California Institute Of Technology

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract This project will focus on chondroitin sulfate glycosaminoglycans (CS GAGs), a class of polysaccharides that plays important roles in development, immunity, viral invasion, cancer, and central nervous system (CNS) injury. CS GAGs undergo spatiotemporally regulated sulfation, giving rise to diverse regiospecific sulfation patterns. However, efforts to identify functions for specific sulfation motifs have been hampered by the structural complexity of CS GAGs and a lack of tools to study them. In this grant, we will combine the power of organic chemistry and neurobiology to overcome these challenges and identify novel functions for specific motifs in the CNS. The broad objectives of this program are to: (1) advance a fundamental understanding of the structure-function relationships of CS GAGs; (2) identify new functions for specific CS sulfation motifs in the brain, building on our recent discoveries in neuroplasticity, memory, remyelination and immunity; and (3) develop new chemical approaches to study and manipulate GAG-mediated processes, with the goal of reducing neuroinflammation and stimulating plasticity and neuronal repair. During the last granting period, we developed new chemical tools to modulate specific GAG sulfation motifs and generated conditional knockout mice lacking the CS-A and CS-E motifs in the brain. Our studies revealed exciting new functions for CS 4-O-sulfation in the regulation of perineuronal nets (PNNs), specialized ECM structures that restrict plasticity, and social memory. We also found that the CS-E motif contributes to neuroinflammation and axon remyelination. In the present grant, we will build on these exciting findings and continue to develop new molecules for manipulating CS sulfation (Aims 1a, 1b, and 3b) and ECM remodeling (Aim 1). Using these approaches, we will study how CS sulfation regulates signaling pathways important for excitatory synaptogenesis (Aim 2a). We will also investigate the impact of CS sulfation on the polarization of immune cells toward pathogenic phenotypes that drive neuroinflammation (Aim 3a) and the maturation of cells critical for axon remyelination (Aim 4a). Finally, we will explore the potential to use our sulfation and ECM remodeling agents to control PNNs, attenuate neuroinflammation, and promote remyelination (Aims 2b, 3b, and 4b). These studies are expected to advance a fundamental understanding of GAGs and expand current paradigms for how CS GAGs are viewed, demonstrating that specific motifs act as sequence-specific ligands and actively regulate processes important for neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis. If successful, these studies could ultimately identify novel therapeutic targets or strategies for stimulating synaptic plasticity and neuronal repair.

NIH Research Projects · FY 2026 · 2024-05

Project Summary - Catalytic nitrogen fixation and C–N bond constructions mediated by iron and copper as models of biocatalysis and tools for organic synthesis This R35 application requests support to expand research funded via NIGMS in the Peters laboratory during the past five years (JCP as PI: R01-070757; JCP as co-investigator (Greg Fu as PI): R01-109194), where we have focused on multi-electron (e.g., 2e–, 4e–, 6e–) catalytic transformations involving nitrogen-containing species. Continued studies of Fe-mediated nitrogen reduction (N2R) catalysts are proposed as functional models of biocatalytic N2R mediated by nitrogenase enzymes. Well-defined catalytically functional Fe model systems can test the viability of specific Fe–NxHy intermediates and pathways en route to ammonia, constraining mechanistic hypotheses, while also providing spectroscopic signatures that guide spectroscopic assignment of enzymatic intermediates. Our proposed studies address outstanding questions in the field, including the role of protoncoupled electron transfer (PCET) steps that may level the turnover-limiting potential of N2R. Exciting new tools to electrochemically and photochemically initiate PCET steps to Fe–NxHy species are described, as are studies to control catalytic selectivity (NH3 vs N2H4 vs H2) via new reagent and catalyst development motivated by mechanistic data. These varied pursuits promise increasingly efficient models of biocatalytic N2R. New research on photoinduced, Cu-catalyzed C–N (and other C–C and C–heteroatom) couplings is also described. As most all medicines and drug candidates contain at least one carbon–nitrogen bond, the continued development of diverse methods for the formation of C–N bonds is needed. Our discovery of photoinduced, Cu-catalyzed C–X couplings (in partnership with the Fu laboratory) has led to a fascinating range of catalytic N- alkylations and related couplings that include (but are not limited to) secondary alkyl bromides and iodides, and activated tertiary chlorides, as electrophiles, in combination with a range of N–nucleophiles (e.g., anilines, amines, amides, carbazoles). These methods include examples of enantioconvergent couplings. We now describe research towards well-defined copper(I) complexes featuring chelating LX-type ligands that engender favorable photophysical properties (i.e., long-lived and strongly reducing excited states) to engage, via electron transfer, unactivated alkyl chlorides, thus furnishing organic radical R× intermediates key in C–N cross-couplings. This research may lead to more universally applicable copper(I) photoreductants in this coupling chemistry. Finally, oxidative catalytic pathways to C–N bond constructions are also proposed, including C–H aminations and aziridinations, directly from amine (RR’NH) precursors. Such transformations most typically require preoxidized nitrogen sources. Based on our recent ammonia oxidation (AO) catalysis studies, (2 NH3 « N2 + 6H+ + 6 e–), we hypothesize oxidative transformations directly from amine substrates to form C–N bonds mediated by polypyridyl iron complexes.

NIH Research Projects · FY 2026 · 2024-04

Implantable neuroelectronic interfaces for recording and modulating brain activity can treat drug-resistant neurological diseases; however, traditional electrode implantation requires invasive open-skull surgery and poses considerable risks, such as intracortical bleeding and infection, and inevitably damages the brain. To address these issues, this proposal aims to create a platform for endovascular delivery of chronically-stable probes for robust recording and modulation of neural activity. Previous works have demonstrated the feasibility of endovascular implantation and recording by developing flexible neural probes that can be delivered into sub-100-micron cortical vessels to acutely record local field potentials and single-unit activity in anesthetized rats. In this proposal, the capabilities of the endovascular probes will be expanded by first achieving controllable implantation into other vessels, followed by systematic studies and characterizations of endovascular recording and stimulation, with a focus on single-unit activity in anesthetized rats, and finally manufacturing stretchable endovascular probes for long-term implantation and recording in awake, behaving rats. This proposal is significant because it will develop a platform technology that can be readily extended to the detection and treatment of other chronic and progressive neurological diseases, and serves as the foundation for the clinical translation of minimally invasive neuroelectronic interfaces to neurology, neurosurgery, and interventional radiology practice..

