2024

July 19th
June 26th

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.



June 19th

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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May 29th

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.


May 15th

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.

April 17th

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.



March 13

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.