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Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) have proven themselves as one of the key in vivo techniques of modern neuroscience, allowing for unprecedented access to cellular manipulations in living animals. With respect to astrocyte research, DREADDs have become a popular method to examine the functional aspects of astrocyte activity, particularly G-protein coupled receptor (GPCR)-mediated intracellular calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) dynamics. With this method it has become possible to directly link the physiological aspects of astrocytic function to cognitive processes such as memory. As a result, a multitude of studies have explored the impact of DREADD activation in astrocytes on synaptic activity and memory. However, the emergence of varying results prompts us to reconsider the degree to which DREADDs expressed in astrocytes accurately mimic endogenous GPCR activity. Here we compare the major downstream signaling mechanisms, synaptic, and behavioral effects of stimulating Gq-, Gs-, and Gi-DREADDs in hippocampal astrocytes of adult mice to those of endogenously expressed GPCRs.

Background: Systemic inflammation accompanies HIV-1 infection, resulting in microbial translocation from different tissues. We investigated interactions between lentivirus infections, neuroinflammation and microbial molecule presence in the brain. Methods: Brain tissues from adult humans with (n = 22) and without HIV-1 (n = 11) infection as well as adult nonhuman primates (NHPs) with (n = 11) and without (n = 4) SIVmac251 infection were investigated by RT-PCR/ddPCR, immunofluorescence and western blotting. Studies of viral infectivity, host immune gene expression and viability were performed in primary human neural cells. Findings: Among NHPs, SIV DNA quantitation in brain showed increased levels among animals with SIV encephalitis (n = 5) that was associated with bacterial genomic copy number as well as CCR5 and CASP1 expression in brain. Microbial DnaK and peptidoglycan were immunodetected in brains from uninfected and SIV-infected animals, chiefly in glial cells. Human microglia infected by HIV-1 showed increased p24 production after exposure to peptidoglycan that was associated CCR5 induction. HIV-1 Vpr application to human neurons followed by peptidoglycan exposure resulted in reduced mitochondrial function and diminished beta-III tubulin expression. In human brains, bacterial genome copies (250–550 copies/gm of tissue), were correlated with increased bacterial rRNA and GroEL transcript levels in patients with HIV-associated neurocognitive disorders (HAND). Glial cells displayed microbial GroEL and peptidoglycan immunoreactivity accompanied by CCR5 induction in brains from patients with HAND. Interpretation: Increased microbial genomes and proteins were evident in brain tissues from lentivirus-infected humans and animals and associated with neurological disease. Microbial molecule translocation into the brain might exacerbate neuroinflammatory disease severity and represent a driver of lentivirus-associated brain disease.

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron–glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

Oligodendrocyte development is accompanied by defined changes in the state of chromatin that are brought about by chromatin remodeling complexes. Many such remodeling complexes exist, but only a few have been studied for their impact on oligodendrocytes as the myelin-forming cells of the central nervous system. To define the role of the PBAF remodeling complex, we focused on Pbrm1 as an essential subunit of the PBAF complex and specifically deleted it in the oligodendrocyte lineage at different times of development in the mouse. Deletion in late oligodendrocyte progenitor cells did not lead to substantial changes in the ensuing differentiation and myelination processes. However, when Pbrm1 loss had already occurred in oligodendrocyte progenitor cells shortly after their specification, fewer cells entered the pre-myelinating state. The reduction in pre-myelinating cells later translated into a comparable reduction in myelinating oligodendrocytes. We conclude that Pbrm1 and, by inference, the activity of the PBAF complex is specifically required at the transition from oligodendrocyte progenitor to pre-myelinating oligodendrocyte and ensures the generation of normal numbers of myelinating oligodendrocytes.

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

Fil: Belkacemi, Thabet. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Niermann, Alexander. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Hofmann, Laura. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Wissenbach, Ulrich. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Research Triangle Park. Neurobiology Laboratory; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Leidinger, Petra. Universität des Saarlandes. Institut für Humangenetik; Alemania Fil: Backes, Christina. Universität des Saarlandes. Klinische Bioinformatik; Alemania Fil: Meese, Eckart. Universität des Saarlandes. Institut für Humangenetik; Alemania Fil: Keller, Andreas. Universität des Saarlandes. Klinische Bioinformatik; Alemania Fil: Bai, Xianshu. Universität des Saarlandes. Molekulare Physiologie; Alemania Fil: Scheller, Anja. Universität des Saarlandes. Molekulare Physiologie; Alemania Fil: Kirchhoff, Frank. Universität des Saarlandes. Molekulare Physiologie; Alemania Fil: Philipp, Stephan E. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Weissgerber, Petra. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Flockerzi, Veit. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Beck, Andreas. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Beck, Andreas. Universität des Saarlandes. Molekulare Physiologie; Alemania Abstract: Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) have proven themselves as one of the key in vivo techniques of modern neuroscience, allowing for unprecedented access to cellular manipulations in living animals. With respect to astrocyte research, DREADDs have become a popular method to examine the functional aspects of astrocyte activity, particularly G-protein coupled receptor (GPCR)-mediated intracellular calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) dynamics. With this method it has become possible to directly link the physiological aspects of astrocytic function to cognitive processes such as memory. As a result, a multitude of studies have explored the impact of DREADD activation in astrocytes on synaptic activity and memory. However, the emergence of varying results prompts us to reconsider the degree to which DREADDs expressed in astrocytes accurately mimic endogenous GPCR activity. Here we compare the major downstream signaling mechanisms, synaptic, and behavioral effects of stimulating Gq-, Gs-, and Gi-DREADDs in hippocampal astrocytes of adult mice to those of endogenously expressed GPCRs.

Background Astrocytes play a central role in maintaining brain energy metabolism, but are also tightly connected to the pathogenesis of Alzheimer’s disease (AD). Our previous studies demonstrate that inflammatory astrocytes accumulate large amounts of aggregated amyloid-beta (Aβ). However, in which way these Aβ deposits influence their energy production remain unclear. Methods The aim of the present study was to investigate how Aβ pathology in astrocytes affects their mitochondria functionality and overall energy metabolism. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils for 7 days and analyzed over time using different experimental approaches. Results Our results show that to maintain stable energy production, the astrocytes initially increased their mitochondrial fusion, but eventually the Aβ-mediated stress led to abnormal mitochondrial swelling and excessive fission. Moreover, we detected increased levels of phosphorylated DRP-1 in the Aβ-exposed astrocytes, which co-localized with lipid droplets. Analysis of ATP levels, when blocking certain stages of the energy pathways, indicated a metabolic shift to peroxisomal-based fatty acid β-oxidation and glycolysis. Conclusions Taken together, our data conclude that Aβ pathology profoundly affects human astrocytes and changes their entire energy metabolism, which could result in disturbed brain homeostasis and aggravated disease progression.

Huntington’s disease (HD) is a severely debilitating, autosomal dominant neurodegenerative disease with a fatal outcome. There is accumulating evidence of a prominent role of glia in the pathology of HD, and we investigated this by conducting single nuclear RNA sequencing of human post mortem brain in 4 regions; caudate nucleus, frontal cortex, hippocampus and cerebellum. Across 127,205 nuclei from HD patients and age and sex matched controls, we found an altered heterogeneity of glia in HD. We describe prominent changes in gene expression and abundance of certain subtypes of astrocytes, microglia, oligodendrocyte precursor cells and oligodendrocytes between HD and control samples, and these differences are widespread across brain regions. Furthermore, we highlight two possible mechanisms that characterise the glial contribution to disease pathology. Firstly, we show that upregulation of molecular chaperones represents a cross-glial signature in HD, which likely reflects an adaptive response to the aggregation of Huntingtin protein and might promote cell survival. Secondly, we show an oligodendrocyte-specific upregulation of the calmodulin-dependent 3’,5’-cyclic nucleotide phosphodiesterase 1A (PDE1A) in HD compared to controls, which could cause dysfunction to key cellular functions through its downregulation of the important second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Our results support the hypothesis that glia have an independent role in the pathology of HD, and we show that all types of glia are affected in the disease. As glia are selfrenewing and therefore more tractable for treatment than neurons, our findings may be of therapeutic relevance.

