is why studies on AD are now focusing on the presymptomatic phase of the disease. Retrospective studies with follow-up covering decades are the only way to study the real presymptomatic phase by describing AD incident cases. Based on these studies, AD subjects present cognitive impairment several years before clinical diagnosis. Moreover, retrospective neuroimaging studies on AD converters subjects highlight the presence of morphological modifications occurring at the level of temporo-parietal regions at least 5-7 years before diagnosis. Whereas, morphological data are now available several years before AD diagnosis, for more recent MRI methodology such as rest fMRI and DTI data no such hindsight are available. However, recent studies indicate that modifications of functional and structural networks are present on cognitively normal subjects at risk for AD. Finally, the link between modifications of the connectome and the emergence of cognitive symptoms is not yet fully understood.

Our project will consider structural (through morphological MRI and diffusion MRI) and functional (through rest fMRI) networks modifications underlying cognitive decline in elderly subjects. Moreover, most previous studies described separately structural and functional networks, whereas no one could ignore the constraint of one on the other. A recent framework based on graph theory could facilitate the exploration of the interaction between structural and functional modifications. We will use here this methodology to first describe separately structural and functional modifications of the topological connectome and to finally integrate these different modalities in one statistical famework. This project is based on two large cohorts of elderly subjects (3City and AMImage studies) with 6 to 10 years follow-up. Based on this multiple time point cognitive assessment, episodic memory and executive function decline will be evaluated for each subject allowing excluding false impaired subjects frequently included in cross-sectional analysis. The results of this project should give some information on the link between cerebral modifications and the emergence of cognitive symptoms in aging subjects, which characterize the presymptomatic phase of AD.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

The characterization of brain network modifications at a functional and morphological level of cognitively decliner elderly would increase knowledge on the pathophysiology of presymptomatic phase of AD which could constitute the suitable temporal window to apply pharmacological treatment or to start preventive therapy.

January 1st, 2015 – December 31th, 2017 (3 years)

100 000 €

Centre de Recherche de l’ICM, INSERM, Paris – Paris

Endo-lysosomal dysfunctions in Alzheimer’s disease

SUMMARY OF PROPOSED RESEARCH

The endo-lysosomal compartment plays a major role in Alzheimer’s disease (AD). Indeed, 1) APP is processed along this route; 2) Risk factors for AD are key players in endocytosis (BIN1, PICALM, SORL1); 3) We and others have shown that cholesterol increases such as that one detected in AD brains promote APP endocytosis and Aß production; 4) Neurons bearing apparently enlarged endosomes have been described as the earliest morphological change observed in AD and Down syndrome (DS) before the clinical symptoms (Cataldo et al., 2000). Individuals with DS are at high risk for AD (45% prevalence at 60 years against around 3% in the general population).

However, our preliminary data acquired with electron microscopy (EM) and super-resolution microscopy (STED) show endosome aggregates rather than enlarged endosomes both in AD and DS cellular models, suggesting local clustering of endosomes. However, both EM and STED microscopies only provide still images of fixed cells. Our project aims at understanding the mechanisms underlying aggregate formation (excess of endosomes, altered trafficking or a deficit in homotypic fusion) by directly imaging the dynamics of endosome aggregation using a recently developed full-field multi-color super-resolution microscopy allowing imaging of individual organelles at the 100-nm scale in live cells (Brunstein et al., 2013).

We will first study endosome morphology using EM and STED microscopy and their dynamics by live super-resolved microscopy in neurons and glial cells (astrocytes and microglia) treated with cholesterol that recapitulate several phenotypes of sporadic AD (increased APP endocytosis and Aß production, endosomal dysmorphology, reduced APP vesicular transport in neurons (Marquer et al. in revision)) as well as fibroblasts, lymphoblastoids, iPS cells and derived neurons from individuals with DS. In parallel, we will analyze the morphology of endosomal compartments in mouse models of AD and DS and postmortem brain samples using EM. We will test the involvement of the cytoskeleton in endo-lysosomal dysfunctions, drawing on what was observed at the immunological synapse during antigen presentation. Finally, we will test molecules interacting with actin and tubulin networks, and key proteins involved in the interaction between the endosomes and the cytoskeleton.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Endo-lysosomal morphological changes appear as the earliest hallmark observed in postmortem brains from AD patients and individuals with DS. The current hypothesis suggests a defect in endosome fusion leading to enlarged endosomes containing high levels of Aß. In cellular models of AD and DS, using high resolution microscopies, we found that endosomes are not enlarged but rather clustered, forming aggregates of normally-sized endosomes. Our project aims at providing relevant and comprehensive data necessary for deciphering the underlying mechanism(s) leading to abnormal endosome aggregation, while redirecting the search for potential drug targets. Access to super-resolved live imaging through collaboration will bring original findings to this field.

