Stanford University

NIH Research Projects · FY 2026 · 2016-07

PROJECT SUMMARY Maintaining genome integrity is paramount for all cells, yet the essential cellular process of gene transcription introduces myriad opportunities for cells to incur damage to their genome. For example, transcription can lead to the formation of R-loops, three-stranded structures that contain an RNA-DNA hybrid and a region of single- stranded DNA. R-loops occur throughout the genome of mammalian cells and regulate various aspects of gene expression. They are typically dynamic, but some sequences and cellular perturbations can lead to stable R-loops that can stall transcription, block DNA replication, or cause DNA breaks via incompletely understood mechanisms. Moreover, some of these R-loops can be excised from the genome and accumulate in the cyto- plasm, where they can trigger innate immune responses and even cell death. Thus, cells carefully regulate R- loop formation and turnover. The long-term goal of this research program is to understand how cells identify deleterious R-loops (as dis- tinct from regulatory ones), how problematic R-loops are removed from the genome, and how these functions are disrupted in human diseases. Data suggest that stabilized R-loops become susceptible to processing (i.e., excision from the genome, followed by gap repair) when less mutagenic pathways such as unwinding of the RNA-DNA hybrid or degradation of the RNA component on the chromosome are unavailable or too slow. The objective of this application is to identify the pathway(s) that excises problematic R-loops from the genome and to define the sites and sequence characteristics of problematic R-loops. Aim 1 will investigate which DNA repair factors are involved in R-loop processing and determine how the nucleases that process these structures are recruited to the sites where R-loops are excised. Studies are also proposed to determine whether R-loop excision leaves a single-stranded gap in the DNA, to define the sites of nuclease processing and gap formation, and to identify the polymerases responsible for repairing these gaps. Aim 2 will characterize the genomic and sequence features of problematic R-loops and develop a novel, modu- lar experimental system to directly test hypotheses about the determinants of R-loop processing. These stud- ies will take advantage of new cell biological methods that have been developed to study R-loop processing and cutting-edge genomic approaches developed to map the spatial distribution of R-loops and R-loop pro- cessing products throughout the genome. Elucidating the features of problematic R-loops and how the cell copes with them has the potential to illumi- nate our understanding of how cells maintain genome stability and how this may go awry in cancer and other diseases states. This will lay a strong foundation for strategies to manipulate R-loop processing for therapeutic ends.

NIH Research Projects · FY 2025 · 2016-06

ABSTRACT Oculocerebrorenal syndrome of Lowe is a rare X-linked disorder characterized by bilateral congenital cataracts and glaucoma, renal tubular dysfunction, and mental retardation. Mutations in OCRL1, an inositol polyphosphate 5-phosphatase, cause Lowe syndrome. Despite the identification of OCRL1 as the causal gene underlying this rare disease, there is currently no therapy for Lowe syndrome. Children with Lowe syndrome often go blind due to glaucomatous optic neuropathy. We have developed a novel CRISPR-based approach to transcriptionally regulate the inositol enzymes that control the substrate of OCRL. We have developed a nuclease-deficient Cas protein that allows activation or inactivation of endogenous genes. Our preliminary data shows a novel hypercompact CasMINI for gene editing in a single viral package that allows “Plug-and-Play” delivery. We also established a nuclease-deficient CRISPR-CasMINI (dCasMINI) for gene transcriptional activation of protein expression, with functional rescue in vivo. We also established human Lowe syndrome-based human iPS stem cells, mouse and zebrafish models, and viral delivery approaches for in vivo therapies. We hypothesize that CRISPR-Cas based rescue strategies for inositol 5-phosphatases will restore the imbalance due to the loss of OCRL in Lowe syndrome. Our aims are to (1) transcriptionally activate a compensatory 5-phosphatase that degrades PI(4,5)P2, (2) generate exon-skipping transcripts to reduce OCRL loss phenotypes in human iPS models of Lowe syndrome, (3) use CRISPR-Pass strategy with both adenosine base editors and prime editors to correct OCRL gene mutations in Lowe patient derived cells. We anticipate this work will provide critical insights into how CRISPR-Cas can be used to rescue the loss of OCRL and provide important translation approaches for Lowe syndrome and then expanded to treat other forms of inherited genetic eye diseases.

NIH Research Projects · FY 2026 · 2016-06

Project Summary Evolutionary adaptation is the central concept in biology. My lab focuses on the inference of adaptation from genomic data and on the study of rapid evolution on ecological timescales in real time. These approaches include (i) inference of adaptation from well resolved population genomic and phylogenetic data, (ii) experimental evolution and quantification of fitness effects of naturally occurring variants in yeast, (iii) adaptation from standing variation on seasonal and ecological time scales in Drosophila. All projects aim at increasing resolution and mechanistic understanding of the adaptive process.

