Cell Reports

The uterine endometrium is capable of scarless regeneration under coordinated estrogen and progesterone signaling across the menstrual cycle. Obesity suppresses progesterone production, leading to chronic estrogen exposure and increased endometrial hyperplasia (EH) risk. To define how obesity alters endometrial cell states, endometrial tissues from control and EH-predisposed mice fed either a control diet or a high-fat diet (HFD) were analyzed by single-cell RNA sequencing and tissue phenotyping. HFD reprogrammed endometrial stroma towards an inflammatory, pro-fibrotic state, reducing progesterone receptor-network-associated Aldh1a2+ fibroblasts and expanding estrogen receptor-network-associated Gsn fibroblasts. HFD further impaired macrophage recruitment and promoted hyperplastic epithelial signatures, consistent with increased disease severity in an EH mouse model. Stromal deletion of Estrogen Receptor established stromal estrogen signaling as a driver of HFD-induced extracellular matrix (ECM) accumulation. Collectively, these findings identify HFD-driven fibroblast reprogramming as a central mechanism linking estrogen dominance to stromal fibrosis, defective immune clearance, and heightened EH susceptibility. We propose that, in response to progesterone, fibroblast-mediated ECM remodeling is vital to normal endometrial homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/713224v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@125d0f7org.highwire.dtl.DTLVardef@1ba1714org.highwire.dtl.DTLVardef@41314borg.highwire.dtl.DTLVardef@b4585_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO HFD-induced estrogen dominance disrupts endometrial fibroblast homeostasis to predispose the endometrium to diseaseThis study demonstrates that HFD drives estrogen-dependent reprogramming of stromal fibroblasts, characterized by inflammation, stromal ECM accumulation and fibrosis, and a post-ovulatory shift from PGR-network-associated Aldh1a2+ Fibroblasts toward increasing ER-network-associated Gsn+ Fibroblasts. These fibroblast changes are accompanied by a reduction in endometrial macrophages and a transcriptomic shift of HFD epithelium toward hyperplastic epithelium seen in a mouse model of EH. Figure made with BioRender. C_FIG

Pancreatic beta cells have the unique function of synthesizing and secreting high amounts of the inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA). The mechanism of GABA secretion, whether vesicular or channel-mediated, is debated. Our study reveals surprising temporal complexity in the pattern of islet GABA secretion. We used insulin secretion modulators to demonstrate that GABA release is not directly correlated with insulin secretion. VGAT reporter mice also showed that beta cells do not express the requisite vesicular GABA transporter (VGAT) for vesicular GABA release. Instead, GABA is secreted from the cytosol in pulses by the LRRC8A/D isoform of the volume regulatory anion channel (VRAC). We further demonstrate the dynamic coordination of GABA release with calcium influx in beta cells and dependence on beta cell depolarization. These results suggest a model where GABA is released during the peaks of beta cell calcium oscillations to provide feedback which strengthens and reinforces the oscillation waveform.

Following their engagement towards differentiation, tissue stem cells often transit through a precursor state that is difficult to define because of its transient nature; similarly, the precise role of lineage precursors in implementation of tissue architecture and function is unknown. In the present work, we used two mouse models of deficient feedback regulation to characterize precursors of the pituitary corticotrope lineage that regulates the stress response. Both the POMC knockout and adrenalectomized mouse models develop glucocorticoid deficiency and compensatory accumulation of corticotrope precursors that have so far eluded characterization. We found that pre-corticotrope differentiation depends on the lineage-specific factor Tpit and is repressed by glucocorticoids. We identified brain-derived neurotrophic factor (BDNF) as the signal that engages pituitary stem cells towards differentiation in these models as well as in normal pituitary development. A glucocorticoid-sensitive BDNF autocrine loop active in pre-corticotropes turns these cells into signaling hubs for maintenance of pituitary-adrenal homeostasis. HighlightsO_LIPituitary lineage precursors expand in conditions of deficient feedback regulation C_LIO_LIBDNF mobilizes pituitary stem cells during establishment of tissue size and architecture C_LIO_LICorticotrope precursors are a signaling hub for tissue homeostasis C_LI

