ASN Neuro

Phagocytic and immune-like cells have been observed in the satellite envelope of neuronal somata in peripheral sensory ganglia of many species for several decades. These cells likely play an important role in normal function of sensory neurons and they may also play an important role in neuronal dysfunction and neurodegeneration seen with neuropathy. Recent findings have described a satellite macrophage population transcriptomically similar to microglia in peripheral ganglia of some mammalian species. The function of these cells, and the mechanisms by which they may influence neurons in neuropathy are unclear. We sought to understand the phenotype and localization of these cells in the human dorsal root ganglion (hDRG) using large-scale single nucleus and spatial transcriptomic datasets from individuals with and without a history of peripheral diabetic neuropathy. We observed a large population of macrophages that express classical microglia makers such as TMEM119 and P2RY12 in the hDRG, as previously described. Our findings confirm that these microglia-like cells (MLCs) localize to the satellite envelope around neuronal somata, yet are transcriptomically distinct from all glial cell types characterized in the hDRG. These MLCs exhibit changes in abundance and localization with diabetic painful neuropathy (DPN) in both the hDRG and sural nerves suggesting that they are not exclusively localized to the DRG. We conclude that microglia-like cells are likely the resident tissue macrophage (RTM) of the hDRG, and perhaps the peripheral nervous system (PNS) given their localization to the sural nerve and other ganglia, where they are predicted to regulate homeostatic neuronal functions and response to injury. HighlightsO_LIMLCs are likely the RTM of hDRGs C_LIO_LIMLCs localize to the satellite envelope and recede with Nageotte nodule formation C_LIO_LIMLC activation state and signaling shift with diabetic neuropathy C_LIO_LIMLCs are also present in other ganglia and sural nerve C_LI

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1-7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

BackgroundMicroglial heterogeneity is a fundamental feature of brain homeostasis and pathology. The purpose of this study was to investigate the complexity of microglial plasticity by characterizing specialized oligodendrocyte-like microglial subsets. MethodsThe study was performed utilizing single-cell transcriptomics analyses and immunofluorescence staining to identify and profile microglial subpopulations. Additionally, spatial transferring and morphological analyses were conducted to determine the anatomical distribution and structural features of these specific cells. ResultsWe identified a distinct microglial subset termed dual-phenotype microglia (DPM), which co-expresses microglial and oligodendrocyte markers. DPM consisted of two subtypes with distinct functions: myelin-associated DPM (mDPM) and neuron-associated DPM (nDPM). Spatial and morphological evaluations revealed that mDPMs were sparsely distributed across the whole brain and exhibited a highly ramified architecture, whereas nDPMs were enriched in the hippocampal dentate gyrus. Mechanistically, we found that mDPM function was driven by the Sox10 regulon to modulate myelin maintenance and axonal ensheathment, while nDPM was orchestrated by Glis2, facilitating essential neuron-glia crosstalk and synaptic regulation. Furthermore, we demonstrated that nDPM and mDPM were predicted to undergo significant alterations in multiple sclerosis and Alzheimers disease. Notably, mDPMs were selectively enriched in active multiple sclerosis lesions, revealing that DPM were closely related to neuropsychiatric disorders. ConclusionsBy comprehensively characterizing the morphology, molecular signatures, and spatial logic of these oligodendrocyte-like microglial subsets, our study elucidated the complexity of microglial plasticity. These findings provided new insights into their diverse roles in central nervous system health and disease. Graphical abstractIdentification, Molecular Profiling, and Functional Modeling of Dual-Phenotype Microglia (DPM). (1) Discovery: Identification of the dual-phenotype microglia (DPM) population through single-cell transcriptomics. (2) Molecular Signatures: The transcriptomic identity of DPM subtypes is governed by specific regulatory networks. (3) Distribution & Pathology: Spatial mapping reveals divergent anatomical logic and disease relations for DPM subtypes. (4) Mechanism/Theory: A proposed functional model of mDPMs as "metabolic relay" and support units. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/724239v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@b7db1dorg.highwire.dtl.DTLVardef@9265e7org.highwire.dtl.DTLVardef@1605d82org.highwire.dtl.DTLVardef@19b048f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oligodendrocyte-enriched cortical organoids (OCOs) are a powerful platform for modeling oligodendrogenesis in a human cellular context. However, neuronal activity is impaired in conventional culture media, limiting assessment of neuronal function in conjunction with oligodendrocyte biology. To address this, we used a modified BrainPhys medium termed neuronal activity medium (NAM) and defined the optimal developmental window for NAM exposure to generate OCOs with robust neuronal activity (NAM-OCOs). Stage-specific exposure to NAM, prior to oligodendrocyte expansion, leads to enhanced structural maturation, as evidenced by increased organoid size, heightened synaptogenesis, and upregulation of transcripts associated with neuronal complexity. Further, NAM-OCOs display increased cellular heterogeneity, including greater representation of GABAergic interneurons while preserving oligodendrocyte development and maturation. Altogether, our studies demonstrate that stage-specific exposure to an activity-permissive environment enhances neuronal activity, establishing an OCO model which integrates neuronal activity with oligodendrocyte development and maturation. HighlightsO_LIIncreased neuronal activity in oligodendrocyte-enriched cortical organoids (OCOs) C_LIO_LIStage-specific Neuronal Activity Medium (NAM) optimizes activity C_LIO_LINAM-OCOs display increased cellular heterogeneity and neuronal maturation C_LIO_LIOligodendrogenesis is preserved in NAM-OCOs C_LI eTOC blurbIn this article, Chung et al enhance neuronal activity in oligodendrocyte-enriched cortical organoids (OCOs) through stage-specific exposure to Neuronal Activity Medium (NAM). OCOs exposed to NAM display elevated cellular heterogeneity, structural maturation, and synaptogenesis, while preserving oligodendrocyte development and maturation. These results establish an increasingly comprehensive OCO model for studying neuronal function and oligodendrogenesis.

