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Patel, S.

Publications and source records attributed to Patel, S..

At least 37 records · Page 2Linked to original sources

Chronic Stress Exacerbates Long-term Microvascular Network Dysfunction Following Brain Trauma

BackgroundPreexisting factors are among the strongest predictors of recovery following traumatic brain injury (TBI), with chronic stress closely associated with permanent disability and worse long-term outcomes. While chronic cerebrovascular dysfunction is linked to these poor trajectories, as impaired blood flow regulation drives secondary disease progression, the mechanisms regulating this vascular failure remain incompletely understood. Crucially, how premorbid chronic stress and TBI disrupt the fundamental coordination of the cerebrovascular network post-injury remains unknown. MethodsTo elucidate this coordination at rest and in response to increased neuronal activity, we used a model of three repeated moderate closed cortical impacts comorbid with chronic stress induced by social isolation (SI) post-weaning. Our previously developed vascular analysis pipeline (NOVAS3D) was employed to estimate changes in vascular radii across cerebrovasculature proximal to neuronal activation. Arteries and veins were annotated in the imaged volumes to allow for blood flow simulations. ResultsUsing graph-based network analysis, we demonstrate that TBI, when compounded with chronic stress, critically disrupts the long-range coordination of the capillary network. Specifically, the functional coordination of radius changes between nearby, non-adjacent capillaries was reduced by 40{+/-}20% in TBI+SI mice relative to controls. Consequently, simulations estimated that the vascular networks in TBI+SI mice experienced a 68{+/-}7% reduction in arterial red blood cell velocity (VRBC) responses to neuronal activation. These network-wide impairments were fundamentally driven by severely blunted vessel reactivity, including a 28{+/-}7% decrease in the magnitude of arteriolar dilations and a 47{+/-}7% decrease in the magnitude of arteriolar constrictions. ConclusionThese findings provide a mechanistic foundation for worse clinical outcomes seen in TBI patients with comorbid chronic stress, identifying arteriolar reactivity and long-range capillary coordination as critical therapeutic targets to mitigate secondary injury and improve long-term recovery.

neuroscience↗

ASS1 deficiency defines a therapeutic vulnerability in Philadelphia chromosome-positive acute lymphoblastic leukaemia

Amino acid deprivation with L-asparaginase is a cornerstone of treatment in acute lymphoblastic leukaemia (ALL), but clinical challenges limit its use in adults. Deficiency in the enzyme argininosuccinate synthase (ASS1-low) confers arginine auxotrophy, defining a dependence on extracellular arginine, and represents an analogue metabolic vulnerability that is targetable through arginine deprivation. We analysed transcriptomic data across >550 adult B-ALL cases to establish clinico-biologic characteristics of low ASS1 expression identifying the Philadelphia chromosome-positive (Ph+) ALL subgroup as a stereotypical arginine auxotroph within molecularly diverse B-ALL. Functionally, arginine deprivation with pegargiminase induced robust apoptosis in both Ph+ ALL cell lines and primary samples in an ASS1-dependant manner and was highly effective as a monotherapy treatment in independent Ph+ ALL patient derived xenografts. Mechanistically, arginine deprivation induces endoplasmic reticulum stress mediated apoptosis which was orthogonal to tyrosine kinase inhibition (TKI) mediated outcomes. Using in vitro and in vivo models of non-genetically mediated TKI-resistance, we demonstrate pegargiminase and TKI combinations robustly eradicates TKI-resistant leukaemia. Thus, we establish ASS1 deficiency (ASS1-low) as a therapeutically actionable vulnerability in Ph+ ALL and a strategy to bypass TKI-resistance, supporting clinical evaluation of arginine deprivation as a novel adjunct to chemotherapy-free treatment.

cancer biology↗

Brain B Vitamin Status and One-Carbon Metabolism Gene Polymorphisms are Associated with Cognitive Impairment in Alzheimer's and Parkinson's Disease

