Biology subjects

Dong, Y.

Publications and source records attributed to Dong, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Discovery of A Small Molecule non-IMiD Degrader of ZBTB7A for the Treatment of β-hemoglobinopathies

Sickle cell disease and {beta}-thalassemia, two major {beta}-hemoglobinopathies, pose significant clinical challenges globally. Current treatments often face limitations in efficacy and tolerability. The transcription factor ZBTB7A has emerged as a promising therapeutic target for reactivating fetal hemoglobin expression. Here, we report the discovery and characterization of SH6, a small molecule non-IMiD degrader of ZBTB7A. SH6 induces fetal hemoglobin in erythroid cell lines in a CRBN and ZBTB7A-dependent manner, and it is capable of inducing fetal hemoglobin expression in healthy donor, SCD and {beta}-thalassemia patient CD34+ cell derived erythroid cells. The efficacy of SH6 is confirmed in a xenotransplantation humanized mouse model. SH6 outperforms currently available therapeutic agents in vitro, and shows synergy with hypomethylating agents. SH6 exhibits a favorable in vivo toxicity profile. Our findings establish SH6 as a promising therapeutic lead candidate for further optimization towards clinical development for treatment of sickle cell disease and {beta}-thalassemia.

cell biology↗

bioRxiv2025

Impact of Membrane Fluidity on α-syn Fibril Structures and Neuronal Pathology

Conformational variations in -syn fibrils are thought to underlie the distinct clinical features of synucleinopathies, including Lewy body dementia (LBD), Parkinsonss disease (PD), and multiple system atrophy (MSA), suggesting that distinct fibril structures act as molecular fingerprints linked to disease phenotype. While the origins of these conformational variations remain unclear, increasing evidence points to membranes as key modulators of fibrils conformations. In this study, we investigated how age-related alterations in membrane composition and fluidity influence -syn fibril formation and cellular outcomes. Using complex mixture membranes that mimic normal neuronal membranes and their age-related modifications in fatty acid chains, we found that -syn fibrils grown with these membranes displayed distinct 2D ssNMR spectral patterns compared to lipid-free -syn fibrils, reflecting differences in rigid fibril cores. Moreover, fibrils grown with age-related membranes exhibited weaker membrane association than those grown with normal neuronal membranes. These membrane-associated fibrils induce stronger neuronal pathologies than lipid-free fibrils, though the severity differed in intraneuronal aggregation and inflammation responses. Overall, our findings provide new insights into how age-related changes in membrane composition shape -syn fibril structure and pathogenicity, strengthening the link between membrane dynamics and amyloid-driven neurodegeneration.

molecular biology↗

bioRxiv2025

Resolving the estrogen paradox in hereditary retinal degeneration: Esr1 activation suppresses Tnf-α signaling as a photoreceptor self-protection mechanism

Retinitis pigmentosa (RP) is an inherited retinal degenerative disorder characterized by progressive photoreceptor loss and irreversible blindness. Increasing evidence implicates neuroinflammation as a contributor to photoreceptor degeneration extending beyond the initial genetic insult. Although estrogen has been reported to exert anti-inflammatory effects in the central nervous system, its role in RP remains controversial, with some studies suggesting a paradoxical exacerbation of retinal pathology. To address this discrepancy, we identify estrogen receptor alpha (Esr1) as a central immunoregulatory hub in RP. Transcriptomic analyses of rd1 and rd10 revealed upregulation of estrogen-responsive and inflammatory pathways, with Esr1 expression markedly elevated during degeneration. TUNEL assays demonstrated that systemic estradiol (E2) exerted divergent effects, protective in rd1 yet deleterious in rd10, whereas selective pharmacological activation of Esr1 with propyl pyrazole triol (PPT) consistently reduced photoreceptor death, preserved dark-adapted ERG responses, and downregulated inflammatory mediators including Tnf-, Cx3cl1/Cx3cr1, Cd68, and Iba1. Mechanistically, Esr1 activation repressed microglial Tnf transcription and disrupted a self-sustaining Cx3cl1/Cx3cr1-Tnf- signaling loop driving microglial recruitment, activation and neurotoxicity in the outer nuclear layer (ONL). Targeted interventions confirmed tumor necrosis factor receptor 1 (Tnfr1) as the principal mediator of Tnf-induced photoreceptor death: selective inhibition with R7050 conferred superior protection compared with broad-spectrum Tnf- inhibitors (etanercept, infliximab). Cx3cr1 blockade likewise suppressed microglial activation and improved visual outcomes. Collectively, our findings establish Esr1 activation as not merely an external intervention but the amplification of an intrinsic self-protective program, positioning Esr1, Tnfr1, and Cx3cr1 as actionable therapeutic targets to suppress neuroinflammation and preserve vision in RP and related retinal disorders.