January 1st, 2015 – December 31th, 2017 (3 years)

100 000 €

INSERM – Lille

Alternative splicing in Alzheimer and Parkinson’s

SUMMARY OF PROPOSED RESEARCH

Recently, next-generation sequencing (RNAseq) and proteome studies suggest that errors in alternative splicing, an essential post-translational process which allows multiple mRNA and protein isoforms from the same transcript, contributes to protein aggregation in Alzheimer’s disease (AD) and Pakinson’s disease (PD). More specifically, a direct association between accumulation of the splicing factor U1 small nuclear ribonucleoprotein (U1 snRNP) and cytoplasmic tangle-like inclusions and dysfunctional amyloid processing has been observed in AD. Recent transcriptome studies from our labs and others also provided support for deregulation of splicing variants, for example genes involved in the Eukaryotic Initiation Factor 2 (EIF2) pathways, in protein aggregation and neuronal death in AD and PD. In the proposed project, we aim to understand the relevance of alternative RNA splicing and translation in sporadic AD and PD pathogenesis and the association with cognitive decline. We hypothesize that deregulation of spliceosome and EIF2 signaling appear at early stages in AD and PD and contribute to protein aggregation, neurodegeneration and dementia in these disorders.

Using RNAseq and microarray analysis, we aim 1) define the frequency of U1 snRNP and EIF2 signaling deregulation in postmortem brain tissue samples and peripheral blood mononuclear cells (PBMC) of well-characterized early stage sporadic AD and PD patients and 2) identify key disturbed regulators of alternative splicing in early and late stage AD and PD. Using qPCR, western blotting techniques and immunohistochemistry, we will investigate 3) the deregulation of identified transcripts in independent sample sets and protein isoforms and their association with protein aggregation and cognitive decline in AD and PD. The studies in brain and PBMC samples of AD and PD patients will be complemented 4) by functional evaluation of deregulated transcripts in two other model systems, SH-SYHY human cell line and corresponding transgenic mouse models (e.g. MAPT and SNCA). We expect to generate lists of spliced transcripts common to AD and PD pathologies and knowledge regarding the biological consequences of alternative splicing in AD and PD. The combined expertise of the cross-border partners VUmc and Univ Lille will provide a strong novel contribution to AD and PD biomedical research by defining if transcriptome deregulations can be shared between brain and peripheral compartments.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Understanding the molecular mechanisms elicited by different risk factors of AD is mandatory to advance beyond the current insufficient tools for diagnosing AD and therapy. Although recent indications that alternative splicing plays a crucial role in AD and PD pathology, little concrete information is known. In this project we will tackle this problem by studying the frequency and biological consequences of aberrant splicing in AD and PD patient postmortem brain tissue and PBMC and model systems. The data generated in this project may form the basis for identification of errors in the translation of gene activity in AD and related disorders, which may serve as diagnostic biomarkers or novel drug targets for early intervention studies to delay the progression of pathology.

