Biology subjects

Gan, L.

Publications and source records attributed to Gan, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Pathogenic tau inhibits synaptic plasticity by blocking eIF4B-mediated local protein synthesis

Activity-dependent modulation of synaptic strength is critical for encoding memories and it is inhibited in tauopathies including Alzheimers disease (AD) and Frontotemporal lobar degeneration with tau inclusions (FTLD-tau). Pathogenic tau accumulates in neurons where it obstructs synaptic plasticity. How tau blocks synaptic plasticity leading to memory loss is unclear. Here, we show that FTLD-tau inhibits plasticity by blocking activity-dependent protein synthesis in dendrites. In the plasticity-associated translatome, we identified a subset of downregulated translated mRNAs in FTLD-tau neurons that encode postsynaptic plasticity regulators. Protein synthesis was blocked by FTLD-tau binding to eIF4B which caused eIF4B dissociation from the translation initiation complex and reduced dendritic eIF4B levels. Inhibiting the tau-eIF4B interaction or enhancing eIF4B levels in FTLD-tau neurons restored local protein synthesis and synaptic plasticity. Together, this suggests that pathogenic tau binding to eIF4B disables the local synthesis of plasticity-related proteins that drive synapse strengthening and memory formation.

neuroscience↗

bioRxiv2025

High-Throughput Screening and Initial SAR Studies Identify a Novel Sub-micromolar Potent Human cGAS Inhibitor

Cyclic GMP-AMP synthase (cGAS) has emerged as a promising therapeutic target of several human diseases, including Alzheimers disease (AD) and other neurodegenerative disorders. As a cytosolic DNA sensor, cGAS generates an innate immune response to promote neuroinflammation by producing an endogenous agonist of the stimulator of interferon genes (STING), 23-cyclic GMP-AMP (cGAMP), which activates the cGAS-STING pathway. We have performed a high-throughput screening of a chemical library containing over 300K small molecules at the Fisher Drug Discovery Resource Center (DDRC), Rockefeller University (RU), to identify multiple hit inhibitors of human (h)-cGAS. We used a modified Kinase Glo(R) Luminescent Kinase assay, which was earlier developed at RU and later used by multiple groups, including ours, to perform primary screening of the library using h-cGAS. The hit candidates bearing novel scaffolds are structurally diverse and exhibited in vitro activity in the low micromolar range. RU-0610270 or compound (cpd) 1, a sulfonamide derivative, is one of the most potent hits (IC50=1.88 {micro}M), selected for hit expansion and structure-activity relationship (SAR) analysis. We synthesized new analogs of cpd 1 and evaluated them in vitro against h-cGAS to identify cpd 6 (IC50=0.66 {micro}M) as the most potent hit analog. We further profiled cpd 6 and found that it modestly inhibited cGAMP levels by 29% at 30 {micro}M in THP1 cells without detectable toxicity, and by 76% at 100 {micro}M, albeit with a moderate decrease ([~]20%) in cell viability. These results highlight a novel chemical series with promising in vitro activity, providing a starting point for the development of selective and potent human cGAS inhibitors for clinical use.

neuroscience↗

bioRxiv2025

NSF is required for diverse endocytic modes by promoting fusion and fission pore closure in secretory cells

The ATPase N-ethylmaleimide-sensitive factor (NSF), known for disassembling SNARE complexes, plays key roles in neurotransmitter release, neurotransmitter (AMPA, GABA, dopamine) receptor trafficking, and synaptic plasticity, and its dysfunction or mutation is linked to neurological disorders. These roles are largely attributed to SNARE-mediated exocytosis. Here, we reveal a previously unrecognized role for NSF: mediating diverse modes of endocytosis--including slow, fast, ultrafast, overshoot, and bulk--by driving closure of both fusion and fission pores. This function was consistently observed across large calyx nerve terminals, small hippocampal boutons, and chromaffin cells using capacitance recordings, synaptopHluorin imaging, electron microscopy, and multi-color pore-closure imaging. Results were robust across four NSF inhibitors, gene knockout, knockdown, and specific mutations. These findings establish NSF as a central regulator of membrane fission, kiss-and-run fusion, endocytosis, and exo-endocytosis coupling--offering new mechanistic insights into its diverse physiological and pathological roles in synaptic transmission, receptor trafficking, and neurological diseases.

