Cell Reports

Insufficient insulin secretion relative to insulin demand is a key feature of type 2 diabetes (T2D). While the defects of insulin-producing {beta}-cells in T2D are well defined, little is known about how {beta}-cells progress from the functionally normal state to the decompensated state during the natural history of this disease. Here, we provide evidence that workload-induced {beta}-cell overstimulation precipitates {beta}-cell failure in T2D. We employ scRNA-seq to define workload-induced changes to {beta}-cell transcriptional states, identifying a novel compensating state that is distinct from the stressed state of decompensated {beta}-cells. We demonstrate a key role for the chromatin-modifying enzyme Lysine-specific demethylase 1 (Lsd1) in restraining workload-induced {beta}-cell state transitions, indicating epigenomic control of {beta}-cell state. Experimental manipulations that promote the compensating state accelerate {beta}-cell failure in mouse models of diabetes. Altogether, these findings show that the compensatory response of the {beta}-cell to increased workload becomes maladaptive over time and contributes to the pathogenesis of T2D.

The HIV reservoir that establishes early upon infection and persists in tissues remains the primary barrier to a functional cure. While progress has been made to study the reservoir in blood compartments and specific cell types, knowledge gaps remain on the tissue microenvironment that facilitates persistence. The development of a novel immunoPET/CT-guided spatial transcriptomics pipeline has enabled the localization of foci of viral infection in tissues, rare events that are challenging to sample. Prior studies leveraging the pipeline have characterized the viral microenvironment (VME) gene signatures, cell types and interactions, and host transcriptome gene drivers of viral persistence. Detailed characterization of the VME revealed multiple shared features with certain tumor microenvironments (TMEs), suggesting shared immunoregulatory and survival mechanisms. In this study, we apply the immunoPET/CT-guided spatial transcriptomics pipeline in the SIVmac239/rhesus macaque model to define immune mechanisms underlying persistent versus transient HIV/SIV reservoirs. We utilize a systematic approach to highlight correlates between the VME and TME transcriptional programs, gene pathways, local tissue neighborhoods, cell-cell interactions, and host transcriptomic drivers. Analysis of broad transcriptional programs revealed SIV-localized spatial enrichment of genes associated with multiple cancer subtypes. The persistent reservoir was characterized by gene pathways of "cold" TMEs (e.g., epithelial to mesenchymal transition, TGF{beta} activation) whereas the transient reservoir was comparable to "hot" TMEs with cytotoxic immune activation. Cell-cell interaction analysis identified regulatory T cells as a key mediator of interactions in both persistent and transient reservoirs. Machine learning identified KRT8, EPCAM, and RRM2, genes with known roles in mediating carcinogenesis, among the top host transcriptomic drivers of the TME phenotype of the persistent VME. Collectively, the findings of this study provide novel and transformative insights on key mechanisms of HIV/SIV persistence and reveal potential targets for immunotherapeutic strategies aimed at reservoir disruption or clearance towards a functional HIV cure.

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.

The preBotzinger Complex (preBotC) within the medulla oblongata contains neuronal circuits critical for generating the mammalian respiratory rhythm, but the functional connectivity among its core excitatory and inhibitory populations remains debated. Defining this connectivity requires disentangling synaptic interactions of functionally identified excitatory and inhibitory preBotC neurons with various electrophysiological phenotypes. We applied a novel synaptic conductance inference method to whole-cell recordings from genetically specified VgluT2-expressing (excitatory) and VGAT-expressing (inhibitory) preBotC neurons active in the rhythmic medullary slice in vitro, which contains core inhibitory-excitatory circuitry with an excitatory rhythmogenic kernel. We found that this circuitry consists of a self-exciting inspiratory VgluT2 population coupled to inspiratory and expiratory VGAT populations that interact reciprocally through inhibition. The functional inhibitory connectome is more complex than previously understood. However, compared with functional synaptic interactions inferred from recordings in the preBotC in situ, the neuronal synaptic conductance profiles in the rhythmic slice reveal a functionally reduced inhibitory connectome, characterized by prominent tonic expiratory inhibition and phasic inspiratory inhibition, without the characteristic multiphasic structure in situ. These results indicate that the functional excitatory and inhibitory circuit interactions within the preBotC isolated in vitro, although reduced relative to more intact states in situ, are intrinsically designed to generate coordinated inspiratory and expiratory population activity. Tonic expiratory phase inhibition together with inspiratory phasic inhibition serves to regulate excitability and phase transitions of the excitatory rhythmogenic kernel.

