Biology subjects

Saha, S.

Publications and source records attributed to Saha, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Rehabilitation drives functional reorganization of intact corticospinal-supraspinal projections following partial spinal cord injury

Spinal cord injury (SCI) disrupts corticospinal tract (CST) connectivity and impairs skilled voluntary movement. However, most human SCIs are anatomically incomplete, allowing spared CST pathways to engage in rehabilitation-mediated plasticity to promote functional recovery. How voluntary rehabilitation engages and reorganizes the supraspinal targets of the intact CST remains incompletely understood. Here, we combined unilateral pyramidotomy (uPyX) in male and female mice with continuous voluntary complex-wheel running to test whether fine motor-dependent rehabilitation drives supraspinal CST plasticity. uPyX mice rapidly resumed wheel running after a transient deficit. In contrast to lesion-only controls, rehabilitation significantly improved skilled forelimb performance on the horizontal ladder rung task. Immunohistochemical c-Fos labeling confirmed that complex-wheel running robustly activated the intact forelimb CST in motor cortex. Whole-brain CST projection mapping using intersectional viral vector tracing revealed targeted supraspinal reorganization localized to medullary motor nuclei. Three nuclei - the lateral paragigantocellular reticular nucleus (LPGi), gigantocellular reticular nucleus, alpha part (GiA), and ventral medullary reticular nucleus (MdV) - exhibited significant lesion- and/or rehabilitation-induced increases in CST innervation. Rehabilitation-driven CST sprouting correlated with regional c-Fos activation, indicating activity-dependent remodeling. Notably, CST projection density in the MdV, critical for skilled forelimb control, correlated with functional recovery. These findings identify a set of spinally-projecting medullary nuclei as key sites of rehabilitation-induced CST plasticity and highlight the MdV as a potential mediator of restored motor function. This work defines how voluntary rehabilitation reorganizes spared corticospinal pathways and provides targets for optimizing activity-based interventions after SCI. Significance StatementEffective rehabilitation after spinal cord injury (SCI) must harness the plasticity of spared motor pathways, yet the supraspinal circuits that support rehabilitation-mediated recovery remain unknown. Using a model that preserves voluntary motor engagement, we show that continuous fine motor-dependent rehabilitation activates intact corticospinal neurons and drives highly specific remodeling of their supraspinal terminals. Rehabilitation selectively strengthens CST inputs to motor regions of the medulla, particularly the ventral medullary reticular nucleus (MdV), and CST plasticity within this region predicts enhanced behavioral recovery. These findings highlight the MdV as a central locus by which rehabilitation re-establishes descending control of the impaired limb, providing mechanistic insight to guide targeted, circuit-based rehabilitation therapies for incomplete SCI.

neuroscience↗

bioRxiv2025

Preclinical immunogenicity of the LP.8.1-adapted BNT162b2 COVID-19 vaccine

SARS-CoV-2 evolution toward antigenically distinct lineages drives escape from host immunity. JN.1 lineage derivatives have recently dominated the global epidemiologic landscape. In preclinical models, an LP.8.1-adapted BNT162b2 vaccine elicited higher serum neutralizing antibody responses against contemporary, circulating JN.1 sublineages, including the currently dominant XFG, as compared to JN.1-, KP.2- and XEC-adapted vaccines. These findings supported the selection of an LP.8.1-adapted vaccine for the composition of the 2025-26 COVID-19 vaccine formula.

immunology↗

bioRxiv2025

Orthographic training finetunes a neuronal letter position code in primate visual cortex

Reading is an acquired skill that enhances brain activity in human visual cortex and is thought to repurpose preexisting ventral visual circuitry for the fast parallel processing of letter strings. However, the neuronal code underlying position-invariant written word recognition remains elusive. Here, we examined this issue in a macaque monkey model before and after orthographic training. Based on prior simulations of reading acquisition in a convolutional neural network model of the ventral visual pathway, we hypothesized the existence of neurons sensitive to both specific letters and their ordinal position within the word, which should be enhanced by orthographic training. By wirelessly recorded neural activity from the inferior temporal (IT) cortex of macaque monkeys trained on orthographic tasks over five successive days, we indeed discovered IT neurons tuned to letters and sensitive to either ordinal or retinotopic positions. Ordinal units existed prior to training, but their responses were enhanced after training, matching with behavioral improvements in word recognition. These findings support the neuronal recycling hypothesis and demonstrate, at the single-cell level, how reading refines pre-existing neural circuits to facilitate fluent word recognition.