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Cell-cell interactions mediated by cell-surface proteins (CSPs) are central to human physiology, controlling assembly and maintenance of organs and tissues. The human genome has about 3700 genes encoding CSPs. In collaboration with a group at Stanford, one of the P.I.s conducted in vitro extracellular “interactome” screens that defined binding partners for 550 human CSPs, and he is currently working on a global in vitro screen. Such screens, which use soluble extracellular domain (ECD) fusion proteins, are most useful for single- transmembrane (TM) CSPs. Many CSPs with multiple TM domains, such as G protein-coupled receptors, have ECDs composed of discontinuous loops, making it difficult to express them in a soluble form. There are about 1700 genes encoding such CSPs. Identification of binding partners for multi-span CSPs will require the use of screens in which intact proteins are expressed on cellular or vesicular membranes. We plan to develop technology for a “library-on-library” cell-based screen for CSP interactions that can identify many binding partners in a single experiment. We hope to use this method to define interactions for CSPs expressed in the human nervous system. Our proposed method uses a new technology, developed by the other P.I., for making engineered extracellular vesicles, called eVLPs, that display single CSPs at high density on their surfaces. We plan to make a pool of eVLPs, each of which encapsulates a unique RNA barcode. Each eVLP will display one member of a library of target CSPs. In parallel, we will generate a cell library in which each cell expresses one CSP from the library. The eVLP pool will be mixed with the cell library, and we will select cells that have fused with an eVLP whose displayed CSP binds to the CSP on the cell surface. Fusion after binding is catalyzed by a mutant VSVG protein, and leads to expression of a fluorescent marker. We will then analyze the sorted cells by single-cell RNA sequencing. By recovering the barcode sequence, which identifies the CSP on the donor eVLP, and the sequence of the CSP expressed by the recipient cell, we will identify candidate CSP interactions that can later be validated using other methods. We plan to attain these objectives of this application through the following Specific Aims. Aim 1: Efficiently incorporate barcode RNAs into eVLPs. Aim 2: Use eVLPs to conduct pilot screens to identify known ligand-receptor pairs. We will determine whether interactions between a CSP on an eVLP and a CSP on recipient cells can lead to membrane fusion and allow sorting and sequencing of cells transduced by the eVLP. We will conduct a small pilot screen to determine if the cell-based screen can identify interactions that we previously found through in vitro screens. The expected outcome of the proposed research will be the development of a new method for library-on-library cell-based interactome screening. This will have a significant positive impact, in that it will allow our group and others to identify many new ligand-receptor interactions in the nervous system and elsewhere. Some of these interactions might define new therapeutic targets.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Both currently expressed transcription factors and inherited epigenetic states contribute to cell identity. However, the causal relationships between transcription factor action and changes in strongly acting chromatin states remain a central question in genomics of development. Despite much correlative data, it is poorly understood how to predict at what sites a chromatin state will prohibit transcription factor action, and under what conditions a transcription factor’s action will alter chromatin states, either transiently or long-term. This proposal takes advantage of Runx family transcription factors as illuminating tools to probe the rules underlying this relationship. The early stages of T cell development can be a particularly useful framework to do this. Runx family factors, especially Runx1 and Runx3, are vital for T cell development from the initiation of the T-lineage gene expression program. The proposed work is based on our recent evidence that despite nearly constant sustained levels of Runx activity across several stages of early T-cell development, the deployment of Runx factors to specific targets changes sharply at the specific transition of T cell development when the cells undergo lineage commitment. Either chromatin changes or changing partner factors could be responsible. Our latest results also indicate that Runx protein expression levels play a strong role in the timing of entry into the T-cell program, as higher levels promote access to key genomic sites prematurely, raising the hypothesis that Runx binding biophysics play a role in developmental timing. At some Runx-activated loci the appearance of new Runx binding sites is linked with major transformations in chromatin 3D interaction patterns of chromatin. The early T-cell development system is highly accessible through in vitro differentiation systems, well characterized as matched to in vivo development, and there is a rich body of data about the programmed expression and deployment of other transcription factors that may be potential Runx interaction partners, making this an ideal system to dissect cause-effect relationships between Runx factors and the epigenome. We propose to discover the rules through which Runx factors choose stage-specific target sites, by comparing Runx actions in cells as they go through successive transitions of early T cell development. In the first aim, we will develop a fine-scale, genome-wide temporal atlas for the binding of Runx factors relative to epigenetic states, both under normal conditions and under conditions when Runx levels are experimentally raised or lowered. This will characterize the epigenetic conditions that are most restrictive for Runx action and the conditions under which Runx can alter them. In the second aim, we will define the reciprocal impacts of Runx and transcription factors representing its candidate stage-specific interaction partners, examining how each affects the binding of the other and how different partners may compete for limited Runx. The third aim will focus on experimentally dissecting cis-elements needed for Runx control of Bcl11b and Ets1, where developmental regulation reflects large-scale chromatin as well as local factor binding changes.

