Dec 17
Shah
Anti-viral defence by an mRNA ADP-ribosyltransferase that blocks translation
The CmdTAC system, a bacterial anti-phage defense mechanism, was investigated where the toxin CmdT is activated during infection by the phage capsid protein Gp23. Immunoprecipitation and mass spectrometry revealed Gp23 as the trigger that displaces CmdC, leading to CmdA degradation by ClpP protease and the release of CmdT. In vitro and in vivo experiments showed CmdT specifically ADP-ribosylates the N6 position of adenine in GA dinucleotides on single-stranded mRNA which were confirmed via RNA immunoprecipitation, HPLC, and mass spectrometry. This modification blocks translation, demonstrated by radiolabeled methionine and cysteine incorporation assays and in vitro translation inhibition. RNA sequencing revealed reduced expression of phage late-stage genes, preventing virion production and phage propagation. These results uncover a novel mechanism of bacterial defense through mRNA-targeting ADP-ribosylation, which blocks phage propagation. These results are crucial in eukaryotic cmdTAC homologues studies for a possible anti-viral mechanism, however, how the translation is blocked by cmdT, is yet to be discovered.
Hannah
Adult skull bone marrow is an expanding and resilient haematopoietic reservoir
Age-related decline in bone marrow (BM) function is well-established, but the functional heterogeneity between different BM compartments remains poorly understood. This paper directly compares skull and femoral BM in mice and humans, using in vivo imaging, flow cytometry, and single-cell RNA sequencing. The research reveals that skull BM undergoes lifelong expansion driven by VEGF-VEGFR2 signaling, a key regulator of vascular growth and lifelong angiogenesis, serving as a critical source of hematopoietic cells for systemic circulation and meningeal/CNS immunity. Computed tomography (CT) scans of 36 human skulls further support the finding of BM expansion with age. The skull BM's function as a resilient hematopoietic reservoir is highlighted by its resistance to aging hallmarks (reduced adipogenesis and inflammation) and its distinct responses to pregnancy, stroke, and other conditions, unlike femoral BM. Although skull BM's resilience and expansion offer potential for clinical therapies, more research is crucial to understand the underlying mechanisms and translate these findings to human applications.
Sean
Cellular ATP demand creates metabolically distinct subpopulations of mitochondria
Aish
Differentiation fate of a stem-like CD4 T cell controls immunity to cancer
Nucleosome flipping drives kinetic proofreading and processivity by SWR1
Histones are fundamental components of chromatin organization and transcriptional regulation. The SWR1 chromatin remodeling complex reorganizes histones by specifically replacing canonical H2A-H2B dimers with Htz1-H2B dimers; however, the mechanism driving this process is unknown. To determine how yeast SWR1 specifically incorporates Htz1-H2B dimers into nucleosomes, the authors of this study designed several single-molecule fluorescence resonance energy transfer (smFRET) assays. These experiments revealed that SWR1 flips the two faces of the nucleosome to exchange H2A-H2B for Htz1-H2B, with the kinetics for nucleosome flipping determined by histone composition. The authors then used their smFRET findings to guide analysis of intermediate states identified in cyro-EM data, which confirmed their previous findings. This study reveals that nucleosome flipping by SWR1 can drive the specificity of its function, and this mechanism could regulate histone modifications in other contexts.
AJM
Rhythmic IL-17 production by γδ T cells maintains adipose de novo lipogenesis
Mammal's metabolic rate oscillates over a 24 hour period in a circadian rhythm. How immune cells sense and contribute to this rhythm are underappreciated despite their importance to preserving metabolic homeostasis. Herein, the authors identified a subset of adipose tissue T cells that express known “clock” genes across different day periods and produce IL-17 in a rhythmic, time dependent manner in vivo and ex vivo. Adipocyte-specific IL-17 receptor deficiency disrupted de novo lipogenesis, resulting in whole-body metabolic dysfunction. Overall, the authors elucidated a novel rhythmic expression pattern of IL-17 in adipose tissue that is crucial to maintaining metabolic homeostatic cycles.
Dec 3
Shah
A lymphocyte chemoaffinity axis for lung, non-intestinal mucosae and CNS
The research article identifies a novel lymphocyte affinity mechanism involving GPR25, lymphocytes receptor and its ligand CXCL17, primarily expressed in non-intestinal mucosae tissues (NIMTs), respiratory tract and central nervous system (CNS). In these tissues, the GPR25-CXCL17 axis directs lymphocyte homing/migration playing a vital role in immune regulation and tolerance. Single-cell transcriptomic analyses and bulk RNA sequencing revealed cell- specific and high expression of GPR25 in various immune cells except the myeloid cells in these tissues. While experiments in mice confirmed CXCL17 dependent GPR25 mediated lymphocyte homing specifically in these tissues. The GPR25-CXCL17 axis highlights its crucial role in immune homoeostasis, tolerance and immune-related diseases making it a potential clinical target.
Hannah
Targeting immune–fibroblast cell communication in heart failure
This research uncovers novel insight into the cellular mechanism driving cardiac fibrosis, a significant contributor to one of the world's leading health concern affecting approximately 1 in 4 people known as heart failure. Using CITE-seq, Multiome, and spatial transcriptomics, the study identified a novel fibroblast population marked by fibroblast activator protein (FAP) and periostin (POSTN) expression that contributes to cardiac fibrosis independently of myofibroblasts. These FAP/POSTN fibroblasts were shown to be critically regulated by cardiac fibroblasts via interleukin-1β (IL-1β) signaling from CCR2+ monocytes and macrophages. This finding was validated during in vivo experiments which blocked IL-1β signaling and demonstrating improved cardiac function and decreased fibrosis. Due to ongoing debate revolving the most accurate model of cardiac fibroblasts and fibrosis, the researchers utilized various in vivo and in vitro models to evaluate cardiac fibrosis; moreover, they conclude mouse models of cardiac injury are superior to human fibroblast cultures for representing the human disease phenotype. Although the study is limited by their sample size, they created a strong foundation to further investigate the mechanisms by which fibroblasts communicate with cells regulating inflammation and highlight the therapeutic potential of targeting this inflammation to treat cardiac fibrosis for improved heart function.
Sean
Cellular ATP demand creates metabolically distinct subpopulations of mitochondria
Chromatin remodelling drives immune cell–fibroblast communication in heart failure.
The nucleus is the center of transcriptional programs that are key mediators of cellular response to stress under chronic disease conditions, such as heart failure. Previous work has identified BRD4, a critical mediator of stress-activated chromatin remodeling, as central to cardiac fibroblast dysfunction; however, the mechanism for BRD4-induced fibroblast activation is unknown. In this study, the authors performed scRNA-seq and scATAC-seq of mouse hearts, and they identified that Cx3cr1+ myeloid cells produced high levels of Il1b to induce fibroblast dysfunction during heart failure. Cx3cr1-induced deletion of Brd4 improved heart failure measures and demonstrated that this effect was Brd4-dependent. This study identified an important new mechanism regulating heart failure and highlights the role of transcriptional regulators in chronic disease.
AJM
Nuclear release of eIF1 restricts start-codon selection during mitosis
Introduction: Alternative translation is a predominant regulatory mechanism during mitosis as transcription is globally inhibited. Recent advances in protein biology has made investigation possible and necessary.
Preliminary results: We performed translation initiation-site sequencing, revealing a global shift in start-codon selection during mitosis. This change was correlated with an increase in eIF1 associated with 40S ribosomes, a translation factor with preference for canonical start codons. Thus, we propose the hypothesis: alternative translation during mitosis prefers canonical start codons due to increased eIF1 association to translation machinery.
Novelty: We have demonstrated a novel angle for mitotic regulation with detrimental affects to cell cycling.
Aim 1: Identify decisive properties for translation during mitosis.
Working hypothesis: High-efficiency translation sites will be dominated by main ORFs. We will perform feature selection from sequence information from the differentially translated genes identified in our preliminary data. We will validate the preference using luciferase assays to compare the translation rate of modified sequences. We will further perform gene-set enrichment on differentially translated genes to determine the cellular impact of alternative translation during mitosis.
Aim 2: Investigate eIF1 regulatory network under mitosis.
Working hypothesis: mitosis promotes eIF1 association with 40S to promote alternative translation. We will perform an extensive search into the regulatory status of eIF1 and its at the mRNA and protein level, as well as phosphorylation status and cellular localization across mitosis.