neuroscience↗

bioRxiv2025

Single-cell phenomics through integrated imaging and molecular profiling

Single-cell technologies such as transcriptomics, microscopy, and flow cytometry have revolutionized the study of cellular identity and function. While each of these technologies is powerful on its own, their full potential lies in their integration, enabling multimodal profiling of the same cell and revealing how distinct modalities influence one another. Here, we introduce IRIS (Interconnected Robotic Imaging and Single cell transcriptomics), a deterministic single-cell platform technology that seamlessly couples high-resolution microscopy with droplet-based single-cell RNA sequencing. IRIS enables precise cell positioning, multimode imaging across brightfield and fluorescent channels, and subsequent molecular capture from the same cell, directly linking high-resolution morphological features to matched transcriptomes. We validate IRIS by recovering cell cycle progression states and transcriptional programmes associated with canonical morphologies and demonstrate its discovery power by molecularly resolving two nuclear-ER architectures within naive CD8+ T cells, each defined by distinct gene expression profiles and functional markers. IRIS establishes an integrative single-cell phenomics framework, opening new avenues for dissecting how cellular form relates to molecular state and function.

immunology↗

bioRxiv2025

Small molecule activator of phosphatase PP2A remodels scaffold PR65 structural dynamics to promote holoenzyme assembly

Small molecule activators of protein phosphatase 2A (PP2A), hereafter SMAPs, have attracted substantial interest, for their potential to inhibit cancer cell proliferation by targeting PR65, the scaffold subunit of the PP2A heterotrimer. PR65 is a uniquely flexible and stable molecule composed of 15 tandem HEAT (Huntingtin, Elongation factor 3-PP2A-TOR1) repeats. We characterized the binding sites and interactions of two SMAPs ATUX-8385 and DT-061 with PR65 and evaluated effects on PR65 structural dynamics using docking and molecular dynamics simulations. We initiated SMAP-bound PR65 simulations starting from two binding sites: S1, determined by cryo-electron microscopy for DT-061 bound to PP2A, on the inner helices of the HEAT repeats 2 and 3 (2i and 3i); and S2, predicted by docking of ATUX-8385 onto PR65, on 4i and 5i and outer helices 5o and 6o consistent with footprinting experiments. S2 proved to be a stable site for both SMAPs when initiating the simulations at S2. However, neither DT-061 nor ATUX-8385 demonstrated stable binding to S1. DT-061 rapidly dissociated from S1 to settle instead at a neighboring site S4 overlapping with our previously identified S3 for PR65 in extended form, suggesting that binding to S1 may be a 2-step process: an initial binding to PR65 alone, either to S3/S4 or S2, followed by movement to S3/S4, and then an induced relocation to S1 upon complexation with the regulatory and catalytic subunits. Targeted in silico mutagenesis showed that mutations at S2 and S4 destabilized SMAP binding to the PR65 (subunit). Heterotrimeric PP2A simulations showed that S3 and S4 binding were not persistent upon complexation. Together, these results corroborate our findings. Furthermore, preferentially stabilized a relatively extended PR65 conformation that would accommodate, if not promote, the assembly of the catalytic and regulatory subunits to prompt the activation of the trimeric phosphatase.

biophysics↗

bioRxiv2025

Characterization of a water-soluble formulation of Passiflora incarnata and its effect on sperm motility: A novel male-centric contraceptive formulation

