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.