Obesity is a major risk factor for severe influenza A virus (IAV) infection, however, the innate immune mechanisms underlying this increased vulnerability remain unclear. Here, we identify significant defects in natural killer (NK) cell antiviral responses in mice with diet-induced obesity. In lean mice, NK cells are critical for protection as NK cell depletion during IAV infection led to increased lung viral load, morbidity, and mortality. In contrast, in obese mice NK cell depletion had minimal impact on viral replication or survival. Notably, IAV infection in obese mice recapitulated the phenotype observed in NK cell-depleted lean mice, indicating that obesity is associated with preexisting NK cell dysfunction. Following IAV infection, obese NK cells in the lung were functionally impaired with diminished activation (CD69+), cytokine production (IFN-{gamma}), and cytolytic activity (Granzyme B) accompanied by defects in the mTOR signaling pathway and reduced glycolytic and oxidative metabolism. Bulk and spatial lipidomics revealed obesity and infection-driven remodeling of the lung lipidome. We observed increased triglyceride accumulation, abundance of long-chain free fatty acids, and a shift toward monounsaturated phospholipid species, reshaping the lung microenvironment that coincides with NK cell metabolic dysfunction. Consistent with this lipid-rich environment, obese NK cells sustained high expression of the lipid transporter CD36 post-IAV infection and accumulation of intracellular lipids (LipidTOX+), consistent with mechanisms known to suppress NK cell function. Notably, short-term weight loss (4 weeks) was sufficient to restore NK cell metabolism, antiviral function, and survival following IAV infection. These findings uncover a lipid-associated mechanism regulating NK cell function and show it plays a critical role in defense against infection and that it is dysfunctional in obesity. We suggest that targeting immunometabolism could lead to new antiviral therapies and potentially improve vaccine efficacy, especially in high-risk populations such as obesity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/710186v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@313263org.highwire.dtl.DTLVardef@1e31111org.highwire.dtl.DTLVardef@758ba6org.highwire.dtl.DTLVardef@12372d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ability for an organism to encode fear memories is necessary for survival. Once a threat is no longer present, organisms must suppress, or extinguish, this fear memory in favor of other adaptive behaviors. In rodents, the infralimbic cortex (IL) is a locus critical for the extinction of cued fear memory. While this role has been known for decades, the circuit mechanisms underlying its recruitment are largely unknown. By using a combination of immunohistochemistry, neural tagging, in vivo calcium imaging and optogenetics, and optogenetics-assisted brain slice electrophysiology, we revealed that the dynamic activity and plasticity of IL inhibitory interneurons is critical for encoding fear extinction. Specifically, after fear conditioning, IL parvalbumin interneurons exhibit increased activity and plasticity, driving enhanced freezing. After fear extinction, however, IL somatostatin interneurons exhibit extinction cue-associated activity and plasticity, and their activity facilitates extinction memory encoding through inhibition of parvalbumin interneuron activity and disinhibition of IL principal neurons. Further, glutamatergic projections from the basolateral amygdala undergo experience- and cell type-specific plasticity that is required to drive the dynamic recruitment of IL parvalbumin and somatostatin interneurons after fear conditioning and extinction, respectively. Overall, these results reveal the mechanisms of cued fear extinction encoding and highlight critical roles for local IL microcircuit computations in these roles.

Cancer testis antigens are widely expressed in human malignancies. Melanoma-Associated Antigens (MAGE) A3 and A6 have been proposed to modulate protein turnover and metabolism in cancer cells. However, the substrate specificity of MAGE-A3/6 and the impact on cancer cell behavior remain poorly understood. Although previous research has identified binding partners, a molecularly validated target for MAGE-A3/6-mediated proteasomal degradation has not been described. In this study, we redefine the substrate specificity of MAGE-A3/6 and present a mechanistic framework for substrate binding, polyubiquitination, and subsequent degradation. We identify BPTF-Associated Protein of 18kDa (BAP18) as a bona fide novel substrate of MAGE-A3/6 and demonstrate its direct regulation via a molecularly defined substrate-degron-E3-adaptor interaction. The degradation of BAP18 by MAGE-A3/6 underlies phenotypic alterations in cancer cells, such as enhanced migratory capacity. This previously unrecognized molecular link is observed in both cancer cell lines and human cancer tissues, supporting a role as a fundamental oncogenic process. The discovery of a molecularly defined interaction between MAGE-A3/6 and their substrate enables systematic investigation into oncogenic protein degradation in human cancers and may inform future therapeutic strategies that leverage the molecular function of aberrantly reexpressed germline proteins in cancer.