Rapid termination of odor responses is essential for accurate olfactory coding in insects, where odorant receptors function as ligand-gated ion channels rather than G protein-coupled receptors. However, the mechanisms that restore olfactory receptor neuron (ORN) excitability after stimulation remain poorly understood. Here, we identify DmTMEM16O (CG6938), an arthropod-specific member of the TMEM16 (Anoctamin) family, as a key regulator of ORN response termination in Drosophila melanogaster. DmTMEM16O is highly enriched in Orco-positive ORNs in both larval and adult olfactory organs. Loss of DmTMEM16O prolongs odor-evoked neuronal activity, increasing decay time constants and causing persistent depolarization. DmTMEM16O mutant ORNs fail to resolve repeated odor stimulation and show impaired temporal coding, accompanied by reduced behavioral responses to both attractive and aversive odorants. Cell-specific rescue demonstrates that DmTMEM16O acts within ORNs to accelerate response termination. Although DmTMEM16O does not exhibit detectable Ca2+-activated chloride channel activity in heterologous cells, our results support a model in which it increases membrane conductance during the decay phase of the response, thereby shortening the membrane time constant and promoting rapid repolarization. This function is consistent with a role for chloride influx in insect ORNs, in contrast to mammalian systems where TMEM16B-mediated chloride efflux amplifies depolarization. Together, our findings identify DmTMEM16O as a lineage-specific regulator of ORN dynamics that enables precise temporal coding in insect olfaction.

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

An immune engram is a recently described phenomenon in which neuronal populations encode functional aspects of an immune challenge. Here we investigate an immune engram arising from respiratory infection with influenza A virus, demonstrating a molecular mechanism with differential influence over behavioral and immunological aspects of the engram. We first define a cellular response to acute non-neurotropic influenza A/Puerto Rico/8/1934 (PR8) infection by mapping cFos+ cells and microglia morphology across brain regions. In the posterior insula, this response has an early peak at 3 days post infection. Using a cre-dependent excitatory chemogenetic system in TRAP2 mice, we capture an engram at this same region and infection timepoint. Activation of this PR8 engram results in anxiety behavior and increased transcriptional expression of cytokines in lung tissue but not spleen tissue. We further explore how pulmonary signals contribute to this PR8 engram. Using tissue-specific, cre-dependent expression of diphtheria toxin fragment in Calcacre mice, we ablate Calca-expressing cells including pulmonary neuroendocrine cells in respiratory tissue. Loss of Calca-expressing cells prevents changes in synaptic engulfment by microglia in the insula during PR8 infection without altering the cellular response to infection in pulmonary tissue. Signaling of calcitonin gene related peptide (CGRP), a peptide encoded by Calca, can be blocked with the small molecule CGRP receptor antagonist rimegepant. Using rimegepant during acute PR8 infection we again demonstrate that loss of Calca signaling prevents the cellular response to PR8 infection in the insula. Finally, applying rimegepant alongside the chemogenetic system in TRAP2 mice we show that CGRP receptor antagonism during engram formation prevents anxiety behavior but not peripheral gene expression changes resulting from PR8 engram activation.