neuroscience↗

bioRxiv2025

Single-cell transcriptome sequencing for opening the blood-brain barrier through specific mode electroacupuncture stimulation

BackgroundThe blood-brain barrier (BBB) interferes with the treatment of central nervous system disorders owing to the complexity of its structure and restrictive function. Thus, it is challenging to develop central nervous system drug delivery strategies. Specific mode electroacupuncture (EA) stimulation can effectively open the BBB in rats. MethodsHere, we used single-cell RNA sequencing (scRNA-seq) to comprehensively map the cell population in the Sprague-Dawley rat cerebral cortex. ResultsWe identified 23 cell subsets and eight types of cells in the brain by cell annotation. scRNA-seq revealed transcriptional changes in the cerebral cortex under EA. ConclusionsOur findings offer valuable insights into the molecular and cellular modifications in the brain resulting from EA intervention and serve as a resource for drug delivery across healthy and diseased states. Innovative approaches to enhance BBB opening will lead to more effective therapeutic plans and enhanced drug delivery.

neuroscience↗

bioRxiv2025

Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

Tumor-derived extracellular vesicles primarily carry PD-L1 via exosomes, which interact with PD-1 receptors on T cells, impacting immune responses in the tumor microenvironment and beyond, leading to a more extensive immunosuppressive landscape. However, the mechanisms governing exosomal PD-L1 sorting and secretion remain elusive. In this study, we identified Munc13-4 as a crucial regulator of exosomal PD-L1 sorting and secretion. Deletion of Munc13-4 in breast tumors enhances T cell-mediated anti-tumor immunity, suppresses tumor growth, and improves the efficacy of immune checkpoint inhibitors. Our results illustrate how Munc13-4 collaborates with HRS, Rab27, and SNAREs to facilitate PD-L1 sorting and secretion via exosomes. The cryo-EM structure of the Munc13-4-Rab27a complex provide new insights into its role in exosome secretion. Importantly, we discovered that Munc13-4 has a novel role in sorting PD-L1 onto exosomes, which relies on the formation of a ternary complex with PD-L1 and HRS. In addition, IFN{gamma} stimulation modifies Munc13-4 and HRS, establishing a dynamic regulatory mechanism that enables tumor cells to adapt to immune pressure by modulating PD-L1 sorting. Using a specially designed peptide to disrupt the Munc13-4-PD-L1 interaction and impede PD-L1 sorting significantly enhances anti-tumor immunity and slows tumor growth in vivo. These results highlight the potential of targeting the Munc13-4-PD-L1 axis to suppress tumor immune evasion.

cell biology↗

bioRxiv2025

Isl1 Controls Axon Pathfinding of Retinal Ganglion Cells in the Binocular Visual Pathway

Axons of retinal ganglion cells (RGCs) face a choice to project ipsilaterally or contralaterally at the optic chiasm. This decision is crucial for the establishment of binocular vision in mammals. The transcription factor ZIC2 is well known as a key determinant of ipsilateral RGC identity, however the transcriptional programs controlling contralateral RGC identity have only begun to be elucidated more recently. Here we show that inactivation of the LIM-HD transcription factor ISL1 results in an expansion of the ZIC2+ ventrotemporal (VT) domain of the retina and an increase in ipsilateral projections from outside the VT retina. RNA-Seq, gene expression and CUT&Tag analyses show that ISL1 regulates RGC laterality through a set of unique downstream effectors as well as a few common ones, as other transcription factors. Our data also show that RGC axons misrouted at the midline in Isl1-null mutants could innervate the appropriate eye-specific regions of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC), in agreement with earlier studies that have shown that eye specific targeting of visual nuclei in the brain is independent of laterality decisions at the optic chiasm.