IntroductionDisturbances in one-carbon metabolism and homocysteine (Hcy) regulation have been implicated in Alzheimers disease (AD) and Parkinsons disease (PD), yet direct evidence from human brain tissue and the contribution of genetic variation remain limited. We investigated whether B-vitamin-related metabolic deficits and polymorphisms in one-carbon metabolism pathways contribute to cognitive impairment in AD and PD. MethodsWe quantitated metabolites related to B vitamins and one-carbon metabolism in post-mortem frontal cortex (n=136) and putamen (n=68) from clinically and neuropathologically characterized healthy controls, AD dementia (AD-D), PD with dementia (PD-D), PD with mild cognitive impairment (PD-MCI), and cognitively normal PD (PD-CN). Genotyping targeted variants related to one-carbon metabolism and B vitamins. Multivariable regression models controlled for demographic, clinical, metabolic, and tissue-handling covariates. ResultsAD-D and PD-D shared a convergent cortical signature of reduced biotin, pyridoxal-5-phosphate, thiamine monophosphate, pantothenic acid, and betaine. Elevated Hcy and reduced tetrahydrofolate were specific to AD-D and to PD-D during acute levodopa exposure. PD-MCI exhibited deficits in putamen resembling cortical dementia-associated changes. Self-reported B-vitamin supplementation attenuated several abnormalities, including complete normalization of levodopa-associated Hcy and folate disturbances in PD-D. Across subjects, multiple B vitamins showed strong associations with Hcy and betaine, indicating coordinated impairment of all three Hcy-metabolizing pathways. Genotyping analysis identified polymorphisms in MTRR, TYMS, MTR, NFE2L2, BHMT, and MTHFR that were differentially distributed across cognitive subgroups or associated with AD-related pathology scores. Composite genetic and metabolic burden scores were elevated in cognitively impaired groups but not in cognitively normal groups. ConclusionDementia in AD and PD is characterized by convergent B-vitamin deficiencies and genetic susceptibilities that disrupt Hcy metabolism. These findings provide a mechanistic explanation for levodopa-related Hcy accumulation in vulnerable PD subjects and identify B-vitamin supplementation as a potentially modifiable factor relevant to cognitive decline.

neuroscience↗

Inhibition of miR-1307 Reverses Resistance to Cisplatin in Drug-Resistant Oral Squamous Cell Carcinoma

BackgroundChemo-resistance remains a major clinical challenge in Oral Squamous Cell Carcinoma (OSCC), attributed to the intrinsically resistant cells. Although tumour-derived extracellular vesicles (EVs) have been implicated in cell-cell communication, their role in propagating chemo-resistance remains poorly defined. This study aims to identify salivary EV-associated miRNAs capable of predicting chemoresistance and to delineate the role of miR-1307-5p in modulating CSC-driven therapeutic refractoriness. MethodsSalivary EV-derived expression profile of miR-1307-5p was assessed by qPCR in chemo resistant OSCC patients and further validated in TCGA small RNA sequencing datasets. Expression was validated by qPCR and correlated with clinicopathological outcomes. Functional assays including cell-cycle analysis, apoptosis, migration/invasion, 3D spheroids, angiogenesis, and CAM assays were performed in miR-1307-5p inhibited CD44 CSC subpopulation compared to its vehicular control. Transcriptomic profiling cross-referencing with TCGA was conducted to identify potential novel targets of miR-1307-5p. Chemo-sensitisation was assessed by treating the knockdown chemo resistant cells with low dose cisplatin and validating it using in-vitro functional assays and orthotopic xenograft model. ResultsmiR-1307-5p was significantly elevated in salivary EVs of chemo resistant OSCC patients and correlated with poor overall survival (p = 0.03). The miRNA was markedly enriched in endogenously resistant CD44 CSCs. Silencing of miR-1307-5p induced G2/M arrest, triggered apoptosis, impaired invasion, and reduced angiogenesis both in-vitro and in ex-vivo assays. Transcriptomic profiling, TCGA validation, and integrative pathway analysis identified key oncogenic hubs which converge on PI3K-AKT, MAPK/ERK, and YAP signalling pathways governing EMT. Inhibition of miR-1307-5p restored cisplatin sensitivity in resistant CSCs, with low-dose cisplatin producing substantial tumour suppression in-vitro and in-vivo. Reduced CD44 expression in xenograft models confirmed CSC reprogramming. EVs from anti-miR-treated cells confer chemo sensitisation upon uptake by resistant CSCs. Xenograft models substantiated that EVs can initiate tumour formation and that EV-mediated delivery of anti-miR-1307-5p drives significant tumour regression. ConclusionThis study identifies salivary EV-derived miR-1307-5p as a clinically relevant biomarker of chemoresistance in OSCC and reveals its mechanistic role in sustaining CSC-driven therapeutic failure. Targeting miR-1307-5p offers a promising avenue for restoring cisplatin sensitivity and developing exosome-based therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/709730v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19f88e0org.highwire.dtl.DTLVardef@d36b95org.highwire.dtl.DTLVardef@3c2579org.highwire.dtl.DTLVardef@c04ef5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Concordant transcriptional and morphological remodeling revealed by in vivo Perturb-CLEAR