NIH Research Projects · FY 2026 · 2024-01

SUMMARY Ultrasound is among the world’s most widely used biomedical imaging technologies due to its low cost and ability to visualize deep tissues with high spatial and temporal resolution. However, ultrasound has had a relatively small role in molecular and cellular imaging due to a lack of contrast agents and reporter genes connected to specific aspects of cellular function such as gene expression and intracellular signaling. To address this limitation, we are developing acoustic biomolecules – proteins that can be imaged with ultrasound. These constructs are based on gas vesicles (GVs) – a unique class of air-filled protein nanostructures from buoyant photosynthetic microbes, which we introduced as imaging agents for ultrasound in 2014 (Nature Nano. 9:311) and as acoustic reporter genes for commensal microbes in 2018 (Nature 554:86). Since our last renewal, we took the next major step of demonstrating that GVs can function as reporter genes in mammalian cells (Science 365:1469, 2019 and Nature Biotech. 2023), learned how to turn GVs into dynamic biosensors of intracellular protease activity (Nature Chem. Biol. 16:988, 2020), developed methods to detect GV-expressing cells down to single-cell sensitivity (Nature Methods 18:945, 2021), demonstrated their utility as injectable contrast agents in a disease context (ACS Nano 14:12210, 2020), and made several other advances in understanding and engineering GVs and accompanying ultrasound methods and applications. Much work remains to be done to develop GVs as targeted nanoscale contrast agents and reporter genes with broad utility in biology and medicine. Our proposed next steps will enable specific applications of GVs in biomedical research and clinically relevant contexts. These next steps include developing GVs as both targeted nanoscale contrast agents and acoustic reporter genes, focusing on biomedically impactful applications in (1) labeling tumors for intraoperative ultrasound imaging and (2) visualizing the migration and proliferation of primary immune cells during immunotherapy. We will support the future clinical translation of these applications by establishing standardized protocols for high quality GV production, characterizing and optimizing their in vivo tolerability and immunogenicity and developing new nonlinear pulse sequences allowing ultrasound to nondestructively image lower doses of injected GVs and smaller numbers of GV-expressing cells. Successful completion of this work will result in unprecedented capabilities for ultrasonic molecular and cellular imaging and lay the foundation for developing clinical nanoscale and cell-based diagnostics and therapeutics.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Multiple sequential waves of T-cell development in the fetal and post-natal thymus give rise to discrete T cell subsets, each with its own functional properties. Compared with all subsequent waves, the first wave of T-cell differentiation proceeds very rapidly and gives rise to unique lineages of innate lymphocytes and innate-like γδT cells. These cell-intrinsic characteristics of first-wave fetal thymocytes suggests that they have their own separate developmental program. T-cell development is coordinated by a dynamic set of transcription factors (TFs) with stage-specific activity, through a complex network of regulatory interactions. Significant progress has been made in recent years in understanding the T-cell differentiation program; however, whether first-wave fetal thymocytes use the same T-lineage differentiation program is unknown. Through direct comparison of the transcriptional program of murine adult and first-wave fetal thymocytes by single-cell RNA-seq, as well as reanalysis of a public scRNA-seq atlas of thymic organogenesis (GSE107910; Kernfeld et al, 2018), I observed that the first wave of fetal T-cell development occurs simultaneously along two separate trajectories. One trajectory follows the well-established T-cell development pathway, while the other is used exclusively by first- wave fetal thymocytes and gives rise to innate lymphocytes and innate-like γδT cells. Cells on this trajectory appear to use a distinct differentiation program consisting of many TFs typically associated with innate lymphocyte development—including PLZF and Lmo4—alongside shared T/innate TFs, such as TCF1, GATA3, and Bcl11b; as well as GATA1 and Meis1, two TFs with no known role in differentiation of either lineage, and the pan-fetal hematopoiesis regulator Lin28b. I hypothesize that this computationally-inferred population consists of immediate precursors of innate lymphoid and innate-like γδT-lineage cells, and that their differentiation is guided by this unique TF network. In Aim 1, I will fate map the progeny of this hypothesized innate lymphocyte/innate-like γδT cell precursor. Within thymocytes, it is the only population that expresses Gata1; therefore I will use Gata1CreERT2 x Rosa26lsl-tdT mice to fate map the progeny of Gata1- expressing cells at embryonic gestational day E13.5. Additionally, I will use clonal barcoding and scRNA-seq at multiple timepoints to construct a full lineage hierarchy of fetal thymocyte differentiation, and I will perform scATAC-seq to determine whether lineage priming is occurring in this population or in an upstream precursor at the level of chromatin accessibility. In Aim 2, I will use CRISPR/Cas9 to delete constituent factors in the innate lymphocyte/innate-like γδT-lineage program in fetal progenitors and assess the impact of each on developmental speed and lineage choice. I will also perform scRNA-seq on TF-deleted cells in order to identify the effect each TF has on the overall program, including how it regulates other members of the program. In this way, I will use perturbations to construct a network of the regulatory interactions dictating the characteristic developmental speed and lineage choice of first-wave thymocytes.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY The hippocampus plays a critical role in the formation of episodic memories. The current predominant hypothesis is that memories are gradually established across distributed cortical networks under the influence of hippocampal activity. Dopamine influences memory processing by signaling the motivational salience of behaviorally relevant stimuli and by modulating the plasticity of cortical and hippocampal circuits. However, our understanding of the impact of hippocampal inputs and dopaminergic modulation on cortical circuits during memory processing remains incomplete. Here we propose to use two-photon imaging to characterize the evolution of hippocampal inputs, dopamine dynamics, and neuronal responses in the auditory cortex throughout the formation of associative auditory memories. In addition, we will measure the plasticity of hippocampal inputs and their modulation by dopamine in vivo. Finally, we propose to use a combination of optogenetic and pharmacological perturbations of the hippocampal inputs and dopamine levels in the auditory cortex to determine their functional role in the learning and consolidation of auditory memories. Characterizing hippocampal-cortical interactions and their neuromodulation is of fundamental importance for understanding the circuit mechanisms underlying memory formation and can provide the foundation for elucidating how these mechanisms are perturbed in memory disorders.