Under normal physiological condition the body needs to fight bacterial infection, clear old dead or dying cells as well as regenerate new ones. Phagocytosis is the immunological process required for cells to digest and destroy particulates thereby maintaining a systemic homeostasis. Phagocytosis involves four steps of recognition, engulfment, formation of phagosome, degradation and elimination. In the nervous system, the phagocytes are the glial cells which support the system framework, primarily microglia. Spinal cord injury is an irreparable nervous system injury and restoration of function is very difficult. One strategy which could aid repair and regeneration is to enhance the phagocytic process thereby removing cell debris and bacteria from the injury site. Glial cells are involved in phagocytosis and clearing the spinal cord injury area. Understanding this process may improve strategies to develop therapy, particularly those that involve cell transplantation such as peripheral nerve glia. Within the nervous system, the main phagocytic glia, which has been studied, in the central nervous system (CNS) are astrocytes and microglia, and in the peripheral nervous system (PNS), the main glia are Schwann cells for most nerves, with olfactory ensheathing cells (OECs) in the olfactory nerve. In this study, my first objective was to compare the CNS and PNS cells like astrocytes, microglia, OECs and trigeminal Schwann cells (TgSCs) for their antimicrobial response to bacterial infection (S. aureus). Results showed live bacteria could be isolated from all glia after 24 h in culture, and microglia, OECs and TgSCs exhibited better protection against intracellular S. aureus survival than astrocytes. All glial types responded to the bacteria by cytokine secretion, but overall, OECs secreted the lowest level of cytokines. Debris clearance is crucial for neural regeneration and OECs have the ability to phagocytose both bacteria and cell debris. OECs and SCs are being considered as cell transplantation therapies in spinal cord injury for the purpose of repairing the damaged nervous system. Thus, understanding the genetic expression in between OECs and SCs during the process of phagocytosis of bacteria (S. aureus) and axonal debris is essential. Results showed OECs responded to S. aureus with protein modification and phosphorus metabolic processes involved with immune response, and leukocyte mediated immunity signalling mechanisms for bacterial engulfment. SCs responded to cell debris with pathways associated with actin filament-based processes, cytoskeleton organization, and FC gamma receptor dependent phagocytosis. These findings demonstrate the differences between OECs and SCs during phagocytic responses to bacteria and cell debris. Overall, OECs expressed a low gene expression profile compared to SCs. Discovering novel compounds that can stimulate OECs to phagocytose bacteria and cell debris more efficiently may improve the therapeutic potential of the cells. Potentially, if OECs could be pre-stimulated with a drug to enhance OEC phagocytosis, that could be useful in spinal cord injury regeneration. Therefore to identify compounds that would enhance the phagocytosis of glial cells, drug discovery assays were performed. Following a preliminary compound screening, a synthetic compound and a natural compound was identified. For a third compound, a FDA approved drug was selected to test their effects on glial cells. As the process and analyses, required to identify compounds, that stimulate glial cell phagocytic activity has not been previously established, development and optimization of a protocol for phagocytosis detection was performed. Phagocytosis measurement in glial cells based on live imaging and fixed widefield imaging was performed to make a replicable drug discovery testing method which could be used on cell lines and primary cells both. Using this method results like cytotoxicity, morphological changes and phagocytosis of various targets can be reliably performed. Testing the compounds with the protocol, demonstrated that significant stimulation of phagocytosis activity could be detected. All three compounds tested showed positive phagocytosis increase on cell lines. The synthetic compound showed positive increase in phagocytosis on primary OECs and TgSCs but not in astrocytes and microglia for S. aureus pHrodo BioParticles. Testing further, using the synthetic compound, in the presence of bacterial stimulus, showed no significant upregulation in cytokines but some positive upregulation in phagocytotic genes, in the compound stimulated wells against controls. The natural compound showed increase in phagocytosis only in microglia but not in other glial cells (OECs, TgSCs and astrocytes). The FDA compound Liraglutide showed significant increase in phagocytosis in all glial cells (astrocytes, microglia, OECs and TgSCs). Testing the synthetic compound on the phagocytosis of Beta (β) amyloid peptide, in OECs, astrocytes and microglia, showed an increase in peptide uptake in compound stimulated OECs as compared to non-stimulated ones. Astrocyte and microglia did not show any difference. In conclusion, this thesis gives us an understanding of the difference between CNS and PNS phagocytosis. While the different glial cells share many properties, OECs exhibit several characteristics which make them favourable candidates for transplantation therapies. Compound testing revealed that phagocytic activity can be stimulated and opens up avenues for identification of compounds that could be used to enhance therapeutic activity of glial cells in SCI and possibly in Alzheimer’s diseases research.

Funding: We thank Aino Järvelin and Jeffrey Y. Lee for help with the conceptualization and discussions of this review. This work was funded by a Wellcome Investigator Award 209412/Z/17/Z and Wellcome Strategic Awards (Micron Oxford) 091911/B/10/Z and 107457/Z/ 15/Z to I.D. D.S.G. is funded by Medial Sciences Graduate Studentships, University of Oxford. R.O.T. is funded by iNOVA4Health—UIDB/04462/2020 and EXPL/BIA-CEL/1484/ 2021. Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses whose plasticity is regulated efficiently by mRNA transport and localized translation. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, mRNA localization has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial subtypes containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing, for example, synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed, using cutting-edge tools already available for neurons. publishersversion published

IntroductionAstrocyte-synapse bi-directional communication is required for neuronal development and synaptic plasticity. Astrocytes structurally interact with synapses using their distal processes also known as leaflets or perisynaptic astrocytic processes (PAPs). We recently showed that these PAPs are retracted from hippocampal synapses, and involved in the consolidation of fear memory. However, whether astrocytic synaptic coverage is affected when memory is impaired is unknown.MethodsHere, we describe in detail an electron microscopy method that makes use of a large number of 2D images to investigate structural astrocyte-synapse interaction in paraformaldehyde fixed brain tissue of mice.Results and discussionWe show that fear memory-induced synaptic activation reduces the interaction between the PAPs and the presynapse, but not the postsynapse, accompanied by retraction of the PAP tip from the synaptic cleft. Interestingly, this retraction is absent in the APP/PS1 mouse model of Alzheimer’s disease, supporting the concept that alterations in astrocyte-synapse coverage contribute to memory processing.