Assessing reproducibility across different molecular profiling studies is a persistent methodological challenge (Zhang et al., 2009; Sweeney et al., 2017; Ioannidis, 2005). Differences in platform technology, cohort composition, analytical pipelines, and feature definitions often make it difficult to interpret cross-study comparisons based solely on gene-identity overlap. In this study, we conducted a retrospective computational analysis of seven publicly available analytical datasets (including alternative analytical pipelines applied to the same cohort) derived from five biologically independent peripheral blood transcriptomic and DNA methylation cohorts, comprising 3,487 samples (1,824 Parkinsons disease cases and 1,663 controls). Reproducibility was evaluated using gene-identity overlap, enrichment-based comparisons, and a permutation-based framework to assess directional consistency of effect estimates across datasets. We also tested the robustness of results by varying false discovery rate thresholds and applying alternative probe-to-gene collapsing strategies. All analyses were performed using reproducible workflows implemented in R and Python with fixed random seeds. Across independent cohorts, gene-identity overlap was generally limited, with enrichment ratios close to one, especially when datasets were generated using different platforms. In several datasets, limited numbers of statistically significant features further constrained overlap-based comparisons. In contrast, directional consistency showed greater stability. High levels of directional consistency were observed across independent cohort comparisons when restricted to overlapping statistically significant features and remained stable across statistical thresholds (90.0% at FDR < 0.05 and 82.8% at FDR < 0.10). When evaluated across the full shared gene universe without conditioning on statistical significance, directional consistency was substantially lower ([~]30 to 32%) but remained significantly above permutation-based null expectations. Permutation testing confirmed that the observed directional consistency exceeded what would be expected by chance. A combined analysis including methodological replicates (n [&ge;] 3 datasets) showed 98.3% directional consistency; however, this estimate includes non-independent analytical pipelines applied to the same cohort and reflects analytical stability rather than independent biological replication. Rather than introducing a new statistical method, this study examines how commonly used reproducibility metrics behave under crossstudy heterogeneity and identifies their practical limitations and appropriate use boundaries.

