Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies

Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions.     

Reactive and Senescent Astroglial Phenotypes as Hallmarks of Brain Pathologies

Astrocytes, as the most abundant glial cells in the central nervous system, are tightly integrated into neural networks and participate in numerous aspects of brain physiology and pathology. They are the main homeostatic cells in the central nervous system, and the loss of astrocyte physiological functions and/or gain of pro-inflammatory functions, due to their reactivation or cellular senescence, can have profound impacts on the surrounding microenvironment with pathological outcomes. Although the importance of astrocytes is generally recognized, and both senescence and reactive astrogliosis have been extensively reviewed independently, there are only a few comparative overviews of these complex processes. In this review, we summarize the latest data regarding astrocyte reactivation and senescence, and outline similarities and differences between these phenotypes from morphological, functional, and molecular points of view. A special focus has been given to neurodegenerative diseases, where these phenotypic alternations of astrocytes are significantly implicated. We also summarize current perspectives regarding new advances in model systems based on astrocytes as well as data pointing to these glial cells as potential therapeutic targets.     

Design Principles for Cationic,Astrocyte-Targeted Probes

The brain's astrocytes play key roles in normal and pathological brain processes. Targeting small molecules to astrocytes in the presence of the many other cell types in the brain will provide useful tools for their visualization and manipulation. Herein, we explore the functional consequences of synthetic modifications to a recently described astrocyte marker composed of a bright rhodamine-based fluorophore and an astrocyte-targeting moiety. We altered the nature of the targeting moiety to probe the dependence of astrocyte targeting on hydrophobicity, charge, and pK_a when exposed to astrocytes and neurons isolated from the mouse cortex. We found that an overall molecular charge of +2 and a targeting moiety with a heterocyclic aromatic amine are important requirements for specific and robust astrocyte labeling. These results provide a basis for engineering astrocyte-targeted molecular tools with unique properties, including metabolite sensing or optogenetic control.     

Not Just a Bystander: The Emerging Role of Astrocytes and Research Tools in Studying Cognitive Dysfunctions in Schizophrenia

Cognitive dysfunction is one of the core symptoms in schizophrenia, and it is predictive of functional outcomes and therefore useful for treatment targets. Rather than improving cognitive deficits, currently available antipsychotics mainly focus on positive symptoms, targeting dopaminergic/serotoninergic neurons and receptors in the brain. Apart from investigating the neural mechanisms underlying schizophrenia, emerging evidence indicates the importance of glial cells in brain structure development and their involvement in cognitive functions. Although the etiopathology of astrocytes in schizophrenia remains unclear, accumulated evidence reveals that alterations in gene expression and astrocyte products have been reported in schizophrenic patients. To further investigate the role of astrocytes in schizophrenia, we highlighted recent progress in the investigation of the effect of astrocytes on abnormalities in glutamate transmission and impairments in the blood–brain barrier. Recent advances in animal models and behavioral methods were introduced to examine schizophrenia-related cognitive deficits and negative symptoms. We also highlighted several experimental tools that further elucidate the role of astrocytes. Instead of focusing on schizophrenia as a neuron-specific disorder, an additional astrocytic perspective provides novel and promising insight into its causal mechanisms and treatment. The involvement of astrocytes in the pathogenesis of schizophrenia and other brain disorders is worth further investigation.     

Rethinking Intellectual Disability from Neuro- to Astro-Pathology

Neurodevelopmental disorders arise from genetic and/or from environmental factors and are characterized by different degrees of intellectual disability. The mechanisms that govern important processes sustaining learning and memory, which are severely affected in intellectual disability, have classically been thought to be exclusively under neuronal control. However, this vision has recently evolved into a more integrative conception in which astroglia, rather than just acting as metabolic supply and structural anchoring for neurons, interact at distinct levels modulating neuronal communication and possibly also cognitive processes. Recently, genetic tools have made it possible to specifically manipulate astrocyte activity unraveling novel functions that involve astrocytes in memory function in the healthy brain. However, astrocyte manipulation has also underscored potential mechanisms by which dysfunctional astrocytes could contribute to memory deficits in several neurodevelopmental disorders revealing new pathogenic mechanisms in intellectual disability. Here, we review the current knowledge about astrocyte dysfunction that might contribute to learning and memory impairment in neurodevelopmental disorders, with special focus on Fragile X syndrome and Down syndrome.     

