Biology subjects

Sarkar, S.

Publications and source records attributed to Sarkar, S..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

Neoadjuvant Therapy Differentially Remodels Carcinoma and Tumor Microenvironment in Pancreatic Ductal Adenocarcinoma: Insights from Spatially Resolved Transcriptomics

PurposeNeoadjuvant therapy (NAT) is increasingly being used for pancreatic ductal adenocarcinoma (PDAC) treatment. However, its distinct effects on carcinoma cells and the tumor microenvironment (TME) are not fully understood. This study employs spatial transcriptomics and single-cell RNA sequencing to investigate how NAT differentially remodels PDACs carcinoma cells and TME. Experimental DesignWe used spatial transcriptomics to compare gene expression profiles in carcinoma cells and the TME between NAT-treated and NAT-naive PDAC patients and correlated with their clinicopathologic features. Complementary single-nucleus RNA sequencing (snRNA-seq) analysis was conducted to validate our findings and identify cell types driving NAT-induced gene expression alterations. ResultsWe found NAT not only induces apoptosis and inhibits proliferation in carcinoma cells but also significantly remodels the TME. Notably, NAT induces a coordinated upregulation of multiple key complement genes (C3, C1S, C1R, C4B and C7) in the TME, making the complement pathway one of the most significantly affected pathways by NAT. Patients with higher TME complement expression following NAT exhibit improved overall survival; more immunomodulatory and neurotrophic cancer-associated fibroblasts (CAFs); more CD4+ T cells, monocytes and mast cells; and lower immune exhaustion gene expression. snRNA-seq analysis demonstrates C3 complement upregulation specifically in CAFs but not in other stroma cell types. ConclusionsOur findings indicate that NAT may reduce immunosuppression in PDAC by enhancing complement production and signaling within the TME. These findings suggest that local complement dynamics could serve as a novel biomarker for prognosis, evaluating treatment response and resistance, and guiding therapeutic strategies in NAT-treated PDAC patients. Translational RelevanceAs neoadjuvant therapy (NAT) increasingly becomes the preferred approach in treating resectable and borderline resectable pancreatic ductal adenocarcinoma (PDAC), there is a growing demand for novel biomarkers specifically tailored for post-NAT PDAC patients. Our study focused on how NAT differentially remodels the tumor cells and the tumor microenvironment (TME). We demonstrate that NAT can enhance local complement production and signaling in PDACs TME, which is associated with reduced immune exhaustion and improved overall survival. Our results highlight the importance of local complement dynamics in influencing treatment response, resistance, and overall clinical outcomes in PDAC. This new mechanism provides new opportunities in biomarker development which could facilitate more accurate prognostication and precise treatment stratification, particularly for patients undergoing NAT in PDAC.

genomics↗

bioRxiv2023

Suppression of the Integrated Stress Response in Islet β Cells Decreases Risk of Autoimmune Diabetes

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing {beta} cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2 kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves {beta} cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in {beta} cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in {beta} cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-{beta}H1 human {beta} cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances {beta} cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

cell biology↗

bioRxiv2023

Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs. Translational ImpactWe developed injectable hydrogels that provide loco-regional, controlled release of protein therapeutics. Local delivery is especially suitable for potent, protein-based drugs like chemokines and monoclonal antibodies whose systemic toxicities can be life threatening. Our hydrogel is equipped with slowly hydrolyzing linkers for transient payload coupling, prolonging its therapeutic window and minimizing the need for repeat surgeries in a clinical setting. A range of release profiles, spanning days to over a month, and a broad compatibility to therapeutically relevant, recombinant proteins provide clinicians with flexible therapeutic options to suit a variety of circumstances.

bioengineering↗

bioRxiv2023

IFNγ modestly contributes to disease progression in the Ndufs4(-/-) model of Leigh syndrome while IP10 is dispensable

Leigh syndrome (LS) is the most common pediatric presentation of genetic mitochondrial disease. LS is a multi-system disease characterized by severe neurologic and metabolic abnormalities. The defining feature of the disease is the presence of symmetric, bilateral, progressive necrotizing lesions in the brain stem, cerebellum, and basal ganglia. The pathogenic mechanisms underlying disease initiation and progression in LS have yet to be elucidated. Recent evidence demonstrates that the immune system plays a key role in LS pathogenesis. Treatment with the macrophage-depleting Csf1r inhibitor pexidartinib prevents disease in the Ndufs4(-/-) mouse model of LS, but the mechanisms leading to immune activation and governing disease progression remain to be elucidated. In recent work, the cytokines IFN{gamma} and IFN{gamma}-induced protein 10 (IP10) were found to be significantly elevated in Ndufs4(-/-) brainstem. Given their role as macrophage-activating factors, here we sought to assess the role of IFN{gamma} and IP10 in LS using by generating Ndufs4(-/-)/Ifng(-/-) and Ndufs4(-/-)/IP10(-/-) double knockout lines. We find that IP10 alone does not significantly impact the onset or progression of disease in the Ndufs4(-/-) model, while IFN{gamma} loss significantly, but modestly, improves survival. These data indicate that IFN{gamma} contributes to pathology, but that IFN{gamma} and IP10 are both dispensable for overall disease course of LS. Our findings support some role for IFN{gamma} targeting therapies in the management of mitochondrial disease, but suggest they may provide only modest benefits, at least in LS.