NIH Research Projects · FY 2026 · 2023-12

The neural crest is a versatile cell population that holds great promise for the purposes of regenerative medicine due to its ability to form a multitude of diverse progeny ranging from the peripheral nervous system to the craniofacial skeleton and portions of the heart. The “cardiac neural crest” arises from the dorsal hindbrain and has the unique potential to form ectomesenchymal derivatives of the heart like the outflow tract septum and a subpopulation of ventricular cardiomyocytes Our preliminary data have uncovered a cardiac crest specific gene regulatory circuit that can reprogram other neural crest populations to cardiac crest fates and have revealed a requirement for cardiac crest-derived cells in adult heart regeneration in zebrafish. Here, we propose to elucidate the role of cardiac-specific subcircuit genes and their targets in acquisition of particular cell fates in the embryonic heart. To extend this to adult stages, we will examine gene regulatory changes that accompany loss of regenerative in mammals and examine the possible role of TGFβ and downstream genes in cardiac neural crest-derived cells therein. As the cardiac crest is a critically important embryonic cell population for normal formation and function of the heart, these studies hold the promise of uncovering novel potential target genes involved in cardiovascular birth defects and repair. Aim 1: Effects of “reprogramming” trunk neural crest identity to a cardiac crest fate. We will use single cell RNA-seq and single cell (sc) ATAC-seq to characterize transcriptional and epigenetic changes that occur in reprogrammed embryonic trunk crest cells over time and trace the fates of reprogrammed cells compared to endogenous cardiac neural crest cells. Aim 2: Role of Tgif1 and co-expressed putative downstream genes in outflow tract development. By coupling loss of function analysis with single cell RNA-seq, we will examine gene expression differences after depletion of Tgif1 as well as other co-expressed genes, including Twist1, FoxC2, and FoxP1. We will test their order of expression and whether they are downstream effectors of Tgif1 by testing the regulatory relationships between these genes. Finally, we will examine the long term effects of their loss of function on development of the cardiovascular system to identify key genes involved in cardiac neural crest fate acquisition. Aim 3: Exploring the role of cardiac neural crest-derived cells in mammalian heart regeneration. Newborn mice can regenerate their hearts after damage from post-natal (P) days 1 – 7. Our preliminary RNA- seq data suggest that there are profound gene regulatory changes that occur in cardiac neural crest derived cells between P1 and P7/8, including an upregulation of genes associated with the TGFβ pathway. Using scRNA-seq coupled with scATAC-seq, we will prepare a careful time course of changes in postnatal cardiac neural crest-derived heart cells under control and cryo-damage conditions and test whether genetic ablation of the neural crest blocks regenerative ability and if inhibition of the TGFβ pathway promotes heart regeneration.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Small-intestine microbes play pivotal roles in human health, and can affect (and be affected by) factors such as probiotics, diet, drugs, supplements, and botanicals. Probiotics, including engineered probiotics, act in the small intestine and may support or compete with commensals and pathogens, as well as improve micronutrient absorption. In contrast, an altered SI microbiota has been implicated in impairing the efficacy of supplemental nutrition. Despite these important roles, studies of the human small-intestine microbiome are challenging because this region of the gut is difficult to access and because the sequencing tools used to analyze stool microbiomes are inappropriate for host-dominated mucosal samples. Critical gaps include knowing which microbes colonize the human small-intestine mucosa, where those colonizers originate from, and which microbial genes and/or environmental features enable them to persist there. These gaps must be addressed in humans because mouse models of the small-intestine microbiome are severely hampered by coprophagy. One long- standing hypothesis is that microbes from the oral cavity transit the acidic gauntlet of the stomach and colonize the small intestine. However, this hypothesis remains contentious and unproven because it is difficult to differentiate colonizers from dead microbes and endoscopies are known to introduce oral contaminants. The primary goal of this R21 exploratory study is to document the first definitive observation of oral microbes translocating and colonizing the SI. To accomplish this goal, we will utilize a combination of two innovative technologies we have optimized for studying host-rich microbiomes: (1) quantitative sequencing for measuring the absolute abundances of microbial taxa and differentiating it from contamination and (2) a microbial enrichment method that enables the construction of high-quality microbial metagenome-assembled genomes directly from host-rich samples. Identifying a definitive example of live oral-derived microbes colonizing the SI will open new avenues for more complete characterization of human intestinal microbes and a framework for further study of the relationship between oral and small-intestine microbial communities. For example, prebiotics intended to nourish beneficial small-intestine microbes and probiotics can be rationally designed to not inadvertently promote growth of oral pathobionts. Similarly, probiotic strains can be engineered to better compete with the oral pathogens capable of colonizing the small intestine and also take advantage of administered prebiotics. Ultimately, the findings from this R21 will improve the microbiome field’s understanding of how oral microbes can affect interventions such as specialized diets, drugs, prebiotics, probiotics, dietary supplements, and botanicals.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY. Nitrogen fixation by the metalloenzyme nitrogenase supports nearly 50% of the global population and is the only biological pathway for nitrogen reduction. Nitrogenase consists of two proteins, the obligate reductase Fe-protein and the catalytic MoFe-protein, both of which are rapidly inactivated by oxygen. Biochemical, crystallographic, and spectroscopic studies have yielded seminal insights into the structures and functions of purified nitrogenase proteins, but a unified understanding of the enzymatic mechanism is still unrealized. These proteins are expressed in a small subset of prokaryotes termed diazotrophs, including aerobes and anaerobes, which inhabit a diverse array of environments. Despite this evolutionary demonstration of compatibility between nitrogen fixation and a variety of metabolisms, robust heterologous expression of the nitrogenase proteins has not been achieved. Instead, endogenous expression within the free-living soil bacterium Azotobacter vinelandii remains the most widely used system for the purification and study of nitrogenase. Several peculiar features of this obligate aerobe have been annotated under nitrogen-limited conditions, to wit, the formation of an intracytoplasmic membrane network, but a comprehensive investigation of these features and their relationship to the nitrogenase proteins is lacking. The primary hypothesis of this proposal is that interactions with cellular ultrastructures and as yet unidentified binding partners in vivo significantly regulate and promote nitrogenase activity. My goal is to identify and characterize these states of nitrogenase in diazotrophic organisms by training in and applying emerging cryoelectron tomography (cryoET) methodologies and performing experiments outlined in two Aims. Aim 1: Revealing the architecture of the nitrogenase interactome in A. vinelandii with mass spectrometry and single particle (SP) cryoEM. Aim 2: Determining in situ structures of nitrogenase complexes in conjunction with cellular features using cryogenic correlative light and electron microscopy (cryoCLEM/ET) and sub-tomogram averaging (STA). As a postdoctoral fellow I have gained expertise in anaerobic SP cryoEM to obtain high resolution structures of nitrogenase. These skills will provide a foundation for the proposed research. However, I seek training in the growing field of cryoET for the study of in situ structures. Throughout the outlined aims, I will train in genetic manipulation of non-model organisms, mass spectrometry, and cryoET from my advisory committee, the Caltech CryoEM and proteomic facilities, and national facilities while continuing to expand my mastery of SP cryoEM and metalloenzyme chemistry. In my independent career, I will apply this training to the continued study of nitrogenase and other anaerobic systems relevant to environmental and human health. The proposed aims will yield the first comprehensive investigation of nitrogenase in situ with the increasingly high resolution technique of cryoET, and will provide insight not only into cellular regulation of nitrogen fixation, but also structures of true turnover states within the cell thus fundamentally moving our understanding of this important biogeochemical process forward.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Deep proteomic and metabolomic profiling of biological tissues is an overarching goal of modern biological and biomedical research. It remains a key – and conspicuous – missing component from the full spectrum of -omics that mainly capitalize on next-generation sequencing of DNA and RNA. Today's existing methodologies for proteomic and metabolomic analysis currently lag far behind the capabilities of tools for genomics and transcriptomics, both in terms of depth-of-coverage and throughput. The proposed effort will meet this challenge by advancing revolutionary new methods for label-free single-molecule proteomic and metabolomic profiling and combining them with novel methods for sub-cellular spatial analysis. Protein concentrations within a mammalian cell span ~8 orders of magnitude; in human blood serum this increases to ~11 orders. Yet, within these immensely complex milieus, even the most sparsely expressed proteins are important. Cellular signaling, gene regulation, early responses to exogenous biological stimuli, and disease onset all generally result in the expression of small copy numbers of proteins. It is thus essential both to discover rare cellular proteins and to attain holistic proteomic maps – but these goals remain far beyond present technological capabilities. Further, deciphering the instantaneous state of an organism's proteome – and, especially, observing its post-translational modifications (PTMs) as they dynamically evolve in response to cellular function, stress, and disease – will provide transformational knowledge for many fields. Deep proteome discovery will tackle the cellular proteome's complexity, allowing identification of proteins over its entire dynamic range of concentration – from the most prolifically expressed cellular proteins to those only sparsely expressed with a few copies per cell. This project's success will enable deep spatial profiling of the cellular proteome and metabolome with high throughput and, thereby, discovery of rare cellular proteins and metabolites. It will fundamentally change the resolution of protein analysis down to the level of individual molecules in subcellular compartments. Its achievement will complete the constellation of single-cell -omics, thereby broadly advancing research worldwide in fields that span from fundamental biology to the frontiers of clinical medicine. In the proposed effort, existing and well-validated techniques for spatially-resolved tissue sampling will be pushed downward into the sub-cellular realm. Scaling these methods downward is feasible now solely because of the single-molecule resolution of the proposed approach. This project builds upon a significant body of recent efforts focused upon creating instrumentation for deep profiling of the single-cell proteome. The effort proposed here will further advance these achievements – and will incorporate high-resolution tissue-sampling methods to deliver, with minimal loss, biological analytes to instrumentation enabling single-molecule analysis. Pursued together, these efforts will enable the first realization of spatial proteomics with sub-cellular resolution.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Deep proteomic and metabolomic profiling of biological tissues is an overarching goal of modern biological and biomedical research. It remains a key – and conspicuous – missing component from the full spectrum of -omics that mainly capitalize on next-generation sequencing of DNA and RNA. Today's existing methodologies for proteomic and metabolomic analysis currently lag far behind the capabilities of tools for genomics and transcriptomics, both in terms of depth-of-coverage and throughput. The proposed effort will meet this challenge by advancing revolutionary new methods for label-free single-molecule proteomic and metabolomic profiling and combining them with novel methods for sub-cellular spatial analysis. Protein concentrations within a mammalian cell span ~8 orders of magnitude; in human blood serum this increases to ~11 orders. Yet, within these immensely complex milieus, even the most sparsely expressed proteins are important. Cellular signaling, gene regulation, early responses to exogenous biological stimuli, and disease onset all generally result in the expression of small copy numbers of proteins. It is thus essential both to discover rare cellular proteins and to attain holistic proteomic maps – but these goals remain far beyond present technological capabilities. Further, deciphering the instantaneous state of an organism's proteome – and, especially, observing its post-translational modifications (PTMs) as they dynamically evolve in response to cellular function, stress, and disease – will provide transformational knowledge for many fields. Deep proteome discovery will tackle the cellular proteome's complexity, allowing identification of proteins over its entire dynamic range of concentration – from the most prolifically expressed cellular proteins to those only sparsely expressed with a few copies per cell. This project's success will enable deep spatial profiling of the cellular proteome and metabolome with high throughput and, thereby, discovery of rare cellular proteins and metabolites. It will fundamentally change the resolution of protein analysis down to the level of individual molecules in subcellular compartments. Its achievement will complete the constellation of single-cell -omics, thereby broadly advancing research worldwide in fields that span from fundamental biology to the frontiers of clinical medicine. In the proposed effort, existing and well-validated techniques for spatially-resolved tissue sampling will be pushed downward into the sub-cellular realm. Scaling these methods downward is feasible now solely because of the single-molecule resolution of the proposed approach. This project builds upon a significant body of recent efforts focused upon creating instrumentation for deep profiling of the single-cell proteome. The effort proposed here will further advance these achievements – and will incorporate high-resolution tissue-sampling methods to deliver, with minimal loss, biological analytes to instrumentation enabling single-molecule analysis. Pursued together, these efforts will enable the first realization of spatial proteomics with sub-cellular resolution.