The effect of sortilin inhibition on acute inner retinal neurodegeneration induced by optic nerve crush was investigated. Pharmacological sortilin inhibition using intravitreal delivery of a polyclonal antibody or a small-molecule inhibitor was evaluated in C57BL/6JRj male mice subjected to unilateral crush. Inner retinal thickness was evaluated by optical coherence tomography, and retinal ganglion cell density was determined in retinal flat mounts. Furthermore, the effect of constitutive sortilin deficiency was examined using Sort1-/- mice. Changes in protein and mRNA levels of sortilin, p75NTR, and associated injury markers were analyzed. Neither pharmacological inhibition or constitutive loss of sortilin protected against inner retinal thinning or retinal ganglion cell loss following optic nerve crush. A transient 1.4-fold increase in p75NTR mRNA was observed early after injury, accompanied by a two-fold increase in protein levels. While sortilin expression remained largely unchanged, sortilin deficiency was associated with an altered baseline retinal state, including increased GFAP, p75NTR, and proBDNF levels. Following optic nerve crush, the induction of p75NTR was significantly attenuated in sortilin-deficient retinas compared with wild type, without affecting the extent of RGC degeneration. In summary, sortilin inhibition does not preserve inner retinal structure following optic nerve crush, but modulates glial activation, inflammatory signaling, and proneurotrophin dynamics. These findings indicate that sortilin-dependent pathways are not key drivers of optic nerve crush-induced neurodegeneration but may be more relevant in disease contexts characterized by chronic stress and neuroinflammation.

Motoneurons are under strong pressure to maintain stable motor output throughout an individual life, through homeostatic regulation of their electrical properties. Dysregulated spinal motoneuron excitability has long been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent work in SOD1G93A mice suggests that the homeostatic response of motoneurons becomes dysregulated as cellular processes are disrupted by the disease, causing fluctuations in motoneuron electrical properties. Yet, few studies directly test whether ALS motoneurons respond differently than wild type motoneurons to a common chronic perturbation. Here, we used in vivo electrophysiology to test whether motoneurons from pre-symptomatic SOD1G93A mice modulate excitability differently than wild type motoneurons in response to the same homeostatic perturbation: chronic inhibition exerted by the benzodiazepine diazepam. Using linear mixed-effects statistical models, we assessed whether diazepam treatment differentially modulated passive properties, firing behavior, spike properties, and/or synaptic inputs in SOD1G93A versus wild type motoneurons. We identified a significant genotype x treatment interaction effect selectively for properties related to passive membrane integration and spike initiation, including membrane time constant, peak input resistance, and recruitment current. In contrast, firing gain, spike waveform characteristics, and synaptic inputs were largely unaffected. These findings indicate that sustained inhibitory perturbation selectively triggered overactive intrinsic compensatory mechanisms in SOD1G93A motoneurons rather than inducing widespread changes in firing or synaptic transmission. Together, our results provide direct evidence for over-active homeostatic control of motoneuron excitability and support a view of motoneuron dysfunction in ALS as a problem of altered feedback regulation rather than simply hyper- or hypo-excitability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/725609v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@25f125org.highwire.dtl.DTLVardef@faf2c9org.highwire.dtl.DTLVardef@15993a8org.highwire.dtl.DTLVardef@1ed006a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Homeostatic plasticity of intrinsic excitability (IE) in the visual system has been essentially shown at the cortical level but whether thalamic nuclei also express homeostatic plasticity of IE is unknown. We show here that 4 days of monocular deprivation (MD) at eye opening induces a homeostatic change in IE in dorsal lateral geniculate nucleus (dLGN) neurons. Neurons recorded in the dLGN region activated by the deprived eye are more excitable than neurons recorded in the dLGN region activated by the open eye. No significant changes were observed following 7 days of MD, however. Enhanced excitability in neurons from the deprived side after 4 days of MD was associated with a reduced Kv1-dependent LTP-IE, a smaller voltage ramp, and a reduced inter-spike interval, suggesting that Kv1 channels are down-regulated in deprived dLGN neurons. Furthermore, the ankyrin G signal of the axon initial segment was larger in deprived dLGN neurons compared with open ones, indicating that Nav1 channel number also undergoes homeostatic regulation, and Kv1.1 channel signals were lower in deprived neurons compared to open ones. In addition, electrical coupling was found to be strengthened in neurons displaying enhanced IE following either brief (4 days) or long (10 days) MD. These results suggest that homeostatic and Hebbian plasticity in the dLGN share common expression mechanisms involving the regulation of Kv1 channels, Nav1 channels and electrical coupling between relay neurons.