Multiple sclerosis is an autoimmune disease of the central nervous system. Yet, the autoimmune targets are still undefined. The extracellular e1 sequence of KCNJ10, the inwardly rectifying potassium channel 4.1, has been subject to fierce debate for its role as a candidate autoantigen in multiple sclerosis. Inwardly rectifying potassium channel 4.1 is expressed in the central nervous system but also in peripheral tissues, raising concerns about the central nervous system-specificity of such autoreactivity. Immunization of C57Bl6/J female mice with the e1 peptide (amino acids 83–120 of Kir4.1) induced anti-e1 immunoglobulin G- and T-cell responses and promoted demyelinating encephalomyelitis with B cell central nervous system enrichment in leptomeninges and T cells/macrophages in central nervous system parenchyma from forebrain to spinal cord, mostly in the white matter. Within our cohort of multiple sclerosis patients (n = 252), 6% exhibited high anti-e1 immunoglobulin G levels in serum as compared to 0.7% in the control cohort (n = 127; P = 0.015). Immunolabelling of inwardly rectifying potassium channel 4.1-expressing white matter glia with the anti-e1 serum from immunized mice increased during murine autoimmune neuroinflammation and in multiple sclerosis white matter as compared with controls. Strikingly, the mouse and human anti-e1 sera labelled astrocytoma cells when N-glycosylation was blocked with tunicamycin. Western blot confirmed that neuroinflammation induces Kir4.1 expression, including its shorter aglycosylated form in murine experimental autoencephalomyelitis and multiple sclerosis. In addition, recognition of inwardly rectifying potassium channel 4.1 using mouse anti-e1 serum in Western blot experiments under unreduced conditions or in cells transfected with the N-glycosylation defective N104Q mutant as compared to the wild type further suggests that autoantibodies target an e1 conformational epitope in its aglycosylated form. These data highlight the e1 sequence of inwardly rectifying potassium channel 4.1 as a valid central nervous system autoantigen with a disease/tissue-specific post-translational antigen modification as potential contributor to autoimmunity in some multiple sclerosis patients.

The spinal cord is an elongated nervous structure that together with the brain forms the central nervous system. It relays sensory and motor information between the brain and the body, thus controlling most somatic and autonomic body functions. In recent years, great progress has been made in creating digital atlases for the mouse brain, covering regions, cell composition and connectivity. For the spinal cord, however, such atlases do not yet exist. In this dissertation, I present the first versions of high-resolution 3D atlases of the spinal cords for five mammalian species: mouse, rat, marmoset, rhesus monkey, and human. First, I summarize the current state of the efforts to understand the spinal cord and its organization principles from anatomy (cytoarchitecture) to cell classifications and connectome principles. I then describe how the different properties of the spinal cord can help us in reconstructing the various spinal structures. Next, I present a workflow for data-driven reconstruction that can be used to create 3D models of the spinal cord of any species based on available annotated 2D microscopy images. I used data from Sengul, Watson, et al., 2012 to generate 3D reference volumes of the cytoarchitecture of the 5 mammalian species: mouse, rat, marmoset, rhesus monkey and human. Every 3D reference volume model comprises six levels of the spinal cytoarchitecture and defines their locations, shapes, and volumes. This allows us to compare the spinal cord of these 5 species. I find that all reconstructed spinal cords are remarkably similar, showing far fewer differences than the brains of the respective species. For the mouse spinal cord, I use data from the Allen Institute for Brain Science to generate a cell atlas for the three major spinal cell types: interneurons, motoneurons, and glial cells. The mouse spinal cell atlas comprises the densities and positions of 11,7 million cells in total with 2.7 million interneurons, about 31 thousand motoneurons, and 9.0 million glial cells. Next, I discuss how recent transcriptomic data can be used to assign a gene expression profile to each neuron in the spinal cell atlas. I present a preliminary analysis of RNA-sequencing and in situ RNA hybridization data aiming to develop a workflow for labeling of spinal cells with appropriate transcriptome profiles. Finally, I summarize the results of my dissertation as well as consequences and limitations of the underlying assumptions. This discussion ends with an outlook for how the atlas can be developed further in the future.

Physical activity (PA) promotes the proliferation of neural stem cells and enhances neurogenesis in the dentate gyrus resulting in hippocampal circuit remodeling and cognitive enhancement. Nonetheless, knowledge of other neural progenitors affected by PA and the mechanisms through which they could contribute to circuit plasticity and cognitive enhancement are still poorly understood. In this work we demonstrated that NG2-glia, also known as oligodendrocyte progenitor cells, show enhanced proliferation and differentiation in response to voluntary PA in a brain region-dependent manner in adult mice. Surprisingly, preventing NG2-glia differentiation during enhanced PA abolishes the exercise-associated cognitive improvement without affecting neurogenesis or baseline learning capacity. Thus, here we provided new evidence highlighting the requirement of oligodendrogenesis for exercise induced-cognition enhancement., publishedVersion

Exposure to stress during early development may lead to altered neurobiological functions, thus increasing the risk for psychiatric illnesses later in life. One potential mechanism associated with those outcomes is the disruption of glial density and morphology, despite results from rodent studies have been conflicting. To address that we performed a systematic review and meta-analysis of rodent studies that investigated the effects of prenatal stress (PNS) and early life stress (ELS) on microglia, astrocyte, and oligodendrocyte density and morphology within the offspring. Our meta-analysis demonstrates that animals exposed to PNS or ELS showed significant increase in microglia density, as well as decreased oligodendrocyte density. Moreover, ELS exposure induced an increase in microglia soma size. However, we were unable to identify significant effects on astrocytes. Meta-regression indicated that experimental stress protocol, sex, age, and type of tissue analyzed are important covariates that impact those results. Importantly, PNS microglia showed higher estimates in young animals, while the ELS effects were stronger in adult animals. This set of data reinforces that alterations in glial cells could play a role in stress-induced dysfunctions throughout development.

Leucine-rich repeat kinase 2 (LRRK2) is a complex GTPase/kinase orchestrating cytoskeletal dynamics and multiple steps of the endolysosomal pathway through interaction with a host of partners and phosphorylation of a subset of Rab GTPases. Mutations in LRRK2 cause late-onset Parkinson's disease (PD) and common variants in the locus containing LRRK2 have been associated with sporadic PD, progressive supranuclear palsy as well as a number of inflammatory diseases. This review encompasses the major discoveries in the field of LRRK2 pathobiology, from the initial gene cloning to the latest progress in LRRK2 inhibition as a promising therapeutic approach to fight neurodegeneration.

