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Brain Structure & Function Jun 2024In this novel large-scale multiplexed immunofluorescence study we comprehensively characterized and compared layer-specific proteomic features within regions of interest...
In this novel large-scale multiplexed immunofluorescence study we comprehensively characterized and compared layer-specific proteomic features within regions of interest of the widely divergent dorsolateral prefrontal cortex (A46) and primary visual cortex (A17) of adult rhesus monkeys. Twenty-eight markers were imaged in rounds of sequential staining, and their spatial distribution precisely quantified within gray matter layers and superficial white matter. Cells were classified as neurons, astrocytes, oligodendrocytes, microglia, or endothelial cells. The distribution of fibers and blood vessels were assessed by quantification of staining intensity across regions of interest. This method revealed multivariate similarities and differences between layers and areas. Protein expression in neurons was the strongest determinant of both laminar and regional differences, whereas protein expression in glia was more important for intra-areal laminar distinctions. Among specific results, we observed a lower glia-to-neuron ratio in A17 than in A46 and the pan-neuronal markers HuD and NeuN were differentially distributed in both brain areas with a lower intensity of NeuN in layers 4 and 5 of A17 compared to A46 and other A17 layers. Astrocytes and oligodendrocytes exhibited distinct marker-specific laminar distributions that differed between regions; notably, there was a high proportion of ALDH1L1-expressing astrocytes and of oligodendrocyte markers in layer 4 of A17. The many nuanced differences in protein expression between layers and regions observed here highlight the need for direct assessment of proteins, in addition to RNA expression, and set the stage for future protein-focused studies of these and other brain regions in normal and pathological conditions.
PubMed: 38943018
DOI: 10.1007/s00429-024-02819-y -
Nature Communications Jun 2024Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in...
Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in health and disease. Most LD studies in vivo rely on staining methods, providing only a snapshot. We therefore developed a LD-reporter mouse by labelling the endogenous LD coat protein perilipin 2 (PLIN2) with tdTomato, enabling staining-free fluorescent LD visualisation in living and fixed tissues and cells. Here we validate this model under standard and high-fat diet conditions and demonstrate that LDs are highly abundant in various cell types in the healthy brain, including neurons, astrocytes, ependymal cells, neural stem/progenitor cells and microglia. Furthermore, we also show that LDs are abundant during brain development and can be visualized using live imaging of embryonic slices. Taken together, our tdTom-Plin2 mouse serves as a novel tool to study LDs and their dynamics under both physiological and diseased conditions in all tissues expressing Plin2.
Topics: Animals; Perilipin-2; Lipid Droplets; Brain; Mice; Neurons; Gene Knock-In Techniques; Mice, Transgenic; Female; Luminescent Proteins; Male; Astrocytes; Diet, High-Fat; Mice, Inbred C57BL; Neural Stem Cells; Microglia
PubMed: 38942786
DOI: 10.1038/s41467-024-49449-w -
Microbes and Infection Jun 2024Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus responsible for severe infections of the central nervous system. Although neurons are predominantly...
Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus responsible for severe infections of the central nervous system. Although neurons are predominantly targeted, specific involvement of microglia in pathogenesis of TBE is not yet fully understood. In this study, the susceptibility of human microglia to TBEV is investigated, focusing on productive infection and different immune responses of different viral strains. We investigated primary human microglia and two immortalized microglial cell lines exposed to three TBEV strains (Hypr, Neudörfl and 280), each differing in virulence. Our results show that all microglia cultures tested support long-term productive infections, regardless of the viral strain. In particular, immune response varied significantly with the viral strain, as shown by the differential secretion of cytokines and chemokines such as IP-10, MCP-1, IL-8 and IL-6, quantified using a Luminex 48-plex assay. The most virulent strain triggered the highest cytokine induction. Electron tomography revealed substantial ultrastructural changes in the infected microglia, despite the absence of cytopathic effects. These findings underscore the susceptibility of human microglia to TBEV and reveal strain-dependent variations in viral replication and immune responses, highlighting the complex role of microglia in TBEV-induced neuropathology and contribute to a deeper understanding of TBE pathogenesis and neuroinflammation.
PubMed: 38942136
DOI: 10.1016/j.micinf.2024.105383 -
Cell Jun 2024Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through...
Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.
PubMed: 38942014
DOI: 10.1016/j.cell.2024.05.058 -
Phytomedicine : International Journal... Jun 2024Neuropathic pain (NP) due to nerve injury, disrupts neural plasticity by triggering the release of inflammatory mediators. Alongside the hypothesis that...
BACKGROUND
Neuropathic pain (NP) due to nerve injury, disrupts neural plasticity by triggering the release of inflammatory mediators. Alongside the hypothesis that neuro-inflammation contributes to this disruption, Andrographolide (Andro), a traditional bioactive compound derived from Andrographis paniculata, has garnered attention for its potent anti-inflammatory properties. However, whether Andro could ameliorate NP by regulating neuroinflammation remains unknown.
PURPOSE
This study aimed to investigate whether and how Andro regulates neuroinflammation and alleviates NP.
