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Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms mediating these effects are poorly understood. Here we identify the glucocorticoid receptor (GR) target gene, serum- and glucocorticoid-inducible kinase 1 (SGK1), as one such mechanism. Using a human hippocampal progenitor cell line, we found that a small molecule inhibitor for SGK1, GSK650394, counteracted the cortisol-induced reduction in neurogenesis. Moreover, gene expression and pathway analysis showed that inhibition of the neurogenic Hedgehog pathway by cortisol was SGK1-dependent. SGK1 also potentiated and maintained GR activation in the presence of cortisol, and even after cortisol withdrawal, by increasing GR phosphorylation and GR nuclear translocation. Experiments combining the inhibitor for SGK1, GSK650394, with the GR antagonist, RU486, demonstrated that SGK1 was involved in the cortisol-induced reduction in progenitor proliferation both downstream of GR, by regulating relevant target genes, and upstream of GR, by increasing GR function. Corroborating the relevance of these findings in clinical and rodent settings, we also observed a significant increase of SGK1 mRNA in peripheral blood of drug-free depressed patients, as well as in the hippocampus of rats subjected to either unpredictable chronic mild stress or prenatal stress. Our findings identify SGK1 as a mediator for the effects of cortisol on neurogenesis and GR function, with particular relevance to stress and depression.

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin α in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

Glia have been implicated in Alzheimer’s disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2 -dependent DAM and identified a previously undiscovered Serpina3n + C4b + reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2- R47H and TREM2 -R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences. Single-nucleus RNA sequencing in a mouse model of Aβ accumulation and postmortem brain tissue from people with Alzheimer’s disease reveals substantial species-specific differences in transcriptional signatures, but both point to the contribution of glia and the importance of TREM2.

The nucleus contains the cell's genetic material, which directs cellular activity via gene regulation. The physical barrier of the nuclear envelope needs to be permeable to a variety of macromolecules and signals. The most prominent gateways for the transport of macromolecules are the nuclear pore complexes (NPCs). The NPC is the largest multiprotein complex in the cell, and is composed of multiple copies of ∼30 different proteins called nucleoporins. Although much progress has been made in dissecting the NPC structure in vertebrates and yeast, the molecular architecture and physiological function of nucleoporins in plants remain poorly understood. In this review, we summarize the current knowledge regarding the plant NPC proteome and address structural and functional aspects of plant nucleoporins, which support the fundamental cellular machinery.

Upon oxidative stress and aging, Nrf2 (NFE2-related factor2) triggers antioxidant defense genes to defends against homeostatic failure. Using human(h) or rat(r) lens epithelial cells (LECs) and aging human lenses, we showed that a progressive increase in oxidative load during aging was linked to a decline in Prdx6 expression. DNA binding experiments using gel-shift and ChIP assays demonstrated a progressive reduction in Nrf2/ARE binding (−357/−349) of Prdx6 promoter. The promoter (−918) with ARE showed a marked reduction in young vs aged hLECs, which was directly correlated to decreased Nrf2/ARE binding. A Nrf2 activator, Sulforaphane (SFN), augmented Prdx6, catalase and GST π expression in dose-dependent fashion, and halted Nrf2 dysregulation of these antioxidants. SFN reinforced Nrf2/DNA binding and increased promoter activities by enhancing expression and facilitating Nrf2 translocalization in nucleus. Conversely, promoter mutated at ARE site did not respond to SFN, validating the SFN-mediated restoration of Nrf2/ARE signaling. Furthermore, SFN rescued cells from UVB-induced toxicity in dose-dependent fashion, which was consistent with SFN’s dose-dependent activation of Nrf2/ARE interaction. Importantly, knockdown of Prdx6 revealed that Prdx6 expression was prerequisite for SFN-mediated cytoprotection. Collectively, our results suggest that loss of Prdx6 caused by dysregulation of ARE/Nrf2 can be attenuated through a SFN, to combat diseases associated with aging.