Relationship between Neuronal Damage/Death and Astrogliosis in the Cerebral Motor Cortex of Gerbil Models of Mild and Severe Ischemia and Reperfusion Injury

Neuronal loss (death) occurs selectively in vulnerable brain regions after ischemic insults. Astrogliosis is accompanied by neuronal death. It can change the molecular expression and morphology of astrocytes following ischemic insults. However, little is known about cerebral ischemia and reperfusion injury that can variously lead to damage of astrocytes according to the degree of ischemic injury, which is related to neuronal damage/death. Thus, the purpose of this study was to examine the relationship between damage to cortical neurons and astrocytes using gerbil models of mild and severe transient forebrain ischemia induced by blocking the blood supply to the forebrain for five or 15 min. Significant ischemia tFI-induced neuronal death occurred in the deep layers (layers V and VI) of the motor cortex: neuronal death occurred earlier and more severely in gerbils with severe ischemia than in gerbils with mild ischemia. Distinct astrogliosis was detected in layers V and VI. It gradually increased with time after both ischemiae. The astrogliosis was significantly higher in severe ischemia than in mild ischemia. The ischemia-induced increase of glial fibrillary acidic protein (GFAP; a maker of astrocyte) expression in severe ischemia was significantly higher than that in mild ischemia. However, GFAP-immunoreactive astrocytes were apparently damaged two days after both ischemiae. At five days after ischemiae, astrocyte endfeet around capillary endothelial cells were severely ruptured. They were more severely ruptured by severe ischemia than by mild ischemia. However, the number of astrocytes stained with S100 was significantly higher in severe ischemia than in mild ischemia. These results indicate that the degree of astrogliosis, including the disruption (loss) of astrocyte endfeet following ischemia and reperfusion in the forebrain, might depend on the severity of ischemia and that the degree of ischemia-induced neuronal damage may be associated with the degree of astrogliosis.     

Effects on Metabolism in Astrocytes Caused by cGAMP,Which Imitates the Initial Stage of Brain Metastasis

The second messenger 2′3′-cyclic-GMP-AMP (cGAMP) is thought to be transmitted from brain carcinomas to astrocytes via gap junctions, which functions to promote metastasis in the brain parenchyma. In the current study, we established a method to introduce cGAMP into astrocytes, which simulates the state of astrocytes that have been invaded by cGAMP around tumors. Astrocytes incorporating cGAMP were analyzed by metabolomics, which demonstrated that cGAMP increased glutamate production and astrocyte secretion. The same trend was observed for γ-aminobutyric acid (GABA). Conversely, glutamine production and secretion were decreased by cGAMP treatment. Due to the fundamental role of astrocytes in regulation of the glutamine–glutamate cycle, such metabolic changes may represent a potential mechanism and therapeutic target for alteration of the central nervous system (CNS) environment and the malignant transformation of brain carcinomas.     

Endothelin ETB Receptor-Mediated Astrocytic Activation: Pathological Roles in Brain Disorders

In brain disorders, reactive astrocytes, which are characterized by hypertrophy of the cell body and proliferative properties, are commonly observed. As reactive astrocytes are involved in the pathogenesis of several brain disorders, the control of astrocytic function has been proposed as a therapeutic strategy, and target molecules to effectively control astrocytic functions have been investigated. The production of brain endothelin-1 (ET-1), which increases in brain disorders, is involved in the pathophysiological response of the nervous system. Endothelin B (ET_B) receptors are highly expressed in reactive astrocytes and are upregulated by brain injury. Activation of astrocyte ET_B receptors promotes the induction of reactive astrocytes. In addition, the production of various astrocyte-derived factors, including neurotrophic factors and vascular permeability regulators, is regulated by ET_B receptors. In animal models of Alzheimer’s disease, brain ischemia, neuropathic pain, and traumatic brain injury, ET_B-receptor-mediated regulation of astrocytic activation has been reported to improve brain disorders. Therefore, the astrocytic ET_B receptor is expected to be a promising drug target to improve several brain disorders. This article reviews the roles of ET_B receptors in astrocytic activation and discusses its possible applications in the treatment of brain disorders.     

