New Therapeutic Platforms for Targeting Metal Nutrient Homeostasis Vulnerabilities in Cancer

Aberrant cell proliferation in cancer is highly dependent on nutrient status, and the Chang lab and others have identified transition metal signaling processes that forge new links between copper and cancer. For example, while copper is an essential nutrient for all cells, tumor cells display a heightened requirement for this metal nutrient, and elevated copper concentrations have been reported in the tumors or serums of animal models and patients with several cancers. One central player in cellular copper homeostasis is the metallochaperone Atox1. Elevated Atox1 expression is observed in human blood, breast, and skin cancer samples, with high Atox1 levels in melanoma correlating with poor patient prognosis, highlighting the value of Atox1 as a novel therapeutic target for cancer. We have developed a novel chemoproteomics platform to target copper homeostasis machinery as a cancer vulnerability by developing a novel inhibitor for Atox1. Our inhibitor discovery strategy utilizes activity-based protein profiling (ABPP), a powerful and versatile chemoproteomics method for the high-throughput, unbiased discovery of new covalent ligands to engage and functionally modulate our chosen target, Atox1. Screening of a library of 700+ small molecule fragments in collaboration with the Nomura lab yielded numerous candidate inhibitor compounds that were able to covalently label Atox1 in a dose-dependent manner. Using biochemical assays, we demonstrate two candidate molecules were functional inhibitors of Atox1 copper binding, and further cell assays showed one inhibitor selectively inhibited proliferation of A375 melanoma cells, but not non-cancerous primary melanocytes. Additionally, this molecule was able to reduce cell migration at sub-EC50 dosages as measured by a cellular scratch wound healing assay. Currently we are working to obtain an x-ray crystal structure of the Atox1-inhbitor complex in collaboration with the Rosenzweig lab. In parallel, we have pursued in silico structural studies via computational docking using the Schrödinger CovDock software. Structures of Atox1-inhibitor complexes, whether achieved by protein x-ray crystallography and/or computational docking, will identify key charged residues, hydrogen-bond donors/acceptors, and hydrophobic residues to establish structure-activity relationships (SARs) of ligand binding and guide modifications to inhibitor structure. Future inhibitors will be tested using our previously optimized experimental workflow. This study uses a range of chemical biology methods to identify and optimize inhibitors for the copper protein Atox1 that can be applied as both a cancer therapeutic, as well as a chemical tool in the study of cellular copper homeostasis. The chemoproteomics method developed in this study will be generalizable to the discovery of covalent ligands to target any metal-binding protein and will inform future efforts to develop small molecule modulators of key metalloproteins useful in the treatment of disease.

Physical drivers of structural order and disorder in biological tissues

Tissues exist in fluctuating environments and must actively maintain their structure through programs of self-organization. However, the fundamental limits of structural order at the tissue scale have not been defined. Here we combine theory and experiments to demonstrate that tissues exist as dynamic structural ensembles with properties that follow Boltzmann statistics, and therefore, are defined by a characteristic level of disorder. The statistical properties of tissue ensembles emerge from three parameters: underlying interfacial energies (enthalpy), geometric constraints (structural degeneracy or entropy), and mechanical fluctuations (activity) of the tissue. We measured the structural distribution for reconstituted organoids, comprising aggregates of human mammary epithelial cells derived from breast reduction mammoplasty surgeries. We show that distribution follow Boltzmann statistics, and engineer the tissue structural ensemble through a systematic perturbation of mechanics, geometry or dynamics. We further demonstrate how this structural ensemble becomes disrupted during breast cancer progression.  Specific pathways downstream of breast cancer driver genes alter the structural ensemble by altering interfacial mechanics and consequently, increasing tissue structural disorder. Among15 cancer-associated genetic changes, PIK3CA most significantly altered the interfacial mechanics of the breast cancer cell of origin–luminal epithelial cells–in a manner that promotes tissue configurations that are primed for invasion. We identify molecular perturbations that restore a normal structural ensemble by correcting luminal epithelial cell interfacial mechanics or decreasing global tissue activity. Overall, this statistical mechanical framework reveals that configurational entropy imposes a theoretical maximum limit to structural order and can promote the emergence of tissue structures that are primed for invasion in cancer.

Structural insights into respiration across the tree of life

Background Respiration is an essential metabolic process that provides energy to all free-living eukaryotes. Even photosynthetic organisms, which convert sunlight into chemical energy at the base of all food chains, require mitochondrial respiration. Thus, photosynthesis and respiration are the most fundamental biological processes fueling life on Earth. Understanding the molecular details of these two mechanisms has significant societal implications, as they affect atmospheric compositions, crop efficiency and food production. Although we have a detailed biochemical and structural understanding of photosynthesis, our understanding of respiration has lagged significantly outside a handful of mammals, yeast and bacteria. This gap has been driven by challenges in purifying respiratory complexes from organisms with low mitochondrial content. When I started my postdoc, we lacked detailed characterizations of respiratory complexes from plants or protists—major “kingdoms” of the tree of life—strongly hindering our knowledge of respiration and our ability to design novel agricultural and environmental strategies.

Accomplishments Applying novel biochemical and structural approaches, I obtained the first high-resolution structures of three respiratory complexes from the plant kingdom^(1,2) using cryo-electron microscopy (cryoEM). I also played a major role in obtaining the first high-resolution structures and functional characterization of respiratory chain from Tetrahymena, part of the major protist branch⁽³⁾.

To overcome long-standing challenges of purifying these complexes, I developed a sample-sparing strategy. Rather than pursuing a traditional full biochemical purification before cryoEM data acquisition, I carry out a partial biochemical purification followed by a full “computational purification” with cryoEM image-processing algorithms. This requires significantly smaller amounts of sample and allows us to obtain multiple structures from a single cryoEM dataset. My work on plant respiratory complexes showed this pipeline can be successfully used for large membrane complexes, opening the door to a new era of structural biology for challenging macromolecular assemblies. This same strategy was used to obtain the reconstructions for the Tetrahymena complexes.

My plant structures revealed plant-specific features of respiratory complex I, III₂ and IV’s structures. They solved controversies on the subunit composition of a domain of complex I, and of the entire complex IV. Further, my analysis of the conformational flexibility of complex III₂ dismantled a long-held belief in the field and called for the re-interpretation of existing data. The structures also generated mechanistic hypotheses on plant-specific aspects of respiration.

Our structures of Tetrahymena’s respiratory complex I, III₂ and IV₂ revealed unanticipated functional and structural diversity in eukaryotes’ core metabolism. With their novel modules, architectures and inter-complex interactions, the Tetrahymena complexes diverge from all previously studied organisms in major ways. This work exposed how much we still do not know about respiration and how much our focus on mammalian and yeast models has biased our understanding.

