bims-musmir Biomed News
on microRNAs in muscle
Issue of 2025–12–21
fourteen papers selected by
Katarzyna Agnieszka Goljanek-Whysall, University of Galway
  1. Skeletal Muscle HSF1 Alleviates Age-Associated Sarcopenia and Mitochondrial Function Decline via SIRT3-PGC1α Axis.
  2. The effects of tissue inflammation on cancer cachexia.
  3. Pharmacological Activation of Mitophagy Confers Neuroprotective Benefits for Amyotrophic Lateral Sclerosis.
  4. CircAMOTL1 promotes adipose lipolysis and browning in cancer cachexia through miR-211-5p-mediated TET2 activation.
  5. TDP-43 directly inhibits mRNA accumulation in neurites through modulation of mRNA stability.
  6. TMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis.
  7. CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk.
  8. Dysregulated TFEB-autophagy-lysosome pathway links acute COVID-19 immunopathology to Long COVID sequelae.
  9. The impact of cellular senescence on aging skeletal muscle.
  10. Identification of competing endogenous RNA networks involved in phrenic nerve stimulation preventing mechanical ventilation induced diaphragm dysfunction.
  11. Hv1 inhibition rescues AD pathology by restoring microglial mitochondrial function and enhancing mitochondrial transfer.
  12. Fhod3 in zebrafish supports myofibril stability during growth of embryonic skeletal muscle.
  13. Influenza A infection accelerates disease-associated microglia formation during physiological aging.
  14. Protocol for applying expansion microscopy to the study of mammalian neuromuscular junctions.
  1. Adv Sci (Weinh). 2025 Dec 16. e10368
    Skeletal Muscle HSF1 Alleviates Age-Associated Sarcopenia and Mitochondrial Function Decline via SIRT3-PGC1α Axis.
    Jun Zhang, Min Hu, Xia Wu, Mingwei Guo, Ying Ma, Jin Qiu, Siqi Wang, Yuxiang Cao, Yinzhao Zhong, Fangfang Chen, Yiwen Wang, Wei Wei, Yan Lu, Yong Zhang, Junjie Xiao, Zhenji Gan, Cheng Hu, Xinran Ma, Lingyan Xu.
      Age-related sarcopenia, characterized by progressive loss of skeletal muscle mass and strength, impacts metabolic health and quality of life in the elderly. Heat shock factor 1 (HSF1) is a transcription factor that orchestrates cellular responses to various stresses, while its role in sarcopenia remains unknown. Here, HSF1 mRNA expression was decreased in muscles of aged mice and humans, correlating negatively with the atrophic gene and positively with the mitochondrial gene. Aged HSF1 muscle-specific knockout mice exhibited severe muscle atrophy and reduced endurance capacity, partially due to smaller fast fibers and mitochondrial dysfunction in slow fibers, as well as impaired systemic metabolic performance. In contrast, HSF1 overexpression in skeletal muscle improved these functions. Mechanistically, via RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), it is revealed that HSF1 transcriptionally activated Sirtuin3 (SIRT3) for the deacetylation of both PGC1α1 and PGC1α4 isoforms of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), in skeletal muscle, enhancing mitochondrial function and muscle hypertrophy in vivo and in vitro, and inducing fibronectin type III domain-containing protein 5 (FNDC5)/Irisin for tissue crosstalk. Thus, HSF1 regulates skeletal muscle functions and systemic energy homeostasis via the SIRT3-PGC1α axis, representing a potential therapeutic target for sarcopenia and metabolic disorders.
    Keywords:  aging; mitochondria; muscle atrophy; oxidative function; sarcopenia
    DOI:  https://doi.org/10.1002/advs.202510368
  2. Biochim Biophys Acta Mol Basis Dis. 2025 Dec 17. pii: S0925-4439(25)00494-6. [Epub ahead of print] 168144
    The effects of tissue inflammation on cancer cachexia.
    Benjamin R Pryce, Haiming L Kerr.