pathology↗

bioRxiv2023

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice

Microglia play diverse pathophysiological roles in Alzheimers disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-{beta} (A{beta}) deposition. Mice expressing CD33M have increased A{beta} levels, mo7re diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to control 5XFAD mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

neuroscience↗

bioRxiv2023

Rocket-miR, a Translational Launchpad for miRNA-based Antimicrobial Drug Development

Developing software tools that leverage biological datasets to accelerate drug discovery is an important aspect of bioinformatics research. Here we present a novel example: a web application called Rocket-miR that applies an existing bioinformatics algorithm (IntaRNA) to predict cross-species miRNA-mRNA interactions and identify human miRNAs with potential antimicrobial activity against antibiotic resistant bacterial infections. Rocket-miR is the logical extension of our prior finding that human miRNA let-7b-5p impairs the ability of the ubiquitous opportunistic pathogen P. aeruginosa to form biofilms and resist the bactericidal effect of beta lactam antibiotics. Rocket-miRs point and click interface enables researchers without programming expertise to predict additional human-miRNA-pathogen interactions. Identified miRNAs can be developed into novel antimicrobials effective against the 24 clinically relevant pathogens, implicated in diseases of the lung, gut and other organs, that are included in the application. The manuscript incorporates three case studies contributed by microbiologists that study human pathogens to demonstrate the usefulness and usability of the application. Rocket-miR is accessible at the following link: http://scangeo.dartmouth.edu/RocketmiR/. ImportanceAntimicrobial resistant infections contribute to millions of deaths worldwide every year. In particular, the group of bacteria collectively known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.) pathogens are of considerable medical concern due to their virulence and exceptional ability to develop antibiotic resistance. New kinds of antimicrobial therapies are urgently needed to treat patients for whom existing antibiotics are ineffective. The Rocket-miR application predicts targets of human miRNAs in bacterial and fungal pathogens, rapidly identifying candidate miRNA-based antimicrobials. The applications target audience is microbiologists that have the laboratory resources to test the applications predictions. The Rocket-miR application currently supports 24 recognized human pathogens that are relevant to numerous diseases including CF, COPD, urinary tract infections and pneumonia. Furthermore, the application code was designed to be easily extendible to other human pathogens that commonly cause hospital-acquired infections.

bioinformatics↗

bioRxiv2023

The molecular prognostic score, a classifier for risk stratification of high-grade serous ovarian cancer

The clinicopathological parameters such as residual tumor, grade, FIGO score are often used to predict the survival of ovarian cancer patients, but the 5-year survival of high grade serous ovarian cancer (HGSOC) still remains around 30%. In recent years, a gene expression based molecular prognostic score (mPS) was developed that showed improved prognosis in several cancers including ovarian cancer. The feature extraction using LASSO-Cox regression was applied on the training data with 10-fold cross validation to obtain 20 predictor genes along with the coefficients to derive mPS. The mPS based prognosis of HGSOC patients was validated using the log-rank test and receiver operator characteristic curve. The AUC of 20 gene-based mPS in predicting the 5-year overall survival was around 0.7 in both the training (n=491) and test datasets (n=491). It was also validated across HGSOC patients (n=7542), data collected from the Ovarian Tumor Tissue Analysis (OTTA) consortium. The mPS showed significant impact (adjusted HR = 6.1, 95% CI of HR= 3.65-10.3; p <0.001) on prognosis of HGSOC. The performance of mPS for the prognosis of survival of HGSOC was substantially better than conventional parameters: FIGO (adjusted HR=1.1, 95% CI=0.97-1.2, p=0.121), residual disease (adjusted HR=1.3, 95% CI= 1.13-1.4, p<0.001), and age (adjusted HR=1.2, 95% CI= 0.98-1.6, p=0.08). It was found that focal-adhesion, Wnt and Notch signaling pathways were significantly (p<0.001) upregulated, whereas antigen processing and presentation (p<0.001) was downregulated in high risk HGSOC cohorts based on mPS stratification. The molecular prognostic score derived from 20-gene signature is found to be the novel robust prognostic marker of HGSOC. It could potentially be harnessed in clinical settings to determine the overall survival of ovarian cancer. The high risk HGSOC patients based on mPS stratification could be benefited from alternative therapies targeting Wnt/ Notch signaling pathways and also immune evasion. Author summaryThe 20-gene signature based molecular prognostic score (mPS) was found to be associated with risk stratification and hence, predicting the overall survival time of HGSOCs. It was applicable in all training HGSOC datasets and across RNA sequencing platforms that also includes the previous reported studies in HGSOC cohorts (Millstein et al., 2020; Talhouk et al., 2020). The 20-gene signature based mPS for the prognosis of overall survival of HGSOC outperformed conventional parameters: age, residual disease and FIGO score. The high or increased risk group of HGSOC based on our mPS stratification was found to have dysregulated pathways of Wnt, Notch, Akt signaling, and antigen presentation. Thus, treatments targeting these pathways might be beneficial for high risk HGSOC and hence anticipated to improve the over-all survival of HGSOC.