Universal principles of nucleosome recognition

Nucleosomes are the fundamental repeating units of chromatin, which facilitate genomic DNA packaging and serve as signal integration hubs for genome templated processes. Previously, using a nucleosome affinity proteomics screen, we demonstrated that over 50% of proteins that interacted with nucleosomes were specifically recruited by a group of acidic amino acids on the nucleosome disk surface. Remarkably the remaining surfaces participated only minimally in nucleosome binding. The acidic patch binding preference has been further confirmed by dozens of high-resolution structures of nucleosome complexes published in recent years. Moreover, mutations in the nucleosome acidic patch were identified in various cancer types. To investigate how these oncohistone mutations and mutations of individual amino acids in and around the acidic patch affect nucleosome binding proteome-wide, we conducted a 2^nd generation nucleosome interactome proteomics screen. We created a library of fifteen-point mutant nucleosomes, twelve to alanine scan the acidic patch surface and three to investigate whether the oncohistone mutations perturb nucleosome interactomes in cancer patients. Our new screen establishes the hierarchy of importance for the acidic patch side chains and demonstrates the severity of oncohistone mutations, which are far more disruptive for chromatin binding than the corresponding alanine mutations. We describe footprinting of nucleosome acidic patch binding at amino acid resolution for hundreds of proteins representing diverse nuclear functions. We observed components of large protein complexes exhibiting consistent acidic patch footprint trends, confirming the robustness of our method. A surprising example is demonstrated by two parolog histone H3K36 demethylases, KDM2A and KDM2B, which exist in two different heterochromatin complexes and show strong and no acidic patch dependence, respectively. The KDM2 family of JmjC-containing domain demethylases regulate gene expression and are commonly misregulated in blood cancers. To gain mechanistic insight and to confirm paralog-specific nucleosome acidic patch interactions, we solved high-resolution cryo-EM structures of both KDM2A and KDM2B bound nucleosomes. Our structures for the first time demonstrate how the JmjC domain demethylases engage and function on nucleosomes. Both KDM2A and KDM2B unwrap DNA and shield the positive charge of the newly exposed histones to access the H3K36 sequence for demethylation. However, only KDM2A interacts with the acidic patch and shows severely decreased activity in demethylating acidic patch neutralized nucleosomes. Interestingly, although KDM2A binding is disrupted by all three oncohistone mutations tested in the screen, its activity is decreased in a charge swap (E/K) but not in a charge neutralized (E/Q) nucleosomes. Overall, our screen deciphers acidic patch binding at amino acid resolution and reveals an exceptional sensitivity in assessing paralog-specific differences in nucleosome recognition as well as the potential deleterious consequences of oncohistone mutations on histone demethylase activity by KDM2A.

Colonization and transmission after antibiotic perturbation in the human gut microbiome.

 As a postdoctoral fellow working with Dmitri Petrov and David Relman at Stanford University, I am studying ecology and evolution in the human gut microbiome. I aim to uncover the ecological determinants of strain colonization: although we encounter new microbes every day, very few introduced microbes become long-term residents of the gut microbiome. I tracked strain dynamics in a longitudinal household cohort of 48 healthy adults from 22 households over two months, during which one subject in each household took a 5-day course of the antibiotic, ciprofloxacin. Individuals had heterogeneous responses to antibiotic perturbation: in some subjects, the gut microbiome rapidly returned to its initial composition after antibiotics, but in others, the microbiome transitioned after antibiotic exposure to an alternative stable state that persisted through months of follow-up sampling. In one household, a strain of Bacteroides stercoris transmitted between cohabiting partners after antibiotics and maintained a relative abundance above 50 percent through subsequent follow-up sampling, suggesting that strain transmission can play a major role in shaping the post-antibiotic community.

To test how ecological factors affect the dynamics of strain transmission that I observed in vivo, I have derived ex vivo microbial communities from pre- and post-antibiotic samples from each household and performed systematic community mixtures to measure the effect of antibiotic perturbation on strain acquisition. Post-antibiotic communities that had experienced greater losses in diversity after antibiotic perturbation were more susceptible to colonization, suggesting that antibiotics clear certain niches in microbial communities that allow new microbes to colonize. I am continuing to use in vitro communities to dissect the ecological interactions that underpin these colonization events. My long-term goal is to build an independent research program that combines genomic technologies with eco-evolutionary theory to understand how microbial communities assemble, function, and evolve.

The Impacts of Coevolution on Ecological Structure and Coexistence in Multispecies Communities.

Ecological communities are natural organizations of multiple species and their interactions. These interactions are partially determined by a species’ traits. As these traits are shaped by evolution and adaptation more generally, this suggests a role for the adaptive process in understanding ecological communities. That said, adaptation does not occur absent of the ecological context and forms a feedback loop with ecology. I model and analyze this eco-evolutionary feedback loop to determine its influence on ecological communities. I create mathematical models of species evolution and seek eco-evolutionary stability, then empirically test these models and their results in natural and artificial systems.

The primary theme of my research is how coevolution shapes ecological communities. Coevolution is necessarily a major component of evolution in an ecological community as individuals of different species reciprocally interact with each other, whether through competition, predation/parasitism, or mutualism. I have focused primarily on competitive eco-evolutionary dynamics at different scales. I worked on projects that determined the limits to niche packing, examined the relationship between a community’s species richness and the evolutionary dynamics of its species, explored the impact clade-level adaptations have on community composition, and analyzed how the balance of stabilizing and disruptive selection facilitates ecological coexistence. Going forward, I plan to add properties like specialization and individual variation to further refine eco-coevolutionary processes in competitive communities. Recently, I have begun to explore mutualistic interactions. I am examining the ecological and environmental conditions that lead to the evolution of mutualism using theory and a greenhouse experiment of the plant-microbial system. Using different types of microbes both separately and together will allow me to tease out the nuances in the mutualism evolution. I am also modelling how coevolution and cospeciation between guilds structures mutualistic networks.

 Another research theme of mine is understanding behavior as an adaptive trait and its impact on ecology. Behavior is plastic, changing with the context in which an individual finds itself. Ergo, it is potentially adaptive both within and between individuals’ lifetimes, providing a unique effect on population and community ecology. I have done work on the effects that adaptive vigilance behavior has on predator-prey community structure. My main interest though is the eco-evolutionary dynamics of cooperative behavior. Cooperation as an adaptation should significantly impact a species’ population dynamics as fitness varies depending upon with whom an individual chooses to cooperate. Previously, I have shown that cooperation should lend instability to a species’ population dynamics due to a mismatch between behavioral and population dynamics. Going forward, I plan to derive more precise eco-evolutionary dynamics of cooperation. Of particular interest is the transition from parasocial to eusocial. I plan to explore the role that parental dominance along with ecological and fitness opportunities play a role in the parasocial to eusocial transition. My work also suggests that intergroup competition facilitates this transition as groups with higher average fitness better escape competition and persist. Incorporating these and other processes like demographic stochasticity, relatedness, and hierarchy should derive a more accurate and precise understanding of cooperative eco-evolutionary dynamics.