The principle that form follows function has long guided thinking in biology and architecture alike. In the nervous system, however, form does more than reflect function: neuronal morphology actively constrains input patterns, synaptic integration, and circuit wiring. During postnatal neurodevelopment, dendritic architectures are assembled and remodeled through genetically encoded programs and activities, transforming molecular programs into circuit architecture. However, dendritic morphogenesis has been difficult to quantify at scale while systematically testing how genetic variants, including neurodevelopmental disorder (NDD) risk genes, alter these structures in vivo. We developed Perturb-CLEAR, which integrates pooled CRISPR screening and whole-mount imaging to quantify brain-wide cytoarchitecture, and paired it with Perturb-seq to link structural phenotypes to transcriptomic changes. Applying Perturb-CLEAR to the developing mouse cortex revealed morphogenesis trajectories accompanied by transcriptomic dynamics. Moreover, systematic perturbation of NDD risk genes uncovered gene-specific multimodal phenotypes. Adnp perturbation remodels basal dendrites in L4/5 IT (intratelencephalic) neurons but not other dendritic compartments or cell types, alongside consistent transcriptional shifts. Combined morphology and transcriptome analyses link NDD risk genes to concordant multimodal cellular phenotypes in the developing brain, highlighting diverse paths of perturbation effect propagation across modalities.

neuroscience↗

Mitochondrial Transplantation in the Eye: A Review and Evaluation of Surgical Approaches

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

cell biology↗

Feeder-Free Generation of Lymphatic Endothelial Cells from Human Induced Pluripotent Stem Cells

AbstractThe lymphatic system is essential for maintaining fluid homeostasis, lipid transport and supporting immune function. Despite its central role in health and disease, advancements in understanding human lymphatic vasculature has been constrained, in part because primary human LECs are difficult to access and study in disease-relevant contexts. This study describes an efficient and scalable feeder-free method to differentiate human iPSCs into lymphatic endothelial cells (LECs) that are transcriptionally and phenotypically similar to primary fetal LECs. An iPSC-derived LEC system overcomes a drawback of primary cells by enabling precise genetic perturbations, supporting study of lymphatic diseases of interest in a human context. By grounding our approach in in vivo stages of lymphangiogenisis, we describe a staged protocol that recapitulates the key milestones of lymphatic development. We first adapted a published method to differentiate human iPSCs into venous endothelial cells (VECs) and then initiate transdifferentiation of VECs into LECs. Using immunocytochemistry, qPCR, as well as flow cytometry, we demonstrated expression of lymphatic-specific markers in the differentiated population. We further characterized our induced VECs (iVECs) and LECs (iLECs) through bulk RNA sequencing analysis and compared the populations to pseudobulk VEC and LEC transcriptomic datasets generated from human fetal heart endothelia at 12, 13 and 14 weeks of gestation. Through this work, we expanded the repertoire of approaches for accessing LECs, with the goal of accelerating discoveries in lymphatic biology and therapeutics. Abstract summary image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/712968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a9a406org.highwire.dtl.DTLVardef@4faec6org.highwire.dtl.DTLVardef@15b4e73org.highwire.dtl.DTLVardef@17b9c36_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