NIH Research Projects · FY 2025 · 2023-09

Probing the neural computations underlying goal-directed decision-making in humans with single-neuron recordings MPIs: Dr. John P. O’Doherty and Dr. Ueli Rutishauser PROJECT SUMMARY Flexible goal-directed decision-making is a core aspect of higher-order adaptive biological intelligence. A number of psychiatric disorders involve impairments in goal-directed decision-making, yet the current lack of even a basic understanding of how goal-directed action selection is implemented at the neuronal level in humans hinders our ability to pinpoint these neuropsychiatric dysfunctions. In particular, it is completely unknown how goals, and the stimuli and actions that need to be selected from in order to pursue them, are represented at the level of single neurons, nor how goals get selected from available possible goals. Here we will characterize the functional contribution of human ventromedial prefrontal (vmPFC), dorsal anterior cingulate (dACC) and pre-supplementary motor area (pre-SMA) in these processes. We will first test the longstanding proposal never tested at the neuronal level in humans that the value of stimuli is especially represented by neurons in vmPFC, while the value of actions are more represented by neurons in dorsal cortical areas such as the pre-SMA. We will then address how goals themselves are represented. In the real world, animals including humans need to select a goal before any action is performed. Thus, there is a hierarchical process of goal selection followed by action selection. We hypothesize that the vmPFC plays a specific role in goal-valuation and selection, while neurons in dorsomedial areas including pre-SMA and dACC will play more of a role in valuing and selecting the actions that implement the chosen goal. Most decision-making studies focus either on action or stimulus selection, but don’t address how goals get selected in the first place. We will use bespoke behavioral tasks to allow us to distinguish between these different goal and action-related computations and analyze single neuron data simultaneously collected from these brain areas through the lens of computational reinforcement-learning models. The significance of this proposal is that we will gain for the first time, a comprehensive understanding of the functions of the human PFC in goal-directed decision-making at cellular resolution while shedding light on the neural mechanisms of goal-representation and selection which hitherto has been virtually unstudied. Consequently, the proposed project is highly significant in terms of the potential impact that will be made toward understanding the distinct role of different human PFC subregions in goal-directed decision-making.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract This proposal focuses on the synergy of experiments and computations in (1) understanding fundamental reactivity in organic and organometallic transformations and (2) enabling reaction design for the discovery of methods for enantioselective spirocycle synthesis and radical hydroamination of tri-substituted alkenes. Two distinct approaches to using aryl and alkyl nitriles for the synthesis of enantioenriched quaternary stereocenters and nitrogen-containing heterocycles, respectively, are proposed. The medicinal properties of molecules bearing quaternary centers have drawn particular attention, as a significant positive correlation exists between the number of stereogenic centers with clinical success. However, the construction of these structural motifs with a high degree of stereocontrol, especially in the synthesis of spiro centers, remains a significant challenge in organic synthesis. The goal of the proposed research is to access sterically congested, spirocyclic quaternary centers in a stereoselective manner by means of nickel-catalyzed acylation reactions of lactones (K99). Reaction optimization and substrate scope studies will first be performed to establish the desired reactivity with aryl nitriles. Upon validating the desired reactivity with aryl nitriles experimentally, computations will be performed to understand the reaction mechanism and help guide substrate scope expansion to alkyl nitriles. Secondly, the chemical properties of aryl nitriles will be applied toward the synthesis of nitrogen-containing heterocycles (R00), which are considered critically important as more than half of the top 20 best-selling small molecule drugs and nearly 60% of all small molecule drugs possess them. Traditional approaches to C–N bond formation rely on transition-metal catalyzed transformations, such as Chan-Lam coupling, Buchwald–Hartwig amination, and Ullmann reaction. Given that most of these metal-catalyzed reactions require the use of high temperatures and pre-functionalized starting materials, the transition to radical-based C–N bond formation, which can use mild conditions and simpler starting materials, is highly desirable. My goal is to establish an independent research program focused on studying boryl radical chemistry for the generation of nitrogen-centered radicals and regiodivergent hydroamination of trisubstituted alkenes (R00). This project will involve 1) initial computations on key mechanistic steps to help identify NHC boranes that allow for regioselective formation of azepine and isoquinoline scaffolds and 2) experimental testing of computational predictions, reaction optimization, and substrate scope studies. Overall, computational analyses of reaction mechanism and the origins of stereo- or regioselectivity in the aforementioned transformations will provide a platform to expand the utility of Ni catalysis for enantioselective synthesis of spirocyclic scaffolds and the application of boryl radical chemistry for C–N bond formation.

