Awardees

The overarching goal of this proposal is to gain a deeper understanding of oligodendrocyte progenitor cell (OPC) phagocytosis and determine how it compares to the phagocytic ability of microglia and its relevance for neurological disease. Oligodendrocyte progenitor cells (OPCs) are a pool of progenitors found in the adult brain that give rise to mature, myelinating oligodendrocytes throughout life. Recent work has established that OPCs function as phagocytes and engulf synapses within both the developing and adult brain. This challenges the long-held notion that the sole function of OPCs is to generate new oligodendrocytes and establishes a role for OPCs in synapse pruning, similar to microglia. However, there is currently no understanding of how OPC phagocytosis impacts their progenitor function or how their phagocytic function compares to microglia. It is also unknown if OPCs function as phagocytes in the context of neurodegeneration. I will now use a combination of in vitro and in vivo phagocytosis assays and live cell imaging to compare OPC and microglial phagocytosis under steady-state conditions and during neurodegeneration and determine how phagocytosis impacts OPC function as progenitor cells. In Aim 1, I will use in vitro phagocytosis assays coupled with live cell imaging, OPC differentiation analyses, and RNA-sequencing to compare microglia and OPC phagocytosis of synaptic substrates and determine how phagocytosis impacts OPC differentiation. In Aim 2, I will utilize an animal model relevant to multiple sclerosis (MS) where the Schafer lab has shown early synapse loss and AAV-driven inhibition of complement C3 deposition to determine if OPCs function as phagocytes in the context of neurodegeneration, and if this phagocytosis is complement C3-dependent. The proposed studies will build on emerging work that challenges the current thinking that microglia are the primary phagocytes of the CNS. It will reveal the role of OPC phagocytosis in modulating the differentiation capacity of OPCs. It will also determine if OPC phagocytosis is dependent on the deposition of the complement protein C3. These results will have implications for neurodegenerative diseases, as lack of OPC differentiation is observed in multiple neurodegenerative states. Additionally, complement is now a target for therapeutic intervention in a variety of neurological diseases, so understanding how this pathway regulates other cells of the brain is essential for the effective use of these therapeutics. Finally, the experiments outlined here combine my strength in OPC biology from graduate school with the expertise in microglia and mechanisms of phagocytosis in my postdoctoral lab. Together, these studies will give me ideal training to achieve my goal of becoming an independent researcher studying glia-glia interactions in neurodegeneration.

Physical frailty, characterized by decreased physiologic reserve and increased vulnerability to stressors, and cognitive impairment, ranging from mild impairment to dementia, often co-occur in older adults. Both are associated with considerable adverse health outcomes, high healthcare costs, and substantial caregiver burden, and highly prevalent in U.S. community-dwelling older adults. However, for older adults receiving long-term care in nursing homes, data is scarce on the prevalence of the two conditions over their stay. Community-based studies suggest heterogeneous clinical presentation of physical frailty, which may have implications for its management. It is unknown if such heterogeneity is similar in older nursing home residents and if it is influenced by cognitive impairment. Further, physical frailty and cognitive impairment share risk factors and predict future onset of one another but the mechanism of this complex interplay remains unclear. Lastly, depression is strongly correlated with both conditions, yet findings regarding the impact of antidepressants on the progression of physical frailty and cognitive impairment are inconsistent. This proposed F99/K00 project seeks to address these gaps by two specific aims with population longitudinal data and advanced statistical methods. Aim 1 (dissertation research) focuses on older nursing home residents and will describe the prevalence of physical frailty and cognitive impairment; identify subgroups of physical frailty and examine the variation of subgroups by cognitive impairment levels; and delineate the developmental trajectories of physical frailty and cognitive impairment and examine the correlations between trajectories. Aim 2 (post-doctoral research) expands to older adults in the community and will assess the reciprocal association between physical frailty and cognitive impairment; quantify the impact of cumulative exposure to antidepressants on trajectories of physical frailty, cognitive impairment and depressive symptoms; and examine the effect of depressive symptoms as a mediator of physical frailty on cognitive impairment with causal mediation analysis. Methodological innovations include the use of latent class analysis, group-based trajectory models, structural equation models (autoregressive cross-lagged panel analysis; autoregressive latent trajectory model), and causal mediation. This proposal is directly relevant to the growing aging population in the U.S., including those residing in the nursing homes and those living in the community, since it uses the national nursing home database Minimum Data Set 3.0 (Aim 1) and the nationally-representative Health and Retirement Study linked to Medicare Part D Drug Event Files and the Harmonized Cognitive Assessment Protocol (Aim 2). This project will shed light on the concurrent progression of age-related physical and cognitive conditions. Results will inform future work to develop diagnostic tools and prediction models to facilitate timely identification of older adults at risk for accelerated functional decline, and implement care tailored to older adults’ needs to effectively delay the onset of negative health outcomes, enhance quality of life, and foster a healthy longevity.

