Astrocyte-neuron Communication Research Articles

Astrocyte-neuron communication through the complement C3-C3aR pathway in Parkinson’s disease

Neuroinflammation and autoimmunity are pivotal in the pathogenesis of neurodegenerative diseases. Complement activation and involvement of astrocyte-neuron C3/C3aR pathway have been observed, yet the mechanisms influencing α-synuclein (α-syn) pathology and neurodegeneration remain unclear. In this study, elevated levels of complement C3 were detected in the plasma of α-syn PFF-induced mice and the substantia nigra of A53T transgenic mice. Colocalization of complement C3 with astrocytes was also observed. Overexpression of complement C3 exacerbated motor dysfunction, dopaminergic neuron loss, and phosphorylated α-syn expression in mice injected with α-syn preformed fibrils (α-syn PFFs). Conversely, downregulation of complement C3 protected α-syn PFF-induced mice. Molecular investigations revealed that inhibition of Toll-like receptor 2 (TLR2) or NF-κB reduced complement C3 expression in primary astrocytes following α-syn PFF treatment. Astrocyte-neuron communication via the C3/C3aR pathway influenced α-syn PFF-induced neuronal apoptosis and α-syn pathology, potentially through modulation of GSK3β. These findings underscore the critical role of astrocyte-neuron communication via the C3/C3aR pathway in PD pathogenesis, highlighting its potential as a therapeutic target.

Astrocyte modulation of synaptic plasticity mediated by activity-dependent Sonic hedgehog signaling.

The influence of neural activity on astrocytes and their reciprocal interactions with neurons has emerged as an important modulator of synapse function. Astrocytes exhibit activity-dependent changes in gene expression, yet the molecular mechanisms by which they accomplish this have remained largely unknown. The molecular signaling pathway, Sonic hedgehog (Shh), mediates neuron-astrocyte communication and regulates the organization of cortical synapses. Here, we demonstrate that neural activity stimulates Shh signaling in cortical astrocytes and upregulates expression of Hevin and SPARC, astrocyte derived molecules that modify synapses. Whisker stimulation and chemogenetic activation both increase Shh activity in deep layers of the somatosensory cortex, where neuron-astrocyte Shh signaling is predominantly found. Experience-dependent Hevin and SPARC require intact Shh signaling and selective loss of pathway activity in astrocytes occludes experience-dependent structural plasticity. Taken together, these data identify Shh signaling as an activity-dependent, neuronal derived cue that stimulates astrocyte interactions with synapses and promotes synaptic plasticity.

Dynamics in Redox-Active Molecules Following Ischemic Preconditioning in the Brain.

It is well known that the brain is quite vulnerable to oxidative stress, initiating neuronal loss after ischemia-reperfusion (IR) injury. A potent protective mechanism is ischemic preconditioning (IPC), where proteins are among the primary targets. This study explores redox-active proteins' role in preserving energy supply. Adult rats were divided into the control, IR, and IPC groups. Protein profiling was conducted to identify modified proteins and then verified through activity assays, immunoblot, and immunohistochemical analyses. IPC protected cortex mitochondria, as evidenced by a 2.26-fold increase in superoxide dismutase (SOD) activity. Additionally, stable core subunits of respiratory chain complexes ensured sufficient energy production, supported by a 16.6% increase in ATP synthase activity. In hippocampal cells, IPC led to the downregulation of energy-related dehydrogenases, while a significantly higher level of peroxiredoxin 6 (PRX6) was observed. Notably, IPC significantly enhanced glutathione reductase activity to provide sufficient glutathione to maintain PRX6 function. Astrocytes may mobilize PRX6 to protect neurons during initial ischemic events, by decreased PRX6 positivity in astrocytes, accompanied by an increase in neurons following both IR injury and IPC. Maintained redox signaling via astrocyte-neuron communication triggers IPC's protective state. The partnership among PRX6, SOD, and glutathione reductase appears essential in safeguarding and stabilizing the hippocampus.

1H-NMR metabolomics investigation of CSF from children with HIV reveals altered neuroenergetics due to persistent immune activation.

HIV can invade the central nervous system (CNS) early during infection, invading perivascular macrophages and microglia, which, in turn, release viral particles and immune mediators that dysregulate all brain cell types. Consequently, children living with HIV often present with neurodevelopmental delays. In this study, we used proton nuclear magnetic resonance (1H-NMR) spectroscopy to analyze the neurometabolic profile of HIV infection using cerebrospinal fluid samples obtained from 17 HIV+ and 50 HIV- South African children. Nine metabolites, including glucose, lactate, glutamine, 1,2-propanediol, acetone, 3-hydroxybutyrate, acetoacetate, 2-hydroxybutyrate, and myo-inositol, showed significant differences when comparing children infected with HIV and those uninfected. These metabolites may be associated with activation of the innate immune response and disruption of neuroenergetics pathways. These results elucidate the neurometabolic state of children infected with HIV, including upregulation of glycolysis, dysregulation of ketone body metabolism, and elevated reactive oxygen species production. Furthermore, we hypothesize that neuroinflammation alters astrocyte-neuron communication, lowering neuronal activity in children infected with HIV, which may contribute to the neurodevelopmental delay often observed in this population.

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.

Pharmacological Activation of Piezo1 Channels Enhances Astrocyte-Neuron Communication via NMDA Receptors in the Murine Neocortex.

The Piezo1 mechanosensitive ion channel is abundant on several elements of the central nervous system including astrocytes. It has been already demonstrated that activation of these channels is able to elicit calcium waves on astrocytes, which contributes to the release of gliotransmitters. Astrocyte- and N-methyl-D-aspartate (NMDA) receptor-dependent slow inward currents (SICs) are hallmarks of astrocyte-neuron communication. These currents are triggered by glutamate released as gliotransmitter, which in turn activates neuronal NMDA receptors responsible for this inward current having slower kinetics than any synaptic events. In this project, we aimed to investigate whether Piezo1 activation and inhibition is able to alter spontaneous SIC activity of murine neocortical pyramidal neurons. When the Piezo1 opener Yoda1 was applied, the SIC frequency and the charge transfer by these events in a minute time was significantly increased. These changes were prevented by treating the preparations with the NMDA receptor inhibitor D-AP5. Furthermore, Yoda1 did not alter the spontaneous EPSC frequency and amplitude when SICs were absent. The Piezo1 inhibitor Dooku1 effectively reverted the actions of Yoda1 and decreased the rise time of SICs when applied alone. In conclusion, activation of Piezo1 channels is able to alter astrocyte-neuron communication. Via enhancement of SIC activity, astrocytic Piezo1 channels have the capacity to determine neuronal excitability.

Abstract TP30: Exercise Postconditioning After Ischemic Stroke: Neuroprotection Through NGF/TRPV1-Mediated Astrocyte-Neuron Interaction

Introduction: Astrocytes activation and interaction with neurons potentially affect stroke outcomes. Transient receptor potential vanilloid 1 (TRPV1) participated in reducing brain injury and nerve growth factor (NGF) affected TRPV1 activation. The present study investigated neuroprotection induced by exercise postconditioning (PostE) and elucidated the mechanistic regulation of astrocytes-neuron crosstalk via NGF/TRPV1 pathway. Methods: Adult Sprague-Dawley rats received 2 h middle cerebral artery (MCA) occlusion, followed by 24 h of reperfusion. The treadmill exercise was then initiated 24 h after reperfusion for 3 days (PostE). Brain damage was determined by infarct volume, edema and apoptotic proteins expression (Bcl-2, BAX and Caspase-3). Neuroplasticity was determined by dendritic spine staining and protein expression of BDNF, GAP-43, PSD-95 and synaptophysin. Neuronal TRPV1 expression, astrocyte proliferation and astrocytic NGF expression was assessed with immunofluorescence. Primary astrocyte-neuron Transwell cultivation were used to determine the secretion of astrocytic NGF activating neural TRPV1. Interaction between TRPV1 and NGF or TRPV1 and A-kinase anchoring protein150 (AKAP150) were detected by immunofluorescence and Co-IP. Results: PostE significantly decreased brain damage and induced neuroplasticity in ischemic rats. PostE induced astrocyte proliferation and astrocytic NGF secretion, which, in turn, activated TRPV1 expression in neuron. Either NGF or TRPV1 inhibitor eliminated the neuroplasticity, while NGF inhibitor decreased TRPV1 expression, suggesting the potential regulating role of astrocytic NGF on TRPV1. Primary cells cultivation demonstrated a NGF/TRPV1 and TRPV1/AKAP150 interplays showing astrocyte-neuron communication. Conclusion: PostE induced neuroprotection, which was mediated by the activation of astrocytes and their interaction with neurons through the NGF/TRPV1 pathway, highlighting the significance of astrocyte-neuron crosstalk in post-stroke recovery processes.

Astrocyte ryanodine receptors facilitate gliotransmission and astroglial modulation of synaptic plasticity.

Intracellular Ca2+-signaling in astrocytes is instrumental for their brain "housekeeping" role and astroglial control of synaptic plasticity. An important source for elevating the cytosolic Ca2+ level in astrocytes is a release from endoplasmic reticulum which can be triggered via two fundamental pathways: IP3 receptors and calcium-induced calcium release (CICR) mediated by Ca2+-sensitive ryanodine receptors (RyRs). While the physiological role for glial IP3 became a focus of intensive research and debate, ryanodine receptors received much less attention. We explored the role for ryanodine receptors in the modulation of cytosolic Ca2+-signaling in the cortical and hippocampal astrocytes, astrocyte-neuron communication and astroglia modulation of synaptic plasticity. Our data show that RyR-mediated Ca2+-induced Ca2+-release from ER brings substantial contribution into signaling in the functional microdomains hippocampal and neocortical astrocytes. Furthermore, RyR-mediated CICR activated the release of ATP and glutamate from hippocampal and neocortical astrocytes which, in turn, elicited transient purinergic and tonic glutamatergic currents in the neighboring pyramidal neurons. The CICR-facilitated release of ATP and glutamate was inhibited after intracellular perfusion of astrocytes with ryanodine and BAPTA and in the transgenic dnSNARE mice with impaired astroglial exocytosis. We also found out that RyR-mediated amplification of astrocytic Ca2+-signaling enhanced the long-term synaptic potentiation in the hippocampus and neocortex of aged mice. Combined, our data demonstrate that ryanodine receptors are essential for astrocytic Ca2+-signaling and efficient astrocyte-neuron communications. The RyR-mediated CICR contributes to astrocytic control of synaptic plasticity and can underlie, at least partially, neuroprotective and cognitive effects of caffein.

Astrocyte-Neuron Interactions Contributing to Amyotrophic Lateral Sclerosis Progression.

Amyotrophic lateral sclerosis (ALS) is a complex disease impacting motor neurons of the brain, brainstem, and spinal cord. Disease etiology is quite heterogeneous with over 40 genes causing the disease and a vast ~90% of patients having no prior family history. Astrocytes are major contributors to ALS, particularly through involvement in accelerating disease progression. Through study of genetic forms of disease including SOD1, TDP43, FUS, C9orf72, VCP, TBK1, and more recently patient-derived cells from sporadic individuals, many biological mechanisms have been identified to cause intrinsic or glial-mediated neurotoxicity to motor neurons. Overall, many of the normally supportive and beneficial roles that astrocytes contribute to neuronal health and survival instead switch to become deleterious and neurotoxic. While the exact pathways may differ based on disease-origin, altered astrocyte-neuron communication is a common feature of ALS. Within this chapter, distinct genetic forms are examined in detail, along with what is known from sporadic patient-derived cells. Overall, this chapter highlights the interplay between astrocytes and neurons in this complex disease and describes the key features underlying: astrocyte-mediated motor neuron toxicity, excitotoxicity, oxidative/nitrosative stress, protein dyshomeostasis, metabolic imbalance, inflammation, trophic factor withdrawal, blood-brain/blood-spinal cord barrier involvement, disease spreading, and the extracellular matrix/cell adhesion/TGF-β signaling pathways.

The role of calcium dynamics with amyloid beta on neuron-astrocyte coupling

Amyloid beta ($A\beta$) plaques are associated with neurodegenerative diseases such as Alzheimer's disease. Due to the involvement of $A\beta$ plaques in the functioning of the brain; cognitive decline disrupts calcium homeostasis in nerve cells and causes abnormal calcium ions ($Ca^{2+}$) signaling patterns. In consequence, there is enhanced neuronal excitability, compromised synaptic transmission, and decreased astrocytic function. Neuron-astrocyte coupling through calcium dynamics with different neuronal functions has been studied. Key signaling molecules in this process include $Ca^{2+}$, which control several cellular functions, including neurotransmission and astrocytic regulation. The mathematical model for neuron-astrocyte communication has been developed to study the importance of calcium dynamics in signal transduction between the cells. To understand the wide role of mitochondria, NCX, and amyloid beta with various necessary parameters included in the model, $Ca^{2+}$ signaling patterns have been analyzed through amplitude modulation and frequency modulation. The results of the current model are simulated and analyzed using XPPAUT. The findings of the current study are contrasted with experimental data from an existing mathematical model that illustrates the impact of calcium oscillation frequency and amplitude modulations in nerve cells.

Building transformers from neurons and astrocytes.

Glial cells account for between 50% and 90% of all human brain cells, and serve a variety of important developmental, structural, and metabolic functions. Recent experimental efforts suggest that astrocytes, a type of glial cell, are also directly involved in core cognitive processes such as learning and memory. While it is well established that astrocytes and neurons are connected to one another in feedback loops across many timescales and spatial scales, there is a gap in understanding the computational role of neuron-astrocyte interactions. To help bridge this gap, we draw on recent advances in AI and astrocyte imaging technology. In particular, we show that neuron-astrocyte networks can naturally perform the core computation of a Transformer, a particularly successful type of AI architecture. In doing so, we provide a concrete, normative, and experimentally testable account of neuron-astrocyte communication. Because Transformers are so successful across a wide variety of task domains, such as language, vision, and audition, our analysis may help explain the ubiquity, flexibility, and power of the brain's neuron-astrocyte networks.

Increased expression of Nav1.6 of reactive astrocytes in the globus pallidus is closely associated with motor deficits in a model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease in elderly people, which is characterized by motor disabilities in PD patients. Nav1.6 is the most abundant subtype of voltage-gated sodium channels (VGSCs) in the brain of adult mammals and rodents. Here we investigated the role of Nav1.6 in the external globus pallidus (GP) involved in the pathogenesis of motor deficits in unilateral 6-OHDA(6-hydroxydopamine)lesioned rats. The results show that Nav1.6 is dramatically increased in reactive astrocytes of the ipsilateral GP in the middle stage, but not different from the control rats in the later stage of the pathological process in 6-OHDA lesioned rats. Furthermore, the down-regulation of Nav1.6 expression in the ipsilateral GP can significantly improve motor deficits in 6-OHDA lesioned rats in the middle stage of the pathological process. The electrophysiological experiments show that the down-regulation of Nav1.6 expression in the ipsilateral GP significantly decreases the abnormal high synchronization between the ipsilateral M1 (the primary motor cortex) and GP in 6-OHDA lesioned rats. Ca2+ imaging reveals that the down-regulation of Nav1.6 expression reduces the intracellular concentration of Ca2+ ([Ca2+ ]i) in primary cultured astrocytes. These findings suggest that the increased Nav1.6 expression of reactive astrocytes in the GP play an important role in the pathogenesis of motor dysfunction in the middle stage in 6-OHDA lesioned rats, which may participate in astrocyte-neuron communication by regulating [Ca2+ ]i of astrocytes, thereby contributing to the formation of abnormal electrical signals of the basal ganglia (BG) in 6-OHDA lesioned rats.

Cortical astrocytes modulate dominance behavior in male mice by regulating synaptic excitatory and inhibitory balance.

Social hierarchy is established as an outcome of individual social behaviors, such as dominance behavior during long-term interactions with others. Astrocytes are implicated in optimizing the balance between excitatory and inhibitory (E/I) neuronal activity, which may influence social behavior. However, the contribution of astrocytes in the prefrontal cortex to dominance behavior is unclear. Here we show that dorsomedial prefrontal cortical (dmPFC) astrocytes modulate E/I balance and dominance behavior in adult male mice using in vivo fiber photometry and two-photon microscopy. Optogenetic and chemogenetic activation or inhibition of dmPFC astrocytes show that astrocytes bidirectionally control male mouse dominance behavior, affecting social rank. Dominant and subordinate male mice present distinct prefrontal synaptic E/I balance, regulated by astrocyte activity. Mechanistically, we show that dmPFC astrocytes control cortical E/I balance by simultaneously enhancing presynaptic-excitatory and reducing postsynaptic-inhibitory transmission via astrocyte-derived glutamate and ATP release, respectively. Our findings show how dmPFC astrocyte-neuron communication can be involved in the establishment of social hierarchy in adult male mice.

Astrocytes adjust the dynamic range of cortical network activity to control modality-specific sensory information processing

Cortical neuron-astrocyte communication in response to peripheral sensory stimulation occurs in a topographic-, frequency-, and intensity-dependent manner. However, the contribution of this functional interaction to the processing of sensory inputs and consequent behavior remains unclear. We investigate the role of astrocytes in sensory information processing at circuit and behavioral levels by monitoring and manipulating astrocytic activity invivo. We show that astrocytes control the dynamic range of the cortical network activity, optimizing its responsiveness to incoming sensory inputs. The astrocytic modulation of sensory processing contributes to setting the detection threshold for tactile and thermal behavior responses. The mechanism of such astrocytic control is mediated through modulation of inhibitory transmission to adjust the gain and sensitivity of responding networks. These results uncover a role for astrocytes in maintaining the cortical network activity in an optimal range to control behavior associated with specific sensory modalities.

Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

The nuclear estrogen receptor (ER) and G-protein-coupled ER (GPER1) play a crucial role during brain development and are involved in dendrite and spine growth as well as synapse formation. Soybean isoflavones, such as genistein, daidzein, and S-equol, a daidzein metabolite, exert their action through ER and GPER1. However, the mechanisms of action of isoflavones on brain development, particularly during dendritogenesis and neuritogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone-augmented estradiol mediated dendrite arborization in Purkinje cells. Such augmentation was suppressed by co-exposure with ICI 182,780, an antagonist for ERs, or G15, a selective GPER1 antagonist. The knockdown of nuclear ERs or GPER1 also significantly reduced the arborization of dendrites. Particularly, the knockdown of ERα showed the greatest effect. To further examine the specific molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite outgrowth of Neuro-2A cells. The knockdown of ERα most strongly reduced isoflavone-induced neurite outgrowth compared with ERβ or GPER1 knockdown. The knockdown of ERα also reduced the mRNA levels of ER-responsive genes (i.e., Bdnf, Camk2b, Rbfox3, Tubb3, Syn1, Dlg4, and Syp). Furthermore, isoflavones increased ERα levels, but not ERβ or GPER1 levels, in Neuro-2A cells. The co-culture study of Neuro-2A cells and astrocytes also showed an increase in isoflavone-induced neurite growth, and co-exposure with ICI 182,780 or G15 significantly reduced the effects. In addition, isoflavones increased astrocyte proliferation via ER and GPER1. These results indicate that ERα plays an essential role in isoflavone-induced neuritogenesis. However, GPER1 signaling is also necessary for astrocyte proliferation and astrocyte-neuron communication, which may lead to isoflavone-induced neuritogenesis.

Promotion of astrocyte-neuron glutamate-glutamine shuttle by SCFA contributes to the alleviation of Alzheimer's disease

The brain is particularly susceptible to oxidative damage which is a key feature of several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease. The shuttling of glutathione (GSH) precursors from astrocytes to neurons has been shown to be instrumental for the neuroprotective activity. Here, we revealed that short chain fatty acids (SCFA), which have been related to AD and PD, could promote glutamate-glutamine shuttle to potentially resist oxidative damage in neurons at cellular level. Furthermore, we performed nine-month-long dietary SCFA supplementations in APPswe/PS1dE9 (APP/PS1) mice, and showed that it reshaped the homeostasis of microbiota and alleviated the cognitive impairment by reducing Aβ deposition and tau hyperphosphorylation. Single-cell RNA sequencing analysis of the hippocampus revealed SCFA can enhance astrocyte-neuron communication including glutamate-glutamine shuttle, mainly by acting on astrocyte in vivo. Collectively, our findings indicate that long-term dietary SCFA supplementations at early aging stage can regulate the neuroenergetics to alleviate AD, providing a promising direction for the development of new AD drug.

CXCL8 delivered by plasma-derived exosomes induces the symptoms of post-traumatic stress disorder through facilitating astrocyte-neuron communication

Extracellular vesicles (EVs) play an important role in post-traumatic stress disorder (PTSD). This study is aimed to investigate the possible molecular mechanism of CD63 mediating CXCL8 delivery via EVs to affect astrocyte-neuron communication in PTSD. The neuron-derived EVs (NDEVs) and astrocyte-derived EVs (ADEVs) were isolated from plasma in PTSD patients. Next, the uptake of EVs by neurons was assessed. Following determination of the interaction between CD63 and CXCL8, gain- and loss-of-function experiments were performed in astrocytes. Finally, a PTSD mouse model was established using the single prolonged stress and electric foot shock to confirm the effects of plasma-derived EVs delivering CXCL8 on anxiety- and depression-like behaviors in PTSD mice. EVs derived from plasma of PTSD patients aggravated anxiety- and depression-like behaviors in PTSD mice. CXCL8 was a key gene upregulated in both NDEVs and ADEVs from plasma of PTSD patients, which could be delivered into EVs by CD63. Meanwhile, CXCL8 was also highly expressed in plasma-derived EVs. In vivo experiments also verified that plasma-derived EVs could enhance astrocyte-neuron communication by delivering CXCL8, and silencing of CXCL8 ameliorated anxiety- and depression-like behaviors in PTSD mice. Taken together, CD63 promotes delivery of CXCL8 via EVs to induce PTSD by enhancing astrocyte-neuron communication, suggesting the potential of CD63 mediating delivery of CXCL8 via EVs as a therapeutic target for PTSD.

Abstract 36: Astrocyte-derived Small Extracellular Vesicles Mediate Astrocyte-neuron Communication To Promote Neurite Outgrowth After Stroke In Astrocyte-vesicle Reporter Mice

As an integral part of the neuron-glia system, astrocytes directly contact and communicate to neurons. Small extracellular vesicles (sEVs) are essential mediators of intercellular communication for both physiological and pathological processes. Using astrocyte-sEV reporter mice, we investigated how endogenous astrocyte-derived sEVs communicate to neurons after ischemic stroke. The reporter mice were generated by crossing a CD63-green fluorescent protein (GFP) floxed mouse line with a GFAP-Cre ERT2 mouse line. Mice were subjected to permanent middle cerebral artery occlusion (n=10). To activate Cre recombinase, tamoxifen was injected daily for 4 days starting 1 day after stroke. Animals were euthanized 14 days after stroke. Coronal brain and spinal cord slices were used for immunofluorescent staining with antibodies against GFAP for astrocytes and SMI32 for neurons. Confocal microscopic imaging analysis showed that ischemia dramatically (p<0.01) increased GFP signal intensity in the brain peri-infarct area (58.4±3.6 A.U.) and in the stroke-impaired gray matter of the spinal cord (42.9±2.8 A.U.), compared to that in normal brain (31.7±1.8 A.U) and spinal cord (34.3±2.7 A.U). Neuronal internalization of astrocyte-sEVs measured as colocalization of GFP with SMI32 neurons in the brain and spinal cord was concomitantly and significantly increased in the stroke mice compared with that in normal mice (stroke brain 0.14±0.01% vs 0.07±0.01% in normal, and spinal cord 0.10±0.01% vs 0.06±0.01% in normal, p<0.05), which was confirmed by electron microscopy. In addition, GFP positive sEVs isolated from ischemic brain of the reporter mice significantly promoted neurite outgrowth of primary cultured mouse embryonic cortical neurons, compared to sEVs isolated from non-ischemic brain (p<0.001). Collectively, our data for the first time provide in vivo evidence that endogenous astrocyte-derived sEVs communicate with neurons under non-ischemic condition, while stroke robustly augments this communication, and thereby promotes neurite remodeling during stroke recovery.