Understanding selection for and maintenance of mixed mating using vertebrate and invertebrate model organisms.

Most eukaryotic organisms engage in sexual reproduction where gametes from two separate individuals of opposite sexes must fuse for fertilization (outcrossing). Despite the prevalence of this reproductive strategy, how outcrossing is maintained has been a key question in the field of evolutionary biology for over a century. Theoretical modeling for the last sixty years has suggested that sexual reproduction should be difficult to sustain over evolutionary time, due to the overwhelming costs of outcrossing and the production of males. Experiments and field observations have yet to reconcile the abundance of outcrossing with theoretical predictions. My primary research objective is to understand why more species, especially vertebrates, are not mixed mating hermaphrodites. Mixed mating species (those that outcross and self-fertilize, i.e., self) such as the nematode Caenorhabditis elegans and the mangrove rivulus fish Kryptolebias marmoratus offer an exemplary opportunity to study the conditions that select for and maintain outcrossing over self-fertilization. In this seminar I will share what I discovered about the only known vertebrate that can self-fertilize (the mangrove rivulus fish), and how I plan to use C. elegans nematodes to explore the finer details of mixed mating. The mangrove rivulus fish exist predominantly as self-fertilizing hermaphrodites but males, which result from hermaphrodite sex change, occur in varying abundances across their geographical range. Levels of heterozygosity and frequencies of outcrossing between hermaphrodites and males also differ among populations, raising the questions of why outcrossing and male abundance varies. We hypothesized that heterozygous progeny derived from outcrossing would have higher fitness than homozygous progeny derived from selfing, especially under stressful conditions. We predicted that increased heterozygosity would correspond with lower mortality, higher growth, and greater reproductive success, trends that would be amplified under suboptimal conditions. To test this hypothesis, fish with varying levels of heterozygosity experienced control conditions or stressors common to their native habitat (high/low salinity, tidal flux). There were significant treatment effects. High salinity animals showed greater mortality, lower fecundity, and lower rates of sex change than those in low salinity or controls. Tidal animals were consistently smaller and less fecund than controls, but mortality was unaffected. Contrary to our predictions, only fecundity was affected by heterozygosity, consistent with outbreeding depression instead of the predicted inbreeding depression. Intriguingly, sex change explained survival. Those animals that changed sex to male survived significantly more in all treatments compared to animals that remained hermaphrodites. We were the first to document sex change that confers greater survival at the expense of reproductive success. Currently, I am using the model organism C. elegans to explore how outcrossing and selfing evolve when exposed to multiple bacteria in an ecologically relevant microbiome community. This builds on previous work of experimental evolution with singular strains of bacteria. Additionally, to better represent how outcrossing and/or selfing is maintained in natural environments, I plan to add the additional treatment of migration among populations to assess how new alleles interact with microbial communities and their hosts to select for outcrossing and/or selfing.

Genetic variation of putative myokine signaling is dominated by biological sex and sex hormones

Proteins secreted from skeletal muscle, termed myokines, allow muscle to play an integral role in coordinating physiologic homeostasis. These factors exert critical roles in the regulation of metabolic homeostasis, exercise improvements, inflammation and cancer, but the genetic architecture, regulation and functions of myokines remains inadequately understood. Having in mind that genetic sex contributes critically to nearly every physiologic outcome, it is essential to consider when relating specific mechanisms to complex genetic and metabolic interactions. We aimed to leverage natural genetic correlation structure of gene expression both within and across tissues to understand how muscle interacts with metabolic tissues. Specifically, we performed a survey of genetic correlations focused on myokine gene regulation, muscle cell composition, cross-tissue signaling and interactions with genetic sex in humans. To address these questions, we made use of the human skeletal muscle gene and metabolic tissue data available through the Genotype-Tissue Expression (GTEx) project, loss of function mice models, and single cell data obtained from bibliography.

While expression levels of a majority of myokines and cell proportions within skeletal muscle showed little relative differences between males and females, nearly all significant cross-tissue enrichments operated in a sex-specific or hormone-dependent fashion. Steroid hormone receptors, in particular estradiol receptor alpha (ESR1), is highlighted as a key regulator of myokines and potentially interacting with biologic sex for proteins such as myostatin. These sex- and hormone-specific effects were consistent across key metabolic tissues: liver, pancreas, hypothalamus, intestine, heart, visceral and subcutaneous adipose tissue. To characterize the role of ESR1 signaling on myokine expression, we generated male and female mice which lack estrogen receptor α specifically in skeletal muscle (MERKO) and integrated with human data. These analyses highlighted potential mechanisms of sex-dependent myokine signaling conserved between species, such as myostatin enriched for divergent substrate utilization pathways between sexes.

Several other putative sex-dependent mechanisms of myokine signaling were uncovered, such as muscle-derived TNFA enriched for stronger inflammatory signaling in females compared to males. Generation of pseudo-single-cell maps of muscle composition also highlighted GPX3 as a male-specific link between glycolytic fiber abundance and hepatic inflammation. Some consideration should be mention when interpreting these findings. While inter-tissue regression analyses have been informative to dissect mechanisms of endocrinology, observations can be subjected to spurious or latent relationships in the data. While causality for inter-organ signaling can be inferred statistically, the only methods to provide definitive validation for new mechanisms is in experimental settings. Collectively, we provide a population genetics framework for inferring muscle signaling to metabolic tissues in humans. We further highlight sex and estradiol receptor signaling as critical variables when assaying myokine functions and how changes in cell composition are predicted to impact other metabolic organs.