METHODS
The analgesic effects of Andro on NP were evaluated using both the spinal nerve ligation (SNL) and formalin rat models. A combination of network pharmacology, RNA sequencing, and experimental validation was employed to elucidate the underlying mechanism behind Andro's analgesic effects. Additionally, various techniques such as functional ultrasound, immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR), patch clamp, and electron microscopy were employed to investigate the specific neural cell types, neural functions, and changes in neural plasticity influenced by Andro.
RESULTS
Network pharmacology analysis unveiled the crucial roles played by shared targets of Andro and pain in regulating pain-related inflammation, including microglia activation, neuroinflammation, immune modulation, and synaptic transmission. Furthermore, we confirmed Andro's superior efficacy in pain relief compared to the traditional analgesic drug, Gabapentin. In these models, Andro was observed to modulate the haemodynamic response triggered by SNL. Transcriptome analysis and molecular docking studies indicated the involvement of major histocompatibility complex class II (MHCII) genes (Db1, Da, and Bb). Electron microscopy revealed improvements in synaptic ultrastructure, and electrophysiological investigations showed a selective reduction in glutamatergic transmission in neuropathic rats after following Andro treatment. The integration of systems pharmacology analysis and biological validation collectively demonstrated that the mechanism of pain relief involves immune modulation, enhancement of synaptic plasticity, and precise regulation of excitatory neurotransmission.
CONCLUSION
In conclusion, this study has demonstrated that Andro, by targeting MHCII genes, may serve as a promising therapeutic candidate for neuropathic pain.
PubMed: 38941815
DOI: 10.1016/j.phymed.2024.155823 -
The Journal of Clinical Investigation Jun 2024STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential...
STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential immunofluorescence (SeqIF) and the Ivy Glioblastoma Atlas, STING expression was found in myeloid populations and in the perivascular space. The STING agonist 8803 increased median survival in multiple preclinical models of glioblastoma, including QPP8, an immune checkpoint blockade-resistant model, where 100% of mice were cured. Ex vivo flow cytometry profiling during the therapeutic window demonstrated increases in myeloid tumor trafficking and activation, alongside enhancement of CD8+ T cell and NK effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and iNOS, while decreasing immunosuppressive CD206 and arginase. In humanized mice, where tumor cell STING is epigenetically silenced, 8803 therapeutic activity was maintained, further attesting to myeloid dependency and reprogramming. Although the combination with a STAT3 inhibitor did not further enhance STING agonist activity, the addition of anti-PD-1 antibodies to 8803 treatment enhanced survival in an immune checkpoint blockade-responsive glioma model. In summary, 8803 as a monotherapy demonstrates marked in vivo therapeutic activity, meriting consideration for clinical translation.
Topics: Animals; Glioblastoma; Tumor Microenvironment; Mice; Membrane Proteins; Humans; Cell Line, Tumor; Brain Neoplasms
PubMed: 38941297
DOI: 10.1172/JCI175033 -
Proceedings of the National Academy of... Jul 2024TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane....
TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane. While the functions of TMEM16F have been extensively characterized in multiple cell types, the role of TMEM16F in the central nervous system remains largely unknown. Here, we sought to study how TMEM16F in the brain may be involved in neurodegeneration. Using a mouse model that expresses the pathological P301S human tau (PS19 mouse), we found reduced tauopathy and microgliosis in 6- to 7-mo-old PS19 mice lacking TMEM16F. Furthermore, this reduction of pathology can be recapitulated in the PS19 mice with TMEM16F removed from neurons, while removal of TMEM16F from microglia of PS19 mice did not significantly impact tauopathy at this time point. Moreover, TMEM16F mediated aberrant phosphatidylserine exposure in neurons with phospho-tau burden. These studies raise the prospect of targeting TMEM16F in neurons as a potential treatment of neurodegeneration.
Topics: Animals; Anoctamins; Phosphatidylserines; Neurons; tau Proteins; Mice; Tauopathies; Humans; Microglia; Phosphorylation; Mice, Transgenic; Disease Models, Animal; Phospholipid Transfer Proteins; Brain; Mice, Knockout
PubMed: 38941274
DOI: 10.1073/pnas.2311831121 -
Molecular Medicine Reports Aug 2024Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia...
Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO) were measured. To investigate the significance of the NOD‑like receptor protein 3 (NLRP3) inflammasome, NLRP3 mice were used, and the expression levels of NLRP3, caspase‑1, full‑length gasdermin D (GSDMD‑FL), GSDMD‑N domain (GSDMD‑N), IL‑1β and IL‑18 were evaluated. In addition, Z‑YVAD‑FMK, a caspase‑1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO and OCR, and decreased PbtO in response to high glucose treatment. Furthermore, hyperglycemia‑induced microglial pyroptosis was confirmed by detection of increased levels of caspase‑1, GSDMD‑N, IL‑1β and IL‑18 and a decreased level of GSDMD‑FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase‑1 also reduced the expression levels of GSDMD‑N, IL‑1β and IL‑18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.