      Cancer cachexia is characterized by a significant loss in body weight due to the wasting of skeletal muscle and adipose tissue. Systemic inflammation has long been associated with cachexia, with various tumor secreted factors shown to correlate with as well as cause tissue wasting. In addition to systemic inflammation, it has become increasingly appreciated that inflammation occurs in specific tissues in cancer cachexia, with tissues such as muscle, adipose, liver and brain being affected. While several studies have shown that this local tissue inflammation contributes to cachexia, there is evidence that some aspects of the inflammatory response may play a protective role to mitigate tissue wasting. Here, we will review the findings on local tissue inflammation in cachexia, comparing the impacts of such inflammation on tissue wasting and cachexia progression overall. Furthermore, we discuss the methods used to mitigate inflammation in various tissues and highlight the outcomes on the cachectic phenotype. Collectively, understanding how inflammation contributes to cachexia in each tissue will ultimately influence how therapies can be designed to treat cachexia while minimizing possible adverse side effects.
    Keywords:  Cancer Cachexia; Inflammation; Macrophage
    DOI:  https://doi.org/10.1016/j.bbadis.2025.168144
  3. Aging Dis. 2025 Dec 14.
    Pharmacological Activation of Mitophagy Confers Neuroprotective Benefits for Amyotrophic Lateral Sclerosis.
    Sen Huang, Ang Li, Yixia Ling, Xinyu Yang, Jiayu Wang, Jing Yuan, Dajiang Qin, Xiaoli Yao.
      Amyotrophic lateral sclerosis (ALS) is a rare and devastating neurodegenerative disease characterized by the progressive degeneration of motor neurons in the brain and spinal cord, for which no cure currently exists. Previous studies have shown that abnormal mitochondrial homeostasis and defective mitophagy occur in neurodegenerative diseases, including ALS. Here, we provide evidence that PINK1-Parkin-dependent mitophagy is impaired in multiple ALS mouse models, including the SOD1G93A, TDP43A315T, and rNLS8 strains, leading to the accumulation of damaged mitochondria in affected motor neurons. These findings suggest that mitophagy may be a druggable target for ALS treatment. A classical mitophagy agonist, urolithin A (UA) was used in this study. UA-induced mitophagy antagonizes ALS pathologies in the ALS SOD1G93A transgenic C. elegans model in a pink-1 (PTEN-induced kinase 1)- and pdr-1 (Parkinson's disease-related 1)-dependent manner. Furthermore, pharmacological activation of mitophagy by UA improves locomotor behavior, delays motor neuron degeneration and reduces neuroinflammation in ALS SOD1G93A transgenic mice. In conclusion, our results establish impaired mitophagy as a hallmark of ALS motor neuron degeneration and demonstrate that its pharmacological activation offers a neuroprotective strategy with therapeutic potential.
    DOI:  https://doi.org/10.14336/AD.2025.1224
  4. J Biol Chem. 2025 Dec 12. pii: S0021-9258(25)02904-7. [Epub ahead of print] 111052
    CircAMOTL1 promotes adipose lipolysis and browning in cancer cachexia through miR-211-5p-mediated TET2 activation.
    Zuoyou Ding, Zhige Zhang, Jun Han, Ruizhao Dong, Ziang Yang, Guohao Wu, Qiulin Zhuang.
