bims-microg 2025-03-02 papers

bims-microg

Biomed News

on Microglia in health and disease

Issue of 2025–03–02
twenty-two papers selected by
Marcus Karlstetter, Universität zu Köln

Integration and functionality of human iPSC-derived microglia in a chimeric mouse retinal model.
Generation of an Inducible Destabilized-Domain Cre Mouse Line to Target Disease Associated Microglia.
Effects and mechanisms of long-acting glucagon-like peptide-1 receptor agonist semaglutide on microglia phenotypic transformation and neuroinflammation after cerebral ischemia/reperfusion in rats.
From Genetics to Neuroinflammation: The Impact of ApoE4 on Microglial Function in Alzheimer's Disease.
Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent Treg-microglia crosstalk.
Accumulation of Damaging Lipids in the Arf1-Ablated Neurons Promotes Neurodegeneration through Releasing mtDNA and Activating Inflammatory Pathways in Microglia.
IL-2/anti-IL-2 complexes attenuates neuroinflammation and neurodegeneration in mice of experimental Parkinson's disease.
Microglial pyroptosis induced by SENP7 via the cGAS/STING/IRF3 pathway contributes to neuronal apoptosis.
Metabolic reprogramming in astrocytes prevents neuronal death through a UCHL1/PFKFB3/H4K8la positive feedback loop.
Hippocampal P2X4 receptor induces type 1 diabetes rats with neuropathic pain through microglial-derived neuroinflammation and neuronal damage.
Quantification and correlation of amyloid-β plaque load, glial activation, GABAergic interneuron numbers, and cognitive decline in the young TgF344-AD rat model of Alzheimer's disease.
Interferon-gamma receptor signaling regulates innate immunity during Staphylococcus aureus craniotomy infection.
HIV-1 gp120 Interactions with Nicotine Modulate Mitochondrial Network Properties and Amyloid Release in Microglia.
Increased ocular plasma cells induce damaging α-synuclein+ microglia in autoimmune uveitis.
Comparing Alzheimer's genes in African, European, and Amerindian induced pluripotent stem cell-derived microglia.
Fructose induces inflammatory activation in macrophages and microglia through the nutrient-sensing ghrelin receptor.
Microglia-Mediated Synaptic Dysfunction Contributes to Chemotherapy-Related Cognitive Impairment.
Transcriptomic Analysis Divulges Differential Expressions of Microglial Genes After Microglial Repopulation in Mice.
25-Hydroxycholesterol modulates microglial function and exacerbates Alzheimer's disease pathology: mechanistic insights and therapeutic potential of cholesterol esterification inhibition.
Matrix Stiffness Regulates Interleukin-10 Secretion in Human Microglia (HMC3) via YAP-Mediated Mechanotransduction.
ADAR1 Promotes NUPR1 A-to-I RNA Editing to Exacerbate Ischemic Brain Injury by Microglia Mediated Neuroinflammation.
Glioblastoma- derived exosomes (GBM-Exo) regulate microglial M2 polarization via the RAC1/AKT/NRF2 pathway.

J Neuroinflammation. 2025 Feb 27. 22(1): 53

Integration and functionality of human iPSC-derived microglia in a chimeric mouse retinal model.

Chun Tang, Qi-Qi Zhou, Xiu-Feng Huang, Ya-Yi Ju, Bi-Lin Rao, Zhi-Cong Liu, Yi-An Jia, Zhan-Pei Bai, Qing-Yang Lin, Lin Liu, Jia Qu, Jun Zhang, Mei-Ling Gao.

   INTRODUCTION: Microglia, the resident immune cells of the central nervous system, play a pivotal role in maintaining homeostasis, responding to injury, and modulating neuroinflammation. However, the limitations of rodent models in accurately representing human microglia have posed significant challenges in the study of retinal diseases.
METHODS: PLX5622 was used to eliminate endogenous microglia in mice through oral and intraperitoneal administration, followed by transplantation of human induced pluripotent stem cell-derived microglia (hiPSC-microglia, iMG) into retinal explants to create a novel ex vivo chimeric model containing xenotransplanted microglia (xMG). The number and proportion of xMG in the retina were quantified using retinal flat-mounting and immunostaining. To evaluate the proliferative capacity and synaptic pruning ability of xMG, the expression of Ki-67 and the phagocytosis of synaptic proteins SV2 and PSD95 was assessed. The chimeric model was stimulated with LPS, and single-cell RNA sequencing (scRNA-seq) was used to analyze transcriptomic changes in iMG and xMG. Mouse IL-34 antibody neutralization experiments were performed, and the behavior of xMG in retinal degenerative Pde6b-/- mice was examined.
RESULTS: We demonstrated that xenotransplanted microglia (xMG) successfully migrated to and localized within the mouse retina, adopting homeostatic morphologies. Our approach achieved over 86% integration of human microglia, which maintained key functions including proliferation, immune responsiveness, and synaptic pruning over a 14-day culture period. scRNA-seq of xMG revealed a shift in microglial signatures compared to monoculture iMG, indicating a transition to a more in vivo-like phenotype. In retinal degenerative Pde6b-/- mice, xMG exhibited activation and migrated toward degenerated photoreceptors.
CONCLUSION: This model provides a powerful platform for studying human microglia in the retinal context, offering significant insights for advancing research into retinal degenerative diseases and developing potential therapeutic strategies. Future applications of this model include using patient-derived iPSCs to investigate disease-specific microglial behaviors, thereby enhancing our understanding of microglia-related pathogenesis.

Keywords:  Chimeric; HiPSCs; Microglia; Retinal explant; Xenotransplantation

DOI:  https://doi.org/10.1186/s12974-025-03393-8
Glia. 2025 Feb 23.

Generation of an Inducible Destabilized-Domain Cre Mouse Line to Target Disease Associated Microglia.

Caden M Henningfield, Minh Ngo, Kaitlin M Murray, Nellie E Kwang, Kate I Tsourmas, Jonathan Neumann, Zachary A Pashkutz, Shimako Kawauchi, Vivek Swarup, Thomas E Lane, Grant R MacGregor, Kim N Green.