Passiflora incarnata (Passionflower) has a long history of use in traditional medicine across South America and Europe, primarily for its sedative and hypnotic effects. Recent evidence suggests that its bioactive constituents can modulate cellular signaling pathways. However, its impact on reproductive physiology, particularly sperm motility, remains underexplored. This study aimed to investigate the effects of an aqueous P. incarnata formulation on sperm motility and viability, with the goal of evaluating its potential as a plant-based, non-hormonal contraceptive agent. An aqueous extract of P. incarnata was prepared and subjected to phytochemical analysis to quantify polyphenols, flavonoids, and carbohydrate content using standard assays. Antioxidant activity was assessed via free radical scavenging assays. The presence of bioactive macromolecules was confirmed by liquid chromatography-mass spectrometry (LC-MS). Goat spermatozoa, isolated from the caudal epididymis, were treated with the formulation. Sperm motility (horizontal and vertical) was evaluated microscopically and spectrophotometrically, respectively. Sperm viability was assessed using standard viability staining. Phytochemical screening revealed high polyphenol (16.85 mg gallic acid equivalent/g) and flavonoid (2.5 mg quercetin equivalent/g) content, along with notable antioxidant activity (87.25%). LC-MS confirmed the presence of diverse plant macromolecules. Treatment with the P. incarnata formulation significantly reduced both horizontal (28.5%) and vertical (20%) sperm motility (both p < 0.0001), while sperm viability remained unaffected. The water solubility of the formulation supports its development as a topical contraceptive gel. The aqueous P. incarnata formulation modulates sperm motility without compromising cell viability, likely via biochemical pathway regulation. These findings highlight its potential as a novel, non-hormonal, plant-based contraceptive.

physiology↗

bioRxiv2025

Calcitonin-Driven Reactivation of the Hippo Tumor-Suppressor Cascade Attenuates YAP/TAZ Oncogenic Signaling in Glioblastoma

Activation of WT calcitonin receptor (WT CTR) by Calcitonin (CT) acts as growth inhibitory axis in GBM, while the patient-derived loss-of-function CTR mutants promote tumor growth and lower patient survival. The precise mechanisms that orchestrate CTR activation, modulate its downstream signaling dynamics, and give rise to structural anomalies in mutant variants remain incompletely defined and warrant comprehensive investigation. Here, we found that salmon CT treatment suppressed the growth of patient-derived glioma stem cells (GSCs) by activating the Hippo pathway through the CTR/cAMP/PKA/LATS1 signaling cascade, while simultaneously inhibiting the oncogenic YAP/TAZ transcription factors. However, GSCs expressing a phosphorylation-resistant YAP mutant were refractory to growth inhibition by CT in vitro. GSCs, unlike differentiated glioma cells (DGCs), expressed high levels of CTR thus making them suitable for CT-based therapy. Furthermore, the intranasal delivery of salmon CT inhibited glioma growth initiated by GSCs in an intracranial orthotopic mouse model, resulting in increased survival in the mice. In addition, unlike the WT, the CTR mutants failed to activate the Hippo pathway. A large-scale, microsecond-long all-atom molecular dynamics simulation study of mutant CTR systems revealed remarkable changes in CTR interactions with the CT and G-GTPase domains. Packing and conformational dynamics data from simulation studies of the WT and mutant CTR systems could explain the perturbed cAMP/PKA signaling that may compromise downstream signaling. Together, we demonstrate that the CT/CTR axis inhibits glioma by activating the Hippo pathway, provide evidence for the structural defects in the loss-of-function CTR mutants, and propose a CT-based therapeutic strategy for GBM. Statement of significanceCT/CTR axis targets YAP/TAZ through Hippo pathway activation. Patient-derived GSCs express higher CTR levels, and intranasally delivered CT suppresses GSC-initiated tumors. All-atom molecular dynamics simulation uncovers structural perturbations in the CTR mutants.

cancer biology↗

bioRxiv2025

Metabolically driven differentiation of human pluripotent stem cells to generate robust and functional lymphatic endothelial cells

