Astrocyte Heterogeneity Research Articles

Astrocyte Diversity: Current Insights and Future Directions.

Astrocytes make up 20-40% of glial cells within the central nervous system (CNS) and provide several crucial functions, ranging from metabolic and structural support to regulation of synaptogenesis and synaptic transmission. Although these cells are morphologically and functionally complex, astrocytes have been historically regarded as homogenous cell populations and studied as one population of cells. Fortunately, recent evidence in RNA profiling and imaging data has begun to refute this view. These studies suggest heterogeneity of astrocytes across brain regions, differing in many aspects such as morphology, function, physiological properties, developmental origins, and response to disease. Increased understanding of astrocyte heterogeneity is critical for investigations into the function of astrocytes in the brain and neuro-glia interactions. Furthermore, insights into astrocyte heterogeneity can help better understand their role in neurological disorders and potentially produce novel approaches to treating these diseases.

Heterogeneity of Astrocytes in Grey and White Matter.

Astrocytes are a diverse and heterogeneous type of glial cells. The major task of grey and white matter areas in the brain are computation of information at neuronal synapses and propagation of action potentials along axons, respectively, resulting in diverse demands for astrocytes. Adapting their function to the requirements in the local environment, astrocytes differ in morphology, gene expression, metabolism, and many other properties. Here we review the differential properties of protoplasmic astrocytes of grey matter and fibrous astrocytes located in white matter in respect to glutamate and energy metabolism, to their function at the blood-brain interface and to coupling via gap junctions. Finally, we discuss how this astrocytic heterogeneity might contribute to the different susceptibility of grey and white matter to ischemic insults.

Discrimination of Prion Strain Targeting in the Central Nervous System via Reactive Astrocyte Heterogeneity in CD44 Expression.

Prion diseases or transmissible spongiform encephalopathies are fatal, progressive, neurodegenerative, protein-misfolding disorders. Prion diseases may arise spontaneously, be inherited genetically or be acquired by infection and affect a variety of mammalian species including humans. Prion infections in the central nervous system (CNS) cause extensive neuropathology, including abnormal accumulations of misfolded host prion protein, vacuolar change resulting in sponge-like (spongiform) appearance of CNS tissue, neurodegeneration and reactive glial responses. Many different prion agent strains exist and these can differ based on disease duration, clinical signs and the targeting and distribution of the neuropathology in distinct brain areas. Reactive astrocytes are a prominent feature in the prion disease affected CNS as revealed by distinct morphological changes and upregulation of glial fibrillary acidic protein (GFAP). The CD44 antigen is a transmembrane glycoprotein involved in cell-cell interactions, cell adhesion and migration. Here we show that CD44 is also highly expressed in a subset of reactive astrocytes in regions of the CNS targeted by prions. Astrocyte heterogeneity revealed by differential CD44 upregulation occurs coincident with the earliest neuropathological changes during the pre-clinical phase of disease, and is not affected by the route of infection. The expression and distribution of CD44 was compared in brains from a large collection of 15 distinct prion agent strains transmitted to mice of different prion protein (Prnp) genotype backgrounds. Our data show that the pattern of CD44 upregulation observed in the hippocampus in each prion agent strain and host Prnp genotype combination was unique. Many mouse-adapted prion strains and hosts have previously been characterized based on the pattern of the distribution of the spongiform pathology or the misfolded PrP deposition within the brain. Our data show that CD44 expression also provides a reliable discriminatory marker of prion infection with a greater dynamic range than misfolded prion protein deposition, aiding strain identification. Together, our data reveal CD44 as a novel marker to detect reactive astrocyte heterogeneity during CNS prion disease and for enhanced identification of distinct prion agent strains.

Subregional differences in astrocytes underlie selective neurodegeneration or protection in Parkinson's disease models in culture.