      Cancer cachexia is characterized by profound adipose tissue loss and metabolic remodeling, yet the regulatory mechanisms driving adipocyte dysfunction remain incompletely understood. Through whole-transcriptome sequencing of human subcutaneous adipose tissue, we identified circAMOTL1 as one of the most significantly upregulated circRNAs in cachectic patients. circAMOTL1 expression positively correlated with weight-loss severity and demonstrated strong diagnostic performance for cachexia. Functional studies revealed that circAMOTL1 promotes lipolysis and white adipose browning in adipocytes, as evidenced by increased expression of ATGL, HSL, UCP1, and PGC-1α, elevated free fatty acid release, and enhanced mitochondrial content. Silencing circAMOTL1 produced the opposite phenotype. Mechanistically, circAMOTL1 localized predominantly in the cytoplasm and acted as a molecular sponge for miR-211-5p, thereby relieving miR-211-5p mediated repression of TET2. Luciferase reporter, RIP, and FISH assays confirmed the circAMOTL1/miR-211-5p/TET2 regulatory interaction. Gain- and loss-of-function experiments demonstrated that TET2 is essential for circAMOTL1-induced lipolysis and thermogenic remodeling. In vivo, adipose-targeted recombinant AAV overexpression of circAMOTL1 in a C26 tumor-bearing mouse model induced adipose wasting and upregulated lipolytic and browning markers, phenocopying cachexia-associated adipose remodeling. These findings identify circAMOTL1 as a critical regulator of adipocyte metabolism in cancer cachexia. By modulating the circAMOTL1/miR-211-5p/TET2 axis, it drives lipolysis and thermogenic reprogramming of white adipose tissue. circAMOTL1 therefore represents a promising biomarker and a potential therapeutic target for preventing or attenuating cachexia-associated adipose tissue loss.
    Keywords:  Cancer cachexia; TET2; adipose tissue; circAMOTL1; lipolysis
    DOI:  https://doi.org/10.1016/j.jbc.2025.111052
  5. EMBO J. 2025 Dec 15.
    TDP-43 directly inhibits mRNA accumulation in neurites through modulation of mRNA stability.
    Charlie Moffatt, Ankita Arora, Katherine F Vaeth, Bryan B Guzman, Gurprit Bhardwaj, Audrey Hoelscher, Levi B Gifford, Holger A Russ, Daniel Dominguez, J Matthew Taliaferro.
      The subcellular localization of many mRNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for most of these RNAs, the mechanisms that govern their localization are unknown. Here, using subcellular fractionation and single-molecule RNA FISH, we found that loss of TDP-43 results in increased accumulation of hundreds of mRNAs in neurites. Using high-throughput functional assays in cells and high-throughput binding assays in vitro, we subsequently identified specific regions within these mRNAs that mediate their TDP-43-dependent localization and interaction with TDP-43. We found that the same regions also mediated TDP-43-dependent mRNA instability, suggesting a mechanism by which TDP-43 regulates mRNA localization. ALS-associated mutations in TDP-43 resulted in similar mRNA mislocalization phenotypes as did TDP-43 loss in mouse dorsal root ganglia and human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of mRNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.
    Keywords:  ALS; RNA Localization; RNA Stability; RNA Trafficking; TDP-43
    DOI:  https://doi.org/10.1038/s44318-025-00653-4
  6. Nat Commun. 2025 Dec 17.
    TMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis.
    Massimo Vetralla, Lena Wischhof, Asrat Kahsay, Vanessa Cadenelli, Enzo Scifo, Beijia Xie, Miriana Sbrissa, Maëlle Sandhira Habert, Dan Ehninger, Rosario Rizzuto, Daniele Bano, Diego De Stefani.
      The bidirectional transport of Ca2+ into and out of mitochondria regulates metabolism, signaling, and cell fate. While influx is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, efflux mechanisms are more diversified, involving Na⁺ or H⁺ exchange pathways. We here demonstrate that TMEM65 is a fundamental component of the Ca2+ efflux machinery of mitochondria. Its overexpression specifically enhances Na⁺- and Li⁺-dependent mitochondrial Ca²⁺ extrusion. This effect is inhibited by CGP-37157 and does not depends on NCLX, currently considered the bona fide mitochondrial Na+/Ca2+ exchanger. Its downregulation chronically elevates basal [Ca²⁺]mt and impairs efflux upon stimulation. In Caenorhabditis elegans, deletion of TMEM65 homologs compromises embryonic development under mild thermal stress, causing necrotic lesions that are suppressed by genetic inhibition of MCU-1. These findings highlight a molecular component that may be relevant in pathological settings in which excessive mitochondrial Ca2+ accumulation critically contribute to degenerative pathways.