genomics↗

bioRxiv2023

Tempered IL-2 Signals Program PD-1 Checkpoint Blockade-Responsive Stem-like Exhausted T Cells During Priming

Stem-like progenitor exhausted CD8 T cells are critical for maintaining long-term resistance during chronic infections and cancer, and represent an important checkpoint blockade immunotherapy target for functional reinvigoration and disease control. Hence, there is vigorous interest in understanding the ontogenesis of TCF-1Hi stem-like exhausted CD8 T cells, and in defining the signals that promote their development. Here, we show that virus-specific TCF1HiGzmBLo CD8 T cells develop during early stages of chronic viral infection, and are progressively deprogrammed from the stem-like lineage towards terminal differentiation by strong IL-2 signals. In vivo fate-tracking studies show that strong T cell-intrinsic IL-2 signals through the high affinity heterotrimeric IL-2R/{beta}/{gamma} receptor drive skewed development of terminally differentiated TCF-1LoGzmBHi cells, which largely die in chronic antigenic environment. In contrast, tempered IL-2 signals through the intermediate affinity IL-2R{beta}/{gamma} heterodimer, or delayed priming in diminished IL-2 milieu support preferential development of TCF-1Hi stem-like exhausted CD8 T cells, capable of long-term persistence and potent responsiveness to PD-1 therapy in later stages of chronic viral infection. In human tumors as well, single cell RNA-seq analyses of tumor infiltrating lymphocytes from melanoma, human papillomavirus+ head and neck cancer and lung cancer patients revealed an inverse relationship between IL-2 signaling signature and T cell stemness. Moreover, melanoma patients with enriched IL-2 signaling signature showed poor responses to checkpoint blockade immunotherapy. Collectively, these findings bear relevance to clinical immunotherapies of chronic viral infections and cancers, and support exogenous IL-2 signal manipulation prior to therapy as a strategy for augmenting long-term clinical outcomes. ONE SENTENCE SUMMARYProgramming of exhausted T cell fates by differential IL-2

immunology↗

bioRxiv2023

Single-Molecule FRET Illuminates Structural Subpopulations and Dissects Crucial Molecular Events During Phase Separation of a Prion-Like Low Complexity Domain

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies that permit us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

biophysics↗

bioRxiv2023

Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity

Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the genome-wide activity of the DNA methylation maintenance machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.

genomics↗

bioRxiv2023

PLGA-based dual-loaded nanoformulation of DIM and TMZ - An advanced clinical strategy for brain cancer treatment in a combinatorial approach

Glioblastoma multiforme (GBM) is a highly aggressive form of primary brain tumor in adults, which unfortunately has an abysmal prognosis and poor survival rates. Even though several FDA-approved multimodal treatments for targeting GBM are available, the effectiveness in most patients are not satisfactory. The reason behind this poor success rate is mainly attributed to insufficient drug distribution to the tumor site across the blood-brain barrier (BBB) and induction of resistance for single-drug based therapies. Chemotherapy with Temozolomide (TMZ) having a median overall survival of around 12-15 months, envisages the urgent necessity for more effective treatment strategies. Based upon these facts, in this study, we have developed a novel approach for repurposing TMZ along with inhibition of EGFR, which overexpressed in GBM, to achieve our goal. PLGA-based nanoencapsulation of both TMZ and 3,3-diindoyl methane (DIM), an EGFR inhibitor, in a combinatorial approach enhances the delivery of them together. Their synergistic mode of actions, significantly enhances the cytotoxic effect of TMZ in vitro and in vivo. Moreover, the dual-loaded nanoformulation works more efficiently than their individually packed nanoparticles on DNA damage and apoptosis, resulting in a several-fold reduction in tumor burden, systemic drug toxicity, and increased survival. These findings suggest the preclinical potential of this new treatment strategy.