Parkinson's disease (PD) is characterized by the selective degeneration of dopamine (DA) neurons of the substantia nigra pars compacta (SN), while the neighboring ventral tegmental area (VTA) is relatively spared. The mechanisms underlying this selectivity are not fully understood. Here, we demonstrate a vital role for subregional astrocytes in the protection of VTA DA neurons. We found that elimination of astrocytes in vitro exposes a novel vulnerability of presumably protected VTA DA neurons to the PD mimetic toxin MPP+ , as well as exacerbation of SN DA neuron vulnerability. Conversely, VTA astrocytes protected both VTA and SN DA neurons from MPP+ toxicity in a dose dependent manner, and this protection was mediated via a secreted molecule. RNAseq analysis of isolated VTA and SN astrocytes demonstrated a vast array of transcriptional differences between these two closely related populations demonstrating regional heterogeneity of midbrain astrocytes. We found that GDF15, a member of the TGFβ superfamily which is expressed 230-fold higher in VTA astrocytes than SN, recapitulates neuroprotection of both rat midbrain and iPSC-derived DA neurons, whereas its knockdown conversely diminished this effect. Neuroprotection was likely mediated through the GRFAL receptor expressed on DA neurons. Together; these results suggest that subregional differences in astrocytes underlie the selective degeneration or protection of DA neurons in PD.

Astrocyte Heterogeneity: Impact to Brain Aging and Disease.

Astrocytes, one of the largest glial cell population in the central nervous system (CNS), play a key function in several events of brain development and function, such as synapse formation and function, control of neurotransmitters release and uptake, production of trophic factors and control of neuronal survival. Initially described as a homogenous population, several evidences have pointed that astrocytes are highly heterogeneous, both morphologically and functionally, within the same region, and across different brain regions. Recent findings suggest that the heterogeneity in the expression profile of proteins involved in astrocyte function may predict the selective vulnerability of brain regions to specific diseases, as well as to the age-related cognitive decline. However, the molecular mechanisms underlying these changes, either in aging as well as in brain disease are scarce. Neuroinflammation, a hallmark of several neurodegenerative diseases and aging, is reported to have a dubious impact on glial activation, as these cells release pro- and anti-inflammatory cytokines and chemokines, anti-oxidants, free radicals, and neurotrophic factors. Despite the emerging evidences supporting that reactive astrocytes have a duality in their phenotype, neurotoxic or neuroprotective properties, depending on the age and stimuli, the underlying mechanisms of their activation, cellular interplays and the impact of regional astrocyte heterogeneity are still a matter of discussion. In this review article, we will summarize recent findings on astrocyte heterogeneity and phenotypes, as well as their likely impact for the brain function during aging and neural diseases. We will focus on the molecules and mechanisms triggered by astrocyte to control synapse formation in different brain regions. Finally, we will discuss new evidences on how the modulation of astrocyte phenotype and function could impact the synaptic deficits and glial dysfunction present in aging and pathological states.

Astrocytes in Neuropathologies Affecting the Frontal Cortex.

To an increasing extent, astrocytes are connected with various neuropathologies. Astrocytes comprise of a heterogeneous population of cells with region- and species-specific properties. The frontal cortex exhibits high levels of plasticity that is required for high cognitive functions and memory making this region especially susceptible to damage. Aberrations in the frontal cortex are involved with several cognitive disorders, including Alzheimer’s disease, Huntington’s disease and frontotemporal dementia. Human induced pluripotent stem cells (iPSCs) provide an alternative for disease modeling and offer possibilities for studies to investigate pathological mechanisms in a cell type-specific manner. Patient-specific iPSC-derived astrocytes have been shown to recapitulate several disease phenotypes. Addressing astrocyte heterogeneity may provide an improved understanding of the mechanisms underlying neurodegenerative diseases.

Region-Restrict Astrocytes Exhibit Heterogeneous Susceptibility to Neuronal Reprogramming.

SummaryThe adult CNS has poor ability to replace degenerated neurons following injury or disease. Recently, direct reprogramming of astrocytes into induced neurons has been proposed as an innovative strategy toward CNS repair. As a cell population that shows high diversity on physiological properties and functions depending on their spatiotemporal distribution, however, whether the astrocyte heterogeneity affect neuronal reprogramming is not clear. Here, we show that astrocytes derived from cortex, cerebellum, and spinal cord exhibit biological heterogeneity and possess distinct susceptibility to transcription factor-induced neuronal reprogramming. The heterogeneous expression level of NOTCH1 signaling in the different CNS regions-derived astrocytes is shown to be responsible for the neuronal reprogramming diversity. Taken together, our findings demonstrate that region-restricted astrocytes reveal different intrinsic limitation of the response to neuronal reprogramming.