Purinergic 2X7 receptor (P2X7R) is considered to play a critical role in neurological diseases, including epilepsy, and has also been proposed as a potential marker for neuroinflammation. This study aimed to validate the binding properties of the novel P2X7R radiotracer, [3H]JNJ-64413739, in rat brain using in vitro autoradiography, and additionally to explore spatial and temporal changes in P2X7R binding levels in a rat model of temporal lobe epilepsy using intrahippocampal administration of kainic acid (KA). Saturation of [3H]JNJ-64413739 to brain sections yielded a KD of approximately 3 nM, with full saturation around 10 nM. The radiotracer was displaced with a structurally different P2X7R ligand, JNJ-47965567, indicating high affinity and specificity to rat P2X7R. In post epileptic rats, region-specific [3H]JNJ-64413739 binding revealed a bilateral increase in the hippocampal formation and its subregions few days after status epilepticus, peaking at day 30, and remained stable at this high level until day 90. Similar temporal profiles were identified in subcortical regions such as the thalamus. Interestingly, no change in binding was observed in the temporal and piriform cortices until day 30 where a dramatic increase occurred. Also, in the corpus callosum, significant increase was detected 30 days after the seizure. These results show that P2X7R binding, likely reflecting inflammation, is increased at delayed time points and exhibit region-specific patterns that is different from acute effects. Our findings suggest that P2X7R may contribute to sustained neuroinflammation and may be involved in those changes leading to epileptogenesis and the development of chronic epilepsy. Highlights[3H]JNJ-64413739 binds specifically to the purinergic P2X7 receptor (P2X7R) and saturates in the rat brain. P2X7R binding increases in a region- and time-dependent manner following status epilepticus. P2X7R binding remains elevated during chronic epilepsy in all examined brain regions. P2X7R is considered a link between early seizures and sustained neuroinflammation and epileptogenesis.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

Among mammals, spiny mice (Acomys spp.) exhibit the unique capacity to regenerate parts of their nervous system. Studying this phenomenon has the potential to reveal new targets that can slow or halt human neurodegenerative disorders. Unfortunately, research tools (e.g., transgenic lines, gene delivery vehicles) are lacking compared to those available for other rodent models. Here, we tested systemic adeno-associated viral vectors (AAVs) in Acomys dimidiatus and identified three promising candidates: X1.1, CAP-Mac, and MaCPNS1. Characterizing their tropism following intravenous delivery, we found that in the brain, MaCPNS1 and X1.1 primarily transduced astrocytes. In the peripheral nervous system, MaCPNS1 efficiently transduced dorsal root ganglia, axon bundles of the ear pinnae, and enteric neurons throughout the gastrointestinal tract. As a proof-of-concept, we used MaCPNS1 to chemogenetically modulate the activity of enteric neurons, successfully decreasing gastric motility in vivo and increasing colonic motility ex vivo. We expect these findings to enable functional studies of the uniquely regenerative nervous system of Acomys, which may in turn help advance neuroregenerative therapeutics for humans. Summary StatementIdentification of an AAV tool to efficiently deliver transgenes to the central and peripheral nervous systems of spiny mice enables functional studies of the nervous system in a mammalian model of regeneration.

Temporal lobe epilepsy (TLE) is a heterogeneous disorder with most clinical presentations involving unilateral or bilateral hippocampal seizure onsets. Antiseizure medications are often ineffective for TLE, and epilepsy surgery can have variable outcomes. Risk factors for TLE are readily identifiable and typically precede chronic epilepsy, providing a window of opportunity for preventative treatments. However, there are currently no clinically approved anti-epileptogenic therapies. In this study, we investigate the role of Wnt signaling in epileptogenesis using two mouse TLE models, the intrahippocampal kainate model of unilateral TLE (IHK), and the intraperitoneal kainate model of bilateral TLE (IPK). We specifically examined adult-born immature dentate granule cells as these cells have been heavily implicated in the pathogenesis of TLE and clinical TLE is typically initiated in adulthood. We observed that adult-born immature dentate granule cells undergo pathological morphological changes during epileptogenesis in both the IHK and IPK models of TLE. When compared across epileptogenic zones, however, these changes differed between the two models. Wnt signaling also decreased in these cells in epileptic mice during the epileptogenic period. When mice were treated with SB415286, a highly selective Wnt activator, Wnt signaling in immature dentate granule cells was restored to baseline levels and pathological remodeling changes were reduced in both models. These data therefore suggest that a reduction in Wnt signaling in immature dentate granule cells plays an etiological role in epileptogenesis, and that restoring Wnt signaling using Wnt activating drugs or alternative agents may have therapeutic potential as an anti-epileptogenic strategy in TLE.

