Cell Reports

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

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.

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.

The brain stores information by changing the strength of its synapses, a process that has at least two phases: Late long-term potentiation (L-LTP) is thought to result from the consolidation of early LTP (E-LTP), just as long-term memory requires the prior establishment of short-term memory. Recently, inhibitory avoidance experiments under CaMKII inhibition have challenged this notion, demonstrating long-term fear memory without measurable short-term memory. Here we use optogenetic activation and inhibition of CaMKII during induction of spike-timing-dependent potentiation (tLTP) to dissect the signaling pathways. While CaMKII activation in CA1 neurons was sufficient to induce E-LTP, growth of the postsynaptic density and spine neck expansion, we found that CaMKII-induced LTP does not give rise to L-LTP. Conversely, inhibition of CaMKII during tLTP induction prevented E-LTP, but FOS and L-LTP were still expressed, driven by CaMKK and PKM{zeta}. Thus, both long-term memory and L-LTP form in the absence of CaMKII activation.

Follicular helper T (Tfh) cells orchestrate germinal centre-derived humoral immunity by providing essential help to B cells. Despite their key role in humoral immunity, the metabolic processes that guide Tfh differentiation and functions in human remain poorly understood. In this study, we used a human ex vivo Tfh differentiation model to investigate how key metabolic pathways influence Tfh cell differentiation and helper function. Human naive CD4 T cells were differentiated into Tfh cells, and glycolysis was selectively inhibited during early differentiation to assess its effects on cell fate, function, and transcriptomic landscape. Unexpectedly, early glycolytic inhibition enhanced Tfh cell differentiation but significantly impaired their function, including reduced IL-21 secretion and diminished B cell help. Transcriptomic analyses further revealed downregulation of fatty acid metabolic pathways when glycolysis was inhibited. To better understand this link, we disrupted fatty acid synthesis and oxidation and observed a marked decline in helper functions, including IL-21 and IFN-{gamma} production. Interestingly, acetate supplementation partially restored IFN-{gamma} secretion in glycolysis inhibited conditions, but not IL-21, suggesting that some functional requirements cannot be compensated by alternative metabolic sources. These findings identify glycolysis during early differentiation as a key regulator of human Tfh cells fate and reveal a glycolysis-dependent fatty acid metabolic axis that selectively controls Tfh function. This metabolic checkpoint provides mechanistic insight into tuning Tfh cells and Tfh-driven humoral immunity in vaccination and autoimmunity.

High-fat diet (HFD) feeding disrupts systemic glucose metabolism, yet the underlying neural mechanisms remain incompletely understood. Here, we demonstrate that glucose-excited (GE) neurons in the ventromedial hypothalamus (VMHGE) are essential for acute glucose regulation and that their function is compromised by HFD via structural synaptic remodeling. We found that HFD feeding suppresses canonical Wnt signaling and downregulates R-spondin 1 (RSPO1), a Wnt enhancer, in the VMH. This Wnt inhibition leads to a loss of dendritic spines and blunted glucose-sensing in VMHGE neurons. Conversely, central administration of RSPO1 restores Wnt/{beta}-catenin signaling, promotes synaptogenesis, and recovers neuronal glucose responsiveness. Consequently, RSPO1 treatment ameliorates HFD-induced glucose intolerance by enhancing peripheral glucose utilization. These findings identify the RSPO1-Wnt signaling axis as a critical regulator of VMH neuronal plasticity and metabolic homeostasis, providing a mechanistic link between diet-induced synaptic pathology and systemic metabolic dysfunction. Highlights- Glucose-excited neurons in VMH were labeled with TRAP - VMH glucose-excited neurons regulates systemic glucose metabolism - Wnt signaling regulates synaptogenesis in VMH and maintain neuronal glucose-sensitivity - R-spondin1 recovers VMH neuronal glucose sensitivity in HFD fed obese mice