    DOI:  https://doi.org/10.1038/s41467-025-67647-y
  7. J Biomed Sci. 2025 Dec 15. 32(1): 107
    CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk.
    Hyun-Jun Kim, Seon-Wook Kim, Jun-Hyeong Kim, Sang-Hoon Lee, Kyoung-Hwan Joo, Soo Young Lee, Hyunju Lee, Jung-Joon Min, Sang-Hee Cho, Da-Woon Jung, Darren R Williams.
       BACKGROUND: Advanced metastasis produces cachexia, a complex skeletal muscle wasting syndrome that accounts for one-third of patient deaths. There is currently no approved drug therapy for cancer cachexia. Cancer-associated fibroblasts (CAF) within tumors have been hypothesized to contribute to cachexia, but the detailed mechanism(s) are unknown.
    METHODS: Myotubes were treated with conditioned media (CM) from CAF or CAF activated by cancer cells. Upregulated chemokines in the cancer-activated CAF CM were identified by cytokine array. The effects of chemokine neutralization were investigated using in vitro myotube cultures and in vivo mouse models. The mechanism of action was characterized by in vivo RNA Seq and validated in human muscle cells. Immunostaining delineated the chemokine expression pattern in a patient tumor type highly associated with cachexia.
    RESULTS: Cancer-activated CAF induced myotube atrophy. CXCL5 was as the major chemokine highly upregulated in the cancer-activated CAF. CXCL5 treatment alone induced myotube atrophy and inhibited myogenic ERK1/2 signaling, similar to cancer-activated CAF treatment. CXCL5 neutralization inhibited cachexia in mice co-injected with HCT 116 colon cancer cells and CAF. RNA Seq showed that CXCL5 neutralization upregulated hypertrophy-related PI3K-AKT-MyoG signaling and remodeled the muscle ECM. CXCL5 neutralization ameliorated muscle wasting induced by CXCL5 and IL-6 co-treatment, and also prevented atrophy in cancer-activated CAF CM-treated human myotubes. CAF were the major detectable source of CXCL5 in a patient tumor highly associated with cachexia.
    CONCLUSION: CAF contribute to cachexia via cancer cell crosstalk that upregulates CXCL5 secretion. CXCL5 neutralization offers a novel therapeutic approach to maintain muscle mass in cancer patients.
    Keywords:  CXCL5; Cancer cachexia; Cancer-associated fibroblasts; Crosstalk; Skeletal muscle wasting; Tumor microenvironment
    DOI:  https://doi.org/10.1186/s12929-025-01192-0
  8. Front Immunol. 2025 ;16 1708364
    Dysregulated TFEB-autophagy-lysosome pathway links acute COVID-19 immunopathology to Long COVID sequelae.
    Magdalena Gabig-Cimińska.
      SARS-CoV-2 disrupts cellular homeostasis, including the autophagy-lysosome pathway (ALP), a critical component of innate immunity and viral clearance. By subverting autophagy, SARS-CoV-2 proteins such as ORF3a, ORF7a, and NSP6 inhibit autophagosome-lysosome (APG-L) fusion, generating "incomplete autophagy" that permits viral persistence and drives hyperinflammation. Transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, has emerged as a central player in the host response to coronavirus infection. TFEB orchestrates the expression of genes required for lysosomal function and autophagic flux while also shaping immune processes, including cytokine production, interferon-stimulated gene expression, and inflammasome clearance. This mini review synthesizes current knowledge on the TFEB-ALP axis in COVID-19 pathogenesis, highlighting its influence on acute immunopathology and its potential contribution to post-acute sequelae (Long COVID). Restoring TFEB activity and autophagic flux may counteract SARS-CoV-2 evasion strategies and restrain aberrant inflammatory responses. Harnessing the TFEB-autophagy pathway as a host-directed therapeutic strategy could help rebalance immune homeostasis, limit tissue damage during acute infection, and mitigate persistent inflammatory sequelae in Long COVID.