NIH Research Projects · FY 2025 · 2016-05

Project Summary The cellular environment is both powerful and complex, depending both on structural organization from the micron scale down to the nanometer scale, as well as on the dynamic time-dependence of a huge array of enzymes, the nanomachines of the cell, and their work on proteins, oligonucleotides, and small molecules. Visible fluorescence microscopy has been a useful tool capable of non-invasively exploring cellular behavior, but the diffraction-limited resolution of conventional imaging has severely restricted the information obtainable on structures on a scale below ~200 nm. Because the primary biomolecular players in cells are in the size range on the order of 10 nm, comprehensive measurements are needed on this size scale in living systems. Super- resolution microscopy, either based on single-molecule fluorescence imaging and control of the emitting concentration, or on stimulated emission depletion, has solved this problem by enabling access to nanoscale position information down to the 10-40 nm regime and below. In addition, the complementary method of single- molecule tracking provides access to the details of motions of cellular components such as motor-driven transport or the motion of DNA or RNA. Combined with advanced three-dimensional (3D) imaging, single-particle tracking allows the full motion of specific cellular players to be observed in their actual context at high speed. It is a primary thrust of this work to develop and enhance both 3D super-resolution imaging and 3D single-particle tracking in cells by pushing the boundaries of both approaches and inventing new strategies to overcome technical limitations, which will lead to unprecedented spatial and temporal information in fixed and living cells. Research in the Moerner laboratory broadly seeks to address the limitations of super-resolution imaging and single-particle tracking in cells by physical and mathematical analysis as well as by invention of new methods. The deep motivation here is to ask the fundamental question: how can the information available from each single molecule be maximized? Two key new microscopes are under development: 3D imaging over large axial ranges using pupil plane phase modulations and a tilted light sheet, and a correlative method to use cryogenic single-molecule fluorescence localizations to annotate cryo-electron tomography reconstructions. The methodological developments of this research will be applied to a variety of critical problems in cell biology by continuing established collaborations and by developing new collaborations with well-known biologists. The bacterium, Caulobacter crescentus, remains as a useful model system for cellular development needing elucidation of the superstructures and motions of biomolecules to understand the origins of asymmetric division. The Toxoplasma gondii parasite is another fascinating organism which needs exploration with super- resolution methods. The organization of chromatin on all scales remains to be fully understood. These and other cell biology questions with implications for both normal and diseased function will be explored by the application of the advanced imaging methods of this research program.

NIH Research Projects · FY 2025 · 2016-04

PROJECT SUMMARY Marfan syndrome (MFS), caused by mutations in the fibrillin-1 gene, is the most common inherited connective tissue disorder. Patients typically develop aortic root aneurysms with ensuing aortic dissection and rupture remaining the leading cause of death. Without prophylactic surgery, life-expectancy is reduced to age 40 years. Novel targeted drug therapies have been absent, likely due to limitations in mechanistic understanding. Thus, there is an urgent need to dissect the pathway(s) involved in MFS aortic wall extracellular matrix (ECM) breakdown resulting in aneurysm formation. We hypothesize that distinct embryonic origins of SMCs populating the aortic root contribute to aortic root-specific aneurysm pathophysiology. In the recent funding period, we created and characterized induced-pluripotent stem cells (iPSCs) from MFS patients and differentiated them into SMCs from each embryologic origin. We learned that the microRNA, miR-29b and related downstream genes were dependent on SMC embryologic origin. After applying high-throughput proteomics, we also discovered that iPSC-derived SMCs from MFS patients express distinct, lineage-specific proteomic profiles affecting critical biologic processes including ECM synthesis/degeneration and SMC contraction/motility. Utilizing single-cell RNA sequencing, we identified a distinct, disease-specific SMC cluster that develops transcriptomic alterations regionally in both murine models and human aortic root aneurysms. In complimentary MFS Fbn1C1041G/+ mouse in vivo studies, we discovered several regional upstream TGF-β-dependent triggers that ultimately increase matrix metalloproteinase-mediated ECM remodeling, including: (a) Ras (GTPase) signaling; (b) NADPH activation leading to reactive oxygen species production; and (c) androgen potentiation of TGF-β signaling in male mice. Drug blockade of each discovered pathway reduced aneurysm formation. In this renewal application, we rigorously expand our research progress by proposing several innovative experiments to study the relative contributions of SMC lineages to regional ECM remodeling, investigate the upstream triggers and pathologic role of modulated SMCs during aneurysm formation, and advance the translation of disease-specific iPSC modeling for novel pathway discovery. Specific Aim 1 seeks whether interactions between MFS SHF- and neural crest-SMCs induce synthetic SMC phenotype and ECM remodeling. We investigate the novel role of reduced SHF-SMC mannose receptor 2 (MRC2) on ECM composition, proteolysis and SMC contractile function. We will also elucidate the effect of ECM biomechanical properties on lineage-specific SMC responses, including SMC transcriptomic modulation and single cell mechanical properties. Specific Aim 2 strives to determine the effects of modulated SMC populations on ECM remodeling in vitro using our innovative MFS decellularized matrix scaffolds. Novel lineage-tracing transgenic mouse models will be utilized to identify modulated SMC embryologic origin and role in aneurysm progression in vivo.