NIH Research Projects · FY 2024 · 2023-09

Probing the neural computations underlying goal-directed decision-making in humans with single-neuron recordings MPIs: Dr. John P. O’Doherty and Dr. Ueli Rutishauser PROJECT SUMMARY Flexible goal-directed decision-making is a core aspect of higher-order adaptive biological intelligence. A number of psychiatric disorders involve impairments in goal-directed decision-making, yet the current lack of even a basic understanding of how goal-directed action selection is implemented at the neuronal level in humans hinders our ability to pinpoint these neuropsychiatric dysfunctions. In particular, it is completely unknown how goals, and the stimuli and actions that need to be selected from in order to pursue them, are represented at the level of single neurons, nor how goals get selected from available possible goals. Here we will characterize the functional contribution of human ventromedial prefrontal (vmPFC), dorsal anterior cingulate (dACC) and pre-supplementary motor area (pre-SMA) in these processes. We will first test the longstanding proposal never tested at the neuronal level in humans that the value of stimuli is especially represented by neurons in vmPFC, while the value of actions are more represented by neurons in dorsal cortical areas such as the pre-SMA. We will then address how goals themselves are represented. In the real world, animals including humans need to select a goal before any action is performed. Thus, there is a hierarchical process of goal selection followed by action selection. We hypothesize that the vmPFC plays a specific role in goal-valuation and selection, while neurons in dorsomedial areas including pre-SMA and dACC will play more of a role in valuing and selecting the actions that implement the chosen goal. Most decision-making studies focus either on action or stimulus selection, but don’t address how goals get selected in the first place. We will use bespoke behavioral tasks to allow us to distinguish between these different goal and action-related computations and analyze single neuron data simultaneously collected from these brain areas through the lens of computational reinforcement-learning models. The significance of this proposal is that we will gain for the first time, a comprehensive understanding of the functions of the human PFC in goal-directed decision-making at cellular resolution while shedding light on the neural mechanisms of goal-representation and selection which hitherto has been virtually unstudied. Consequently, the proposed project is highly significant in terms of the potential impact that will be made toward understanding the distinct role of different human PFC subregions in goal-directed decision-making.

NIH Research Projects · FY 2025 · 2023-09

Project Summary mRNA holds great therapeutic potential as an agent to induce expression of proteins in cells. Precise delivery of mRNA to diseased cell types could have numerous applications, from correcting pathogenic mutations by gene editing to inducing killing in diseased cell types to cell state reprogramming. Despite this potential, it remains challenging to deliver mRNA to specific subsets of cells. A general method to safely deliver mRNA cargo to target cell types within a patient’s body would unlock the full therapeutic potential of mRNA. Living cells are ideal delivery vehicles because they can be programmed to use sensing and logic to specifically deliver mRNA to target cell populations. Towards this goal, our lab has recently developed delivery cells capable of exporting mRNA in synthetic export vehicles (synEVs). These particles can transfer mRNA cargo to receiver cells, where it is expressed. In this proposal, we aim to create, optimize, and integrate elements to generate an ideal cell-based mRNA delivery platform. In Aim 1, we will engineer primary human T cells, a clinically relevant cell type, as delivery cells. In Aim 2, we will develop strategies to control targeting specificity by engineering conditional sending and pseudotyping. In Aim 3, we will demonstrate cell-cell delivery and functionality of mRNA in vivo by delivering a circuit that induces cell death to tumor cells, which should bypass the immunosuppression that limits traditional immune cell-based therapies. Together, this work will expand our capabilities in cell-cell mRNA delivery and enable a novel, cell-based tumor killing strategy that circumvents immunosuppression. Further, while we focus on applications related to cancer in this proposal, this work will establish a platform with broad utility across biomedicine, including for in vivo cell reprogramming and genome editing.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract: Enzymes, supported by their macromolecular structure, can catalyze chemical transformations with exquisite control, delivering products with high selectivity. The mechanisms that enzymes can support, however, are limited, making chemical technologies often the preferred method for synthesis, regardless of cost, toxicity, and environmental burden. Herein are two proposals to expand the activity of enzymes beyond their natural function for synthesizing medicinally important functional groups. Specifically, heme enzymes and methionine sulfoxide reductases are tasked for the first time with performing aminations and oxidations, respectively, toward synthesizing chiral trifluoroethylamines, sulfoxides, and sulfoximines, motifs known to bestow function and drug- like properties to therapeutics and clinical candidates. By engineering enzymes to catalyze reactions beyond their natural capabilities, we are boldly pushing the boundaries of biology and chemistry. This innovation has the potential to revolutionize the way we make molecules and introduce new chemical reactions that can be performed in living organisms. Furthermore, these efforts will unlock activities never before seen in biocatalysis, expanding the repertoire of genetically encoded chemical transformations. The success of this proposal will afford high-value molecules and biorenewable catalysts that may lead to the discovery of new medicines and strategies to regulate biology, with the ultimate objective of informing and improving human health.