Microbiota-derived extracellular vesicles link intestinal dysbiosis to neuroimmune activation in long COVID

Post COVID-19 condition (Long COVID, LC) is frequently accompanied by persistent neurological symptoms, but the mechanisms linking intestinal dysbiosis to neuroinflammation remain unclear. Here we identify gut microbiota-derived extracellular vesicles (GMEVs) as functional mediators linking LC-associated dysbiosis to systemic and neuroimmune inflammation. In a longitudinally characterized cohort, individuals with LC and neurological symptoms exhibit a persistent intestinal microbiome signature. Transplantation of LC-associated microbiota into germ-free mice induces intestinal barrier disruption and neuroinflammatory phenotypes. GMEVs from individuals with LC activate inflammasome-associated programs and impair epithelial barrier function, promote inflammatory responses in macrophages, and induce coordinated pro-inflammatory transcriptional programs in human induced pluripotent stem cell (iPSC)-derived microglia. Chronic oral administration of LC-derived GMEVs remodels the microbiota and induces intestinal and systemic inflammation with glial activation in vivo. Together, these findings support a vesicle-centered framework in which microbiota-derived extracellular vesicles translate dysbiosis into sustained immune and neuroimmune activation in a post-viral inflammatory state.

microbiology↗

Distinct mechanistic pathways of early tauopathy revealed by MAPT mutations

Tau pathology underlies a broad spectrum of neurodegenerative disorders, collectively termed tauopathies, yet these diseases exhibit striking heterogeneity in their biological mechanisms and clinical outcomes. The basis for this heterogeneity remains poorly understood. Here, we address this question using knock-in mouse models expressing two distinct frontotemporal dementia-associated tau mutations to define how different tau variants drive divergent pathogenic programs in vivo. We find that the two mutations give rise to fundamentally different trajectories of tau pathogenesis. One trajectory is marked by progressive tau hyperphosphorylation and cytoskeletal destabilization occurring in the absence of detectable tau seed formation. In contrast, an alternative trajectory is characterized by tau hypophosphorylation, early seed formation, and alterations in nucleotide metabolism and chromatin organization, without overt cytoskeletal disruption. With aging, tau in this latter pathway transitions to a hyperphosphorylated state and forms mature fibrillar aggregates. Genetic enhancement of {beta}-amyloid selectively accelerates fibril formation, particularly in the model exhibiting early seeding. Together, these findings demonstrate that distinct tau mutations can engage separable pathogenic mechanisms, providing a biological framework for the heterogeneity observed across tauopathies, and highlighting the need for mechanism-informed therapeutic strategies and patient stratification. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/705716v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@11eb652org.highwire.dtl.DTLVardef@153cc53org.highwire.dtl.DTLVardef@8b670eorg.highwire.dtl.DTLVardef@1afaad_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractSchematic representation comparing two trajectories of tau pathology. The S305N mutation promotes a 4R isoform shift, cytoskeletal damage and synapse loss, and accumulation of soluble hyperphosphorylated tau. Tau remains soluble even at old ages. In contrast, the P301S mutation generates hypophosphorylated, seed-competent tau that forms fibrils. The effect of amyloid is slow in the S305N, but results in accelerated acceleration of pathology in the P301S. Figure made with Biorender.com.

neuroscience↗

Macrophage Phagocytic Impairment is associated with Dysbiosis of the Respiratory Microbiome in Frail older adults

