Circadian and Brain State Modulation of Network Hyperexcitability in Alzheimer’s Disease

Abstract Network hyperexcitability is a feature of Alzheimer’ disease (AD) as well as numerous transgenic mouse models of AD. While hyperexcitability in AD patients and AD animal models share certain features, the mechanistic overlap remains to be established. We aimed to identify features of network hyperexcitability in AD models that can be related to epileptiform activity signatures in AD patients. We studied network hyperexcitability in mice expressing amyloid precursor protein (APP) with mutations that cause familial AD, and compared a transgenic model that overexpresses human APP (hAPP) (J20), to a knock-in model expressing APP at physiological levels (APPNL/F). We recorded continuous long-term electrocorticogram (ECoG) activity from mice, and studied modulation by circadian cycle, behavioral, and brain state. We report that while J20s exhibit frequent interictal spikes (IISs), APPNL/F mice do not. In J20 mice, IISs were most prevalent during daylight hours and the circadian modulation was associated with sleep. Further analysis of brain state revealed that IIS in J20s are associated with features of rapid eye movement (REM) sleep. We found no evidence of cholinergic changes that may contribute to IIS-circadian coupling in J20s. In contrast to J20s, intracranial recordings capturing IIS in AD patients demonstrated frequent IIS in non-REM (NREM) sleep. The salient differences in sleep-stage coupling of IIS in APP overexpressing mice and AD patients suggests that different mechanisms may underlie network hyperexcitability in mice and humans. We posit that sleep-stage coupling of IIS should be an important consideration in identifying mouse AD models that most closely recapitulate network hyperexcitability in human AD

Implication de nouvelles cibles microgliales, Cst7 et Clec7a/Dectin-1, dans le développement et la progression de la maladie d'Alzheimer

Alzheimer’s Disease (AD) is the most common form of dementia in the elderly. Effective threapeutics are still very needed for this neurodegenerative disorder. Interestingly, almost all risk genes for sporadic AD are highly expressed in microglia demonstrating their crucial role in this disorder.Microglial cells are brain resident immune cells. They play pivotal roles in neuroinflammatory processes. However, whether microglia play beneficial and/or detrimental roles in AD progression remains heavily debated and, in addition, their roles in the early stages of the pathology are still poorly understood.Previous study from our lab identified several microglial genes dysregulated in AD early stages. We focused our work on two main targets : Cst7 and Clec7a. Indeed, while the microglial roles of these two genes are poorly known, their peripheral functions (cytokines production, phagocytosis and protein degradation) put them under the spotlight for early involvment in AD.The main objectives of this thesis project were : (i) to caracterize AD early stage development in the APP(swe)/PS1(dE9) model; then (ii) to study the targets’ implication in AD initiation, (iii) to unravel their microglial functions, and finally (iv) to demonstrate their relevance in Humans.As a whole our data support an early contribution of microglia to AD progression in the APP(swe)/PS1(dE9) mouse model and point to two specific genes that may represent potential early biomarkers and/or therapeutic targets.La maladie d’Alzheimer (MA), première cause de démence chez les personnes âgées, est une maladie neurodégénérative pour laquelle il n'existe toujours pas de stratégie thérapeutique efficace. Singulièrement, une grande partie des facteurs de risques des formes sporadiques de la MA sont associés à des gènes microgliaux. Les microglies sont les cellules immunitaires résidentes du cerveau, et jouent un rôle pivot dans les processus neuroinflammatoires. Néanmoins, leurs rôles dans les différentes phases de la MA, et en particulier durant la phase précoce constituant un enjeu thérapeutique majeur, restent encore très mal connus.Des travaux réalisés dans notre laboratoire ont permis d'identifier deux cibles moléculaires particulières, Cst7 et Clec7a, qui du fait de leur dérégulation précoce et de leurs rôles à la périphérie (production de cytokines, phagocytose et dégradation de protéines) représentent des cibles pertinentes dans les phases précoces de la MA.Les objectifs de mon projet de thèse ont donc été : (i) de caractériser le modèle d'étude APP(swe)/PS1(dE9) au stade précoce; puis (ii) de déterminer l'implication des deux cibles dans le développement de la MA et (iii) d'élucider leurs rôles microgliaux; enfin (iv) j'ai cherché à démontrer l’intérêt de ces cibles chez l'Homme.L'ensemble de ces travaux ont permis de démontrer l'existence d'une réaction microgliale précoce dans le modèle APP(swe)/PS1(dE9) et de mettre en avant deux gènes microgliaux, pour l'instant peu étudiés, qui pourraient représenter des biomarqueurs précoces et/ou des cibles thérapeutiques innovantes