Androgen excess drives metabolic and reproductive complications in polycystic ovary syndrome (PCOS), affecting 10-15% of women globally. Aldo-keto reductase 1C3 (AKR1C3) converts inactive precursors from both the classic and the recently identified 11-oxygenated androgen pathways, generating testosterone and 11-ketotestosterone, respectively, which exert comparable androgen receptor activation. Both circulate in similar concentrations in premenopausal women while 11-ketotestosterone is predominant after menopause and in PCOS. Here, we show that adipocytes are a major site of AKR1C3 and androgen receptor expression, with increased expression in women and individuals with obesity. Using human female adipose tissue explants, we find a much higher activation of 11-oxygenated over classic androgens, observing a decrease in 11-oxygenated but not classic androgen activation by AKR1C3 inhibition. Correspondingly, we demonstrate that AKR1C3 inhibitor treatment in premenopausal women selectively disrupts the activation of 11-oxygenated androgens. Pharmacological targeting of AKR1C3 provides a novel strategy to alleviate systemic and intra-adipose 11-oxygenated androgen excess. One Sentence SummaryInhibition of the androgen-activating enzyme AKR1C3 results in a major decrease in 11-oxygenated but not classic androgens in women.

Obesity alters systemic metabolism and immune function, yet how obesity and tumor progression regulate extracellular vesicle (EV) composition and function within the tumor microenvironment remains unclear. Using a preclinical model of diet-induced obesity (DIO) and triple-negative breast cancer (TNBC), we investigated how obesity and tumor stage shape the proteomic composition of EVs from visceral adipose tissue (VAT-EVs) and mammary tumors (tumor-EVs), and how these EVs regulate immune and tumor cell metabolism. Orthotopically transplanted metM-Wntlung tumors were classified as early ([~]0.5 cm3) or late ([~]1.0 cm3), and EV proteomes were analyzed by mass spectrometry. At early stages, tumor-EVs from DIO mice, compared with control lean mice, were depleted in immune-related proteins, whereas VAT-EVs were enriched in mitochondrial and fatty acid oxidation proteins. In contrast, at later stages, tumor-EVs from DIO mice were enriched in lipid metabolism and oxidative stress-associated proteins, while VAT-EVs exhibited loss of mitochondrial proteins consistent with metabolic dysfunction. Functionally, tumor-EVs and VAT-EVs differentially regulated CD8 T cell mitochondrial activity and cytokine production and induced distinct, stage-dependent metabolic reprogramming in non-aggressive epithelial-like (E-Wnt) versus mesenchymal-like (M-Wnt) tumor cells. These findings suggest that obesity and tumor progression dynamically reshapes EV cargo, enabling EV-mediated metabolic reprogramming that may contribute to immune suppression and TNBC progression.

TNFR superfamily members such as 4-1BB sustain T cell responses to control virus infections or tumors. However, the precise role of 4-1BB during an acute infection remains incompletely understood. Here we used mixed bone marrow chimeras and transcriptome analysis to show that intrinsic 4-1BB signaling in lung T cells during influenza A virus (IAV) infection induces the transcriptional coregulator PR domain containing 16 (Prdm16), known for its role in regulating mitochondrial biology in other cell types. T cell-specific deletion of Prdm16 reduced the number of Ag-specific CD8 T cells, with a larger effect on T cells in the lung parenchyma compared to the vasculature or lymphoid tissues. Conversely, Prdm16 overexpression in T cells increased effector and memory CD8 T cell accumulation during IAV infection. Single nuclei transcriptomics suggested that Prdm16 allows the accumulation of T cells with high protein translation and mitochondrial activity. Prdm16 increased genes associated with oxidative phosphorylation and mitophagy. Consistently, Prdm16 overexpressing cells had more compact mitochondrial cristae, which has been associated with more efficient electron transport. Prdm16 also repressed some genes, including Herpes virus entry mediator, which can inhibit T cell responses through B and T lymphocyte attenuator. These findings reveal a 4-1BB-Prdm16 axis that is induced in T cells during viral infection to support T cell accumulation and memory formation.

Lung cancer is the most common cause of cancer-related death in the U.S. and globally. Cigarette smoking remains the leading risk factor for lung cancer, in part by inducing loss-of-function mutations in tumor suppressor genes, including TP53. While most cancers share a set of common "hotspot" mutations in p53, lung cancer exhibits an additional, distinct cluster of hotspot mutations. This cluster is typified by the missense mutations TP53:p.V157F and TP53:p.R158L. While canonical hotspot mutations cause broad misfolding of p53 or eliminate specific DNA contact residues, mechanistic studies of the lung cancer mutants reported here demonstrate that they retain the ability to bind the same genomic sites as wild-type p53. Despite actively binding to traditional p53 target genes, the lung cancer mutants are defective in activating transcription. To our knowledge, this represents the first demonstration of functional inactivation of the p53 tumor suppressor at a point after DNA binding, but prior to target gene activation. Relevant to the sequential inactivation of each p53 allele during cancer progression, the lung cancer mutants block the activity of a wild-type p53 allele when co-expressed in a dominant negative manner. Identification of this loss-of-function mechanism has key implications for therapeutic strategies aimed at restoring p53 function in lung cancer.