Viral infections are one of the main environmental factors triggering type 1 diabetes (T1D). Pancreatic alpha cells are more resistant than beta cells to diabetogenic viruses, partially explaining their survival in T1D. Similarly, bats have enhanced viral resistance, suggesting putative convergent evolution in antiviral mechanisms. Herein, we compared global gene expression in bat fibroblasts under basal conditions or exposed to double-stranded RNA to human alpha and beta cells and found that alpha cells exhibit greater similarity than beta cells to the antiviral responses of bat cells, as well as stronger intrinsic resistance to viral infection. Interferon-stimulated gene 15 (ISG15), a key regulator of antiviral responses in humans and bats, has higher expression in alpha compared to beta cells in five single-cell RNASeq datasets from human islet cells and in human induced pluripotent stem cell (hiPSC)-derived alpha-like cells. ISG15 knockdown in human insulin-producing EndoC-{beta}H1 cells and human islets increases apoptosis under basal conditions and after IFN exposure, exacerbates IFN responses and increases cell death and viral production after infection with the diabetogenic virus coxsackievirus B1, while its overexpression protects EndoC-{beta}H1 cells from the virus. Collectively, the present results demonstrate that alpha cells but not beta cells have similarities with the virus resistance gene program present in bats and identify ISG15 as an important factor for islet cells to cope with viral and diabetogenic stresses.

The pituitary gland operates as an organized signaling network in which endocrine cell populations coordinate hormone secretion, through homotypic and heterotypic interactions, yet the contribution of spontaneous intrinsic activity in shaping population-level dynamics remains poorly understood. Using geometric analysis of population trajectories -- including subspace alignment, manifold separation, and directed coupling metrics -- we identified two classes of spontaneous oscillatory signals associated with distinct cell populations exhibiting asymmetric geometric dominance and a reproducible temporal lag. Our results support that spontaneous activity generates a self-sustained oscillator exhibiting transient bistability, linked to increased physiological demand, with slow oscillations reflecting the properties of an excitatory resonator capable of self-oscillating dynamics without external drive. A low-rank recurrent neural network model recapitulated the empirical geometric landscape under three coupling conditions, confirming that directed population coupling underlies the observed coordination. These findings suggest that intrinsic population dynamics play a central role in coordinating pituitary secretion, with implications for understanding hormonal dysregulation in secretory adenomas and other pituitary disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/716480v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@193954aorg.highwire.dtl.DTLVardef@2e4299org.highwire.dtl.DTLVardef@1165736org.highwire.dtl.DTLVardef@1b79cd5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Structural and functional distinctions between homotypic and heterotypic interactions have been widely described in the pituitary endocrine system. However, whether functional differences in intrinsic calcium time-series dynamics are relevant to pulsatile hormone secretion remains unexplored. Here, we classify the spontaneous activity underlying both homotypic and heterotypic interactions and characterise their synchrony. We find that heterotypic interactions exhibit transient bistability, consistent with a Hopf-type oscillator regime, in which slow oscillations drive secretory output according to physiological demand. C_FIG

The metastatic process initiates with collective cell invasion into surrounding tissues and axillary nodes, and subsequent colonization at a distant site. Previously, we found collective invasion is augmented during the G2 cell cycle phase, facilitated through Aurora kinase A (AURKA)-mediated centrosome polarization in the leader cell. Here, we identify cell cycle-associated gene signatures as overrepresented in axilla and liver metastatic sites, with AURKA expression strongly correlated with breast cancer metastasis signatures, and pan-cancer patient survival. Then, we show GFP-AURKA expression endows breast epithelia cells with the ability to form metastatic outgrowths within immune-incompetent chicken embryos. Multi-parametric imaging of wound closure assays reveals phenotypes enabled by, and dependent upon AURKA expression. We discover leader cells express AURKA and acquire front-polarized centrosomes, which differentiates them from other cells in the migrating group. Ectopic expression of GFP-AURKA induces a leader cell phenotype. Conversely, inhibition of AURKA activity alters actin dynamics, promotes turnover of cell contacts, and reduces coordination within migrating groups. Specifically, AURKA interacts with the actin regulator EPLIN, and AURKA inhibition localizes EPLIN to lamellipodia and away from E-cadherin-positive contacts. Inhibiting these necessary roles for AURKA may provide a critical barrier against the metastatic spread of human breast carcinoma cells.