Alternatives: Given eIF1 is known to be autoregulated via its weak translation site, ectopic upregulation will decrease endogenous eIF1 translation; further, the rapid change in translation suggests protein modification rather than translation. Thus, particular interest will focus on the protein dynamics and alternations of this and other translation regulators.
Overall: This research will validate the novel concept of alternative translation regulation in mitosis and investigate regulatory mechanisms underlying this crucial switch.
Nov 19
Hannah
Selective utilization of glucose metabolism guides mammalian gastrulation
This paper investigates the role of glucose metabolism in guiding mammalian gastrulation, a critical stage of embryonic development. The researchers challenge the traditional view that only transcription factors and morphogens dictate cell fate and morphology during this process, proposing instead that localized glucose metabolism acts as a crucial developmental regulator. Using single-cell imaging, stem cell models, and tissue explants, the researchers discovered two distinct waves of glucose uptake during gastrulation: the first in the epiblast utilizes the hexosamine biosynthetic pathway to drive cell fate acquisition, while the second wave in the mesoderm uses glycolysis to guide cell migration. These glucose-mediated processes were linked to high extracellular signal-regulated kinase (ERK) activity, suggesting an essential connection between glucose metabolism and signaling pathways that drive successful tissue patterning during mammalian gastrulation. This study provides a framework for understanding the intricate interplay between metabolic activity and signaling in early development, challenging our understanding of the housekeeping nature of cellular metabolism which future researchers should continue to explore to uncover the precision of these mechanisms.
Shah
Temporal BMP4 effects on mouse embryonic and extraembryonic development
The researchers studied the temporal effects of bone morphogenetic protein 4 (BMP4) signaling during early mouse embryonic development, specifically its role in coordination of embryonic and extraembryonic tissue differentiation from embryonic days E6.5 to E8.0. This strategy has been lacking previously limiting the understanding of proper signaling involved in tissue specific embryonic differentiation. Using single-cell RNA sequencing and genetic perturbations, the researchers mapped the differentiation path of chorion progenitor cells and ectoplacental cone (EPC) cells which are crucial precursors in the development of placenta. The results showed that early BMP4 signals from chorion progenitors within the extraembryonic ectoderm are crucial for specific development of mesoderm and primordial germ cells (PGCs), while later BMP4 signals from the embryo limit PGC expansion, promotes allantois differentiation and differentiation of EPC into trophoblast giant cells (TGCs) and spongiotrophoblast/glycogen cells (SpT-Gly)—crucial for formation and function of placenta. This study highlights the time and origin dependent functions of BMP4 to be crucial factor deciding the fate of cells in different tissue types to ensure proper development of embryo and placenta.
Sean
The lipid globotriaosylceramide promotes germinal center B cell responses and antiviral immunity
The lipid globotriaosylceramide (Gb3) promotes a high level of cross-protection by leading to a higher expression of subdominant epitopes. Gb3 is a glycosphingolipid expressed on B cells recruited to germinal centers at high levels. Using a series of KO mice of critical synthesis (A4Galt) and degradation (Gla) enzymes in the Gb3 pathways, researchers tested for differences in affinity maturation across strains. Researchers observed that the higher expressions of Gb3, Gla-KO mice, produced a higher population of subdominant epitope antibodies. This was crucial as these mice were better at neutralizing heterologous forms of influenza. This means that in future research, Gb3 could act as an adjuvant for vaccinations, increasing the antibody repertoire against mutated strains of Influenza.
The genetic architecture of protein stability
KK
Retrotransposons are co-opted to activate hematopoietic stem cells and erythropoiesis
Background: During mammalian pregnancy, increased blood volume and oxygen consumption create significant hematopoietic stress. This stress is addressed through increased hematopoiesis in the bone marrow and in extramedullary sites; however, well-known factors such as erythropoietin signaling and pregnancy-related hormones do not fully explain the mechanisms behind increased hematopoiesis during pregnancy.
Preliminary data: To identify additional mechanisms contributing to increased hematopoiesis during pregnancy, we performed RNA-sequencing on sorted splenic hematopoietic stem cells (HSCs) from pregnant mice, and we identified a significant increase in retrotransposon activation during pregnancy. We treated pregnant and nonpregnant mice with reverse transcriptase inhibitors to block retrotransposons and observed a decrease in hematopoiesis exclusively in pregnant mice. This led us to hypothesize that pregnancy activates retrotransposons in HSCs to enhance hematopoiesis.
Innovation: This project has identified a novel mechanism which could enhance hematopoiesis during pregnancy.
Approach: We will test our hypothesis through the following specific aims:
Aim 1: Characterize the impact of pregnancy-induced retrotransposons on HSC proliferation. Working hypothesis: pregnancy induces specific retrotransposons to induce hematopoiesis in HSCs. We will screen for specific retrotransposons that induce hematopoiesis using bone marrow reconstitution assays in bone marrow depleted for these elements.
Aim 2: Identify the signaling events that induce HSC retrotransposon activation during pregnancy. Working hypothesis: Retrotransposons are selectively induced in HSCs during pregnancy by specific signals to promote hematopoiesis. We will stimulate HSCs from pregnant and non-pregnant mice with serum from pregnant mice, as well as known hematopoietic factors, and observe the impact on HSC proliferation. We will perform proteomic analysis on mouse and human serum to identify potential factors, then treat HSCs and quantify their impact on retrotransposon expression and cell proliferation.
Pitfalls/alternatives: Another method for identifying retrotransposon activation could be through chromatin accessibility profiling, then identification of transcription factors that bind. It is possible that pregnancy is not the only condition that induces HSC retrotransposon activation and proliferation. We could use additional models such as chronic bleeding or sickle cell anemia that also induce extramedullary hematopoiesis.
Overall impact: This project could identify a novel mechanism for hematopoietic cell proliferation, which could guide future treatment options for anemic patients.
Two-factor authentication underpins the precision of the piRNA pathway
Oct 15
Hannah
Multiple sclerosis (MS) is a leading cause of neurological disability in young adults, yet the mechanisms underlying immune dysregulation in its pathology remain poorly understood. In a twin study involving 12 pairs—one twin with MS and the other either with subclinical neuroinflammation (SCNI) or healthy—a combination of single-cell RNA (scRNA-seq) and T cell receptor (TCR) sequencing was used to analyze peripheral CD8+ T cells from both blood and cerebrospinal fluid. The study revealed consistent immune and metabolic alterations in CD8+ T cells across both the prodromal and clinical stages of MS. These findings, validated by brain tissue analysis and two independent cohorts, demonstrated heightened expression of genes related to immune responses and energy metabolism. Despite a small cohort, the twin-based design controlled for genetic background, revealing CD8+ T cells' proinflammatory role in MS and offering insights into therapeutic targets for immune dysregulation.
Shah
Environment-independent distribution of mutational effects emerges from microscopic epistasis
Sean
Expression of Nr5a2 is critical for morula development. Using a series a maternal, zygotic and maternal/zygotic knockout mice of the Nr5aS transpiration factor (TF), researchers studied the development of the zygote before implantation. From these knockout mice it was determined that Nr5a2 has a greater impact on the development after 8C. Using ATAC-Seq Nr5a2 appeared to be responsible for increasing chromatin accessibility at important TFs. This inhibition caused for morphological amoralities and developmental decay. Researchers point out how their study was limited as Nr5a2 may not act alone, but this specific class of TFs should be studied in the future in relation to morula development.
It has very recently been shown that microglia interact with neurons through their tunneling nanotubes (TNTs), which allow transfer of organelles and vesicles between cells; However, the impact of this connection on neuronal health has not been studied. Herein, the authors demonstrated TNTs facilitate a bidirectional transfer of cytosolic proteins and mitochondria between microglia and neurons. Importantly, the authors utilized neurons that form pathological protein aggregates of alpha-synuclein (α-syn) and tau, which are models of Alzheimer's, dementia, and Parkinson's disease (PD). Neurons burdened with α-syn express higher oxidative stress markers; co-culture with TNT-competent microglia revealed non-specific transfer of fluorescently tagged proteins, allowing α-syn and tau to be degraded in microglia. Further, TNT-dependent microglia transfer of mitochondria improved protein aggregate-induced oxidative stress. Lastly, microglia expressing common disease-associated SNPs of PD revealed disrupted TNT formation. This research implicates microglia TNTs for several neurological protein aggregation diseases, however TNT markers are underdeveloped and basic knowledge of their formation is necessary to rigorously evaluate their contributions to disease etiology.