The lymphatic system plays various crucial but underappreciated roles in fluid transport and immune response in numerous organs and tissue types. Consequently, generation of lymphatic vessels has been postulated as an innovative therapeutic strategy for various diseases. However, there is a lack of efficient and reliable method to differentiate human pluripotent stem cells into lymphatic endothelial cells (LEC) for lymphatic regeneration. Current differentiation methods suffer from poor yield and low lymphatic marker expression, while also having limited clinical applicability due to its reliance on either the embryoid body intermediates or xenogenic supporting cells. Given that LECs exclusively rely on anaerobic and fatty acid metabolism due to the hypoxic environment of the lymph, here we report that the unique lymphatic-specific metabolic pathways can be exploited to promote lymphatic identity in differentiated LECs (dLECs). We show that dLECs express elevated levels of lymphatic markers compared to native endothelial cells, which is up to 15 times higher than the current leading standard of dLECs. Moreover, dLECs can form lymphatic vascular networks in both 2D and 3D, as well as secrete important lymphangiocrine for organ maturation. Upon implantation into double-ligation tail lymphedema and mammary fat pad models, dLECs were able to integrate with the host lymphatic vessels, restore fluid flow, and reduce swelling. Collectively, we show that metabolite supplementation can drive stem cell differentiation into dLECs, which can be incorporated into new alternative methods for personalized therapies and disease modeling, as well as provide a direct therapeutic option for lymphedema and lymphatic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/686405v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1c4caa5org.highwire.dtl.DTLVardef@d42b08org.highwire.dtl.DTLVardef@1554ffcorg.highwire.dtl.DTLVardef@1f64ae6_HPS_FORMAT_FIGEXP M_FIG C_FIG A xeno-free and stepwise differentiation protocol with VEGF-C and sodium acetate induces lymphatic identity in iPSC-derived endothelial cells. Differentiated LECs (dLECs) express key lymphatic markers as verified using bulk RNA-sequencing, qPCR, FACS, and immunostaining. These dLECs secrete important cytokines for lympangiocrine signaling, able to form robust 2D and 3D lymphatic networks, as well as demonstrate in vivo functionality and host integration in murine models.

bioengineering↗

bioRxiv2025

Right-Lateralized Maladaptive Topological Reorganization in Hyperthyroidism

Hyperthyroidism (HT) has been associated with cognitive impairments, but the structural connectomic and molecular mechanisms underlying these deficits remain poorly understood. In this study, we investigated large-scale brain network reorganization using structural connectivity derived from diffusion MRI in patients with 30 hyperthyroidism and 28 matched control participants. Subject level connectomes were analyzed to characterize network topology, with a specific focus on hub architecture, nodal alterations, and hemispheric reorganization. To probe molecular underpinnings, we integrated normative neurotransmitter receptor and transporter density maps with network metrics, assessing their correspondence to observed connectomic disruptions. Individuals with hyperthyroidism exhibited increased structural connectivity, particularly in mid- and long-range connections, alongside disrupted network topology. These changes were characterized by reduced modularity, increased characteristic path length and hub reorganization, most prominently in subcortical and right-hemisphere regions. Importantly, network alterations were functionally relevant, as metrics showed significant associations with clinical and cognitive variables, including FT4 levels, BMI, and perceptuomotor performance in the hyperthyroid group. Spatial neuromolecular correspondence analyses further revealed that DAT, 5-HT2A, and NET were closely aligned with topological changes. Moreover, neurotransmitter-weighted within-module degree z-scores moderately predicted TSH levels in HT. Our findings demonstrate that hyperthyroidism is associated with an increase in redundant, spatially extended white matter connections and a marked right-lateralized asymmetry in network organization. Furthermore, serotonergic (5-HT1a) and dopaminergic (DAT) systems consistently contributed to the modulation of intra-modular hubness, suggesting their role in maladaptive structural reorganization that may underlie perceptuomotor dysfunction in hyperthyroidism.

neuroscience↗

bioRxiv2025

Structural and genetic signatures of two classes of HCV E2 neutralizing face antibodies from non-human primates immunized with a recombinant E1E2

Hepatitis C continues to be a significant public health problem despite advancements in antiviral therapeutics. To eliminate this disease, an effective vaccine against new infections and re-infections is needed. However, to date only one Hepatitis C virus (HCV) envelope protein (E1E2) immunogen, developed by Chiron Inc., has been tested in a Phase I clinical trial (ClinicalTrials.gov identifier NCT00500747). To establish a benchmark for elicitation of broadly neutralizing antibodies (bnAbs) by E1E2, we previously immunized non-human primates (NHPs) with this immunogen and isolated monoclonal nAbs that exhibit neutralization potency comparable to human nAbs. Here we show that NHP nAbs, encoded by germline genes IGHV1-138*01 and IGHV4-NL_5*01 (homologs of human IGHV1-69*10 and IGHV4-59*12, respectively), recognize a relatively E2 conserved region (neutralizing face) proximal to antigenic region 3 (AR3). These NHP AR3-targeting nAbs share highly similar binding modes to human AR3-targeting nAbs, suggesting a similarity in human and NHP immune responses to the same HCV immunogen.