neuroscience↗

bioRxiv2024

Identification and characterization of UBA5 inhibitors

UBA5 is a critical E1-activating enzyme in the UFMylation pathway, a post-translational modification process implicated in neurodegenerative diseases and cancers. In this study, we developed a high-throughput screening (HTS) assay to identify inhibitors of UBA5 from a library of blood-brain barrier (BBB)-permeable compounds. The assay, based on the AMP-Glo kit, enabled the identification of five novel UBA5 inhibitors belonging to three distinct chemical scaffolds with low micromolar IC50 values. These inhibitors demonstrated selectivity for UBA5 over other E1 enzymes, such as UBA1, and showed efficacy in inhibiting endogenous UFMylation in HEK293T cells. The identified inhibitors not only provided valuable tools for studying UFMylation but also represent potential therapeutic candidates for diseases associated with dysregulated UFMylation, such as Alzheimers disease and cancer. Further optimization of these compounds is necessary to enhance their potency and pharmacokinetic properties. Our findings highlight UBA5 as a promising therapeutic target and pave the way for the development of selective inhibitors that can modulate UFMylation in human diseases.

biochemistry↗

bioRxiv2024

Reciprocal regulation of m6A modification and miRNA production machineries via phase separation-dependent and -independent mechanisms

Methyltransferase complex (MTC) deposits N6-adenosine (m6A) onto RNA, whereas microprocessor produces miRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B (MTB) tends to form insoluble condensates with poor activity, with its level monitored by 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promotes solubility and stability of MTB, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behavior, exhibit reduced m6A level. Reciprocally, MTC can recruit microprocessor to MIRNA loci, prompting co-transcriptional cleavage of primary miRNA (pri-miRNAs) substrates. Additionally, pri-miRNAs carrying m6A modifications at their single-stranded basal regions are enriched by m6A readers, which retain microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

plant biology↗

bioRxiv2024

CREB determines the expression of ST2 in Tregs and mediates the balance between type 1 and type 2 immune responses

Regulatory T cells (Tregs) are gatekeepers of immune homeostasis and characterized by expression of Foxp3, which maintains Treg identity. Here we demonstrate that in mice with a Foxp3-specific knockout of CREB, enhanced numbers of Tregs are found in vivo in spleen, lung and colon. These Tregs display a reduced Foxp3 expression, but enhanced expression of the IL-33 receptor (ST-2), IL-10, IL-13, and CREM. CREB deficient Tregs were highly suppressive in vitro and prevented disease activity in CD4 T cell mediated transfer colitis in an IL-10 dependent way. Mechanistically CREB fulfils dual roles in Tregs. First it downregulates Foxp3 expression, however in cooperation with CREM, CREB expression in Tregs alters chromatin accessibility to the ST-2 region and thereby influences T cell specific immune responses mediated by IL-10. Brief summary: Mice with a Foxp3-specific knockout of CREB display enhanced expression of IL-13, IL-10, ST-2 and CREM, which prevents gut inflammation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/601312v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a2d341org.highwire.dtl.DTLVardef@1db6852org.highwire.dtl.DTLVardef@19e024borg.highwire.dtl.DTLVardef@a8c84d_HPS_FORMAT_FIGEXP M_FIG C_FIG Created by Biorender

immunology↗

bioRxiv2024

A host enzyme reduces non-alcoholic fatty liver disease by inactivating intestinal lipopolysaccharide

The incidence of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) has been increasing world-wide. Since gut-derived bacterial lipopolysaccharides (LPS) can travel via the portal vein to the liver and play an important role in producing hepatic pathology, it seemed possible that (1) LPS stimulates hepatic cells to accumulate lipid, and (2) inactivating LPS can be preventive. Acyloxyacyl hydrolase (AOAH), the eukaryotic lipase that inactivates LPS and oxidized phospholipids, is produced in the intestine, liver, and other organs. We fed mice either normal chow or a high-fat diet for 28 weeks and found that Aoah-/- mice accumulated more hepatic lipid than did Aoah+/+ mice. In young mice, before increased hepatic fat accumulation was observed, Aoah-/-mouse livers increased their abundance of Sterol Regulatory Element-Binding Protein 1 (SREBP1) and the expression of its target genes that promote fatty acid synthesis. Aoah-/- mice also increased hepatic expression of CD36 and Fabp3, which mediate fatty acid uptake, and decreased expression of fatty acid oxidation-related genes Acot2 and Ppar-. Our results provide evidence that increasing AOAH abundance in the gut, bloodstream and/or liver may be an effective strategy for preventing or treating MASLD.