KK
Oct 1
Hannah
DNA-sensing inflammasomes cause recurrent atherosclerotic stroke
Stroke ranks as the second leading cause of death globally, and despite its prevalence, effective strategies to prevent stroke recurrence remain elusive. In this study researchers uncovered how DNA-sensing inflammasomes contribute to the destabilization of atherosclerotic plaques after stroke. Using a novel mouse model, they identified that cell-free DNA (cfDNA) released from neutrophil extracellular traps (NETs) activates the AIM2 inflammasome, promoting inflammation and plaque destabilization. Human carotid artery samples validated these findings, showing elevated cfDNA and inflammasome markers in stroke patients compared to asymptomatic individuals. Targeting cfDNA with DNase or inhibiting inflammasome activity significantly reduced stroke recurrence in mice, offering promising therapeutic approaches, though further research is needed to evaluate long-term efficacy in clinical settings.
Shah
The interaction between the epithelium and immune cells is vital for maintaining homeostasis; therefore, disturbance in the interaction is linked to inflammation, autoimmunity, and cancer. The researchers introduced a novel model to capture the interactions between the epithelium and immune cells. The study generated human intestinal immuno-organoids from intestinal resection and integrated autologous tissue-resident memory T cells into IIOs. The scRNA-seq, live imaging and flow cytometry revealed changes in the TRMs leading to CD8 T cell activation which were involved in inflammatory responses after introducing cancer therapeutic drugs in IIOs. IIOs can be used to study immune responses to identify crucial pathways for treating cancer, autoimmune, and infectious diseases as it’s a useful mimicry of in-vivo environment.
Recognition and control of neutrophil extracellular trap formation by MICL
Neutrophils are innate immune cells whose activation is critical to both host defense and autoinflammatory diseases. Previous work has shown that the neutrophil lectin receptor MICL was associated with exacerbated joint inflammation, and they sought to characterize its role in disease. Using mouse models of rheumatoid arthritis, the authors found that MICL depletion increased disease severity, and they also identified functional anti-MICL antibodies in samples from human patients with autoimmune diseases. Chemical inhibition of neutrophil functions demonstrated that MICL was regulating NET formation, and they identified MICL binding directly to NETs to activate additional neutrophils. This study thoroughly characterized the role of MICL in neutrophil activation and identified a novel pattern recognition receptor.
Sean
Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells
AJM
IgE plasma cells are transcriptionally and functionally distinct from other isotypes
Introduction: Type I hypersensitivity is elicited by IgE immunoglobulins and can result in a range of severe reactions including anaphylaxis, dermatitis, angioedema, and asthma. High resolution study of allergen-specific IgE B cells have been difficult due to their low frequency across tissues. Herein, we will employ our novel procedure, TRAPnSeq, to capture IgE-secreting B cells across type I hypersensitivity establishment.
Preliminary results: We previously published the utility of TRAPnSeq to capture antigen-specific antibody-secreting B cells (ASCs) for downstream analysis, including single-cell RNA-Seq. Transcriptional analysis of allergen house dust mite-specific IgE-secreting cells in the bone marrow (BM) revealed a greater dependence on endoplasmic reticulum (ER) stress compared to other antigen-specific ASCs, suggesting a higher rate of antibody production. Thus, we propose the hypothesis: long-lived allergen-specific IgE secreting B cells enforce type I hypersensitivity through higher production of antibody compared to other isotypes.
Novelty: We propose a novel hypothesis that considers B cell isotypes as functionally distinct populations that explains why IgE type I hypersensitivity overcomes other allergen-specific antibody types despite lower cell frequencies. This hypothesis can be robustly assessed using our high-resolution TRAPnSeq technique.
Aim 1: Establish antibody secretion rate of antigen-specific isotypes.
We will use DropMap to measure antibody production to restimulated Derp1-specific IgE and IgG1 B cells isolated from the same sensitized mice. Further, we will measure antibody isotype proportions in mouse sera following challenge in parallel to overall B cell isotype proprotions over time to type I hypersensitivity establishment.
Aim 2: Establish ER stress levels as a driver for differential antibody production rate
We will dose sensitized mice with titrated ER stress inhibitors (4-PBA, Rapamycin, Toyocamycin) and track antibody isotype proportions in mouse sera.
KK
Phage-triggered reverse transcription assembles a toxic repetitive gene from a noncoding RNA
Sept 17
HL
This study addresses the knowledge gap in understanding how cardiomyocytes, rather than immune cells alone, initiate inflammation after myocardial infarction, the most common cause of death in the world. Using spatial transcriptomics in mice and humans, the authors discovered that cardiomyocytes in the infarct borderzone trigger a type I interferon response, forming clusters of interferon-induced cells (IFNICs). By selectively inhibiting IRF3 in cardiomyocytes, the formation of these IFNICs is prevented which reduces the risk of heart rupture and improves survival. However, inhibiting IRF3 in cardiomyocytes without disrupting its essential role in immune cells is a major challenge of these efforts. Future research should focus on refining methods which block the cardiomyocyte driven response while maintaining necessary immune activity with aims to develop treatment for post-infarction heart failure.
ST
4E (P-eIF4E) is involved in translation of specific mRNA through the 5' UTR region during fasting or a ketogenic diet which results in tumorigenesis; however, the physiological role of 4E (P-eIF4E) during fasting was not previously understood. Researchers aimed to understand the pathways elicited by fasting involved in remodeling the translatome in relation to cancer. Desired outcomes were analyzed using mice in both fasted and fed conditions where the liver lysates were used for PolyRibo-seq and qPCR. The results showed remodeling of translatome during fasting or a ketone diet due to increased fatty acids (specifically linoleic acid) acting as a substrate (signaling molecules) activating AMPK which results in phosphorylation of 4E (eIF4E) via MNK activation. 4E (P-eIF4E) remodels translatome via 5' UTR by downregulating global translatome and upregulating translation of specific mRNA encoding for Hmgcs2 and PPAR-α, involved in lipid metabolism and ketogenesis (utilized by tumor cells for its growth and survival), a major determinant for pancreatic cancer formation. Furthermore, the researchers used a combination of a ketogenic diet with eFT508 (clinical inhibitor of MNK) which inhibited phosphorylation of 4E (eIF4E) and inhibited pancreatic tumor growth. The data suggested 4E (P-eIF4E), a potential target for future clinical treatment of cancer that utilizes ketone bodies as energy source and a major target to monitor cellular metabolic remodeling at varying nutritional conditions.
Colorectal cancer (CRC) is the second deadliest form of cancer, and current precision medicine tools for determining treatment and prognosis are inadequate. To address this issue, the authors of this study characterized the mutational and transcriptomic features of CRC in a cohort of over 1000 patients and developed novel tools for tumor classification and prognosis. Using whole-genome and whole-transcriptome sequencing, they performed in-depth characterization of driver genes, CRC-associated variants, and mutational signatures. The authors then clustered tumor transcriptomes to classify tumor subtypes, identified transcriptomic pathway signatures from each subtype, and incorporated these signatures into a high-performing prognostic model. Though this study did not perform any true mechanistic analysis of CRC, it highlights the power of integrated multi-omics studies to characterize human disease.
AJM
July 26
KP: Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis
Localized subsets of fibroblasts at tissue injury sites have been suggested to contribute to pathologic fibrosis, but their specific origin and role as drivers of disease were not well understood; the researchers previously distinguished alveolar fibroblasts as one subset and sought to determine their involvement in fibrosis. When Cthrc1, a pro metastasis gene that emerges after lung injury, was partially ablated in mouse lung tissue, fibrosis was significantly reduced. When Tgfbr2, a key signaling gene in fibrosis, was conditionally deleted from alveolar fibroblasts, there was a nearly complete inhibition of fibrosis. These findings suggest that alveolar fibroblasts are a dominant source of pathologic fibroblasts following fibrotic lung injury via TGFβ signaling and that CTHRC+ fibroblasts are significant players in fibrogenesis. While these findings imply alveolar fibroblasts as therapeutic targets, single-cell RNAseq data showed that some fibrotic fibroblasts did not express CTHRC1, so future work should determine whether CTHRC uniquely contributes to the development of pulmonary fibrosis.