Thousands of human deaths occur annually due to Japanese encephalitis (JE), caused by Japanese encephalitis virus. During the virus infection of the central nervous system, reactive gliosis, uncontrolled inflammatory response, and neuronal cell death are considered as the characteristic features of JE. To date, no specific treatment has been approved to overcome JE, indicating a need for the development of novel therapies. In this article, we focused on basic biological mechanisms in glial (microglia and astrocytes) and neuronal cells that contribute to the onset of neuroinflammation and neuronal cell damage during Japanese encephalitis virus infection. We also provided comprehensive knowledge about anti-JE therapies tested in clinical or pre-clinical settings, and discussed recent therapeutic strategies that could be employed for JE treatment. The improved understanding of JE pathogenesis might lay a foundation for the development of novel therapies to halt JE. Abbreviations AKT: a serine/threonine-specific protein kinase; AP1: activator protein 1; ASC: apoptosis-associated speck-like protein containing a CARD; ASK1: apoptosis signal-regulated kinase 1; ATF3/4/6: activating transcription factor 3/4/6; ATG5/7: autophagy-related 5/7; BBB: blood-brain barrier; Bcl-3/6: B-cell lymphoma 3/6 protein; CCL: C-C motif chemokine ligand; CCR2: C-C motif chemokine receptor 2; CHOP: C/EBP homologous protein; circRNA: circular RNA; CNS: central nervous system; CXCL: C-X-C motif chemokine ligand; dsRNA: double-stranded RNA; EDEM1: endoplasmic reticulum degradation enhancer mannosidase alpha-like 1; eIF2-ɑ: eukaryotic initiation factor 2 alpha; ER: endoplasmic reticulum; ERK: extracellular signal-regulated kinase; GRP78: 78-kDa glucose-regulated protein; ICAM: intercellular adhesion molecule; IFN: interferon; IL: interleukin; iNOS: inducible nitric oxide synthase; IRAK1/2: interleukin-1 receptor-associated kinase 1/2; IRE-1: inositol-requiring enzyme 1; IRF: interferon regulatory factor; ISG15: interferon-stimulated gene 15; JE: Japanese encephalitis; JEV: Japanese encephalitis virus; JNK: c-Jun N-terminal kinase; LAMP2: lysosome-associated membrane protein type 2; LC3-I/II: microtubule-associated protein 1 light chain 3-I/II; lncRNA: long non-coding RNA; MAPK: mitogen-activated protein kinase; miR/miRNA: microRNA; MK2: mitogen-activated protein kinase-activated protein kinase 2; MKK4: mitogen-activated protein kinase kinase 4; MLKL: mixed-linage kinase domain-like protein; MMP: matrix metalloproteinase; MyD88: myeloid differentiation factor 88; Nedd4: neural precursor cell-expressed developmentally downregulated 4; NF-κB: nuclear factor kappa B; NKRF: nuclear factor kappa B repressing factor; NLRP3: NLR family pyrin domain containing 3; NMDAR: N-methyl-D-aspartate receptor; NO: nitric oxide; NS2B/3/4: JEV non-structural protein 2B/3/4; P: phosphorylation. p38: mitogen-activated protein kinase p38; PKA: protein kinase A; PAK4: p21-activated kinase 4; PDFGR: platelet-derived growth factor receptor; PERK: protein kinase R-like endoplasmic reticulum kinase; PI3K: phosphoinositide 3-kinase; PTEN: phosphatase and tensin homolog; Rab7: Ras-related GTPase 7; Raf: proto-oncogene tyrosine-protein kinase Raf; Ras: a GTPase; RIDD: regulated IRE-1-dependent decay; RIG-I: retinoic acid-inducible gene I; RIPK1/3: receptor-interacting protein kinase 1/3; RNF11/125: RING finger protein 11/125; ROS: reactive oxygen species; SHIP1: SH2-containing inositol 5ʹ phosphatase 1; SOCS5: suppressor of cytokine signaling 5; Src: proto-oncogene tyrosine-protein kinase Src; ssRNA = single-stranded RNA; STAT: signal transducer and activator of transcription; TLR: toll-like receptor; TNFAIP3: tumor necrosis factor alpha-induced protein 3; TNFAR: tumor necrosis factor alpha receptor; TNF-α: tumor necrosis factor-alpha; TRAF6: tumor necrosis factor receptor-associated factor 6; TRIF: TIR-domain-containing adapter-inducing interferon-β; TRIM25: tripartite motif-containing 25; VCAM: vascular cell adhesion molecule; ZO-1: zonula occludens-1.

Neuroglial apoptosis and neuroinflammation play an important role in epileptogenesis. The aim of this study is to evaluate neuronal and glial apoptosis in association with neuroinflammation in brain epileptic focus and inflammatory changes in blood in patients with focal drug-resistant epilepsy (DRE). Pathological changes in the temporal lobe in epilepsy (histology, transmission electron microscopy), levels of apoptotic and neuroinflammatory proteins: active caspase-3 (immunohistochemistry), full-length form caspase-3, caspase-9, FAS, FAS-L, NF-kB, TNF-α, p53 (Western blot), and cytokine levels in blood: IL-1β, IL-2, IL-4, IL-7, TNF-α, etc. (multiplex analysis) were studied. In the present work, ultrastructural and immunohistochemical apoptotic signs were found in neurons and oligodendrocytes in the temporal lobe of DRE patients. Levels of proinflammatory cytokines that play a role in apoptosis (TNF-α, FAS, NF-kB) were increased. The blood concentration of IL-4, IL-7, TNF-α is increased and IL-2 is reduced. Oligodendroglial apoptosis has been shown to play an important role in DRE pathogenesis and to explain demyelination. Thus, a comprehensive analysis of revealed changes in the blood and brain in DRE patients showed the neuroinflammation in the epileptic focus, which was combined with the development of apoptosis of glial cells and neurons. This creates conditions for the development of drug resistance and the epilepsy progression.

BACKGROUND: The formation and retrieval of fear memories depends on orchestrated synaptic activity of neuronal ensembles within the hippocampus, and it is becoming increasingly evident that astrocytes residing in the environment of these synapses play a central role in shaping cellular memory representations. Astrocyte distal processes, known as leaflets, fine-tune synaptic activity by clearing neurotransmitters and limiting glutamate diffusion. However, how astroglial synaptic coverage contributes to mnemonic processing of fearful experiences remains largely unknown.METHODS: We used electron microscopy to observe changes in astroglial coverage of hippocampal synapses during consolidation of fear memory in mice. To manipulate astroglial synaptic coverage, we depleted ezrin, an integral leaflet-structural protein, from hippocampal astrocytes using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing. Next, a combination of Föster resonance energy transfer analysis, genetically encoded glutamate sensors, and whole-cell patch-clamp recordings was used to determine whether the proximity of astrocyte leaflets to the synapse is critical for synaptic integrity and function.RESULTS: We found that consolidation of a recent fear memory is accompanied by a transient retraction of astrocyte leaflets from hippocampal synapses and increased activation of NMDA receptors. Accordingly, astrocyte-specific depletion of ezrin resulted in shorter astrocyte leaflets and reduced astrocyte contact with the synaptic cleft, which consequently boosted extrasynaptic glutamate diffusion and NMDA receptor activation. Importantly, after fear conditioning, these cellular phenotypes translated to increased retrieval-evoked activation of CA1 pyramidal neurons and enhanced fear memory expression.CONCLUSIONS: Together, our data show that withdrawal of astrocyte leaflets from the synaptic cleft is an experience-induced, temporally regulated process that gates the strength of fear memories.

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 µl) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

Parkinson’s disease (PD) is a progressive neurodegenerative disease of both the central and peripheral / enteric nervous systems. Oxidative stress and neuroinflammation are associated with the pathogenesis of PD, suggesting that anti-oxidative and anti-inflammatory compounds could be neuroprotective agents for PD. Eucommia ulmoides (EU) is a traditional herbal medicine which exerts neuroprotective effects by anti-inflammatory and anti-oxidative properties. Our previous study showed that treatment with chlorogenic acid, a component of EU, protected against neurodegeneration in the central and enteric nervous systems in a PD model. In this study, we examined the effects of EU extract (EUE) administration on dopaminergic neurodegeneration, glial response and α-synuclein expression in the substantia nigra pars compacta (SNpc), and intestinal enteric neurodegeneration in low-dose rotenone-induced PD model mice. Daily oral administration of EUE ameliorated dopaminergic neurodegeneration and α-synuclein accumulation in the SNpc. EUE treatment inhibited rotenone- induced decreases in the number of total astrocytes and in those expressing the antioxidant molecule metallothionein. EUE also prevented rotenone-induced microglial activation. Furthermore, EUE treatment exerted protective effects against intestinal neuronal loss in the PD model. These results suggest that EU exerts neuroprotective effects in the central and enteric nervous systems of rotenone-induced parkinsonism mice, in part by glial modification.