Interleukin 6 (IL-6) is a major mediator of the septic cytokine storm. It activates the transcription factor STAT3, resulting in pathological vascular leakage, leukocyte adhesion, and coagulopathy, leading to acute multiorgan dysfunction and potentially death. Sepsis survivors have a high risk of developing chronic cardiovascular disease, cognitive impairment, and reduced overall survival, a condition knonwn as post-intensive care syndrome (PICS). Using endothelial-specific mouse knockouts of SOCS3 (SOCS3iEKO), the main negative regulator of the IL-6/STAT3 pathway, our lab demonstrated that increased endothelial STAT3 activation led to mortality after a single lipopolysaccharides (LPS) challenge that was non-lethal in wild-type mice. This mortality was associated with kidney failure, leukostasis, and vascular leak in lungs and brain. New in vitro data strongly suggest that the endothelium responds to LPS with an increase in IL-6 expression that acts in an autocrine fashion to promote strong barrier function loss. Together, these findings potentially explain the systemic vascular leak that we observed in critically ill patients with high circulating IL-6 levels.
The risk of developing PICS is associated with the severity of the acute response. Thus, we propose that overactivation of STAT3 within the endothelium may lead to chronic sequelae in sepsis survivors. Previous studies have shown that shock leads to acute changes in DNA methylation. We hypothesize that overactivation of STAT3 leads to altered DNA methylation, driving long-term vascular dysfunction after shock. We measured the changes in DNA methylation in the kidney endothelium of SOCS3iEKO and control mice challenged or not with LPS using Infinium Methylation EPIC arrays. We identified 2596 differentially methylated positions (DMP) induced by LPS. When comparing control LPS vs SOCS3iEKO mice after LPS treatment, we identified a total of 3564 DMP. Gene ontology analysis showed an enrichment of genes involved in inflammation, cytokine signaling, and leukocyte adhesion, suggesting that the acute inflammatory response may lead to long-lasting changes in endothelial inflammation. To determine if endothelial cells in culture respond to IL-6 signaling specifically by inducing long-term epigenetic changes, HUVEC were treated with a combination of IL-6+R or saline for 6h, 24h or 72h prior to the methylomics assays. Unbiased clustering showed a separation between treated and control cells after 72h, but not 6h or 24h. We observed 431 CpG sites were significantly modified. GO analysis showed enrichment of pathways directly linked with the regulation of cell adhesion, proliferation, and cytokine signaling. To determine the stability of these changes in the levels of DNA methylation, we treated HUVEC with IL-6+R for 72h, followed by 96h in the absence of IL-6+R. We identified 40% DMP sites with a mean difference of less than 2% when comparing washed (72+96h) vs non-washed cells (72h alone), suggesting that many changes in the endothelial methylome may remain in place for prolonged periods. In summary, persistent activation of endothelial IL-6 signaling leads to severe organ damage and endothelial DNA methylation changes. These differences may regulate pathways associated with reduced long-term survival and chronic organ damage, potentially providing a novel therapeutic target to treat and/or prevent PICS.

Studying Basic Adeno-associated Viral Vector Biology to Maximize its Therapeutic Potential.

Recombinant adeno-associated viral vectors (rAAV) are a simple yet potent vehicle for delivering DNA to a wide range of tissue and cell types. rAAVs contain a single-stranded DNA vector genome (VG) encased in a 25nm unenveloped capsid that delivers the VG to host nuclei. VGs then become double stranded and concatenate into large episomes that can remain transcriptionally active for the life of the host cell. The potential for long-term gene expression without integration into the host genome makes rAAVs an ideal gene therapy vector. However, several limitations prevent rAAV’s application to a broad spectrum of diseases. For example, current platforms require large doses to achieve benefits, and adverse effects—including deaths—that can occur at these doses have raised safety concerns for rAAV’s clinical use. Another shortcoming of rAAV is its 4.8kb packaging limit. This size can accommodate many genes’ cDNA plus a short viral promoter, but the ubiquitous and high-expression-level nature of these promoters often result in off-target tissue expression and/or an immune response. To enable inclusion of larger coding sequences and tailored regulatory elements, rAAV’s natural ability to concatenate can be exploited, but the inefficiency of this approach requires large doses. Overcoming these limitations to achieve long-term transgene expression at appropriate levels from a minimal rAAV dose will require basic knowledge of how VGs are processed and regulated.

AAV has been studied for over 50 years and is FDA approved for clinical use, yet the mechanisms establishing its expression in the host cell – a central process in gene therapy – are unknown. The simple nature of the rAAV particle – carrying no enzymes within it – implies that host factors are co-opted to execute all steps of VG processing and expression regulation. Astoundingly, zero of the factors mediating VG concatenation and stable episome formation have been identified. For my postdoctoral studies, I am combining super-resolution and live cell imaging approaches with genomic methods to identify these factors and investigate previously understudied areas of AAV Vectorology. These new approaches enable quantitative studies at individual VG resolution and on unprecedented time scales from minutes to weeks post-infection. In this talk, I will present results from a genome-wide screen based on split-transgene reconstitution that implicate inhibitory functions of several DNA damage repair (DDR) factors in VG concatenation. Validation studies suggest that multiple DDR pathways are simultaneously active on VGs, and inhibition of a specific subset of factors greatly increases the size, number, and kinetics of growing rAAV episomes. By visualizing cellular factors recruited to VGs, I show that, contrary to previous thinking, the rAAV episome is under a constant state of maintenance, continuously recruiting DDR factors over long durations. In the future, my research group will study the intersection of host/pathogen response mechanisms with rAAV vector technologies to gain insight into fundamental human biology and directly inform strategies toward increasing the safety, efficacy, and longevity of rAAV gene therapies. As a first-generation college student, I will emphasize training others from this group, who are uniquely positioned to bring diverse perspectives to gene therapy.

Cytoskeletal regulation of a transcription factor by DNA mimicry.

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. In contrast, the principal effectors of Hedgehog signaling, the Gli transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How Gli is tethered to microtubules remains unclear. We uncover DNA mimicry by the ciliary kinesin Kif7 as a mechanism for the recruitment of Gli to microtubules, wherein the coiled-coil dimerization domain of Kif7, characterized by striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in Gli, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. Gli increases the Kif7-microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive Gli tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We re-tooled the unique coiled-coil-based Gli-Kif7 interaction for inhibiting the nuclear and cilium localization of Gli. This strategy can be potentially exploited for downregulating erroneously activated Gli in human cancers.