Well-differentiated and dedifferentiated liposarcoma (WDLPS and DDLPS) exhibit markedly different clinical behaviors, with DDLPS showing greater aggressiveness, higher recurrence and metastasis rates, and worse outcomes. Using single-nucleus multiome sequencing, epigenomic profiling, and spatial transcriptomics, we characterized cellular and epigenetic heterogeneity between these subtypes at single-cell and spatial resolution. We found distinct phenotypic states reflecting altered lineage differentiation and plasticity: DDLPS is dominated by early-differentiated progenitor-like cells, sclerotic WDLPS displays broader mesenchymal lineage plasticity, and adipocytic WDLPS contains abundant committed adipocytes. The DDLPS immune microenvironment was dominated by immunosuppressive macrophages, whereas WDLPS harbored more T cells and inflammatory macrophages. Notably, sclerotic WDLPS displayed intermediate cellular and molecular features, suggesting it may represent a distinct WDLPS subtype. Importantly, we identified novel gene regulatory circuits underlying each state, including FABP4/PPARG programs in adipocytic WDLPS, GLI2/TCF7L2/RBPJ/KLF7 programs in sclerotic WDLPS, and KLF7/FOSL2/SP3/GLI2/RBPJ programs in DDLPS. H3K27ac-marked enhancers were enriched near adipocytic marker genes in WDLPS and mesenchymal markers in DDLPS. Together, these findings reveal the cellular heterogeneity of tumor and immune compartments across liposarcoma subtypes and identify regulatory programs driving their differentiation states. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/713651v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@1c84ee1org.highwire.dtl.DTLVardef@1b2ad42org.highwire.dtl.DTLVardef@18ce5a6org.highwire.dtl.DTLVardef@138f615_HPS_FORMAT_FIGEXP M_FIG C_FIG

Local RNA regulation is essential for synaptic plasticity, yet the full repertoire of RNA species and associated isoforms within specific synaptic compartments in vivo has yet to be determined. Existing RNA profiling approaches lack the spatial and temporal precision needed to resolve RNA repertoires restricted to cell-type specific synaptic compartments. To overcome this challenge, we developed PSD-95-Halo-seq, a light-induced proximity labelling technique that, when combined with long-read sequencing, selectively captures all post-synaptic full length RNA species in the excitatory post-synaptic compartment. Here, we applied this approach to investigate sex differences in RNA expression within excitatory synapses following exposure to an associative fear learning task in C57/Bl6 mice. We found dramatic sex differences in pseudogene and protein coding RNA expression, which are most abundant in females, with males exhibiting multiple noncoding RNA classes, including lncRNA, snoRNA, and rRNA. Females generally showed more 3' UTR expression, and there was widespread differential exon usage following fear conditioning, including 179 isoforms in males, 69 in females, with no significant gene-level differential expression, indicating that behavioural state modifies sex-specific isoform usage rather than overall transcript abundance. Our discovery that synaptic RNA composition is dynamic, sexually dimorphic, and profoundly shaped by experience, offers new insight into previously inaccessible mechanisms underlying sex differences in fear-related learning and memory. PSD-95-Halo-seq is therefore a powerful method for the precise spatiotemporal identification of compartment-and cell-type-specific RNA. One sentence take-awaySynapse-targeted proximity labelling and Long-Read sequencing demonstrate that excitatory post-synapses encode experience through sex-specific, isoform-level RNA remodeling.