Involvment of new microglial genes in Alzheimer's disease development, focus on Cst7 and Clec7a/Dectin-1

La maladie d’Alzheimer (MA), première cause de démence chez les personnes âgées, est une maladie neurodégénérative pour laquelle il n'existe toujours pas de stratégie thérapeutique efficace. Singulièrement, une grande partie des facteurs de risques des formes sporadiques de la MA sont associés à des gènes microgliaux. Les microglies sont les cellules immunitaires résidentes du cerveau, et jouent un rôle pivot dans les processus neuroinflammatoires. Néanmoins, leurs rôles dans les différentes phases de la MA, et en particulier durant la phase précoce constituant un enjeu thérapeutique majeur, restent encore très mal connus.Des travaux réalisés dans notre laboratoire ont permis d'identifier deux cibles moléculaires particulières, Cst7 et Clec7a, qui du fait de leur dérégulation précoce et de leurs rôles à la périphérie (production de cytokines, phagocytose et dégradation de protéines) représentent des cibles pertinentes dans les phases précoces de la MA.Les objectifs de mon projet de thèse ont donc été : (i) de caractériser le modèle d'étude APP(swe)/PS1(dE9) au stade précoce; puis (ii) de déterminer l'implication des deux cibles dans le développement de la MA et (iii) d'élucider leurs rôles microgliaux; enfin (iv) j'ai cherché à démontrer l’intérêt de ces cibles chez l'Homme.L'ensemble de ces travaux ont permis de démontrer l'existence d'une réaction microgliale précoce dans le modèle APP(swe)/PS1(dE9) et de mettre en avant deux gènes microgliaux, pour l'instant peu étudiés, qui pourraient représenter des biomarqueurs précoces et/ou des cibles thérapeutiques innovantes.Alzheimer’s Disease (AD) is the most common form of dementia in the elderly. Effective threapeutics are still very needed for this neurodegenerative disorder. Interestingly, almost all risk genes for sporadic AD are highly expressed in microglia demonstrating their crucial role in this disorder.Microglial cells are brain resident immune cells. They play pivotal roles in neuroinflammatory processes. However, whether microglia play beneficial and/or detrimental roles in AD progression remains heavily debated and, in addition, their roles in the early stages of the pathology are still poorly understood.Previous study from our lab identified several microglial genes dysregulated in AD early stages. We focused our work on two main targets : Cst7 and Clec7a. Indeed, while the microglial roles of these two genes are poorly known, their peripheral functions (cytokines production, phagocytosis and protein degradation) put them under the spotlight for early involvment in AD.The main objectives of this thesis project were : (i) to caracterize AD early stage development in the APP(swe)/PS1(dE9) model; then (ii) to study the targets’ implication in AD initiation, (iii) to unravel their microglial functions, and finally (iv) to demonstrate their relevance in Humans.As a whole our data support an early contribution of microglia to AD progression in the APP(swe)/PS1(dE9) mouse model and point to two specific genes that may represent potential early biomarkers and/or therapeutic targets

Image Processing for Bioluminescence Resonance Energy Transfer Measurement—BRET-Analyzer

A growing number of tools now allow live recordings of various signaling pathways and protein-protein interaction dynamics in time and space by ratiometric measurements, such as Bioluminescence Resonance Energy Transfer (BRET) Imaging. Accurate and reproducible analysis of ratiometric measurements has thus become mandatory to interpret quantitative imaging. In order to fulfill this necessity, we have developed an open source toolset for Fiji—BRET-Analyzer—allowing a systematic analysis, from image processing to ratio quantification. We share this open source solution and a step-by-step tutorial at https://github.com/ychastagnier/BRET-Analyzer. This toolset proposes (1) image background subtraction, (2) image alignment over time, (3) a composite thresholding method of the image used as the denominator of the ratio to refine the precise limits of the sample, (4) pixel by pixel division of the images and efficient distribution of the ratio intensity on a pseudocolor scale, and (5) quantification of the ratio mean intensity and standard variation among pixels in chosen areas. In addition to systematize the analysis process, we show that the BRET-Analyzer allows proper reconstitution and quantification of the ratiometric image in time and space, even from heterogeneous subcellular volumes. Indeed, analyzing twice the same images, we demonstrate that compared to standard analysis BRET-Analyzer precisely define the luminescent specimen limits, enlightening proficient strengths from small and big ensembles over time. For example, we followed and quantified, in live, scaffold proteins interaction dynamics in neuronal sub-cellular compartments including dendritic spines, for half an hour. In conclusion, BRET-Analyzer provides a complete, versatile and efficient toolset for automated reproducible and meaningful image ratio analysis