The northern tree shrew (Tupia belangeri): Elucidating a complex visual system using functional ultrasound imaging, electrophysiology, and non-invasive viral delivery

The central goal of my work has been to understand the role of functionally defined brain areas along the tree shrew visual hierarchy in processing visual object representation. Importantly, tree shrews have a substantially more complex visual system than rodents and a clear experimental advantage over primate models. The neural mechanisms underlying visual inference is one of the central mysteries of neuroscience, getting at the heart of how different brain areas work together to construct a coherent percept. Understanding this process requires separately and simultaneously interrogating multiple nodes along the visual hierarchy, as well as the ability to experimentally manipulate specific nodes. We have studied this question in a proto-primate model using recently developed techniques for whole-brain functional imaging and modulation. Existing animal models present methodological challenges: in primates, which allow the probing of complex cognition, it is difficult to record from large ensembles of neurons across multiple brain areas or manipulate genetically defined cell populations; in rodents, rudimentary cortical organization and behavioral repertoire limits the modeling of visual processing. For these reasons, I chose to study visual organization in the tree shrew, an animal with high visual acuity and considerable cognitive abilities. First, I have established the brain wide organization of the visual system using a non-invasive imaging technique – functional ultrasound imaging, which allows measurements of evoked activity across the entire brain, much like fMRI, but with greatly improved spatial and temporal resolution. Next, I have targeted high density silicon probe recordings (neuropixels) to various nodes of this network (e.g. V1, V2, and IT) and measured the local activity in response to a host of visual stimuli used in classical visual physiology studies. I have also developed techniques to manipulate populations of genetically defined cells in a non-invasive and spatially targeted way in each of these regions using acoustically targeted delivery of promotor-specific virus carrying inhibitory DREADDs by transiently disrupting the blood brain barrier. This allows specific and reversible control, by decreasing the excitability of genetically defined excitatory cell populations at different nodes in this hierarchy with the systemic administration of CNO or other DREADD receptor agonists. Finally, I have developed custom behavior systems to test the behavioral effects of these targeted manipulations in the tree shrew on several different behaviors. This work provides valuable new information to researchers and clinicians about the function and computations of sensory transformations along cortical networks. Furthermore, these studies lend important insight into the consequences of disruption of the typical balance of excitation and inhibition within these areas which is associated with several neurological disorders including autism, schizophrenia, and epilepsy, to name a few. We have begun to extend this work to non-invasive neuromodulation for circuit-level treatments of neuropsychiatric disorders in a collaboration with Mike Halassa’s lab at MIT. Here, using acoustically targeted chemogenetics targeted to medio-dorsal thalamus (MD) in tree shrew, and  propose that MD neurostimulation in schizophrenia may enhance PFC activity,  boost cognition, and alleviate symptoms of this debilitating disease.

Extrinsic control and intrinsic computation in the hippocampal CA1 circuit

In understanding circuit operations, a key issue is the extent to which neuronal spiking reflects local computation or responses to upstream inputs. Because pyramidal cells in CA1 do not have local recurrent projections, it is currently assumed that firing in CA1 is inherited from its inputs – thus, entorhinal inputs provide communication with the rest of the neocortex and the outside world, whereas CA3 inputs provide internal and past memory representations. Several studies have attempted to prove this hypothesis, by lesioning or silencing either area CA3 or the entorhinal cortex and examining the effect of firing on CA1 pyramidal cells. Despite the intense and careful work in this research area, the magnitudes and types of the reported physiological impairments vary widely across experiments. At least part of the existing variability and conflicts is due to the different behavioral paradigms, designs and evaluation methods used by different investigators. Simultaneous manipulations in the same animal or even separate manipulations of the different inputs to the hippocampal circuits in the same experiment are rare.

To address these issues, I simultaneously silenced the major inputs feeding into CA1 to address the question of whether CA1 spiking can be sustained by local computations or merely reflects responses to upstream inputs. I used optogenetic silencing of unilateral and bilateral medial entorhinal cortex (mEC), of the local CA1 region, and performed bilateral pharmacogenetic silencing of the entire CA3 region. I combined this with high spatial resolution recording of local field potentials (LFP) in the CA1-dentate axis and simultaneously collected firing pattern data from thousands of single neurons. Each experimental animal had up to two of these manipulations being performed simultaneously. Silencing the mEC largely abolished extracellular theta and gamma currents in CA1, without affecting firing rates. In contrast, CA3 and local CA1 silencing strongly decreased firing of CA1 neurons without affecting theta currents. While unilateral mEC silencing did not alter CA1 place fields, remapping of place cells was observed for the other manipulations, or when multiple manipulations were performed simultaneously. Yet, the ability of the CA1 circuit to support place field activity persisted, maintaining the same fraction of spatially tuned place fields, and reliable assembly expression as in the intact mouse. Thus, intrinsic excitatory-inhibitory circuits within CA1 can generate neuronal assemblies in the absence of external inputs, although external synaptic inputs are critical to reconfigure (remap) neuronal assemblies.

To contrast these results during awake behavior, I am currently working on a follow-up project to examine how CA1 assemblies are affected by these input manipulations during sleep. During slow wave sleep and consummatory behaviors, hippocampal network events called sharp wave ripple oscillations (SWRs) coordinate the sequential activation of hippocampal neuronal assemblies, i.e., replay, that represent past trajectories. My preliminary results reveal the exciting finding that even small manipulations, such as unilateral mEC silencing, that were ineffective in impacting place cells during behavior, can alter these sharp-wave ripple assemblies activated during sleep. This suggests different circuit mechanisms are at play to generate sequences during sleep versus waking brain states.

Auditory processing and modulation in the auditory cortext

My talk has two parts: neural responses to complex sounds in mouse auditory cortex and modulation of human auditory cortical responses during vocal production. In the first part of my talk (Kim et al., 2020), I will elaborate on neural response properties in mouse primary auditory cortex (A1), a hub that communicates with subcortical and cortical regions. Acoustic information conveyed from the auditory thalamus is modified along intracortical horizontal pathways in A1. A1 neurons respond to characteristic frequencies through direct thalamocortical projects and to non-characteristic frequency stimuli via intracortical horizontal pathways because thalamocortical axons project toward restricted portions of A1 without diverging. Thus, A1 neurons respond to complex sounds carrying both characteristic and non-characteristic frequencies differently from pure tones conveying a single frequency. Spectral integration in A1 can be examined using in vivo whole-cell recordings, an intracellular higher resolution recording method, exemplifying subthreshold and spiking receptive fields. This study contrasted the specific properties of receptive fields with subthreshold and spiking responses employing both pure tones and complex stimuli in the mouse primary auditory cortex. The key finding of this study was that spectral tuning in receptive fields deduced from complex sounds for spiking and subthreshold responses is more selective than that in receptive fields derived from pure tones by almost a 1-octave. This finding suggests that the excitatory and inhibitory weighing for each neuron’s spectral integration is modified by dynamic cortical activity resulting from inputs of spectrotemporally complex stimuli, and that responses to pure tones cannot fully enlighten us about the function of A1 neurons in natural environments.