NIH Research Projects · FY 2024 · 2023-09

PROPOSAL SUMMARY/ABSTRACT Establishing proper cell identities and tissue architecture during early embryogenesis is crucial for a successful pregnancy. Coordination of these processes is integral to patterning the mammalian embryos as they increase complexity from the implantation stages. However, how this tight coordination is regulated remains poorly understood. A high rate of mortality seen in human embryos during the first 2-3 weeks post-fertilization is a major cause of early pregnancy loss, yet the essential cellular, molecular, and mechanical changes remain almost entirely uncharacterized. Rather than merely a structural component that provides physical support, recent studies have shown that the extracellular matrix (ECM) has emerging roles in regulating cell fate specification and morphogenesis. My recent work reveals that the embryonic basement membrane (BM), a specialized ECM, plays an essential role in patterning the early post-implantation mammalian embryo. My preliminary results lay the foundation of my proposal to determine how the BM coordinates the collective cell behaviors and facilitates pattern formation at critical developmental stages of early mammalian embryogenesis. We will apply novel technologies, including 4D quantitative imaging and single-cell spatial genomics, combined with in vivo, in vitro, and in silico approaches to test the hypothesis that the BM facilitates embryo patterning through coordinating cell fate specification and tissue morphodynamics. In my first aim, we will comprehensively map the cell behaviors and the morphodynamics of the developing embryos with 3D quantitative imaging and timelapse imaging approaches. We will define the role of the BM through genetic manipulations in stem cell-derived embryo-like models as well as in the natural embryos. In my second aim, we will map the BM organization and apply single-cell spatial transcriptomics approaches to generate in situ fate maps. We will functionally test how the BM regulates cell identities by quantitatively defining gene expression patterns with BM architecture and validate the findings with loss-of-function analyses. Next, we will build mathematical models that integrate cell identities and cell dynamics with the BM mechanics to uncover mechanisms of pattern formation. In my third aim, we will apply the technologies and tools developed in the previous two aims to explore the roles of the BM in shaping the formation of germ layers during gastrulation. Overall, my proposed work will increase our understanding of how extracellular cues facilitate a successful pregnancy and could inspire novel therapeutic approaches to prevent early pregnancy loss. The training provided by this award will allow me to acquire the necessary skills to develop my independent research program to apply a quantitative systems-level approach to uncover the dynamics, functions, and regulations that shape the embryos at critical stages of early pregnancy.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Numerous aging-associated neurodegenerative diseases are rooted in the misfolding of proteins and the generation of toxic protein aggregates, with Alzheimer's disease (AD) being the most prevalent form of dementia characterized by the aggregation of the amyloid beta peptide (Aβ) and hyperphosphorylated tau. Few effective strategies are currently available for most of these diseases. In this proposal, we will test the hypothesis that novel molecular chaperones can be evolved to recognize and target aggregation-prone proteins and ameliorate their associated toxicity, thus providing a new approach to intervene in protein misfolding diseases. Using Aβ as the model substrate, our specific goal is to provide proof-of-principle for a new platform for the directed evolution of molecular chaperones that can protect Aβ sequences from aggregation. The efficacy of the evolved chaperones in ameliorating Aβ toxicity will be tested in vitro and in vivo. The proposed studies will establish a new, facile, and broadly applicable approach to evolve novel molecular chaperones tailored to specific aggregation-prone target proteins and thus broadly impact diverse diseases rooted in protein misfolding. The evolved TMMCs are expected to achieve a specificity that is normally unattainable by other approaches, such as small molecules or general chaperone upregulation, and can supplement the reduced capacity of the cellular chaperone network during aging to protect neuronal cells from toxic protein aggregates.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Nitrogen-containing functionalities constitute one of the largest classes within health-relevant small molecules. Therefore, the development of new methods to construct carbon–nitrogen bonds remains paramount among opportunities for innovation in chemical synthesis. The Haber–Bosch process is arguably the most important synthetic catalytic process, wherein atmospheric nitrogen (N2) is reduced to ammonia (NH3) by an iron catalyst under high temperature and pressure. In contrast, nature’s N2 fixation catalysts, nitrogenases, operate at ambient conditions using a bimetallic active site. As such, many well-defined coordination complexes have been developed to study key bond-forming steps in N2 reduction, with the goal of designing more efficient catalysts for the synthesis of ammonia and other value-added compounds. While there has been some success in catalytic N2 silylation to give tris(trialkylsilyl)amines, there are no examples of the analogous catalytic process for amine synthesis through carbon–nitrogen bond-formation from N2. Current strategies for the direct conversion of N2 amines require multistep synthetic sequences of metal–N2 complexes with organic electrophiles and subsequent product release under harsh conditions. To circumvent these limitations, the proposed research employs metallocene-based catalysts to promote proton-coupled electron-transfer pathways for radical functionalization of metal–N2 catalysts. Enabled by a renaissance in organic radical generation, easy access to a suite of abundant olefins, and by exploiting complementary reactivity modes of metal–N2 catalysts, a diverse range of primary, secondary, and aryl amine products can be synthesized. The research plan outlines specific approaches that will deliver fundamental insights into reactions of metal–N2 complexes with organic radicals and metal nitrides, commonly proposed intermediates in N2 reduction, with alkenes. These studies will provide the foundation for the realization of a catalytic synthesis of medicinally relevant functional groups from the most abundant source of nitrogen—N2.

NIH Research Projects · FY 2025 · 2023-08

Proposal Summary The enteric nervous system (ENS) is a vital part of the peripheral nervous system and is responsible for the control of critical gut functions like peristalsis and gastrointestinal secretion. Abnormal development of the ENS can lead to life threatening disorders like Hirschsprung’s disease, characterized by the absence of innervating neurons and glia in the gut. Seminal experiments in chick have shown that two distinct neural crest cell populations innervate the gut and give rise to the ENS: the vagal and the sacral neural crest. Although extensive research has been done on the vagal neural crest’s contribution to the ENS, very little is known about the role of the sacral neural crest in the post-umbilical gut. We aim to address this gap in knowledge by using modern-day molecular biology techniques to gain granular understanding of sacral neural crest- derivatives in the post-umbilical gut, characterize their unique gene signatures, and determine the requirement of sacral-specific transcriptional regulators in ENS development. We hypothesize that the sacral neural crest contributes to unique derivatives within the post-umbilical gut, distinct from vagal-derived structures, and that these derived-cells are under the regulation of a novel gene regulatory scheme. Ultimately, this work will address a lack of understanding of the sacral neural crest in ENS development and shed light on the etiology of ENS-derived congenital disorders. Aim 1: Retroviral mediated lineage analysis of the chick sacral neural crest: Previous work in quail-chick chimeras had multiple disadvantages like traumatic surgery and cross-species artifacts. Here we will implement our technique of replication incompetent avian (RIA) retroviruses for comparative cell lineage analysis of vagal and sacral-derived cells in the post-umbilical gut, visualize interactions between the two populations, and perform clonal analysis of sacral neural crest cells. Aim 2: Transcriptional profiling of sacral neural crest-derived cells in the post-umbilical gut: In order to identify the gene signature of sacral-derived structures of the ENS and regulators of their differentiation, we propose single cell RNA sequencing of sacral neural crest derivatives across multiple time points to characterize the transcriptional profile of sacral neural crest entry into the gut and neuronal differentiation. Upon identification of transcriptional regulators, we will perform conditional loss-of-function analysis to examine their role in regulating the sacral neural crest’s neuronal differentiation.

