Mechanistic multiscale modelling of energy metabolism in human astrocytes reveals the impact of morphology changes in Alzheimer's Disease.

Astrocytes with their specialised morphology are essential for brain homeostasis as metabolic mediators between blood vessels and neurons. In neurodegenerative diseases such as Alzheimer's disease (AD), astrocytes adopt reactive profiles with molecular and morphological changes that could lead to the impairment of their metabolic support and impact disease progression. However, the underlying mechanisms of how the metabolic function of human astrocytes is impaired by their morphological changes in AD are still elusive. To address this challenge, we developed and applied a metabolic multiscale modelling approach integrating the dynamics of metabolic energy pathways and physiological astrocyte morphologies acquired in human AD and age-matched control brain samples. The results demonstrate that the complex cell shape and intracellular organisation of energetic pathways determine the metabolic profile and support capacity of astrocytes in health and AD conditions. Thus, our mechanistic approach indicates the importance of spatial orchestration in metabolism and allows for the identification of protective mechanisms against disease-associated metabolic impairments.

Normal and Pathological NRF2 Signalling in the Central Nervous System.

The nuclear factor erythroid 2-related factor 2 (NRF2) was originally described as a master regulator of antioxidant cellular response, but in the time since, numerous important biological functions linked to cell survival, cellular detoxification, metabolism, autophagy, proteostasis, inflammation, immunity, and differentiation have been attributed to this pleiotropic transcription factor that regulates hundreds of genes. After 40 years of in-depth research and key discoveries, NRF2 is now at the center of a vast regulatory network, revealing NRF2 signalling as increasingly complex. It is widely recognized that reactive oxygen species (ROS) play a key role in human physiological and pathological processes such as ageing, obesity, diabetes, cancer, and neurodegenerative diseases. The high oxygen consumption associated with high levels of free iron and oxidizable unsaturated lipids make the brain particularly vulnerable to oxidative stress. A good stability of NRF2 activity is thus crucial to maintain the redox balance and therefore brain homeostasis. In this review, we have gathered recent data about the contribution of the NRF2 pathway in the healthy brain as well as during metabolic diseases, cancer, ageing, and ageing-related neurodegenerative diseases. We also discuss promising therapeutic strategies and the need for better understanding of cell-type-specific functions of NRF2 in these different fields.

The Multifaceted Neurotoxicity of Astrocytes in Ageing and Age-Related Neurodegenerative Diseases: A Translational Perspective.

In a healthy physiological context, astrocytes are multitasking cells contributing to central nervous system (CNS) homeostasis, defense, and immunity. In cell culture or rodent models of age-related neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD) and Parkinson's disease (PD), numerous studies have shown that astrocytes can adopt neurotoxic phenotypes that could enhance disease progression. Chronic inflammatory responses, oxidative stress, unbalanced phagocytosis, or alteration of their core physiological roles are the main manifestations of their detrimental states. However, if astrocytes are directly involved in brain deterioration by exerting neurotoxic functions in patients with NDDs is still controversial. The large spectrum of NDDs, with often overlapping pathologies, and the technical challenges associated with the study of human brain samples complexify the analysis of astrocyte involvement in specific neurodegenerative cascades. With this review, we aim to provide a translational overview about the multi-facets of astrocyte neurotoxicity ranging from in vitro findings over mouse and human cell-based studies to rodent NDDs research and finally evidence from patient-related research. We also discuss the role of ageing in astrocytes encompassing changes in physiology and response to pathologic stimuli and how this may prime detrimental responses in NDDs. To conclude, we discuss how potentially therapeutic strategies could be adopted to alleviate or reverse astrocytic toxicity and their potential to impact neurodegeneration and dementia progression in patients.

Microglia phenotypes are associated with subregional patterns of concomitant tau, amyloid-ß and α-synuclein pathologies in the hippocampus of patients with Alzheimer's disease and dementia with Lewy bodies.