Age-related diseases arise from prolonged exposure to genetic and/or environmental factors, ultimately leading to cumulative and irreversible degeneration of tissues and the organism as a whole. We previously reported that accumulation of mitochondrially-generated aldehydes (i.e., 4-hydroxynonenal and acetaldehyde) causes mitochondrial dysfunction and accelerates the progression of age-related diseases. However, the contribution of mitochondrial aldehyde metabolism to aging (via aldehyde dehydrogenase 2, ALDH2) remains elusive. Here, we provide a comprehensive analysis of aldehyde metabolism and mitochondrial bioenergetics across different tissues in aging mice. We also address how mitochondrial function is influenced by the highly prevalent human inactivating ALDH2 E504K point mutation (ALDH2E504K) during aging. The liver metabolism was relatively resilient to aging, showing enhanced ALDH2 activity and improved mitochondrial coupling. Strikingly, aging-associated liver resilience was lost in ALDH2E504K mice. Aged hearts exhibited mixed outcomes including impaired mitochondrial basal respiration, improved ADP-driven respiration, and decreased ALDH2 detox capacity. The ALDH2E504K mutation exacerbated the already impaired cardiac ALDH2 detox capacity in aging. Strikingly, aging brain displayed pronounced vulnerability, with decreased ALDH2 activity, impaired mitochondrial bioenergetics and defective ALDH2 detox capacity. These changes were paralleled by impaired cognitive and behavioral functions in aged mice. As proof of concept, either the presence of ALDH2E504K mutation or acute ethanol challenge worsened cognitive and behavioral dysfunction in aging mice. Finally, we assessed in vitro efficacy of pharmacological ALDH2 activation in aging tissues. Collectively, these findings unravel the contribution of ALDH2E504K mutation to mitochondrial metabolism during aging; highlighting the detrimental synergy between genetic ALDH2 deficiency and aging in brain metabolism and physiology.

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.

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.

Dopamine is essential for basal ganglia function. Striatal dopamine release can be triggered by dopamine cell firing, but also by coordinated cholinergic interneuron activity, which stimulates dopamine release via presynaptic nicotinic acetylcholine receptors on dopamine axons. While acetylcholine-dependent dopamine release is well-documented ex vivo and under artificial optogenetic stimulation in vivo, its role during natural behavior has remained unclear. One possible natural driver of acetylcholine-dependent dopamine release is thalamic input, which provides strong excitatory drive to cholinergic interneurons. To examine whether thalamic input provokes acetylcholine-dependent dopamine release during behavior, we performed simultaneous fiber photometry recordings of striatal dopamine (GRAB-rDA3m) and thalamic axon activity (gCaMP8m) in the dorsomedial (DMS) and dorsolateral striatum (DLS) of mice learning the accelerating rotarod, a striatal-dependent task that demands precise and effortful motor control. Recordings were obtained on- and off-task and across days of training to capture the full arc of learning. Dopamine transients in DMS, but not DLS, were frequently coupled to peaks in thalamic axon activity via an acetylcholine-dependent mechanism. The occurrence of these thalamic-evoked dopamine transients depended on learning, task engagement, and the recent history of striatal dopamine activity, but did not appear to signal motor errors. Together, these findings establish thalamic input as a physiological driver of acetylcholine-dependent dopamine release. Moreover, they reveal that striatal sensitivity to this local release mechanism is dynamically gated by dopaminergic history, providing a compelling framework for understanding how local and soma-triggered dopamine signals are coordinated to support learning.