cancer biology↗

bioRxiv2023

Migration coupled with recombination explains disparate HIV-1 anatomical compartmentalization signals

The evolution of HIV-1 in a host is shaped by many evolutionary forces, including recombination of virus genomes and the potential isolation of viruses into different tissues with compartmentalized evolution. Recombination and compartmentalization have opposite effects on viral diversification, with the former causing global mixing and the latter countering it through spatial segregation of the virus population. Therefore, recombination and compartmentalization together give rise to complex evolutionary dynamics that convolutes their individual effects in standard, bifurcating phylogenetic trees. Although there are various theoretical methods available to infer the presence of recombination or compartmentalization individually, there is little knowledge of their combined effect. To study their interaction and whether that could explain the many disparate results that have been described in the HIV-1 literature, we developed an age-structured forward-time evolutionary model that includes compartments, migration, and recombination. By tracking the evolutionary history of individual virus variants in an Ancestral Recombination Graph (ARG) and resolving the ARG into standard bifurcating trees, we reexamined 771 anatomical tissue pairs infected with HIV-1. Remarkably, we found that recombination can make the resulting bifurcating tree appear more compartmentalized than the virus population actually is. However, we found migration between all 771 tissue pairs, typically at 2.8-4.9x10-3 taxa-1 day-1. Thus, while different point mutations may arise in different parts of the body, migration eventually brings these variants together and recombination merges them into a relatively homogeneous cloud of a universally evolving quasispecies. Modelling this process, we explain the many different results previous research found among distinct anatomical tissues. We also show that the popular Slatkin-Maddison test comes to different results about compartmentalization at the same migration rate depending on the sample size. SIGNIFICANCEWhether HIV-1 sampled in different anatomical tissues are compartmentalized or not has been a long-standing quest because it relates to both clinical and biological insight. Previous studies of this question have reported disparate results from the same as well as different tissue comparisons. Here, we consolidate and explain these contrasting results with a new evolutionary model that includes compartments, migration, and recombination. We show that no anatomical tissues are fully compartmentalized, and that the migration of HIV-1 between the tissues allows recombination to homogenize the diversity that may arise in separate tissues of an infected person.

evolutionary biology↗

bioRxiv2023

DNA damage-induced interaction between a lineage addiction oncogenic transcription factor and the MRN complex shapes a tissue-specific DNA Damage Response and cancer predisposition

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA Damage Response (DDR) programs. However, some cells, in skin for example, are normally exposed to high levels of DNA damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Here we show, using melanoma as a model, that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a non-transcriptional role in shaping the DDR. On exposure to DNA damaging agents, MITF is phosphorylated by ATM/DNA-PKcs, and unexpectedly its interactome is dramatically remodelled; most transcription (co)factors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks, and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement, high MITF levels are associated with increased SNV burden in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of ATM/DNA-PKcs-phosphorylated MITF. Our data suggest that a non-transcriptional function of a lineage-restricted transcription factor contributes to a tissue-specialised modulation of the DDR that can impact cancer initiation.

molecular biology↗

bioRxiv2023

Development of Drosophila as a metazoan model to study inorganic polyphosphate biology

Polyphosphate (polyP) exists in all life forms; however, its biological functions in metazoans are understudied. Here, we explored Drosophila, to our knowledge, as the first genetic model to explore polyP biology in metazoans. We established biochemical and in situ methods to detect, quantify, and visualise polyP in Drosophila. We then engineered a FLYX system to deplete polyP in subcellular compartments in a tissue-specific manner. Using these tools, we demonstrated a spatiotemporal and subcellular compartment-specific regulation of polyP levels in various developmental stages and tissue types. We discovered that polyP is crucial for Drosophila hemolymph clotting and proper developmental timing, consistent with an evolutionarily conserved role as exogenous polyP also accelerates mammalian blood clotting. Further, the transcriptomics analysis of polyP-depleted larvae demonstrates the impact of polyP on several cellular processes, including translation. These observations underscore the utility of the toolkit we developed to discover previously unknown polyP functions in metazoans.

cell biology↗

bioRxiv2023

Astroglia-derived TIMP-1 alleviates cognitive deficits by restoring synaptic plasticity in Alzheimer's disease