    Keywords:  COVID-19; Long COVID; SARS-CoV-2; autophagy-lysosome pathway (ALP); host-directed therapy; lysosomal organelle; transcription factor EB (TFEB)
    DOI:  https://doi.org/10.3389/fimmu.2025.1708364
  9. Front Cell Dev Biol. 2025 ;13 1719279
    The impact of cellular senescence on aging skeletal muscle.
    Michael Kamal, Ryan Bevington, Amanda Johnson, Gianni Parise.
      Skeletal muscle is a highly plastic tissue that relies on its resident muscle stem cell population, known as satellite cells (MuSC), for its timely repair and regeneration. During aging, there is a decline in muscle regenerative capacity that is largely attributed to the loss of MuSC content and function. These aberrations are thought to contribute to the aging-related decline in skeletal muscle mass and strength. Cellular senescence, which is characterized by a state of irreversible cell cycle arrest and the presence of a senescence-associated secretory phenotype (SASP), has emerged as a potential factor in the dysfunction of MuSCs with aging. Much effort has recently been made to examine the detrimental effects of senescence on skeletal muscle as well as identify therapeutic approaches to selectively eliminate these cells and improve the aging phenotype. Here, we discuss the current understanding of aging-related MuSC impairments and the underlying mechanisms that link cellular senescence to the decline in muscle regenerative capacity.
    Keywords:  SASP; aging; cellular senescence; regeneration; satellite cells; senolytics; skeletal muscle
    DOI:  https://doi.org/10.3389/fcell.2025.1719279
  10. Sci Rep. 2025 Dec 15.
    Identification of competing endogenous RNA networks involved in phrenic nerve stimulation preventing mechanical ventilation induced diaphragm dysfunction.
    Mingming Zhang, Fangyuan Li, Min Wang, Lei Li, Wenyan Hao, Xuejiao Duan, Dong Zhang.
      Ventilator-induced diaphragmatic dysfunction (VIDD) is characterized by diaphragmatic atrophy and contractile failure, leading to prolonged intensive care unit (ICU) stays and increases mortality. While phrenic nerve stimulation (PNS) has demonstrated efficacy in mitigating VIDD by preserving diaphragmatic activity, its underlying molecular mechanisms remain unclear. This study aimed to elucidate the role of competing endogenous RNA (ceRNA) networks in PNS-mediated protection against VIDD through integrated miRNA-Seq and RNA-Seq analyses in a rabbit model. Eleven adult male New Zealand white rabbits were divided into control (n = 4), MV (n = 3) and PNS groups (n = 4). MV and PNS groups underwent 24 h of MV, with intermittent bilateral transvenous PNS applied only to the PNS group. Differentially expressed (DE) analysis of mRNAs, miRNAs and circRNAs across pairwise group comparisons was performed via RNA-seq and miRNA-seq. Functional enrichment analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) identified key pathways. Potential miRNA targets and interacting circRNAs were computationally predicted. An integrated ceRNA network was constructed using major DE RNAs to identify PNS-associated core regulators. CeRNA network was validated by quantitative real-time polymerase chain reaction (RT-qPCR) and dual-luciferase assays. High-throughput sequencing revealed significant dysregulation of miRNAs, circRNAs and mRNAs in the diaphragm following MV which was partially reversed by PNS. Bioinformatic screening identified a ceRNA network, wherein two key miRNAs emerged: miR-500-3p (targeting RAB37, an autophagy-related gene) and miR-133b-3p (targeting L-selectin, a cell adhesion molecule regulating immune responses and fibrosis). Both miRNAs were down-regulated after MV and restored by PNS. Computational prediction also identified five circRNAs (circRNA_12437, 24673, 14127, 14942, 12463) as putative sponges for these miRNAs, although this interaction lacks experimental confirmation. Dual-luciferase assays confirmed direct binding of miR-500-3p to RAB37 and miR-133b-3p to L-selectin, functionally linking them to PNS-mediated VIDD protection. Enrichment analyses indicated that DE genes were predominantly enriched in phagosome activity and cell adhesion molecule pathways. Collectively, these findings suggest that PNS preserves diaphragmatic function by modulating ceRNA networks to suppress excessive autophagy and immune cell infiltration. This study identifies the first PNS-responsive ceRNA network in VIDD pathogenesis. Our data highlight the potential critical roles of miR-500-3p-RAB37 and miR-133b-3p-L-selectin axes in regulating autophagy and immune responses. These results provide mechanistic insights and suggest potential therapeutic targets for diaphragm dysfunction.