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), known as PKR, is a key antiviral kinase activated by double-stranded RNA (dsRNA) typically produced during viral replication. Upon activation, PKR phosphorylates eIF2α, leading to the inhibition of translation and viral replication. However, many viruses have evolved mechanisms to counteract PKR activity. In Cardioviruses, the leader protein (L), a short peptide cleaved from the N-terminus of the viral polyprotein, not only inhibits PKR but also blocks interferon production and disrupts nucleocytoplasmic trafficking (NCT). L disrupts NCT by recruiting host RSK kinases to the nuclear pore complex (NPC), where RSK phosphorylates FG-nucleoporins, thereby impairing NCT. L mutations that affect NCT disruption also impact PKR inhibition, suggesting a mechanistic link between NCT and PKR activity. Recombinant TMEV and EMCV viruses designed to disrupt NCT through different mechanisms exhibited some extent of PKR inhibition, supporting the link between NCT disruption and PKR inhibition. Immunostaining and live-cell imaging revealed that, during mitosis and after L-induced NCT disruption, a fraction of PKR maps to the nucleoli, where PKR remains inactive despite its recruitment by dsRNA-like structures. This suggests that nucleolar sequestration contributes to PKR inhibition. Additionally, L-mediated NCT disruption leads to the release of nuclear RNA-binding proteins (nRBPs) into the cytosol, which may bind or modify viral dsRNA, further preventing PKR activation. Collectively, these results highlight nucleocytoplasmic trafficking as a critical regulatory mechanism governing PKR activation. Thus, beyond the specific action of cardiovirus L protein, our study reveals that interference with host nucleocytoplasmic transport can significantly impact the subcellular localization and functional regulation of immune effectors such as PKR.

Liquid–liquid phase separation (LLPS) has emerged as a central paradigm for understanding how membraneless organelles compartmentalize diverse cellular activities in eukaryotes 1 – 3 . Here we identify a superfamily of plant guanylate-binding protein (GBP)-like GTPases (GBPLs) that assemble LLPS-driven condensates within the nucleus to protect against infection and autoimmunity. In Arabidopsis thaliana , two members of this family—GBPL1 and GBPL3—undergo phase-transition behaviour to control transcriptional responses as part of an allosteric switch that is triggered by exposure to biotic stress. GBPL1, a pseudo-GTPase, sequesters catalytically active GBPL3 under basal conditions but is displaced by GBPL3 LLPS when it enters the nucleus following immune cues to drive the formation of unique membraneless organelles termed GBPL defence-activated condensates (GDACs) that we visualized by in situ cryo-electron tomography. Within these mesoscale GDAC structures, native GBPL3 directly bound defence-gene promoters and recruited specific transcriptional coactivators of the Mediator complex and RNA polymerase II machinery to massively reprogram host gene expression for disease resistance. Together, our study identifies a GBPL circuit that reinforces the biological importance of phase-separated condensates, in this case, as indispensable players in plant defence. A family of plant guanylate-binding protein-like GTPases controls phase separation and assembly of condensates, thereby forming a circuit that regulates transcriptional responses to biotic stress.

Down-regulation and mutations of the nuclear-architecture proteins lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule \"Remodelin\" that improved nuclear architecture, chromatin organization, and fitness of both human lamin A/C–depleted cells and HGPS-derived patient cells and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular, and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture and suggest alternative strategies for treating laminopathies and aging.

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases. A set of ready-to-use tools for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq facilitates the implementation of single-cell technologies in clinical settings and the construction of single-cell tumor atlases.

Loss of function of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene is associated with many human cancers. In the cytoplasm, PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K) signaling pathway. PTEN also accumulates in the nucleus, where its function remains poorly understood. We demonstrate that SUMOylation (SUMO, small ubiquitin-like modifier) of PTEN controls its nuclear localization. In cells exposed to genotoxic stress, SUMO-PTEN was rapidly excluded from the nucleus dependent on the protein kinase ataxia telangiectasia mutated (ATM). Cells lacking nuclear PTEN were hypersensitive to DNA damage, whereas PTEN-deficient cells were susceptible to killing by a combination of genotoxic stress and a small-molecule PI3K inhibitor both in vitro and in vivo. Our findings may have implications for individualized therapy for patients with PTEN-deficient tumors.