  The function of microglia during progression of Alzheimer's disease (AD) can be investigated using mouse models that enable genetic manipulation of microglial subpopulations in a temporal manner. We developed mouse lines that express either Cre recombinase (Cre) for constitutive targeting, or destabilized-domain Cre recombinase (DD-Cre) for inducible targeting from the Cst7 locus (Cst7DD-Cre) to specifically manipulate disease associated microglia (DAM) and crossed with Ai14 tdTomato cre-reporter line mice. Cst7Cre was found to target all brain resident myeloid cells, due to transient developmental expression of Cst7, but no expression was found in the inducible Cst7DD-Cre mice. Further crossing of this line with 5xFAD mice combined with dietary administration of trimethoprim to induce DD-Cre activity produces long-term labeling in DAM without evidence of leakiness, with tdTomato-expression restricted to cells surrounding plaques. Using this model, we found that DAMs are a subset of plaque-associated microglia (PAMs) and their transition to DAM increases with age and disease stage. Spatial transcriptomic analysis revealed that tdTomato+ cells show higher expression of disease and inflammatory genes compared to other microglial populations, including non-labeled PAMs. These models allow either complete cre-loxP targeting of all brain myeloid cells (Cst7Cre), or inducible targeting of DAMs, without leakiness (Cst7DD-Cre).

Keywords:  Alzheimer's disease; disease associated microglia; inflammation; microglia

DOI:  https://doi.org/10.1002/glia.70004
Brain Circ. 2024 Oct-Dec;10(4):10(4): 354-365

Effects and mechanisms of long-acting glucagon-like peptide-1 receptor agonist semaglutide on microglia phenotypic transformation and neuroinflammation after cerebral ischemia/reperfusion in rats.

Rulin Mi, Huifeng Cheng, Rui Chen, Bo Bai, An Li, Fankai Gao, Guofang Xue.

   BACKGROUND: The optimal method for addressing cerebral ischemic stroke involves promptly restoring blood supply. However, cerebral ischemia-reperfusion injury (CIRI) is an unavoidable consequence of this event. Neuroinflammation is deemed the primary mechanism of CIRI, with various activation phenotypes of microglia playing a pivotal role. Research has demonstrated that long-lasting agonists of the glucagon-like peptide-1 receptor can suppress neuroinflammation and microglial activation.
METHODS: A transient middle cerebral artery occlusion (tMCAO) rat model was established to investigate the effects of semaglutide. Neurological impairments were evaluated utilizing modified neurological severity score on days 1, 3, and 7 postinterventions. Brains were stained with 2,3,5-Triphenyltetrazolium Chloride to determine infarct volume. To assess the expression of various microglia activation phenotypes and neuroinflammatory biomarkers, we utilized immunohistochemistry and immunoblotting.
RESULTS: The study demonstrated that semaglutide in the tMCAO model could decrease neurological deficit scores and reduce the size of cerebral infarcts. In addition, we observed low levels of cluster of differentiation 68 (CD68, an indicator of M1 microglial activation) and tumor necrosis factor alpha (a pro-inflammatory mediator). Moreover, the results indicated a rise in the levels of CD206 (an indicator of M2 activation) and transforming growth factor beta (an anti-inflammatory mediator), while simultaneously reducing P65 levels in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling cascade.
CONCLUSION: In the CIRI model, semaglutide exhibits notable neuroprotective effects on rats, reducing neuroinflammation through the regulation of microglia phenotype transformation and inhibition of NF-κB activation.

Keywords:  Cerebral ischemia-reperfusion; M1/M2 microglia; glucagon-like peptide-1; neuroinflammation; nuclear factor kappa-light-chain-enhancer of activated B-cell signaling pathway

DOI:  https://doi.org/10.4103/bc.bc_38_24
Cells. 2025 Feb 07. pii: 243. [Epub ahead of print]14(4):

From Genetics to Neuroinflammation: The Impact of ApoE4 on Microglial Function in Alzheimer's Disease.

Daniela Dias, Camila Cabral Portugal, João Relvas, Renato Socodato.

  Alzheimer's disease (AD) is a debilitating neurodegenerative disorder marked by progressive cognitive decline and memory loss, impacting millions of people around the world. The apolipoprotein E4 (ApoE4) allele is the most prominent genetic risk factor for late-onset AD, dramatically increasing disease susceptibility and accelerating onset compared to its isoforms ApoE2 and ApoE3. ApoE4's unique structure, which arises from single-amino-acid changes, profoundly alters its function. This review examines the critical interplay between ApoE4 and microglia-the brain's resident immune cells-and how this relationship contributes to AD pathology. We explore the molecular mechanisms by which ApoE4 modulates microglial activity, promoting a pro-inflammatory state, impairing phagocytic function, and disrupting lipid metabolism. These changes diminish microglia's ability to clear amyloid-beta peptides, exacerbating neuroinflammation and leading to neuronal damage and synaptic dysfunction. Additionally, ApoE4 adversely affects other glial cells, such as astrocytes and oligodendrocytes, further compromising neuronal support and myelination. Understanding the ApoE4-microglia axis provides valuable insights into AD progression and reveals potential therapeutic targets. We discuss current strategies to modulate ApoE4 function using small molecules, antisense oligonucleotides, and gene editing technologies. Immunotherapies targeting amyloid-beta and ApoE4, along with neuroprotective approaches to enhance neuronal survival, are also examined. Future directions highlight the importance of personalized medicine based on individual ApoE genotypes, early biomarker identification for risk assessment, and investigating ApoE4's role in other neurodegenerative diseases. This review emphasizes the intricate connection between ApoE4 and microglial dysfunction, highlighting the necessity of targeting this pathway to develop effective interventions. Advancing our understanding in this area holds promise for mitigating AD progression and improving outcomes for those affected by this relentless disease.

Keywords:  Alzheimer’s disease (AD); amyloid-beta clearance; apolipoprotein E4 (ApoE4); microglia; neurodegeneration; neuroinflammation; therapeutic strategies

DOI:  https://doi.org/10.3390/cells14040243
Nat Neurosci. 2025 Feb 27.

Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent Treg-microglia crosstalk.

Saef Izzy, Taha Yahya, Omar Albastaki, Hadi Abou-El-Hassan, Michael Aronchik, Tian Cao, Marilia Garcia De Oliveira, Kuan-Jung Lu, Thais G Moreira, Patrick da Silva, Masen L Boucher, Leah C Beauchamp, Danielle S LeServe, Wesley Nogueira Brandao, Ana Carolina Durão, Toby Lanser, Federico Montini, Joon-Hyuk Lee, Joshua D Bernstock, Megha Kaul, Gabriel Pasquarelli-do-Nascimento, Kusha Chopra, Rajesh Krishnan, Rebekah Mannix, Rafael M Rezende, Francisco J Quintana, Oleg Butovsky, Howard L Weiner.