neuroscience↗

bioRxiv2025

Metabolic Immunosuppression Mediated by Proliferative CD168+TAMs in Colon Cancer

The role of non-monocyte-derived, in situ proliferating tumor associated macrophages (TAMs) in tumor progression has been underappreciated. Proliferative TAM subsets likely represent critical mediators in establishing the immunosuppressive microenvironment, given the susceptibility of TAMs to be hijacked by malignant cells for pro-tumorigenic functions. However, the regulatory networks governing their self-renewal circuitry and lineage bifurcation remained undefined. Combining single cell RNA sequencing of human and murine colon tumors, we identified CD168+ TAMs, a hyperproliferative subpopulation with FOXM1-dependent maintenance of replicative competence. Spatial multi-omics and functional validation revealed that this subset exhibited peritumoral localization and underwent immunosuppressive polarization through cholesterol-coordinated CX3CL1-CX3CR1 signaling. Colon cancer-specific lipid metabolic rewiring elevates glycerophospholipid and cholesterol flux, with PLA2G7-mediated glycerophospholipid catabolism promoting CX3CR1 expression in TAMs. Thus, the CX3CR1 signaling-driven immunosuppressive effect of CD168+ TAM is an integrated response to tumor-specific metabolic features. Targeting the CD168+ TAMs may enable therapeutic strategies for metabolic-immune reshaping in colon cancers.

cancer biology↗

bioRxiv2025

A comprehensive RNA virome from molluscan transcriptomes reveals extensive diversity and modular genome evolution

Mollusca is the second-largest animal phylum and an important marine food resource for humans. While DNA viruses that threaten molluscan aquaculture have received much attention, molluscan RNA viromes remain poorly explored. Here, based on 223 molluscan metatranscriptomes covering eight animal classes, we identified 80 species-level RNA viruses spanning three viral phyla and nine viral families. Phylogenetic result combined structural modeling found a major increase in the number of Pisuviricota-related lineages. Extensive modular evolution in viral genomes was observed including gene rearrangements and co-evolution of capsid and RdRP genes. Host prediction linked 78% of the RNA viruses to a diverse range of eukaryotes, including vertebrates, invertebrates, and algae. These findings expand the known diversity of RNA viruses in molluscs and shed light on their phylogenetic relationships, highlighting molluscs as valuable models for RNA virus evolution. Graphical abstractRNA virus diversity within 223 molluscan transcriptome samples were analyzed based on RdRP identification. Viruses belonging to the phylum Pisoviricota were the most abundant; co-evolution between RdRP and capsid proteins of molluscan Picornavirales was revealed, and a complex virus-host interaction network was constructed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/673676v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1ce8fc0org.highwire.dtl.DTLVardef@3f9068org.highwire.dtl.DTLVardef@121cc2forg.highwire.dtl.DTLVardef@1e431a0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIComprehensive metatranscriptomic analysis found extensive RNA viral diversity in molluscs. C_LIO_LIPisuviricota dominate the molluscan RNA virome, substantially broadening known viral lineages. C_LIO_LIModular genome evolution involving gene rearrangements and conserved structural features characterize molluscan RNA viruses. C_LIO_LIMolluscs potentially serve as major ecological reservoirs for diverse RNA viruses infecting various eukaryotic hosts. C_LI

bioinformatics↗

bioRxiv2025

Segmented poly(A) tails with microRNA target sites confer tissue-specific regulation for mRNA therapeutics

Targeted delivery and controlled expression of mRNA-LNPs are critical for the development of safe and effective mRNA medicines. However, efficient post-delivery regulation of mRNA-LNP expression remains challenging. In this study, we engineered segmented poly(A) tail variants that function as gene-specific regulatory elements for synthetic mRNAs. Specifically, we inserted microRNA target sites (MTS) for miR-122 or miR-142 at various positions of the poly(A) tail of synthesized luciferase reporter mRNA. These modifications significantly reduced luciferase expression in non-target tissues in vitro and in vivo, demonstrating position-dependent selective expression control. Furthermore, by incorporating triple-MTS sequences for miR-142, miR-126, and miR-148a in all possible combinations at the 5 end of the poly(A) tail, we identified triple-MTS arrangements that simultaneously decrease luciferase activities in three non-target hepatic cell types, while preserving robust expression in hepatocytes. The results highlighted the importance of MTS insertion order for optimal mRNA silencing. These triple-MTS modules significantly expanded the utility of single miRNA-responsive elements, enabling cell-type selective regulation for mRNA therapeutics. By complementing tissue-tropic delivery LNPs, our mRNA cargo regulatory elements have the potential to improve tissue and cell type selectivity as a novel platform for post-delivery regulation of mRNA-LNP.

