Deciphering the spectrum of astrocyte diversity: Insights into molecular, morphological, and functional dimensions in health and neurodegenerative diseases

Astrocytes are the most abundant and morphologically complex glial cells that play active roles in the central nervous system (CNS). Recent research has identified shared and region-specific astrocytic genes and functions, elucidated the cellular origins of their regional diversity, and uncovered the molecular networks for astrocyte morphology, which are essential for their functional complexity. Reactive astrocytes exhibit a wide range of functional diversity in a context-specific manner in CNS disorders. This review discusses


Introduction
The central nervous system (CNS) is composed of neurons and a group of non-neuronal cells including glial cells.Therefore, to explore how the CNS executes its functions, it is important to examine both neuronal and glial cells.Astrocytes, which comprise the majority of glial cells, are morphologically complex and ubiquitously populate the entire CNS, interacting with other CNS-resident cells (Allen and Lyons, 2018).Astrocytes play crucial roles in maintaining neural circuit functionality, including ion homeostasis, clearance of neurotransmitters, formation and elimination of synapses, formation of the neurovascular unit, and production of neurotrophic factors in health and disease (Khakh and Deneen, 2019;Sofroniew, 2020).Unlike neurons, which are extremely diverse within the CNS, astrocytes have traditionally been considered homogeneous.However, recent studies using multiple methods, including RNA sequencing, have revealed that astrocytes exhibit differences between several CNS regions (Chai et al., 2017;John Lin et al., 2017;Boisvert et al, 2017;Lanjakornsiripan et al., 2018;Bayraktar et al., 2020;Batiuk et al., 2020).These findings underscore the potential diversity of astrocytes and suggest that their J o u r n a l P r e -p r o o f complex morphology may facilitate multifaceted functions across different CNS regions.
Nonetheless, a comprehensive molecular assessment of astrocyte diversity, similarity, or morphology across the CNS has yet to be conducted.
In addition to their essential roles in the healthy CNS, astrocytes display reactive states in CNS disorders, traditionally referred to as astrocyte reactivity.This reactivity has been regarded as passive and homogeneous and has been described only for the evaluation of disease severity.However, accumulating evidence suggests that astrocyte reactivity is diverse and context-dependent, resulting in a wide variety of changes such as alterations in molecular profile, morphology, and function.These changes can significantly affect the progression of neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) (Yu et al., 2020;Sofroniew, 2020).
Targeting astrocytes to reduce mutant superoxide dismutase 1 (SOD1) expression markedly prolonged the survival of SOD1-ALS mice with reduced astrocyte reactivity (Yamanaka et al., 2008).This highlights the need to understand the molecular underpinnings of the functional alterations of astrocytes in specific CNS disorders.This review focuses on recent advances in understanding the mechanisms of molecular and morphological diversity of astrocytes in the healthy CNS and their relation to the diverse functional roles of astrocytes in neurodegenerative diseases such as AD and ALS.