Neuroinflammation plays a crucial role in traumatic brain injury (TBI), contributing to both damage and recovery, yet no effective therapy exists to mitigate central nervous system (CNS) injury and promote recovery after TBI. In the present study, we found that nasal administration of an anti-CD3 monoclonal antibody ameliorated CNS damage and behavioral deficits in a mouse model of contusional TBI. Nasal anti-CD3 induced a population of interleukin (IL)-10-producing regulatory T cells (Treg cells) that migrated to the brain and closely contacted microglia. Treg cells directly reduced chronic microglia inflammation and regulated their phagocytic function in an IL-10-dependent manner. Blocking the IL-10 receptor globally or specifically on microglia in vivo abrogated the beneficial effects of nasal anti-CD3. However, the adoptive transfer of IL-10-producing Treg cells to TBI-injured mice restored these beneficial effects by enhancing microglial phagocytic capacity and reducing microglia-induced neuroinflammation. These findings suggest that nasal anti-CD3 represents a promising new therapeutic approach for treating TBI and potentially other forms of acute brain injury.

DOI: https://doi.org/10.1038/s41593-025-01877-7
Adv Sci (Weinh). 2025 Feb 28. e2414260

Accumulation of Damaging Lipids in the Arf1-Ablated Neurons Promotes Neurodegeneration through Releasing mtDNA and Activating Inflammatory Pathways in Microglia.

Xu Li, Shuhan Jin, Danke Wang, Ying Wu, Xiaoyu Tang, Yufan Liu, Tiange Yao, Shoufa Han, Lin Sun, Yuetong Wang, Steven X Hou.

  Lipid metabolism disorders in both neurons and glial cells have been found in neurodegenerative (ND) patients and animal models. However, the pathological connection between lipid droplets and NDs remains poorly understood. The recent work has highlighted the utility of a neuron-specific Arf1-knockout mouse model and corresponding cells for elucidating the nexus between lipid metabolism disorders and amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). In this study, it is found that Arf1 deficiency first induced surplus fatty acid synthesis through the AKT-mTORC1-SREBP1-FASN axis, which further triggered endoplasmic reticulum (ER)-mitochondrial stress cascade via calcium flux. The organelle stress cascade further caused mitochondrial DNA (mtDNA) to be released into cytoplasm. Concurrently, the FASN-driven fatty acid synthesis in the Arf1-deficient neurons might also induce accumulation of sphingolipids in lysosomes that caused dysfunction of autophagy and lysosomes, which further promoted lysosomal stress and mitochondria-derived extracellular vesicles (MDEVs) release. The released MDEVs carried mtDNA into microglia to activate the inflammatory pathways and neurodegeneration. The studies on neuronal lipid droplets (LDs) and recent studies of microglial LDs suggest a unified pathological function of LDs in NDs: activating the inflammatory pathways in microglia. This finding potentially provides new therapeutic strategies for NDs.

Keywords:  Arf1 ablation; accumulation of damaging lipids; mtDNA release; neurodegeneration

DOI:  https://doi.org/10.1002/advs.202414260
Brain Res Bull. 2025 Feb 23. pii: S0361-9230(25)00085-1. [Epub ahead of print] 111273

IL-2/anti-IL-2 complexes attenuates neuroinflammation and neurodegeneration in mice of experimental Parkinson's disease.

Lanxin Li, Weiwei Gao, Ning Ren, Lei Chen.

  Parkinson's disease (PD) is the second most common neurodegenerative disease, with motor and non-motor symptoms being its main clinical manifestations. Neuroinflammation has been shown to involve in pathogenesis of PD. Regulatory T cells (Tregs) in PD exhibited reduction in number and suppressive activity. Existing methods to increase the Tregs remains challenging for clinical application because of the difficulty in Tregs expanding or serious side-effects. Therefore, new approaches still need to be explored to balance the amount and activity of Tregs. In this study, we assessed the protective effects of IL-2/anti-IL-2 complexes (IL-2C) on mouse models of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). And the results showed that IL-2C significantly increased the number of Tregs both in spleen and brain, accompanied by reduced nigral dopaminergic neuron loss and behavioral defects. Besides, IL-2C also attenuated neuroinflammation as observed by diminished glial activation, fewer infiltration of CD4+ and CD8+ T cells and reduced pro-inflammatory cytokines releasing in the nigral region. Moreover, the protective effects of IL-2C were abolished by pre-treatment of anti-CD25 antibody (PC61), which was used to delete the Tregs. In summary, our results demonstrate that IL-2C-induced Tregs expansion attenuates the dopaminergic neurons loss and the neuroinflammatory response in vivo, suggesting that IL-2C maybe a promising therapeutic target for PD.

Keywords:  Astrocytes; Dopaminergic neuron; IL-2/anti-IL-2 complexes; Microglia; Parkinson's disease; Regulatory T cell

DOI:  https://doi.org/10.1016/j.brainresbull.2025.111273
Cytokine. 2025 Feb 24. pii: S1043-4666(25)00040-7. [Epub ahead of print]189 156893

Microglial pyroptosis induced by SENP7 via the cGAS/STING/IRF3 pathway contributes to neuronal apoptosis.

Lin Liu, Fei Xiao, Jinyue Yang, Hanqing Yao, Ke Hua.