Advanced age and frailty are risk factors for pulmonary infections, which are the leading cause of death in older adults. The mechanism behind increased susceptibility to infection is poorly understood, but interactions between innate immunity and the respiratory microbiome may be implicated. We investigated changes to monocyte-derived macrophage function, and the respiratory microbiome in healthy young adults, healthy older adults, and frail older adults. We found that macrophage phagocytosis and efferocytosis is impaired in frail older adults, associated with elevated pro-inflammatory cytokine secretion and a failure to regulate expression of phagocytic receptors CD14 and CD36. Pre-treatment of macrophages with a PPAR{gamma} agonist restored phagocytosis, implicating this pathway in cellular dysfunction. Accompanying respiratory microbiome showed reduced bacterial diversity and richness in older adults, which was more advanced in frail older adults. These results are consistent with a macrophage predation defect in the lungs during frailty which may impact bacterial diversity, and may be improved via manipulation of the PPAR{gamma} pathway. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/703495v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1c53e8forg.highwire.dtl.DTLVardef@8e34bcorg.highwire.dtl.DTLVardef@28345dorg.highwire.dtl.DTLVardef@185d93d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

Forest Owlet or Farmer's owlet: Scale-dependent habitat selection reveals conditional compatibility between Forest Owlet conservation and traditional agroforestry in Gujarat, India

Understanding how species that are threatened with extinction utilise human-modified landscapes is essential for evidence-based conservation. We investigated multi-scale habitat selection by the Forest Owlet (Athene blewitti), an Endangered species, endemic to central India with fewer than 1000 mature individuals, in the Dangs district of Gujarat, the westernmost extent of its range. Using a hierarchical Bayesian occupancy framework, we examined how forest cover and three agricultural land-use types (dry agriculture with trees, dry agriculture without trees, and intensive agriculture) affected occupancy across three nested spatial scales: regional (81 km2), landscape (4 km2), and territory (0.25 km2). At the regional scale, the forest x agriculture interaction term was significantly negative ({beta} = -6.82, 95% CI: -9.87 to -1.59), indicating that owlets favour agroforestry-dominated regions over forest-dominated landscapes. Conversely, at the landscape scale, a significant positive interaction ({beta} = 1.36, 95% CI: 0.41-2.50) revealed synergistic benefits from forest-agriculture mosaics. Agriculture type strongly influenced landscape-scale occupancy: dry agriculture with trees showed positive effects ({beta} = 1.17, 95% CI: 0.43-2.02), whereas dry agriculture without trees had significant negative effects ({beta} = -1.19, 95% CI: -2.28 to -0.29). These findings demonstrate that Forest Owlets are not forest-obligate specialists but occupy complex agroforestry mosaics, requiring multi-scale conservation strategies. We propose that the traditional Malki agroforestry system, which incentives tree retention on farmland, offers conditional compatibility with Forest Owlet conservation, provided that mature cavity-bearing trees and small forest patches are explicitly protected.

ecology↗

Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

Liquid biopsies can enable cancer detection and monitoring yet remain limited by low concentrations of ctDNA. To address this limitation, we previously introduced a monoclonal antibody (mAb) "priming agent" that transiently increased the concentration of ctDNA in blood. Here, we investigated the molecular features that drive this effect. In a panel of novel mAbs that bound cfDNA, both those targeting dsDNA and those targeting mononucleosomes increased the concentration of ctDNA. mAbs with high avidity to dsDNA performed best, suggesting dsDNA as the key binding target. One agent preserved short, biologically informative molecules of cfDNA typically depleted at baseline. The Fc domain was dispensable, as F(ab)2 fragments retained priming activity. Leveraging these insights, we engineered single-chain agents using the dsDNA binding domain sso7d, expanding priming strategies beyond immunoglobulins. This study identifies the molecular features and general design principles for mAb-based priming agents to enhance recovery and detection of ctDNA. SignificanceLow concentration of ctDNA limits the sensitivity of liquid biopsies in many applications. This study shows how engineered antibodies and other dsDNA-binding molecules can inhibit clearance of ctDNA from the bloodstream and increase concentration of ctDNA in a blood draw, and identifies the key features that enable this activity. Modulation of cfDNA in the bloodstream using these agents can increase recovery of ctDNA and improve the performance of liquid biopsies for cancer detection.