bioengineering↗

bioRxiv2025

Angptl5 restricts primitive hematopoiesis by modulating retinoic acid signaling in zebrafish

Homeostasis is essential for hematopoiesis, and its dysregulation can lead to severe pathological conditions. Retinoic acid (RA) is a key regulator that exerts concentration-dependent effects on both embryonic and adult hematopoiesis. However, the mechanisms that modulate RA signaling in hematopoietic processes remain poorly understood. Using zebrafish as a model, we identified angiopoietin-like protein 5 (Angptl5) as a critical regulator of hematopoietic homeostasis. Loss of Angptl5 function resulted in myeloid hyperplasia in the anterior lateral plate mesoderm (ALPM) and anterior expansion of erythroid progenitors in the posterior lateral plate mesoderm (PLPM)-- phenotypes consistent with attenuated RA signaling. Molecular analyses confirmed impaired RA signaling in angptl5{Delta}10/{Delta}10 mutants, and exogenous RA supplementation fully rescued the hematopoietic defects. Mechanistically, we found that Angptl5 transcriptionally activates retinol dehydrogenase dhrs9 through its interaction with Integrin 6l{beta}5. Our findings establish Angptl5 as a novel and essential regulator of embryonic hematopoiesis and reveal a previously unrecognized mechanism controlling hematopoietic homeostasis. These insights position Angptl5 as a potential therapeutic target for hematological disorders.

developmental biology↗

bioRxiv2025

Cross-ancestry information transfer framework improves protein abundance prediction and protein-trait association identification

Genetics-informed proteome-wide association studies (PWAS) provide an effective way to map the complex molecular landscape of biological mechanisms for complex diseases. PWAS relies on an ancestry-matched reference panel to model protein expression using genetic variants as features and determine the proteins impact on phenotype. However, reference panels from underrepresented populations remain relatively limited. In this study, we developed an analytic framework that borrows information from potentially multiple ancestries to boost the protein abundance prediction accuracy in an underrepresented population. We illustrate the frameworks utility and reproducibility through application to PWAS in East Asians: BioBank Japan (BBJ), Korean Genome and Epidemiology Study (KoGES), and Taiwan Biobank (TWB). An ensemble of information-sharing approaches was integrated to build the Multi-Ancestry-based Best-performing Model (MABM). MABM substantially improved the prediction performance with higher performance observed in both cross-validation and an external validation dataset (Tongji-Huaxi-Shuangliu Birth Cohort). Leveraging the BBJ, we identified three times as many significant PWAS associations with MABM as with the baseline Lasso model. Notably, 47.5% of the MABM specific associations were reproduced in independent East Asian datasets with concordant effect sizes. Furthermore, MABM enhanced gene/protein prioritization for downstream functional validation by (1) confirming a greater number of well-established gene/protein-trait associations and (2) identifying previously uncharacterized trait-associated genes. The benefits of MABM were further validated in additional ancestries and demonstrated in brain tissue-based PWAS, underscoring its broad applicability. Our findings close critical gaps in multi-omics research, develop a new reference resource of genetic models of protein abundance, and facilitate trait-relevant protein discovery in underrepresented populations.

genomics↗

bioRxiv2025

Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

In planta gene expression via exogenous mRNAs has a wide range of potential biotechnological applications from genome editing to plant protection and to circumventing tissue culture. Yet, regarding mRNA delivery into intact plant cells, the purity, stability and translational efficiency of exogenous mRNAs appear as obstacles for effective exogenous mRNA application. Our study investigates the potential of exogenous mRNAs in mediating protein synthesis in plant cells. We applied unformulated, unmodified green fluorescent protein (GFP)-coding mRNA to monocot (Triticum aestivum, Hordeum vulgare, Zea mays) crops and dicot model plant Arabidopsis thaliana and observed GFP signals in all species, confirming the successful uptake and translation of mRNA into protein. This approach opens possibilities for broader, GM-free applications in plant science.