            In the second part of my talk, I will focus on the human auditory cortex, in particular describing how its activity is modulated when processing feedback during speech production (Kim et al., 2022). When studying the mechanisms setting firing rates in the auditory cortex, one should consider feedback mechanisms modulating the auditory cortical activity. The importance of understanding how auditory feedback is processed during speech production is underscored by the many diseases and conditions where abnormal feedback processing is thought to contribute to observed speech dysfunctions. Such conditions include autism, stuttering, apraxia of speech, spasmodic dysphonia, cerebellar ataxia, schizophrenia, dementia, and Parkinson’s disease. In all these conditions, dysfunction is suspected in the brain networks involved in speech sensorimotor integration. A neural phenomenon called speaking-induced suppression (SIS) characterizes auditory feedback processing exceedingly well. SIS is the suppression of auditory cortex response during speaking compared to listening to self-produced speech. This effect is thought to arise from a comparison of incoming auditory feedback with a prediction of that feedback derived from efference copy of motor cortex activity during speaking. SIS quantifies the accuracy of the match between feedback predictions produced internally in the brain and the actual incoming auditory feedback. A huge SIS implies an excellent match. Yet, a diminished SIS denotes a poor one, an impairment in predicting auditory feedback. It can depict unusual patterns in patients having multiple diseases. This study examined SIS in Alzheimer’s disease (AD). The critical finding revealed that SIS was absent in AD patients, suggesting that they were impaired in predicting auditory feedback during speech. This finding has implications for measuring speech motor control network deficits in AD patients. In future, I plan to examine the synaptic mechanisms underlying normal and abnormal SIS in animal models of vocal disorders.

Homeostatic plasticity establishes the specificity of associative taste memory

My research investigates the plasticity mechanisms underlying memory processing within neuronal networks. The current view holds that learning-induced long-term potentiation (LTP) – a Hebbian form of synaptic plasticity – strengthens the synaptic connections essential for memory formation. Nonetheless, computational models predict that LTP is inherently destabilizing and, if left unchecked, would trigger positive feedback in neuronal signaling that pervasively increases postsynaptic strength. The nonspecific strengthening of synaptic connections would hinder faithful memory acquisition and result in maladaptive behaviors. Synaptic scaling is a form of homeostatic plasticity that supports optimal neuronal firing by globally and bidirectionally adjusting postsynaptic strength in response to changing neuronal activities. It is posited to constrain learning-induced runaway LTP and maintain relative differences in synaptic weights, thereby ensuring precise and stable memory formation. While compelling on theoretical grounds, how synaptic scaling enables memory processing has not been tested experimentally. To tackle this question, I first generated an arsenal of virally expressed molecular tools that allowed me to perturb synaptic scaling in vivo. I then examined the impacts of disrupting synaptic scaling on associative memory using the conditioned taste aversion (CTA) paradigm, in which rodents acquired a strong aversion to the novel tastant previously paired with induction of sickness. I found that after conditioning, animals initially generalized the learned aversion to other benign tastants, and this nonspecific aversion was sculpted into a more specific aversive memory in the course of hours. To identify neuronal populations that subserved generalized aversion, I employed a genetic labeling method and imaged active neurons during memory encoding. In the gustatory cortex (GC) – the brain region crucial for CTA acquisition – I found that neurons activated by conditioning were robustly reactivated in the event of generalized aversion; chemogenetically silencing this subset of neurons diminished the generalization of conditioned aversion. My data thus suggest that conditioning-active GC neurons could undergo changes in synaptic plasticity pivotal in the transition from generalized to specific taste memory. Indeed, ex vivo electrophysiology revealed an evident correlation between the expression of generalized aversion and the amplitude of miniature excitatory postsynaptic currents (mEPSCs) – the physiological marker of postsynaptic strength – in these conditioning-active neurons. Strikingly, ablating synaptic scaling in conditioning-active GC neurons caused a uniform, unbridled increase in the postsynaptic strength that was reminiscent of runaway LTP, and behaviorally prevented the transition from generalized to specific taste aversion. Together, my work has unveiled a novel role of synaptic scaling in memory processing and demonstrated that synaptic scaling constrains the learning-induced increases in synaptic weights in the cortex to ensure memory fidelity. Motivated by this work, I am currently establishing an in vivo calcium imaging method to longitudinally track network-wide taste-evoked neuronal activities in awake animals throughout the CTA paradigm. I aim to further understand (1) how neuronal representations of sensory stimuli are reorganized within the cortex during memory generalization, and (2) how memory traces of associative taste memory are homeostatically stabilized over a longer timescale. Ultimately, I will examine whether these homeostatic processes are compromised in animal models of autism spectrum disorder (ASD).

In mammals, before visual information is transmitted to the brain, an intricate dance of visual processing takes place in the retinal circuit. Each of the cell classes in the retina—photoreceptor, horizontal, bipolar, amacrine, and ganglion cells— has multiple cell types, with the diversity of types especially large in the inner retina, where amacrine cells and ganglion cells each have over 30 types. These cell types are responsible for constraining and highlighting the visual information that mammals can use for vision. Despite great leaps in our understanding about the functions of individual cell types in the retina, many cell types’ roles in visual processing and behavior remain unknown. My long-term goals are to elucidate how circuit diversity contributes to visual information processing and determine how retinal circuits contribute to brain function and behavior in neurodiverse contexts.

I have established new approaches for evaluating the functional properties of large populations of interneurons using advanced computational methods and imaging techniques. Using two-photon imaging of fluorescent sensors of glutamate, calcium and a novel voltage sensor, I have uncovered previously unknown properties of bipolar cells, horizontal cells and amacrine cells. In my talk, I will focus on two recent findings from my work at the University of Tuebingen in Thomas Euler’s and Philipp Berens’ labs.

For the first part of the talk, I will discuss the representation of motion in the second stage of retinal processing in mouse retinal bipolar cells. We found that some bipolar cells are radially direction selective, preferring the origin of small object motion trajectories. This motion sensitivity had only previously been described in neurons downstream of bipolar cells. Using a glutamate sensor, we directly observed bipolar cells synaptic output and found that there are radial direction selective and non-selective bipolar cell types, the majority being selective, and that radial direction selectivity relies on properties of the center-surround receptive field. Thus, motion vs. non-motion information diverges at the level of bipolar cells. As bipolar cells provide excitation to most amacrine and ganglion cells, their radial direction selectivity may contribute to motion processing throughout the visual system.

In the second part, I will discuss ongoing research to understand information processing in the most diverse and least-well understood retinal interneuron class, the amacrine cells. One challenge in studying responses of the >40 types of amacrine cells is the fact that most of them lack axons and signal primarily through their dendrites; thus recordings of their somatic activity do not necessarily capture their many functional roles. To overcome this challenge, we performed a comprehensive survey of chromatic responses in GABAergic amacrine cells at sub-cellular resolution using 2-photon calcium imaging in mouse retina. We presented color noise stimuli calibrated to green- and UV-sensitive mouse photoreceptors to obtain chromatic receptive fields of individual subcellular regions of interest and identified functional groups with diverse color preferences and response polarities. Our data suggest that amacrine cells play an important role in diversifying the representation of chromatic information in the inner retina.