Astrocyte Heterogeneity in the Adult Central Nervous System.

Astrocytes are the most abundant cell type in the central nervous system (CNS), providing critical roles in the overall maintenance and homeostasis. Over 100 years ago, Cajal first showed morphological depictions of different astrocyte populations. Surprisingly, to date astrocytes remain classified in two groups based on their morphological and neuroanatomical positioning. However, accumulating evidence over the past few years is showing that astrocytes are highly diverse throughout the CNS. Astrocyte heterogeneity is not surprisingly, as these cells interact with all other cells in the CNS. Like neurons, astrocytes may also have subpopulations that vary in their functionality. In this mini review, we will explore some of the recent evidence in the adult CNS of astrocyte diversity. First, we will review the very little literature on healthy adult astroglia heterogeneity, followed by the identification of different subpopulations in disease states and how this varies between human and mouse. Exploring this new area of neuroscience will hopefully provide researchers with a new perspective on astrocytes and their heterogeneity throughout the CNS.

Intracortical astrocyte subpopulations defined by astrocyte reporter Mice in the adult brain.

Although historically regarded as a homogeneous cell population, astrocytes in different brain regions exhibit differences in their physiological properties, such as gap-junction coupling, glutamate uptake dynamics, and intracellular Ca2+ response. Recent in vivo RNA profiles have further demonstrated the molecular heterogeneity of astrocytes in the adult CNS. Astrocyte heterogeneity exists not only inter-regionally but also intra-regionally. Despite the characteristic laminal organization of cortical layers and multiple sources of radial glia progenitors for (astro)gliogenesis, the molecular profile and functional properties of astroglial subpopulations in the adult cerebral cortex remain essentially undefined. Using two astrocyte reporter mouse lines: eaat2-tdTomato and Bac aldh1l1-eGFP, we identified tdT- eGFP+ , tdTlow eGFP+ , and tdThigh eGFP+ astroglial subpopulations (in an approximate 1:7:2 ratio) within the cortex. The tdT- eGFP+ astrocyte population is selectively localized at layers I-II and exhibits increased resting membrane potential and membrane resistance but reduced functional expression of the potassium channel Kir4.1. We also isolated individual astrocyte subpopulations through fluorescence activated cell sorting (FACS) and examined their transcriptome differences by RNA-seq. We found that the whole-genome transcriptional profiles of tdT- eGFP+ astrocytes are drastically different from that of tdTlow eGFP+ and tdThigh eGFP+ astrocytes. Particularly, elevated levels of several nonastrocyte genes that are typically specific to other glial cells, such as mog, mobp, Iba1, and pdgfrα, are observed in tdT- eGFP+ astrocytes, suggesting a less-specific molecular identity of these astrocytes. Overall, our study has unveiled molecular differences between adult cortical astroglial subpopulations, shedding new light on understanding their unique functions in the adult cortex.

Phenotypic heterogeneity of astrocytes in motor neuron disease.

Accumulating evidence has shown that astrocytes do not just support the function of neurons, but play key roles in maintaining the brain environment in health and disease. Contrary to the traditional understanding of astrocytes as static cells, reactive astrocytes possess more diverse functions and phenotypes than previously predicted. In the present focused review, we summarize the evidence showing that astrocytes are playing profound roles in the disease process of amyotrophic lateral sclerosis. Aberrantly activated astrocytes in amyotrophic lateral sclerosis rodents express microglial molecular markers and provoke toxicities to accelerate disease progression. In addition, TIR domain–containing adapter protein–inducing interferon‐β‐dependent innate immune pathway in astrocytes also has a novel function in terminating glial activation and neuroinflammation. Furthermore, heterogeneity in phenotypes and functions of astrocytes are also observed in various disease conditions, such as other neurodegenerative diseases, ischemia, aging and acute lesions in the central nervous system. Through accumulating knowledge of the phenotypic and functional diversity of astrocytes, these cells will become more attractive therapeutic targets for neurological diseases.