Transdifferentiation of Schwann cells is essential for functional peripheral nerve regeneration after injury. By activating a repair program, Schwann cells promote functional axonal regeneration and remyelination. However, chronic denervation, aging, metabolic diseases, or chronic inflammatory processes reduce the transdifferentiation capacity and thus diminish peripheral nerve repair. It was recently described that the sphingosine-1-phosphate receptor (S1PR) agonist Fingolimod enhances the Schwann cell repair phenotype by activation of dedifferentiation markers and concomitant release of trophic factors resulting in enhanced neurite growth. Since Fingolimod targets four out of five S1PRs (S1P1, S1P3-5) possibly leading to non-specific adverse effects, identification of the main receptor(s) responsible for the observed phenotypic changes is mandatory for future specific treatment approaches. Our experiments revealed that S1P3 dominates and that along with S1P1 acts as the responsible receptor for Schwann cell transdifferentiation as revealed by the combinatory application of specific agonists and antagonists. Targeting both receptors reduced the expression of myelin-associated genes, increased PDGF-BB representing enhanced trophic factor expression likely to result from c-Jun induction. Furthermore, we demonstrated that S1P4 and S1P5 play only a minor role in the adaptation of the repair phenotype. In conclusion, modulation of S1P1 and S1P3 could be effective to enhance the Schwann cell repair phenotype and thus stimulate proper nerve repair.

Astrocytes are a group of cells with specific morphological and functional characteristics that vary in different regions of the brain. During postnatal development, astrocytes migrate to the regions of interest in the brain. Astrocytes interact with neurons to protect, support and maintain normal cellular homeostasis during physiological ageing of the central nervous system (CNS), including the brain, spinal cord and retina. Mature astrocytes retain most of the genes expressed in progenitor cells to keep their proliferative potential. In the last few decades, astrocytes have been reported to serve an important role in age-related neurodegenerative diseases..

Introduction: Vanishing white matter (VWM) is a leukodystrophy that leads to neurological dysfunction and early death. Astrocytes are indicated as therapeutic target, because of their central role in VWM pathology. Previous cell replacement therapy using primary mouse glial precursors phenotypically improved VWM mice. Aims: The aim of this study was to determine the translational potential of human stem cell-derived glial cell replacement therapy for VWM. We generated various glial cell types from human pluripotent stem cells in order to identify a human cell population that successfully ameliorates disease hallmarks of a VWM mouse model. The effects of cell grafts on motor skills and VWM brain pathology were assessed. Results: Transplantation of human glial precursor populations improved the VWM phenotype. The intrinsic properties of these cells were partially reflected by cell fate post-transplantation, but were also affected by the host microenvironment. Strikingly, the spread of transplanted cells into the white matter versus the gray matter was different when grafted into the VWM brain as compared to a healthy brain. Conclusions: Transplantation of human glial cell populations can have therapeutic effects for VWM. For further translation to the clinic, the microenvironment in the VWM patient brain should be considered as an important moderator of cell replacement therapy.

Microglia play a critical role in the neuroimmune response, but little is known about the role of microglia in sleep following an inflammatory trigger. Nevertheless, decades of research have been predicated on the assumption that an inflammatory trigger increases sleep through microglial activation. We hypothesized that mice (n = 30) with depleted microglia using PLX5622 (PLX) would sleep less following the administration of lipopolysaccharide (LPS) to induce inflammation. Brains were collected and microglial morphology was assessed using quantitative skeletal analyses and physiological parameters were recorded using non-invasive piezoelectric cages. Mice fed PLX diet had a transient increase in sleep that dissipated by week 2. Subsequently, following a first LPS injection (0.4 mg/kg), mice with depleted microglia slept more than mice on the control diet. All mice were returned to normal rodent chow to repopulate microglia in the PLX group (10 days). Nominal differences in sleep existed during the microglia repopulation period. However, following a second LPS injection, mice with repopulated microglia slept similarly to control mice during the dark period but with longer bouts during the light period. Comparing sleep after the first LPS injection to sleep after the second LPS injection, controls exhibited temporal changes in sleep patterns but no change in cumulative minutes slept, whereas cumulative sleep in mice with repopulated microglia decreased during the dark period across all days. Repopulated microglia had a reactive morphology. We conclude that microglia are necessary to regulate sleep after an immune challenge.

Pain is a complex entity that can be described in several dimensions, such as, acute pain and chronic pain, characterized by its duration. After the tissue injury, the activation of the sensory nervous tissue occurs from where the different pro-inflammatory mediators are released with the consequent nociceptive transmission that characterizes the genesis of inflammatory pain. The main objective of this study was to review the role of the opioid system, using morphine and MOR opioid receptors as paradigm, in the antinociceptive modulation of inflammatory pain, by means of the formalin test as a model. Various pieces of evidence are compiled that establish the fundamental role of morphine in the inflammatory pain. Morphine has noticeable antinociceptive efficacy in to decrease the inflammatory pain. This review demonstrates the fundamental role that morphine plays in inflammatory pain states and that it could serve as the basis for a new pharmacotherapy of inflammatory pain.

Neuropathic pain remains a clinical challenge due to its complex and not yet fully understood pathomechanism, which result in limited analgesic effectiveness of the management offered, particularly for patients with acute, refractory neuropathic pain states. In addition to the introduction of several modern therapeutic approaches, such as neuromodulation or novel anti-neuropathic drugs, significant efforts have been made in the repurposing of well-known substances such as phenytoin. Although its main mechanism of action occurs at sodium channels in excitable and non-excitable cells and is well documented, how the drug affects the disturbed neuropathic interactions at the spinal cord level and how it influences morphine-induced analgesia have not been clarified, both being crucial from a clinical perspective. We demonstrated that single and repeated systemic administrations of phenytoin decreased tactile and thermal hypersensitivity in an animal model of neuropathic pain. Importantly, we observed an increase in the antinociceptive effect on thermal stimuli with repeated administrations of phenytoin. This is the first study to report that phenytoin improves morphine-induced antinociceptive effects and influences microglia/macrophage activity at the spinal cord and dorsal root ganglion levels in a neuropathic pain model. Our findings support the hypothesis that phenytoin may represent an effective strategy for neuropathic pain management in clinical practice, particularly when combination with opioids is needed.

Acute inflammation can represent an effective immune response to the corporeal presence of organisms of exogenous origin or damaged cells of intrinsic origin, both of which are potentially injurious. Very often, such inflammation persists long after the triggering stimulus no longer poses a threat. Such a chronic condition can ultimately be more harmful than the original triggering agent. The means by which metallic compounds can provoke undesirable and extended inflammatory changes within the nervous system are diverse. This short review classifies the likely mechanisms underlying their toxicity by taking into account their known chemical properties and also by reflecting on other injurious events that commonly occur together with inflammation. The brevity of this survey does not allow focusing exhaustively on each individual metal but rather seeks to uncover some intracellular susceptible biochemical loci, based on the chemistry of each class of metals. Developmental and senescence-related characteristics are considered as these stages of life often involve especial vulnerabilities. Another feature addressed is that while research is generally conducted on purified and well-defined agents, exposures in the real world generally involve diverse mixtures in which the individual toxicity of a component can be enhanced or suppressed by the presence of other chemicals.