The Sedentary Lifestyle and Masticatory Dysfunction: Time to Review the Contribution to Age-Associated Cognitive Decline and Astrocyte Morphotypes in the Dentate Gyrus

As aging and cognitive decline progresses, the impact of a sedentary lifestyle on the appearance of environment-dependent cellular morphologies in the brain becomes more apparent. Sedentary living is also associated with poor oral health, which is known to correlate with the rate of cognitive decline. Here, we will review the evidence for the interplay between mastication and environmental enrichment and assess the impact of each on the structure of the brain. In previous studies, we explored the relationship between behavior and the morphological features of dentate gyrus glial fibrillary acidic protein (GFAP)-positive astrocytes during aging in contrasting environments and in the context of induced masticatory dysfunction. Hierarchical cluster and discriminant analysis of GFAP-positive astrocytes from the dentate gyrus molecular layer revealed that the proportion of AST1 (astrocyte arbors with greater complexity phenotype) and AST2 (lower complexity) are differentially affected by environment, aging and masticatory dysfunction, but the relationship is not straightforward. Here we re-evaluated our previous reconstructions by comparing dorsal and ventral astrocyte morphologies in the dentate gyrus, and we found that morphological complexity was the variable that contributed most to cluster formation across the experimental groups. In general, reducing masticatory activity increases astrocyte morphological complexity, and the effect is most marked in the ventral dentate gyrus, whereas the effect of environment was more marked in the dorsal dentate gyrus. All morphotypes retained their basic structural organization in intact tissue, suggesting that they are subtypes with a non-proliferative astrocyte profile. In summary, the increased complexity of astrocytes in situations where neuronal loss and behavioral deficits are present is counterintuitive, but highlights the need to better understand the role of the astrocyte in these conditions.     

Key Role of Astrocytes in Postnatal Brain and Retinal Angiogenesis

Angiogenesis is a key process in various physiological and pathological conditions in the nervous system and in the retina during postnatal life. Although an increasing number of studies have addressed the role of endothelial cells in this event, the astrocytes contribution in angiogenesis has received less attention. This review is focused on the role of astrocytes as a scaffold and in the stabilization of the new blood vessels, through different molecules release, which can modulate the angiogenesis process in the brain and in the retina. Further, differences in the astrocytes phenotype are addressed in glioblastoma, one of the most devastating types of brain cancer, in order to provide potential targets involved in the cross signaling between endothelial cells, astrocytes and glioma cells, that mediate tumor progression and pathological angiogenesis. Given the relevance of astrocytes in angiogenesis in physiological and pathological conditions, future studies are required to better understand the interrelation between endothelial and astrocyte signaling pathways during this process.     

Extracellular Vesicles Taken up by Astrocytes

Extracellular vesicles (EVs) are composed of lipid bilayer membranes and contain various molecules, such as mRNA and microRNA (miRNA), that regulate the functions of the recipient cell. Recent studies have reported the importance of EV-mediated intercellular communication in the brain. The brain contains several types of cells, including neurons and glial cells. Among them, astrocytes are the most abundant glial cells in the mammalian brain and play a wide range of roles, from structural maintenance of the brain to regulation of neurotransmission. Furthermore, since astrocytes can take up EVs, it is possible that EVs originating from inside and outside the brain affect astrocyte function, which in turn affects brain function. However, it has not been fully clarified whether the specific targeting mechanism of EVs to astrocytes as recipient cells exists. In recent years, EVs have attracted attention as a cell-targeted therapeutic approach in various organs, and elucidation of the targeting mechanism of EVs to astrocytes may pave the way for new therapies for brain diseases. In this review, we focus on EVs in the brain that affect astrocyte function and discuss the targeting mechanism of EVs to astrocytes.     