Electrical transmission is mediated by intercellular channels that cluster into structures known as gap junctions (GJ). In vertebrates, GJ channels are encoded by the gene family of connexin (Cx) proteins that assemble as hexamers, termed hemichannels, in the pre- and postsynaptic membranes, and that subsequently dock to form GJ channels. Auditory contacts on the fish Mauthner cells serve as model to study the properties and organization of vertebrate electrical synapses. Electrical transmission at these synapses is mediated by multiple co-existing GJs at which the presence of intercellular channels is regulated by a molecular scaffold. Zebrafish contain four homologs of the neuronal Cx36: Cx35.5 and Cx35.1 (gjd2a and b, respectively), and Cx34.1 and Cx34.7 (gjd1a and b). Cx mutations suggested that GJs are formed by heterotypic channels made of presynaptic Cx35.5 and postsynaptic Cx34.1. Using transgenic fish in which Cxs were tagged, we found that a second Cx, Cx34.7, is present together with Cx34.1 on the postsynaptic side at some but not all GJs at these terminals. When exogenously expressed, both Cx34.1 and Cx34.7 formed heterotypic functional channels with Cx35.5, each with substantially different voltage-dependent properties, indicating they can serve differential functions. However, we previously demonstrated that electrical transmission is lost in Cx34.1 but not Cx34.7 null mutants, suggesting that Cx34.7 cannot compensate for the loss of Cx34, despite the intrinsic ability of Cx34.1 and Cx34.7 to create functional channels. The findings reveal an unanticipated functional organization in the electrical synapse, where Cx34.1 is obligatory and Cx34.7 accessory, roles that appear to be defined by the postsynaptic molecular scaffold, with two postsynaptic Cxs possibly assembling under specific functional contexts. Thus, our results indicate that electrical synapses share an organizational motif with chemical synapses, akin to how they combine postsynaptic receptor types to modify synaptic function.

Retinal ganglion cells (RGCs) degenerate in optic neuropathies like glaucoma and traumatic optic nerve injury leading to irreversible vision loss. Higher levels of homeostatic Ca2+ and canonical Ca2+ regulated signaling promote RGC survival in animal models of glaucoma and optic nerve injury. Mitochondrial dysfunction is also a hallmark of degenerating neurons, including RGCs. Here, we investigate the intersection of mitochondrial function, Ca2+ homeostasis, and cellular resilience by performing an optic nerve crush model of RGC degeneration while monitoring and manipulating mitochondrial Ca2+ levels (mito-Ca2+). We find that mito-Ca2+ is predicative of RGC survival in that surviving RGCs are enriched for higher homeostatic mito-Ca2+ levels. Mitochondrial dysfunction was observed where mito-Ca2+ was reduced in RGCs after injury, regardless of survival. We then examined the importance of higher mito-Ca2+ in surviving RGCs by altering mito-Ca2+ levels and Ca2+ transit using pharmacological and AAV-mediated approaches. Paradoxically, treatment to decrease mito-Ca2+ increased survival to ONC. We then manipulated mito-Ca2+ permeability by altering the expression levels of the mitochondrial calcium uniporter (MCU) pore forming subunit that allows Ca2+ to enter mitochondria from the cytoplasm. Overexpressing MCU reduced RGC survival to injury, while shRNA knockdown of MCU increased RGC survival. These results reveal a complex relationship between mito-Ca2+ and RGC degeneration and suggest that well-surviving RGCs may be under chronic mitochondrial stress due to higher homeostatic mito-Ca2+ levels.