The influence of astrocyte-secreted cytokines on neuronal health in Alzheimers disease (AD) is poorly understood, despite their increasing recognition as potential molecules for therapeutic targeting. Recently, we demonstrated that an anti-inflammatory cytokine, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is robustly secreted by astrocytes early in response to amyloid-{beta} (A{beta}) but is reduced in its prolonged presence. Here, we found strikingly diminished levels of TIMP-1 in the brain of six-month-old 5xFAD mice concomitant with high levels of A{beta}, versus wild-type mice. Intracerebroventricular injection of TIMP-1 in 5xFAD mice ameliorated their cognitive functions. TIMP-1 not only ensured neuronal viability against apoptosis and aberrant autophagy in the AD model by binding to neuronal CD63 receptors, but also conferred synapse-specific effects. Synaptosomal analysis revealed TIMP-1 elevates dendritic spine size and protein levels, likely by promoting post-synaptic long-term potentiation in hippocampus, independent of pre-synaptic activity. TIMP-1 induced brain-derived neurotrophic factor (BDNF) and BDNF-mediated post-synaptic signaling. Therefore, we identify TIMP-1 as a multifunctional cytokine with distinct protective mechanisms-of-action on neurons and propose it as a promising therapeutic candidate in AD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/533245v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@187adadorg.highwire.dtl.DTLVardef@9b6ae1org.highwire.dtl.DTLVardef@185e352org.highwire.dtl.DTLVardef@10f2995_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug resistant Mycobacterium tuberculosis

Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor. SignificanceIsoniazid (INH) is a critical frontline antibiotic to treat Mycobacterium tuberculosis (Mtb) infections. INH efficacy is limited by its suboptimal penetration of the Mtb-containing lesion and by the prevalence of clinical INH-resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated by katG mutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistant katG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causes Mtb to become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitize Mtb to sub-lethal doses of INH, such as those achieved at the infection site.

microbiology↗

bioRxiv2023

Peripheral macrophages causally contribute to disease onset and progression in the Ndufs4(KO) model of Leigh syndrome

Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common paediatric presentation of genetic mitochondrial disease. LS is a multi-system disorder with severe neurologic, metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Recently, we demonstrated that high-dose pexidartinib, a CSF1R inhibitor, prevents LS CNS lesions and systemic disease in the Ndufs4(-/-) mouse model of LS. While the dose-response in this study implicated peripheral immune cells, the immune populations involved have not yet been elucidated. Here, we used a targeted genetic tool, deletion of the colony stimulating factor 1 receptor (CSF1R) macrophage super-enhancer FIRE (Csf1r{Delta}FIRE), to specifically deplete microglia and define the role of microglia in the pathogenesis of LS. Homozygosity for the Csf1r{Delta}FIRE allele ablates microglia in both control and Ndufs4(-/-) animals, but onset of CNS lesions and sequalae in the Ndufs4(-/-), including mortality, are only marginally impacted by microglia depletion. The overall development of necrotizing CNS lesions is not altered, though microglia remain absent. Finally, histologic analysis of brainstem lesions provides direct evidence of a causal role for peripheral macrophages in the characteristic CNS lesions. These data demonstrate that peripheral macrophages play a key role in the pathogenesis disease in the Ndufs4(-/-) model.

pathology↗

bioRxiv2023

DYRK1A interacts with Tuberous Sclerosis complex and promotes mTORC1 activity

DYRK1A, a ubiquitously expressed kinase, is linked to the dominant intellectual developmental disorder, microcephaly and Down syndrome in humans. It regulates numerous cellular processes such as cell cycle, vesicle trafficking and microtubule assembly. DYRK1A is a critical regulator of organ growth; however, how it regulates organ growth is not fully understood. Here, we show that the knockdown of DYRK1A results in reduced cell size, which depends on mTORC1. Using proteomic approaches, we found that DYRK1A interacts with the Tuberous sclerosis complex (TSC) proteins, namely TSC1 and TSC2, which negatively regulate mTORC1 activation. Further, we show that DYRK1A phosphorylates TSC2 at T1462, a modification known to inhibit TSC activity and promote mTORC1 activity. We also found that the reduced cell growth upon knockdown of DYRK1A can be rescued by overexpression of RHEB, an activator of mTORC1. Our findings suggest that DYRK1A inhibits TSC complex activity through inhibitory phosphorylation on TSC2, thereby promoting mTORC1 activity. Further, using the Drosophila neuromuscular junction as a model, we show that the mnb, the fly homologues of DYRK1A, is rescued by RHEB overexpression, suggesting a conserved role of DYRK1A in TORC1 regulation.

molecular biology↗