Topics: Animals; Pyroptosis; Microglia; Ischemic Stroke; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Oxygen; Male; Hyperglycemia; Inflammasomes; Mice, Inbred C57BL; Caspase 1; Disease Models, Animal; Mice, Knockout; Interleukin-1beta; Phosphate-Binding Proteins; Oxygen Consumption; Gasdermins
PubMed: 38940333
DOI: 10.3892/mmr.2024.13270 -
Journal of Integrative Neuroscience Jun 2024The majority of neuromyelitis optica spectrum disorders (NMOSD) patients are seropositive for aquaporin-4 (AQP4)-specific antibodies [also named neuromyelitis optica...
OBJECTIVES
The majority of neuromyelitis optica spectrum disorders (NMOSD) patients are seropositive for aquaporin-4 (AQP4)-specific antibodies [also named neuromyelitis optica immunoglobulin G antibodies (NMO-IgG)]. Although NMO-IgG can induce pathological changes in the central nervous system (CNS), the immunological changes in the CNS and peripheral tissue remain largely unknown. We investigated whether NMO-IgG binds to tissue expressing AQP4 and induces immunological changes in the peripheral tissue and CNS.
METHODS
C57BL/6 female mice were assigned into an NMOSD or control group. Pathological and immunological changes in peripheral tissue and CNS were measured by immunostaining and flow cytometry, respectively. Motor impairment was measured by open-field test.
RESULTS
We found that NMO-IgG did bind to astrocyte- and AQP4-expressing peripheral tissue, but induced glial fibrillary acidic protein and AQP4 loss only in the CNS. NMO-IgG induced the activation of microglia and modulated microglia polarization toward the classical (M1) phenotype, but did not affect innate or adaptive immune cells in the peripheral immune system, such as macrophages, neutrophils, Th17/Th1, or IL-10-producing B cells. In addition, NMOSD mice showed significantly less total distance traveled and higher immobility time in the open field.
CONCLUSIONS
We found that injection of human NMO-IgG led to astrocytopathic lesions with microglial activation in the CNS. However, there were no significant pathological or immunological changes in the peripheral tissues.
Topics: Animals; Neuromyelitis Optica; Mice, Inbred C57BL; Immunoglobulin G; Aquaporin 4; Female; Humans; Mice; Disease Models, Animal; Microglia; Autoantibodies; Astrocytes; Glial Fibrillary Acidic Protein; Central Nervous System
PubMed: 38940087
DOI: 10.31083/j.jin2306119 -
Sheng Li Xue Bao : [Acta Physiologica... Jun 2024The purpose of the study was to investigate the mechanism of TFEB activator 1 (TA1) improving the autophagic degradation of oligomeric amyloid-β (oAβ) in microglia,...
The purpose of the study was to investigate the mechanism of TFEB activator 1 (TA1) improving the autophagic degradation of oligomeric amyloid-β (oAβ) in microglia, and to explore the therapeutic effect of TA1 on an in vitro model of microglia in Alzheimer's disease (AD). Primary microglia were exposed to 1 μmol/L oAβ for 0, 3, 12, and 24 h respectively to construct the in vitro model of microglia in AD. In order to explore the therapeutic effect of TA1, primary microglia were co-treated with 1 μmol/L oAβ and 1 μmol/L TA1 for 12 h. To determine the autophagy flux, the above cells were further treated with 100 nmol/L Bafilomycin A1 for 1 h before fixation. Fluorescent probes were used to detect the endocytosis or degradation of oAβ by microglia. The autophagic flux was determined by infection of lentivirus mCherry-EGFP-LC3. The nuclear TFEB intensity, the autophagosomes number, and the colocalization ratio of oAβ with lysosome-associated membrane protein 1 (LAMP1) or microtubule-associated protein light chain 3 (LC3), were detected by immunofluorescence assay. Expressions of autophagy-related-genes, including Lamp1, Atg5, and Map1lc3b, were detected by qRT-PCR. Results showed that prolonged oAβ exposure inhibited the endocytosis and degradation of oAβ by microglia. Meanwhile, the number of autophagosomes and autophagy flux in microglia decreased after 12 h of oAβ treatment. We further found that the nuclear expression of autophagy regulator TFEB decreased after 12 h of oAβ exposure, resulting in the decrease of autophagy genes, thus leading to the damage of autophagic degradation of oAβ. Therefore, long-term oAβ exposure was considered to construct the in vitro model of microglia in AD. After TA1 treatment, the nuclear expression of TFEB in cells was obviously upregulated. TA1 treatment upregulated the expressions of autophagy-related genes, leading to the recovery of autophagy flux. TA1 also recovered the endocytosis and degradation of oAβ by microglia. In conclusion, TA1 could improve oAβ clearance by microglia in AD by upregulating microglial TFEB-mediated autophagy, suggesting TA1 as a potential therapeutic drug for AD.
Topics: Microglia; Amyloid beta-Peptides; Autophagy; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Alzheimer Disease; Cells, Cultured; Mice
PubMed: 38939931
DOI: No ID Found