Loss-of-function mutations in the X-linked CDKL5 gene lead to a severe neurodevelopmental disorder characterized by early-onset epilepsy, known as CDKL5 Deficiency Disorder (CDD). Despite its clinical significance, the physiological substrates of the serine/threonine kinase CDKL5 and its roles in neuronal development remain poorly understood. To address this, we performed quantitative phosphoproteomics analysis in Cdkl5 knockout (KO) mouse brains, identifying 22 CDKL5 substrates involved in diverse cellular functions. Among these, we focused on the neuronal RNA-binding proteins (nELAVLs) ELAVL2, ELAVL3, and ELAVL4, as these represented the only evolutionarily conserved phosphorylation and are known regulators of neuronal differentiation. Through kinase assays and individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), we found that CDKL5 phosphorylates S119/131 in ELAVL2/3/4, promoting their cytoplasmic localization and enhancing their binding to target mRNAs at 3UTRs. Loss of CDKL5 activity in neurons caused reduced new protein synthesis, as measured by puromycin incorporation; this phenotype was rescued by knockdown of the nELAVL inhibitor long non-coding RNA, RNY3, revealing an essential function of CDKL5 in enhancing protein synthesis via nELAVL phosphorylation. To investigate the in vivo functions of nELAVL phosphorylations, we generated Elavl2/3/4 phosphomutant mice and found that collectively nELAVL phosphorylations are required for viability. Proteomic and transcriptomic analyses of Elavl2/3 homozygous phosphomutants, which exhibited sub-viability, revealed compensatory upregulation of ELAVL4 and synaptic proteins. Functionally, in Elavl2/3/4 triple heterozygous mice Neuropixels recordings in the primary visual cortex showed deficits in receptive field properties and orientation tuning, revealing the role of nELAVL phosphorylation for accurate cortical circuit formation. Our study uncovers a crucial role for CDKL5 in regulating nELAVL-mediated protein synthesis and the development of cortical circuits.

Songbirds exhibit remarkable seasonal neuroplasticity, with song control nuclei undergoing seasonal cycles of extreme and rapid neuronal death and regeneration. While adult neurogenesis in these systems is well-characterized, the dynamics and functional significance of astrocytic turnover remain unknown. Here, we examined the fate of neural progenitor cell progeny born during seasonally-induced reactive proliferation and identified a rapid astrocytic turnover event in HVC following seasonal neuronal loss. Using lineage-specific and proliferation labeling, we characterized a previously undescribed SOX2-positive neural progenitor-like population within the avian parenchyma beyond the canonical ventricular zone niche. These parenchymal astrocyte precursor cells (pAPCs) proliferate at quantifiable, steady levels under homeostatic conditions, yet as a proliferative cell pool dramatically expand following non-injury induced neuronal death. Beyond their proliferative potential, pAPCs demonstrate capabilities suggestive of self-renewal and generation of astrocytes and neurons. The coordinated response of canonical neural progenitor cells and the newly-described pAPCs generates new astrocytes that persist throughout re-establishment of homeostasis, all of which together likely facilitate subsequent circuit regrowth. These findings reveal extensive astrocyte plasticity in the adult avian telencephalon and establish a foundation for understanding how astrocytes and their precursors--both within and beyond their canonical niches--contribute to neural circuit remodeling and behavioral maintenance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/717664v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@15437e6org.highwire.dtl.DTLVardef@221e58org.highwire.dtl.DTLVardef@1f65df2org.highwire.dtl.DTLVardef@191ca3c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LISubstantial astrocyte turnover following a natural, extreme neuronal death event C_LIO_LIDiscovery of proliferative SOX2 positive precursor cells within the avian parenchyma C_LIO_LIThese parenchymal astrocyte precursor cells (pAPCs) proliferate even in homeostasis C_LIO_LIThe proliferative pAPC pool expands during the natural neuronal death event C_LIO_LIDynamics in both NPCs and pAPCs contribute to homeostasis return and potentially enable circuit regrowth C_LI