Aggregates of misfolded -synuclein (Syn) and neuroinflammation are pathological features of Parkinsons disease (PD). These, misfolded conformations of Syn promote cytokine and chemokine signaling in the surrounding microenvironment by triggering activation of glial cells through pattern recognition receptors. Microglia and astrocytes act as innate mediators of the neuroimmune response in the brain by regulating inflammatory signaling via paracrine and autocrine forms of cell communication. Extracellular vesicles (EVs) represent a form of glial cell to cell communication that can regulate the glial neuroimmune responses depending on the phenotype of the donor cell. Research has shown that the contents of EVs can be altered via pharmacologically altering the donor cell which offers a potential avenue for the regulation of inflammation. As such, we analyzed enriched mouse cortical primary astrocytes and characterized their response to Syn exposure in the absence and presence of microglia-derived EVs. Using trans-resveratrol, a naturally occurring polyphenol implicated for its anti-inflammatory properties, as our pharmacological agent to generate an anti-inflammatory microglial-derived EV phenotype we found that EVs derived from resveratrol-treated microglia decreased the production of proinflammatory molecules in enriched astrocytes exposed to Syn. Sequencing of EV miRNAs revealed two miRNAs (miR-5099 and miR-115) with significant up-regulation in resveratrol EVs compared to control EVs. Astrocytes transfected with corresponding miRNA mimics prior to Syn exposure showed a dramatic decrease in inflammatory biomarker production. These findings show that microglia-derived EVs and their specific miRNA cargo can attenuate Syn-directed inflammation in astrocytes and may serve as a novel therapeutic for proteinopathies like PD.

Sympathetic preganglionic neurons (SPNs) distribute signals widely across paravertebral ganglia, yet the reliability of spike propagation along their predominantly unmyelinated axons remains poorly defined. We examined temperature- and activity-dependent modulation of SPN axonal conduction using an ex vivo adult mouse thoracic sympathetic chain preparation. Population compound action potentials (CAPs) were evoked by supramaximal stimulation of T10 ventral roots and recorded from branching axons in interganglionic compared to unbranching axons in the splanchnic nerve. At physiological temperature (36{degrees}C), scaled CAP magnitude was reduced by [~]50% relative to 22{degrees}C, with preferential loss of slower-conducting axonal components. These reductions are consistent with substantial temperature-dependent decreases in effective axonal recruitment, likely reflecting conduction failure in a large fraction of SPNs. Losses were more pronounced in interganglionic pathways, suggesting increased vulnerability in branching projections. To assess activity-dependent effects, stimuli were delivered at 1, 5, and 20 Hz with focus on 5 and 20 Hz stimulus trains (20s duration). The overall time-course of train-evoked depression was similar across temperatures; however, the underlying axonal populations differed. At 22{degrees}C, slower-conducting axons exhibited marked frequency-dependent depression, whereas at 36{degrees}C the remaining faster-conducting axons displayed facilitation, particularly at 20 Hz. Slower-conducting responses also showed post-train potentiation at physiological temperature. These findings indicate that SPN axonal conduction is not uniformly reliable and is strongly modulated by temperature and activation history. Preferential vulnerability of slow-conducting, likely small-diameter and branching axons identifies axonal conduction as a physiologically regulated site of gain control in sympathetic output.