    Keywords:  Autophagy; Competitive endogenous RNA network; Immune cell infiltration; Phrenic nerve stimulation; Ventilator-induced diaphragmatic dysfunction
    DOI:  https://doi.org/10.1038/s41598-025-31787-4
  11. Exp Mol Med. 2025 Dec 17.
    Hv1 inhibition rescues AD pathology by restoring microglial mitochondrial function and enhancing mitochondrial transfer.
    Jiayuan Lin, Huayun Han, Kexin Wu, Xingyu Wu, Juwen Shen, Yiqing Mo, Qiansen Zhang, Huaiyu Yang, Zhihua Yu.
      Hyperphosphorylated tau aggregation and neuroinflammation are hallmark pathologies of Alzheimer's disease (AD), with microglia playing a critical role in modulating these processes through maintaining immune homeostasis and clearing pathological tau, both of which depend on mitochondrial health. However, the mechanisms underlying microglial mitochondrial dysfunction in AD remain poorly understood, limiting therapeutic development. Hydrogen voltage-gated channel 1 (Hv1), expressed in microglia within the central nervous system, regulates intracellular pH and reactive oxygen species generation. Here we observe that Hv1 is upregulated in activated microglia in AD mouse models. Remarkably, Hv1 contributes to electron transport chain abnormalities, leading to mitochondrial oxidative stress, loss of mitochondrial membrane potential, impaired ATP production and deficient mitophagy in tau pathology. These deficits impair tau clearance through phagocytosis and autophagy but can be significantly reversed by the Hv1-specific inhibitor YHV98-4. Furthermore, YHV98-4 enhances microglia-to-neuron mitochondrial transfer, promoting the delivery of functional mitochondria to rescue neuronal damage and improve cognitive function. Collectively, our study underscores the pivotal role of Hv1 in microglial mitochondrial dysfunction in AD and identifies YHV98-4 as a promising therapeutic candidate.
    DOI:  https://doi.org/10.1038/s12276-025-01593-z
  12. Dev Dyn. 2025 Dec 19.
    Fhod3 in zebrafish supports myofibril stability during growth of embryonic skeletal muscle.
    Aubrie Russell, Tracy Eng, Jeffrey D Amack, David Pruyne.
       BACKGROUND: Actin filament organization in cardiomyocytes critically depends on the formin Fhod3, but a role for Fhod3 in skeletal muscle development has not yet been described.
    RESULTS: We demonstrate here that in zebrafish mutated for one of two fhod3 paralog genes, fhod3a, skeletal muscle of the trunk appears normal through 2 days post-fertilization, but afterward exhibits myofibril damage, including gaps between myofibrils and myofibril fragmentation. Despite the progressive nature of the myofibril damage, fhod3a mutants differ from muscular dystrophy models in that damage is exacerbated by inhibition of muscle activity, and fhod3a mutants show no evidence of sarcolemma disruption. Rather, myofibril damage appears to coincide with growth of the contractile apparatus. We find neither the second fhod3 paralog, fhod3b, nor the related fhod1 contribute to embryonic skeletal muscle development, but fish individually mutated for fhod3a, fhod3b, or fhod1 are viable and appear grossly normal as adults. This may reflect redundancy in adults, as all three are expressed in many adult organs.
    CONCLUSIONS: These results indicate a fhod3-encoded formin is dispensable for initial myofibril assembly in skeletal muscle but promotes myofibril stability during muscle fiber growth. This is the first demonstration in a vertebrate that Fhod3 contributes to skeletal muscle development.