   BACKGROUND: Maternal anesthetic exposure may exacerbate significant neurocognitive risks in the immature brains of fetuses. However, the mechanisms through which sevoflurane exposure during pregnancy results in cognitive impairments in offspring remain unclear.
METHODS: Pregnant C57BL/6 mice (gestational day 14) were intervented with 2.5 % sevoflurane for 6 h. Morris water maze test and context fear conditioning test were utilized to evaluate the cognitive function of the offspring. BV2 cells were stimulated with LPS-ATP to evaluate the impacts of SENP7 on microglial pyroptosis. A co-culture experiment was conducted to investigate the apoptosis of mouse hippocampal neuronal cells induced by BV2 cells. The regulatory roles of SENP7 in the cGAS/STING/IRF3 pathway were assessed using an immunoprecipitation SUMOylation assay, along with Western blot analysis.
RESULTS: Sevoflurane exposure during pregnancy resulted in cognitive impairments in offspring mice, which were associated with the upregulation of SENP7, Iba1, Caspase1, and GSDMD-N proteins, as well as the downregulation of NeuN and TH proteins in the brains of the offspring. The knockdown of SENP7 inhibited the elevation of GSDMD-N, Caspase1, and NLRP3 protein levels, subsequently reducing the concentrations of IL-1β and IL-18 in BV2 cells induced by LPS-ATP. Furthermore, SENP7 facilitated the activation of the cGAS/STING/IRF3 axis by regulating the deSUMOylation of cGAS, which triggered microglial pyroptosis and subsequently led to neuronal apoptosis.
CONCLUSION: Maternal exposure to sevoflurane increased the expression of SENP7 in the brains of offspring and resulted in detrimental effects on cognitive function. This phenomenon was associated with neuronal apoptosis triggered by microglial pyroptosis, which was regulated by SENP7 through the cGAS/STING/IRF3 pathway.

Keywords:  Microglial pyroptosis; Neuronal apoptosis; SENP7; Sevoflurane; cGAS/STING/IRF3 pathway

DOI:  https://doi.org/10.1016/j.cyto.2025.156893
Cell Death Differ. 2025 Feb 27.

Metabolic reprogramming in astrocytes prevents neuronal death through a UCHL1/PFKFB3/H4K8la positive feedback loop.

Junjun Xiong, Xuhui Ge, Dishui Pan, Yufeng Zhu, Yitong Zhou, Yu Gao, Haofan Wang, Xiaokun Wang, Yao Gu, Wu Ye, Honglin Teng, Xuhui Zhou, Zheng Wang, Wei Liu, Weihua Cai.

Astrocytic metabolic reprogramming is an adaptation of metabolic patterns to meet increased energy demands, although the role after spinal cord injury (SCI) remains unclear. Analysis of single-cell RNA sequencing (scRNA-seq) data identified an increase in astrocytic glycolysis, while PFKFB3, a key regulator of glycolytic flux, was significantly upregulated following SCI. Loss of PFKFB3 in astrocytes prohibited neuronal energy supply and enhanced neuronal ferroptosis in vitro and expanded infiltration of CD68+ macrophages/microglia, exacerbated neuronal loss, and hindered functional recovery in vivo after SCI. Mechanistically, deubiquitinase UCHL1 plays a crucial role in stabilizing and enhancing PFKFB3 expression by cleaving K48-linked ubiquitin chains. Genetic deletion of Uchl1 inhibited locomotor recovery after SCI by suppression of PFKFB3-induced glycolytic reprogramming in astrocytes. Furthermore, the UCHL1/PFKFB3 axis increased lactate production, leading to enhanced histone lactylation and subsequent transcription of Uchl1 and several genes related to glycolysis, suggesting a glycolysis/H4K8la/UCHL1 positive feedback loop. These findings help to clarify the role of the UCHL1/PFKFB3/H4K8la loop in modulation of astrocytic metabolic reprogramming and reveal a potential target for treatment of SCI.

DOI: https://doi.org/10.1038/s41418-025-01467-x
Neuroscience. 2025 Feb 22. pii: S0306-4522(25)00166-6. [Epub ahead of print]

Hippocampal P2X4 receptor induces type 1 diabetes rats with neuropathic pain through microglial-derived neuroinflammation and neuronal damage.

Xuanwei Zhang, Wenyu Zhang, Yan Li, Guangda Liang, Shirong Peng, Yi Shen, Xingwei Wu, Kaiyu Nie, Zhi Xiao, Xingfeng Liu.

  Diabetic neuropathic pain (DNP) is a serious complication of diabetes, characterized by spontaneous burning pain, hyperalgesia or allodynia, and is associated with severely reduced quality of life. The purinergic P2X4 receptor (P2X4R) plays an essential role in neuropathic pain. In this study, we investigated the roles of hippocampal P2X4R in type 1 diabetes (T1D) rats with DNP. The reduced body weight, elevated blood glucose, and reduced mechanical withdrawal threshold (MWT) were manifested in DNP rats. The increased hippocampal P2X4R enhanced the release of TNF-α, IL-1β, IL-6, which may be related to activated microglia, thereby inducing the development of DNP, and these changes were attenuated by P2X4R antagonist. Our findings suggest that in the state of T1D, hippocampal P2X4R was elevated and enhanced reactive microglia, thereby aggravating the release of pro-inflammatory cytokines and neuronal damage to aggravate hyperalgesia.

Keywords:  Astrocytes; Diabetic neuropathic pain; Hippocampus; Microglia; P2X4 receptor

DOI:  https://doi.org/10.1016/j.neuroscience.2025.02.048
Front Aging Neurosci. 2025 ;17 1542229

Quantification and correlation of amyloid-β plaque load, glial activation, GABAergic interneuron numbers, and cognitive decline in the young TgF344-AD rat model of Alzheimer's disease.

Anett Futácsi, Kitti Rusznák, Gergely Szarka, Béla Völgyi, Ove Wiborg, Boldizsár Czéh.

   Background: Animal models of Alzheimer's disease (AD) are essential tools for investigating disease pathophysiology and conducting preclinical drug testing. In this study, we examined neuronal and glial alterations in the hippocampus and medial prefrontal cortex (mPFC) of young TgF344-AD rats and correlated these changes with cognitive decline and amyloid-β plaque load.
Methods: We compared TgF344-AD and non-transgenic littermate rats aged 7-8 months of age. We systematically quantified β-amyloid plaques, astrocytes, microglia, four different subtypes of GABAergic interneurons (calretinin-, cholecystokinin-, parvalbumin-, and somatostatin-positive neurons), and newly generated neurons in the hippocampus. Spatial learning and memory were assessed using the Barnes maze test.
Results: Young TgF344-AD rats had a large number of amyloid plaques in both the hippocampus and mPFC, together with a pronounced increase in microglial cell numbers. Astrocytic activation was significant in the mPFC. Cholecystokinin-positive cell numbers were decreased in the hippocampus of transgenic rats, but calretinin-, parvalbumin-, and somatostatin-positive cell numbers were not altered. Adult neurogenesis was not affected by genotype. TgF344-AD rats had spatial learning and memory impairments, but this cognitive deficit did not correlate with amyloid plaque number or cellular changes in the brain. In the hippocampus, amyloid plaque numbers were negatively correlated with cholecystokinin-positive neuron and microglial cell numbers. In the mPFC, amyloid plaque number was negatively correlated with the number of astrocytes.
Conclusion: Pronounced neuropathological changes were found in the hippocampus and mPFC of young TgF344-AD rats, including the loss of hippocampal cholecystokinin-positive interneurons. Some of these neuropathological changes were negatively correlated with amyloid-β plaque load, but not with cognitive impairment.