NIH Research Projects · FY 2025 · 2023-08

Cells are constantly exposed to exogenous and endogenous agents that chemically damage the genome. During DNA replication, this chemical DNA damage can introduce mutations, chromosomal rearrangements, and chromosome mis-segregation events that contribute to progression of cancer and ageing. DNA interstrand cross- links (ICLs) are highly toxic DNA lesions that covalently link the two strands of DNA and block unwinding by the replicative CDC45/MCM2-7/GINS (CMG) helicase. These lesions are generated by cancer chemotherapeutics, endogenous metabolites, and microbiome toxins. Deficits in ICL repair cause the bone marrow failure and cancer predisposition syndrome Fanconi anemia (FA). The products of genes implicated in FA participate in a common ICL repair pathway that is activated when CMG collides with an ICL. Replication fork stalling at the ICL initiates nucleolytic incisions that convert the ICL into a DNA double strand break (DSB). The DSB is itself a potential source of genome instability that must be repaired by homologous recombination. In previous work, we used Xenopus egg extracts to demonstrate that certain ICLs are repaired by an alternative pathway that is also activated upon CMG collision with an ICL. In this pathway, the NEIL3 glycosylase cleaves an N-glycosyl bond in the cross-link, resolving the ICL without DSB formation but generating a labile abasic (AP) site. Our work indicated that the NEIL3 pathway is the preferred response for resolving a subset of ICLs, though the FA pathway can process these lesions when NEIL3 is inactivated. We further showed that the AP site produced by NEIL3 forms a DNA-protein cross-link with the HMCES protein, which stabilizes the AP site and regulates mutagenic DNA synthesis past the AP site. These results indicate that multiple functionally distinct pathways can cooperate to promote efficient replication-coupled repair of DNA damage. In this proposal we will use approaches spanning biochemistry, molecular biology, and cell biology to investigate how repair mechanisms are coordinated at the replication fork during repair of physiologically- and clinically-relevant DNA lesions. In Aim 1, we will determine the mechanisms of repair for an ICL formed by a bacteria toxin implicated in cancer progression, providing new insight as to how the chemical structure of an ICL influences repair. In Aim 2, we will explore the repair of ICLs by the NEIL3/HMCES pathway, including examining how this pathway is activated and how it regulates ICL repair outcomes. In Aim 3, we will examine how HMCES regulates AP site metabolism and contributes to genome stability in cells. These experiments will provide a deeper understanding of how different biochemical repair activities are integrated at stalled replication forks. This work has the potential to inform therapeutic interventions that modulate replication-coupled repair to sensitize cancer cells to chemotherapy or halt progression of diseases caused by DNA repair deficiencies.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Mitochondria are essential organelles of endosymbiotic origin which have evolved to play critical roles in eukaryotic physiology. Mitochondrial activity is dependent on proteins embedded in the outer mitochondrial membrane (OMM), which mediate mitochondrial-cytoplasmic communication, and critical aspects of cellular function such as apoptosis and the innate immune response. a-helical proteins are an important subset of OMM proteins. However, how they get inserted into the lipid bilayer of the OMM in mammalian cells has until recently been unclear. Work by myself, in collaboration with Rebecca Voorhees and Jonathan Weissman’s labs, has identified the OMM resident protein MTCH2, a defining member of a novel family of insertases, as both necessary and sufficient for the insertion of a-helical proteins into the OMM. MTCH2 is a diverged member of the solute carrier family 25 (SLC25), and has evolved to exploit the canonical transporter fold for insertion. Bioinformatic analysis reveals that MTCH2 has a homolog in peroxisomes, and orthologs across holozoa, suggesting a common mechanism for a-helical protein insertion across membranes and eukaryotes. Building on this finding, this proposal aims to develop a comprehensive understanding of how a-helical proteins are correctly targeted to the OMM and inserted into the lipid bilayer across eukaryotes. This work will address a fundamental question in cell biology, as the OMM proteome has evolved to support increasingly more complex functions in higher eukaryotes. a-helical proteins are defined by the presence of one or more transmembrane domains (TAs), though their TMs can vary significantly in number and a range of biophysical characteristics such as hydrophobicity. This proposal aims to first develop a deeper understanding of MTCH2 function. First, in vivo and biochemical techniques will be combined to map the route a substrate TM takes through MTCH2 into the lipid bilayer, directly testing whether MTCH2’s conserved hydrophilic groove has a direct role in this process. Second, this work will establish whether structural homologs of MTCH2 have retained their insertase ability across eukaryotes. The biochemical skills and knowledge acquired by defining the molecular basis for MTCH2 will be combined with systematic functional genomics to establish how a-helical proteins get targeted to the OMM, and whether other factors besides MTCH2 are required to support the biogenesis of this diverse class. Cumulatively, this proposal will provide important insights into the mechanisms that have evolved to support eukaryotic life. Further, intimate knowledge of the machinery that governs a-helical OMM protein insertion will be critical for developing new treatments associated with outer mitochondrial membrane protein dysregulation, including neurodegenerative diseases such as Parkinson’s and Alzheimer’s.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Many social behaviors, such as defense and aggression, are innate- requiring no prior experience to be expressed and presumably ‘hardwired’ into neural circuits. Interestingly, however, these ‘hardwired’ behaviors vary in expression among individuals and can be altered by experience. What are the neural circuits and mechanisms that support such flexible expression of innate behaviors? The ventrolateral, ventromedial hypothalamus (VMHvl) controls innate social behaviors including aggression and social defense, but whether VMHvl encodes individual differences in the expression of these behaviors, or how VMHvl may mediate experience-dependent behavioral changes is not understood. An experience that exposes individual differences in innate behavior and induces experience-dependent changes to aggression and defense behavior is chronic social defeat stress (CDS). CDS induces persistent changes to VMHvl activity- but the cell- types, circuits, and mechanisms involved in these transformations are unidentified. This proposal will use CDS to produce a range of social behavior decisions and dissect the cell-types, circuits, and synaptic mechanisms that mediate these decisions using multiple levels of analysis. This includes in-vivo imaging of individual neurons to characterize the activity dynamics of VMHvl throughout the course of CDS, electrophysiological recordings to uncover the cellular and synaptic signatures of CDS in VMHvl circuits, and in-vivo optogenetic manipulation during behavior to perturb VMHvl CDS-plasticity. This proposal constitutes significant technical and conceptual training in circuit-mapping, in-vivo recordings, in-vivo circuit manipulation, cell-type and input- specific synaptic plasticity, and computational and quantitative methods of analysis. Together, completion of this research will elucidate the experience-dependent transformations to VMHvl circuit dynamics that occur throughout social stress, and relate these transformations to individual differences in stress outcome. Examining these relationships will provide insight to the mechanisms underlying stress-plasticity, which provide insight to stress-related mental health disorders.