plant biology↗

bioRxiv2025

MOH: a novel multilayer multi-omics heterogeneous graph for single-cell clustering

Cell clustering is crucial in single-cell multi-omics research for identifying distinct cellular populations. Although there has been progress in integrating multi-omics data for clustering, combining more than two types of omics data remains challenging due to the diversity and heterogeneity of these datasets. Traditional approaches typically use heterogeneous graphs that integrate only two types of omics data, constructing graphs with genes and cells as nodes and a single type of edge representing their relationships. However, this method has limitations as it overlooks cell-cell interactions and struggles to capture complex cellular dynamics. Additionally, the graph structure must be redesigned whenever new omics data are introduced, limiting the scalability of these models. To address these issues, we introduce MOH, a novel single-cell clustering algorithm based on a multilayer multi-omics heterogeneous graph. MOH integrates three key single-cell omics types: scRNA-seq, scATAC-seq, and spatial transcriptomics. It constructs a multilayer heterogeneous graph to simultaneously extract and enhance representations from all three omics layers, incorporating both intra-layer and inter-layer edges to capture association and similarity relationships. This enriched representation leads to an accurate clustering results. Extensive experiments show that MOH outperforms six state-of-the-art methods on unsupervised clustering metrics, offering a precise and comprehensive analysis with consistent improvements across all evaluation criteria. Moreover, downstream analyses validate the results, revealing novel biological insights into immune disorder complications in cancer, cancer drug repurposing, and new signaling pathways, which merit further investigation and validation.

bioinformatics↗

bioRxiv2025

Aging-enhanced accumulation of fibroblasts excludes oligodendrocytes in demyelinated lesions

Fibroblast dysregulation contributes to pathological fibrosis and aberrant repair. Emerging evidence suggest that fibroblasts accumulate in lesions following central nervous system injury, but whether and how they influence oligodendrocyte repair responses, including in aging, is uncertain. Here we report that fibroblasts accumulate in the parenchyma of spinal cord white matter lesions of 6 - 10 week old young mice after lysolecithin-induced demyelination. This was first observed through immunofluorescence microscopy that employed several markers attributed to fibroblasts, including platelet-derived growth factor-{beta}, collagen type 11, -smooth muscle actin, periostin and fibronectin; and by the use of platelet-derived growth factor-{beta} TdTomato reporter transgenic mice. Single-nucleus and spatial transcriptomics of lysolecithin lesions established the presence of fibroblasts in lysolecithin lesions and delineated them from closely related pericytes. CellChat ligand - receptor analyses highlight fibroblasts in the lysolecithin environment as a major source of input of signals for microglia/macrophages and oligodendrocyte precursor cells, with numerous reciprocal interactions. The infiltration of fibroblasts was promoted by microglia/macrophages, as anticipated by their temporal representation in lysolecithin lesions, and by tissue culture experiments where the migration of fibroblasts was enhanced by macrophages. Of particular relevance to spontaneous regenerative events in lysolecithin demyelination, areas of fibroblast accumulation were devoid of oligodendrocyte precursor cells. In tissue culture, oligodendrocyte precursor cells were excluded from fibroblast domains. Moreover, fibroblast accumulation after lysolecithin injury was enhanced with increasing age, a known detriment to the capacity to remyelinate after injury, and exclusion of oligodendrocyte precursor cells from fibroblast areas of 48 - 52 week mice exceeds that occurring in younger 6 - 10 weeks animals. Finally, by mining a publicly available single-nucleus RNA database of multiple sclerosis, we found fibroblasts in the edge of chronic active and chronic inactive lesions and in lesion core, and fewer in periplaque or normal white matter. We identified several communication networks between fibroblasts, microglia/macrophages and oligodendrocyte precursor cells in these MS lesions. Our collective results demonstrate a role of fibroblasts in demyelination-associated neuropathology, which is exacerbated by aging, and highlight the importance of regulating fibroblasts to promote effective CNS repair.

neuroscience↗

bioRxiv2025

Remodeling of the immune-metabolic landscape triggered by long-term high-altitude exposure