Background/ObjectivesNeuroinflammation-driven iron dysregulation and neurotoxic astrocyte polarization are increasingly recognized as interconnected pathological mechanisms in neurodegenerative diseases. Systemic inflammation triggered by strenuous exercise or infection can engage the central nervous system and astrocytic inflammatory responses and perturb iron homeostasis; however, targeted nutritional strategies to counteract these processes remain limited. Inflamate(R) is a multi-component botanical supplement comprising boswellic acids, astilbin, xanthohumol, and cinnamaldehyde, each with documented anti-inflammatory properties. However, whether this combined formulation can modulate the inflammatory-iron metabolic axis and astrocyte phenotypic polarization remains unexplored. This study aimed to investigate the effects of Inflamate(R) on LPS-induced pro-inflammatory gene expression, iron metabolism-related gene regulation, and A1/A2 astrocyte phenotypic polarization in mouse astrocytes. MethodsMouse astrocytes (AWT) were pre-treated with Inflamate(R) (0.0375 g/mL) or DMSO vehicle for 24 h, followed by lipopolysaccharide (LPS; 1 g/mL) stimulation for an additional 24 h. The non-cytotoxic working concentration was determined by morphological assessment, CCK-8 cell viability, and LDH cytotoxicity assays. Expression of 14 target genes spanning pro-inflammatory mediators (NOS2, IL6, C3, COX2, PLA2g15, SOCS3), iron metabolism regulators (FTH1, Hepcidin, TFRC, SLC40A1, RGMa, RGMb), and astrocyte polarization markers (S100A10, GFAP) was quantified by qRT-PCR. ResultsUnder normal culture conditions, Inflamate(R) did not significantly alter the expression of any target gene except S100A10, confirming the absence of baseline cytotoxicity or transcriptional homeostatic perturbation. Upon LPS stimulation, Inflamate(R) selectively suppressed NOS2 (approximately 64% reduction, p < 0.0001), IL6 (approximately 37% reduction, p < 0.0001), and C3 (approximately 47% reduction, p < 0.0001), while COX2, PLA2g15, and SOCS3 remained unaffected. Concurrently, Inflamate(R) significantly reduced LPS-induced Hepcidin expression to approximately 17% of the control level (p < 0.05) and attenuated FTH1 upregulation (p < 0.01), without altering the expression of iron transporters (TFRC, SLC40A1) or BMP-SMAD pathway components (RGMa, RGMb). Furthermore, Inflamate(R) upregulated the neuroprotective A2 marker S100A10 under both basal (p < 0.05) and LPS-stimulated conditions (p < 0.01), while the general reactivity marker GFAP remained unchanged. ConclusionsInflamate(R) exerts a selective, multi-target modulatory effect at the transcriptional level in LPS-stimulated astrocytes, encompassing suppression of the iNOS-NO and IL-6 signaling axes, attenuation of inflammation-driven hepcidin-ferritin iron dysregulation via the IL-6-STAT3 pathway, and promotion of a phenotypic shift from neurotoxic A1 toward neuroprotective A2 astrocyte polarization. Given that the IL-6-JAK-STAT3-hepcidin axis is also activated during exercise-induced systemic inflammation, these findings suggest that Inflamate(R) may represent a targeted nutritional strategy for preserving CNS iron homeostasis and supporting neuroprotective astrocyte function in both neurodegenerative and exercise-related neuroinflammatory contexts. Further validation in in vivo neurodegenerative and exercise models, including protein-level analyses, is warranted to confirm these transcriptional findings.

Multipotent mesenchymal stem cells (MSCs) including bone marrow stromal cells (BMSCs) have shown analgesic efficacy in recent years. Studies suggested that the therapeutic effect of MSCs was mediated by their secreted small extracellular vesicles (sEVs) mainly exosomes. The present study evaluated the antihyperalgesic effect of BMSC-related sEVs in a mouse model of neuropathic pain involving chronic constriction injury of the infraorbital nerve (CCI-ION). Our separation protocol generated EV particles mostly sized in the range of exosomes (30-170 nm) and express exosome marker proteins CD9, CD81, and Tsg101, suggesting their endosome origin. We show that intravenous injection of BMSC-related sEVs attenuated pain hypersensitivity induced by CCI-ION as indicated by decreased mechanical hypersensitivity (von Frey test) and reduced aversion to noxious stimulation (conditioned place avoidance test). The antihyperalgesic effect of sEVs was observed in both female and male animals, and the effect was dose-dependent. sEVs from NAIVE serum-treated BMSC cultures produced short-lasting antihyperalgesia in male but not female mice, suggesting a subtle sex difference. The antihyperalgesia of sEVs from BMSC culture was blocked by the pretreatment of the culture with GM4869, the antagonist of exosome secretion, suggesting that the effect was not related to other co-isolated soluble mediators but mediated by MSC-derived exosomes. Interestingly, the prior injury condition in which sEVs were isolated favors the pain-relieving effect of sEVs. sEVs isolated from the serum of BMSC-treated animals receiving tendon ligation (TL) injury attenuated hyperalgesia for 24 h, while sEVs from the serum of BMSC-treated NAIVE animals only attenuated hyperalgesia at 3 h after injection. sEVs from the BMSC culture treated with the serum of TL rats were antihyperalgesic, but sEVs from the BMSC culture treated with the serum of naive animals were ineffective. Our results indicate that BMSC-related sEVs produced antihyperalgesia similar to that produced by BMSCs. The results suggest that the interactions between BMSCs and injury conditions are crucially important for producing efficacious sEVs/exosomes and support that the effect of sEVs could be optimized by priming BMSCs with injury-related conditions.