January 1st, 2015 – December 31th, 2017 (3 years)

100 000 € (et 50 000 € versés par ISAO)

Collège de France, INSERM, Paris – Paris

Targeting astroglial hemichannels in Alzheimer disease

SUMMARY OF PROPOSED RESEARCH

Our project aims at understanding the contribution of astroglial connexins (Cxs), membrane proteins forming gap junction channels and hemichannels, to neuronal dysfunction and disease progression in a murine model of Alzheimer’s disease (AD), the APP_swe/PS1_dE9 mouse. The expression of Cxs, a characteristic feature of astrocytes, is modified in AD patients and in APP_swe/PS1_dE9 mice in the vicinity of Aβ plaques. Their hemichannel function is chronically activated in mice bearing Aβplaques leading to gliotransmitter (ATP, glutamate and D-serine) release that can affect neuronal functions and survival. We thus hypothesize that a pathological dialogue between neurons and astrocytes, linked to gliotransmitter release through activated astroglial hemichannels, contributes to the neuropathology in particular near amyloid plaques. Thus, we propose to block this pathological process by suppressing Cx43 hemichannel activity in APP_swe/PS1_dE9 mice using two complementary strategies: a genetic approach with the astroglial-targeted knocking-out of Cx43 gene in APP_swe/PS1_dE9 mice and a pharmacological strategy consisting in the chronic oral delivery of an hemichannel blocker that crosses the blood brain barrier. We will analyze whether such hemichannel silencing will reduce the amyloid pathology and improve neuronal function and behavior. In both models, we will examine: i) the Aβ load, the extent of reactive gliosis, the recruitment and activation of microglial cells as well as the neuronal damages by immunohistochemical and biochemical approaches; ii) the neuronal activity and plasticity (LTP) by electrophysiological recordings in hippocampal acute slices and iii) the behavioral performances tested in memory tasks using Morris water maze and star-maze. Thus, our project should lead to the identification of novel potential non-neuronal targets to reduce the neuronal alterations associated to the amyloid pathology.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Our project concerns AD in the framework of a neuroprotective strategy that targets glial cells. By blocking connexin hemichannels that are chronically activated in a murine model of AD and that release neurotoxic gliotransmitters, we expect to reduce their deleterious effects on neuronal integrity, synaptic activity and behavioral deficits. Its completion, in particular by treating the selected AD model mouse with a blood brain barrier permeant hemichannel blocker, will determine whether glial connexins represent therapeutical targets to improve neuronal functions in AD.

January 1st, 2015 – December 31th, 2016 (2 years)

100 000€

Université Aix-Marseille – Marseille

MT5-MMP is a new target in Alzheimer’s disease

SUMMARY OF PROPOSED RESEARCH

Objective: Our global objective is to better understand some of the proteolytic pathways involved in Alzheimer’s disease (AD) pathogenesis. Consistent preliminary data indicate that membrane type 5-matrix metalloproteinase (MT5-MMP) controls -secretase activity and/or the bioavailability of its substrate the amyloid precursor protein (APP), and the accumulation of the amyloid beta peptide (Aβ) during the prodromal-like phase of the pathology in the 5xFAD mouse model of AD.

Hypothesis: MT5-MMP is a new important pro-amyloidogenic factor in Alzheimer’s pathogenesis and the modulation of its activity and/or interactions with other proteins may alleviate pathology and cognitive decline in AD.

Experimental design and methods. The following specific aims will constitute the backbone of the research project:

Aim 1. To study the impact of MT5-MMP deficiency on the progression of amyloid pathology in bigenic 5xFAD/MT5-MMP^-/- (TgMT5^-/-) mice compared to 5xFAD (Tg) mice. Biochemical and histopathological analyses on Aβ and APP metabolism will be performed at different stages of the pathology (pre-symptomatic, prodromal-like, moderate symptomatic and severe symptomatic). At symptomatic stages of the pathology we will evaluate potential long-term neuroprotective effects of MT5-MMP deficiency by assessing neuronal loss, and associative and working memory.

Aim 2. To study the impact of MT5-MMP deficiency on neuroinflammation in TgMT5^-/- mice compared to Tg mice. Since MT5-MMP potentiates the inflammatory response to IL-1β in the PNS, we will inject this cytokine in the brain to evaluate a possible impairment of its signalling in the CNS of TgMT5^-/- mice. To address this question, we will measure by qPCR the expression of M1 (“pro-inflammatory”) and M2 (“anti-inflammatory”) genes that characterize classical and alternative microglial activation. The specificity of gene changes will be confirmed at the protein level.