Certain viruses in the picornaviridae family, specifically enterovirus-D68 (EV68), have emerged as global health concerns over the last decade with severe symptomatic infections with EV68 able to result in long lasting neurological deficits and death. There are currently no US Food and Drug Administration approved drugs for any non-polio enterovirus, highlighting the need to develop strategies against these lethal enteroviral strains. One particularly attractive class of potential drugs are small molecules inhibitors, which can act as direct-acting antiviral (DAA) inhibitors towards the conserved active site of EV68 3C protease. This main viral protease is a cysteine protease conserved in the 3C family, responsible for cleaving eight sites along the viral polyprotein, which is essential for viral propagation. DAAs designed to target 3C proteases can potentially achieve robust inhibition across enterovirus species. However, as drug resistance in viruses can be prevalent, it is paramount to design inhibitors less susceptible to resistance mutations. It was demonstrated previously in the Schiffer Lab that when bound to protease, viral substrates occupy a conserved three-dimensional volume called the substrate envelope. It was also demonstrated that inhibitors that extend beyond the substrate envelope are more susceptible to drug resistance mutations. By utilizing the substrate envelope and cocrystal structures of the proteases, DAAs designed to fit within the three-dimensional consensus volume as naturally occurring substrates will interact primarily with functionally important residues and be less susceptible to drug resistance mutations. The central hypothesis of this proposal is that cocrystallization of EV68 3C protease with its natural substrates will enable the calculation of the substrate envelope to inform on substrate specificity, which will also aid in the design of robust pan-3C-protease inhibitors. In Aim 1, I will determine the cocrystal structures of EV68 3C protease bound to viral substrates. I will then use these structures to elucidate the molecular mechanism of substrate specificity for EV68 3C protease and calculate the substrate envelope. These data will aid in small- molecule design to create DAAs with improved resilience to mutations that can confer drug resistance. In Aim 2, I will design and test novel DAAs that target EV68 3C protease. I will first characterize previously designed inhibitors for other 3C and 3C-like proteases with the substrate envelope to establish a starting compound based on potency. Inhibitors based on the scaffold will be designed, synthesized, and tested in a FRET-based enzyme inhibition assay. Crystallization of novel potent compounds with EV68 3C and their characterization within the substrate envelope will assess inhibitors’ susceptibility to drug resistance mutations. Overall, this study aims to develop a robust, novel compounds with resistance-thwarting protease inhibition against the emerging pathogen that is EV68.