Glial cell transplantation using olfactory ensheathing cells (OECs) holds a promising approach for treating spinal cord injury (SCI). However, integration of OECs into the hostile acute secondary injury site requires interaction and response to macrophages. Immunomodulation of macrophages to reduce their impact on OECs may improve the functionality of OECs. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), known for their immunomodulatory and neuroprotective functions, have provided improved outcomes in SCI animal models. Thus, VEGF and PDGF modulation of the SCI microenvironment may be beneficial for OEC transplantation. In this in vitro study, the effect of VEGF and PDGF on macrophages in an inflammatory condition was tested. Combined VEGF + PDGF reduced translocation nuclear factor kappa B p65 in macrophages without altering pro-inflammatory cytokines. Further, the ability of OECs to phagocytose myelin debris was assessed using macrophage-conditioned medium. Conditioned medium from macrophages incubated with PDGF and combined VEGF + PDGF in inflammatory conditions promoted phagocytosis by OECs. The growth factor treated conditioned media also modulated the expression of genes associated with nerve repair and myelin expression in OECs. Overall, these results suggest that the use of growth factors together with OEC transplantation may be beneficial in SCI therapy.

Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/β-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release.

Humans with post-traumatic stress disorder (PTSD) exhibit sleep disturbances that include insomnia, nightmares, and enhanced daytime sleepiness. Sleep disturbances are considered a hallmark feature of PTSD; however, little is known about the cellular and molecular mechanisms regulating trauma-induced sleep disorders. Using a rodent model of PTSD called “Single Prolonged Stress” (SPS) we examined the requirement of the brain-type fatty acid binding protein Fabp7, an astrocyte expressed lipid-signaling molecule, in mediating trauma-induced sleep disturbances. We measured baseline sleep/wake parameters and then exposed Fabp7 knock-out (KO) and wild-type (WT) C57BL/6N genetic background control animals to SPS. Sleep and wake measurements were obtained immediately following the initial trauma exposure of SPS, and again 7 days later. We found that active-phase (dark period) wakefulness was similar in KO and WT at baseline and immediately following SPS; however, it was significantly increased after 7 days. These effects were opposite in the inactive-phase (light period), where KOs exhibited increased wake in baseline and following SPS, but returned to WT levels after 7 days. To examine the effects of Fabp7 on unconditioned anxiety following trauma, we exposed KO and WT mice to the light–dark box test before and after SPS. Prior to SPS, KO and WT mice spent similar amounts of time in the lit compartment. Following SPS, KO mice spent significantly more time in the lit compartment compared to WT mice. These results demonstrate that mutations in an astrocyte-expressed gene (Fabp7) influence changes in stress-dependent sleep disturbances and associated anxiety behavior.

In this review article, we present the major insights from and challenges faced in the acquisition, analysis and modeling of astrocyte calcium activity, aiming at bridging the gap between those fields to crack the complex astrocyte “Calcium Code”. We then propose strategies to reinforce interdisciplinary collaborative projects to unravel astrocyte function in health and disease.

Spinal motor nerves are essential for proper organismal locomotion and survival. Motor nerves consist of bundles of motor axons ensheathed by layers of connective tissue and supporting glial cells. Remarkably, in most vertebrates these motor nerves are able to naturally regenerate following injury, though full functional recovery in mammals is limited. The same glial cells that support the motor nerve during development are activated to allow for efficient and effective regeneration. However, the cellular and molecular interactions that underlie successful motor nerve regeneration remain poorly understood. Studies have classically focused on the regenerative responses of Schwann cells and macrophages; however, little is known about the injury responses of other cells that make up the motor nerve. Recently, studies from our lab identified that in the early hours following spinal motor nerve injury in zebrafish, perineurial glia form a glial bridge across the injury site and phagocytose axonal debris. Though perineurial glia are essential for successful motor nerve development and regeneration after injury, very little is known about their injury response. In this dissertation, using zebrafish as a model organism, I present the first known signaling pathway to drive perineurial glial bridging after spinal motor nerve injury, explore cellular interactions between perineurial glia and Schwann cells, and establish tools for future studies to continue to elucidate the signals that drive successful regeneration. This work fills gaps in our knowledge about the cellular and molecular interactions that occur between essential glial cells to drive injury responses that are necessary for successful motor nerve regeneration and presents potential targets for future therapeutics.

Suitable in vivo and in vitro models are instrumental for the development of new drugs aimed at improving symptoms or progression of multiple sclerosis (MS). The cuprizone (CPZ)-induced murine model has gained momentum in recent decades, aiming to address the demyelination component of the disease. This work aims at assessing the differential cytotoxicity of CPZ in cells of different types and from different species: human oligodendroglial (HOG), human neuroblastoma (SH-SY5Y), human glioblastoma (T-98), and mouse microglial (N-9) cell lines. Moreover, the effect of CPZ was investigated in primary rat brain cells. Cell viability was assayed by oxygen rate consumption and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based (MTT) method. Our results demonstrated that CPZ did not cause death in any of the assayed cell models but affected mitochondrial function and aerobic cell respiration, thus compromising cell metabolism in neural cells and neuron-glia co-cultures. In this sense, we found differential vulnerability between glial cells and neurons as is the case of the CPZ-induced mouse model of MS. In addition, our findings demonstrated that reduced viability was spontaneous reverted in a time-dependent manner by treatment discontinuation. This reversible cell-based model may help to further investigate the role of mitochondria in the disease, and study the molecular intricacies underlying the pathophysiology of the MS and other demyelinating diseases., Los modelos in vivo e in vitro adecuados son fundamentales para el desarrollo de nuevos fármacos destinados a mejorar los síntomas o la progresión de la esclerosis múltiple (EM). El modelo murino inducido por cuprizona (CPZ) ha ganado impulso en las últimas décadas, con el objetivo de abordar el componente de desmielinización de la enfermedad. Este trabajo tiene como objetivo evaluar la citotoxicidad diferencial de CPZ en células de diferentes tipos y de diferentes especies: oligodendroglial humano (HOG), neuroblastoma humano (SH-SY5Y), glioblastoma humano (T-98) y microglial de ratón (N-9). líneas celulares. Además, se investigó el efecto de CPZ en células cerebrales primarias de rata. La viabilidad celular se ensayó mediante el consumo de oxígeno y mediante el método basado en bromuro de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT). Nuestros resultados demostraron que CPZ no causó la muerte en ninguno de los modelos celulares ensayados, pero afectó la función mitocondrial y la respiración celular aeróbica, comprometiendo así el metabolismo celular en las células neurales y los cocultivos de neuronas y glías. En este sentido, encontramos una vulnerabilidad diferencial entre las células gliales y las neuronas, como es el caso del modelo de EM en ratón inducido por CPZ. Además, nuestros hallazgos demostraron que la reducción de la viabilidad se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. comprometiendo así el metabolismo celular en las células neurales y los cocultivos de neurona y glía. En este sentido, encontramos una vulnerabilidad diferencial entre las células gliales y las neuronas, como es el caso del modelo de EM en ratón inducido por CPZ. Además, nuestros hallazgos demostraron que la reducción de la viabilidad se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. comprometiendo así el metabolismo celular en las células neurales y los cocultivos de neurona y glía. En este sentido, encontramos una vulnerabilidad diferencial entre las células gliales y las neuronas, como es el caso del modelo de EM en ratón inducido por CPZ. Además, nuestros hallazgos demostraron que la reducción de la viabilidad se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. nuestros hallazgos demostraron que la viabilidad reducida se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. nuestros hallazgos demostraron que la viabilidad reducida se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes.