LZ: Molecular definition of the endogenous Toll-like receptor signaling pathways
Toll-like receptor (TLR) pathways are critical to the innate immune system but not well understood. This study aims to define TLR pathway activities by tagging the critical protein MyD88 in human and mouse macrophages before lipopolysaccharide treatment. Using microscopy and proteomic analysis, researchers found that activated TLRs induce the formation of many myddosomes with organized MyD88-proximal proteins at all stages of the TLR signaling pathway. Thus, they conclude that the entire TLR signaling pathway occurs within the myddosome. As this study was limited to macrophages, future research should validate its conclusions on myddosome role and structure with other cell types.
PL: Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis
Maintaining platelet homeostasis is essential for proper blood clotting and immune defense. In this paper, researchers discovered that innate immune guard cells called plasmacytoid dendritic cells (pDCs) are essential in platelet homeostasis. In order to keep platelet homeostasis, stable levels of megakaryocytes and megakaryocyte progenitors are required. pDCs release IFN-alpha when encountering a fragmenting megakaryocyte, which promotes MKP proliferation and restabilizes levels. The discovery of pDCs’ role in megakaryopoiesis opens up new therapeutic strategies and medicines to treat patients with platelet production disorders and platelets hyperplasia under viral infections.
Endoplasmic reticulum–plasma membrane contact gradients direct cell migration
HL: dsRNA formation leads to preferential nuclear export and gene expression
Understanding the dynamic interplay between the lesser-studied antisense RNA (asRNA) and sense RNA is crucial for comprehending how cellular processes respond to environmental changes. The deregulation of long non-coding RNAs (lcnRNAs), which include asRNAs, has been linked with diseases such as cancer and neurodegenerative disorders. Researchers use RNA co-immunoprecipitation (RIP) with a dsRNA-specific antibody J2 to map and analyze interactions between asRNAs and their corresponding sense mRNAs during transcription. They discovered that asRNAs anneal with sense mRNAs to form double-stranded RNA (dsRNA), facilitating their export from the nucleus to the cytoplasm and enhancing gene expression via the Mex67-Mtr2 pathway in yeast. Although it may be challenging to distinguish between functional and spurious antisense transcripts, this discovery reveals a new layer of gene regulation and broadens our understanding of RNA biology, with implications for disease treatment.
Single-cell nascent RNA sequencing unveils coordinated global transcription
The human mitochondrial mRNA structurome reveals mechanisms of gene expression
Capturing totipotency in human cells through spliceosomal repression
Kavya : Neuroimmune cardiovascular interfaces control atherosclerosis
The central nervous system uses the outer connective tissue of arteries (lamina adventitia) as a principal channel to reach peripheral tissue, but the role of innervation and neuroimmune interactions in atherosclerosis has been historically overlooked because plaques are not directly innervated. This study investigated whether the peripheral nervous system (PNS) interacts with plaque-associated adventitial leukocytes to influence atherosclerosis progression. Using an Apoe-deficient mouse model, researchers found that atherogenesis induces the formation of a unique neuroimmune-cardiovascular network. Therapeutic intervention in this artery-brain circuit (ABC) reduced plaque volume; however, the makeup of the ABC is complex and highly variable, especially with age. Therefore, future research should identify specific neurons targeted by the discovered sensor channels to reveal direct targets for therapeutic intervention.
Hannah: Specification of CNS macrophage subsets occurs postnatally in defined niches
There is limited knowledge on the ontogenetic relationships, transcriptional programs, and molecular signals guiding the development and differentiation of central nervous system (CNS) macrophage subsets. This study aimed to clarify the origins, developmental pathways, and specific anatomical niches of microglia, meningeal, and perivascular macrophages, which are crucial for CNS immune function and advancing treatments for neurological disease. To investigate the CNS macrophage subsets, transgenic mouse models and human fetal brain cells were assessed via single-cell RNA sequencing, immunohistochemistry, and fate-mapping techniques. The findings revealed that CNS macrophage specification occurs through a stepwise process during perinatal stages within their anatomical niches, influenced by local cues. While studying only specific markers may limit the findings broad application, the potential to develop new therapies and improve outcomes for CNS disorders like Alzheimer's Disease is highly compelling in today's medical landscape.
Stephen: Obesity induces PD-1 on macrophages to suppress anti-tumour immunity
Obesity is an important clinical risk factor for many cancers with a rising prevalence around the world; however, cancer patients with obesity tend to show strong responses to PD-1 checkpoint blockade immunotherapy. Macrophages play key roles in both tumors and adipose tissue, and may fit into this framework. In both diet-induced obese cancer mouse models and macrophage cell lines exposed to obesity-associated cytokines and hormones, increased PD-1 expression along with impairments in phagocytosis, antigen presentation, and T cell activation are observed. PD-1 is also correlated with inflammatory increases in glycolysis as a negative feedback response to immune activation in gene ontology. With anti-PD-1 treatment reversing all the above impairments and significantly reducing tumor size in vivo compared to non-obese models, PD-1 appears to act as a metabolic mediator of anti-tumor immunity with enhanced activity in obesity. Other immune moderator cells may also have obesity-associated PD-1 expression but were not considered in this study; thus, future work should broaden the scope beyond macrophages and study the potential of pathogenic PD-1 expression as an indicator for immune therapy efficacy.
T cell tolerance’s involvement in atherosclerosis is not yet understood; this study investigates the involvement of T cells and T cell receptors (TCRs) in atherosclerosis pathogenesis in wild-type and atherosclerosis induced mice. Researchers collected peripheral tissues and used scRna-seq and TCR sequencing to identify T cell sub-type distribution and TCR diversity. They found that CD8+ cells clonally expanded in atherosclerotic conditions, signaling tolerance mechanisms in atherosclerosis are altered. These findings suggest that atherosclerosis may exhibit autoimmune-like features, with broad implications for understanding the disease. While this study relies on mouse models and may not completely translate to human atherosclerosis, scRna-seq analysis showed a large overlap in transcriptomes responsible for CD8+ clonal expansion in coronary and carotid arteries.
Currently, there is limited knowledge on why statin has pleiotropic benefits besides lowering cholesterol. This paper aims to explain one of statin’s benefits: reducing inflammation. Researchers observed that high-fat mice had reduced lesions and necrotic core area and increased efferocytosis rates when receiving a combined treatment of statin and CD47-SIRP(alpha) blockade. Further exploration of statin’s effect on CD47 expression with in vitro smooth muscle cells stimulated with TNF-alpha shows that when treated with statin, the smooth muscle cells were found with reduced CD47 levels, leading to the researchers suspecting that statin might impact NF-kappaB1’s function in the cell, a key transcription factor in producing CD47 proteins. Statin was observed not to affect lipid levels or apoptosis generation rate. This new knowledge could inspire more studies to explore additional statin benefits, possibly finding more use for statins than just lowering cholesterol.
Alyssa: Bridge RNAs direct programmable recombination of target and donor DNA
Genomic rearrangements are essential across organisms and have provided the backbone of many critical technologies for scientific development. One such genomic rearrangement rely on IS110 insertion sequences, which are a poorly understood family of mobile genetic elements that encode their own transposase. Herein, we aim to support that IS110 sequences use a ncRNA that uniquely endows a bispecific guide molecule for high efficacy rearrangement.
Preliminary results: Our small RNA-Seq of an artificial IS110 plasmid confirmed that an ncRNA is transcribed from a circularized IS110 excision product. Mapping of ncRNAs from various IS110s revealed two stem-loop structures with little homology across species and are specific to each unique IS110 transposases. To establish the importance of this, we artificially studied recombination of IS621 with a mutated ncRNA, revealing each stem is specific for either target or donor sequences and necessary for targeted recombinase. Thus, this grant will address our hypothesis that ncRNA of IS110 species encodes specificity for both target and donor DNA to permit highly accurate recombination.
Novelty: IS110-transposase-ncRNA system represents a novel backbone to develop the first bispecific guide molecule for genetic modifications.
Aim 1: targeted mutation of ncRNA will drive transposition of IS110 elements.
Through iterative mutations of artificial plasmid IS110s, we will demonstrate that each loop endows specificity for either the donor region or target for specific transposition that can be applied to in vitro systems. We will track insertions genome-wide in the E.coli genome to define specify and rate.
Aim 2: IS110 backbone can provide DNA rearrangement of inputted DNA sequences.
We will insert GFP into an IS110 backbones plasmid and track genome-wide insertions specificity for application in various cell lines.