The goal of this project is to determine the mechanism by which cell death results from transcriptional inhibition. The consensus model in the field posits that cell death following transcriptional inhibition results from the loss of specific mRNA species and subsequent loss of protein. By targeting such a core cellular process, transcriptional inhibition is thought to overwhelm cellular control and lead to unavoidable cell death. This death process, defined as Accidental Cell Death (ACD), is not controlled by the cell, and does not result from the use of defined effector molecules. Contrary to the conventional model, we found that, rather than induce ACD, cell death following transcriptional inhibition results from a previously undescribed regulated apoptotic signal. Furthermore, we found that RNA Pol II degradation, rather than loss of mRNA production, resulted in cell death. Our data suggests a new model, whereby degradation of Pol II induces a signal that leaves the nucleus and is received by the mitochondria to initiate apoptosis. To identify genes that regulate a pro-apoptotic signal following transcriptional inhibition, we performed a genome-wide CRISPR screen. Genome-wide CRISPR screens often fail to identify death regulatory genes, making it difficult to elucidate mechanisms of cell death. To overcome this, we developed a novel experimental strategy that allowed us to identify genes whose knockout modulated the cell death rate following transcriptional inhibition. Based on the results of our screen, in Aim 1 we will test the hypothesis that the alternative splicing regulator PTBP1 facilitates altered splicing and nuclear export of regulatory pre-mRNA, and that this activity is required for cell death following transcriptional inhibition. We will use live cell microscopy to establish the functional role of PTBP1 nuclear export. We will use SLAM-seq and RIP-seq to quantify PTBP1 activity following transcriptional inhibition. Our screen also identified BCL2L12 as the critical apoptotic effector gene for transcriptional inhibition. In Aim 2, we will test the hypothesis that BCL2L12 activates apoptosis following transcriptional inhibition in an isoform-specific manner. We will perform a series of functional genetics experiments to characterize the role of BCL2L12 in the apoptotic response. By describing a new mechanistic model by which transcriptional inhibition induces cell death, we will improve our understanding of how to effectively use transcriptional inhibitors therapeutically. Ultimately, we hope our work will improve our ability to predict which patients will best respond to transcriptional inhibitors and help identify novel treatment strategies.

"Small noncoding RNAs <70 nucleotides long (sncRNAs) regulate diverse biological processes, including development and epigenetic inheritance. For example, in the germ cells of male placental mammals PIWI-interacting RNAs (piRNAs) and microRNAs (miRNAs) guide Argonaute proteins to silence transposable elements and mRNAs, thereby ensuring the development of functional sperm. As sperm mature, piRNAs and miRNAs are replaced by tRNA fragments, which are believed to mediate epigenetic inheritance. Methods to clone and identify sncRNAs have played a key role in understanding their biogenesis and function. Notably, the strategies typically used to sequence Argonaute protein-associated small RNAs only capture RNAs with 5′-monophosphate (P) and 3′ hydroxyl (OH) termini. Consequently, they fail to detect tRNA fragments or other sncRNAs with different terminal groups. Methods that convert all termini to 5′-P and 3′-OH can capture diverse sncRNA types but fail to retain knowledge of the original terminal groups, information critical to deducing sncRNA biogenesis and function. To fill this gap, we have developed Terminus-Specific Ligation and sequencing of sncRNAs (TSL-Seq), a high-throughput sequencing framework that captures diverse sncRNAs while identifying the terminal groups of each RNA. Here, I propose to: 1. Maximize the efficiency of the TSL-Seq cloning procedure; 2. Use TSL-Seq to capture the diversity of mouse sncRNAs in developing and mature male germ cells; 3. Use TSL-Seq to capture the diversity of mouse sncRNAs in female germ cells and zygotes. Optimizing TSL-seq will decrease the amount of input RNA required, allowing characterization of sncRNAs from rare cell types or mutant tissues. This project will produce the first comprehensive catalog of sncRNAs in developing mouse sperm and oocytes, uncover paternally and maternally deposited sncRNAs, and reveal the fates of sncRNAs after fertilization before the activation of zygotic transcription"

Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer with standard therapy involving neoadjuvant chemotherapy, surgical management, and radiation therapy. However, the high recurrence rate and low pathological complete response of TNBC suggest that radioresistance is a critical factor in the diminished therapeutic efficiency of the current treatment strategy. There is limited literature exploring the specific pathways responsible for radiation resistance in TNBC, but most data support the role of limiting reactive oxygen species (ROS) accumulation. Our lab has studied the role of Vascular Endothelial Growth Factor (VEGF) binding to Neuropilin-2 (NRP2) and initiating several cancer stem cell properties. Preliminary data indicate that radiation enriches for NRP2 expressing cells and using a function-blocking antibody specific to VEGF/NRP2 with irradiation decreases cell viability compared to either treatment alone in a TNBC organoid. The central hypothesis of this proposal is that VEGF/NRP2 induces radioresistance by altering redox homeostasis and can be targeted for better therapeutic outcomes in TNBC. This proposal will seek to investigate the possible role of NRP2 in regulating NOS2 transcription and its contribution to mitigating ROS accumulation. I will also use single-cell RNA sequencing technology to identify the subpopulations of TNBC that are radioresistant and whether they utilize the NRP2/NOS2 signaling axis. Another aspect of this proposal is to observe the effectiveness of a function-blocking antibody of NRP2 with radiation using an in vivo model. I plan to identify if this approach reduces the radioresistant clones in TNBC. The completion of this proposal will heighten the understanding of radioresistance in TNBC and identify a novel molecular pathway responsible for this phenotype.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by amyloid beta plaques and neurofibrillary tangles in the brain along with inflammation both in the brain and systemically. This has led to the theory of microbial communities or infections as causative in the development of neuroinflammation, immunosenescence, and inflamm-aging seen in AD. Our own research has demonstrated a decrease in gut microbiota with anti-inflammatory properties and higher abundances of pro-inflammatory gut microbiota in AD elders. However, it is unclear how the AD microbiome exerts effects on the central nervous system. To address this gap in knowledge we have performed gut microbiome profiling, analysis of immune cell populations in blood, serum cytokine profiling, and cognitive assessments of AD elders at 90-day intervals. This analysis identified changes in B cell populations with an increased abundance of class-switched and decreased abundance of naïve B cells at levels of greater cognitive impairment. To better understand how the microbiome may control AD progression, we propose to investigate the connection between the AD microbiome and the adaptive immune system with a focus on regulation of the intestinal epithelium by commensal gut bacteria. Specifically, we intend to use stool and plasma samples collected from our AD cohort to measure makers of intestinal permeability and determine whether metabolites secreted by the AD gut microbiome cause disruptions in the intestinal epithelium. We will directly study the disruptive effects of AD stool by applying stool supernatants to intestinal epithelial cells, quantifying changes in epithelial permeability using established assays, and determining whether specific taxa depleted in AD are sufficient to cause epithelial disruption. In our previously published data, we have observed the loss of the phytoestrogen-metabolizing bacteria, Adlercreutzia equolifaciens (AE), in the microbiome of AD elders. My preliminary studies reveal that a metabolic product of AE, (S)-equol, prevents epithelial damage in the setting of inflammation. Therefore, we aim to determine whether AE or its metabolic products protect the intestinal epithelium. To untangle the role of the AD microbiome on our observed changes in class switched and naïve B cells, I have collected preliminary data which demonstrates that colonization of mice with the microbiome of AD elders promotes B cell class switching when compared with colonization of cognitively impaired elders without AD. This application proposes to expand this finding and characterize the changes in the adaptive immune system caused by the AD microbiome. This continuing work will further establish the connection between AD related neurocognitive decline, the microbiome, and immune system.