Plectin, a high-molecular-mass cytolinker, is abundantly expressed in the central nervous system (CNS). Currently, a limited amount of data about plectin in the CNS prevents us from seeing the complete picture of how plectin affects the functioning of the CNS as a whole. Yet, by analogy to its role in other tissues, it is anticipated that, in the CNS, plectin also functions as the key cytoskeleton interlinking molecule. Thus, it is likely involved in signalling processes, thereby affecting numerous fundamental functions in the brain and spinal cord. Versatile direct and indirect interactions of plectin with cytoskeletal filaments and enzymes in the cells of the CNS in normal physiological and in pathologic conditions remain to be fully addressed. Several pathologies of the CNS related to plectin have been discovered in patients with plectinopathies. However, in view of plectin as an integrator of a cohesive mesh of cellular proteins, it is important that the role of plectin is also considered in other CNS pathologies. This review summarizes the current knowledge of plectin in the CNS, focusing on plectin isoforms that have been detected in the CNS, along with its expression profile and distribution alongside diverse cytoskeleton filaments in CNS cell types. Considering that the bidirectional communication between neurons and glial cells, especially astrocytes, is crucial for proper functioning of the CNS, we place particular emphasis on the known roles of plectin in neurons, and we propose possible roles of plectin in astrocytes.

Zika virus (ZIKV) is a neurotropic flavivirus recently linked to congenital ZIKV syndrome in children and encephalitis and Guillain-Barré syndrome in adults. Neurotropic viruses often use axons to traffic to neuronal or glial cell somas where they either remain latent or replicate and proceed to infect new cells. Consequently, it has been suggested that axon degeneration could represent an evolutionarily conserved mechanism to limit viral spread. Whilst it is not known if ZIKV transits in axons, we previously reported that ZIKV infection of glial cells in a murine spinal cord-derived cell culture model of the CNS is associated with a profound loss of neuronal cell processes. This, despite that postmitotic neurons are relatively refractory to infection and death. Here, we tested the hypothesis that ZIKV-associated degeneration of neuronal processes is dependent on activation of Sterile alpha and armadillo motif-containing protein 1 (SARM1), an NADase that acts as a central executioner in a conserved axon degeneration pathway. To test this, we infected wild type and Sarm1 homozygous or heterozygous null cell cultures with ZIKV and examined NAD+ levels as well as the survival of neurons and their processes. Unexpectedly, ZIKV infection led to a rapid SARM1-independent reduction in NAD+. Nonetheless, the subsequent profound loss of neuronal cell processes was SARM1-dependent and was preceded by early changes in the appearance of β-tubulin III staining. Together, these data identify a role for SARM1 in the pathogenesis of ZIKV infection, which may reflect SARM1's conserved prodegenerative function, independent of its NADase activity.

Excess glucocorticoid exposure affects emotional and cognitive brain functions. The extreme form, Cushing's syndrome, is adequately modelled in the AdKO(2.0) mouse, consequential to adrenocortical hypertrophy and hypercorticosteronemia. We previously reported that the AdKO(2.0) mouse brain undergoes volumetric changes that resemble closely those of Cushing's syndrome human patients, as well as changes in expression of glial related marker proteins. In the present work, the expression of genes related to glial and neuronal cell populations and functions was assessed in regions of the anterior brain, hippocampus, amygdala and hypothalamus. Glucocorticoid target genes were consistently regulated, including CRH mRNA suppression in the hypothalamus and induction in amygdala and hippocampus, even if glucocorticoid receptor protein was downregulated. Expression of glial genes was also affected in the AdKO(2.0) mouse brain, indicating a different activation status in glial cells. Generic markers for neuronal cell populations, and cellular integrity were only slightly affected. Our findings highlight the vulnerability of glial cell populations to chronic high levels of circulating glucocorticoids.

International audience; Cell therapy is a promising strategy in the field of regenerative medicine; however, several concerns limit the effective clinical use, namely a valid cell source. The gastrointestinal tract, which contains a highly organized network of nerves called the enteric nervous system (ENS), is a valuable reservoir of nerve cells. Together with neurons and neuronal precursor cells, it contains glial cells with a well described neurotrophic potential and a newly identified neurogenic one. Recently, enteric glia is looked at as a candidate for cell therapy in intestinal neuropathies. Here, we present the therapeutic potential of the ENS as cell source for brain repair, too. The example of stroke is introduced as a brain injury where cell therapy appears promising. This disease is the first cause of handicap in adults. The therapies developed in recent years allow a partial response to the consequences of the disease. The only prospect of recovery in the chronic phase is currently based on rehabilitation. The urgency to offer other treatments is therefore tangible. In the first part of the review, some elements of stroke pathophysiology are presented. An update on the available therapeutic strategies is provided, focusing on cell- and biomaterial-based approaches. Following, the ENS is presented with its anatomical and functional characteristics, focusing on glial cells. The properties of these cells are depicted, with particular attention to their neurotrophic and, recently identified, neurogenic properties. Finally, preliminary data on a possible therapeutic approach combining ENS-derived cells and a biomaterial are presented

Summary Bipolar disorder (BD) is characterized by cyclical mood shifts. Studies indicate that BD patients have a peripheral pro-inflammatory state and alterations in glial populations in the brain. We utilized an in vitro model to study inflammation-related phenotypes of astrocytes derived from induced pluripotent stem cells (iPSCs) generated from BD patients and healthy controls. BD astrocytes showed changes in transcriptome and induced a reduction in neuronal activity when co-cultured with neurons. IL-1β-stimulated BD astrocytes displayed a unique inflammatory gene expression signature and increased secretion of IL-6. Conditioned medium from stimulated BD astrocytes reduced neuronal activity, and this effect was partially blocked by IL-6 inactivating antibody. Our results suggest that BD astrocytes are functionally less supportive of neuronal excitability and this effect is partially mediated by IL-6. We confirmed higher IL-6 in blood in a distinct cohort of BD patients, highlighting the potential role of astrocyte-mediated inflammatory signaling in BD neuropathology., Highlights • Bipolar disorder astrocytes are functionally less supportive of neuronal activity • Bipolar disorder astrocytes response to IL-1β is transcriptionally distinct • IL-6 secretion in bipolar disorder astrocytes reduces neuronal activity • Bipolar disorder patients show higher circulating levels of IL-6 in blood, In this article, Gage and collaborators show that astrocytes differentiated from induced pluripotent stem cells generated from bipolar disorder patients are functionally less supportive of neuronal activity. Bipolar disorder astrocytes' response to pro-inflammatory cytokines is characterized by a unique transcriptional response and increased IL-6 secretion that directly and negatively impacted on neuronal activity. Increased peripheral IL-6 was confirmed in a distinct clinical cohort highlighting the potential role of astrocyte-mediated inflammatory signaling in the neuropathology of bipolar disorder.

To facilitate analyses of purinergic signaling in peripheral nerve glia, we review recent literature and catalog purinergic receptor mRNA expression in cultured mouse Schwann cells (SCs). Purinergic signaling can decrease developmental SC proliferation, and promote SC differentiation. The purinergic receptors P2RY2 and P2RX7 are implicated in nerve development and in the ratio of Remak SCs to myelinating SCs in differentiated peripheral nerve. P2RY2, P2RX7, and other receptors are also implicated in peripheral neuropathies and SC tumors. In SC tumors lacking the tumor suppressor NF1, the SC pathway that suppresses SC growth through P2RY2‐driven β‐arrestin‐mediated AKT signaling is aberrant. SC‐released purinergic agonists acting through SC and/or neuronal purinergic receptors activate pain responses. In all these settings, purinergic receptor activation can result in calcium‐independent and calcium‐dependent release of SC ATP and UDP, growth factors, and cytokines that may contribute to disease and nerve repair. Thus, current research suggests that purinergic agonists and/or antagonists might have the potential to modulate peripheral glia function in development and in disease., MAIN POINTS SC purinergic receptors are implicated in suppressing SC proliferation, increasing SC differentiation, and elevating intracellular Ca2+ during nerve development in response to injury and/or in disease.P1R, P2XR, and P2YR receptors expressed in SCs and adjacent neurons need further study.