Image Processing for Bioluminescence Resonance Energy Transfer Measurement—BRET-Analyzer

International audienceA growing number of tools now allow live recordings of various signaling pathways and protein-protein interaction dynamics in time and space by ratiometric measurements, such as Bioluminescence Resonance Energy Transfer (BRET) Imaging. Accurate and reproducible analysis of ratiometric measurements has thus become mandatory to interpret quantitative imaging. In order to fulfill this necessity, we have developed an open source toolset for Fiji—BRET-Analyzer—allowing a systematic analysis, from image processing to ratio quantification. We share this open source solution and a step-by-step tutorial at https://github.com/ychastagnier/BRET-Analyzer. This toolset proposes (1) image background subtraction, (2) image alignment over time, (3) a composite thresholding method of the image used as the denominator of the ratio to refine the precise limits of the sample, (4) pixel by pixel division of the images and efficient distribution of the ratio intensity on a pseudocolor scale, and (5) quantification of the ratio mean intensity and standard variation among pixels in chosen areas. In addition to systematize the analysis process, we show that the BRET-Analyzer allows proper reconstitution and quantification of the ratiometric image in time and space, even from heterogeneous subcellular volumes. Indeed, analyzing twice the same images, we demonstrate that compared to standard analysis BRET-Analyzer precisely define the luminescent specimen limits, enlightening proficient strengths from small and big ensembles over time. For example, we followed and quantified, in live, scaffold proteins interaction dynamics in neuronal sub-cellular compartments including dendritic spines, for half an hour. In conclusion, BRET-Analyzer provides a complete, versatile and efficient toolset for automated reproducible and meaningful image ratio analysis

Microglia in Alzheimer Disease: Well-Known Targets and New Opportunities

International audienceMicroglia are the resident macrophages of the central nervous system. They play key roles in brain development, and physiology during life and aging. Equipped with a variety of molecular sensors and through the various functions they can fulfill, they are critically involved in maintaining the brain's homeostasis. In Alzheimer disease (AD), microglia reaction was initially thought to be incidental and triggered by amyloid deposits and dystrophic neurites. However, recent genome-wide association studies have established that the majority of AD risk loci are found in or near genes that are highly and sometimes uniquely expressed in microglia. This leads to the concept of microglia being critically involved in the early steps of the disease and identified them as important potential therapeutic targets. Whether microglia reaction is beneficial, detrimental or both to AD progression is still unclear and the subject of intense debate. In this review, we are presenting a state-of-knowledge report intended to highlight the variety of microglial functions and pathways shown to be critically involved in AD progression. We first address both the acquisition of new functions and the alteration of their homeostatic roles by reactive microglia. Second, we propose a summary of new important parameters currently emerging in the field that need to be considered to identify relevant microglial targets. Finally, we discuss the many obstacles in designing efficient therapeutic strategies for AD and present innovative technologies that may foster our understanding of microglia roles in the pathology. Ultimately, this work aims to fly over various microglial functions to make a general and reliable report of the current knowledge regarding microglia's involvement in AD and of the new research opportunities in the field

De nouvelles techniques pour dévoiler le rôle des cellules gliales du cerveau

International audienceBrain function relies on complex interactions between neurons and different types of glial cells, such as astrocytes, microglia and oligodendrocytes. The relatively young field of "gliobiology" is thriving. Thanks to various technical innovations, it is now possible to address challenging biological questions on glial cells and unravel their multiple roles in brain function and dysfunction.L’exécution des fonctions cérébrales requiert des interactions optimales entre les neurones et les différents types de cellules gliales (astrocytes, microglies et oligodendrocytes). Le domaine de la gliobiologie, qui s’intéresse aux cellules gliales, est en pleine expansion. Les innovations techniques permettent désormais d’aborder des questions biologiques complexes quant aux rôles de ces cellules dans le fonctionnement physiologique et pathologique du cerveau. Dans cette synthèse, nous décrivons comment certaines de ces avancées techniques nous ont permis d’en apprendre davantage sur les origines et les rôles fonctionnels des cellules gliales. Nous illustrons également comment ces techniques et les découvertes qui en ont découlé, peuvent être transposées en clinique et pourraient, dans un futur proche, offrir des nouvelles perspectives thérapeutiques

Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons

International audienceNeuronal hippocampal cultures are simple and valuable models for studying neuronal function. While embryonic cultures are widely used for different applications, mouse postnatal cultures are still challenging, lack reproducibility and/or exhibit inappropriate neuronal activity. Yet, postnatal cultures have major advantages such as allowing genotyping of pups before culture and reducing the number of experimental animals. Herein we describe a simple and fast protocol for culturing and genetically manipulating hippocampal neurons from P0 to P3 mice. This protocol provides reproducible cultures exhibiting a consistent neuronal development, normal excitatory over inhibitory neuronal ratio and a physiological neuronal activity. We also describe simple and efficient procedures for genetic manipulation of neurons using transfection reagent or lentiviral particles. Overall, this method provides a detailed and validated protocol allowing to explore cellular mechanisms and neuronal activity in postnatal hippocampal neurons in culture

Analysis of CX3CR1 haplodeficiency in male and female APPswe/PSEN1dE9 mice along Alzheimer disease progression

International audienceMicroglia, the resident immune cells of the brain, have recently emerged as key players in Alzheimer Disease (AD) pathogenesis, but their roles in AD remain largely elusive and require further investigation. Microglia functions are readily altered when isolated from their brain environment, and microglia reporter mice thus represent valuable tools to study the contribution of these cells to neurodegenerative diseases such as AD. The CX3CR1+/eGFP mice is one of the most popular microglia reporter mice, and has been used in numerous studies to investigate in vivo microglial functions, including in the context of AD research. However, until now, the impact of CX3CR1 haplodeficiency on the typical features of Alzheimer Disease has not been studied in depth. To fill this gap, we generated APPswe/PSEN1dE9:CX3CR1+/eGFP mice and analyzed these mice for Alzheimer's like pathology and neuroinflammation hallmarks. More specifically, using robust multifactorial statistical and multivariate analyses, we investigated the impact of CX3CR1 deficiency in both males and females, at three typical stages of the pathology progression: at early stage when Amyloid-β (Aβ) deposition just starts, at intermediate stage during Aβ accumulation phase and at more advanced stages when Aβ plaque number stabilizes. We found that CX3CR1 haplodeficiency had little impact on the progression of the pathology in the APPswe/PSEN1dE9 model and demonstrated that the APPswe/PSEN1dE9:CX3CR1+/eGFP line is a relevant and useful model to study the role of microglia in Alzheimer Disease. In addition, although Aβ plaques density is higher in females compared to age-matched males, we show that their glial reaction, inflammation status and memory deficits are not different

The Shank3Venus/Venus knock in mouse enables isoform-specific functional studies of Shank3a

International audienceBackground Shank3 is a scaffolding protein essential for the organization and function of the glutamatergic postsynapse. Monogenic mutations in SHANK3 gene are among the leading genetic causes of Autism Spectrum Disorders (ASD). The multiplicity of Shank3 isoforms seems to generate as much functional diversity and yet, there are no tools to study endogenous Shank3 proteins in an isoform-specific manner. Methods In this study, we created a novel transgenic mouse line, the Shank3 Venus/Venus knock in mouse, which allows to monitor the endogenous expression of the major Shank3 isoform in the brain, the full-length Shank3a isoform. Results We show that the endogenous Venus-Shank3a protein is localized in spines and is mainly expressed in the striatum, hippocampus and cortex of the developing and adult brain. We show that Shank3 Venus/+ and Shank3 Venus/Venus mice have no behavioral deficiency. We further crossed Shank3 Venus/Venus mice with Shank3 ΔC/ΔC mice, a model of ASD, to track the Venus-tagged wild-type copy of Shank3a in physiological (Shank3 Venus/+ ) and pathological (Shank3 Venus/ΔC ) conditions. We report a developmental delay in brain expression of the Venus-Shank3a isoform in Shank3 Venus/ΔC mice, compared to Shank3 Venus/+ control mice. Conclusion Altogether, our results show that the Shank3 Venus/Venus mouse line is a powerful tool to study endogenous Shank3a expression, in physiological conditions and in ASD