Astrocyte diversity in healthy CNS
J o u r n a l P r e -p r o o f 2.1.Exploring shared and region-specific molecular signatures of astrocytes and the underlying mechanism Recent advances in research technologies such as RNA sequencing have revealed astrocyte diversity within a single region or across multiple CNS regions (Chai et al., 2017;John Lin et al., 2017;Boisvert et al, 2017;Lanjakornsiripan et al., 2018;Bayraktar et al., 2020;Batiuk et al., 2020).Astrocytes in different regions of the mouse CNS show differences in gene and protein expression, morphology, and Ca 2+ signaling properties (Chai et al., 2017).Astrocyte diversity has also been observed in specific CNS regions (Boisvert et al, 2017;Lanjakornsiripan et al., 2018;Batiuk et al., 2020).However, comprehensive studies on the diversity and similarities of astrocytes throughout the CNS are lacking.Furthermore, the mechanisms underlying the astrocytic diversity remain unclear.To address these gaps, previously, my colleagues and I investigated 13 major CNS regions (Figure 1A).First, we conducted whole-brain imaging of astrocytes to assess their density.We found 2-fold differences in astrocyte density between the 13 CNS regions compared to neurons (Figure 1B (i) and (ii)).This may reflect the importance of astrocytes in maintaining homeostasis.
However, when assessing the expression of astrocytic markers such as S100β, Aldh1l1, and GFAP, significant differences in protein expression patterns were observed across these regions (Figure 1B (iii)), suggesting functional diversity among astrocytes of different regions.Subsequently, through astrocyte-specific and bulk tissue RNA sequencing of the 13 CNS regions (Figure 1C and D) we identified 825 commonly shared genes enriched in astrocytes (Figure 1E).Pathway analysis revealed the core functions of astrocytes in lipid, carbohydrate, neurotransmitter, and potassium ion metabolism (Figure 1F).Additionally, J o u r n a l P r e -p r o o f gene expression variability analysis of astrocytes in 13 CNS regions showed region-specific patterns, and cluster analysis classified them into three groups based on their anatomical proximity (cerebrum, cerebellum, brainstem, and spinal cord) (Figure 1G).What is the cause of this similarity in astrocytes?Further upstream analysis of these genes identified numerous transcription factors and nuclear receptors, suggesting that the functions of astrocytes in maintaining homeostasis were encoded (Figure 2A and E).What is the mechanism underlying astrocyte diversity?Single-cell RNA sequencing (scRNA-seq) of astrocytes of the cerebral cortex, hippocampus, and striatum, which were classified into the same group using cluster analysis, revealed the existence of seven subclusters at different ratios in these three regions (Figure 2B).Upstream analysis of these subclusters identified subcluster-specific extracellular signaling molecules (Figure 2C).Moreover, regionspecific genes in astrocytes correlated with the results from the bulk tissue (Figure 2D).The tissue microenvironment, which refers to the cellular and biochemical factors that surround astrocytes, includes neighboring cells, extracellular matrix components, and signaling molecules such as cytokines, growth factors, and neurotransmitters.These elements collectively influence astrocyte behavior, gene expression, and the functional state.
Collectively, these results strongly suggest that the microenvironment influences the mechanism underlying the region-specific diversity of astrocytes (Figure 2E).Morphological complexity is a key feature of astrocytes that supports their physiological functions (Khakh and Deneen, 2019).Although genes involved in astrocyte morphology, such as neuroligins and HEPACAM, have been reported (Stogsdill et al., 2017;Baldwin et al., 2021), no systematic research has identified genes associated with the morphological complexity of astrocytes in an unbiased manner.My colleagues and I previously analyzed astrocyte morphology across 13 regions using 10 distinct morphological parameters and observed morphological diversity (Figure 3A).Subsequently, these morphological parameters were correlated with the gene profiles of astrocytes from the same regions using a weighted correlation network analysis (WGCNA).This analysis revealed that gene networks were closely linked to astrocyte territory size, which represented the morphological complexity of astrocytes (Figure 3B and C).Notably, this gene set included genes associated with AD risk, such as Fermt2 (Figure 3C).Fermt2 and Ezr were focused on as candidate genes.Fermt2 encodes a protein that links the extracellular matrix to the cytoskeleton.This linkage is crucial for cellular adhesion, signal transduction, and cell shape maintenance, motility, and survival (Gahmberg and Grönholm, 2022).In astrocytes, Fermt2 is essential for preserving the structural integrity and functional connectivity within the CNS.Ezr encodes a member of the Ezrin-Radixin-Moesin (ERM) family, which connects the actin cytoskeleton to the plasma membrane.Ezr regulates cell shape and surface dynamics, and plays a significant role in astrocyte morphological complexity and interactions with other cells and the extracellular environment (Fehon et al., 2023).To test the hypothesis that these astrocyte morphology-related genes regulate morphological complexity, my colleagues and I developed an astrocyte-specific gene knockdown method J o u r n a l P r e -p r o o f using CRISPR/Cas9 (Figure 3D).By knocking out the expression of the candidate genes Fermt2 and Ezr in the hippocampus of wild-type mice (Figure 3E), we observed a decrease in astrocyte territory size (Figure 3F), impaired spatial memory (Figure 3G), elevated c-Fos expression in hippocampal CA1 neurons (Figure 3H), and reduced synaptic colocalization between the pre-and post-synapses (Figure 3I).These findings are consistent with those of recent studies (Badia-Soteras et al., 2022;Popov et al., 2023) that explored the role of Ezr in behavior and aging.Badia-Soteras et al. demonstrated that alterations in Ezr expression affect cognitive function and behavioral outcomes, linking astrocyte morphology to broader physiological and pathological states.Similarly, Popov et al. highlighted how changes in astrocyte morphology influence aging processes, emphasizing the importance of Ezr in maintaining astrocyte function.Overall, these findings establish a functional link between astrocyte morphology-related genes, astrocyte morphological complexity, and neuronal circuit output (Figure 3J).Such alterations may provide insight into how astrocytes contribute to the underlying mechanisms of CNS disorders.