immunology↗

bioRxiv2025

Diverse ancestral myosin motors generate and segregate distinct types of nanocluster-rich domains at the plasma membrane

Molecular organization of the plasma membrane (PM) at nano and micron scales is critical for its function in all living cells. This emerges not only from the self-assembly of lipids and proteins but also from active forces originating in the underlying cytoskeletal cortex. These forces drive membrane molecules into non-equilibrium steady state patterns such as nanoclusters. However, the molecular agents connecting membrane organization with cytoskeletal dynamics and stresses have remained unknown. Here we show that two classes of ubiquitous ancestral non-muscle myosins are deployed for the organization of different types of membrane components. Inner-leaflet localized Class I myosins link outer-leaflet GPI-anchored molecules to juxta-membrane actin-filaments, whereas the more cortically-localized Class II myosins operate on transmembrane proteins endowed with actin-binding capacity. Consistent with an active Flory Huggins theory for phase separation, these observations show that the distinct motor-driven membrane molecules generate spatially segregated mesoscale domains, enriched in nanoclusters derived from different myosin classes. Moreover, chemically reversible post-translational modifications such as palmitoylation enable concatenation of these domains by enhancing affinity of the membrane domain constituents for each other. We anticipate that the segregation potential of the ATP-fueled cell membrane is made available for the crucial purpose of modulating information transduction because it can be regulated in space and time during the construction of signaling cascades, underpinning functional plasma membrane organization.

cell biology↗

bioRxiv2025

Proton FLASH Exposure Preserves Gut Commensal Microbiomes and Spares Intestinal Stem Cells

Emerging evidence shows Proton FLASH radiotherapy can spare normal tissues while maintaining anti-tumor efficacy. However, its impact on intestinal stem cell (ISC) populations and the gut microbiome remains unclear. This is critical, as the gut microbiome influences ISC radiosensitivity. In a mouse model of radiation-induced gastrointestinal syndrome, FLASH-irradiated mice exhibited better survival and less crypt-villus damage compared to mice exposed to conventional proton irradiation. Using scRNA-sequencing, we demonstrated that proton FLASH exposure using pulsed pencil beam scanning spares two distinct ISC populations--Lgr5+ CBCs and a Clu+, Mif+, Fabp2+, Anxa2+ revival stem cell (revSC) population--by modulating oxidative stress and cell cycle progression. Analysis of alpha and beta diversity demonstrated that FLASH modulates gut microbiota composition without compromising overall species richness. Notably, FLASH-irradiated mice had higher abundances of Alistipes sp. and Akkermensia sp., both known for protective effects on ISCs and the intestinal mucosa. The critical role of microbiome in FLASH-mediated sparing effect against radiation toxicity was further confirmed by fecal microbiota transplantation, where FLASH-donor microbiota demonstrated reduced lethality in recipients exposed to proton irradiation with conventional dose rate. Our findings highlight the crucial role of the microbiome in the FLASH-mediated sparing of the mucosal epithelium.

bioengineering↗

bioRxiv2025

Macrophage-derived WNT regulates tumor immune microenvironment to reduce colitis-associated colon cancer

Prolonged colonic inflammation and ulcerative colitis lead to colon cancer. The rapid growth and treatment-resistant nature of these tumors are primarily influenced by an immunosuppressive tumor microenvironment, which is led by tumor-associated macrophages (TAMs). However, factors influencing or regulating the immunosuppressive nature of TAMs have not been sufficiently studied. In this manuscript, we use a mouse model of colitis-associated colorectal cancer (CRC) to demonstrate that WNT expression in TAMs regulates their immunosuppressive function by inhibiting Glycogen synthase kinase-3 beta (GSK-3{beta}) within the macrophages, possibly through an autofeedback loop. GSK3{beta} is a positive regulator of PD-1 and PDL1 expression in macrophages and promotes an immunosuppressive microenvironment. Therefore, GSK-3{beta} inhibition alters the immunosuppressive nature of the immune microenvironment and effectively controls tumor growth. In Csf1r-iCre; Porcnfl/fl mice, the absence of macrophage-derived WNT promotes tumor growth in the model of colitis-associated colon cancer. Absence of macrophage-derived WNT stabilizes GSK-3{beta} in macrophages and promotes an immunosuppressive tumor microenvironment. We also show that pharmacological inhibition of GSK-3{beta} in a macrophage-specific manner, achieved by systemic delivery of a lipo-GSK3{beta} inhibitor, effectively inhibits tumor growth. Therefore, this manuscript demonstrates for the first time that the macrophage-specific modulation of GSK3{beta} can be a potential target to treat colitis-associated colon cancer.