Overall, this work will provide critical understanding of IS110 elements and potentiate it as the first bispecific genetic modification system that requires fewer genomic elements in a plasmid.
Keaton: Structural mechanism of bridge RNA-guided recombination
Background: Though rapid technological advancement has led to the development of a number of genome-editing techniques, these techniques are limited by issues including low efficacy, complex design, and off-target effects, preventing their broader implementation.
Preliminary data: To identify novel candidates for genome engineering, we studied IS110-family elements, the simplest autonomous transposable elements. These elements encode a recombinase and a small non-coding bridge RNA, and we recently discovered that the bridge RNA guides recombination through linking the donor and target DNA in a unique mechanism. We hypothesize that IS110-family elements can be programmed for effective and efficient genomic engineering.
Innovation: In our preliminary study, we characterized a novel mechanism for genomic recombination from the simplest known autonomous transposable elements. This project also aims to engineer this mechanism into a new method for next-generation genome engineering.
Approach: We will test our hypothesis in vivo through the following specific aims:
Aim 1: Determine the ability of IS110-family elements to perform effective site-specific editing. Working hypothesis: IS110-family elements can induce programmable editing at specific sites. We will design targeted bridge RNAs for various genome sites and transfect them into target cells. We will verify their expression using RNA sequencing and recombination using DNA sequencing. We will perform Sanger sequencing to measure off target editing and indels.
Aim 2: Characterize the impact of IS110-family element insertion on eukaryotic cell viability. Working hypothesis: Prokaryotic IS110-family elements will not negatively impact cell viability when inserted into eukaryotic cells. We will use lentiviral vectors to incorporate IS110-family elements into the genomes of target cells encoding a fluorescent reporter. We will compare the viability and proliferation of these cells compared to controls expressing non-targeting IS110-family elements or other genome editing technologies (such as Cre or CRISPR-Cas9).
Pitfalls/alternatives: Aim 1 focuses on the efficacy of IS110-family elements to perform site-specific recombination. We could additionally test the ability of this technique to insert donor DNA into target sites, including establishing a size limit for DNA insertion. We could also compare editing efficacy and accuracy between types of edits, such as insertions, deletions, or inversions.
Overall impact: The successful completion of this project will result in the development and implementation of a novel technique for genome editing which could improve the efficacy and accessibility of these technologies.
Kavya: Plasma extracellular vesicle tau and TDP-43 as diagnostic biomarkers in FTD and ALS
Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and corticobasal degeneration exist on a spectrum of overlapping symptoms and molecular pathology, but diagnosis of molecular pathology is only possible postmortem, which makes early identification impossible and indicates an urgent need for specific biomarkers. This disease continuum is characterized by TAR DNA-binding protein (TDP-43) and frontotemporal lobar degeneration (FTLD) driven by 3R and 4R isoform tau pathology; this study turned to plasma extracellular vesicles (EVs) because they are known to transport tau and TDP-43. After plasma collection from 704 total patients across conditions and isolation of EVs, researchers found that TDP-43 levels were significantly higher in patients with ALS and behavioral-variant FTD compared to control, distinct ratios of 3R and 4R were associated with specific forms of FTLD, and certain combinations of both TDP-34 and tau ratios correlated with higher disease severity. These results suggest plasma EV biomarkers as a minimally invasive tool for identifying and distinguishing between FTD spectrum disorders, and future studies should collect pre-onset measurements to determine how early these biomarkers are significant and to inform early intervention therapies. With that, mutations linked to FTD progression affect EVs by hindering their secretion and therefore marker expression levels, making EV-based markers unviable for patients with such mutations.
Stephen: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs
Paternal obesity as an independent risk factor for early-onset obesity in children is not well understood, especially given the protection against environmental factors granted to spermatozoa by the blood-testis barrier. Spermatogenesis does not, however, conclude within the protected seminiferous tubules, and spermatozoa are susceptible to external stimuli when reaching maturity in the epididymis. Male mice at 6 weeks challenged with a high fat diet (HFD), versus mice allowed to recover to baseline physiology and evacuate potentially affected spermatozoa, produced male offspring with increased incidence of insulin resistance and glucose intolerance. RNAseq and transcriptomic analyses showed upregulation of mitochondrial tRNA and rRNA fragments in mature spermatozoa following HFD, suggesting that increased transcription in response to diet-induced mitochondrial dysfunction may cause the observed intergenerational metabolic symptoms. There may be other sources of epigenetic influence that were not directly ruled out in this study, therefore future work should include a broader study of other germline influences on early embryonic transcription, as well as a focus on lifestyle changes and paternal epigenetic inheritance.
Hannah: DNA mismatch and damage patterns revealed by single-molecule sequencing
Understanding the mechanisms of DNA repair and the consequences of DNA damage is crucial for studying genetic disorders and the development of certain cancers. Current sequencing methods, including NanoSeq, can profile mutations in double stranded DNA (dsDNA), however, utilizing sequencers to analyze DNA after amplification renders the degree of fidelity of identifying single stranded DNA (ssDNA) mutations and precursor events before amplification. Since single-strand mutation events are not represented by the current DNA-sequencing technologies, a single-molecule, long-read sequencing method, HiDEF-seq (Hairpin Duplex Enhanced Fidelity sequencing) has been created to distinguish base substitution in either one or both DNA strands while maintaining single-molecule fidelity. In this study, 134 samples from diverse tissues were analyzed to derive single-strand mismatch and damage signatures, including those from individuals with cancer predisposition syndromes, revealing correspondences with known double-strand mutational signatures and distinct mismatch patterns in tumors deficient in both mismatch repair and polymerase proofreading. This paper suggests that profiling ssDNA mismatches and damage provides a real-time view of the originating errors, potentially bridging the gap in studying mutagenesis, with HiDEF-seq being the first tool of its kind. Although HiDEF-seq does not currently achieve single-molecule fidelity for insertions and deletions due to high error rates, examining ssDNA and expanding this technology is advantageous as it approaches mutation development at their single-stranded state rather than at their end point double-stranded state, offering an early context for researching mutation development in critical areas.
Alyssa: A body–brain circuit that regulates body inflammatory responses
A recent flourish of papers has demonstrated peripheral nervous system regulation of innate immune responses; however, brain regions that aid these interactions have been difficult to identify. Herein, the authors pinpoint vagal neurons within the brainstem that respond to circulating cytokines levels. Utilizing an inducible Cre system under the early-activation gene FOS and adeno-associated virus (AAV9) loading system, the authors claim inhibition or overactivation of these vagal neurons impact the ratio of pro-inflammatory:anti-inflammatory plasma cytokine levels. This work demonstrates an inflammation-responsive region in the vagus nerve and claims it to be a brain dial that tightly regulates immune activity. However, their work lacks a rigorous evaluation of the impact of their manipulations on the peripheral nervous system (PNS); thus, more work is needed to integrate this brain region into the established knowledge of the PNS-immune circuit to substantiate their claims.
Keaton: Profiling phagosome proteins identifies PD-L1 as a fungal-binding receptor
Background: Phagocytosis is a critical component of immunity in which phagocytic cells engulf and internalize potentially pathogenic microorganisms. Phagocytosis can lead to diverse actions including microbial killing, activation of signaling pathways, and antigen presentation for engagement of the adaptive immune response. Despite the importance of these processes, the receptors and signaling pathways that determine pathogen-specific responses from the phagosome are unknown.
Preliminary data: To identify pathogen-induced changes to the phagocytic proteome, we developed a proximity-labelling assay using APEX2-expressing microbes which we called PhagoPL. Using PhagoPL in bone marrow-derived macrophages (BMDMs), we profiled the proteomes of phagosomes containing yeast, Gram-negative, and Gram-positive bacteria, and we identified distinct proteomic profiles for each pathogen. Unexpectedly, we found that the protein PD-L1 was enriched uniquely within yeast-containing phagosomes, which we confirmed via fluorescent microscopy. This led us to hypothesize that PD-L1 functions as a fungal receptor in phagosomes.
Innovation: Previous studies have performed proteomic analysis of phagosomes using bead- or fractionation-based assays; however, technological limitations inhibited their capacity to identify changes to phagosome receptor composition. Our PhagoPL assay is the first technique capable of identifying pathogen-specific changes to the phagocytic proteome. Using PhagoPL, we may have identified PD-L1 as a novel receptor for fungal pathogens as well as a novel function for PD-L1.