Hardwired to attack? Understanding the developmental and cellular mechanisms for innate social behaviors in the medial amygdala

Innate social behaviors are crucial for survival, thus shared across animal species. In humans, psychiatric disorders with deficits in social interactions, e.g., autism spectrum disorder, can be observed during child development and have been associated with amygdala dysfunction. There is still a lack of understanding of the circuitry and developmental mechanisms for the generation of social behaviors. We have focused on the murine medial amygdala (MeA) as it receives conspecific pheromone inputs and projects to hypothalamic regions. The MeA GABAergic cells have been shown to be sufficient for the production of social behaviors including aggression and mating. Given that these diverse social behaviors differ in their sensory trigger and behavioral outcomes, can the neuronal substrates for these behaviors be distinct? Taking a developmental approach, we have previously characterized two MeA GABAergic neuronal subpopulations, marked by the expression of the transcription factors Foxp2 and Dbx1 which originate from the same embryonic region. The Foxp2+ and Dbx1-derived subpopulations are spatially, molecularly and physiologically distinct. Interestingly, I have now observed that these two subpopulations receive anatomically distinct inputs and differ in their processing of social conspecific information. The MeA Foxp2+ cells are uniquely processing male sensory cues and are functionally relevant for aggression, while Dbx1-derived cells process multiple sensory stimuli, particularly female cues. These neuronal responses take place even with no/minimal social experience. To determine the extent to which these neuronal responses are hard-wired, I investigated the social tuning of Foxp2+ cells across development by recording the neuronal activity to social cues in juvenile mice at multiple postnatal days, providing a developmental understanding on how neuronal responses are established. In conclusion, developmentally distinct MeA neuronal subpopulations differ in their anatomical circuitry, are differentially relevant for processing conspecific sensory cues and mediating social behaviors.

Neural circuit mechanisms of pathological high frequency oscillations

Our brain has the remarkable ability to encode memories of our environment and experiences. This requires the precise coordination of neuronal activity, from the firing of specific cells, located in particular circuits, in distinct regions of the brain. Dysfunction in the regulation of this coordinated activity can lead to the development of severe neurological disorders. Epilepsy is characterized by excessive neuronal activity in the brain, epitomized by the occurrence of spontaneous recurrent seizures. While most clinical treatments for epilepsy target seizure reduction, less is known about the effects of the abnormal activity that is continuously present in the epileptic brain presented as interictal spikes. Although studies have found the presence of interictal spikes can disrupt learning and memory processes, the underlying mechanism of how this disruption occurs remains to be determined.

I have developed a novel mouse model of temporal lobe epilepsy (TLE) using targeted knockout of the β3 subunit of the GABA_A receptor, deficits of which have been implicated in epilepsy in humans, in CA1 pyramidal cells of the hippocampus. Mice receiving this focal knockout develop prominent interictal spikes as well as spontaneous seizures. Unlike other mouse models of TLE, these mice do not display excessive neuronal loss at the time when interictal spikes and spontaneous recurrent seizures are first detected, although increased neuroinflammation was observed in the hippocampus. This allowed for the investigation of a largely intact neuronal circuit. I utilized 24-hour video EEG monitoring, 2-photon calcium imaging, silicon probe recording, ex-vivo slice electrophysiology, and computational modeling to investigate the development and properties of these interictal spikes. I found that they are pathological high frequency oscillations (pHFOs) comprised of highly synchronous pyramidal cell firing which disrupt the ability of the cells to encode spatial information. In addition, these pHFOs, while present only at the epileptic focus where the β3 subunit knockout occurred, can interfere with hippocampal network activity outside of the focus. Further investigation showed that these pHFOs emerge from a specific deficit in inhibitory synaptic transmission from PV interneurons to pyramidal cells located in the deep layer of the CA1 and are triggered by CA2 input.

These findings demonstrate how alterations in particular microcircuits can have a profound impact on function at the cellular and network level and ultimately lead to epilepsy. In addition, this newly developed mouse model provides a powerful tool for use in future research looking to identify novel avenues for targeted therapeutic intervention in temporal lobe epilepsy.

Bridging the Gap: Engineering Connexin Proteins as Novel Neuromodulatory tools

Coordinated electrical communication (synchrony) between neurons is a key determinant of neural circuit function in both normal physiology and disease states; nevertheless, methodologies capable of selectively regulating distinct circuits without affecting the surrounding context of brain activity remain sparce. To address this limitation, as a postdoctoral research fellow at Duke University, I sought to rationally engineer the docking features of a pair of connexin proteins to act as novel and docking-selective electrical synapses (gap junctions, GJs) capable of selectively synchronizing  neurons.

To engineer connexin docking features, I first developed the FETCH (flow enabled tracking of connexosomes in HEK cells) method, which uses flow cytometry to determine docking interactions between connexin hemichannels by identifying dual-labeled annular gap junctions (AGJs), the products of gap junction docking and turnover. FETCH is the only known method that specifically captures docking compatibility as opposed to functional conductivity and makes strides in throughput, speed and ease over standard connexin evaluation techniques. I demonstrated that this method characterizes homotypic (same-type) and heterotypic (multi-type) pairings of several human connexin isoforms and I used the method to evaluate docking characteristics of Morone Americana connexin34.7 (Cx34.7) and connexin35 (Cx35), the targets for engineering.

Using FETCH, mutagenic library screening and computational modeling, I generated Cx34.7 and Cx35 hemichannels that dock with each other, but not with themselves nor other major connexins expressed in the mammalian brain. These hemichannels were validated in vivo by demonstrating that they facilitate communication between two neurons in Caenorhabditis elegans (worms) and recode a learned behavioral preference. Additionally, in vivo functionality was validated in mice using two experimental paradigms: phase-amplitude coupling in a prelimbic microcircuit and the modulation of a stress adapted behavior via expression across a long-range monosynaptic projection. Thus, I demonstrated the utility of engineered gap junction devices and established a genetically encoded, translational approach, ‘Long-term integration of Circuits using connexins’ (LinCx), for context-precise circuit-editing with unprecedented spatiotemporal specificity.

Elucidating the role of nucleated red blood cells to uncover knowledge of immune regulation during pregnancy

Red blood cells (RBCs) are not typically considered active mediators of immune responses. This dogma stems from the fact that RBC precursors discard organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. In contrast, in non-mammalian vertebrates, mature RBCs retain their organelles and orchestrate immunological processes. Intriguingly, nucleated RBCs (nRBCs) circulate in human fetuses and neonates. Due to high evolutionary pressure for successful reproduction, circulation of nRBCs during pregnancy is likely important. However, the role of nRBCs in utero remains unknown, leaving a critical gap in knowledge. To define the role of human nRBCs during pregnancy, I queried single cell RNA-sequencing data and found that transcriptomics support nRBCs as putative mediators of antimicrobial and immunotolerant responses.