NIH Research Projects · FY 2025 · 2016-03

Control of gene expression in space and time plays an important role in enabling cells to “know” where they are in the developing embryo and what to become, a process often referred to as cellular specification. Decades of research have demonstrated numerous layers of regulation in control of gene expression, at both the transcriptional and post-transcriptional level, which coordinate this process. Translational control of gene expression has, on the contrary, received less experimental attention. Most notably, the prevailing dogma has been that at the level of protein production, the ribosome - although an immensely complex molecular machine- possesses a constitutive rather than regulatory function in translating mRNAs. Our findings have established a new field of study by demonstrating that ribosomes are highly regulatory in control of the expression of developmental gene regulatory networks underlying tissue patterning and formation of the mammalian body plan. In our most recent studies, we have identified entire biological pathways in embryonic stem cells represented by the translational preferences of specific ribosomes, that differ in the composition of their ribosomal proteins (RPs) or the interaction of novel ribosome-associated proteins (RAPs) that we have recently identified that directly associate with mammalian ribosomes. We have further shown ribosome heterogeneity in proximity to key cellular organelles as a mechanism to control localized protein production within subcellular space. These findings change our understanding of gene regulation and open a new portal of study into an additional layer of gene expression vital to control of cell specification, tissue patterning, and embryonic development. In this proposal we will undertake a highly multidisciplinary approach to characterize this novel regulatory code for translational control of the circuitry of key developmental networks. In Aim1 we will extend our new roadmap of ribosome heterogeneity indicated by the presence of distinct ribosomes during primary human ES cellular differentiation to an organismal level. In particular, we will leverage novel genetic tools to study ribosome biology in-vivo. Using this approach, we will delineate the mechanisms by which a single RP can control a paramount step in embryonic development, namely sustained paraxial mesoderm formation, and its role in translational control of the WNT signaling pathway, which reflects a novel step in the regulation of a major signaling pathway in development. In Aim2 we will undertake a systems level approach to characterize the role of ribosomes as key regulators of cell fate transitions. We will utilize novel technologies to forcibly and inducibly remove specific RPs selectively from cytoplasmic ribosomes for the first time and assess their individual functions on stem cells differentiation down the mesoderm and endoderm lineages. In Aim3 we will functionally characterize alternative RP paralogs in mammary-glad development for which our compelling preliminary data indicate that they translate distinct subsets of mRNAs during the switch to lactation. We hypothesize that translation control is required to synthesize copious milk proteins critical for neonate sustenance. Defining at a more basic level the specificity and dynamics of ribosome-mediated control gene regulation will be invaluable for our understanding of how deregulations in the ribosome alter accurate control of gene expression underlying human congenital birth defects.

NIH Research Projects · FY 2026 · 2015-12

PROJECT SUMMARY Cardiac fibrosis contributes to the progression of heart failure. Currently, there is no FDA-approved drug specifically targeting cardiac fibrosis due to the lack of understanding of the underlying mechanism and a reliable drug discovery platform. Here, we will use a novel multiplexing methodology of creating a "cell village" by pooling multiple patients' induced pluripotent stem cell (iPSC) lines in a dish to map the genetic basis of interindividual differences in response to a profibrotic cocktail. In Aim 1, we will co-culture 100 iPSC lines from 60 healthy donors and 40 dilated cardiomyopathy (DCM) patients in 10 distinct "cell villages," where each "cell village" contains ten independent patient-specific iPSC lines. Next, we will differentiate each "cell village" into iPSC-derived cardiomyocytes (iPSC-CMs), endothelial cells (iPSC-ECs), and cardiac fibroblasts (iPSC-CFs), and then generate 3D engineered heart tissues (EHTs). Finally, we will perform a single-cell multiomics sequencing analysis of the "cell villages" to understand the impact of internal genetic predisposition and external profibrotic stimuli on EHTs. In Aim 2, we will perform high throughput screening to identify novel small molecules for treating cardiac fibrosis using novel iPSC-CF reporter lines. The antifibrotic effects of the drug hits will be validated in primary CFs and EHTs. We will perform RNA- and ATAC- sequencing to determine the downstream signaling pathways of the newly identified compounds. In Aim 3, we will validate the therapeutic effects of antifibrotic compounds in mice models of dilated cardiomyopathy. The cardiac function will be measured by echocardiography, and fibrosis severity will be assessed by histology at multiple time points. We will also perform single-nuclei multiome and spatial transcriptomics to delineate the impact of therapeutic drug treatment in each cell type cluster and cell-cell interactions in the heart based on the 10x Genomics platform. Collectively, we expect to identify new signaling pathways and novel compounds for patients with cardiac fibrosis and heart failure.