bioengineering↗

A Course-Undergraduate Research Experience (CURE) to explore the effect of structural variants on gene expression in C. elegans balancers

Bioinformatics, a discipline at the crossroads of Biology and Computational Sciences, also referred to as Computational Biology, is nowadays widely spread in research programs. However, implementing any Bioinformatics projects requires the ability to comprehend biological concepts and apply computational approaches, and rare are the undergraduate programs offering such multi-disciplinary training. In addition, understanding the dynamic between Biology research projects and Bioinformatics analyses is challenging with no real-life experience. Course-based undergraduate research experience (CURE) courses are innovative programs that allow more students to acquire research experience and provide the perfect setting to introduce students to applied bioinformatics. As a part of the Bachelor of Health Sciences of the Cumming School of Medicine at the University of Calgary (Canada), a CURE applied bioinformatics was implemented in the Winter of 2023 to 2025. Students investigated the effect of structural variants (SVs, genetic variants larger than 50 bp) on gene expression in the model organism Caenorhabditis elegans (a hermaphrodite 1-mm long roundworm). The students detected and characterized SVs by analyzing genome and transcriptome sequencing data of C. elegans strains called balancers, as they are known to carry large genomic variations balancing regions of the genome by limiting recombination and allowing maintenance of lethal mutations. They used Galaxy, a public web-based supercomputing resource, but also a local High-Performance computing system, and R, to report different effects of SVs on gene expression and splicing. Students research explained the molecular mechanism behind the uncoordinated phenotype caused by the reciprocal translocation eT1(III;V) and uncovered unexpected effects on gene expression on an understudied gene. We evaluated the courses impact on student learning journeys and showed that the CURE favored students understanding of the Bioinformatics field and fostered their research interest. We provide here guidelines to facilitate the CURE implementations to improve access for undergraduate students to bioinformatics research experiences.

bioinformatics↗

Astrocytic response to traumatic brain injury to rescue neuronal mitochondrial dysfunction through mitochondrial transfer

As highly dynamic organelles, mitochondria play an essential role in neuronal survival and synaptic function. Excitotoxicity is as a critical factor that promotes mitochondrial dysfunction after traumatic brain injury (TBI). Intercellular mitochondrial transfer and exogenous mitochondrial transplantation are emerging concepts to understand mitochondrial trafficking in response to mitochondrial dysfunction; however, robust in vivo evidence remains limited on the extent of these processes in the central nervous system (CNS). There is a significant knowledge gap in our understanding of mitochondrial transfer mechanisms under both normal physiological conditions and after experimental TBI. Mouse lines expressing mitochondrial green-fluorescent dendra-2 (mtD2) and GFP (mtGFP) targeted to inner and outer mitochondrial membranes, respectively, were used to study astrocyte-specific (Aldh1l1-CreER; mtD2f/f - AmtD2 and Aldh1l1-CreER; mtGFPf/f - AmtGFP) and neuron-specific (CamK2aCre; mtD2f/f - NmtD2 and CamK2aCre; mtGFPf/f - NmtGFP) mitochondrial dynamics and bioenergetics in acute TBI and excitotoxicity. At 24 hrs following TBI, neurons in the NmtD2 mouse brain exhibited rapid and significant alterations in mitochondrial morphology, including changes in total mitochondrial volume, volume distribution, and sphericity. Synaptic neuronal (SN) mitochondria display robust deficits in mitochondrial bioenergetics and complex protein levels while non-synaptic neuronal (NSN) mitochondria show State III bioenergetics and complex proteins at control levels. These findings are accompanied by a marked increase in astrocyte-derived mitochondria (AmtGFP) transfer to neurons at 24 hrs post-injury, compared to control animals, but no increase in transfer to neuronal synapses. While TBI also altered astrocytic mitochondrial morphology in the cortex, astrocytic mitochondrial bioenergetics remained preserved. Single-cell RNA-seq analysis of astrocytes revealed significant transcriptional reprogramming following TBI, characterized by the upregulation of genes associated with mitochondrial homeostasis and the machinery for organelle trafficking. In vitro co-cultures of primary cortical astrocytes and neurons demonstrated that astrocytes can transfer mitochondria to neurons via direct contact and that NMDA-mediated excitotoxicity further enhanced this astrocyte-to-neuron mitochondrial transfer. Furthermore, astrocytic-derived extracellular vesicles containing mitochondria (EV-mito) deliver mitochondria to neurons and EV-mediated mitochondrial transfer significantly ameliorated NMDA-induced mitochondrial dysfunction in primary cortical neurons. Together, these findings show that astrocytes take on a TBI-related phenotype that facilitates dynamic changes in mitochondrial networks and mitochondrial trafficking to neurons. Astrocytic transfer of respiratory-competent mitochondria support is an intrinsic neuroprotective response to injury that supports mitochondrial function in neuronal soma, dendrites, and axons but not at the neuronal synapse. Finally, we show therapeutic potential of exogenous mitochondrial transfer, particularly via EV-mito, for treating neurological disorders associated with excitotoxicity, such as TBI.