Metabolic dysregulation in obesity reshapes immune function, but how lipid signals drive immune suppression remains unclear. Here, we identify a FABP4-PD-L1 axis that links lipid metabolism to immune checkpoint regulation in monocytes and macrophages. Single-cell transcriptomics revealed a distinct FABP4high immunosuppressive macrophage subset enriched under high-fat diet (HFD) conditions, characterized by impaired antigen presentation and elevated PD-L1 expression. Mechanistically, palmitic acid (PA) induces FABP4 and promotes PD-L1 palmitoylation, leading to its stabilization on the cell surface independent of transcriptional regulation. FABP4 is essential for this process, which enables PD-L1 surface stabilization, immunosuppression and mammary tumor progression. In humans, a conserved CD14intCD16 monocyte population exhibits elevated FABP4-PD-L1 signaling and correlates with obesity and invasive breast cancer. These findings establish PD-L1 as a metabolically regulated protein and reveal a mechanism by which lipid excess drives immune evasion, suggesting that targeting FABP4 may enhance responses to immune checkpoint blockade. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/717546v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1cce278org.highwire.dtl.DTLVardef@2848b1org.highwire.dtl.DTLVardef@bc9c25org.highwire.dtl.DTLVardef@af5e3c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFABP4 defines a lipid-responsive, immunosuppressive monocyte/macrophage subset C_LIO_LIFABP4 links lipid sensing to PD-L1 expression in macrophages C_LIO_LIFABP4 enables palmitic acid-dependent PD-L1 palmitoylation and stabilization C_LIO_LIFABP4-PD-L1 signaling correlates with obesity and invasive breast cancer in humans C_LI

Triple-negative breast cancer (TNBC) is characterized by aggressive progression and poor prognosis, partly due to abnormal angiogenesis. While the metabolic reprogramming of tumor cells is well characterized, the metabolic regulation of tumor-associated endothelial cells (ECs) remains unclear. Here, we identified the mitochondrial deacylase SIRT5, which has established tumor-promoting roles in TNBC cells, as a key regulator of endothelial metabolic homeostasis and tumor angiogenesis. SIRT5-deficient host mice showed significant defects in supporting the growth of orthotopic SIRT5-proficient mammary tumor transplants, and the resulting neoplasms showed defects in tumor vascularization. In a 3D microfluidic vessel-on-chip model, SIRT5 loss compromised vascular barrier integrity and EC sprouting. Mechanistically, SIRT5-deficient ECs exhibited diminished mitochondrial respiratory capacity but apparently normal glycolysis. SIRT5 loss also caused increased mitochondrial reactive oxygen species levels, and a mitochondrial antioxidant rescued the endothelial cell defects following SIRT5 loss, indicating that SIRT5-mediated mitochondrial redox homeostasis in the tumor microenvironment is necessary to maintain vascular function. Orthotopic co-transplantation of TNBC and EC cells with or without SIRT5 knockdown demonstrated that endothelial SIRT5 promotes increased tumor growth in vivo. These results suggest that targeting SIRT5 offers a potential therapeutic strategy to disrupt tumor angiogenesis and suppress TNBC progression by targeting the metabolic vulnerabilities of the tumor endothelium.

Microbial steroid metabolism represents an underappreciated extension of the vertebrate endocrine system, with growing evidence that host-associated microbes contribute to the diversity and bioavailability of sex steroids within human tissues. Emerging studies have linked microbial androgen metabolism to urinary microbiome composition and to resistance to androgen deprivation therapy (ADT) in prostate cancer. While microbial pathways capable of converting steroid precursors such as cortisol to androgens, via the steroid-17,20-desmolase pathway, such as DesG-mediated interconversion of androstenedione to testosterone have been reported, the diversity of enzymes mediating downstream androgen interconversion remains incompletely defined. Here, we investigate the androgen-forming capabilities of anaerobic bacteria from the male genitourinary microbiome, focusing on NADPH-dependent 17{beta}-hydroxysteroid dehydrogenases (17{beta}-HSDHs) that catalyze interconversion of androstenedione and testosterone. We isolated androgen-forming bacterial strains from human male urine and identified a previously uncharacterized 17{beta}-HSDH encoded by Peptoniphilus obesi, demonstrated that this enzyme catalyzes the NADPH-dependent reduction of androstenedione to testosterone and the reverse oxidation reaction. Sequence similarity searches further identified a homologous 17{beta}-HSDH in Anaerococcus, which was synthesized and functionally validated, revealing conserved activity despite low sequence identity to the previously characterized urinary tract enzyme DesG. The enzymes were found to have broad substrate specificity for C19 and C18 17keto- and 17{beta}-hydroxysteroids. Together, these findings expand the known diversity of microbial 17{beta}-HSDHs and identify previously unrecognized androgen-forming activities within the genitourinary microbiome. ImportanceMicrobial steroid-transforming pathways may provide a mechanism by which commensal anaerobes contribute to androgen availability in the genitourinary tract. By identifying novel 17{beta}-hydroxysteroid dehydrogenases from Peptoniphilus and Anaerococcus, genera repeatedly associated with prostate cancer, this study provides mechanistic insight into how microbial steroid metabolism may influence hormone-driven disease.