High-grade serous carcinoma (HGSC) is the most common and aggressive form of ovarian cancer. Advanced HGSCs exhibit pronounced cellular heterogeneity, including a subset of cancer-propagating cells (CPCs, also known as cancer stem cells) that are highly tumorigenic and display stem cell-associated properties such as self-renewal and chemoresistance. In contrast, a substantial fraction of HGSC cells is non-tumorigenic. The role of these non-cancer-propagating cells (non-CPCs) and their relationship to CPCs remain poorly understood. Here, we demonstrate that neoplastic cells expressing the intermediate filament protein keratin 5 (KRT5) represent bona fide CPCs. KRT5 cells form cancer organoids over successive passages, are tumorigenic in serial dilution xenograft assays, and are resistant to the antineoplastic agents, doxorubicin and cisplatin. Single-cell lineage-tracing experiments show that KRT5 CPCs give rise to KRT5- cells. KRT5 and KRT5- populations exhibit distinct gene expression profiles, with KRT5- cells characterized by expression of SPP1, which encodes the secreted factor osteopontin (OPN). Treatment with OPN enhances HGSC organoid growth and chemoresistance, whereas SPP1 knockdown reverses these effects. Together, these findings support a model in which HGSC contains two hierarchically related cell populations: KRT5, OPN-responsive CPCs and KRT5-, non-tumorigenic cells that form a niche producing OPN. Targeting pathways that sustain both stem-like tumor cells and their supportive niche may enable reduced dosing of highly toxic chemotherapeutic agents while enhancing therapeutic efficacy in HGSC.

