Reversal of Postnatal Brain Astrocytes and Ependymal Cells towards a Progenitor Phenotype in Culture.

Astrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFß pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by "milking of the brain", it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal.

Network-level encoding of local neurotransmitters in cortical astrocytes.

Astrocytes, the most abundant non-neuronal cell type in the mammalian brain, are crucial circuit components that respond to and modulate neuronal activity through calcium (Ca2+) signalling1-7. Astrocyte Ca2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales-from fast, subcellular activity3,4 to slow, synchronized activity across connected astrocyte networks8-10-to influence many processes5,7,11. However, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon astrocyte imaging while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca2+ activity-propagative activity-differentiates astrocyte network responses to these two main neurotransmitters, and may influence responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over a minutes-long time course, contributing to accumulating evidence that substantial astrocyte-neuron communication occurs across slow, network-level spatiotemporal scales12-14. These findings will enable future studies to investigate the link between specific astrocyte Ca2+ activity and specific functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

Lineage-specific changes in mitochondrial properties during neural stem cell differentiation.

Neural stem cells (NSCs) reside in discrete regions of the adult mammalian brain where they can differentiate into neurons, astrocytes, and oligodendrocytes. Several studies suggest that mitochondria have a major role in regulating NSC fate. Here, we evaluated mitochondrial properties throughout NSC differentiation and in lineage-specific cells. For this, we used the neurosphere assay model to isolate, expand, and differentiate mouse subventricular zone postnatal NSCs. We found that the levels of proteins involved in mitochondrial fusion (Mitofusin [Mfn] 1 and Mfn 2) increased, whereas proteins involved in fission (dynamin-related protein 1 [DRP1]) decreased along differentiation. Importantly, changes in mitochondrial dynamics correlated with distinct patterns of mitochondrial morphology in each lineage. Particularly, we found that the number of branched and unbranched mitochondria increased during astroglial and neuronal differentiation, whereas the area occupied by mitochondrial structures significantly reduced with oligodendrocyte maturation. In addition, comparing the three lineages, neurons revealed to be the most energetically flexible, whereas astrocytes presented the highest ATP content. Our work identified putative mitochondrial targets to enhance lineage-directed differentiation of mouse subventricular zone-derived NSCs.

Machine learning approach for recognition and morphological analysis of isolated astrocytes in phase contrast microscopy.

Astrocytes are glycolytically active cells in the central nervous system playing a crucial role in various brain processes from homeostasis to neurotransmission. Astrocytes possess a complex branched morphology, frequently examined by fluorescent microscopy. However, staining and fixation may impact the properties of astrocytes, thereby affecting the accuracy of the experimental data of astrocytes dynamics and morphology. On the other hand, phase contrast microscopy can be used to study astrocytes morphology without affecting them, but the post-processing of the resulting low-contrast images is challenging. The main result of this work is a novel approach for recognition and morphological analysis of unstained astrocytes based on machine-learning recognition of microscopic images. We conducted a series of experiments involving the cultivation of isolated astrocytes from the rat brain cortex followed by microscopy. Using the proposed approach, we tracked the temporal evolution of the average total length of branches, branching, and area per astrocyte in our experiments. We believe that the proposed approach and the obtained experimental data will be of interest and benefit to the scientific communities in cell biology, biophysics, and machine learning.

Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells.

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Spatial organization of astrocyte clones: The role of developmental progenitor timing.

Astrocytes represent a diverse and morphologically complex group of glial cells critical for shaping and maintaining nervous system homeostasis, as well as responding to injuries. Understanding the origins of astroglial heterogeneity, originated from a limited number of progenitors, has been the focus of many studies. Most of these investigations have centered on protoplasmic and pial astrocytes, while the clonal relationship of fibrous astrocytes or juxtavascular astrocytes has remained relatively unexplored. In this study, we sought to elucidate the morphological diversity and clonal distribution of astrocytes across adult cortical layers, with particular emphasis on their ontogenetic origins. Using the StarTrack lineage tracing tool, we explored the characteristics of adult astroglial clones derived from single and specific progenitors at various embryonic stages. Our results revealed a heterogeneous spatial distribution of astroglial clones, characterized by variations in location, clonal size, and rostro-caudal dispersion. While a considerable proportion of clones were confined within specific cortical layers, others displayed sibling cells crossing layer boundaries. Notably, we observed a correlation between clone location and developmental stage at earlier embryonic stages, although this relationship diminished in later stages. Fibrous astrocyte clones were exclusively confined to the corpus callosum. In contrast, protoplasmic or juxtavascular clones were located in either the upper or lower cortical layers, with certain clones displayed sibling cells distributed across both regions. Our findings underscore the developmental origins and spatial distribution of astroglial clones within cortical layers, providing new insights into the interplay between their morphology, clonal sizes, and progenitor heterogeneity.

A concerted neuron-astrocyte program declines in ageing and schizophrenia.