Neuronal remodeling is a conserved, late developmental mechanism to refine neural circuits. Although remodeling typically occurs with remarkable spatiotemporal precision, its underlying molecular mechanisms remain poorly understood. In the Drosophila mushroom body (MB) circuit, {gamma}-Kenyon cells ({gamma}-KCs) undergo stereotyped remodeling during metamorphosis, in which they prune their larval vertical and medial axonal branches and subsequently regrow a medial, adult-specific branch. Our previous transcriptional profiling of developing {gamma}-KCs revealed dynamic expression of Defective proboscis extension response (Dpr) proteins and their binding partners, Dpr-interacting proteins (DIPs), members of the Immunoglobulin (Ig) superfamily. Despite their established roles in neurodevelopment, how Dpr/DIPs function - given their lack of intracellular domains - remains unclear. Here, we show that overexpression of Dpr4 in developing {gamma}-KCs cell-autonomously inhibits axon pruning. Strikingly, this effect is branch-specific: the vertical axonal branch fails to prune, while the medial branch prunes normally. To our knowledge, this represents the first demonstration of branch-specific control of pruning in this system. Moreover, the adult medial branch regrows normally, indicating that pruning and regrowth are independently regulated at the level of individual branches. We demonstrate that this unique branch-specificity arises from trans-neuronal interactions between Dpr4 in {gamma}-KCs and DIP-{theta} in dopaminergic neurons that selectively innervate the vertical larval MB lobe. Furthermore, our findings suggest that this phenotype relies on an Ig2 domain of a Dpr family member, implying the involvement of a third binding partner. Leveraging this robust overexpression phenotype to probe downstream mechanisms, we find that loss of the transmembrane adhesion protein N-Cadherin suppresses the Dpr4-induced pruning defect. Together, our findings highlight the local impact of Dpr/DIP-mediated trans-neuronal interactions on the spatial regulation of remodeling, and provide genetic evidence implicating N-Cadherin as a potential downstream mediator of Dpr/DIP function within a developing neural circuit.

Novel (nua) Kinase 1 (NUAK1) encodes a serine-threonine protein kinase, mutations in which are associated with autism spectrum disorder. Direct phosphorylation targets of NUAK1 have been elusive hindering mechanistic understanding of its role in brain development. Here, we characterize autism-associated NUAK1 variants and show their differential impact on catalytic activity and subcellular distribution. We engineered ATP-analog sensitive NUAK1 and utilized its specificity towards bulky analogs to identify over 30 hitherto unknown direct phosphorylation targets of NUAK1. We demonstrate that Pleckstrin Homology and Sec7-domain containing protein 3 (PSD3) is a bona fide phosphorylation target of NUAK1. A guanine exchange factor (GEF) for ARF6 GTPase, PSD3 is phosphorylated by NUAK1 at S476. Expression of phosphodeficient PSD3 leads to aberrant activation of ARF6 and generation of PI(4,5)P2 that accumulates in intracellular vesicles. In neurons, phosphomutant PSD3 leads to enhanced spine maturation in an ARF6 dependent fashion. This study reveals direct neuronal substrates of an autism risk gene NUAK1, and delineates a mechanism by which PSD3 phosphorylation regulates ARF6 activation and spine maturation.