Aim 3. The functional interactions of MT5-MMP with key elements of the amyloidogenic cascade. We will use heterologous cell systems, cell biology, biochemical and gain/loss of MT5-MMP function approaches to characterize a possible tri-molecular complex formed by MT5-MMP/APP/and -secretase, which may underpin the pro-amyloidogenic role of MT5-MMP.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Although the proteolytic processes that govern Aβ production/accumulation are among the most promising targets against AD pathogenesis, many aspects of their functioning remain still elusive, therefore hampering the chances of developing efficient anti-amyloid therapies without side effects. Our studies are aimed at better understanding the mechanisms of action involved in the metabolism of APP/Aβ under the prism of MT5-MMP. This proteinase, with emerging roles in pathophysiology and barely studied in AD, appears to modulate APP trafficking and/or -secretase activity, without impacting on the physiological activity of the latter. We expect that the proposed research project will help: i) to better understand fundamental mechanisms of the amyloid cascade and ii) to ultimately develop MT5-MMP modulating agents that could alleviate amyloid pathology in AD.

January 1st, 2015 – December 31th, 2017 (3 years)

100 000€

INSERM – Lille

A2A receptor modulation in APP transgenic mice

SUMMARY OF PROPOSED RESEARCH

Adenosine A_2A receptors (A_2ARs) are G-protein-coupled receptors found upregulated in the post-mortem brains of AD patients. Impact of this upsurge remains ill-defined but several lines of evidence support that such A_2AR dysfunction contributes to AD physiopathology. Indeed: 1) Consumption of caffeine, a non-selective A_2AR antagonist, reduces AD risk and improves the phenotype in APP and Tau (our previous LECMA/AFI grant) transgenic mice; 2) Selective A_2AR blockade is beneficial in a Tau transgenic mouse model from the memory, synaptic and pathological points-of-view (our previous LECMA/AFI grant). Additionally, some of our preliminary data suggest that A_2AR blockade also improves memory in APP mice; 3) Forebrain neuronal A_2AR upsurge leads to memory deficits in rats. However so far, to which extent A_2AR modulation impacts amyloïdogenesis and related impairments is not known. The present project thus aims to get a comprehensive insight on the impact of neuronal A_2AR dysregulation in AD regarding cardinal symptoms and amyloidogenesis as well as to establish a basis for A_2A-based disease-modifying therapeutic intervention.

To achieve these aims, we will establish: 1) the effects of neuronal A_2ARs upsurge in APP/PS1dE9 mice towards amyloidogenesis processes and associated memory/neuro-inflammatory alterations, thanks to a new and original in-house developed transgenic (“Tet-Off”) mouse model and 2) establish the impact of A_2AR blockade in APP/PS1dE9 mice using the specific water-soluble antagonist MSX-3, found to be beneficial in Tau transgenic animals (our previous LECMA/AFI grant). We anticipate that increased A_2AR expression drives an early onset or induces a potentiation of the pathological features in APP/PS1 transgenic mice and that MSX-3 treatment blocking A_2AR will be protective.

Our project is original as the impact of A_2A modulation in the context of amyloidogenesis remains unknown. In our previous LECMA/AFI grant, we demonstrated a beneficial impact of A_2AR blockade in Tau mice (see report). Demonstrating relationships between A_2AR, amyloidogenesis and related detrimental processes may prove the high pathophysiological importance of A_2AR dysregulation in AD and support the clinical development of A_2AR antagonists as therapeutic candidates for AD.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Epidemiological studies report that caffeine, a non-selective A_2AR antagonist, reduces AD risk. We recently demonstrated that A_2AR blockade improves Tau mouse phenotype from the memory to the pathological point-of-views. However, impact of A_2AR modulation towards amyloidogenesis and associated impairments is far from being understood. Therefore, demonstrating detrimental impact of A_2AR dysregulation as well as the protective effects of their blockade in a mouse model of amyloidogenesis will push forward A_2AR-based trials in AD patients. Given the existence of A_2AR-targeting drugs that are already clinically tested, our project has a strong translational value and is an important basis to develop tolerability studies and clinical trials in AD patients in a reasonable time-window.

January 1st, 2015 – December 31th 2016 (2 years)

70 000 € (et 30 000 € versés par AFI)