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA sequencing to analyse the prefrontal cortex of 191 human donors aged 22-97 years, including healthy individuals and people with schizophrenia. Latent-factor analysis of these data revealed that, in people whose cortical neurons more strongly expressed genes encoding synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the synaptic neuron and astrocyte program (SNAP). In schizophrenia and ageing-two conditions that involve declines in cognitive flexibility and plasticity1,2-cells divested from SNAP: astrocytes, glutamatergic (excitatory) neurons and GABAergic (inhibitory) neurons all showed reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy people of similar age, may underlie many aspects of normal human interindividual differences and may be an important point of convergence for multiple kinds of pathophysiology.

Direct investigations of the effects of nicardipine on calcium channels of astrocytes by Atomic Force Microscopy.

Calcium channel blockers (CCB) of astrocytes can blockade the calcium ions entry through the voltage gated calcium channels (VGCC), and is widely used in the diseases related with VGCC of astrocytes. But many aspects of the interaction mechanisms between the CCB and VGCC of astrocytes still remain unclear due to the limited resolution of the approaches. Herein the effects of the nicardipine (a type of CCB) on VGCC of astrocytes were investigated at very high spatial, force and electrical resolution by multiple modes of Atomic Force Microscopy (AFM) directly. The results reveal that after the addition of nicardipine, the recognition signals of VGCC disappeared; the specific unbinding forces vanished; the conductivity of the astrocytes decreased (the current decreased about 2.9 pA and the capacitance was doubled); the surface potential of the astrocytes reduced about 14.2 mV. The results of electrical properties investigations are consistent with the simulation experiments. The relations between these biophysical and biochemical properties of VGCC have been discussed. All these demonstrate that the interactions between nicardipine and VGCC have been studied at nanometer spatial resolution, at picoNewton force resolution and very high electrical signal resolution (pA in current, pF in capacitance and 0.1 mV in surface potential) level. The approaches are considered to be high resolution and high sensitivity, and will be helpful and useful in the further investigations of the effects of other types of CCB on ion channels, and will also be helpful in the investigations of mechanisms and therapy of ion channelopathies.

Spatial transcriptomics reveal neuron-astrocyte synergy in long-term memory.

Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.

The distinct localization of CDC42 isoforms is responsible for their specific functions during migration.

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), which only differ in their carboxy-terminal sequence, including the CAAX motif essential for their association with membranes. We show that these divergent sequences do not directly affect the range of CDC42's potential binding partners but indirectly influence CDC42-driven signaling by controlling the subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u associates with the leading-edge plasma membrane of migrating cells, where it recruits the Par6-PKCζ complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it regulates N-WASP-mediated endocytosis. Both CDC42 isoforms contribute their specific functions to promote the chemotaxis of neural precursors, demonstrating that their expression pattern is decisive for tissue-specific cell behavior.

A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Primary Astrocytes Purification and Immortalization.

Astrocytes, the most abundant cells in the central nervous system (CNS), are essential for neuronal development, network formation, and overall CNS homeostasis. Primary astrocyte culture has been successfully used as a tool to study astrocyte biology in vitro. In the present protocol, a modified immunopanning method was utilized to obtain and purify primary astrocytes from mouse cortex and spinal cord in a relatively quick and inexpensive way. Purified primary astrocytes were then immortalized through infection of lentivirus expressing the SV40 large T antigens. In addition, we provide protocols to determine the expression levels of astrocyte-specific markers and to perform functional studies measuring the ATP-induced calcium flux in the immortalized astrocytes. Following the described protocols assures that the immortalized astrocytes that one prepares mimic the cell biology of primary astrocytes in culture. Thus, the purification and immortalization protocols for primary astrocytes presented in here provide two models for the studies of astrocyte biology and may be useful for the immortalization of other types of primary cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary astrocyte purification by a modified immunopanning method Support Protocol: Serum-free primary astrocyte culture Basic Protocol 2: Primary astrocyte immortalization Basic Protocol 3: Calcium transient detection in astrocytes.

Comprehensive cell atlas of the first-trimester developing human brain.

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre-oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.

Specialized astrocytes mediate glutamatergic gliotransmission in the CNS.

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

Role of Lipids in Regulation of Neuroglial Interactions.

Lipids comprise an extremely heterogeneous group of compounds that perform a wide variety of biological functions. Traditional view of lipids as important structural components of the cell and compounds playing a trophic role is currently being supplemented by information on the possible participation of lipids in signaling, not only intracellular, but also intercellular. The review article discusses current data on the role of lipids and their metabolites formed in glial cells (astrocytes, oligodendrocytes, microglia) in communication of these cells with neurons. In addition to metabolic transformations of lipids in each type of glial cells, special attention is paid to the lipid signal molecules (phosphatidic acid, arachidonic acid and its metabolites, cholesterol, etc.) and the possibility of their participation in realization of synaptic plasticity, as well as in other possible mechanisms associated with neuroplasticity. All these new data can significantly expand our knowledge about the regulatory functions of lipids in neuroglial relationships.