    Keywords:  Fhod3; formin; muscular dystrophy; sarcomere; skeletal muscle; zebrafish
    DOI:  https://doi.org/10.1002/dvdy.70108
  13. bioRxiv. 2025 Dec 14. pii: 2025.12.11.693336. [Epub ahead of print]
    Influenza A infection accelerates disease-associated microglia formation during physiological aging.
    Rogan A Grant, Constance E Runyan, Grace A Minogue, Joshua S Stoolman, Satoshi Watanabe, Fei Chen, Karolina J Senkow, Luisa Cusick, Maxwell J Schleck, Radmila Nafikova, Ziyan Lu, Elsie G Bunyan, Hiam Abdala-Valencia, Jacob I Sznajder, Robert Vassar, Rudolph Castellani, Changiz Geula, Tamar Gefen, Navdeep S Chandel, Alexander V Misharin, G R Scott Budinger.
      Severe pneumonia is associated with an increased risk of cognitive decline and dementia, particularly in the elderly. Changes in microglia, the most abundant immune cell population in the brain, are also associated with cognitive decline and dementia, including the emergence of a transcriptional cell state referred to as disease-associated microglia (DAM). We sought to test the hypothesis that non-neuroinvasive influenza A virus (IAV) pneumonia results in transcriptional responses in brain microglia that drive premature expansion of DAM. Using bulk and single-cell RNA-sequencing, metabolomics, and spatial transcriptomics, we profiled neuroimmune populations in young, middle-aged, and old male mice during IAV infection and recovery. We observed an increased abundance of DAM, interferon-responsive microglia (IRM), CD4+ T cells, and CD8+ T cells in white matter regions beginning in middle age and persisting in old animals, irrespective of IAV infection. DAM exhibited a metabolic shift toward aerobic glycolysis with disrupted TCA cycling, citrulline depletion, and an elevated itaconate/α-ketoglutarate ratio. Spatial transcriptomic profiling of the human middle frontal gyrus (MFG) in normal agers, SuperAgers, and patients with dementia revealed an analogous accumulation of DAM and CD8+ T cells in white matter. IAV pneumonia induced a transient immunosenescent-like response in microglia, marked by glucocorticoid-responsive gene expression and Ccnd3 upregulation. In response to IAV pneumonia, DAM expanded in middle-aged mice, whereas old mice were elevated at baseline and were largely unaffected by IAV infection. The age-related expansion of DAM was unaffected by pharmacological depletion and repopulation of microglia with a CSF1R antagonist or genetic gain or loss of function of the phagocytic receptor MERTK, suggesting the DAM phenotype is driven by the CNS microenvironment, rather than cell-intrinsic mechanisms. Our findings suggest that IAV pneumonia induces an acute immunosenescence response in microglia and accelerates the age-dependent expansion of DAM in white matter.
    DOI:  https://doi.org/10.64898/2025.12.11.693336
  14. STAR Protoc. 2025 Dec 12. pii: S2666-1667(25)00678-1. [Epub ahead of print]7(1): 104272
    Protocol for applying expansion microscopy to the study of mammalian neuromuscular junctions.
    Abdullah Ramadan, Thomas M D Sheard, Abrar Alhindi, Philippa A Rust, Ross A Jones, Thomas H Gillingwater, Izzy Jayasinghe.
      Expansion microscopy (ExM) is a tissue-swelling technique that enables super-resolution imaging through a specialized preparation process that physically expands stained biomolecules within a fixed sample. Here, we detail a protocol to apply the 4× ExM technique to neuromuscular junctions (NMJs) from both human and mouse muscle preparations. We describe steps for muscle fixation, microdissection, staining, gelation, and digestion. We then detail procedures for expansion, mounting, imaging, analysis, and quantification. This protocol can be used to reveal nanoscale anatomical NMJ features. For complete details on the use and execution of this protocol, please refer to Ramadan et al.1.
    Keywords:  microscopy; model organisms; neuroscience
    DOI:  https://doi.org/10.1016/j.xpro.2025.104272
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