NIH Research Projects · FY 2025 · 2015-09

Project Summary/Abstract This application to renew participation in the renamed Chronic Pancreatitis Clinical Research Consortium (CPCRC) (formerly called the Consortium for the Study of Chronic Pancreatitis, Diabetes and Pancreas Cancer [CPDPC]), will enable the Stanford Clinical Center to continue its productive recruitment and retention of patients into 2 clinical studies of the consortium – 1) Prospective Evaluation of Chronic Pancreatitis for Epidemiologic and Translational Studies (PROCEED), and 2) Pediatric Longitudinal Cohort Study of Chronic Pancreatitis [The International Study Group of Pediatric Pancreatitis: In Search for a Cure (INSPPIRE2). Prospectively collected specimens from these studies will be used to further examine the role of the immune system in recurrent acute pancreatitis and chronic pancreatitis with clinically relevant outcomes. As part of the original Consortium, the Stanford Clinical Center, has demonstrated capacity to effectively recruit and retain patients into 2 primary studies of the CPCRC. We have successfully used prospectively collected samples from the PROCEED cohort to demonstrate key immune signaling pathways to diagnose chronic pancreatitis. In response to the research objectives of the CPCRC as outlined in this RFA, we have formed a strong scientific team with broad complementary expertise in clinical adult and pediatric pancreatitis, endocrinology, radiology, and immunology. In this renewal, we intend to validate these findings and characterize predictive immune signatures of chronic pancreatitis and relevant clinical outcomes with 3 proposed ancillary studies – one of which will use serially collected samples over time for the same patient. We also propose a proof-of-concept immune-based clinical trial to repurpose a commercially available FDA-approved IL-17 inhibitor to prevent progression of recurrent acute pancreatitis to chronic pancreatitis.

NIH Research Projects · FY 2024 · 2015-09

Project Summary/Abstract Emerging and endemic viral pathogens are a persistent threat to human health, the global economy, and national readiness. Viral proteins interact with host proteins to hijack host cells and replicate transmissible viral particles. Human-viral and viral-viral protein-protein interactions (PPls) have been comprehensively characterized for a limited set of viruses, identifying a "PPI profile" for each virus screened. However, these efforts have characterized only a small fraction of the known viruses. A complete viral-human PPI map would be an invaluable resource, enabling analyses of how often interacting viral proteins converge on common human protein targets, cellular functions, or pathways, and which of these interactions are associated with transmissibility or virulence. Combining the viral-human PPI map with human population genetic analyses will enable characterization of the molecular mechanisms underlying extant and ancient epidemics and how these PPIs drive much of human adaptation. In addition, PPI profiles of human viruses could be used to aid screening of animal reservoirs to identify potential threats before a zoonotic spillover occurs. Despite its great promise, characterization of the viral-human PPI map remains a challenge given the throughput of current viral-human PPI screening assays. Current high-throughput assays also lack a quantitative output, meaning that the emergent human-viral PPI map, or viral-viral PPI maps, would be difficult to exploit for important downstream variant scanning or drug screening applications. Here we will use a quantitative sequencing-based protein-protein interaction assay platform to screen for PPls between -24 million viral-human or viral-viral protein pairs. We will further develop this technology into a massively parallel drug screening platform and use it to screen >3 million drug-PPI combinations for small-molecule compounds that promote or antagonize a PPI. The viral-human PPI and drug-PPI maps developed here will be an invaluable resource for a broad research community. In addition, this work will establish new massively parallel and quantitative PPI and drug-PPI screening technologies that will scale with advances in gene synthesis, mutagenesis and sequencing, enabling parallel screening of tens of thousands of gene and gene variants of extant, emerging, and potentially zoonotic viruses.

NIH Research Projects · FY 2024 · 2015-09

PROJECT SUMMARY Heart disease, the most common cause of death, frequently arises from blocking blood flow to cardiac muscle. Blood flow travels to the heart first through coronary arteries and then into a capillary network where oxygen exchange occurs. One approach to treating heart disease has been to expand the capillary network, but this has achieved limited success. Here, we propose to instead expand coronary artery networks and promote the development of collateral arteries, which are a subtype of artery with the potential to form a natural bypass. In the previous funding period, our laboratory discovered cellular and molecular mechanisms driving coronary artery formation in the developing embryo, including how the transcription factor Dach1 supports artery growth through regulating blood flow stimulated cell behaviors (Chang, 2017, Genes and Dev). We also described how the chemokine CXCL12 triggers collateral artery formation in the injured heart during the neonatal growth period (Das, 2019, Cell). We hypothesize that these developmental pathways can be utilized to stimulate adult coronary artery regeneration and provide beneficial outcomes during cardiac injury and disease. Preliminary studies activating Dach1 or CXCl12 in adults shows indications of enhanced recovery following experimental myocardial infarction. We will use the following Aims to further explore their reparative potential. Aim 1 will use tissue clearing, whole organ imaging technology, and computational modeling to define how injury, Dach1 overexpression, and CXCL12 administration alter artery structure and affect blood flow parameters. Aim 2 will use cardiac injury models to intensively study how Dach1- and CXCL12-induced artery growth and collateral development enhance recovery post-myocardial infarction. Aim 3 will delve into the mechanisms by which Dach1 stimulates artery endothelial cell differentiation and morphogenesis. This work is significant because delineating how developmental signals stimulate coronary artery regeneration could ultimately contribute to therapeutic interventions for heart disease. The work is innovative because it takes a new approach to revascularization—targeting artery differentiation rather than just the microvasculature. It also further develops cutting edge experimental techniques such as adult whole organ imaging and a novel in vitro endothelial cell differentiation model, which could ultimately benefit the cardiovascular research community at large. Finally, successful completion of the Aims is ensured by the interdisciplinary environment at Stanford University and collaborative track record between this group of investigators (Drs. Kristy Red-Horse, Kyle Loh, Alison Marsden, and Daniel Bernstein). The proposed work will enhance our knowledge on cardiovascular development and regeneration by illuminating the biology of the hitherto-enigmatic collateral arteries, as well as how transcriptional regulators such as DACH1 determine artery fate.