Keywords:  Barnes maze; CCK+ interneurons; astrocyte; cell number; cholecystokinin; hippocampus; medial prefrontal cortex; microglia

DOI:  https://doi.org/10.3389/fnagi.2025.1542229
J Neuroinflammation. 2025 Feb 22. 22(1): 46

Interferon-gamma receptor signaling regulates innate immunity during Staphylococcus aureus craniotomy infection.

Zachary Van Roy, Gunjan Kak, Rachel W Fallet, Tammy Kielian.

  A craniotomy is a neurosurgical procedure performed to access the intracranial space. In 3-5% of cases, infections can develop, most caused by Staphylococcus aureus biofilm formation on the skull surface. Medical management of this infection is difficult, as biofilm properties confer immune and antimicrobial recalcitrance to the infection and necessitate additional surgical procedures. Furthermore, treatment failure rates can be appreciably high. These factors, compounded with rapidly expanding rates of antimicrobial resistance, highlight the need to develop alternative treatment strategies to target and reverse the immune dysfunction that occurs during biofilm infection. Our recent work has identified CD4+ Th1 and Th17 cells as potent regulators of innate immune cell activation during craniotomy infection. Here, we report the role of IFN-γ, versus other Th1- and Th17-derived cytokines, in programing the immune response to biofilm infection using both global and cell type-specific IFN-γR1-deficient (Ifngr1-/-) mice. Bacterial burdens were significantly higher in Ifngr1-/- relative to WT animals despite few changes in immune cell abundance. Single-cell transcriptomics identified candidate explanations for this phenotype as alterations in cell death pathways, innate immune cell activation, MHC-II expression, and T cell responses were significantly reduced in Ifngr1-/- mice. While caspase-1 activation in PMNs and macrophage/microglial MHC-II expression were regulated by IFN-γ signaling, no phenotypes were observed with either granulocyte- or macrophage/microglia Ifngr1-/- conditional knockout mice, suggestive of redundancy. Instead, a decreased Th1/Th17 ratio was identified in Ifngr1-/- animals that was corroborated by elevated IL-17 levels and correlated with dysfunctional T cell-innate immune communication. Further, Th17 cells were less effective than Th1 cells in promoting S. aureus bactericidal activity in microglia and macrophages. Collectively, this work identifies a key protective role for IFN-γ during craniotomy infection by enhancing macrophage and microglial antibacterial activity. Therefore, controlled programming of IFN-γ responses may represent a novel therapeutic strategy for chronic craniotomy infections.

Keywords:  Biofilm; Craniotomy; Granulocyte; IFN-γR; Infection; Macrophage; Microglia; T cells; Th1; Th17

DOI:  https://doi.org/10.1186/s12974-025-03376-9
Neurochem Res. 2025 Feb 24. 50(2): 103

HIV-1 gp120 Interactions with Nicotine Modulate Mitochondrial Network Properties and Amyloid Release in Microglia.

Alexandru Graur, Natalie Erickson, Patricia Sinclair, Aya Nusir, Nadine Kabbani.

  Human immunodeficiency virus (HIV) infections remain a significant public health burden globally with infected individuals at high risk for cognitive decline and memory loss even on combination antiretroviral therapy. Almost half of HIV infected individuals smoke, which drives poorer health outcomes including a higher dementia rate. Microglia are the brain's immune cells that serve as a persistent HIV reservoir contributing to neuroinflammatory signaling. We examined interactions between the HIV envelope glycoprotein gp120 and nicotine within human microglia cells (HMC3) that endogenously express chemokine receptor 5 (CCR5) and nicotinic acetylcholine receptors (nAChRs). Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI/MS) shows that gp120 alters mitochondria proteins within HMC3 cells. In the presence of nicotine, gp120 increased the expression of mitochondrial prohibitin 2 (PHB2), cytochrome c (cyt c), and mitofusin 2 (MFN2) but decreased fission 1 (FIS1) levels. An analysis of mito-YFP expression confirms that interaction between nicotine and gp120 increases the size and branching of mitochondrial networks. Interaction between nicotine and gp120 is also surprisingly found to promote the release of amyloid precursor protein (APP) peptides from microglia. This was accompanied by visualization of amyloid containing vesicles that colocalized with the autophagy protein LC3B-II in the cell. Taken together, our findings show that interaction between nicotine and gp120 impact microglia in a manner that regulates mitochondrial proteins and network properties and impacts amyloid protein management and release within microglia. These mechanisms may contribute to understanding neuroinflammatory signaling in smokers with HIV.

Keywords:  APP; Amyloid plaques; Autophagy; Nicotinic receptor; Proteomics

DOI:  https://doi.org/10.1007/s11064-025-04357-3
Mucosal Immunol. 2025 Feb 25. pii: S1933-0219(25)00025-X. [Epub ahead of print]

Increased ocular plasma cells induce damaging α-synuclein+ microglia in autoimmune uveitis.

Minghao Li, Meng Feng, Tingting Liu, Songqi Duan, Xuejing Man, Xiaomeng Yuan, Lijie Wang, Yu Sun, Xunbin Wei, Qiang Fu, Baofa Sun, Wei Lin.