Huntington’s disease (HD) is caused by expanded trinucleotide repeats (CAG) in exon 1 of the huntingtin (HTT) gene. Therapies lowering the downstream mutant HTT protein show limited clinical success. New evidence reveals that repeat tract length in the HTT locus, not mutant HTT protein, correlates to disease onset/severity. CAG repeat length is inherited, but further expands due to somatic instability, which contributes to HD. Somatic expansion occurs in non- dividing cells like neurons after transcription, forming a slipped loop that activates mismatch repair (MMR). In MMR, nuclease complexes help recognize the slipped loop and cut the non-slipped strand to create a gap that is filled to expand the repeat. Polymorphisms in MMR complexes are linked to HD onset, and knocking out or altering activity of MMR proteins block expansion or induce contraction in HD models. Yet, the contribution of each MMR protein to CAG expansion, and the effect of their conditional CNS-specific reduction on HD outcomes, is untested. Also, mechanisms favoring contraction over expansion are unknown. This project seeks to define MMR complexes facilitating HTT CAG expansion/contraction using divalent small interfering RNA (siRNA)—which induce potent, CNS-specific silencing of target genes—and antisense oligonucleotides (ASOs)—which can disrupt specific protein-nucleic acid binding in the CNS. Aim 1 will use divalent siRNA to evaluate the effects of MMR silencing on HTT CAG repeat expansion and HD progression. Efficacies of siRNAs targeting each MMR protein have been validated in human and mouse cells. Furthermore, one of these siRNAs was delivered to CNS of an HD mouse model, BAC-CAG (carries human HTT with 120 CAG that undergo expansion), showing target MMR silencing and blocked somatic expansion 2 months later. In Aim 1, divalent siRNA targeting each MMR enzyme will be injected into BAC-CAG mice. Target silencing and HTT CAG repeat expansion will be measured 2 months later. Top siRNA that block expansion will be re- injected into BAC-CAG mice, and the impact on motor behavior, ventricular size, and HD pathology will be explored over 9 months. Aim 2 will develop HTT CAG-targeting ASOs to induce MMR-mediated contraction in HD cells and mice. An initial panel of ASOs targeting HTT CAG repeats was screened in non-transformed HD patient-derived fibroblasts (HDpFs) using a high-throughput format, and ASOs that increase contraction events were identified. To improve contraction rates, ASO chemistries and lengths will be optimized and screened in HDpFs using the same assay. HTT CAG repeat length/instability will be quantified over 40 days to identify leads. Leads will be delivered to HDpFs, in combination with validated siRNA targeting each MMR protein, to identify MMR proteins mediating ASO-induced contraction events. In parallel, in vivo efficacy of leads will be confirmed in BAC-CAG mice. This work will reveal somatic expansion/contraction mechanisms, inform HD therapy design, and provide the fellow with crucial training in therapeutic development, neurobiology, and bioinformatics.

Loss of the breast cancer susceptibility (BRCA1 or BRCA2) genes in hereditary breast and ovarian cancer (HBOC) is characterized by defects DNA repair by homologous recombination (HR) and in the protection of replication forks (known as fork protection (FP)). It is thought that HR and FP deficiencies produce points of vulnerability in cancer cells because they cannot fix or prevent DNA double stranded breaks (DSBs) and therefore cells are sensitive to DNA damaging agents such as to cisplatin and Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). Our recent findings provide a counter model in which these therapies induce single stranded DNA (ssDNA) gaps that sensitize BRCA deficient cells due to a defect in gap suppression (GS). Several BRCA mutant cell models support gaps in mediating response, however, each model of resistance maintains at least two functions. Thus, it is not certain which function underlies the resistance, leaving a knowledge gap that limits clinical insight. The development of effective therapies requires identifying whether HR, FP, and/or GS is the fundamental mediator of response. This goal of this study is to systematically disrupt and retain each function (HR, FP, GS) within BRCA2 to define what function is critical for therapy resistance, elucidate a unified mechanism of resistance, and provide insight into inhibiting pathways of resistance to inform therapeutic choices. To do this we aim to determine the molecular mechanism of GS through mapping the GS domain(s) in BRCA2 (Specific Aim 1). In BRCA2 deficient cells complemented with wild-type vs a series of BRCA mutants that either delete or selectively target well-characterized domains (i.e., HR or FP), protein interacting regions, or DNA binding sites, we will analyze gap induction in our routine DNA fiber and immunofluorescence assays. If not already well characterized, we will assess mutants for HR proficiency in standard assays and FP via examination of nascent strand degradation in DNA fiber assays. We will use CRISPR/CAS9 to make additional mutants in the identified GS domain(s) to further characterize the critical residues mediating GS. We will also test PARPi sensitivity of these mutant expressing cells in order to assess the link of HR, FP, or GS to response. We also aim to determine if apoptosis underlies loss of cell viability in BRCA2 deficient cells following genotoxins (Specific Aim 2). Apoptosis will be measured using standard assays in BRCA2 mutants following treatment with cisplatin or PARPi. In addition, we will treat cells with apoptosis inhibitors and determine if sensitivity to PARPi or cisplatin is suppressed. We will verify the time and dose in which DSBs are induced compared to apoptosis and assess if inhibition of apoptosis reduces DSB formation. The rationale for the proposed research is that BRCA2 deficiency will be most effectively treated by therapies that form gaps, gap formation will be a biomarker of tumor response, and to maximize therapy response, pathways limiting gap formation should be targeted. The insight gained from the experiments proposed will have implications for cancer and provide new opportunities for therapeutic intervention.