Diverse roles of reactive astrocytes in neuroinflammation
Astrocytic dysfunction plays an important role in the development of neurological and psychiatric diseases.Astrocytes react to these diseases through context-specific molecular and functional alterations (Yu et al., 2020), which may have both beneficial and detrimental effects on neuronal function.Reactive astrocytes and neuroinflammation are hallmarks of neurodegenerative diseases such as AD.However, the exact functional contributions of the reactive astrocytes are not fully understood.
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A1/A2 hypothesis and molecular mechanism of reactive astrocytes in CNS disorders
To explore the molecular underpinnings of astrocytic reactivity in neuroinflammation, a concept similar to the M1/M2 hypothesis for microglia has been proposed.The hypothesis suggests that astrocytes may adopt one of the two binary states: neurotoxic (A1) or neuroprotective (A2) (Liddelow et al., 2017).A1-associated genes were elevated in astrocytes of mouse models of inflammation, whereas A2-related genes showed increased expression in astrocytes of mouse models of stroke.A1 astrocytes, which are triggered by inflammatory factors secreted from microglia, such as tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), and C1q, have been identified in several major neurodegenerative diseases, including AD, HD, and ALS.Experiments in SOD1-ALS mice have shown that eliminating TNF-α, IL-1α, and C1q (not exclusively from microglia) reduces A1-related factors and prolongs survival (Guttenplan et al., 2021).However, recent studies regarding RNA-seq and scRNA-seq of astrocytes in AD and HD mice have reported no increase in A1-associated gene expression, indicating the need for further investigation (Diaz-Castro et al., 2019;Jiwaji et al., 2022;Endo et al., 2022).Considering that the A1 and A2 classifications of astrocytes are defined by a limited set of genes, understanding the pathology of astrocytes in CNS disorders solely based on this concept remains controversial (Escartin et al., 2021).A recent study has provided a more insightful perspective on astrocyte reactivity based on scRNA seq.Cameron et al. revealed a wide array of reactive astrocyte subtypes with distinct gene expression profiles in mouse models of injury (Cameron et al., 2024), suggesting that a spectrum of activation states is influenced by the microenvironment and disease.
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Diverse roles of reactive astrocytes in the context of neuroinflammation in AD
In patients with AD and mouse models of AD, astrocytes show reactivity around Aβ plaques with high expression of glial fibrillary acidic protein (GFAP).Single-nucleus RNA sequencing (snRNA-seq) in 5xFAD AD mice revealed a subset of disease-associated astrocytes (DAA) marked by high GFAP expression, which was also observed in aged mice (Habib et al., 2020).This finding suggests potentially shared pathways in astrocytic abnormalities between AD and aging, although the specific roles and functions of DAAs require further investigation.
Under physiological conditions, astrocytes modulate intracellular Ca 2+ signaling in response to external neurotransmitters and adenosine triphosphate (ATP), thereby performing various functions.However, in APP/PS1 AD model mice, astrocytes around Aβ plaques exhibit increased Ca 2+ signaling (Kuchibhotla et al., 2009).Intriguingly, in APP/PS1 mice, an upregulation of the purinergic G protein-coupled (P2Y1) receptor expressed near Aβ plaques led to aberrant Ca 2+ signaling.Administration of a P2Y1 receptor antagonist mitigated this aberrant signaling, thereby enhancing cognitive function in these mice (Reichenbach et al., 2017).Furthermore, deletion of signal transducer and activator of transcription 3 (Stat3), a key transcriptional regulator in reactive astrocytes, not only normalized Ca 2+ signaling but also improved Aβ clearance by microglia and astrocytes, contributing to cognitive improvement (Reichenbach et al., 2019).These findings underscore the significant link between altered Ca 2+ signaling in astrocytes and AD-related neuroinflammation.Jiwaji et al. conducted astrocyte-specific RNA-seq in AD J o u r n a l P r e -p r o o f (APP/PS1) and tauopathy (MaptP301S) mice and identified Nrf2, a transcription factor that reacts to oxidative stress, as a pivotal regulator of diverse genes.Overexpressing Nrf2 specifically in astrocytes of both models attenuated pathological changes and neurodegeneration linked with Aβ and tau pathology (Jiwaji et al., 2022), suggesting its crucial role in modulating neurodegenerative processes.