  Autoimmune uveitis (AIU) is an immune-inflammatory disease that can lead to blindness. However, incomplete understanding of the involved immune cell subsets and their contributions to retinal injury has hindered the development of effective AIU therapies. Using single-cell RNA sequencing and immunofluorescence, we identified α-synuclein+ microglia as the primary subset of damaged ocular cells in the eyes of the experimental autoimmune uveitis (EAU) mouse model. Ocular-infiltrating plasma cells (PCs) were shown to express multiple inflammatory factors, particularly TNF-α, which promoted the production of α-synuclein+ microglia. Studies of heterogeneous PC subtypes revealed that MUC1- PCs represent the primary pathogenic subset, secreting multiple cytokines. Although MUC1+ PCs expressed TGF-β, they exhibited long-lived characteristics and secreted IgG and IgM, thereby prolonging disease progression. Finally, the small G protein Rab1A, also expressed in the PCs of Vogt-Koyanagi-Harada (VKH) patients, was found to mediate autophagy and NF-κB expression, influencing PCs survival and inflammatory responses. Silencing or knocking down Rab1A in PCs inhibited their survival. This study elucidates potential mechanisms underlying the neuroimmune inflammatory response and highlights the previously unrecognized role of infiltrating PCs in AIU, offering novel therapeutic targets for this disease.

Keywords:  Autoimmune uveitis; Inflammatory factors; Ocular immunology; Plasma cells; single-cell RNA sequencing

DOI:  https://doi.org/10.1016/j.mucimm.2025.02.007
Alzheimers Dement. 2025 Feb;21(2): e70031

Comparing Alzheimer's genes in African, European, and Amerindian induced pluripotent stem cell-derived microglia.

Sofia Moura, Luciana Bertholim Nasciben, Aura M Ramirez, Lauren Coombs, Joe Rivero, Derek J Van Booven, Brooke A DeRosa, Kara L Hamilton-Nelson, Patrice L Whitehead, Larry D Adams, Takiyah D Starks, Pedro R Mena, Maryenela Illanes-Manrique, Sergio Tejada, Goldie S Byrd, Mario R Cornejo-Olivas, Briseida E Feliciano-Astacio, Karen Nuytemans, Liyong Wang, Margaret A Pericak-Vance, Derek M Dykxhoorn, Farid Rajabli, Anthony J Griswold, Juan I Young, Jeffery M Vance.

   INTRODUCTION: Genome-wide association studies (GWAS) studies in Alzheimer's disease (AD) demonstrate ancestry-specific loci. Previous studies in the regulatory architecture have only been conducted in Europeans (EUs), thus studies in additional ancestries are needed. Given the prevalence of AD genes expressed in microglia, we initiated our studies in induced pluripotent stem cell (iPSC) -derived microglia.
METHODS: We created iPSC-derived microglia from 13 individuals of either high Amerindian (AI), African (AF), or EU global ancestry, including both AD and controls. RNA-seq, ATAC-seq, and pathway analyses were compared between ancestries in both AD and non-AD genes.
RESULTS: Twelve AD genes were differentially expressed genes (DEGs) and/or accessible between ancestries, including ABI3, CTSB, and MS4A6A. A total of 5% of all genes had differential ancestral expression, but differences in accessibility were less than 1%. The DEGs were enriched in known AD pathways.
DISCUSSION: This resource will be valuable in evaluating AD in admixed populations and other neurological disorders and understanding the AD risk differences between populations.
HIGHLIGHTS: First comparison of the genomics of AI, AF, and EU microglia. Report differences in expression and accessibility of AD genes between ancestries. Ancestral expression differences are greater than differences in accessibility. Good transcriptome correlation was seen between brain and iPSC-derived microglia. Differentially expressed AD genes were in known AD pathways.

Keywords:  ATAC‐seq; Alzheimer's disease; RNA‐seq; diversity; genetic ancestry; genetic regulatory architecture; iPSC‐derived microglia

DOI:  https://doi.org/10.1002/alz.70031
FASEB J. 2025 Feb 28. 39(4): e70412

Fructose induces inflammatory activation in macrophages and microglia through the nutrient-sensing ghrelin receptor.

Zheng Shen, Zeyu Liu, Hongying Wang, Danilo Landrock, Ji Yeon Noh, Qun Sophia Zang, Chih-Hao Lee, Yuhua Z Farnell, Zheng Chen, Yuxiang Sun.

  High fructose corn syrup (HFCS) is a commonly used sweetener in soft drinks and processed foods, and HFCS exacerbates inflammation when consumed in excess. Fructose, a primary component of HFCS; however, it is unclear whether fructose directly activates inflammatory signaling. Growth hormone secretagogue receptor (GHSR) is a receptor of the nutrient-sensing hormone ghrelin. We previously reported that GHSR ablation mitigates HFCS-induced inflammation in adipose tissue and liver, shifting macrophages toward an anti-inflammatory spectrum. Since inflammation is primarily governed by innate immune cells, such as macrophages in the peripheral tissues and microglia in the brain, this study aims to investigate whether GHSR autonomously regulates pro-inflammatory activation in macrophages and microglia upon fructose exposure. GHSR deletion mutants of RAW 264.7 macrophages and the immortalized microglial cell line (IMG) were generated using CRISPR-Cas9 gene editing. After treating the cells with equimolar concentrations of fructose or glucose for 24 h, fructose increased mRNA and protein expression of GHSR and pro-inflammatory cytokines (Il1β, Il6, and Tnfα) in both macrophages and microglia, suggesting that fructose activates Ghsr and induces inflammation directly in macrophages and microglia. Remarkably, GHSR deletion mutants (Ghsrmutant) of macrophages and microglia exhibited reduced inflammatory responses to fructose, indicating that GHSR mediates fructose-induced inflammation. Furthermore, we found that GHSR regulates fructose transport and fructose metabolism and mediates fructose-induced inflammatory activation through CREB-AKT-NF-κB and p38 MAPK signaling pathways. Our results underscore that fructose triggers inflammation, and reducing HFCS consumption would reduce disease risk. Moreover, these findings reveal for the first time that the nutrient-sensing receptor GHSR plays a crucial role in fructose-mediated inflammatory activation, suggesting that targeting GHSR may be a promising therapeutic approach to combat the immunotoxicity of foods that contain fructose.

Keywords:  fructose; growth hormone secretagogue receptor (GHSR); inflammation; macrophage; microglia

DOI:  https://doi.org/10.1096/fj.202402531R
J Neurochem. 2025 Mar;169(3): e70024

Microglia-Mediated Synaptic Dysfunction Contributes to Chemotherapy-Related Cognitive Impairment.

Jingxiong Wang, Hua Zhang, Marc Augenreich, Luis Martinez-Lemus A, Zhenguo Liu, Xunlei Kang, Bo Lu, Hui-Ming Chang, Edward T H Yeh, Juan Cata, Srikant Rangaraju, Heike Wulff, De-Pei Li.