Pregnancy is an astounding immunological feat that requires overriding our intrinsic immune response to mount defense against non-self. Mechanistic details underlying this immune shift are largely undiscovered. In line with my hypothesis that nRBCs contribute to this immune shift and mediate fetal tolerance, I found that conditioned media from nRBCs induces differentiation of regulatory T cells in vitro, suggesting that nRBCs modulate immune activity via a secreted factor. I found that nRBCs express several soluble immunomodulatory factors including cytokine Growth/Differentiation Factor-15 (GDF15). Future studies aim to define the contributions of GDF15 to nRBC-mediated immunosuppression.

I also found that human nRBCs express molecular machinery required for pathogen recognition and response. Unexpectedly, I found that nRBCs constitutively express MHC Class II and co-stimulatory molecules, a hallmark of specialized antigen-presenting cells, as well as pattern recognition receptors capable of detecting pathogens and initiating an antimicrobial response. These data suggest a putative functionality for nRBCs in mediating innate and adaptive immunity in utero. I am currently investigating the antimicrobial immune properties of nRBCs.

Taken together, these findings shed light on an unexpected orchestrator of fetal immune activity. A better understanding of the fetal immune system has potential to help us understand health and disease during pregnancy, as a neonate, and likely every stage of life afterward.

Altered Vδ2+ γδ T cell chromatin accessibility and immune function following reduced in vivo or in vitro malaria exposure.

Almost 500,000 people—predominantly children under age 5—die from Plasmodium falciparum (Pf) malaria each year; however, the short-lived nature of natural anti-malarial immunity remains poorly understood. Natural immunity provides some protection against symptomatic disease in older children and adults, but is unable to eliminate parasite replication—likely due to chronic inflammation that suppresses anti-parasitic responses. Given that historical emphasis on antibody and T cell responses has led to a malaria vaccine with less than 30% efficacy, addressing the contribution of innate cells to Pf immune responses is essential to understanding natural immunity and designing effective vaccines.

Building on increasing evidence supporting an ability for innate cells like monocytes and γδ T cells to adapt their responses to subsequent infections, my work focuses on characterizing mechanisms that contribute to attenuated γδ T cell inflammatory responses following repeated malaria. We hypothesize that repeated Pf exposure leads to epigenetic and transcriptional reprogramming of Vδ2+ γδ T cells and interactions with monocytes that together lead to altered Vδ2+ T cell function. To test this hypothesis, we are utilizing several innovative strategies, including in vitro co-culture systems both in the US and Uganda and cutting-edge epigenetic approaches developed at Stanford. We are leveraging paired samples obtained from ongoing longitudinal cohorts in Tororo, Uganda, in which peripheral blood mononuclear cells have been obtained monthly since 2011 from children with varying malaria exposure.

In order to identify epigenetic and transcriptional mechanisms underlying Vδ2+ T cell dysfunction and to characterize the longevity of this response, we obtained repeated samples from Ugandan children (n=20) at multiple timepoints before and after a district-wide insecticide campaign that dramatically reduced malaria transmission. Paired ATAC-Seq and RNA-Seq experiments utilizing sort-purified Vδ2+ T cells revealed differential chromatin accessibility and gene expression based on prior incidence of clinical malaria. We identified differential chromatin and transcription factor motif accessibility between samples from 2016 (reduced transmission) vs. 2013 (high malaria transmission) at sites associated with immune signaling (e.g., IL-19, CD8, CXCR6, STAT1) and regulation (e.g., BCL2, KLRC1). Analysis of RNA-Seq data is ongoing.

To further characterize altered cell function following ongoing or reduced malaria transmission, including defining the role of cell-cell interactions, we established an in vitro co-culture system. Purified malaria-naïve Vδ2+ cells stimulated with Pf-infected red blood cells (iRBCs) or the phosphoantigen HMBPP produced less TNFα and IFNγ and degranulated less in response to secondary stimulation compared to unstimulated cells. In contrast, stimulation with iRBCs or HMBPP did not impact the ability of the cells to respond to control stimuli, indicating that the reduced response is Pf-specific. Addition of monocytes to malaria-naive Vδ2+ T cells –particularly at higher ratios—reduced responses compared to unstimulated cells or cells stimulated without monocytes. Together, these results support both cell-intrinsic and -extrinsic mechanisms contributing to reduced Vδ2+ T cell responsivity following malaria exposure.

Ultimately, this work could deepen our understanding of mechanisms driving inefficient acquisition of antimalarial immunity—including potential reversibility of functional changes following repeated malaria—and could have applications for novel therapeutic approaches targeting innate immune responses.

Viral infection induced activation of RNA polymerase III drives mRNA isoform switching

For decades, it has been recognized that a single gene can encode many gene products, thereby expanding the coding potential of our genomes. Alternative transcription or alternative splicing can generate many mRNA isoforms from a single gene. Despite the fact that almost every mRNA in our cells undergoes alternative transcription or alternative splicing, the mechanisms that drive the production of one isoform over another (i.e., isoform switching) remain poorly understood. One possible driver of isoform switching are retrotransposons: invasive genetic elements defined by the ability to move throughout the genome through RNA intermediates. Strikingly, 40% of our genome is composed of retrotransposons, yet little is known about their functional roles. A subset of retrotransposons known as short interspersed nuclear elements (SINEs) are robustly transcribed during infection with DNA viruses of the adeno-, polyoma-, parvo-, and herpesviridae families. Recently, we discovered that protein-coding genes proximal to activated SINEs exhibit alternative transcription during infection. Thus, SINE activation may promote isoform switching events to modulate gene expression during infection.

To gain a genome-wide understanding of SINE-dependent isoform switching and determine the functional relevance of these events, I used long-read RNA sequencing to unambiguously capture the isoform diversity of cellular mRNAs during infection with MHV68, a model herpesvirus. I identified hundreds of previously unknown alternative transcription and splicing events, determined their expression levels, and identified isoform switching events dependent on infection-activated SINEs. Intriguingly, several SINE-dependent isoform switching events involve mRNAs that encode immune factors, suggesting a role for SINE activation in modulating cellular anti-viral responses. Through additional work, I also identified a novel viral inducer of SINEs. Overall, this work provides novel insight into the mRNA isoform diversity produced during infection and points to a mechanism that drives isoform switching through the co-option of parasitic elements – perhaps explaining their vast persistence in our genomes.

hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.