Significance of astrocyte morphology-related genes in AD and other CNS disorders
As discussed above, reactive astrocytes are crucial in the pathology of AD.However, the specific alterations in astrocyte morphology in AD and other CNS disorders are not fully understood.By analyzing gene expression in astrocytes from APP/PS1 mice via scRNAseq and astrocytes from AD postmortem tissue using snRNA-seq, it was discovered that genes positively correlated with astrocyte territory size were notably downregulated in AD (Figure 4A).Interestingly, similar gene expression patterns were observed in snRNA-seq data from HD mice (Figure 4B), and a significant decrease in astrocyte territory size was confirmed in mouse models of AD and HD (Figure 4C and D) (Yu et al., 2020;Endo et al., 2022).The significant overlap between astrocyte morphology-related and diseaseassociated genes in various neurological and psychiatric diseases indicated that a decrease in the morphological complexity of astrocytes may represent a common pathological feature across a spectrum of CNS disorders (Figure 4E and F).

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In reactive astrocytes, alterations in the expression of neurotransmitter receptors and transporters compromise synaptic functions.The concept of astrocyte-derived glutamate excitotoxicity is pivotal for understanding the role of astrocytes in the pathophysiology of neurodegenerative diseases, including ALS.A reduction in the astrocyte-specific glutamate transporter-1 (GLT-1) in both patients with sporadic ALS and SOD1-ALS mice (Howland et al., 2002), led to the theory of excitotoxicity due to excess glutamate.Transplantation of glial precursor cells from SOD1 G93A mice into the spinal cord of wild-type rats resulted in motor neuron degeneration, which is associated with reactive astrocytes and reduced GLT-1 expression (Papadeas et al., 2002).Similarly, transplantation of astrocytes generated from induced pluripotent stem cells (iPSCs) of patients with sporadic ALS into the spinal cord of severe combined immunodeficiency (SCID) mice led to analogous motor neuron degeneration, accompanied by decreased GLT-1 expression in astrocytes (Qian et al., 2017).Intriguingly, astrocytes from both SOD1 G93A mice and patients with sporadic ALS have been found to secrete undefined toxic factors into motor neurons in vitro, inducing necroptosis-like cell death, indicating a shared mechanism of astrocyte-induced motor neuron degeneration in both sporadic and familial forms of ALS (Nagai et al., 2007;Re et al., 2014).Further analysis revealed that astrocytes derived from glial precursor cells in the spinal cord of patients with sporadic and familial ALS exhibit upregulation of inflammatory cytokines and related factors (Haidet-Phillips et al., 2011).Additionally, such reactive astrocytes demonstrate neurotoxicity by diminishing the expression of major histocompatibility complex-1 (MHC-I) molecules in motor neurons (Song et al., 2016), J o u r n a l P r e -p r o o f underscoring the role of reactive astrocyte-mediated neuroinflammation as a crucial therapeutic target in ALS.
3.5 Interaction of reactive astrocytes with microglia and T cells: roles of astrocytic transforming growth factor-β1 in ALS In both AD and ALS, T-cell infiltration into the CNS parenchyma has been observed (Heneka et al., 2015;Endo et al., 2016).Functional T cells such as helper, regulatory, and effector memory T cells have been shown to act as modifiers in these diseases (Beers et al., 2006;Chiu et al., 2008;Baruch et al., 2015;Sheean et al., 2018;Gate et al., 2020).