  Chemotherapy-related cognitive impairment (CRCI) significantly impacts cancer survivors. Due to unclear mechanisms, effective treatments for cognitive deficits are lacking. Here, we examined if microglia-mediated deficits in synaptic plasticity drive CRCI. Adult male mice were treated with the chemotherapeutic drugs 5-fluorouracil and leucovorin (5-Fu/LV, intraperitoneal injection, I.P.) on Days 1, 8, and 15 at a dosage of 50 mg/kg for 5-Fu and 90 mg/kg for LV for 3 weeks. Cognitive function was assessed using a novel object recognition (NOR) test 4 weeks after completion of 5-Fu/LV treatment. Compared with vehicle treatment, 5-Fu/LV treatment reduced the preference for exploring novel objects in the NOR test. Treatment with 5-Fu/LV increased the numbers of Iba1-positive microglial and CD68-positive/Iba1-positive microglia with shortened process lengths and diminished endpoints but decreased the number of phagocytotic (≤ 1 FITC-labeled beads) Iba1-positive microglia. Furthermore, 5-Fu/LV treatment reduced the long-term potentiation (LTP) recorded in the hippocampal CA1 region in response to a theta burst stimulation of the CA3-CA1 pathway and decreased the evoked N-methyl-D-aspartic acid receptor (NMDAR)-excitatory postsynaptic currents (NMDAR-EPSCs) in CA1 neurons. Cotreatment with the microglial inhibitor minocycline (33 mg/kg, daily for 3 weeks) restored cognitive deficits and microglial ramification, decreased the number of CD68-positive microglia, and reversed the reductions in LTP and the amplitude of NMDAR-EPSCs in 5-Fu/LV-treated mice. Our data suggest that microglial dysfunction and related synaptic dysfunction contribute to 5-Fu/LV-induced cognitive impairment.

Keywords:  chemotherapy; cognitive deficits; long‐term potentiation; microglia; synaptic transmission

DOI:  https://doi.org/10.1111/jnc.70024
Int J Mol Sci. 2025 Feb 11. pii: 1494. [Epub ahead of print]26(4):

Transcriptomic Analysis Divulges Differential Expressions of Microglial Genes After Microglial Repopulation in Mice.

Muhammad Tariq Hafeez, Hao Gao, Furong Ju, Fujian Qi, Ting Li, Shengxiang Zhang.

  Microglia are key immune cells in the central nervous system (CNS) and maintain hemostasis in physiological conditions. Microglial depletion leads to rapid repopulation, but the gene expression and signaling pathways related to repopulation remain unclear. Here, we used RNA sequencing (RNA-Seq) analysis to profile the transcriptome of microglia-depleted tissue by taking advantage of a conditional genetic microglial depletion model (CX3CR1CreER/+ system). Differential gene expression (DGE) sequencing analysis showed that 1226 genes were differentially up- and downregulated in both groups compared to control. Our data demonstrated that many microglial genes were highly regulated on day 3 after depletion but the numbers of differentially expressed genes were reduced by day 7. Gene ontology (GO) analysis categorized these differentially expressed genes on day 3 and day 7 to the specific biological processes, such as cell proliferation, cell activation, and cytokine and chemokine production. DGE analysis indicated that specific genes related to proliferation were regulated after depletion. Consistent with the changes in transcriptome, the histological analysis of transgenic mice revealed that the microglia after depletion undergo proliferation and activation from day 3 to day 7. Collectively, these results suggest that transcriptomic changes in microglial genes during depletion have a profound implication for the renewal and activation of microglia and may help to understand the regulatory mechanism of microglial activation in disease conditions.

Keywords:  RNA sequencing (RNA-Seq); density; depletion; microglia; proliferation

DOI:  https://doi.org/10.3390/ijms26041494
J Neuroinflammation. 2025 Feb 25. 22(1): 50

25-Hydroxycholesterol modulates microglial function and exacerbates Alzheimer's disease pathology: mechanistic insights and therapeutic potential of cholesterol esterification inhibition.

Hayoung Choi, Haeng Jun Kim, Sang-Eun Lee, Hyun Ho Song, Jieun Kim, Jihui Han, June-Hyun Jeong, Do Yup Lee, Sunghoe Chang, Inhee Mook-Jung.

  This study investigates the role of 25-hydroxycholesterol (25HC), a metabolite produced by cholesterol hydroxylase encoded by the Ch25h gene, in modulating microglial function and its potential implications in Alzheimer's disease (AD) pathology. We demonstrated that 25HC impairs microglial surveillance, reduces phagocytic capacity, and increases the production of pro-inflammatory cytokines. In vivo two-photon microscopy revealed that 25HC administration diminishes microglial response to brain lesions, while flow cytometry confirmed reduced phagocytosis in both in vivo and in vitro models. Additionally, amyloid-beta (Aβ) was shown to upregulate Ch25h expression and elevate 25HC levels in microglia, exacerbating these functional impairments. Mechanistically, 25HC was found to enhance cholesterol esterification, disrupt cell membrane dynamics, and further reduce microglial mobility and phagocytosis. Treatment with Avasimibe, a cholesterol esterification inhibitor, restored membrane dynamics and microglial function, leading to attenuated AD pathology in a 5XFAD mouse model. These findings suggest that 25HC-induced changes in microglial function contribute to AD progression, and targeting cholesterol metabolism could offer therapeutic potential.

Keywords:  25HC; Avasimibe; Aβ; Cholesterol; Disease-associated microglia; Microglia

DOI:  https://doi.org/10.1186/s12974-025-03357-y
Cell Biochem Funct. 2025 Mar;43(3): e70061

Matrix Stiffness Regulates Interleukin-10 Secretion in Human Microglia (HMC3) via YAP-Mediated Mechanotransduction.

Xue Fang, Haiying Jia, Shaoshan Pan, Qian Liu, Qian Wang, Ye Feng, Weiping Ding, Tianzhi Luo.