cancer biology↗

bioRxiv2025

Gain-of-function mutant p53 regulates long-noncoding RNA LINC00643 to modulate HIF1α in glioblastoma

Gain-of-function mutations of p53 (GOF-MUT-p53) act as oncogenes by regulating gene transcription. We screened for the genome-wide transcriptional targets of GOF-MUT-p53 in glioblastoma (GBM) and found that a significant subset of them were long non-coding RNAs (lncRNA). Among these, LINC00643 was strongly repressed by GOF-MUT-p53 but not wild-type p53. LINC00643 was downregulated in GBM and low-grade glioma and correlated with patient survival. LINC00643 and its conserved third exon (Exon3) suppressed GBM cell proliferation, migration, invasion, stem cell self-renewal, and in vivo tumor growth Mechanistically, ChIRP-seq identified HIF1 as a key LINC00643 interactor. Under hypoxia, LINC00643 repressed HIF1 expression and its target genes by interacting with the HIF1 enhancer. Knockdown of GOF-MUT-p53 upregulated LINC00643 and reduces HIF1, revealing a regulatory axis. These findings show extensive regulation of lncRNAs by GOF-MUT-p53 and uncover a novel mechanism by which GOF-MUT-p53 drives GBM through repression of LINC00643 and dysregulation of the HIF1 pathway.

cancer biology↗

bioRxiv2025

Super-resolution Fluorescence Microscopy Reveals Densely Packed EboV Membrane Glycoprotein Clusters in the Cell Plasma-membrane During Ebola Infection

The glycoprotein (GP) of Ebola virus plays a central role in the viral entry into host cells. The detailed study of Ebola glycoprotein in the cellular system is unknown, mainly due to the resolution limit ([~]{lambda} /2) of optical fluorescence microscopes. To overcome this, we propose to use single-molecule localization microscopy (SMLM), which enables the study of the role of glycoproteins at both the single-molecule and ensemble levels. Accordingly, a photoactivable recombinant probe Dendra2 - EboV Glyc is developed by linking Ebola glycoprotein with Dendra2. The transient expression of photoactivable recombinant protein in NIH3T3 cells indicated the dominant presence of glycoprotein in the plasma membrane with regions of densely packed glycoproteins. Super-resolution single-molecule imaging gives direct proof of glycoprotein clusters in the plasma membrane. The corresponding single-molecule analysis using DBSCAN clustering algorithm reveals a dense packing of glycoprotein with an estimated average density of 5345 mol/{micro}m2 compared to the density in other regions of the membrane. The number of glycoproteins is estimated to be as high as 642 molecules per cluster and clusters spread area of {approx} 0.1219 {micro}m2 after 48 hrs post-transfection. Noting that glycoprotein is involved in cellular attachment, endosomal entry, and membrane fusion, the formation of glycoprotein clusters in the membrane is significant in the progression of Ebola pathogenesis. A drug capable of dispersing the glycoproteins clusters could be of therapeutic potential to lessen the intensity of infection and to slow down the disease progression. Statement of SignificanceThe fact that powerful fluorescence microscopes (such as SMLM) are available is a boon to disease biology, specifically to study the function of viral proteins at the cellular level. Ebola, one of the nocturnal viral diseases, is a threat and calls for study at the single-molecule level to understand the dynamics (kinetics and ensemble) of glycoprotein during viral infection. In this respect, the super-resolution microscopy techniques may help reveal the biological mechanism behind the infection process at the single-molecule level. A photoactivable fluorescent probe (Dendra2 - EboV Glyc) containing the gene-of-interest (EboVGP) is developed, and transfection studies are carried out, showing densely packed glycoprotein clusters in the cell plasma membrane with specific density, number, and size. We believe that dispersing these glycoprotein protein clusters could be of therapeutic potential.