The mechanisms that drive myelin damage as seen in demyelinating disorders such as multiple sclerosis remain incompletely understood. Much of our current knowledge is derived from animal models, but interspecies differences limit their relevance in the context of human pathology and could explain why various promising preclinical therapies failed during clinical translation. Human post-mortem organotypic brain slice cultures provide a unique platform to study human myelin biology, as they preserve genetic, cytoarchitectural, pathological and species-specific context. Here, we evaluated myelin integrity in a human post-mortem brain organotypic slice culture model and experimentally induce focal myelin damage. Human post-mortem organotypic slices cultures retain key features throughout the culturing period, but exhibit gradual cellular and myelin loss over time. Myelin fibres within the white matter remain detectable and present preserved structural and chemical integrity up to 13 days in vitro, indicated by the conserved paranodal and nodal organization and stable myelin spectroscopic signature. Delivery of lysophosphatidylcholine using cryogel scaffolds enables focal drug administration throughout the full depth of the slice with minimal diffusion into surrounding tissue and induces localized demyelination after lysophosphatidylcholine application. Similar focal application of the selective Nav1.6 stimulator {beta}-mammal scorpion toxin Cn2 induces subtle myelin destabilization. Overall, our results demonstrate the suitability of a human post-mortem brain organotypic slice culture model as an adequate platform for studying myelin damage in a human disease context.

A growing number of studies indicate the possible involvement of astrocytes in triggering or modulating neurovascular coupling (NVC), i.e. the local dilation of blood vessels in the brain in response to neuronal activity. Astrocytes possess specialized subcellular compartments, named endfeet, that surround arterioles and capillaries, ideally positioned to mediate NVC. Various vasodilators have been shown to contribute to NVC, such as epoxyeicosatrienoic acid (EET), nitric oxide (NO), or prostaglandin E2 (PGE2), but the precise mechanisms underlying NVC and their variability remain to be fully elucidated. In particular, the involvement of astrocytes in this process is controversial. Recent translatome and proteomics data reveal that astrocytes and in particular endfeet are enriched in the proteins of the PGE2 pathway. However, how the latter could contribute to NVC remains to be characterized. Here, we develop a computational model of astrocyte-mediated NVC that recapitulates these findings and describes Ca2+ and PGE2 signaling in astrocytes, NO release by neurons, and arteriole diameter dynamics using ordinary differential equations. The model successfully reproduces the dynamics of arteriole diameter change during hyperemia from in vivo neocortical recordings in awake mice. Our simulations suggest that the astrocyte PGE2 pathway could be responsible for the late response of NVC at the arteriolar level. We further observe that PIP2-derived diacylglycerol plays a major role in driving arteriole diameter dynamics in our model, while phosphatidic acid-derived diacylglycerol, which is calcium-dependent, mainly acts as an amplifier of this response. Finally, a spatial implementation of the model using a simplified astrocyte geometry suggests that NVC is more efficient when synaptic stimulation occurs at the endfoot level rather than at other astrocytic compartments. Overall, this computational study suggests a partial role for astrocyte-mediated PGE2 release in NVC and points to astrocyte perivascular processes as sub-compartments that are ideally positioned and equipped to mediate NVC. Author summaryIn the brain, the local blood flow is regulated to meet neuronal energy demand by modulating the dilation of neighboring blood vessels. The mechanisms driving this process, known as neurovascular coupling (NVC), remain debated and are likely to differ depending on the physiological context. Recent evidence points to astrocytes, a cell type possessing specialized protrusions called "endfeet", that envelop the entire brain vascular tree. Contacts between synapses and endfeet have recently been reported, positioning the latter as ideal mediators of NVC. Here, we developed a computational model that simulates the signaling between neurons, astrocytes, and blood vessels. Our model successfully reproduces experimental recordings of blood vessels dilation in the brains of awake mice. Our simulations suggest that a specific signaling pathway in astrocytes, involving a molecule called prostaglandin E2, is a key driver of the late phase of NVC, occurring a few seconds after neuronal activity. Furthermore, our model indicates that the location of the stimulated synapses matters: signals sent to the astrocyte endfeet are particularly effective at controlling blood flow. This work helps clarify the active role of astrocytes in brain blood flow regulation, a process critical for healthy brain function.