immunology↗

bioRxiv2024

APOE3-R136S mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition

The Christchurch mutation (R136S) on the APOE3 (E3S/S) gene is associated with attenuation of tau load and cognitive decline despite the presence of a causal PSEN1 mutation and high levels of amyloid beta pathology in the carrier1. However, the specific molecular mechanisms enabling the E3S/S mutation to mitigate tau-induced neurodegeneration remain unclear. Here, we replaced mouse ApoE with wild-type human E3 or E3S/S on a tauopathy background. The R136S mutation markedly decreased tau load and protected against tau-induced synaptic loss, myelin loss, and reduction in theta and gamma powers. Additionally, the R136S mutation reduced interferon response to tau pathology in both mouse and human microglia, suppressing cGAS-STING activation. Treating tauopathy mice carrying wild-type E3 with a cGAS inhibitor protected against tau-induced synaptic loss and induced similar transcriptomic alterations to those induced by the R136S mutation across brain cell types. Thus, suppression of microglial cGAS-STING-IFN pathway plays a central role in mediating the protective effects of R136S against tauopathy. One-sentence summaryThe R136S mutation on APOE3 enhances resistance to tau-related pathology and toxicity by downregulating the cGAS-STING-IFN signaling pathway.

neuroscience↗

bioRxiv2024

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer's disease.

Multimodal measurements have become widespread in genomics, however measuring open chromatin accessibility and splicing simultaneously in frozen brain tissues remains unconquered. Hence, we devised Single-Cell-ISOform-RNA sequencing coupled with the Assay-for-Transposase-Accessible-Chromatin (ScISOr-ATAC). We utilized ScISOr-ATAC to assess whether chromatin and splicing alterations in the brain convergently affect the same cell types or divergently different ones. We applied ScISOr-ATAC to three major conditions: comparing (i) the Rhesus macaque (Macaca mulatta) prefrontal cortex (PFC) and visual cortex (VIS), (ii) cross species divergence of Rhesus macaque versus human PFC, as well as (iii) dysregulation in Alzheimers disease in human PFC. We found that among cortical-layer biased excitatory neuron subtypes, splicing is highly brain-region specific for L3-5/L6 IT_RORB neurons, moderately specific in L2-3 IT_CUX2.RORB neurons and unspecific in L2-3 IT_CUX2 neurons. In contrast, at the chromatin level, L2-3 IT_CUX2.RORB neurons show the highest brain-region specificity compared to other subtypes. Likewise, when comparing human and macaque PFC, strong evolutionary divergence on one molecular modality does not necessarily imply strong such divergence on another molecular level in the same cell type. Finally, in Alzheimers disease, oligodendrocytes show convergently high dysregulation in both chromatin and splicing. However, chromatin and splicing dysregulation most strongly affect distinct oligodendrocyte subtypes. Overall, these results indicate that chromatin and splicing can show convergent or divergent results depending on the performed comparison, justifying the need for their concurrent measurement to investigate complex systems. Taken together, ScISOr-ATAC allows for the characterization of single-cell splicing and chromatin patterns and the comparison of sample groups in frozen brain samples.

genomics↗

bioRxiv2024

Alzheimer's disease-linked risk alleles elevate microglial cGAS-associated senescence and neurodegeneration in a tauopathy model

The strongest risk factors for Alzheimers disease (AD) include the {chi}4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H (R47H) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting disease-causing mechanisms. We find that the R47H variant induces neurodegeneration in female APOE4 mice without impacting hippocampal tau load. The combination of APOE4 and R47H amplified tauopathy-induced cell-autonomous microglial cGAS-STING signaling and type-I interferon response, and interferon signaling converged across glial cell types in the hippocampus. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.

neuroscience↗

bioRxiv2024

Deep Learning Analysis on Images of iPSC-derived Motor Neurons Carrying fALS-genetics Reveals Disease-Relevant Phenotypes