Growing evidence indicates that immunological and metabolic outcomes are key mediators of long-term high-altitude exposure (LTHAE) adaption, but the underlying mechanisms remain poorly understood. This study employs plasma metabolomics and peripheral blood single-cell transcriptomic sequencing to analyze the metabolic and immune dynamic regulation in 46 young male lowlanders following a 90-day adaptation period at high altitude. Single-cell analysis shows a pattern of "innate immune activation and adaptive immune suppression" under LTHAE, characterized by facilitated maturation of neutrophils, enhanced cytotoxicity of CD56dim NK cells, and increased immune responsiveness of cDC2 and pDC, while inhibited maturation of plasmablasts and suppressed immune responsiveness of CD8{square}TEM and CD4+ T cells. Plasma metabolic analysis reveals significant alterations, involving enhanced steroid hormone synthesis, unsaturated fatty acid and amino acid metabolism under LTHAE, which in turn are associated with immune remodeling. Moreover, transcriptomic-metabolic integration analysis indicates the molecular mechanisms of enhanced aerobic oxidation efficiency under LTHAE. Collectively, these findings provide integrated insights into immune-metabolic landscape remodeling and suggest potential mutual regulatory relationship between immune and metabolic state following LTHAE, offering a molecular foundation for high-altitude adaptation research.

genomics↗

bioRxiv2025

Receptor Tyrosine Kinase Profiling Identifies Chronic Constitutive Floodgate Oxidative Signaling in Glutathione-Independent Human Mammary Luminal Progenitor Cells

The human mammary epithelium contains a subset of luminal progenitor (LP) cells that are distinct from basal cells in both lineage potential and redox biology. LPs are uniquely equipped to tolerate oxidative stress through glutathione-independent mechanisms and have been implicated as candidate cells of origin in basal-like breast cancers. In this study, we identify the receptor tyrosine kinase (RTK) cKIT (CD117), as a defining feature of LPs and a key mediator of their expansion. cKIT is developmentally restricted to the LP compartment via Polycomb-mediated epigenetic repression in basal and luminal-committed cells. It is expressed in scattered epithelial cells within both ductal and alveolar regions of resting human mammary glands. Using RTK-engineered MCF10A models, we demonstrate that cKIT ligand/stem cell factor (SCF)-activated wildtype cKIT signaling is sufficient to drive proliferation in the absence of epidermal growth factor (EGF) and that cKIT is responsive not only to canonical ligands but also to hydrogen peroxide (H2O2). In primary human LPs, cKIT is rapidly phosphorylated upon exposure to SCF and H2O2, with concomitant AKT activation. These responses are enhanced when cKIT and EGFR signaling are co-engaged, suggesting a cooperative mitogenic program. In mammary gland, phosphorylation of the antioxidant enzyme PRDX1 is selectively detected in LPs, consistent with a floodgate model of redox signaling in which transient oxidative inactivation of peroxiredoxins (PRDXs) facilitates RTK signaling under elevated intracellular reactive oxygen species conditions. Clinically, elevated cKIT expression is associated with shorter progression-free survival in certain basal-like breast cancer, supporting a link between LP-like redox signaling states and aggressive tumor behavior. Together, these findings define a redox-integrated RTK signaling axis centered on cKIT that drives LP expansion and is associated with poor outcomes in a subset of basal breast cancers. This work establishes a mechanistic framework for targeting redox-responsive progenitor populations in both regenerative and oncologic context.

cancer biology↗

bioRxiv2025

Construction of recombinant Bacillus subtilis spores surface-displaying PRRSV ORF1b, GP5, M, and N proteins in tandem and evaluation of their immunogenicity in mice

Porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded RNA virus, is a highly contagious pathogen that causes severe reproductive and respiratory disorders in pigs, leading to significant economic losses in the swine industry worldwide. However, current commercial vaccines provide only limited protection against circulating PRRSV strains, highlighting the urgent need for novel vaccine strategies. B. Bacillus subtilis, a well-characterized probiotic, has emerged as a promising platform for mucosal vaccine delivery due to its safety profile and ability to induce robust immune responses. In this study, we screened B cell and T cell linear epitopes from PRRSV-ORF1b, GP5, M, and N proteins to construct a recombinant tandem antigen (ER), which was displayed on the surface of B. subtilis 168 spores. The resulting recombinant strain, designated B. subtilis BE, was evaluated for immunogenicity in mice via oral administration. The results showed that Oral immunization with B. subtilis BE significantly elevated antigen-specific secretory IgA levels in intestinal contents and IgG levels in serum, indicating potent mucosal and systemic humoral responses. Moreover, B. subtilis BE induced measurable neutralizing antibody titers and enhanced cellular immunity, as evidenced by increased frequencies of CD3+ and CD8+ T cells in mesenteric lymph nodes and upregulation of IFN-, TNF-, and IFN-{gamma} expression in intestinal tissues. Collectively, these findings demonstrate that B. subtilis BE elicits both humoral and cellular immune responses and may serve as a novel oral vaccine candidate against PRRSV, offering a promising alternative for controlling the ongoing epidemic.