Feifei Ren,1 Rongjuan Guo2 1Second Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, People’s Republic of China; 2Department of Neurology, Dongfang Hospital, Beijing University of Chinese Medicine, Beijing 100078, People’s Republic of ChinaCorrespondence: Rongjuan GuoDepartment of Neurology, Dongfang Hospital, Beijing University of Chinese Medicine, No. 6, 1st Section, Fangxingyuan, Fangzhuang, Fengtai District, Beijing City, People’s Republic of ChinaTel/Fax +86 10-6768-9655Email 381160563@qq.comAbstract: Depression is a major disease that can affect both mental and physical health, limits psychosocial functioning and diminishes the quality of life. But its complex pathogenesis remains poorly understood. The dynamic changes of synaptic structure and function, known as synaptic plasticity, occur with the changes of different cellular microenvironment and are closely related to learning and memory function. Accumulating evidence implies that synaptic plasticity is integrally involved in the pathological changes of mood disorders, especially in depressive disorder. However, the complex dynamic process of synaptic plasticity is influenced by many factors. Here, we reviewed and discussed various factors affecting synaptic plasticity in depression, and proposed a specific framework named synaptic microenvironment, which may be critical for synaptic plasticity under pathological conditions. Based on this concept, we will show how we understand the balance between the synaptic microenvironment and the synaptic plasticity network in depression. Finally, we point out the clinical significance of the synaptic microenvironment in depression.Keywords: depression, synaptic plasticity, synaptic microenvironment, glia, influence factors

Dopamine (DA) is a key neurotransmitter in the basal ganglia, implicated in the control of movement and motivation. Alteration of DA levels is central in Parkinson’s disease (PD), a common neurodegenerative disorder characterized by motor and non-motor manifestations and deposition of alpha-synuclein (α-syn) aggregates. Previous studies have hypothesized a link between PD and viral infections. Indeed, different cases of parkinsonism have been reported following COVID-19. However, whether SARS-CoV-2 may trigger a neurodegenerative process is still a matter of debate. Interestingly, evidence of brain inflammation has been described in postmortem samples of patients infected by SARS-CoV-2, which suggests immune-mediated mechanisms triggering the neurological sequelae. In this review, we discuss the role of proinflammatory molecules such as cytokines, chemokines, and oxygen reactive species in modulating DA homeostasis. Moreover, we review the existing literature on the possible mechanistic interplay between SARS-CoV-2-mediated neuroinflammation and nigrostriatal DAergic impairment, and the cross-talk with aberrant α-syn metabolism.

Alzheimer’s disease (AD) is a progressive and degenerative disease producing the most common type of dementia worldwide. The main pathogenetic hypothesis in recent decades has been the well-known amyloidogenic hypothesis based on the involvement of two proteins in AD pathogenesis: amyloid β (Aβ) and tau. Amyloid deposition reported in all AD patients is nowadays considered an independent risk factor for cognitive decline. Vascular damage and blood–brain barrier (BBB) failure in AD is considered a pivotal mechanism for brain injury, with increased deposition of both immunoglobulins and fibrin. Furthermore, BBB dysfunction could be an early sign of cognitive decline and the early stages of clinical AD. Vascular damage generates hypoperfusion and relative hypoxia in areas with high energy demand. Long-term hypoxia and the accumulation within the brain parenchyma of neurotoxic molecules could be seeds of a self-sustaining pathological progression. Cellular dysfunction comprises all the elements of the neurovascular unit (NVU) and neuronal loss, which could be the result of energy failure and mitochondrial impairment. Brain glucose metabolism is compromised, showing a specific region distribution. This energy deficit worsens throughout aging. Mild cognitive impairment has been reported to be associated with a glucose deficit in the entorhinal cortex and in the parietal lobes. The current aim is to understand the complex interactions between amyloid β (Aβ) and tau and elements of the BBB and NVU in the brain. This new approach aimed at the study of metabolic mechanisms and energy insufficiency due to mitochondrial impairment would allow us to define therapies aimed at predicting and slowing down the progression of AD.

Cues in the micro-environment are key determinants in the emergence of complex cellular morphologies and functions. Primary among these is the presence of neighboring cells that form networks. For high-resolution analysis, it is crucial to develop micro-environments that permit exquisite control of network formation. This is especially true in cell science, tissue engineering, and clinical biology. We introduce a new approach for assembling polydimethylsiloxane (PDMS)-based microfluidic environments that enhances cell network formation and analyses. We report that the combined processes of PDMS solvent-extraction and hydrothermal annealing create unique conditions that produce high-strength bonds between solvent-extracted PDMS (E-PDMS) and glass—properties not associated with conventional PDMS. Extraction followed by hydrothermal annealing removes unbound oligomers, promotes polymer cross-linking, facilitates covalent bond formation with glass, and retains the highest biocompatibility. Herein, our extraction protocol accelerates oligomer removal from 5 to 2 days. Resulting microfluidic platforms are uniquely suited for cell-network studies owing to high adhesion forces, effectively corralling cellular extensions and eliminating harmful oligomers. We demonstrate the simple, simultaneous actuation of multiple microfluidic domains for invoking ATP- and glutamate-induced Ca2+ signaling in glial-cell networks. These E-PDMS modifications and flow manipulations further enable microfluidic technologies for cell-signaling and network studies as well as novel applications.

Background Neuron-glial antigen 2 (NG2) cells are a glial cell type tiled throughout the gray and white matter of the central nervous system (CNS). NG2 cells are known for their ability to differentiate into oligodendrocytes and are commonly referred to as oligodendrocyte precursor cells. However, recent investigations have begun to identify additional functions of NG2 cells in CNS health and pathology. NG2 cells form physical and functional connections with neurons and other glial cell types throughout the CNS, allowing them to monitor and respond to the neural environment. Growing evidence indicates that NG2 cells become reactive under pathological conditions, though their specific roles are only beginning to be elucidated. While reactive microglia and astrocytes are well-established contributors to neuroinflammation and the development of epilepsy following CNS infection, the dynamics of NG2 cells remain unclear. Therefore, we investigated NG2 cell reactivity in a viral-induced mouse model of temporal lobe epilepsy. Methods C57BL6/J mice were injected intracortically with Theiler’s murine encephalomyelitis virus (TMEV) or PBS. Mice were graded twice daily for seizures between 3 and 7 days post-injection (dpi). At 4 and 14 dpi, brains were fixed and stained for NG2, the microglia/macrophage marker IBA1, and the proliferation marker Ki-67. Confocal z stacks were acquired in both the hippocampus and the overlying cortex. Total field areas stained by each cell marker and total field area of colocalized pixels between NG2 and Ki67 were compared between groups. Results Both NG2 cells and microglia/macrophages displayed increased immunoreactivity and reactive morphologies in the hippocampus of TMEV-injected mice. While increased immunoreactivity for IBA1 was also present in the cortex, there was no significant change in NG2 immunoreactivity in the cortex following TMEV infection. Colocalization analysis for NG2 and Ki-67 revealed a significant increase in overlap between NG2 and Ki-67 in the hippocampus of TMEV-injected mice at both time points, but no significant differences in cortex. Conclusions NG2 cells acquire a reactive phenotype and proliferate in response to TMEV infection. These results suggest that NG2 cells alter their function in response to viral encephalopathy, making them potential targets to prevent the development of epilepsy following viral infection.