Multiple origins and modularity in the spatiotemporal emergence of cerebellar astrocyte heterogeneity

The morphological, molecular, and functional heterogeneity of astrocytes is under intense scrutiny, but how this diversity is ontogenetically achieved remains largely unknown. Here, by quantitative in vivo clonal analyses and proliferation studies, we demonstrate that the major cerebellar astrocyte types emerge according to an unprecedented and remarkably orderly developmental program comprising (i) a time-dependent decline in both clone size and progenitor multipotency, associated with clone allocation first to the hemispheres and then to the vermis(ii) distinctive clonal relationships among astrocyte types, revealing diverse lineage potentials of embryonic and postnatal progenitors; and (iii) stereotyped clone architectures and recurrent modularities that correlate to layer-specific dynamics of postnatal proliferation/differentiation. In silico simulations indicate that the sole presence of a unique multipotent progenitor at the source of the whole astrogliogenic program is unlikely and rather suggest the involvement of additional committed components.

Ventral midbrain astrocytes display unique physiological features and sensitivity to dopamine D2 receptor signaling

Astrocytes are ubiquitous CNS cells that support tissue homeostasis through ion buffering, neurotransmitter recycling, and regulation of CNS vasculature. Yet, despite the essential functional roles they fill, very little is known about the physiology of astrocytes in the ventral midbrain, a region that houses dopamine-releasing neurons and is critical for reward learning and motivated behaviors. Here, using a combination of whole-transcriptome sequencing, histology, slice electrophysiology, and calcium imaging, we performed the first functional and molecular profiling of ventral midbrain astrocytes and observed numerous differences between these cells and their telencephalic counterparts, both in their gene expression profile and in their physiological properties. Ventral midbrain astrocytes have very low membrane resistance and inward-rectifying potassium channel-mediated current, and are extensively coupled to surrounding oligodendrocytes through gap junctions. They exhibit calcium responses to glutamate but are relatively insensitive to norepinephrine. In addition, their calcium activity can be dynamically modulated by dopamine D2 receptor signaling. Taken together, these data indicate that ventral midbrain astrocytes are physiologically distinct from astrocytes in cortex and hippocampus. This work provides new insights into the extent of functional astrocyte heterogeneity within the adult brain and establishes the foundation for examining the impact of regional astrocyte differences on dopamine neuron function and susceptibility to degeneration.

Functional Astrocyte Heterogeneity and Implications for Their Role in Shaping Neurotransmission.

In recent years, the role of astrocytes in shaping neuronal signaling has come to the forefront of neuroscience research. The development of genetic tools that enable targeted manipulation of astrocytes has revealed a wealth of mechanisms by which they can alter the synaptic strength and intrinsic excitability of neurons in behaviorally relevant ways. In parallel, several studies have demonstrated significant variability in the gene expression and physiology of astrocytes within and between brain regions. Thus, to form an accurate understanding of how astrocytes contribute to neuronal transmission, we must take into consideration the diversity that exists in their intrinsic properties. In this review, we will summarize recent findings on astrocyte heterogeneity and discuss the implications for their interactions with neurons and their effects on neuronal transmission.

Regulation of Astrocyte Functions in Multiple Sclerosis.

Astrocytes play complex roles in health and disease. Here, we review recent findings on molecular pathways that control astrocyte function in multiple sclerosis (MS) as well as new tools for their investigation. In particular, we describe positive and negative regulators of astrocyte-mediated pathogenesis in MS, such as sphingolipid metabolism and aryl hydrocarbon receptor signaling, respectively. In addition, we also discuss the issue of astrocyte heterogeneity and its relevance for the contribution of astrocytes to MS pathogenesis. Finally, we discuss how new genomic tools could transform the study of astrocyte biology in MS.

A review of functional heterogeneity among astrocytes and the CS56-specific antibody-mediated detection of a subpopulation of astrocytes in adult brains.