The cellular alterations of the hippocampus lead to memory decline, a shared symptom between Alzheimer's disease (AD) and dementia with Lewy Bodies (DLB) patients. However, the subregional deterioration pattern of the hippocampus differs between AD and DLB with the CA1 subfield being more severely affected in AD. The activation of microglia, the brain immune cells, could play a role in its selective volume loss. How subregional microglia populations vary within AD or DLB and across these conditions remains poorly understood. Furthermore, how the nature of the hippocampal local pathological imprint is associated with microglia responses needs to be elucidated. To this purpose, we employed an automated pipeline for analysis of 3D confocal microscopy images to assess CA1, CA3 and DG/CA4 subfields microglia responses in post-mortem hippocampal samples from late-onset AD (n = 10), DLB (n = 8) and age-matched control (CTL) (n = 11) individuals. In parallel, we performed volumetric analyses of hyperphosphorylated tau (pTau), amyloid-ß (Aß) and phosphorylated α-synuclein (pSyn) loads. For each of the 32,447 extracted microglia, 16 morphological features were measured to classify them into seven distinct morphological clusters. Our results show similar alterations of microglial morphological features and clusters in AD and DLB, but with more prominent changes in AD. We identified two distinct microglia clusters enriched in disease conditions and particularly increased in CA1 and DG/CA4 of AD and CA3 of DLB. Our study confirms frequent concomitance of pTau, Aß and pSyn loads across AD and DLB but reveals a specific subregional pattern for each type of pathology, along with a generally increased severity in AD. Furthermore, pTau and pSyn loads were highly correlated across subregions and conditions. We uncovered tight associations between microglial changes and the subfield pathological imprint. Our findings suggest that combinations and severity of subregional pTau, Aß and pSyn pathologies transform local microglia phenotypic composition in the hippocampus. The high burdens of pTau and pSyn associated with increased microglial alterations could be a factor in CA1 vulnerability in AD.

MIC-MAC: An automated pipeline for high-throughput characterization and classification of three-dimensional microglia morphologies in mouse and human postmortem brain samples.

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High-throughput molecular approaches such as single-cell RNA-sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC-MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three-dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging-based approach enables automatic 3D-morphology characterization and classification of thousands of individual microglia in situ and revealed species- and disease-specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC-MAC is a precision diagnostic tool that allows a rapid, unbiased, and large-scale analysis of microglia morphological states in mouse models and patient brain samples.

A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples.

Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response to brain injury or disease, a process called reactive gliosis. Precisely how astrocytes contribute to healthy brain function and play differential roles in brain pathology and regeneration remain important areas of investigation. To better understand the properties of astrocytes, more sophisticated approaches for probing their rich and complex anatomical and molecular features are needed to fully determine their contribution to brain physiology. Here we present an efficient and straightforward immunolabeling protocol to obtain high-resolution fluorescence-based images from fixed nonhuman primate (common marmoset Callithrix jacchus) and human brain samples. Importantly, the protocol is useful for obtaining images from samples that have been stored in fixative solutions (such as formalin) for years. This approach is especially useful for three-dimensional, multichannel confocal microscopy and can be optimized for super-resolution techniques such as stimulated emission depletion (STED) microscopy. We also present a strategy for using specific combinations of markers to define the phenotypic variations and cellular/subcellular properties of astrocytes to better predict the function of these cells on their surrounding brain microenvironment.

High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around ß-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

Synergistic actions of microglia and astrocytes in the progression of Alzheimer's disease.

Microglia and astrocytes are essential components of brain homeostasis. Interestingly, when the brain is exposed to adverse conditions, both astrocytes and microglia acquire specialized 'reactive' or 'activated' phenotypes that relate to the characteristics of the insult. In most cases they become important perpetrators of inflammation and potentially neuronal dysfunction. In neurodegenerative diseases such as Alzheimer's disease, the reciprocal interactions between microglia and astrocytes may be particularly important for the development of neuronal pathology and disease states. An important challenge is to understand how microglia and astrocytes inter-communicate at different stages of disease and the importance of this crosstalk on the physiology of surrounding neurons. In this review we focus on the potential roles that microglia and astrocytes fulfill in early to late stages of AD and how their synergistic actions may shape the progression of AD pathology to affect brain health.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

Astrocyte-secreted matricellular proteins in CNS remodelling during development and disease.