Inhibitory input directs astrocyte morphogenesis through glial GABABR.

Communication between neurons and glia has an important role in establishing and maintaining higher-order brain function1. Astrocytes are endowed with complex morphologies, placing their peripheral processes in close proximity to neuronal synapses and directly contributing to their regulation of brain circuits2-4. Recent studies have shown that excitatory neuronal activity promotes oligodendrocyte differentiation5-7; whether inhibitory neurotransmission regulates astrocyte morphogenesis during development is unclear. Here we show that inhibitory neuron activity is necessary and sufficient for astrocyte morphogenesis. We found that input from inhibitory neurons functions through astrocytic GABAB receptor (GABABR) and that its deletion in astrocytes results in a loss of morphological complexity across a host of brain regions and disruption of circuit function. Expression of GABABR in developing astrocytes is regulated in a region-specific manner by SOX9 or NFIA and deletion of these transcription factors results in region-specific defects in astrocyte morphogenesis, which is conferred by interactions with transcription factors exhibiting region-restricted patterns of expression. Together, our studies identify input from inhibitory neurons and astrocytic GABABR as universal regulators of morphogenesis, while further revealing a combinatorial code of region-specific transcriptional dependencies for astrocyte development that is intertwined with activity-dependent processes.

Astrocytes and brain-derived neurotrophic factor (BDNF).

Astrocytes are emerging in the neuroscience field as crucial modulators of brain functions, from the molecular control of synaptic plasticity to orchestrating brain-wide circuit activity for cognitive processes. The cellular pathways through which astrocytes modulate neuronal activity and plasticity are quite diverse. In this review, we focus on neurotrophic pathways, mostly those mediated by brain-derived neurotrophic factor (BDNF). Neurotrophins are a well-known family of trophic factors with pleiotropic functions in neuronal survival, maturation and activity. Within the brain, BDNF is the most abundantly expressed and most studied of all neurotrophins. While we have detailed knowledge of the effect of BDNF on neurons, much less is known about its physiology on astroglia. However, over the last years new findings emerged demonstrating that astrocytes take an active part into BDNF physiology. In this work, we discuss the state-of-the-art knowledge about astrocytes and BDNF. Indeed, astrocytes sense extracellular BDNF through its specific TrkB receptors and activate intracellular responses that greatly vary depending on the brain area, stage of development and receptors expressed. Astrocytes also uptake and recycle BDNF / proBDNF at synapses contributing to synaptic plasticity. Finally, experimental evidence is now available describing deficits in astrocytic BDNF in several neuropathologies, suggesting that astrocytic BDNF may represent a promising target for clinical translation.

Generation of Urine-Derived Induced Pluripotent Stem Cells and Cerebral Organoids for Modeling Down Syndrome.

Down syndrome (DS, or trisomy 21, T21), is the most common genetic cause of intellectual disability. Alterations in the complex process of cerebral cortex development contribute to the neurological deficits in DS, although the underlying molecular and cellular mechanisms are not completely understood. Human cerebral organoids (COs) derived from three-dimensional (3D) cultures of induced pluripotent stem cells (iPSCs) provide a new avenue for gaining a better understanding of DS neuropathology. In this study, we aimed to generate iPSCs from individuals with DS (T21-iPSCs) and euploid controls using urine-derived cells, which can be easily and noninvasively obtained from most individuals, and examine their ability to differentiate into neurons and astrocytes grown in monolayer cultures, as well as into 3D COs. We employed nonintegrating episomal vectors to generate urine-derived iPSC lines, and a simple-to-use system to produce COs with forebrain identity. We observed that both T21 and control urine-derived iPSC lines successfully differentiate into neurons and astrocytes in monolayer, as well as into COs that recapitulate early features of human cortical development, including organization of neural progenitor zones, programmed differentiation of excitatory and inhibitory neurons, and upper-and deep-layer cortical neurons as well as astrocytes. Our findings demonstrate for the first time the suitability of using urine-derived iPSC lines to produce COs for modeling DS.

Molecular diversity of astrocytes.

The tissue environment influences astrocyte form and function in health and disease.

Suppressor of Fused Regulation of Hedgehog Signaling is Required for Proper Astrocyte Differentiation.

Hedgehog signaling is essential for vertebrate development; however, less is known about the negative regulators that influence this pathway. Using the mouse P19 embryonal carcinoma cell model, suppressor of fused (SUFU), a negative regulator of the Hedgehog (Hh) pathway, was investigated during retinoic acid (RA)-induced neural differentiation. We found Hh signaling increased activity in the early phase of differentiation, but was reduced during terminal differentiation of neurons and astrocytes. This early increase in pathway activity was required for neural differentiation; however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of Gli, remained relatively unchanged during differentiation, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hh target genes. Interestingly, these SUFU-deficient cells were unable to differentiate toward neural lineages without RA, and when directed toward these lineages, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout RA-induced differentiation and this was accompanied by the loss of Gli3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, likely acting through Gli3, to reduce Hh signaling during late-stage differentiation.