Depleting functional T-cells has been shown to reduce microglial reactivity and decrease
the expression of neurotrophic factors, thereby accelerating the progression of SOD1 G93A mutation in mice (Beers et al., 2006;Chiu et al., 2008).However, in the context of neuroinflammation in ALS, the mechanism by which astrocytes interact with microglia and T-cells has not yet been fully elucidated.
Our investigation has uncovered astrocytic transforming growth factor-β1 (TGF-β1), one of the major anti-inflammatory cytokines, as a crucial factor influencing the progression of ALS.TGF-βs serve multifaceted roles, including those in the maintenance of immune homeostasis (Li et al., 2006), modulation of neurotrophic responses (Katsuno et al., 2011), and the regulation of microglial development (Butovsky et al., 2014).In AD mice, increasing astrocytic TGF-β1 levels is associated with reduced senile plaque accumulation (Wyss-Coray et al., 1995;Wyss-Coray et al., 2001).Notably, in patients with sporadic J o u r n a l P r e -p r o o f ALS, TGF-β1 protein levels are significantly increased in the serum, plasma, and cerebrospinal fluid (CSF) (Houi et al., 2002;Iłzecka et al., 2002).In a previous study (Endo et al., 2015), TGF-β1 was found to be elevated in the spinal cord astrocytes in patients with sporadic ALS and symptomatic SOD1 G93A mice (Figure 5A and B).To further explore the impact of astrocytic TGF-β1 on pathophysiology of ALS, SOD1 G93A mice were crossbred with transgenic mice engineered to overexpress TGF-β1 specifically within astrocytes (Wyss-Coray et al., 1995).Intriguingly, double-transgenic SOD1 G93A /TGF-β1 mice unexpectedly showed a shortened life span with astrocytic TGF-β1 (Figure 5C).How does astrocytic TGF-β1 accelerate disease progression?Our findings suggest that astrocytic overproduction of TGF-β1 correlates with a marked reduction in the production of neurotrophic factors, notably insulin-like growth factor I (IGF-I), by deactivated microglia.This is accompanied alongside diminished T-cell infiltration with a predominance of interferon γ (IFN-γ) microenvironment over IL-4 in SOD1 G93A /TGF-β1 mice (Figure 5D-F).
This indicates that astrocytic TGF-β1 may suppress the neuroprotective inflammatory responses typically mediated by microglia and T-cells.Conversely, astrocyte-specific reduction of mutant SOD1 slowed disease progression in SOD1 G37R mice (Yamanaka et al., 2008), accompanied by a reduction in TGF-β1 levels within astrocytes and markers of astrocyte reactivity evidenced by GFAP levels, highlighting a detrimental role of astrocytic TGF-β1 in SOD1-ALS mouse models (Figure 5G and H).To translate these findings into experimental therapy, we demonstrated that pharmacological administration of TGF-β signaling inhibitor after disease onset resulted in a modest but significant increase in survival of SOD1 G93A mice (Figure 5I).In summary, astrocytic TGF-β1 is a detrimental J o u r n a l P r e -p r o o f factor in dampening the beneficial microenvironment of microglia and T-cells in ALS (Figure 5J) and might be a potential therapeutic target for ALS (Endo et al., 2015).Recent studies using microglia-specific RNA-seq and scRNA-seq have revealed a subtype of microglia in both AD and SOD1-ALS mouse models.Characterized by elevated expression of APOE and TREM2, genes implicated as risk factors for AD, these microglia are designated as disease-associated microglia (DAM) or the microglial neurodegenerative phenotype (MGnD) (Keren-Shaul et al., 2017;Krasemann et al., 2017).Notably, there is upregulated expression of phagocytosis-associated genes, such as CD11c and CD68, which are reduced in the presence of astrocytic TGF-β1 in SOD1 G93A mice.However, the dichotomy between the neurotoxic and neuroprotective functions of DAM remains unclear.
Intriguingly, altering the genetic background of SOD1 G93A mice from C57BL/6 to BALB/c led to diminished induction of Igf1-positive DAM and accelerated progression of ALS (Komine et al., 2024).This highlights the need for further exploration of the interactions between reactive astrocytes and DAMs within the milieu of ALS and other neurodegenerative diseases to pave the way for novel therapeutic insights.

Future perspectives
This review examines compelling evidence for astrocyte diversity throughout the CNS and the mechanisms underlying this variability.However, significant questions remain unanswered regarding how local cues specific to each region influence astrocyte identity, J o u r n a l P r e -p r o o f the extent to which particular astrocyte populations can be manipulated, and whether the diversity observed in astrocytes is a reversible trait, especially in the context of aging (Baldwin, 2022).These are critical considerations for deepening our understanding of astrocyte pathophysiology in CNS disorders.Advancements in comprehensive gene expression analysis, particularly through RNA sequencing of reactive astrocytes across various neurological disease models, and ATAC sequencing for detailed examination of open chromatin regions, have revealed that changes in gene expression within reactive astrocytes are highly disease-specific and dictated by unique combinations of transcription factors (Burda et al., 2022).This discovery offers promising directions for targeting specific astrocyte subtypes closely linked to specific diseases.Such targeted manipulations have the potential to unravel the complex roles of astrocyte diversity in maintaining CNS health and contributing to CNS disorders, providing clues for novel therapeutic strategies.

Declaration of Competing Interest
None.

2. 2 .
Astrocyte morphological diversity: identification and experimental exploration of the molecular basis for morphological complexity J o u r n a l P r e -p r o o f

Figure captions Figure 1 .
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Figure 2 .
Figure 2. Exploring origins for molecular astrocyte diversity.All data are extracted from

Figure 3 .
Figure 3. Identification and experimental evaluation of the molecular basis for astrocyte