Astrocytes comprise the largest class of glial cells in the mammalian central nerve system (CNS). Although astrocytes were long considered to be a homogeneous population of neuron-supporting cells, recent decades have seen a shift toward the recognition that astrocytes exhibit morphological and functional heterogeneities and serve as essential modulators of brain functions. However, the mechanism underlying astrocyte diversity remains unclear, and the different subpopulations are difficult to identify due to a lack of specific cell markers. In this review, I discuss current knowledge regarding astrocyte heterogeneity and introduce a subpopulation that can be detected via labeling with a chondroitin sulfate-specific antibody (CS56). These CS56-positive astrocytes were found to selectively express tenascin-R (TNR) in the adult mouse cerebral cortex. Further research demonstrated significantly lower levels of glutamate uptake activity and glutamate aspartate transporter expression in TNR-knockdown astrocytes relative to controls, suggesting that the expression and secretion of Tnr by a subpopulation of astrocytes may contribute to region-specific neuron-astrocyte interactions. In summary, these results suggest that CS56-specific antibody and Tnr could be used as novel markers to detect an astrocyte subpopulation in the adult CNS.

Different Mixed Astrocyte Populations Derived from Embryonic Stem Cells Have Variable Neuronal Growth Support Capacities.

Central nervous system injury often leads to functional impairment due, in part, to the formation of an inhibitory glial scar following injury that contributes to poor regeneration. Astrocytes are the major cellular components of the glial scar, which has led to the belief that they are primarily inhibitory following injury. Recent work has challenged this by demonstrating that some astrocytes are required for spinal cord regeneration and astrocytic roles in recovery depend on their phenotype. In this work, two mixed populations containing primarily either fibrous or protoplasmic astrocytes were derived from mouse embryonic stem cells (mESCs). Motoneuron and V2a interneuron growth on live cultures, freeze-lysed cultures, or decellularized extracellular matrix (ECM) from astrocytes were assessed. Both neuronal populations were found to extend significantly longer neurites on protoplasmic-derived substrates than fibrous-derived substrates. Interestingly, neurons extended longer neurites on protoplasmic-derived ECM than fibrous-derived ECM. ECM proteins were compared with in vivo astrocyte expression profiles, and it was found that the ESC-derived ECMs were enriched for astrocyte-specific proteins. Further characterization revealed that protoplasmic ECM had significantly higher levels of axon growth promoting proteins, while fibrous ECM had significantly higher levels of proteins that inhibit axon growth. Supporting this observation, knockdown of spondin-1 improved neurite growth on fibrous ECM, while laminin α5 and γ1 knockdown decreased neurite growth on protoplasmic ECM. These methods allow for scalable production of specific astrocyte subtype-containing populations with different neuronal growth support capacities, and can be used for further studies of the functional importance of astrocyte heterogeneity.

Resolving Astrocyte Heterogeneity in the CNS.

Astrocytes play essential roles in nearly all aspects of brain function from modulating synapses and neurovasculature to preserving appropriate extracellular solute concentrations. To meet the complex needs of the central nervous system (CNS), astrocytes possess highly specialized properties that are optimized for their surrounding neural circuitry. Precisely how these diverse astrocytes types are generated in vivo, however, remains poorly understood. Key to this process is a critical balance of intrinsic developmental patterning and context-dependent environmental signaling events that configures astrocyte phenotype. Indeed, emerging lines of evidence indicate that persistent cues from neighboring cells in the mature CNS cooperate with early patterning events to promote astrocyte diversity. Consistent with this, manipulating Sonic hedgehog (Shh), Notch and fibroblast growth factor (FGF) signaling in the adult brain, have profound effects on the structural, morphological and physiological state of mature astrocytes. These pathways may become disrupted in various neurological diseases and contribute to CNS pathology. This mini-review article focuses on how context-dependent environmental cues cooperate with intrinsic developmental patterning events to control astrocyte diversity in vivo in order to promote healthy brain function.

Molecular and Functional Properties of Regional Astrocytes in the Adult Brain.