neuroscience↗

Dysregulated TGFb-ERK Signaling Drives Aberrant Extracellular Matrix Production in Noonan Syndrome-Associated Pulmonary Valve Stenosis

Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms linking pathogenic variants to the valve pathology remain poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineages--including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. CRISPR-edited iPSCs harboring NS gain-of-function RAS/MAPK and Noonan syndrome with multiple lentigines (NSML) dominant-negative RAS/MAPK variants exhibited early defects in mesodermal and endocardial specification in all genotypes. Additionally, NS-iPSC endocardial cells exhibited defects in endothelial-to-mesenchymal transition (EndMT) specifically towards fibrosa VICs, which was most pronounced in PTPN11N308D (N308D) cells. Single-cell transcriptomics revealed widespread dysregulation of extracellular matrix (ECM) programs in N308D fibrosa VICs, including increased expression of collagens and proteoglycans, as well as dysregulation of multiple genes involved in ECM remodeling. We also detected activation of RAS-MAPK, TGF{beta}, and fibrosis-associated pathways in our transcriptional dataset. Mass spectrometry-based phosphoproteomics confirmed coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity of the TGF{beta} receptor. Functionally, N308D fibrosa VICs exhibited exaggerated upregulation of ECM genes in the presence of TGF{beta}2 ligand, suggesting that these cells are hypersensitive to TGF{beta} stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of BAMBI, a negative regulator of TGF{beta} signaling that was found to be decreased in N308D fibrosa VICs. Lastly, we performed histopathological analyses of stenotic pulmonary valves from two NS infants, which demonstrated marked overproduction and disorganization of ECM, mirroring the findings from our iPSC-based disease model. Together, our data reveal a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGF{beta}, which leads to aberrant downstream signaling and drives the pathological ECM program in NS-associated PVS.

cell biology↗

Multiple-Context Training Enhances Generalization of Cued Fear Extinction and Requires the Dorsal Hippocampus