NIH Research Projects · FY 2026 · 2015-09

PROJECT SUMMARY To understand the neuronal intrinsic control of optic nerve regeneration, we initially conducted an extensive molecular dissection of the PTEN/PI3K/AKT/mTOR complexes signaling network, illuminated their cross- regulating mechanisms, and definitively determined the linear and parallel signals that contribute to optic nerve (ON) regeneration. We then developed a novel Retro-seq platform to label, purify, and single cell sequence regenerating and non-regenerating retinal ganglion cells (RGCs), by which we identified a group of downstream effectors of PTEN deletion that directly promote significant ON regeneration and surprisingly, also produce striking glaucoma neuroprotection. We propose the common target of these pro-regeneration and pro- neuroprotection genes may be the mechanistic nodal point that drives axon regeneration and neuroprotection directly. We will investigate another top gene from the regeneration-associated gene list and its functional related molecules to illustrate the potential new mechanisms of neuronal intrinsic control in adult CNS axon regeneration. Moreover, we will test individual pro-regeneration genes and their combinations in our newly developed acute and chronic mouse SOHU glaucoma models, in hope of identifying the most effective neural repair strategies that promote significant neuroprotection and visual functional recovery. These in-depth focused studies of prominent pro-regeneration and neuroprotection genes and their interactions are likely to illustrate a novel mechanism of axon regeneration, identify the most promising therapeutic targets, and therefore eventually establish translational strategies for safe and effective clinical management of glaucoma and other CNS neurodegeneration patients.

NIH Research Projects · FY 2025 · 2015-08

PROJECT SUMMARY/ABSTRACT The TP53 tumor suppressor gene is mutated in over half of all human cancers, but the mechanisms through which p53 suppresses cancer in vivo remain incompletely understood. Notably, there are no standard-of-care cancer therapies based on the p53 pathway. In this proposal, we strive to deconstruct the pathways through which p53 suppresses cancer to illuminate pathways dysregulated upon p53 loss that could ultimately be targeted therapeutically. Our previous work using transcriptional activation domain mutant mice suggested that the transcriptional programs underlying p53 acute DNA damage responses are not essential for tumor suppression, prompting us to search for new mechanisms of p53-mediated tumor suppression. We thus performed unbiased in vivo shRNA and CRISPR/Cas9 screens for p53 target genes important for tumor suppression. We identified several p53 target genes with tumor suppressor activity, including Zmat3 – the top hit in both screens. We found that Zmat3 expression is highly p53-dependent across contexts and that Zmat3 suppresses lung adenocarcinoma (LUAD) and hepatocellular carcinoma (HCC) in autochthonous mouse models. Zmat3 encodes a zinc finger RNA-binding protein that we found acts by modulating alternative splicing, revealing a new branch of p53-mediated tumor suppression. As recent work has revealed a critical role for alternative splicing in cancer, we hypothesize that studying p53 pathways at the post-transcriptional level, such as through splicing and proteomics analyses, will yield novel insights into p53-mediated tumor suppression. In Theme 1, we propose to identify p53-dependent splicing and proteome changes, including both Zmat3-dependent and Zmat3-independent ones, that could explain tumor suppression in mouse LUAD and HCC. We will also identify genes that cooperate with Zmat3 to suppress cancer downstream of p53. We will test the importance of genes found in these analyses for LUAD and HCC suppression using a quantitative in vivo tumor assay known as Tuba-seq. In Theme 2, we will pursue our observation that p53 repurposes a role in lung regeneration, in which it drives alveolar type 1 cell differentiation upon lung injury, to suppress LUAD. Through single cell (sc)RNA-seq and scATAC-seq analyses, we will ask how p53 status dictates the evolutionary path of KrasG12D-expressing alveolar type 2 cells and how p53 transcriptional programs change with cell state across LUAD evolution in mouse models. We will also ask how cells in the tumor microenvironment (TME) affect cancer cell trajectories in wild-type and p53-deficient tumors. To define genes functionally important for cancer cell state transitions and crosstalk between cancer cells and TME components, we will employ scPerturb-seq. Studies of LUAD evolution, in which we express KrasG12D and analyze subsequent events at both the cancer cell and TME levels through a detailed kinetic analysis, will deconstruct p53-mediated tumor suppression in vivo at an unprecedented molecular depth. Collectively, these studies will provide crucial new insight into how to modulate p53 pathways in therapeutic strategies for cancer.

NIH Research Projects · FY 2025 · 2015-08

ABSTRACT The inaccessibility of human brain tissue at the molecular and cellular level has hindered therapeutic development for psychiatric disorders. Our laboratory has developed neural organoids mimicking specific brain regions from stem cells and pioneered neural assembloids to study interneuron migration and circuit formation. However, in vitro models lack the in vivo interactions and inputs necessary for complex neuropsychiatric disease modeling. We now propose generating assembloids within the in vivo rodent cortex to enhance neuronal maturation and behavioral readouts. Specifically, we will expand transplantation models by integrating glutamatergic and GABAergic neurons to model excitation-inhibition imbalances and early cortical circuit formation. We will establish transplanted forebrain assembloids, characterize their integration and functionality, and apply this model to study severe neurodevelopmental disorders caused by genetic mutations.