Matricellular proteins are secreted, nonstructural proteins that regulate the extracellular matrix (ECM) and interactions between cells through modulation of growth factor signaling, cell adhesion, migration, and proliferation. Despite being well described in the context of nonneuronal tissues, recent studies have revealed that these molecules may also play instrumental roles in central nervous system (CNS) development and diseases. In this minireview, we discuss the matricellular protein families SPARC (secreted protein acidic and rich in cysteine), Hevin/SC1 (SPARC-like 1), TN-C (Tenascin C), TSP (Thrombospondin), and CCN (CYR61/CTGF/NOV), which are secreted by astrocytes during development. These proteins exhibit a reduced expression in adult CNS but are upregulated in reactive astrocytes following injury or disease, where they are well placed to modulate the repair processes such as tissue remodeling, axon regeneration, glial scar formation, angiogenesis, and rewiring of neural circuitry. Conversely, their reexpression in reactive astrocytes may also lead to detrimental effects and promote the progression of neurodegenerative diseases.

ßCTF-correlated burst of hippocampal TNFα occurs at a very early, pre-plaque stage in the TgCRND8 mouse model of Alzheimer's disease.

Tumor necrosis factor-alpha (TNFα) regulates neuronal excitability. We investigated whether alterations in the level of TNFα occur at a time point that precedes the reported seizure-associated hyperexcitability of hippocampal networks in pre-plaque models of Alzheimer's disease (AD). Western blot and ELISA experiments indicated a significant increase in hippocampal TNFα expression in 1-month-old TgCRND8 mice that correlated with levels of the ß-C-terminal fragment (ßCTF) of amyloid-ß protein precursor. CD11b labeling indicated changes in microglial morphology toward an activated state, suggesting that these cells may be a putative source of the observed TNFα increase during this pre-symptomatic stage of AD-like pathology.

Local Ca2+ detection and modulation of synaptic release by astrocytes.

Astrocytes communicate with synapses by means of intracellular calcium ([Ca(2+)](i)) elevations, but local calcium dynamics in astrocytic processes have never been thoroughly investigated. By taking advantage of high-resolution two-photon microscopy, we identify the characteristics of local astrocyte calcium activity in the adult mouse hippocampus. Astrocytic processes showed intense activity, triggered by physiological transmission at neighboring synapses. They encoded synchronous synaptic events generated by sparse action potentials into robust regional (∼12 µm) [Ca(2+)](i) elevations. Unexpectedly, they also sensed spontaneous synaptic events, producing highly confined (∼4 µm), fast (millisecond-scale) miniature Ca(2+) responses. This Ca(2+) activity in astrocytic processes is generated through GTP- and inositol-1,4,5-trisphosphate-dependent signaling and is relevant for basal synaptic function. Thus, buffering astrocyte [Ca(2+)](i) or blocking a receptor mediating local astrocyte Ca(2+) signals decreased synaptic transmission reliability in minimal stimulation experiments. These data provide direct evidence that astrocytes are integrated in local synaptic functioning in adult brain.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.

EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding partner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity.

Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain.

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.

EphA4 signaling regulates phospholipase Cgamma1 activation, cofilin membrane association, and dendritic spine morphology.

Specialized postsynaptic structures known as dendritic spines are the primary sites of glutamatergic innervation at synapses of the CNS. Previous studies have shown that spines rapidly remodel their actin cytoskeleton to modify their shape and this has been associated with changes in synaptic physiology. However, the receptors and signaling intermediates that restructure the actin network in spines are only beginning to be identified. We reported previously that the EphA4 receptor tyrosine kinase regulates spine morphology. However, the signaling pathways downstream of EphA4 that induce spine retraction on ephrin ligand binding remain poorly understood. Here, we demonstrate that ephrin stimulation of EphA4 leads to the recruitment and activation of phospholipase Cgamma1 (PLCgamma1) in heterologous cells and in hippocampal slices. This interaction occurs through an Src homology 2 domain of PLCgamma1 and requires the EphA4 juxtamembrane tyrosines. In the brain, PLCgamma1 is found in multiple compartments of synaptosomes and is readily found in postsynaptic density fractions. Consistent with this, PLC activity is required for the maintenance of spine morphology and ephrin-induced spine retraction. Remarkably, EphA4 and PLC activity modulate the association of the actin depolymerizing/severing factor cofilin with the plasma membrane. Because cofilin has been implicated previously in the structural plasticity of spines, this signaling may enable cofilin to depolymerize actin filaments and restructure spines at sites of ephrin-EphA4 contact.

Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influence dendritic spine morphology and long-term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4-labeled dendritic spines and axon terminals corresponded to 9-19% and 25-40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre- or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP.