Astrocytic Signaling Research Articles

Impaired amygdala astrocytic signaling worsens neuropathic pain-associated neuronal functions and behaviors.

Introduction: Pain is a clinically relevant health care issue with limited therapeutic options, creating the need for new and improved analgesic strategies. The amygdala is a limbic brain region critically involved in the regulation of emotional-affective components of pain and in pain modulation. The central nucleus of amygdala (CeA) serves major output functions and receives nociceptive information via the external lateral parabrachial nucleus (PB). While amygdala neuroplasticity has been linked causally to pain behaviors, non-neuronal pain mechanisms in this region remain to be explored. As an essential part of the neuroimmune system, astrocytes that represent about 40-50% of glia cells within the central nervous system, are required for physiological neuronal functions, but their role in the amygdala remains to be determined for pain conditions. In this study, we measured time-specific astrocyte activation in the CeA in a neuropathic pain model (spinal nerve ligation, SNL) and assessed the effects of astrocyte inhibition on amygdala neuroplasticity and pain-like behaviors in the pain condition. Methods and Results: Glial fibrillary acidic protein (GFAP, astrocytic marker) immunoreactivity and mRNA expression were increased at the chronic (4weeks post-SNL), but not acute (1week post-SNL), stage of neuropathic pain. In order to determine the contribution of astrocytes to amygdala pain-mechanisms, we used fluorocitric acid (FCA), a selective inhibitor of astrocyte metabolism. Whole-cell patch-clamp recordings were performed from neurons in the laterocapsular division of the CeA (CeLC) obtained from chronic neuropathic rats. Pre-incubation of brain slices with FCA (100µM, 1h), increased excitability through altered hyperpolarization-activated current (Ih) functions, without significantly affecting synaptic responses at the PB-CeLC synapse. Intra-CeA injection of FCA (100µM) had facilitatory effects on mechanical withdrawal thresholds (von Frey and paw pressure tests) and emotional-affective behaviors (evoked vocalizations), but not on facial grimace score and anxiety-like behaviors (open field test), in chronic neuropathic rats. Selective inhibition of astrocytes by FCA was confirmed with immunohistochemical analyses showing decreased astrocytic GFAP, but not NeuN, signal in the CeA. Discussion: Overall, these results suggest a complex modulation of amygdala pain functions by astrocytes and provide evidence for beneficial functions of astrocytes in CeA in chronic neuropathic pain.

Purinergic Astrocyte Signaling Driven by TNF-α After Cannabidiol Administration Restores Normal Synaptic Remodeling Following Traumatic Brain Injury

Traumatic brain injury (TBI) is a prevalent form of cranial trauma that results in neural conduction disruptions and damage to synaptic structures and functions. Cannabidiol (CBD), a primary derivative from plant-based cannabinoids, exhibits a range of beneficial effects, including analgesic, sedative, anti-inflammatory, anticonvulsant, anti-anxiety, anti-apoptotic, and neuroprotective properties. Nevertheless, the effects of synaptic reconstruction and the mechanisms underlying these effects remain poorly understood. TBI is characterized by increased levels of tumor necrosis factor-alpha (TNF-α), a cytokine integral for the modulation of glutamate release by astrocytes. In the present study, the potential of CBD in regulating aberrant glutamate signal transmission in astrocytes following brain injury, as well as the underlying mechanisms involved, were investigated using immunofluorescence double staining, enzyme-linked immunosorbent assay (ELISA), western blot analysis, hematoxylin and eosin (H&E) staining, Nissl staining, transmission electron microscopy, and RT-qPCR. In this study, we examined the impact of CBD on neuronal synapses, focusing on the TNF-α-driven purinergic signaling pathway. Specifically, our research revealed that CBD pretreatment effectively reduced the secretion of TNF-α induced by astrocyte activation following TBI. This reduction inhibited the interaction between TNF-α and P2Y1 receptors, leading to a decrease in the release of neurotransmitters, including Ca2+ and glutamate, thereby initiating synaptic remodeling. Our study showed that CBD exhibits significant therapeutic potential for TBI-related synaptic dysfunction, offering valuable insights for future research and more effective TBI treatments. Further exploration of the potential applications of CBD in neuroprotection is required to develop innovative clinical strategies.

Chemogenetic activation of astrocytic Gi signaling promotes spinogenesis and motor functional recovery after stroke.

Ischemic stroke is the leading cause of adult disability. The rewiring of surviving neurons is the fundamental process for functional recovery. Accumulating evidence implicates astrocytes in synapses and neural circuits formation, but few studies have further studied how to enhance the effects of astrocytes on synapse and circuits after stroke and its impacts on post-stroke functional recovery. In this study, we made use of chemogenetics to specifically activate astrocytic Gi signaling in the peri-infarcted sensorimotor cortex at different time epochs in a mouse model of photothrombotic stroke. We found that early activation of astrocytic hM4Di after stroke by CNO modulates astrocyte activity and upregulates synaptogenic molecules including thrombospondin-1 (TSP1) as revealed by bulk RNA-sequencing, but no significant improvement was observed in dendritic spine density and behavioral performance in grid walking test. Interestingly, when the manipulation was initiated at the subacute phase of stroke, the recovery of spine density and motor function could be effectively promoted, accompanied by increased TSP1 expression. Our data highlight the important role of astrocytes in synapse remodeling during the repair phase of stroke and suggest astrocytic Gi signaling activation as a potential strategy for synapse regeneration, circuit rewiring, and functional recovery.

In Vitro Pharmacological Modulation of PIEZO1 Channels in Frontal Cortex Neuronal Networks.

PIEZO1 is a mechanosensitive ion channel expressed in various organs, including but not limited to the brain, heart, lungs, kidneys, bone, and skin. PIEZO1 has been implicated in astrocyte, microglia, capillary, and oligodendrocyte signaling in the mammalian cortex. Using murine embryonic frontal cortex tissue, we examined the protein expression and functionality of PIEZO1 channels in cultured networks leveraging substrate-integrated microelectrode arrays (MEAs) with additional quantitative results from calcium imaging and whole-cell patch-clamp electrophysiology. MEA data show that the PIEZO1 agonist Yoda1 transiently enhances the mean firing rate (MFR) of single units, while the PIEZO1 antagonist GsMTx4 inhibits both spontaneous activity and Yoda1-induced increase in MFR in cortical networks. Furthermore, calcium imaging experiments revealed that Yoda1 significantly increased the frequency of calcium transients in cortical cells. Additionally, in voltage clamp experiments, Yoda1 exposure shifted the cellular reversal potential towards depolarized potentials consistent with the behavior of PIEZO1 as a non-specific cation-permeable channel. Our work demonstrates that murine frontal cortical neurons express functional PIEZO1 channels and quantifies the electrophysiological effects of channel activation in vitro. By quantifying the electrophysiological effects of PIEZO1 activation in vitro, our study establishes a foundation for future investigations into the role of PIEZO1 in neurological processes and potential therapeutic applications targeting mechanosensitive channels in various physiological contexts.

Acute Genetic Damage Induced by Ethanol and Corticosterone Seems to Modulate Hippocampal Astrocyte Signaling.

Astrocytes maintain CNS homeostasis but also critically contribute to neurological and psychiatric disorders. Such functional diversity implies an extensive signaling repertoire including extracellular vesicles (EVs) and nanotubes (NTs) that could be involved in protection or damage, as widely shown in various experimental paradigms. However, there is no information associating primary damage to the astrocyte genome, the DNA damage response (DDR), and the EV and NT repertoire. Furthermore, similar studies were not performed on hippocampal astrocytes despite their involvement in memory and learning processes, as well as in the development and maintenance of alcohol addiction. By exposing murine hippocampal astrocytes to 400 mM ethanol (EtOH) and/or 1 μM corticosterone (CTS) for 1 h, we tested whether the induced DNA damage and DDR could elicit significant changes in NTs and surface-attached EVs. Genetic damage and initial DDR were assessed by immunolabeling against the phosphorylated histone variant H2AX (γH2AX), DDR-dependent apoptosis by BAX immunoreactivity, and astrocyte activation by the glial acidic fibrillary protein (GFAP) and phalloidin staining. Surface-attached EVs and NTs were examined via scanning electron microscopy, and labeled proteins were analyzed via confocal microscopy. Relative to controls, astrocytes exposed to EtOH, CTS, or EtOH+CTS showed significant increases in nuclear γlH2AX foci, nuclear and cytoplasmic BAX signals, and EV frequency at the expense of the NT amount, mainly upon EtOH, without detectable signs of morphological reactivity. Furthermore, the largest and most complex EVs originated only in DNA-damaged astrocytes. Obtained results revealed that astrocytes exposed to acute EtOH and/or CTS preserved their typical morphology but presented severe DNA damage, triggered canonical DDR pathways, and early changes in the cell signaling mediated by EVs and NTs. Further deepening of this initial morphological and quantitative analysis is necessary to identify the mechanistic links between genetic damage, DDR, cell-cell communication, and their possible impact on hippocampal neural cells.

α-Synuclein oligomers potentiate neuroinflammatory NF-κB activity and induce Cav3.2 calcium signaling in astrocytes

BackgroundIt is now realized that Parkinson’s disease (PD) pathology extends beyond the substantia nigra, affecting both central and peripheral nervous systems, and exhibits a variety of non-motor symptoms often preceding motor features. Neuroinflammation induced by activated microglia and astrocytes is thought to underlie these manifestations. α-Synuclein aggregation has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration; however, there is still a lack of critical information about the structural identity of the α-synuclein conformers that activate microglia and/or astrocytes and the molecular pathways involved.MethodsTo investigate the role of α-synuclein conformers in the development and maintenance of neuroinflammation, we used primary quiescent microglia and astrocytes, post-mortem brain tissues from PD patients and A53T α-synuclein transgenic mice that recapitulate key features of PD-related inflammatory responses in the absence of cell death, i.e., increased levels of pro-inflammatory cytokines and complement proteins. Biochemical and -omics techniques including RNAseq and secretomic analyses, combined with 3D reconstruction of individual astrocytes and live calcium imaging, were used to uncover the molecular mechanisms underlying glial responses in the presence of α-synuclein oligomers in vivo and in vitro.ResultsWe found that the presence of SDS-resistant hyper-phosphorylated α-synuclein oligomers, but not monomers, was correlated with sustained inflammatory responses, such as elevated levels of endogenous antibodies and cytokines and microglial activation. Similar oligomeric α-synuclein species were found in post-mortem human brain samples of PD patients but not control individuals. Detailed analysis revealed a decrease in Iba1Low/CD68Low microglia and robust alterations in astrocyte number and morphology including process retraction. Our data indicated an activation of the p38/ATF2 signaling pathway mostly in microglia and a sustained induction of the NF-κB pathway in astrocytes of A53T mice. The sustained NF-κB activity triggered the upregulation of astrocytic T-type Cav3.2 Ca2+ channels, altering the astrocytic secretome and promoting the secretion of IGFBPL1, an IGF-1 binding protein with anti-inflammatory and neuroprotective potential.ConclusionsOur work supports a causative link between the neuron-produced α-synuclein oligomers and sustained neuroinflammation in vivo and maps the signaling pathways that are stimulated in microglia and astrocytes. It also highlights the recruitment of astrocytic Cav3.2 channels as a potential neuroprotective mediator against the α-synuclein-induced neuroinflammation.Graphical

Dimethyl fumarate improves cognitive impairment and neuroinflammation in mice with Alzheimer's disease.

Neuroinflammation substantially contributes to the pathology of Alzheimer's disease (AD), the most common form of dementia. Studies have reported that nuclear factor erythroid 2-related factor 2 (Nrf2) attenuates neuroinflammation in the mouse models of neurodegenerative diseases, however, the detailed mechanism remains unclear. The effects of dimethyl fumarate (DMF), a clinically used drug to activate the Nrf2 pathway, on neuroinflammation were analyzed in primary astrocytes and AppNL-G-F (App-KI) mice. The cognitive function and behavior of DMF-administrated App-KI mice were evaluated. For the gene expression analysis, microglia and astrocytes were directly isolated from the mouse cerebral cortex by magnetic-activated cell sorting, followed by quantitative PCR. DMF treatment activated some Nrf2 target genes and inhibited the expression of proinflammatory markers in primary astrocytes. Moreover, chronic oral administration of DMF attenuated neuroinflammation, particularly in astrocytes, and reversed cognitive dysfunction presumably by activating the Nrf2-dependent pathway in App-KI mice. Furthermore, DMF administration inhibited the expression of STAT3/C3 and C3 receptor in astrocytes and microglia isolated from App-KI mice, respectively, suggesting that the astrocyte-microglia crosstalk is involved in neuroinflammation in mice with AD. The activation of astrocytic Nrf2 signaling confers neuroprotection in mice with AD by controlling neuroinflammation, particularly by regulating astrocytic C3-STAT3 signaling. Furthermore, our study has implications for the repositioning of DMF as a drug for AD treatment.

Remote Ischemia Postconditioning Mitigates Hippocampal Neuron Impairment by Modulating Cav1.2-CaMKIIα-Aromatase Signaling After Global Cerebral Ischemia in Ovariectomized Rats

Brain-derived estrogen (BDE2) is gaining attention as an endogenous neurotransmitter. Recent research has revealed that selectively removing the aromatase gene, the pivotal enzyme responsible for BDE2 synthesis, in forebrain neurons or astrocytes can lead to synaptic loss and cognitive impairment. It is worth noting that remote ischemia post-conditioning (RIP), a non-invasive technique, has been shown to activate natural protective mechanisms against severe ischemic events. The aim of our study was to investigate whether RIP triggers aromatase-BDE2 signaling, shedding light on its neuroprotective mechanisms after global cerebral ischemia (GCI) in ovariectomized rats. Our findings are as follows: (1) RIP was effective in mitigating ischemic damage in hippocampal CA1 neurons and improved cognitive function after GCI. This was partially due to increased Aro-BDE2 signaling in CA1 neurons. (2) RIP intervention efficiently enhanced pro-survival kinase pathways, such as AKT, ERK1/2, CREB, and suppressed CaMKIIα signaling in CA1 astrocytes induced by GCI. Remarkably, inhibiting CaMKIIα activity led to elevated Aro-BDE2 levels and replicated the benefits of RIP. (3) We also identified the positive mediation of Cav1.2, an LVGCC calcium channel, on CaMKIIα-Aro/BDE2 pathway response to RIP intervention. (4) Significantly, either RIP or CaMKIIα inhibition was found to alleviate reactive astrogliosis, which was accompanied by increased pro-survival A2-astrocyte protein S100A10 and decreased pro-death A1-astrocyte marker C3 levels. In summary, our study provides compelling evidence that Aro-BDE2 signaling is a critical target for the reparative effects of RIP following ischemic insult. This effect may be mediated through the CaV1.2-CaMKIIα signaling pathway, in collaboration with astrocyte-neuron interactions, thereby maintaining calcium homeostasis in the neuronal microenvironment and reducing neuronal damage after ischemia.

Astrocytic GPCR signaling in the anterior cingulate cortex modulates decision making in rats.

Decision making is a process of selecting a course of action by assessing the worth or value of the potential consequences. Rat Gambling Task (RGT) is a well-established behavioral paradigm that allows for assessment of the decision-making performance of rats. Astrocytes are emerging as key players in modulating cognitive functions. Using repeated RGTs with short intersession time intervals (48h), the current study demonstrates that Gi pathway activation of astrocytes in the anterior cingulate cortex (ACC) leads to impaired decision-making in consistently good decision-making rats. On the other hand, ACC astrocytic Gq pathway activation improves decision-making in a subset of rats who are not consistently good decision-makers. Furthermore, we show that astrocytic Gq activation is associated with an increase in the L-lactate level in the extracellular fluid of the ACC. Together, these results expand our knowledge of the role of astrocytic GPCR signaling in modulating cognitive functions.

Dynamics of astrocytes Ca2+ signaling: a low-cost fluorescence customized system for 2D cultures.

In an effort to help reduce the costs of fluorescence microscopy and expand the use of this valuable technique, we developed a low-cost platform capable of visualising and analysing the spatio-temporal dynamics of intracellular Ca2+ signalling in astrocytes. The created platform, consisting of a specially adapted fluorescence microscope and a data analysis procedure performed with Imagej Fiji software and custom scripts, allowed us to detect relative changes of intracellular Ca2+ ions in astrocytes. To demonstrate the usefulness of the workflow, we applied the methodology to several in vitro astrocyte preparations, specifically immortalised human astrocyte cells and wild-type mouse cells. To demonstrate the reliability of the procedure, analyses were conducted by stimulating astrocyte activity with the agonist dihydroxyphenylglycine (DHPG), alone or in the presence of the antagonist 2-methyl-6-phenylethyl-pyridine (MPEP).

TRPM7 Mediates Neuropathic Pain by Activating mTOR Signaling in Astrocytes after Spinal Cord Injury in Rats.

In this study, we investigated whether transient receptor melastatin 7 (TRPM7), known as a non-selective cation channel, inhibits neuropathic pain after spinal cord injury (SCI) and how TRPM7 regulates neuropathic pain. Neuropathic pain was developed 4weeks after moderate contusive SCI and TRPM7 was markedly upregulated in astrocytes in the lamina I and II of L4-L5 dorsal horn. In addition, both mechanical allodynia and thermal hyperalgesia were significantly alleviated by a TRPM7 inhibitor, carvacrol. In particular, carvacrol treatment inhibited mechanistic target of rapamycin (mTOR) signaling, which was activated in astrocytes. When rats were treated with rapamycin, an inhibitor of mTOR signaling, neuropathic pain was significantly inhibited. Furthermore, blocking TRPM7 and mTOR signaling by carvacrol and rapamycin inhibited astrocyte activation in lamina I and II of dorsal spinal cord and reduced the level of p-JNK and p-c-Jun, which are known to be activated in astrocytes. Finally, inhibiting TRPM7/mTOR signaling also downregulated the production of pain-related factors such as tumor necrosis factor-α, interleukin-6, interleukin-1β, chemokine (C-C motif) ligand (CCL) 2, CCL-3, CCL-4, CCL-20, chemokine C-X-C motif ligand 1, and matrix metalloproteinase 9 which are known to be involved in the induction and/or maintenance of neuropathic pain after SCI. These results suggest an important role of TRPM7-mediated mTOR signaling in astrocyte activation and thereby induction and/or maintenance of neuropathic pain after SCI.

Brainstem astrocytes regulate breathing and may affect arousal state in rats

Variations in arousal levels can impact respiratory patterns. The mechanisms by which breathing behaviors can influence arousal state is not fully understood. In this study, we investigated the role of astrocytes in the preBötzinger complex (preBötC) in modulating arousal states via breathing in adult conscious rats. Using viral vector tools, we selectively interfered with astrocytic signaling in the preBötC. Rats with inhibited astrocytic signaling exhibited slower breathing rates and behaviors indicative of a calmer state, whereas enhanced purinergic signaling in preBötC astrocytes led to faster breathing and heightened arousal. Our findings reveal a key role for an astrocyte-mediated mechanism in the preBötC that influences both respiratory behaviors and higher-order brain functions like arousal, suggesting a bidirectional link between breathing behaviors and mental states.

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.

The Interplay of Astrocytes and Neurons in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) comprises a complex neurodevelopmental condition characterized by an impairment in social interaction, involving communication deficits and specific patterns of behaviors, like repetitive behaviors. ASD is clinically diagnosed and usually takes time, typically occurring not before four years of age. Genetic mutations affecting synaptic transmission, such as neuroligin and neurexin, are associated with ASD and contribute to behavioral and cognitive deficits. Recent research highlights the role of astrocytes, the brain's most abundant glial cells, in ASD pathology. Aberrant Ca2+ signaling in astrocytes is linked to behavioral deficits and neuroinflammation. Notably, the cytokine IL-6 overexpression by astrocytes impacts synaptogenesis. Altered neurotransmitter levels, disruptions in the blood-brain barrier, and cytokine dysregulation further contribute to ASD complexity. Understanding these astrocyte-related mechanisms holds promise for identifying ASD subtypes and developing targeted therapies.

Astrocytic Ephrin-B1 Regulates Oligodendrocyte Development and Myelination.

Astrocytes have been implicated in oligodendrocyte development and myelination, however, the mechanisms by which astrocytes regulate oligodendrocytes remain unclear. Our findings suggest a new mechanism that regulates astrocyte-mediated oligodendrocyte development through ephrin-B1 signaling in astrocytes. Using a mouse model, we examined the role of astrocytic ephrin-B1 signaling in oligodendrocyte development by deleting ephrin-B1 specifically in astrocytes during the postnatal days (P)14-P28 period and used mRNA analysis, immunohistochemistry, and mouse behaviors to study its effects on oligodendrocytes and myelination. We found that deletion of astrocytic ephrin-B1 downregulated many genes associated with oligodendrocyte development, myelination, and lipid metabolism in the hippocampus and the corpus callosum. Additionally, we observed a reduced number of oligodendrocytes and impaired myelination in the corpus callosum of astrocyte-specific ephrin-B1 KO mice. Finally, our data show reduced motor strength in these mice exhibiting clasping phenotype and impaired performance in the rotarod test most likely due to impaired myelination. Our studies provide new evidence that astrocytic ephrin-B1 positively regulates oligodendrocyte development and myelination, potentially through astrocyte-oligodendrocyte interactions.

The Role of Astrocytes in Parkinson's Disease : Astrocytes in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder with a complex and multifactorial pathogenesis. This chapter delves into the critical role of astrocytes in PD. Once viewed as supporting cells in the central nervous system, astrocytes have emerged as key players in both maintaining neuronal health and contributing to neurodegeneration in PD. Their functions play a dual role in the progression of PD, ranging from protective functions like secretion of neurotrophic factors and clearance of α-synuclein to detrimental functions like promotion of neuroinflammation. This chapter is structured into three primary sections: the morphological and functional organization of astrocytes, astrocytic calcium signaling, and the role of astrocyte heterogeneity in PD. We provide a detailed exploration of astrocytic organelles, bidirectional astrocyte-neuron interactions, and the impact of astrocytic secretions such as antioxidant molecules and neurotrophic factors. Furthermore, we discuss the influence of astrocytes on non-neuronal cells, including interactions with microglia and the blood-brain barrier (BBB). By examining the multifaceted roles of astrocytes, in this chapter, we aim to bridge basic astrocyte biology with the clinical complexities of PD, offering insights into novel therapeutic strategies. The inclusion of astrocyte biology in our broader research approach will aid in the development of more effective treatment strategies for PD.

Roles of astrocytic sonic hedgehog production and its signal for regulation of the blood-brain barrier permeability.

Sonic hedgehog (Shh) is a secreted glycopeptide belonging to the hedgehog family that is essential for morphogenesis during embryonic development. The Shh signal is mediated by two membrane proteins, Patched-1 (Ptch-1) and Smoothened (Smo), following the activation of transcription factors such as Gli. Shh decreases the permeability of the blood-brain barrier (BBB) and plays a key role in its function. In the damaged brain, BBB function is remarkably disrupted. The BBB disruption causes brain edema and neuroinflammation resulting from the extravasation of serum components and the infiltration of inflammatory cells into the cerebral parenchyma. Multiple studies have suggested that astrocyte is a source of Shh and that astrocytic Shh production is increased in the damaged brain. In various experimental animal models of acute brain injury, Shh or Shh signal activators alleviate BBB disruption by increasing tight junction proteins in endothelial cells. Furthermore, activation of astrocytic Shh signaling reduces reactive astrogliosis, neuroinflammation, and increases the production of vascular protective factors, which alleviates BBB disruption in the damaged brain. These findings suggest that astrocytic Shh and Shh signaling protect BBB function in the damaged brain and that target drugs for Shh signaling are expected to be novel therapeutic drugs for acute brain injuries.

Single Cell RNASeq Analysis of AD and Control Post Mortem Brain Samples

Alzheimer’s disease (AD) is a devastative late life disease, molecularly characterized by biochemical lesions of amyloid β plaques and Tau tangles. the 2nd most frequent one worldwide with no early detection methods/cure. I have applied single cell sequencing and analysed post mortem brain samples (using a Chromium machine) from 2 AD and 2 control frontal cortex samples. All the samples had ethics approval, and the RNA was tested for quality. The samples were obtained from the Edenborough MRC brain bank. Very few genes have been linked to far to AD (mainly through GWAS studies [1]). Overall, I quantified about 24,000 nuclei. Using statistical, bioinformatics and computational analyses I have detected significant differential expression changes in astrocyte, neuronal and microglial marker genes. I also conducted network analyses. The percentage of reads passing filter was slightly low (68%). Overall output was a bit lower than optimal. There was still over 1.6Bn clusters generated though, which is within the specification for an S1 flow-cell, and base call quality (quality control) was excellent: 97% >Q30. The read counts mapping to genome occupancy rate was also fairy low (63-81%). The number of barcodes is as follows: sample 1 (AD, Braak I-II) - 6,223 sample 2 (control) 6,262 sample 3 (AD Braak I-II) 5,332 sample 4 (AD, Braak I-II) 5,332 as well. The data is available for download under the GEO (accession number GSE175814). to assess global and cell-type specific gene expression impacts in Alzheimer’s versus control brain samples, we performed a holistic comparison of matching cell populations in AD and controls with the software cell Harmony. To identify possible gene regulatory network underlying transcriptional regulation in these distinct populations, we further explored prior evidenced transcription factor targets relationships from cell Harmony. Importantly, we observe both common and cell-type specific core transcription factors regulators, with both enriched downstream targets and regulated transcription factors. Upregulated networks in Astrocytes were associated with broad regulation by HIF1A, SOX2, NRF1, and RB1. the proportion of endothelial cells was higher in the Alzheimer’s disease samples than the neurological control samples. results reveal that the cell type-specific transcriptomic changes in Alzheimer’s disease are associated with 4 molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signalling in astrocytes and neurons. I also compared the results with microarray data from large cohort studies that examined samples from the prefrontal cortex (Alzheimer’s disease: n = 310; Neurological controls: n = 157) or temporal cortex (Alzheimer’s disease: n = 106; Neurological controls: n = 135). Among the DEGs identified in our snRNA-seq analysis, 1,113 and 764 genes were significantly differentially expressed in the microarray data from the prefrontal cortex and temporal cortex, respectively. subcluster analysis of microglia identified 13 subpopulations; VGF signalling was also found as changed. In the future, imaging of post mortem Alzheimer’s patients may verify brain cellular morphology/cell volume of vascular changes in the Alzheimer’s compared to control samples. My study findings [2] may open new venues to our understanding of the disease underlying molecular processes and cellular signaling. Regarding the glial cell changes, our main conclusion is that the glial cells may be reactive in the disease and not causal.