Amyotrophic lateral sclerosis (ALS) is a devastating condition with very limited treatment options. It is a heterogeneous disease with complex genetics and unclear etiology, making the discovery of disease-modifying interventions very challenging. To discover novel mechanisms underlying ALS, we leverage a unique platform that combines isogenic, induced pluripotent stem cell (iPSC)-derived models of disease-causing mutations with rich phenotyping via high-content imaging and deep learning models. We introduced eight mutations that cause familial ALS (fALS) into multiple donor iPSC lines, and differentiated them into motor neurons to create multiple isogenic pairs of healthy (wild-type) and sick (mutant) motor neurons. We collected extensive high-content imaging data and used machine learning (ML) to process the images, segment the cells, and learn phenotypes. Self-supervised ML was used to create a concise embedding that captured significant, ALS-relevant biological information in these images. We demonstrate that ML models trained on core cell morphology alone can accurately predict TDP-43 mislocalization, a known phenotypic feature related to ALS. In addition, we were able to impute RNA expression from these image embeddings, in a way that elucidates molecular differences between mutants and wild-type cells. Finally, predictors leveraging these embeddings are able to distinguish between mutant and wild-type both within and across donors, defining cellular, ML-derived disease models for diverse fALS mutations. These disease models are the foundation for a novel screening approach to discover disease-modifying targets for familial ALS.

neuroscience↗

bioRxiv2023

DAP12 deficiency alters microglia-oligodendrocyte communication and enhances resilience against tau toxicity

Pathogenic tau accumulation fuels neurodegeneration in Alzheimers disease (AD). Enhancing aging brains resilience to tau pathology would lead to novel therapeutic strategies. DAP12 (DNAX-activation protein 12) is critically involved in microglial immune responses. Previous studies have showed that mice lacking DAP12 in tauopathy mice exhibit higher tau pathology but are protected from tau-induced cognitive deficits. However, the exact mechanism remains elusive. Our current study uncovers a novel resilience mechanism via microglial interaction with oligodendrocytes. Despite higher tau inclusions, Dap12 deletion curbs tau-induced brain inflammation and ameliorates myelin and synapse loss. Specifically, removal of Dap12 abolished tau-induced disease-associated clusters in microglia (MG) and intermediate oligodendrocytes (iOli), which are spatially correlated with tau pathology in AD brains. Our study highlights the critical role of interactions between microglia and oligodendrocytes in tau toxicity and DAP12 signaling as a promising target for enhancing resilience in AD.

neuroscience↗

bioRxiv2023

Sex Chromosomes and Gonads Shape the Sex-Biased Transcriptomic Landscape in Tlr7-Mediated Demyelination During Aging

Demyelination occurs in aging and associated diseases, including Alzheimers disease. Several of these diseases exhibit sex differences in prevalence and severity. Biological sex primarily stems from sex chromosomes and gonads releasing sex hormones. To dissect mechanisms underlying sex differences in demyelination of aging brains, we constructed a transcriptomic atlas of cell type-specific responses to illustrate how sex chromosomes, gonads, and their interaction shape responses to demyelination. We found that sex-biased oligodendrocyte and microglial responses are driven by interaction of sex chromosomes and gonads prior to myelin loss. Post demyelination, sex chromosomes mainly guide microglial responses, while gonadal composition influences oligodendrocyte signaling. Significantly, ablation of the X-linked gene Toll-like receptor 7 (Tlr7), which exhibited sex-biased expression during demyelination, abolished the sex-biased responses and protected against demyelination. One-sentence summaryCell type-specific processes underlying aged demyelination are sex-biased and mediated by Tlr7.

neuroscience↗

bioRxiv2023

Early life high fructose exposure disrupts microglia function and impedes neurodevelopment

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment1-7. An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous systems (CNS) resident professional phagocyte8-10. Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo. Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

immunology↗

bioRxiv2023

Nanoscale analysis of human G1 and metaphase chromatin in situ

The structure of chromatin at the nucleosome level inside cells is mysterious. Here we present in situ electron cryotomography analyses of chromatin in both G1 and metaphase RPE-1 cells. G1 nucleosomes are concentrated in globular chromatin domains and metaphase nucleosomes are concentrated in the chromatids. Classification analysis reveals that canonical mononucleosomes, ordered stacked dinucleosomes, and mononucleosomes with a disordered gyre-proximal density are abundant in both cell-cycle states. Class averages that have more than two stacked nucleosomes or that have side-by-side dinucleosomes are not detected, suggesting that groups of more than two nucleosomes are heterogeneous. Large multi-megadalton structures are abundant in G1 nucleoplasm, but not found in G1 chromatin domains and metaphase chromatin. The macromolecular phenotypes studied here represent a starting point for the comparative analysis of compaction in normal and unhealthy human cells, other cell-cycle states, other organisms, and in vitro chromatin assemblies.

cell biology↗