biophysics↗

bioRxiv2025

Go/z-biased coupling profile of the dopamine D3 receptor

Dopamine receptors are G protein coupled receptors (GPCRs) that serve as key targets for FDA-approved drugs used to treat various neuropsychiatric disorders. Notably, [~]11% of all marketed GPCR-targeting drugs act on dopamine receptors. Five GPCRs mediate the effects of endogenous dopamine and compounds used to treat Parkinsons disease, schizophrenia, and other conditions. However, on-target side effects associated with these medications highlight the need to analyze dopamine receptor signaling to design safer, more effective therapeutics. We characterized the G protein coupling of dopamine D2-like receptors and observed the striking inability of D3R to engage with Gi proteins while effectively activating Go and Gz subtypes. Applying orthogonal cell-based assays that utilize wild-type G proteins both in parental and {Delta}Gi/o/z cells, we conclusively established that D3R does not activate Gi proteins. Further analysis of Gi2:GoA and D2R:D3R chimeras revealed that this selective inability is driven by molecular determinants located within the 5 helix of Gi and the intracellular loop 2 (ICL2) of D3R. Guided by cryo-EM structures, we modeled the interface between these regions to better understand the structural basis of this selectivity. Finally, we treated hippocampal neurons in acute brain slices with selective agonists for D2R and D3R and observed marked differences in their ability to regulate endogenous adenylyl cyclase to produce cAMP, highlighting the neurophysiological significance of our findings.

pharmacology and toxicology↗

bioRxiv2025

Local homeostasis preserves global neural dynamics compensating for structural loss during human lifespan aging

Aging brain undergoes a structural decline over lifespan accompanied by changes in neurotransmitter levels, leading to altered functional markers. Past studies have reported human resting state brain display a remarkable preservation of coordination among neural assemblies stemming from an underlying neuro-computational principles along aging trajectories, however, the true nature of which remains unknown. Here, we identify the computational mechanisms with which neurotransmitters, such as altered GABA and glutamate concentrations, can preserve functional integration across lifespan aging, despite structural decline. We employ multiscale, biophysically grounded modeling, constrained by the empirically derived anatomical connectome of the human brain, where the neurotransmitter concentrations can be free parameters that are algorithmically adjusted to maintain regional homeostasis and optimal working point. The two estimated neurotransmitters can maintain critical firing rates in the brain region and mimic age-associated functional connectivity patterns, consistent with empirical observations. We identified invariant GABA and reduced glutamate as the principle computational mechanism that can explain the topological variation of functional connectivity along lifespan, validated using graph-theoretic metrics. The results are subsequently replicated on three distinct datasets. Thus, the study offers an operational framework that integrates brain network dynamics at macroscopic and molecular scales, to gain insight into age-associated neural disorders.

neuroscience↗

bioRxiv2025

Tracking Chemotherapy-Induced Oxidative Stressin Glioblastoma Cells Using NV-Based QuantumSensors

Glioblastoma (GBM) is a highly aggressive cancer with poor prognosis despite temozolomide(TMZ) treatment. TMZ induces oxidative stress, causing persistent redox alterations. We report the first use of nitrogen-vacancy centers in nanodiamonds as quantum sensors for T1 relaxometry in fixed GBM cells, detecting stable paramagnetic signatures linked to chemotherapy-induced persistent oxidative damage. U87-MG cells treated with TMZ show dose-dependent T1 shortening, consistent with conventional ROS fluorescence assays. TBHP studies confirm that the T1 response reflects accumulated redox adducts. Measurements remain robust across fixation and permeabilization protocols. Since drug-resistant GBM cells preserve antioxidant signatures, unchanged T1 times after treatment may indicate early resistance phenotypes. This fixed-tissue compatible readout could stratify patient response, guide adaptive therapy, and support biopsy-based diagnostics and post-treatment drug analysis in routinely preserved clinical specimens enabling retrospective studies and better outcomes for future personalized oncology decision making pathways.

biophysics↗