Recording changes in biochemical parameters <i>in vivo</i> in the ischemic stroke model

Stroke is a significant and socially insidious disease that ranks second among fatal diseases according to the World Health Organization [1]. Understanding the molecular mechanisms behind the pathogenesis of this ailment will enable the development of more effective preventative measures and treatment strategies to minimize the negative consequences of stroke. Despite the abundance of experimental data, most of which were acquired indirectly, the study of the dynamics of biochemical parameters in brain tissue in real time during the acute phase of ischemic stroke is difficult. The use of genetically-encoded sensors creates novel possibilities for monitoring alterations in different biochemical and metabolic parameters in vivo tissues. In this study, we evaluated pH changes, hydrogen peroxide production (an important type of biologically active ROS), and polysulfide synthesis in various types of brain tissue cells of SHR rats during the development of ischemic stroke in real time using sensors such as SypHer3s (for pH detection), HyPer7 (for H2O2 detection), and PersIc (for polysulfide detection). Middle cerebral artery occlusion was used to simulate an ischemic stroke. The in vivo sensor signals were registered with a fiber optic setup that was created in the laboratory of spectroscopy and nonlinear optics at Moscow State University. The studies revealed that in the acute phase of stroke, acidosis occurred in the cytoplasm of neurons in the caudate nucleus, the epicenter of ischemia. The pH mutated from 7.25±0.08 to 6.7±0.15 within the first few seconds after arterial occlusion initiation. A gradual increase in pH was observed after the initial drop, which persisted throughout reperfusion but did not return to the original value in all animals. In the penumbra zone, a wave-like shift in sensor signal was detected, whereas no change in sensor signal was noted in the healthy hemisphere. Investigation of the dynamics of H2O2 formation in the mitochondrial matrix of caudate neurons revealed minimal sensor oxidation during ischemia/reperfusion in the acute phase of stroke, indicating low ROS production. Nevertheless, a substantial increase in the sensor signal was detected after 24 hours following the surgery. Thus, the confirmation of oxidative stress development in the affected hemisphere differed from the commonly accepted view in terms of its dynamics. Previously, it was believed that excessive production of H2O2 leading to oxidative stress and related brain cell death occurred primarily in the acute phase. However, a comparison of hydrogen peroxide production dynamics in neurons and astrocytes revealed differences between these cell populations. It was discovered that as early as 12 hours after middle cerebral artery occlusion, the sensor signal in astrocytes increased more intensely than in neurons. This trend persisted until the end of the measurements, 40 hours after surgery. The observed distinctions may stem from glial cells’ protective function in counteracting the harmful consequences of hydrogen peroxide on neurons, along with their contribution to maintaining the myelin structure in the brain. Additionally, the role of astrocytes in neuroinflammation development is noteworthy. Reactive sulfur species, in addition to reactive oxygen species, appear to be significant contributors to the development of pathological processes. The PersIc sensor signal measurement did not show any disparities between the caudate nucleus of the healthy hemisphere and the hemisphere affected by stroke development in terms of polysulfide and persulfide appearance detection. However, the area surrounding the core infarction is noteworthy due to the observed bouts of acidosis using the SypHer3s sensor. Our findings suggest a potential association between these bouts, spreading depolarization, changes in calcium concentration, and the development of neuroinflammation. These reactions may ultimately lead to the synthesis of polysulfides, known modulators of inflammatory reactions. Thus, our data provides valuable additions to the existing knowledge on metabolic changes that take place during the progression of ischemic brain injury.

Astrocytic insulin receptor controls circadian behavior via dopamine signaling in a sexually dimorphic manner

Mammalian circadian clocks respond to feeding and light cues, adjusting internal rhythms with day/night cycles. Astrocytes serve as circadian timekeepers, driving daily physiological rhythms; however, it’s unknown how they ensure precise cycle-to-cycle rhythmicity. This is critical for understanding why mistimed or erratic feeding, as in shift work, disrupts circadian physiology- a condition linked to type 2 diabetes and obesity. Here, we show that astrocytic insulin signaling sets the free-running period of locomotor activity in female mice and food entrainment in male mice. Additionally, ablating the insulin receptor in hypothalamic astrocytes alters cyclic energy homeostasis differently in male and female mice. Remarkably, the mutants exhibit altered dopamine metabolism, and the pharmacological modulation of dopaminergic signaling partially restores distinct circadian traits in both male and female mutant mice. Our findings highlight the role of astrocytic insulin-dopaminergic signaling in conveying time-of-feeding or lighting cues to the astrocyte clock, thus governing circadian behavior in a sex-specific manner.