  Microglia, as resident immune cells in the brain, adhere to the extracellular matrix and typically exhibit anti-inflammatory polarization under normal physiological conditions. Despite their pivotal roles, the regulatory effects of extracellular matrix properties on microglial function and the associated molecular mechanisms remain inadequately understood. Here, we elucidate how matrix stiffness modulates interleukin-10 (IL-10) secretion in human microglia (HMC3) via yes-associated protein (YAP)-mediated mechanotransduction. Using soft collagen Ⅰ-coated hydrogels, we observed a substantial reduction in IL-10 secretion, accompanied by a decrease in the expression and nuclear localization of YAP compared to cells adhered to glass substrates. With increasing hydrogel substrate stiffness, the expression and nuclear localization of YAP were enhanced, leading to an elevated secretion of IL-10. Subsequently, to further investigate the relationship between YAP and IL-10, we performed YAP depletion experiments, which revealed that nuclear exclusion of YAP suppressed IL-10 secretion. Interestingly, overexpression of YAP in microglia did not markedly affect IL-10 levels. We seeded YAP-knockdown microglia onto hydrogels of varying stiffness, and no significant differences were observed in IL-10 secretion. Our findings suggested that cytoskeletal polymerization was crucial for the regulation of IL-10 secretion mediated by YAP. Given the crucial role of IL-10 in the tumor microenvironment, we further found shYAP-microglia attenuated the pro-proliferative effect of microglia on gliomas. Besides, when YAP was silenced, actin of human microglia decreased, and their contractility was weakened. In summary, this study identifies YAP as a pivotal molecule in controlling cytokine secretion and sensing matrix stiffness in microglia. These insights offer potential therapeutic avenues for glioma treatment by targeting YAP-mediated pathways in microglial cells.

Keywords:  HMC3; glioma proliferation; human microglia; interleukin‐10; matrix stiffness; yes‐associated protein

DOI:  https://doi.org/10.1002/cbf.70061
Neuromolecular Med. 2025 Feb 26. 27(1): 16

ADAR1 Promotes NUPR1 A-to-I RNA Editing to Exacerbate Ischemic Brain Injury by Microglia Mediated Neuroinflammation.

Guo-Ping Wang, Wen-Juan Li, Ye Li, Ming-Xing Ma, Kai-Kai Guo.

  Microglial cells occupy a crucial position as potential therapeutic targets in the context of ischemic stroke (IS). Nonetheless, the intrinsic mechanisms that govern microglial activation in the aftermath of IS remain incompletely elucidated. ADAR1 p150 plays a significant role in immune regulation and stress responses; however, the specific pathways through which it modulates microglial activation and the subsequent mechanisms that unfold following IS have yet to be clearly delineated. The distal middle cerebral artery occlusion (dMCAO) mouse model was utilized to induce IS. The evaluation of infarct volume was conducted through TTC staining, while neurological function was assessed using the modified Neurological Severity Score (mNSS). To evaluate the expression of ADAR1 and apoptosis-related proteins, immunofluorescence and Western blot techniques were employed. BV2 cells were subjected to oxygen-glucose deprivation followed by reperfusion (OGD/R). Additionally, a co-culture system of BV2 cells and neurons was established, and subsequent assessments of neuronal viability and apoptosis were performed using CCK-8 assays and LDH release assays. ADAR1 p150 expression was significantly upregulated in the brains of ischemic mice, particularly within microglial cells. The overexpression of ADAR1 p150 was found to promote microglial activation and enhance pro-inflammatory responses, whereas the knockdown of ADAR1 p150 yielded the opposite effect. Additionally, the knockdown of ADAR1 p150 in microglia resulted in a marked reduction in neuronal apoptosis within the co-culture system. Rescue experiments indicated that the knockdown of NUPR1 partially reinstated the inflammatory response previously induced by ADAR1 p150 knockdown. Notably, ADAR1 p150 knockdown also inhibited A-to-I RNA editing while simultaneously upregulating NUPR1. Furthermore, the reduction of ADAR1 expression was associated with decreased infarct volume, improved neurological outcomes, and a significant attenuation of neuroinflammation in dMCAO mice. ADAR1 p150 enhances the microglial inflammatory response and neuronal apoptosis in IS by facilitating A to I RNA editing of NUPR1.

Keywords:  ADAR1; Ischemic stroke; Microglia; Neuroinflammation; Neuron apoptosis

DOI:  https://doi.org/10.1007/s12017-025-08841-5
J Neurooncol. 2025 Feb 28.

Glioblastoma- derived exosomes (GBM-Exo) regulate microglial M2 polarization via the RAC1/AKT/NRF2 pathway.

Qionghui Wu, Shanlin Chen, Xiaodong Xie, Hong Yan, Xinli Feng, Gang Su, Zhenchang Zhang.

   PURPOSE: The impact of exosome-mediated communication between glioblastoma and microglia on the formation of an immunosuppressive microenvironment remains to be explored. Tumor-associated macrophages are more likely to adopt an M2-like phenotype within the immunosuppressive environment. Here, we investigate the molecular mechanisms by which glioblastoma-derived exosomes promote microglial M2 polarization through RAC1.
METHODS: The expression of RAC1 in GBM was collected from public databases. A C57BL/6 mouse glioma xenograft model was established using intracranial stereotactic injection. RAC1 expression was validated by qRT-PCR, Western blotting, and immunohistochemistry. Glioblastoma-derived exosomes were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy, and Western blotting for exosome markers, with the content of RAC1 being profiled. RAC1 and AKT inhibitors were used to co-treat microglia with exosomes. Microglial polarization under different treatment conditions was assessed by Western blotting and immunofluorescence.
RESULT: Our study reveals that RAC1 is aberrantly expressed in glioblastoma and is associated with macrophage immune infiltration. GBM-derived exosomes, carrying RAC1, promote the M2 polarization of microglia. In microglia treated with GBM-derived exosomes, inhibition of RAC1 activity suppressed AKT phosphorylation and NRF2 nuclear translocation, while reducing the expression of M2 phenotype markers. Notably, following AKT inhibition, the exosome-induced NRF2 nuclear translocation was also significantly suppressed, highlighting the critical role of RAC1-mediated AKT activation in NRF2 translocation and microglial M2 polarization.
CONCLUSION: Our study demonstrates that RAC1-carrying GBM-exosomes promote M2 polarization of microglia, a process mediated through the RAC1/AKT/NRF2 pathway.

Keywords:  Exosomes; Glioblastoma; RAC1; RAC1/AKT/NRF2

DOI:  https://doi.org/10.1007/s11060-024-04934-6