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

In response to environmental and inflammatory cues, microglia adopt diverse morphologies reflecting their functional state. However, characterizing these morphological alterations in human iPSC-derived microglia (iMGLs) remains limited by reliance on fluorescent labeling, endpoint imaging, and coarse categorical classifications (e.g., ramified vs amoeboid states). Here, we developed a label-free pipeline combining live-cell imaging, Cellpose-SAM segmentation, and CellProfiler-based feature extraction to track iMGL morphology at single-cell resolution over time. Applying this framework to a 24-hour time course of LPS and IFN{gamma} co-stimulation revealed rapid and transient morphological remodeling, with responses peaking within 2-4 hours and partially attenuating thereafter. At single-cell resolution, four morphological states captured the major axes of variation, with co-stimulation driving a pronounced redistribution toward a single "Spread state". Extending the analysis to individual inflammatory stimuli (LPS, IFN{gamma}, IL-1{beta}, IL-6, and TNF) revealed graded but overlapping shifts in state composition, with IFN{gamma} inducing the strongest response, whereas IL-1{beta} showed minimal effects. Importantly, state-level changes alone were insufficient to distinguish stimulus-specific responses. Instead, single-cell density mapping revealed distinct occupancy patterns within a shared morphological landscape. In addition, texture complexity provided an independent feature layer that further separated among treatments, indicating that stimulus identity is represented by continuous, high-dimensional morphological features beyond discrete state assignments. Linking morphology to function, IFN{gamma} produced the largest morphological redistribution and was also the only stimulus to significantly elevate intracellular ROS at 24 h, indicating that morphological remodeling and redox activation can be coordinately engaged by specific inflammatory signals. Together, these findings support a model in which microglia respond to inflammatory stimuli by graded occupancy of a continuous morphological landscape, with each stimulus producing a stimulus-specific occupancy pattern that extends beyond discrete state assignments.

The dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a key mediator of monoaminergic signaling, is expressed in the cortex; however, its cellular distribution and role in cortically dependent behaviors remain elusive. Here, we determined the functional integration of DARPP-32 in motor cortex circuitry using molecular profiling, circuit tracing, patch-clamp electrophysiology, virus-assisted gene targeting and motor behavior analyses. Unlike the significant overlap between DARPP-32 and dopamine receptors in striatal GABAergic medium spiny projection neurons, we found that the majority of DARPP-32-positive cortical neurons express the corticothalamic marker FoxP2 but not dopamine D1 or D2 receptors. Notably, in cortical slices, adenylyl cyclase activation induced a more robust increase in DARPP-32 phosphorylation at threonine 34, a protein kinase A target site, compared to dopamine D1 receptor stimulation. Conditional ablation of DARPP-32 in the motor cortex did not affect basal or psychostimulant-induced motor activity but reduced motor aptitude and compromised overnight retention of motor skill. Concomitantly, the absence of DARPP-32 reduced dendritic spines density and prevented the induction of glutamatergic long-term potentiation in layer 6 motor cortical neurons. Altogether, our study demonstrates a critical role for DARPP-32 in cortical synaptic plasticity, emphasizing its importance in corticothalamic regulation of motor skill learning.

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

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

The neurotransmitter dopamine (DA) is central to synaptic regulation that support diverse behavioral functions, including both learning and forgetting. This multi-functional role of DA is due to receptor specific signaling in specific subcellular environments that remain uncharacterized. Here we utilized proximity labelling proteomics in human cells to characterize the proximal environments of two Drosophila D1-like DA receptors (Dop1R1 and Dop1R2) in basal and DA activation environments. While DA drives both receptors to recruit Beta-Arrestin 2, Dop1R1 alone showed ligand driven recruitment of G-protein Receptor Kinase 2/3, proximity to clathrin mediated endocytosis, and WASH complex mediated endosomal trafficking. Additionally, we show evidence that Dop1R1 and Dop1R2 reside in distinct domains at the cell surface. In vivo disruption of Drosophila orthologs of Dop1R proximal proteins revealed three trafficking proteins, Sec24AB, Krz, and CG13887, that regulate R1-mediated learning, starvation induced attraction to odors, and DA-mediated cAMP responses in memory circuits. In addition to revealing DA receptor trafficking proteins that support learning, our comparative characterization of the cellular environments D1-like receptors offers insights into how DA differentially regulates diverse behavioral and synaptic functions. For TOC only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/728438v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@656e56org.highwire.dtl.DTLVardef@12f0084org.highwire.dtl.DTLVardef@cb05cdorg.highwire.dtl.DTLVardef@e9d623_HPS_FORMAT_FIGEXP M_FIG C_FIG