Hypothalamic Astrocytes as a Specialized and Responsive Cell Population in Obesity

Astrocytes are a type of glial cell anatomically and functionally integrated into the neuronal regulatory circuits for the neuroendocrine control of metabolism. Being functional integral compounds of synapses, astrocytes are actively involved in the physiological regulatory aspects of metabolic control, but also in the pathological processes that link neuronal dysfunction and obesity. Between brain areas, the hypothalamus harbors specialized functional circuits that seem selectively vulnerable to metabolic damage, undergoing early cellular rearrangements which are thought to be at the core of the pathogenesis of diet-induced obesity. Such changes in the hypothalamic brain region consist of a rise in proinflammatory cytokines, the presence of a reactive phenotype in astrocytes and microglia, alterations in the cytoarchitecture and synaptology of hypothalamic circuits, and angiogenesis, a phenomenon that cannot be found elsewhere in the brain. Increasing evidence points to the direct involvement of hypothalamic astrocytes in such early metabolic disturbances, thus moving the study of these glial cells to the forefront of obesity research. Here we provide a comprehensive review of the most relevant findings of molecular and pathophysiological mechanisms by which hypothalamic astrocytes might be involved in the pathogenesis of obesity.     

Heterodimer of A2A and Oxytocin Receptors Regulating Glutamate Release in Adult Striatal Astrocytes

Background: Roles of astrocytes in the modulatory effects of oxytocin (OT) in central nervous system are increasingly considered. Nevertheless, OT effects on gliotransmitter release have been neglected. Methods: In purified astrocyte processes from adult rat striatum, we assessed OT receptor (OTR) and adenosine A2A receptor expression by confocal analysis. The effects of receptors activation on glutamate release from the processes were evaluated; A2A-OTR heteromerization was assessed by co-immunoprecipitation and PLA. Structure of the possible heterodimer of A2A and OT receptors was estimated by a bioinformatic approach. Results: Both A2A and OT receptors were expressed on the same astrocyte processes. Evidence for A2A-OTR receptor-receptor interaction was obtained by measuring the release of glutamate: OT inhibited the evoked glutamate release, while activation of A2A receptors, per se ineffective, abolished the OT effect. Biochemical and biophysical evidence for A2A-OTR heterodimers on striatal astrocytes was also obtained. The residues in the transmembrane domains 4 and 5 of both receptors are predicted to be mainly involved in the heteromerization. Conclusions: When considering effects of OT in striatum, modulation of glutamate release from the astrocyte processes and of glutamatergic synapse functioning, and the interaction with A2A receptors on the astrocyte processes should be taken into consideration.     

Blood–Brain Barrier Dysfunction and Astrocyte Senescence as Reciprocal Drivers of Neuropathology in Aging

As the most abundant cell types in the brain, astrocytes form a tissue-wide signaling network that is responsible for maintaining brain homeostasis and regulating various brain activities. Here, we review some of the essential functions that astrocytes perform in supporting neurons, modulating the immune response, and regulating and maintaining the blood–brain barrier (BBB). Given their importance in brain health, it follows that astrocyte dysfunction has detrimental effects. Indeed, dysfunctional astrocytes are implicated in age-related neuropathology and participate in the onset and progression of neurodegenerative diseases. Here, we review two mechanisms by which astrocytes mediate neuropathology in the aging brain. First, age-associated blood–brain barrier dysfunction (BBBD) causes the hyperactivation of TGFβ signaling in astrocytes, which elicits a pro-inflammatory and epileptogenic phenotype. Over time, BBBD-associated astrocyte dysfunction results in hippocampal and cortical neural hyperexcitability and cognitive deficits. Second, senescent astrocytes accumulate in the brain with age and exhibit a decreased functional capacity and the secretion of senescent-associated secretory phenotype (SASP) factors, which contribute to neuroinflammation and neurotoxicity. Both BBBD and senescence progressively increase during aging and are associated with increased risk of neurodegenerative disease, but the relationship between the two has not yet been established. Thus, we discuss the potential relationship between BBBD, TGFβ hyperactivation, and senescence with respect to astrocytes in the context of aging and disease and identify future areas of investigation in the field.     