Approach:
Aim 1: Identify the fungal ligand for PD-L1. Working hypothesis: PD-L1 binds to fungal ligands in the phagosome. We will perform pull-down assays for PD-L1 in BMDMs cultured with yeast and perform mass spectrometry to identify potential ligands for PD-L1. We will also incubate PD-L1 with whole yeast as well as yeast lysates and perform immunoblotting to confirm the ligand for PD-L1.
Aim 2: Characterize PD-L1 dependent signaling response to yeast. Working hypothesis: PD-L1 initiates a signaling cascade to shape the immune response to phagocytosed fungi. We will perform RNA-sequencing to identify transcriptomic changes to fungal-treated WT or PD-L1 KO BMDMs, and we will perform pathway enrichment analysis to characterize these changes. We will also perform multiplex assays on WT and PD-L1 KO BMDMs to identify differences in cytokine expression as well as phosphoproteomic analysis of the upstream signaling molecules. We will then perform a pulldown to find signalilng molecules bound to PD-L1.
Pitfalls/alternatives: If PD-L1 does not function as a fungal receptor, we can perform analysis of phagocytosed fungi to determine the effect of PD-L1 KO on fungal killing. We could also perform proximity labeling to identify PD-L1 partners in the phagosome and the cytosol.
AJM
Genome organization around nuclear speckles drives mRNA splicing efficiency
Introduction: Nuclear speckles are specific 3D territories with high concentrations of splicing factors; however, their role in splicing has been debated due to their inconsistent proximity to nascent mRNA. Herein, we aim to resolve the impact of genomic DNA proximity to speckles on spicing to address the fundamental knowledge gap of the purpose of these splicing factor-rich structures.
Preliminary results: We validated our previously published speckle proximity score by two independent approaches to assert genomic regions within a cell type have preferential organization relative to speckles, termed speckle-near, intermediate, and speckle-far regions. Preliminary work by seqFISH+ demonstrated that mRNA of genes on the same chromosome but of different speckle proximity have significantly different slicing snRNA densities. Thus, we propose the hypothesis: nuclear speckles are hubs for preferential splicing of key transcripts that assert controlled and efficient splicing of cell-specific genes.
Novelty: We propose a novel hypothesis for the role of the long discovered but mysterious nuclear speckles that would not only address a fundamental question but add a crucial layer of splicing regulation that asserts cell lineage by tightly regulating splicing efficiency by the 3D organization of DNA to nuclear speckles. To combat this hypothesis, we will utilize our recently published method of split-pool recognition of interactions by tag extension (SPRITE) sequencing that enables the mapping of higher-order interactions within the nucleus by cross-linking DNA, RNA, and protein molecules.
Aim 1: Proximity to nuclear speckles increases splicing efficacy.
To investigate differential splicing rate, we will compare speckle-near and speckle-far genomic regions for differences in splicing through the association of snRNAs via SPRITE sequencing. We will enforce that proximity is key for splicing changes by driving mRNA to speckles via plasmid expression of pre-mRNA with embedded hairpin structures that localize with high affinity to a fusion gene of speckle-localized splicing factors and a protein tag.
Aim 2: Splicing efficiency at nuclear speckles asserts cell lineage identity.
We will generate SPRITE profiles of distinct cell types and stimulations to compare speckle-near and speckle-far regions. We will compare speckle proximity and splicing rate across cell types of commonly transcribed genes with known specificity for one cell type. We will further correlate speckle proximity to PolII gene density across cell types to demonstrate highly expressed genes are preferentially located to speckles for increased splicing.
Alternatives: We have demonstrated efficiency in SPRITE generation and analysis. Given the kinetic coupling of mRNA splicing and PolII transcription, we cannot completely account for differences in splicing rate due to transcription in SPRITE data in Aim 1. Thus, as a surrogate, we introduce plasmids that localize to the speckle independent of transcription rate.
Overall, our work will validate the role of speckles in splicing and reveal a novel epigenetic regulatory mechanism of speckle proximity for the splicing efficacy of lineage-affirming genes.
Kavya
Computationally restoring the potency of a clinical antibody against Omicron
The COVID-19 pandemic demonstrated the promise of monoclonal antibody-based drugs as treatments for infectious disease, but the rigor of the Omicron BA.1 variant also highlighted the therapeutic vulnerability we face in addressing escape variants. This study sought to computationally redesign COV2-2130, a class 3 antibody clinically proven to neutralize the Delta variant, to protect against Omicron variants. 376 antibody sequences were computationally identified and experimentally validated; the authors identified the top antibody design that accommodates Omicron escape mutation through 4 amino acid substitutions, without decreasing efficacy against Delta compared to COV2-2130. This paper suggests this computational model as a faster approach to drug development against quickly evolving diseases; however, the current model relies on predicting antibody-antigen interactions based on simulations of antibody-antigen co-structures, rather than human data. Future work should advance machine-learning models via human data to be able to inform predictions based on the presented amino acid sequences, so that this model is applicable in combating new variants.
Hannah
Modulation of insulin secretion by RBFOX2-mediated alternative splicing
Researchers explored the effects of the RNA-binding protein RBFOX2 on alternative splicing mechanisms related to insulin granule docking and exocytosis in pancreatic β cells. They conducted in vivo experiments with Rbfox2-mutant mice and performed RNA sequencing on Rbfox2 knockdown mouse-insulinoma cell line (MIN6), identifying RBFOX2-regulated splicing events in pancreatic islets from both mice and humans with type 2 diabetes (T2D). Phenotypic observations of Rbfox2-mut mice showed fewer docked insulin granules and significant decreases in first-phase insulin secretion, despite absent changes in cell number or insulin content. Comparative analysis of gene expression in islets from obese diabetic and obese non-diabetic mice showed similar rates of mis-splicing events, linking early insulin secretion disruptions to diabetes. RBFOX2 expression, typically high in pancreatic β cells, decreased under diabetic stress in both mice and humans. The study suggests that targeting RBFOX2-mediated splicing could offer new therapeutic strategies for diabetes, however, since RBFOX2 regulates splicing in multiple tissues, altering its function could inadvertently affect splicing in other organs unintentionally.
Stephen/(KK)
Mapping genotypes to chromatin accessibility profiles in single cells
Within the study of cancerous mutations, there is a disconnect between the epigenetic changes in chromatin accessibility and the somatic mutations that drive progression of disease that lacks effective tools for analysis, especially due to the heterogeneity within cancer tissue and between individuals. Building off of the methods of scATAC-seq, the authors developed a droplet-based high-throughput method of assessing genotypes and chromatin accessibility by inferring RNA expression through targeted amplification of genomic DNA called genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This system has been able to separate out with accuracy wild-type and mutated cells in primary human samples: the authors demonstrating this through separation and genotyping of various co-cultures that included cells with mutations in JAK2. Although the authors were able to show successful and highly accurate genotyping and the novel elucidation of increased accessibility to pro-inflammatory genes in JAK2 mutant hematopoietic stem cells, conjoining GoT-ChA with ASAP-seq to observe cell surface protein markers alongside epigenetic data led to a decreased genotyping rate. Future directions of this study may include optimization and refining of GoT-ChA and ASAP-seq combined methods, as well as a deeper exploration of clinical applications through a better understanding of chromatin accessibility with associated biomarkers via use of this method.
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AJM
Mitochondrial transfer mediates endothelial cell engraftment through mitophagy
Introduction: Revascularization of ischemic tissue is a major medical challenge that would significantly improve outcomes in myocardial infarctions and critical limb ischemia. Vascular grafting with autologous endothelial cells (ECs) remains the best option for revascularization; however, the need for co-transplantation with perivascular cell types, particularly mesenchymal stem cells (MSCs), complicates its translatability. Herein, we aim to resolve critical MSC factors that allow successful EC engraftment to address a critical knowledge gap that would potentiate the application of revascularization in human ischemic disease.
Preliminary results: Previously, we demonstrated paracrine factors of MSCs are not sufficient to support EC engraftment. Rather, MSC-derived mitochondria are transferred to ECs through tunneling nanotubes (TNTs), and disruption of MSC TNT formation or mitochondrial mobilization eliminates the benefit of MSC in EC engraftment. Importantly, transferred mitochondria induce ECs mitophagy and mitochondria biogenesis at the transcriptomic level. Thus, our hypothesis is that ECs require exogenous mitochondria from perivascular cells to initiate mitophagy necessary for their revascularization programs. To address this, we propose these two aims:
Aim 1: Mitochondrial transfer to ECs is necessary to support metabolically expensive revascularization programs.