NIH Research Projects · FY 2025 · 2015-08

PROJECT SUMMARY Stroke is the leading cause of death with very limited treatment options. This devastating neurological disease is increasingly viewed as a disease of brain connectivity as a damaged stroke area can affect both local and connected brain regions, causing disruptions in neuronal activity and metabolism network-wide. Recovery of lost function can occur after stroke and is attributed to brain remodeling in areas adjacent to or connected to the infarct. In this proposal, we aim to investigate the role of key brain circuits in post-stroke recovery at the functional, cellular and molecular level, using optogenetics, advanced live imaging and high throughput RNA sequencing techniques. Previously our lab has demonstrated that selective optogenetic neuronal stimulation in the ipsilesional motor cortex (iM1) can activate plasticity mechanisms and promote recovery. Recently we have employed the optogenetic functional MRI technique to systematically map brain-wide changes in neural circuits after stroke. We have identified key circuits altered by stroke and demonstrated two key circuits restored by iM1 stimulations. Our map data also revealed two candidate circuits that were not restored by iM1 stimulations, suggesting that greater recovery could be achieved if we can rescue these circuits by directly stimulating them. In this proposal we aim to investigate key neural circuits we identified from our activation maps and elucidate their role in post-stroke recovery. In Aim1 we will use circuit-specific optogenetic tools and functional behavior tests to interrogate the role of key circuits in post-stroke recovery. This aim will address whether these circuits have beneficial or maladaptive role during post-stroke recovery. In Aim2 we will examine cellular resolution of real-time neuronal activity dynamics in key circuits after stroke using a portable live calcium imaging system. This will elucidate the neural activity dynamics (excitatory and inhibitory) of key circuits at the cellular level, allowing us to identify the temporal profile and the key neuronal populations altered by stroke, and how iM1 stimulations affect these characteristics to enhance recovery. In Aim3 we will investigate the transcriptome of key circuit areas using RNAseq, in order to identify key molecular targets and pathways altered by stroke and by iM1 stimulations. Preliminary RNAseq analysis revealed distinct pathways altered by iM1 stimulations. We aim to perform RNAseq in multiple regions including iM1 (stimulation site) and ipsilesional thalamus (iM1- connected region) to elucidate whether similar pathways are involved, and if we can identify a common molecular signature that drive recovery. We will also perform RNAseq in both sexes in order to ascertain any sex-specific differences that may be present in post-stroke recovery. Together these results will 1) advance the understanding of neural circuit dynamics during post-stroke recovery; and 2) identify key neural circuits/cell types/molecular targets and optimal time window for designing brain stimulation strategies and other therapeutic interventions in future clinical studies.

NIH Research Projects · FY 2025 · 2015-04

Project Summary: Learning and executing motor skills are crucial functions of the brain and involve the coordinated activity of the motor cortex and basal ganglia. Notably, the connections between the primary motor cortex (M1) and the dorsolateral striatum (DLS), a major target of M1 output neurons, are crucially involved in motor learning. Loss- of-function studies, such as DLS lesions or silencing spiny projection neurons (SPNs) impairs learned motor behaviors, and blocking SPN plasticity by deleting NMDA receptors on SPNs prevents mice from learning new motor skills. In addition, in movement disorders, such as Parkinson’s disease and L-DOPA-induced dyskinesia, disruption of ensemble activity of neurons in the DLS or M1 may mediate behavioral deficits. Yet, direct evidence of plasticity and dynamics of corticostriatal synapses during motor learning is surprisingly lacking. One reason for this gap is the widespread and convergent innervation of corticostriatal projections which has made it challenging to assess the function and plasticity of this circuit over the course of motor learning. How corticostriatal synaptic plasticity contributes to motor learning and the formation of motor memory in vivo remains unclear. Motor learning leads to adaptation of neuronal activity patterns in M1 as well as in DLS and their activity becomes more closely associated with learned movements. An intriguing interpretation of these adaptations in neuronal activity is that such behavior-related neurons may represent the neural correlate of motor memory, forming a motor memory engram. Here, we hypothesize that motor learning induces synaptic plasticity in the corticostriatal motor engram neurons, which is crucial for the formation and consolidation of motor memory. In this proposal, using approaches combining such genetic tools to label and manipulate motor engram neurons with electrophysiology, ex vivo and in vivo 2-photon imaging, and single-cell RNA- sequencing, we aim to investigate how corticostriatal circuit adapts during motor learning at molecular, cellular, and circuit levels. The major goals are: 1: To investigate cortical and striatal excitatory synaptic plasticity of motor engram neurons. 2: To examine how motor learning affects the structure and function of corticostriatal projections. 3. To determine the molecular mechanism underlying corticostriatal synaptic plasticity induced by motor learning. Success in the proposed experiments will provide an in-depth, mechanistic understanding of synaptic plasticity and integration in the corticostriatal circuits. Given the fundamental role of synaptic plasticity in the learning and execution of motor skills and maladaptive cortical and striatal synaptic plasticity seen in movement disorders, our findings may further contribute to future strategies to more effectively treat these diseases, such as Parkinson’s disease.