immunology↗

bioRxiv2025

Targeting ZNRF3 and RNF43 to Restore Regeneration and Reverse Metabolic Dysfunction-Associated Steatotic Liver Disease

Liver regeneration and hepatocyte metabolic identity are disrupted in metabolic dysfunction-associated steatotic liver disease (MASLD) and its advanced form, metabolic dysfunction-associated steatohepatitis (MASH), yet the mechanisms of restore liver regeneration and reprogram metabolism for disease reversal remains poorly understood. Here, we show that {beta}-Catenin activity progressively declines in hepatocytes during MASH in both humans and mice, coinciding with impaired regeneration and defective lipid clearance. Targeted deletion of the endogenous WNT pathway inhibitors ZNRF3 and RNF43 in hepatocytes after MASH onset reactivates {beta}-Catenin signaling, leading to robust regression of steatosis, inflammation, and fibrosis, and restoring regenerative capacity across multiple fatty liver disease models. Mechanistically, this therapeutic effect is driven by {beta}-Catenin-dependent induction of the alternative bile acid synthesis pathway without disrupting systemic lipid homeostasis. Importantly, both short-and long-term deletion of ZNRF3/RNF43 restores liver function without triggering tumorigenesis or hepatotoxicity, indicating a safe therapeutic window. These findings reveal that physiological activation of WNT/{beta}-catenin signaling via ZNRF3 and RNF43 offers a viable regenerative and metabolic strategy for reversing fatty liver disease.

cell biology↗

bioRxiv2025

Almost Free Enhancement of Multi-Population PRS: From Data-Fission to Pseudo-GWAS Subsampling

Many multi-population polygenic risk score (PRS) methods have been proposed to improve prediction accuracy in underrepresented populations; however, no single method outperforms other methods across all data scenarios. Although integrating PRS results across multiple methods and populations may lead to more accurate predictions, this approach may be limited by the availability of individual-level tuning data to calculate combination weights. In this manuscript, we introduce MIXPRS, a robust PRS integration framework based on data fission principles, to effectively combine multiple multi-population PRS methods using only genome-wide association study (GWAS) summary statistics from multiple populations. Specifically, MIXPRS employs SNP pruning to mitigate linkage disequilibrium (LD) mismatch between the training GWAS summary statistics and LD reference panels, and utilizes non-negative least squares regression to robustly estimate PRS combination weights. Extensive simulations and real-data analyses involving 22 continuous traits and four binary traits across five populations from the UK Biobank and All of Us datasets demonstrate that MIXPRS consistently outperforms the existing methods in prediction accuracy. Because MIXPRS relies solely on GWAS summary statistics, it enjoys broad accessibility, robustness, and generalizability for underrepresented populations.

genetics↗

bioRxiv2025

Astrocytic CREB regulates transcriptional, neuronal, and behavioral responses to cocaine

Drug addiction is characterized by neuronal adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. Emerging evidence increasingly indicates that astrocytes are also involved in nervous system disorders, including addiction, but the cocaine-induced astrocyte-specific transcriptome has not yet been investigated. We utilized whole cell sorting of astrocytes, RNA-sequencing, and bioinformatic approaches to characterize the astrocyte transcriptome in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, prolonged abstinence, and "relapse" in male mice. We found that astrocytes exhibit robust and contextually-specific transcriptional signatures that converge strongly with human cocaine use disorder. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator of cocaine-induced transcriptional regulation in NAc astrocytes, and CUT&RUN-sequencing mapped increased CREB binding across the astrocyte genome in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes, in combination with several measures of addiction-related behaviors including conditioned place preference and self-administration, revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in astrocytic CREB activity or CPP found in females. Subsequent experiments identify potential molecular mechanisms of astrocytic CREBs influence through modulating astrocytic Ca2+ signaling in response to cocaine. Finally, we show that astrocytic CREB selectively modulates D1-type medium spiny neurons in NAc to control cocaine-related behaviors. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine and that CREB mediates cocaines effects on gene expression in astrocytes, with consequent effects on neuronal activity and rewarding responses to the drug.

neuroscience↗

bioRxiv2025

Engineering gene expression dynamics via self-amplifying RNA with drug-responsive non-structural proteins

The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug- regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.

bioengineering↗