Qizhi Gong

Olfactory dysfunction is a hallmark of COVID-19, yet the mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes widespread sensory impairment remain incompletely understood. We compared the olfactory epithelial tropism of multiple SARS-CoV-2 variants in a mouse model and observed that Alpha and Beta variants exhibited the greatest infectivity for the olfactory epithelium (OE), whereas Omicron rarely infected this tissue despite comparable pulmonary viral titers. We further defined the effects of SARS-CoV-2 on olfactory sensory neurons (OSNs) using immunohistochemistry, single-cell RNA sequencing, spatial gene expression characterization, and functional odor stimulation. The localized infection of sustentacular cells triggered a tissue-wide interferon-stimulated antiviral response that extended beyond infected regions for Alpha and Beta but was largely absent following Omicron infection. Mature OSNs transiently adopted an interferon-responsive state before exhibiting persistent downregulation of odorant signal transduction, mitochondrial, and activity-dependent gene pathways. Consistent with these transcriptional changes, odor stimulation failed to elicit normal activity-dependent gene expression during SARS-CoV-2 infection, indicating impaired neuronal function without widespread neuronal loss. Progressive accumulation of macrophages further indicated sustained inflammatory remodeling of the OE. Together, these findings demonstrate that SARS-CoV-2 infection initiates tissue-wide antiviral signaling in the OE that persistently disrupts OSN function, providing a mechanistic framework for COVID-19-associated anosmia.

The global impact of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), persists in part due to the emergence of new variants. Understanding variant-specific infection dynamics and pathogenesis in murine models is crucial for identifying phenotypic changes and guiding the development of countermeasures. To address the limitations of earlier studies that investigated only a few variants or used small sample sizes, we evaluated clinical disease, infection kinetics, viral titers, cellular localization, and histopathologic changes in the lungs and brains of transgenic B6.Cg-Tg(K18-ACE2)2Prlmn/J (“K18”) and corresponding genetic control (C57BL/6J) mice expressing human angiotensin-converting enzyme 2 (hACE2). Six SARS-CoV-2 variants were assessed: B.1 (WA1-like), alpha, beta, delta, omicron, and omicron XBB.1.5, using cohorts of ≥18 mice. Following intranasal inoculation with B.1, alpha, beta, or delta variants, K18 mice experienced rapid weight loss and reached euthanasia criteria by 5–6 days post-inoculation (dpi). In contrast, K18 mice inoculated with both omicron variants recovered to their starting weight within 4–6 dpi. Infectious SARS-CoV-2 was detected in the oropharynx at 1 and2 dpi, in the lungs at 2, 4, and 6 dpi, and in the brain at 4 and 6 dpi for all variants except omicron. SARS-CoV-2 nucleoprotein was detected, and interstitial pneumonia of varying severity was observed in K18 mice infected with all variants. Brain lesions were identified in mice infected with the B.1, beta, and delta variants 6 dpi. As K18 mice express hACE2 in the brain—a feature not present in humans—we also compared infection dynamics of three variants to those of a mouse-adapted WA1 strain in C57BL/6J mice lacking the human ACE2 gene. C57BL/6J mice did not experience lethal disease, exhibited milder pneumonia, and had no evidence of neuroinvasion despite similar infection kinetics to K18 mice. These findings demonstrate contrasting phenotypes across the two models and reduced tropism and pathology of omicron compared to earlier variants in both models. This comprehensive analysis of SARS-CoV-2 variants in two mouse models provides valuable insights for model and variant selection for future studies.

Potential systemic factors contributing to aging-associated breast cancer (BC) remain elusive. Here, we reveal that the polyploid giant cells (PGCs) that contain more than two sets of genomes prevailing in aging and cancerous tissues constitute 5-10% of healthy female bone marrow mesenchymal stromal cells (fBMSCs). The PGCs can repair DNA damage and stimulate neighboring cells for clonal expansion. However, dying PGCs in advanced-senescent fBMSCs can form “spikings” which are then separated into membraned mtDNA-containing vesicles (Senescent PGC-Spiking Bodies; SPSBs). SPSB-phagocytosed macrophages accelerate aging with diminished clearance on BC cells and protumor M2 polarization. SPSB-carried mitochondrial OXPHOS components are enriched in BC of elder patients and associated with poor prognosis. SPSB-incorporated breast epithelial cells develop aggressive characteristics and PGCs resembling the polyploid giant cancer cells (PGCCs) in clonogenic BC cells and cancer tissues. These findings highlight an aging BMSC-induced BC risk mediated by SPSB-induced macrophage dysfunction and epithelial cell precancerous transition. SIGNIFICANCE Mechanisms underlying aging-associated cancer risk remain unelucidated. This work demonstrates that polyploid giant cells (PGCs) in bone marrow mesenchymal stromal cells (BMSCs) from healthy female bone marrow donors can boost neighboring cell proliferation for clonal expansion. However, the dying-senescent PGCs in the advanced-senescent fBMSCs can form “spikings” which are separated into mitochondrial DNA (mtDNA)-containing spiking bodies (senescent PGC-spiking bodies; SPSBs). The SPSBs promote macrophage aging and breast epithelial cell protumorigenic transition and form polyploid giant cancer cells. These results demonstrate a new form of ghost message from dying-senescent BMSCs, that may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk. Xie et al demonstrate that the polyploid giant cells (PGCs) in the juvenile phase expansion of female bone marrow mesenchymal stromal cells (fBMSCs) can boost neighboring cell proliferation for clonal expansion. However, when fBMSCs enter to the advanced-senescent phase, the dying-senescent PGCs form “spikings” which are then separated into membraned vesicles termed Senescent PGC spiking bodies, SPSBs). The SPSBs carrying fragmented mitochondrial elements and OXPHOS proteins can be phagocytosed by macrophage and breast epithelial cells leading to macrophage aging and breast epithelial protumorigenic transition. The SPSBs are demonstrated to be a new form of post-cell death vesicle from aging BMSCs and may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk.