HIV and cocaine are known to disrupt neuronal signaling and contribute to neurocognitive dysfunction, yet the underlying molecular mechanisms are not clear. In this study, we delineate the underlying molecular mechanism by which HIV and/or cocaine enhance Tau phosphorylation (p-Tau S396), a marker of Tau-mediated neuropathies. Furthermore, we elucidate how these two independent neuropathogenic factors, cocaine and HIV, exploit distinct yet convergent signaling pathways to drive this pathological event. We demonstrate that HIV robustly activates and upregulates RSK1, which functions upstream of AKT and promotes Tau phosphorylation through an AKT-independent mechanism while simultaneously inactivating GSK3{beta} via serine-9 phosphorylation (p-GSK3{beta} S9). However, cocaine not only activates RSK1 but also strongly stimulates AKT1, resulting in sustained GSK3{beta} inhibition and persistent Tau phosphorylation. Notably, Tau phosphorylation persists even under conditions of GSK3{beta} inactivation in both HIV and cocaine exposure, revealing a previously unrecognized GSK3{beta}-independent mechanism of Tau modification. Collectively, these findings identify RSK1 as the primary mediator of Tau phosphorylation upon HIV and/or cocaine exposure, and uncover a novel RSK1-driven, GSK3{beta}-independent pathway contributing to Tauopathy. Through a combination of immunofluorescence, immunoblotting, genetic knockout, and overexpression approaches, we establish RSK1 as a central signaling hub linking the AKT-GSK3{beta} pathway to Tau phosphorylation. We demonstrate that RSK1 operates as a critical upstream regulator of AKT and GSK3{beta} signaling, playing dual roles, both activating AKT and suppressing GSK3{beta}, thereby uncovering a novel layer of pathways that regulates Tau phosphorylation. The reproducibility of these main signaling pathways across SH-SY5Y neurons, mixed cell 3D spheroids, and human brain organoids underscores the robustness and biological relevance of this mechanism. Collectively, these findings reveal mechanistic convergence of HIV and cocaine on RSK1-dependent signaling and provide critical insight into how diverse neuropathic / neuropathological factors remodel neuronal signaling to drive Tau-associated dysfunction. These findings provide novel mechanistic insight into the molecular underpinnings of neuro-HIV and substance abuse associated Tauopathy. By identifying RSK1 as a master regulator and demonstrating that Tau phosphorylation can bypass GSK3{beta} inhibition, our study advances understanding of signaling complexity and highlights new opportunities for therapeutic intervention. Targeting RSK1 may represent a promising strategy to mitigate Tau pathology, induced due to insoluble aggregates of phosphorylated Tau, a common factor promoting cognitive decline not only in individuals with Alzheimers disease but also in those exposed to cocaine or/and infected with HIV. SignificancesThis study demonstrates that exposure to HIV and/or cocaine induces Tau phosphorylation at serine 396 (S396), a well-established marker of Tau pathology, and delineates how these two independent neuropathogenic factors engage distinct yet convergent signaling pathways to drive this pathogenic event. We show that HIV exposure drives robust RSK1 activation, positioning it upstream of AKT to promote Tau phosphorylation via an AKT-independent mechanism, while concurrently suppressing GSK3{beta} activity through serine-9 phosphorylation. In contrast, cocaine, while only moderately activating RSK1, primarily enhances AKT signaling, leading to sustained GSK3{beta} inhibition and increased Tau phosphorylation. Notably, Tau phosphorylation persists even under conditions of GSK3{beta} inactivation in both settings, revealing a previously unrecognized, RSK1-centered, GSK3{beta}-independent pathway of Tau modification. Overall, our findings demonstrate that Tau phosphorylation in the context of HIV infection and cocaine exposure is a complex, multi-layered regulatory process involving multiple signaling nodes. Importantly, we identify RSK1 as a central integrative hub linking viral and substance-induced signaling to downstream Tau pathology. This work advances our understanding of the molecular mechanisms underlying neuroHIV and substance abuse-associated neurodegeneration. Furthermore, it highlights RSK1 as a novel and promising therapeutic target for mitigating Tauopathy in both cocaine-using and non-using people with HIV (PWH). Highlighted pointsO_LIRSK1 acts as a central regulator of Tau phosphorylation, capable of driving this process through a GSK3{beta}-independent mechanism. C_LIO_LIHIV promotes Tau phosphorylation primarily via robust upregulation and activation of RSK1, operating largely independent of AKT1, while concurrently inducing GSK3{beta} inactivation. C_LIO_LIDrugs of abuse, such as cocaine induces Tau phosphorylation through dual activation of AKT1 and RSK1, alongside sustained inactivation of GSK3{beta}. C_LIO_LITau phosphorylation persists despite GSK3{beta} inhibition, revealing a complex AKT1-RSK1 signaling axis and underscoring the dominant role of GSK3{beta}-independent mechanisms in Tau pathology following HIV and cocaine exposure. C_LIO_LIHIV and cocaine engage distinct yet convergent signaling pathways that disrupt neuronal homeostasis and drive tauopathy, providing mechanistic insight into neuroHIV and substance abuse-associated neurodegeneration. C_LIO_LIRSK1 functions as a key upstream modulator of AKT and GSK3{beta} pathways, positively regulating AKT signaling while negatively regulating GSK3{beta} activity. C_LIO_LIRSK1 emerges as a potential therapeutic target, offering new opportunities for intervention in HIV-associated neurocognitive disorders (HAND) and drug-induced neurodegeneration. C_LIO_LIEstablished and characterized H80 cells as a novel neuronal cell model and demonstrated their suitability for studying neuron-specific signaling pathways, including Tau phosphorylation. C_LIO_LIThe conserved and widespread nature of the signaling cascade driving Tau phosphorylation in response to HIV and/or cocaine exposure was validated across multiple model systems, including both 2D neuronal cell cultures and 3D systems such as human brain organoids and spheroids. C_LI Strength of the StudyThis original study provides novel mechanistic insight into how HIV and cocaine, two independent neuropathological factors, converge and diverge on intracellular signaling pathways to regulate Tau phosphorylation. By integrating immunofluorescence, immunoblotting, genetic knockout, and overexpression approaches, we identified RSK1 as a master regulator of Tau phosphorylation. Importantly, we discovered that HIV robustly upregulates and activates RSK1 to promote Tau phosphorylation through an AKT-independent route while simultaneously inactivating GSK3{beta}. On the other hand, cocaine exerts a moderate effect on RSK1 but strongly stimulates AKT to induce GSK3{beta} inactivation and drive Tau phosphorylation. A key strength of this work is the discovery that Tau phosphorylation persists despite GSK3{beta} inactivation, revealing a complex, GSK3{beta}-independent mechanism, involving RSK1 in Tau pathology. Moreover, our study, for the first time, identify RSK1 as an upstream regulator of AKT-GSK3{beta} signaling cascade, enhancing AKT signaling while simultaneously inhibiting GSK3{beta} activity, thereby underscoring the critical role of RSK1 in Tau phosphorylation and associated illnesses, such as HAND and Alzheimers disease. Together, these findings not only advance our understanding of the molecular underpinnings of neuroHIV and substance abuse associated tauopathy but also highlight RSK1 as a promising therapeutic target for not only HIV and cocaine induced neurotoxicity but also other neurodegenerative diseases, such as Alzheimers disease. Another key strength of this study is the establishment and characterization of H80 cells as a novel neuronal model, demonstrating their suitability for investigating neuron-specific signaling pathways, including Tau phosphorylation. The combination of comparative signaling analysis, genetic perturbations, and integrative mechanistic modeling makes this study both conceptually and technically novel, besides broadly relevant to the fields of neurovirology, addiction neuroscience, neurodegeneration, and cognitive impairments.