To this end, we will allow MSC-EC mitochondria transfer as well as artificial mitochondria transfer from other non-perivascular cell types and track EC engraftment success (EC viability, network density of microvessels, tissue perfusion).
To demonstrate transferred mitochondria-induced metabolic change, we will evaluate the recipient ECs’ ATP pool, oxidative phosphorylation capacity, and cell function (migration).
Aim 2: Transferred mitochondria-induced mitophagy is necessary for EC engraftment.
To this end, we will first evaluate the importance of transferred mitochondrial function in EC metabolic changes using depolarized or mtDNA-deficient mitochondria. We will further track their incorporation into the recipient EC’s mitochondria pool as either autonomous structures or mitochondria fusion using MSCs expressing a red mito-tag or a split GFP system.
To demonstrate the initiation of mitophagy, we will track the association of EC or MSC-derived mitochondria with EC’s autophagosome. Finally, we will determine the importance of mitophagy in EC engraftment by inhibiting the mitophagy mediators PINK1 and Parkin in MSCs and EC.
Pitfalls/Alternatives: While autologous mitochondria can be artificially implanted in ECs ex vivo through micropinocytosis, it is possible mitochondrial transfer is continuously needed for proper microvessel biogenesis. If so, we can track MSC-mitochondria transfer across engraftment time and supplement EC-only engraftments with mitophagy-inducing factors in vivo to replicate transferred mitochondria-induced programs.
Overall, this work will address the critical knowledge gap of EC dependence on perivascular cells and potentiate EC engraftment to treat ischemic disease.
Kavya
Interpericyte tunnelling nanotubes regulate neurovascular coupling
Neurovascular coupling is crucial for regulating local blood flow during neuronal activity; while pericytes are known to regulate their microvascular environment, their role in neurovascular coupling was previously unknown. This study used mouse retinas to investigate the in vivo functions of interpericyte tunneling nanotubes (IP-TNTs) — networks that connect two distinct pericytes. The findings revealed that ischemia damaged significant quantities of IP-TNTs and led to impaired dilation. Additionally, capillary pairs connected by IP-TNTs responded to light stimulation by dilating on one end and constricting on the other, contrary to the intuitive belief of only a dilation response. One potential limitation is that this study was conducted on mouse retinas, but results are also being applied to brain diseases. Future studies should aim to replicate these findings in a human capillary model so future therapies can focus on preserving IP-TNTs to restore neurovascular function through neurological conditions like ischemia.
KK
Transient loss of Polycomb components induces an epigenetic cancer fate
Background:
Several key features of cancer development and progression are accumulation of somatic mutations and epigenetic changes, and cancer initiation has long been thought to be caused mainly secondary to somatic "driver mutations". However, epigenetic alterations have been associated with cancer susceptibility and appear very early during tumorigenesis, suggesting that they may be an additional driver of cancer initiation. The proposed work challenges the existing paradigm and seeks to determine whether epigenetic changes are sufficient to drive malignant transformation and tumorigenesis.
Preliminary data:
To determine whether epigenetic changes are sufficient to drive malignant transformation, we developed a model using temperature-sensitive RNAi to transiently or constitutively knock down the highly conserved Polycomb Repressive Complex 1, a critical epigenetic regulator and determinant of cell fate and development, in Drosophila larvae. In our model, both constitutive and transient (24h) KD of ph (encoding the Drosophila Polycomb analog) were sufficient to drive tumorigenesis within 11 days, which DNA-sequencing confirmed was not driven by genomic mutational accumulation. These findings provide key evidence to support our hypothesis that epigenetic alteration is sufficient for cancer initiation, which we will test with the following specific aims:
Approach:
Aim 1: Characterize the phenotypic changes in epigenetically induced tumors. Working hypothesis: Transient Polycomb KD induces tumorigenesis via epigenetic changes. To identify the mechanism through which transient ph KD induces tumorigenesis, we will performi RNA-sequencing, ChIP-sequencing, and ATAC-sequencing to identify transcriptomic and epigenetic changes following transient ph KD compared to constitutive KD and controls. We will identify genes with reversible changes as well as those with irreversible changes, which putatively could drive tumorigenesis in this model. We will perform functional analysis of these genes and identify upstream regulators.
Aim 2: Determine the threshold of Polycomb repression to induce tumorigenesis. Working hypothesis: 24h of ph KD in our model is sufficient to induce irreversible cellular programming. To identify a threshold necessary for tumorigenesis, we will repeat our experiments using transient ph KD for multiple time points, up to a maximum of the 24h from our preliminary data. We will determine the kinetics of Polycomb repression at the RNA and protein levels, as well as their return to normal kinetics. We will also measure the impact of these changes on tumor growth kinetics.
Pitfalls/alternatives: Although epigenetic mechanisms seem to be the most likely drivers of tumorigenesis in this model, it is possible that another mechanism could be involved beyond those we have tested. We could test for ph KD-induced activation of growth factors and other signaling pathways at the protein level. It is also possible that other epigenetic regulators could drive tumorigenesis, which could be valuable to test.
AJM
Ancestral allele of DNA polymerase gamma modifies antiviral tolerance
Mitochondrial recessive ataxia syndrome (MIRAS) is caused by a common European founder mutation in DNA polymerase gamma (POLG1) which primarily impacts mitochondrial DNA and RNA levels. Despite this, the onset of disease pathology is disparant across individuals and often associated with viral infections, suggesting the etiology is dependent on immunodeficiencies rather than metabolic changes. Herein, we aim to elucidate the role of the mitochondrial immune regulation in MIRAS.
Preliminary results: It has been established that MIRAS patients have reduced mtDNA/mtRNA, however, the metabolic and immunologic implications are not understood. In order to investigate this, we have generated a mouse model with the MIRAS POLG1 variant (p.W748S). Preliminary characterization demonstrates reduced POLG1 expression does not cause severe phenotypes as seen in human MIRAS until viral infection. This novel model allows for rigorous investigation of the etiology of MIRAS and implication of mitochondria dysfunction.
Aim 1: Demonstrate impaired immune response to infection elicits MIRAS-associated loss of neurons and liver toxicity
Working hypothesis: Altered immune response is the driving force for MIRAS-associated epilepsy and liver failure.
To this end, we will infect our p.W748S mice with the neurotrophic TBEV to demonstrate accelerated organ death and MIRAS-associated epilepsy. To implicate exacerbated immune responses, we will provide immunosuppressants and assess organ damage.
Aim 2: Demonstrate p.W748S-induced reduction of mtDNA/mtRNA pool limits interferon priming.
Working hypothesis: Reduced mtDNA/mtRNA pools limits interferon priming and exacerbates viral response.
To this end, we will utilize MIRAS patient primary fibroblast and our p.W748S mouse model to characterize metabolic and immune phenotypes in the transcriptome and proteome to pinpoint deficiencies in interferon responses. We will implicate p.W748S-induced loss of mtDNA/mtRNA-priming in exacerbated response to poly(I:C) while controlling for metabolic changes.
KK
CGRP sensory neurons promote tissue healing via neutrophils and macrophages
Background:
Delayed wound healing leads to significant clinical complications, including infection, amputation, increased healthcare costs, and decreased quality of life. Two major factors controlling tissue healing are macrophages and the nervous system, which can modulate the immune system. Recent work has demonstrated that sensory neurons can promote macrophage tissue repair functions; however, the type of sensory neurons directing this process and the mechanism through which they promote healing are unknown.
Preliminary data:
Because pain-sensing nociceptive neurons can regulate immune function and are known to play a role in wound healing, we evaluated the impact of nociceptor ablation on wound healing using Nav1.8cre/Rosa26DTA mice, which lack peripheral nociceptive neurons. Nav1.8cre/Rosa26DTA mice had significantly delayed wound healing and closure, and we observed that the neuropeptide CGRP was present only in wounds from control mice with normal wound healing. Mice lacking the CGRP receptor in myeloid cells also demonstrated delayed wound healing, leading us to hypothesize that nociceptive neurons promote wound healing in macrophages through CGRP. Through our preliminary efforts, we have identified a novel target to promote tissue regeneration, and we will test our hypothesis via the following specific aims:
Approach:
Aim 1: Characterize the effect of CGRP on myeloid cells.