BACKGROUNDMultiple-session fear extinction (FE) models exposure therapy, which also occurs over many sessions. In humans, conducting extinction training across multiple contexts improves fear suppression as compared to a single context, but rodent work delineating boundary conditions and neural mechanisms remains sparse. METHODSWe compared single-context FE (SCFE) versus multiple-context FE (MCFE) in 129S1 and C57BL/6J mice, manipulating context familiarity (familiar vs novel FE contexts). Outcomes included FE acquisition and tests for fear relapse such as recent (2 d) and remote (30 d) recall in trained, extinction, and novel contexts, as well as reinstatement tests. We mapped extinction-related neural activity with dual Fos labeling (TRAPxFos immunofluorescence) and graph analyses of regional co-activation, and used chemogenetic inhibition to selectively silence dorsal (dHP) or ventral hippocampus (vHP) during extinction. RESULTSMCFE produced greater across-session reduction in freezing than SCFE. The MCFE advantage did not require contextual novelty. MCFE also enhanced short- (2 d) and long-term (30 d) recall in a novel context, but did not prevent recovery over time or US-induced reinstatement. Dual-labeling revealed that MCFE strengthened hippocampal-prefrontal co-activation (with relatively weaker prefrontal-amygdala coupling than SCFE). Chemogenetic silencing of dHP, but not vHP, selectively impaired between-session extinction under MCFE and abolished the MCFE benefit; hippocampal silencing did not improve SCFE. CONCLUSIONSConducting extinction across multiple contexts enhances acquisition and recall relative to SCFE, without requiring novelty, and engages distinct hippocampal-prefrontal circuit dynamics. dHP activity is necessary for the MCFE-specific improvement, highlighting a circuit mechanism in which distributing extinction across contexts augments fear reduction.

neuroscience↗

A glucocorticoid-responsive polygenic signature in the anterior cingulate cortex moderates the association of early-life adversity and vulnerability for depression

Stress exposure is a major risk factor for psychopathology, yet how stress mediators shape long-term psychiatric vulnerability in humans remains unclear. Glucocorticoids, central effectors of the stress response, regulate gene expression through tissue-specific transcriptional programs, suggesting that glucocorticoid-responsive networks may shape sensitivity to adversity. Using RNA-sequencing following chronic glucocorticoid exposure in a non-human primate model, we identified a gene co-expression network specific to the anterior cingulate cortex (ACC) that was highly preserved across human post-mortem brain datasets relevant to depression. We derived an expression-based polygenic score (ePGS) reflecting genetic variation in network activity and tested its interaction with adversity in the UK Biobank. The ACC-specific glucocorticoid-responsive ePGS moderated the association between adversity and depressive symptoms in adult females, with the strongest effects for early-life adversity. Network genes were enriched for neurodevelopmental processes and showed stronger co-expression during childhood, highlighting a developmentally sensitive, region-specific mechanism linking stress exposure to depression risk.

neuroscience↗

Liquid liquid phase separation of the intrinsically disordered protein JPT2compartmentalizes components of NAADP-evoked Ca2+ signaling

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-releasing second messenger that activates two-pore channels (TPCs) on endosomes and lysosomes. Rather than binding TPCs directly, NAADP acts through cytoplasmic NAADP-binding proteins (NAADP-BPs) which are essential for endolysosomal Ca2+ release. Here we characterized the properties of two recombinant, purified NAADP-BPs: Jupiter Microtubule Associated Homolog 2 (JPT2) and like-Sm protein 12 (LSM12). In contrast to LSM12, JPT2 is predicted to be an intrinsically disordered protein, a feature confirmed by circular dichroism and NMR spectroscopy. Under conditions of low Na+ concentration or molecular crowding, JPT2 underwent phase separation, as demonstrated by multiple orthogonal approaches. JPT2 condensates displayed liquid-like behavior and efficiently recruited LSM12, a novel fluorescent NAADP analog, as well as tubulin. JPT2 condensates also interacted with polymerized microtubules and lysosomes isolated from human cell lines. These findings reveal an unexpected capability of NAADP-BPs to undergo phase separation, and segregate with components needed for NAADP-dependent Ca2+ release. We speculate that these signaling condensates dictate cellular NAADP sensitivity, desensitization of NAADP responses, as well as NAADP targeting to TPCs at membrane contact sites between acidic organelles and the endoplasmic reticulum. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/698830v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@108ebc4org.highwire.dtl.DTLVardef@ae9eb9org.highwire.dtl.DTLVardef@36b859org.highwire.dtl.DTLVardef@81053a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