Evaluation of Therapeutic Targets in Histological Subtypes of Bladder Cancer

Histologically, bladder cancer is a heterogeneous group comprising urothelial carcinoma (UC), squamous cell carcinoma, adenocarcinomas (ACs), urachal carcinomas (UrCs), and small cell neuroendocrine carcinomas (SCCs). However, all bladder cancers have been treated so far uniformly, and targeted therapy options are still limited. Thus, we aimed to determine the protein expression/molecular status of commonly used cancer targets (programmed cell death 1 ligand 1 (PD-L1), mismatch repair (MMR), androgen and estrogen receptors (AR/ER), Nectin-4, tumor-associated calcium signal transducer 2 (Tacstd2, Trop-2), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and fibroblast growth factor receptor 3 (FGFR3)) to give first insights into whether patients with SCC, AC/UrCs, and squamous-differentiated carcinomas (Sq-BLCA) of the bladder could be eligible for targeted therapies. In addition, for MMR-deficient tumors, microsatellite instability was analyzed. We completed our own data with molecular data from The Cancer Genome Atlas (TCGA). We present ratios for each drug and cumulative ratios for multiple therapeutic options for each nonurothelial subtype. For example, 58.9% of SCC patients, 33.5% of AC/UrCs patients, and 79.3% of Sq-BLCA patients would be eligible for at least one of the analyzed targets. In conclusion, our findings hold promise for targeted therapeutic approaches in selected patients in the future, as various drugs could be applied according to the biomarker status.     

Differential Proteomic Analysis of Astrocytes and Astrocytes-Derived Extracellular Vesicles from Control and Rai Knockout Mice: Insights into the Mechanisms of Neuroprotection

Reactive astrocytes are a hallmark of neurodegenerative disease including multiple sclerosis. It is widely accepted that astrocytes may adopt alternative phenotypes depending on a combination of environmental cues and intrinsic features in a highly plastic and heterogeneous manner. However, we still lack a full understanding of signals and associated signaling pathways driving astrocyte reaction and of the mechanisms by which they drive disease. We have previously shown in the experimental autoimmune encephalomyelitis mouse model that deficiency of the molecular adaptor Rai reduces disease severity and demyelination. Moreover, using primary mouse astrocytes, we showed that Rai contributes to the generation of a pro-inflammatory central nervous system (CNS) microenvironment through the production of nitric oxide and IL-6 and by impairing CD39 activity in response to soluble factors released by encephalitogenic T cells. Here, we investigated the impact of Rai expression on astrocyte function both under basal conditions and in response to IL-17 treatment using a proteomic approach. We found that astrocytes and astrocyte-derived extracellular vesicles contain a set of proteins, to which Rai contributes, that are involved in the regulation of oligodendrocyte differentiation and myelination, nitrogen metabolism, and oxidative stress. The HIF-1α pathway and cellular energetic metabolism were the most statistically relevant molecular pathways and were related to ENOA and HSP70 dysregulation.     

Optogenetic Activation of Astrocytes—Effects on Neuronal Network Function

Optogenetics approach is used widely in neurobiology as it allows control of cellular activity with high spatial and temporal resolution. In most studies, optogenetics is used to control neuronal activity. In the present study optogenetics was used to stimulate astrocytes with the aim to modulate neuronal activity. To achieve this goal, light stimulation was applied to astrocytes expressing a version of ChR2 (ionotropic opsin) or Opto-α1AR (metabotropic opsin). Optimal optogenetic stimulation parameters were determined using patch-clamp recordings of hippocampal pyramidal neurons’ spontaneous activity in brain slices as a readout. It was determined that the greatest increase in the number of spontaneous synaptic currents was observed when astrocytes expressing ChR2(H134R) were activated by 5 s of continuous light. For the astrocytes expressing Opto-α1AR, the greatest response was observed in the pulse stimulation mode (T = 1 s, t = 100 ms). It was also observed that activation of the astrocytic Opto-a1AR but not ChR2 results in an increase of the fEPSP slope in hippocampal neurons. Based on these results, we concluded that Opto-a1AR expressed in hippocampal astrocytes provides an opportunity to modulate the long-term synaptic plasticity optogenetically, and may potentially be used to normalize the synaptic transmission and plasticity defects in a variety of neuropathological conditions, including models of Alzheimer’s disease and other neurodegenerative disorders.