The molecular signature and functional properties of astroglial subtypes in the adult CNS remain largely undefined. By using translational ribosome affinity purification followed by RNA-Seq, we profiled astroglial ribosome-associated (presumably translating) mRNAs in major cortical and subcortical brain regions (cortex, hippocampus, caudate-putamen, nucleus accumbens, thalamus, and hypothalamus) of BAC aldh1l1-translational ribosome affinity purification (TRAP) mice (both sexes). We found that the expression of astroglial translating mRNAs closely follows the dorsoventral axis, especially from cortex/hippocampus to thalamus/hypothalamus posteriorly. This region-specific expression pattern of genes, such as synaptogenic modulator sparc and transcriptional factors (emx2, lhx2, and hopx), was validated by qRT-PCR and immunostaining in brain sections. Interestingly, cortical or subcortical astrocytes selectively promote neurite growth and synaptic activity of neurons only from the same region in mismatched cocultures, exhibiting region-matched astrocyte to neuron communication. Overall, these results generated new molecular signature of astrocyte types in the adult CNS, providing insights into their origin and functional diversity.SIGNIFICANCE STATEMENT We investigated the in vivo molecular and functional heterogeneity of astrocytes inter-regionally from adult brain. Our results showed that the expression pattern of ribosome-associated mRNA profiles in astrocytes closely follows the dorsoventral axis, especially posteriorly from cortex/hippocampus to thalamus/hypothalamus. In line with this, our functional results further demonstrated region-selective roles of cortical and subcortical astrocytes in regulating cortical or subcortical neuronal synaptogenesis and maturation. These in vivo studies provide a previously uncharacterized and important molecular atlas for exploring region-specific astroglial functions.

Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination.

Astrocytes have emerged as essential regulators of function and response to injury in the brain and spinal cord, yet very little is known about regional differences that exist. Here we compare the expression of key astroglial markers (glial fibrillary acidic protein (GFAP) and Aldehyde Dehydrogenase-1 Family Member L1 (ALDH1L1)) across these disparate poles of the neuraxis, tracking their expression developmentally and in the context of demyelination. In addition, we document changes in the astrocyte regulatory cytokine interleukin 6 (IL-6), and its signaling partner signal transducer and activator of transcription 3 (STAT3), in vivo and in vitro. Results demonstrate that GFAP expression is higher in the developing and adult spinal cord relative to brain. Comparisons between GFAP and ALDH1L1 expression suggest elevations in spinal cord GFAP during the early postnatal period reflect an accelerated appearance of astrocytes, while elevations in adulthood reflect higher expression by individual astrocytes. Notably, increases in spinal cord compared to whole brain GFAP were paralleled by higher levels of IL-6 and STAT3. Equivalent elevations in GFAP, GFAP/ALDH1L1 ratios, and in IL-6, were observed in primary astrocyte cultures derived from spinal cord compared to cortex. Also, higher levels of GFAP were observed in the spinal cord compared to the brain after focal demyelinating injury. Altogether, these studies point to key differences in astrocyte abundance and the expression of GFAP and IL-6 across the brain and spinal cord that are positioned to influence regional specialization developmentally and responses occurring in the context of injury and disease.

Heterogeneity in Synaptogenic Profile of Astrocytes from Different Brain Regions.

Astrocytes, the most abundant glial cells in the central nervous system (CNS), comprise a heterogeneous population of cells. However, how this heterogeneity impacts their function within brain homeostasis and response to injury and disease is still largely unknown. Recently, astrocytes have been recognized as important regulators of synapse formation and maturation. Here, we analyzed the synaptogenic property of astrocytes from different regions of the CNS. The effect of conditioned medium derived from astrocytes (astrocyte-conditioned medium (ACM)) from cerebral cortex, hippocampus, midbrain and cerebellum, in synapse formation, was evaluated. Synapse formation was analyzed by quantification of pre- and postsynaptic proteins, synaptophysin, and postsynaptic density protein 95 (PSD-95). ACM from the four regions increased significantly the number of synaptophysin/PSD-95 puncta on neurons from the same and different brain regions. Differences on astrocytic synaptogenic potential between the regions were observed according to ACM protein concentration. Thus, cerebellar astrocytes have higher synaptogenic effect when ACM is less concentrated. Also, heterotypical co-culture assays revealed that neurons from cerebral cortex and midbrain equally respond to ACM, indicating that differences in synapse effect are unlike to be neuron-autonomous. The expression profile of the synaptogenic molecules secreted by astrocytes from distinct brain regions was analyzed by qPCR. Gene expression of glypicans 4 and 6, hevin, and secreted protein-acidic and rich in cysteine (SPARC) greatly varies between astrocytes from different brain regions. Furthermore, in vivo analysis of hevin protein confirmed that variance. These findings highlight the heterogeneity of astrocytes and suggest that their synaptogenic potential may be different in each brain region, mainly due to distinct gene expression profiles.