The olfactory neuroepithelium (OE) is one of the few neuronal tissues where environmental pathogens can gain direct access. Despite this vulnerable arrangement, little is known about the protective mechanisms in the OE to prevent viral infection and its antiviral responses. We systematically investigated acute responses in the olfactory mucosa upon exposure to vesicular stomatitis virus (VSV) via RNA-seq. VSVs were nasally inoculated into C57BL/6 mice. Olfactory mucosae were dissected for gene expression analysis at different time points after viral inoculation. Interferon functions were determined by comparing the viral load in interferon receptor knockout (Ifnar1^−/− and Ifnlr1^−/−) with wildtype OE. Antiviral responses were observed as early as 24 h after viral exposure in the olfactory mucosa. The rapidly upregulated transcripts observed included specific type I as well as type III interferons (Ifn) and interferon-stimulated genes. Genetic analyses demonstrated that both type I and type III IFN signaling are required for the suppression of viral replication in the olfactory mucosa. Exogenous IFN application effectively blocks viral replication in the OE. These findings reveal that the OE possesses an innate ability to suppress viral infection. Type I and type III IFNs have prominent roles in OE antiviral functions.

BackgroundThe olfactory epithelium (OE) is one of the few sites where environmental pathogens can gain direct access to the brain. Despite this vulnerable arrangement, little is known about the protective mechanisms in the OE to prevent viral infection and subsequent entry into the brain.MethodsWe systematically investigated acute responses in the olfactory mucosa (OM) upon exposure to vesicular stomatitis virus (VSV) by RNA-sEq. VSVs were nasally inoculated into C57BL/6 mice. OM were dissected for gene expression analysis at different time points after viral inoculation. Interferon functions were determined by comparing viral gene expression in interferon (Ifn) receptor knockout (Ifnar1^−/− and Ifnlr1^−/−) with wildtype OM.ResultsAntiviral responses were observed as early as 24hrs post viral exposure in the OM. Amongst the rapidly upregulated transcripts observed were specific type I as well as type III Ifns and interferon stimulated genes. Genetic analyses demonstrated that both type I and type III IFN signaling are required for the suppression of viral replication in the OM. Nasal IFNb1 or IFNl2 administration effectively reduces viral load in the OM.Conclusions:OE possesses innate ability to respond and suppress viral infection. Type I and type III IFNs actively participate in the OE’s antiviral functions. Nasal IFN application effectively blocks viral replication in the OE suggesting therapeutic potential against viral insult.

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation.

There is growing recognition regarding the role of intracellular amyloid beta (Aβ) in the Alzheimer’s disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aβ likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aβ and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aβ accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aβ. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aβ. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aβ-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aβ targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aβ, its promotion of ROS, and Aβ metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aβ oligomer formation.

Methyl-CpG binding protein 2 (MeCP2) is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2-binding data, we show that DNA sequence features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported genome-wide association with methylation is in part due to MeCP2’s affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localizes with nucleosomes. Finally, MeCP2 binding downstream of promoters correlates with increased expression in Mecp2-deficient neurons.

During postnatal development, neuronal activity controls the remodeling of initially imprecise neuronal connections through the regulation of gene expression. MeCP2 binds to methylated DNA and modulates gene expression during neuronal development and MECP2 mutation causes the autistic disorder Rett syndrome. To investigate a role for MeCP2 in neuronal circuit refinement and to identify activity-dependent MeCP2 transcription regulations, we leveraged the precise organization and accessibility of olfactory sensory axons to manipulation of neuronal activity through odorant exposure in vivo. We demonstrate that olfactory sensory axons failed to develop complete convergence when Mecp2 is deficient in olfactory sensory neurons (OSNs) in an otherwise wild-type animal. Furthermore, we demonstrate that expression of selected adhesion genes was elevated in Mecp2-deficient glomeruli, while acute odor stimulation in control mice resulted in significantly reduced MeCP2 binding to these gene loci, correlating with increased expression. Thus, MeCP2 is required for both circuitry refinement and activity-dependent transcriptional responses in OSNs.