We will treat macrophages in vitro using CGRP and determine their polarization, maturation, and phagocytic capacity via flow cytometry and RNAseq. We will also characterize macrophage states in vivo using scRNAseq in Nav1.8cre/Rosa26DTA mouse wounds compared to controls.
Aim 2: Determine the efficacy of exogenous CGRP in promoting wound healing.
We will first determine the ability of CGRP to promote wound healing in Nav1.8cre/Rosa26DTA mice. We will treat these mice with exogenous CGRP following injury in both our skin and muscle wound models, and we will measure wound closure and characterize wounds via histology. We will then evaluate CGRP efficacy in disease relevant models including diabetic db/db mice and aged mice, which both have impaired wound healing capabilities.
Pitfalls/alternatives:
If we find that CGRP does not have an effect on macrophage actions in promoting wound healing, we can examine other neurotransmitters released from Nav1.8+ neurons. It is also possible that the nociceptive neurons promote tissue healing by acting on cells other than macrophages, which we can test via scRNA-seq in our model.
4/17
AJM
Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids
Despite routine glucocorticoid (GC) therapy for a broad spectrum of immune-mediated inflammatory diseases, the mechanism of action of GC-induced immunosuppression is incompletely understood. Herein, the authors investigate GC-induced metabolic changes, revealing an immediate increase in the tricarboxylic acid (TCA) cycle, independent of GC-mediated transcription. Co-immunoprecipitation suggests GCs release pyruvate dehydrogenase (PDH) from glucocorticoid receptor (GR), allowing sustained TCA cycle activity and anti-inflammatory metabolite itaconate production. In line, blocking itaconate synthesis in mouse models of arthritis and asthma abolishes the therapeutic benefit of GC treatment. These findings shed light on crucial actions of GCs that could promote innovative therapeutics to lessen the side effects of continuous GC therapy.
KK
Improving prime editing with an endogenous small RNA-binding protein
Background: Although the advent of gene-editing technology has promised great potential to treat genetic diseases, many conditions are caused by point mutations, which current technologies cannot correct reliably or efficiently. Prime editing (PE) is an exciting new technology which uses a mutant Cas9 protein and reverse transcription to insert small edits with much higher precision than other techniques. In spite of its high accuracy, current PE techniques are inefficient, representing a key obstacle preventing their broader application.
Preliminary data: To identify molecular determinants of PE efficiency, we developed two genome-wide screening assays where we measured expression of a reporter gene induced by PE. In both assays, the only gene with a significant positive effect on PE efficiency was La, a ubiquitously expressed RNA-binding protein. This leads us to the hypothesis that La promotes efficient prime editing. Through our novel screening assays, we have identified La as a component which we can use to improve PE technology. We will test our hypothesis through the following specific aims:
Approach:
Aim 1: Characterize the relationship between La expression and PE efficiency. Working hypothesis: La expression exhibits a dose-dependent relationship with PE efficiency. Using a cell line expressing PE components, we will knock down and overexpress La, then perform DNA sequencing to measure the proportion of cells containing the intended edit. We will quantify the relationship of La expression at the protein and RNA levels with editing efficiency.
Aim 2: Define the mechanism for La improvement of PE efficiency. Working hypothesis: La promotes PE efficiency by stabilizing pegRNAs through interactions between its N-terminal domain and the 3' polyU on pegRNAs. To test this hypothesis, we will transfect cells with labeled pegRNAs, then pull down La. We will additionally perform RNA-sequencing at various time points following pegRNA transfection in La-depleted or overexpressing cells to measure pegRNA degradation.
Pitfalls/alternatives: If we determine that La does not promote efficient prime editing, we can study the effects of other genes identified in our screening assay on PE efficiency. If La does not promote PE efficiency through pegRNA stabilization, we can transfect mutant La to identify the critical domains for its role in promoting PE efficiency, then characterize their interactions with RNA, DNA, and other proteins.
AJM
The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation
Background: cGAS is a sensitive DNA sensor that initiates robust innate immunity. Critically, cGAS does not autoreact to cellular genomic DNA despite its nuclear localization, necessitating a tight regulatory mechanism. Herein, we aim to elucidate crucial mechanisms of nuclear cGAS regulation and implicate defective clearance in altered immune homeostasis.
Preliminary data: We were the first to rigorously validate ubiquitin proteasomal system (UPS) as the sole regulator of nuclear cGAS clearance across organisms. We resolved this complex to reveal a highly specific interaction driven by the poorly characterized SPSB3 substrate recognition component. The work in this grant will outline a novel and highly potent regulatory mechanism of nuclear cGAS necessary for cellular homeostasis through SPSB3-UPS.
Approach:
Aim 1: Demonstrate SPSB3 regulatory regions necessary for targeted nuclear cGAS clearance
To this end, we will utilize our expertise in cryo-EM to demarcate regions of interaction between SPSB3 and cGAS. Further, we will establish mutant SPSB3 and cGAS and track their interactions and localization. Lastly, we will establish the conservation of these sites across organisms to evaluate the central role of SPSB3 in nuclear cGAS regulation.
Aim 2: Implicate improper SPSB3-cGAS regulation in immune homeostasis
To this end, we will disrupt SPSB3-mediated cGAS regulation through inhibitors and point mutations in the UPS-SPSB3-cGAS system. Subsequently, we will track cGAS activation and innate immune response across cell types. In parallel, we will utilize publicly available GWAS studies to evaluate the risk of inherited mutations in this SPSB3-UPS-cGAS mechanism for autoimmune diseases.
Pitfalls/Alternatives: Previous work demonstrates SPSB3 to have few disordered regions, thus, we expect cryo-EM to achieve the resolution to annotate specific residues. Further, immunoprecipitation has implicated SPSB3 binding partners for UPS targeting, thus, we can perform molecular dynamic stimulations to predict conformational changes within SPSB3 that may be necessary to achieve cGAS interactions.
In total, this work will map an essential mechanism of cGAS regulation to maintain cellular homeostasis.
notes: lost significance due to lack of background (e.g. cell cycling). novelty section needed. precision not there. missing hypothesis.
KK
Disease-associated astrocyte epigenetic memory promotes CNS pathology
Background: Astrocytes are one of the most abundant cell types in the brain, and they are critical to both homeostasis under normal conditions and pathology in diseases such as multiple sclerosis (MS). While the transcriptomic and epigenetic adaptations of immune cells under chronic inflammatory conditions have been well-characterized in MS, the impact of chronic inflammation on astrocyte function and their subsequent disease contribution is unknown.
Preliminary data:. To characterize the impact of repeated inflammation on astrocyte behavior, we performed RNA-sequencing and ATAC-sequencing on astrocytes from mice treated with multiple intracranial injections of TNFa + IL1b, and we observed enrichment of inflammatory and epigenetic pathways following repeated injections. We identified a causal role for the transcriptional coactivator p300 in a subset of astrocytes in controlling this response, using acetyl-CoA synthesized by Acly. The proposed work aims to understand the mechanism controlling p300 activation in astrocytes during chronic inflammation and the contribution of p300+ astrocytes to MS pathology.
Approach:
Aim 1: Identify the mechanism of astrocyte p300 activation during CNS inflammation.
To identify the causal regulator of astrocyte p300 induction during CNS inflammation, analyze our existing ATAC-sequencing data to identify open motifs in the p300 promoter region and the transcription factors that bind to them. We will then knock out these transcription factors in vitro to determine which induce p300 expression under repeated inflammatory stimulus.
Aim 2: Determine the role of p300+ Acly+ astrocytes in promoting inflammatory CNS pathology.
In parallel, we will characterize the functional changes caused by CNS inflammation in p300+ Acly+ astrocytes using our novel method, FIND-seq, which allows us to perform scRNA-seq on rare cell populations selected using RNA or DNA markers. We will generate a transcriptomic signature for this disease-related astrocyte subset and determine whether it contributes to other inflammatory CNS diseases such as Sjogren's syndrome using publicly available transcriptomic data. We will also perform conditional knockouts of p300 and Acly in astrocytes in models of inflammatory CNS disease such as EAE to determine the role of these genes in disease progression.
Pitfalls/Alternatives:
It is possible that p300 activation in astrocytes is mediated by mechanisms other than transcriptomic induction; therefore, we can also evaluate p300 modifications using proteomics. Additionally, though the EAE model for MS is widely used, it contains significant differences from human disease and may not fully explain observed phenotypes in humans.