Induced Remodelling of Astrocytes In Vitro and In Vivo by Manipulation of Astrocytic RhoA Activity.

Structural changes of astrocytes and their perisynaptic processes occur in response to various physiological and pathophysiological stimuli. They are thought to profoundly affect synaptic signalling and neuron-astrocyte communication. Understanding the causal relationship between astrocyte morphology changes and their functional consequences requires experimental tools to selectively manipulate astrocyte morphology. Previous studies indicate that RhoA-related signalling can play a major role in controlling astrocyte morphology, but the direct effect of increased RhoA activity has not been documented in vitro and in vivo. Therefore, we established a viral approach to manipulate astrocytic RhoA activity. We tested if and how overexpression of wild-type RhoA, of a constitutively active RhoA mutant (RhoA-CA), and of a dominant-negative RhoA variant changes the morphology of cultured astrocytes. We found that astrocytic expression of RhoA-CA induced robust cytoskeletal changes and a withdrawal of processes in cultured astrocytes. In contrast, overexpression of other RhoA variants led to more variable changes of astrocyte morphology. These induced morphology changes were reproduced in astrocytes of the hippocampus in vivo. Importantly, astrocytic overexpression of RhoA-CA did not alter the branching pattern of larger GFAP-positive processes of astrocytes. This indicates that a prolonged increase of astrocytic RhoA activity leads to a distinct morphological phenotype in vitro and in vivo, which is characterized by an isolated reduction of fine peripheral astrocyte processes in vivo. At the same time, we identified a promising experimental approach for investigating the functional consequences of astrocyte morphology changes.

Astrocyte-neuron communication mediated by the Notch signaling pathway: focusing on glutamate transport and synaptic plasticity.

Maintaining glutamate homeostasis after hypoxic ischemia is important for synaptic function and neural cell activity, and regulation of glutamate transport between astrocyte and neuron is one of the important modalities for reducing glutamate accumulation. However, further research is needed to investigate the dynamic changes in and molecular mechanisms of glutamate transport and the effects of glutamate transport on synapses. The aim of this study was to investigate the regulatory mechanisms underlying Notch pathway mediation of glutamate transport and synaptic plasticity. In this study, Yorkshire neonatal pigs (male, age 3 days, weight 1.0-1.5 kg, n = 48) were randomly divided into control (sham surgery group) and five hypoxic ischemia subgroups, according to different recovery time, which were then further subdivided into subgroups treated with dimethyl sulfoxide or a Notch pathway inhibitor (N-[N-(3, 5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester). Once the model was established, immunohistochemistry, immunofluorescence staining, and western blot analyses of Notch pathway-related proteins, synaptophysin, and glutamate transporter were performed. Moreover, synapse microstructure was observed by transmission electron microscopy. At the early stage (6-12 hours after hypoxic ischemia) of hypoxic ischemic injury, expression of glutamate transporter excitatory amino acid transporter-2 and synaptophysin was downregulated, the number of synaptic vesicles was reduced, and synaptic swelling was observed; at 12-24 hours after hypoxic ischemia, the Notch pathway was activated, excitatory amino acid transporter-2 and synaptophysin expression was increased, and the number of synaptic vesicles was slightly increased. Excitatory amino acid transporter-2 and synaptophysin expression decreased after treatment with the Notch pathway inhibitor. This suggests that glutamate transport in astrocytes-neurons after hypoxic ischemic injury is regulated by the Notch pathway and affects vesicle release and synaptic plasticity through the expression of synaptophysin.