Duchenne muscular dystrophy (DMD)—a fatal inherited muscular dystrophy—is caused by loss of Dystrophin, a protein that maintains muscle integrity. Corticosteroids slow DMD progression but cause side effects. Addressing the root cause of DMD may improve patient health without needing corticosteroids. Many DMDcausing mutations disrupt the dystrophin mRNA reading frame, resulting in non-functional protein. Strategies that skip the out-of-frame exon to restore the reading frame and produce semi-functional protein for improved muscle function could correct 64% of DMD mutations. FDA-approved antisense oligonucleotide drugs can skip select exons in dystrophin mRNA, but require lifelong infusions and only work in a small group of patients. Using CRISPR to edit dystrophin would require just one treatment. CRISPR-mediated ablation of splice sites to cause exon skipping can increase Dystrophin in DMD models. Yet, editing in unintended tissues is a safety concern for Cas9 therapies. An ideal platform for DMD would restrict editing to muscle tissue to maximize therapeutic benefit. Efforts to achieve tissue-specific editing often rely on delivery via adeno-associated viruses (AAVs) with tissue tropism; yet, it is rarely absolute. Tissue-specific editing was recently achieved using tissue-specific miRNAs to regulate expression of Cas9 inhibitors [anti-CRISPR (Acr) proteins] via miRNA target sites (TS) in the 3’ UTR of Acr mRNA. When the platform is systemically delivered to mice via AAV, Acr-TS targeted by liver-specific miRNA allows editing only in the liver. Unlike tissue-specific promoters, this Acr-TS strategy could be adapted to one or multiple muscle tissues affected in DMD, as long as muscle-specific (myo)-miRNA can repress an Acr. With support from Erik Sontheimer (CRISPR, Acr), Eric Olson (DMD), Wen Xue (in vivo CRISPR delivery), Phillip Zamore (miRNA), Guangping Gao (AAV), and Zhiping Weng (bioinformatics), this proposal seeks to develop a muscle-specific editing platform to treat DMD. The myo-miRNA, miR-1, can repress an Acr in muscle cell lines to achieve muscle-specific editing. To fine-tune specificity of editing in muscle tissues for DMD, Aim 1 will test the ability of myo-miRs varying in abundance and muscle-type specificity to repress Acr and drive muscle-specific editing in mouse cell lines. The myo-miR construct supporting highest muscle-specific editing will be delivered to a DMD mouse model, and in vivo muscle function as well as dystrophin exon skipping, Dystrophin protein, and miRNA level in muscle tissues and liver will be measured. Aim 2 will test the compatibility of additional Cas9 orthologs in the Acr-TS system to enable targeting of more sequences, and develop a single AAV delivery system for improved safety. An Acr inhibiting the Cas9s to be tested has been identified. The ability of miR-1 to repress this Acr and drive muscle specific editing by each Cas9 will be tested in cells. A single vector encoding the AcrTS system will be designed and packaged into AAV, and muscle-specific editing will be compared to a dual AAV system in mice. This work will develop a flexible, safe, muscle-specific CRISPR platform with the potential to be used for any combination of muscle tissues to treat patients with DMD, or other genetic muscle disorders.