NIH Research Projects · FY 2025 · 2015-04

PROJECT SUMMARY Communication technology for people with severe speech and motor impairments (SSMI) continues to improve, with recent advances being made in the neural control of communication devices. In prior NIDCD- supported research, our research team developed high-performance intracortical brain-computer interfaces (BCIs) to enable point-and-click control of computer cursors and virtual hands via brain activity alone. Despite these advancements, typing interfaces remain limited in communication speed (8 words per minute) due to the inherent slowness of selecting keys one by one with a pointing device. Recently, we set a new communication record with a speech BCI that decoded neural activity evoked by attempted speech into text (62 words per minute). Speech BCIs currently show the most promise for achieving the long-standing goal of restoring fluent communication at conversational speeds to people with SSMI. However, speech BCIs still require further improvements in performance to allow for more widespread adoption. The goals of this project are to improve speech BCI performance using new methods and brain signals in 3 ways: (1) reduce the calibration burden on the user by translating self-recalibrating algorithms we have demonstrated in handwriting to the decoding of speech, (2) improve ease-of-use and speed by leveraging signals related to inner speech (as opposed to motor signals related to attempted speech, which can be slow and difficult for people with SSMI), and (3) improve accuracy by leveraging complementary signals from Broca's area that represent abstract linguistic features of the user's intended message, as opposed to the articulator movements that make it up. Upon completion, this project will advance both the capabilities of speech BCIs for communication and our understanding of the function of speech-related areas of the brain.

NIH Research Projects · FY 2025 · 2015-03

While it has taken a few decades, adeno-associated viral vectors (rAAV) have shown promise in clinical trials resulting in a handful of FDA approved drugs. While there is continued progress a number of limitations persist that hamper the field. For example, there is still an unpredictable discordance when new capsids are tested in animals vs humans as well as a large variation in dose response between individuals treated. In recent years, we have established that some of these differences are not due to DNA delivery into the cell and nucleus but differences in how the vector epigenome is created resulting in differential rates of vector-mediated transcription. The studies revealed that not only does the capsid protein influence the epigenetic state of the vector but that small variations in the capsid sequence can alter the epigenetic outcome and enhancing expression by two orders of magnitude with no or marginal changes in vector nuclear DNA copy number. In this proposal, we plan to study the molecular mechanisms involved in the process by identifying specific proteins and non-coding RNAs that associate with the capsid and vector genomes once in the nucleus. We will perform studies to establish the functionality of these host proteins in setting up the vector epigenome. We will use machine learning technologies to select for capsids that have improved properties for more concordant animal-human predictability and establish how the alterations in the selected capsids affects AAV-epigenomic states in transduced cells and in mice. Finally, we will design strategies to enhance the proximity of the epigenome favorable molecules into the capsid once in the nucleus. These studies will not only provide mechanistic knowledge of how AAV-delivered genomes become chromatinized but also establish new approaches to maximize expression in transduced tissues, and provide better parameters for predicting clinical efficacy from preclinical studies.

NIH Research Projects · FY 2025 · 2015-03

Stanford/UNC Biomimetic U19 Research Center PROJECT SUMMARY/ABSTRACT – CENTER OVERVIEW Infectious diseases continue to pervasively afflict global health and socioeconomic stability despite substantial prevention and treatment initiatives. Respiratory and gastrointestinal pathogens rank amongst the most intractable infectious diseases, particularly notable for recurrent waves of zoonotic coronaviruses and the recent COVID-19 pandemic, engendered by SARS-CoV-2. Overall, an urgent need exists for improved in vitro experimental models of human disease to study pathogenesis and to validate therapeutics. The central mission of the Stanford/UNC Biomimetic U19 Research Center is thus to deploy novel 3-dimensional organoid culture models to elucidate the biology and therapy of respiratory and gastrointestinal infectious pathogens. Our application is a renewal of our prior Stanford NAMSED U19 Research Center and is comprised of two Cores and three research Projects, leveraging complementary and synergistic expertise of our investigators at Stanford University and the University of North Carolina. The Center continues to be led by the Multi-PIs, Calvin Kuo and Manuel Amieva, who also co-lead Core A (Administrative Core). Core B (Organoid Core) is led by Calvin Kuo and provides novel capabilities for lung and GI organoid culture, gene editing and multiplexed screening. The three Projects extensively utilize organoid biomimetics for exploration of GI and respiratory pathogens. Project 1, (PI, Manuel Amieva) investigates H. pylori and Salmonella colonization, competition and invasion in the GI tract, while Project 2 (PI, Harry Greenberg) investigates rotavirus host range, neutralization, and M cell interactions in enteric biomimetics. Project 3 (PI, Ralph Baric) is a new addition and extensively uses organoids to model SARS-CoV-2, other closely related epidemic and pre-epidemic emerging coronaviruses and 1918 H1N1 influenza to reveal common and unique host networks associated with severe pulmonary outcomes. The activities of the Stanford/UNC Biomimetic U19 Research Center reside within three overarching Aims. In Aim 1, our Center performs organoid modeling of the epithelium-pathogen interface to investigate pathogenesis, susceptibility and host range restriction. This employs robust reverse genetics and CRISPR screens to systematically manipulate host versus viral/bacterial compartments, within novel apical-basal polarity modulated distal lung/alveolar, nasal sinus, stomach and intestinal organoid systems. Aim 2 defines how SARS- CoV-2, pre-epidemic coronaviruses, rotavirus and Salmonella can perturb reciprocal cross-talk between tissue epithelium and resident immune cells. This exploits a unique 3D air-liquid interface organoid method preserving GI and lung epithelium en bloc with diverse endogenous infiltrating immune cell types without artificial reconstitution. Lastly, Aim 3 performs organoid-based evaluation of therapeutic candidates against SARS-CoV- 2, pre-epidemic coronaviruses and rotavirus in medium- to high-throughput formats including epithelium and/or immune cells. Overall, the explorations of the Stanford/UNC Biomimetic U19 Research Center directly apply advanced organoid systems to the investigation and therapy of recalcitrant infectious pathogens.