Chemoresistance is the leading cause of poor prognosis in triple-negative breast cancer (TNBC), yet the underlying mechanisms remain unknown. To reveal metabolic drivers of de novo chemoresistance in TNBC, we analyzed pretreatment primary tumor biopsies, employing quantitative proteomics and metabolomics. Chemoresistant TNBCs exhibit hallmarks of oxidative phosphorylation (OXPHOS) and altered nucleotide metabolism linked to overexpression of the mitochondrial sirtuin, SIRT5. Through gain- and loss-of-function studies and stable isotope tracing, we demonstrate that SIRT5 induces a coordinated metabolic switch that redirects glycolysis to the pentose phosphate pathway, thereby augmenting nucleotide pools, while enhancing glutaminolysis to support OXPHOS. Mechanistically, SIRT5 enhances conversion of 6-phospho-D-gluconate to ribulose-5-phosphate through demalonylation of 6-phosphogluconate dehydrogenase (6-PGD), and coordinately activates oncogenic c-MYC to promote glutamine utilization and dependence. Concurrently, SIRT5-induced nucleotide deregulation induces replication stress and hypersensitivity to ATR checkpoint activation, and ATR inhibition synergistically reverses chemoresistance in TNBC. Thus, elevated SIRT5 orchestrates a coordinated metabolic switch to expand nucleotide pools and drive chemoresistance, while producing ATR checkpoint dependence that represents a metabolic vulnerability of SIRT5-overexpressing TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=182 HEIGHT=200 SRC="FIGDIR/small/716852v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1c7a27corg.highwire.dtl.DTLVardef@17cb22borg.highwire.dtl.DTLVardef@1956670org.highwire.dtl.DTLVardef@1786dee_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epithelial-mesenchymal transition (EMT) and glycolytic metabolism are well-characterized drivers of cancer progression and metastasis. However, most primary breast tumors and metastases express E-cadherin and the epithelial phenotype is associated with mitochondrial oxidative metabolism, yet the causality and relevance of these relationships and their underlying mechanisms remain poorly understood. Using a 3D culture model with mechano-stimulation, we found that E-cadherin promotes mitochondrial oxidative phosphorylation (OXPHOS) while reducing oxidative stress. Through pharmacological and genetic manipulations of inflammatory breast cancer (IBC) and/or triple negative breast cancer (TNBC) cell lines, we identified pyruvate carboxylase (PC) as an E-cadherin effector. Critically, restoring PC in E-cadherin-silenced cells rescued mitochondrial oxygen consumption and protection from oxidative stress. Co-expression of E-cadherin and PC was confirmed in breast cancer tissues and experimental lung metastases. Mechanistically, E-cadherin induced PC expression and OXPHOS via AKT-mediated activation of YAP/ /TEAD transcription factors, which are better known as supporting EMT. Clinically relevant AKT and TEAD inhibitors reduced both PC expression and oxidative respiration. Importantly, PC inhibition as monotherapy attenuated or reduced established experimental lung metastasis burden in mice. These findings reveal that E-cadherin-mediated cell-cell adhesions directly support mitochondrial metabolism through AKT-YAP/TEAD-PC signaling, identifying a therapeutic vulnerability in metastatic epithelial TNBC.

Maintaining organismal homeostasis requires mechanisms that coordinate the metabolic needs of individual organs with whole animal nutrient intake. Although nutrient sensors and central pathways regulating hunger have been extensively characterized, how peripheral organ physiology influences nutrient specific appetites remains poorly understood. Here, we identify a previously unrecognized signalling axis, originated in the ovary, that modulates yeast appetite in Drosophila melanogaster. Through a targeted germline RNAi screen, we find that specific perturbations in oogenesis consistently and selectively increase yeast appetite. The manipulations increasing yeast appetite disrupt oogenesis progression, producing a shared signature of increased vitellogenic follicle accumulation and reduced number of mature (stage14) oocytes. This shift is accompanied by decreased expression of the relaxin-like hormone Dilp8, and loss of Dilp8 recapitulates the feeding phenotype. We show that this ovarian regulation of nutrient specific appetite is independent of amino acid state but requires mating, indicating integration with Sex Peptide-mediated reproductive activation. Together, our findings uncover a novel mechanism that couples oogenesis progression to nutrient selection, emphasizing the importance of the ovary as an active regulator of whole organism nutritional decisions. This work provides a conceptual framework for how reproductive tissues communicate their physiological demands to other organs and raises the possibility that analogous ovary-derived signals may shape nutrient specific appetite and metabolic states in other animals. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/711327v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@15e268forg.highwire.dtl.DTLVardef@35ab4dorg.highwire.dtl.DTLVardef@1817872org.highwire.dtl.DTLVardef@10ab453_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Francisco, A. (2026) https://BioRender.com/pwalhtl

Downregulation of major histocompatibility complex class I (MHC-I) molecules is a frequent mechanism of tumor immune evasion, impairing recognition and elimination of cancer cells by cytotoxic CD8+ T lymphocytes. This phenotype, associated with poor prognosis and resistance to immune checkpoint blockade, is often driven by oncogenic pathways that reversibly suppress MHC-I. The MAPK ERK5 and its only upstream activating kinase MEK5 configure a unique intracellular signaling that regulates cell proliferation, differentiation and survival, and it has emerged as an oncogenic driver of different tumors. In this work, we investigated whether the ERK5 pathway contributes to cellular immunity. We used a panel of human cancer cell lines representing three well-established oncogenic contexts linked to reversible MHC-I downregulation (MYCN amplification, PTEN/PIK3CA mutations, and androgen receptor signaling), together with control cellular models exhibiting constitutively high MHC-I expression. We found that MEK5 or ERK5 pharmacologic inhibition or ERK5 targeted degradation increased MHC-I surface and total expression in low-MHC-I cells, without affecting PD-L1 levels. Conversely, ERK5 overexpression impaired MHC-I levels. Moreover, systemic administration of an ERK5 inhibitor also enhanced MHC-I expression in tumor xenografts. Mechanistically, RT-qPCR analysis showed that ERK5 or MEK5 inhibition did not significantly modify transcription of classical HLA-I genes or antigen-processing machinery components, and RNA-seq analysis did not render enrichment of MHC-I transcriptional programs in response to ERK5 inhibition. In contrast, trafficking experiments implicated the ERK5 pathway in the regulation of MHC-I lysosomal degradation, suggesting that ERK5 controls surface MHC-I through post-translational mechanisms. Notably, functional assays were carried out in co-cultures of cancer cells with tumor-specific human CD8+ T cells, where ERK5 inhibition sensitized MYCN-amplified or PTEN/PI3KCA-mutated cancer cells to CD8+ T cell-mediated apoptosis. These results identify ERK5 as a novel regulator of MHC-I expression in cancer cells, by regulating antigen presentation across diverse oncogenic contexts, and support a rationale for the use of ERK5 inhibitors as a strategy to improve the efficacy of immune checkpoint blockade-based immunotherapy.

Neocortical synapses are highly dynamic during brain development, undergoing formation, elimination, and maturation before acquiring properties that support adult cognition. Individual neocortical regions develop at different ages and individual layers within these regions contain distinct neuronal subtypes that process unique patterns of local and long-range synaptic input. To better understand the development of the cortical hierarchy we explored the laminar maturation of glutamatergic synapses across cortical regions. Synapse maturation was associated with the upregulation of the postsynaptic density protein PSD95. This maturation occurred in a region- and layer-specific manner -- layers associated with feedforward pathways develop earlier, while layers associated with higher-order circuits develop later. Our findings highlight adolescence as an important period for the cortex-wide maturation of synapses in cortical layer 1, synapses known to receive top-down feedback from higher-order cortices. We propose that this delayed adolescent maturation of top-down input represents a global signature of cortical development and seemingly acts as the final stage of outside-in brain maturation.

Triple-negative breast cancer (TNBC) relies on metabolic plasticity to sustain growth under diverse microenvironmental conditions. Although growth hormone (GH) signaling has been linked to breast cancer progression, its mechanistic integration with mitochondrial dynamics and metabolic reprogramming remains unclear. Here, we show that GH promotes TNBC progression accompanied by DRP1-associated mitochondrial remodeling, as indicated by sensitivity to Mdivi-1. The MDA-MB-231 line enabled integrated assessment across 2D, 3D, hypoxia, and in vivo xenografts using consistent workflows and readouts. GH increased proliferation and mitochondrial mass without increasing OCR under protein normalization. Instead, GH selectively enhanced glycolytic flux and metabolic flexibility. Inhibition of DRP1 uncoupled GH-induced glycolysis from proliferation, demonstrating that mitochondrial fission is required to link metabolic reprogramming to cell-cycle progression. DRP1 inhibition with Mdivi-1 was associated with altered TP53 and HIF1A expression and extended GH activity to the regulation of a pro-inflammatory tumor microenvironment marked by cxcr4b, il8, and il12. Consistent with these findings, analysis of human TNBC transcriptomes revealed conserved enrichment of mitochondrial, metabolic, and inflammatory pathways. Together, these results support the GH-DRP1 axis as a candidate regulator of mitochondrial dynamics, metabolic plasticity, tumor progression and tumor microenvironment interactions in TNBC.