NIH Research Projects · FY 2025 · 2015-01

Bone healing involves sequential and overlapping biological processes including inflammation, new bone formation and remodeling. Most bone tissue engineering research to date focused on targeting bone-forming cells such as stem cells. Emerging studies highlight the critical roles of immune cells in fracture healing. Macrophages (M𝜙) is one of the first responders to bone defects and can be polarized into pro-inflammatory M1 and pro-regenerative M2 phenotype. Unsolved acute inflammatory phase and delayed M1 to M2 phenotype switch often lead to long-term and chronic inflammation, resulting in delayed bone healing. To enhance bone healing, there remains a lack of strategy that promotes desirable M𝜙 polarization at appropriate timing. Biomaterial scaffolds have been widely used for bone tissue engineering as carriers for cell and growth factors delivery. Recent studies also demonstrate biomaterials compositions impact immune responses, with natural extracellular derived materials promote more pro-regenerative immune response than synthetic materials. However, several key gaps in knowledge remain. First, previous studies were done using soft-tissue defect models only, and how varying biomaterials compositions impact bone healing remains unknown. Second, macroporosity is critical for bone regeneration in vivo, whereas previous work on assessing immune response to biomaterials is limited to conventional nanoporous hydrogels. Third, previous work on the role of T cells in bone healing is limited to a non-critical size long bone fracture model, and no scaffolds were used. T-cell response to scaffold implant in a critical-sized cranial defect model has never been studied before. Last, previous work only studied individual cell type (i.e. immune cell only or stem cell only), yet how biomaterials composition modulates cell-cell crosstalk and subsequent tissue regeneration remains largely unknown. The goal of our original R01 was to assess the potential of µRB scaffolds for enhancing stem cell-based tissue regeneration by focusing on stem cell differentiation. The goal of this renewal R01 application is to harness µRB scaffolds to enhance bone formation through immunomodulation by tuning biomaterial composition, which has never been investigated before. Specifically, we propose to (1) Assess the effect of varying µRB scaffold composition on M𝜙 polarization and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro; (2) Investigate the effect of the varying µRB composition on MSC/M𝜙 crosstalk, and further impacts on MSC-based bone formation and T cell response using a 3D co-culture model in vitro; and (3) Evaluate the effect of the varying µRB scaffold composition on immune responses and bone regeneration in vivo using a mouse critical-size cranial defect model. By working at the interface of biomaterials, immunology, bone disease and biology, stem cells, animal models, and high dimensional single cell analyses, the proposed work will fill in the critical gap of knowledge on the divergent immune response to macroporous scaffolds with tunable compositions and guide optimal scaffold design to enhance critical-size cranial bone repair through immunomodulation.

NIH Research Projects · FY 2025 · 2014-11

ABSTRACT Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically within granulomas for long periods of time. Persistently infected hosts are often asymptomatic and transmit disease to naïve hosts, thereby serving as a critical reservoir for disease. From the bacterial perspective, persistent infection is essential for microbial survival in nature. However, very little is known about the molecular mechanisms involved in persistent Salmonella infections and transmission between mammalian hosts. Increased knowledge of the molecular mechanisms of Salmonella persistence may lead to the ability to eradicate the Salmonella carrier state pharmacologically. Our long-term goal is to understand how Salmonella persists within tissues of mammalian hosts for preventive and therapeutic purposes. The objective of this proposal, which is our next step in pursuit of this goal, is to identify host pathways involved in granuloma dynamics and to determine how Salmonella manipulates host cells for long-term survival. The premise that will be tested in this application is that Salmonella injects virulence factors into granuloma macrophages that both promote an anti-inflammatory state and block specific proinflammatory responses in order to persist in mammalian hosts. We propose to study the molecular mechanisms of persistent Salmonella infections in granulomas of mammalian hosts. Aim 1 will characterize the cellular organization and molecular regulation of granulomas during persistent Salmonella mouse infection, with a particular focus on visualization and analysis of gene expression of granuloma macrophages in tissue sections by spatial transcriptomics. In Aim 2, we will identify mechanisms of Salmonella-dependent manipulation of granuloma macrophages. Aim 3 will characterize the role of the Type 6 secretion system during persistent Salmonella infection. The proposed research is innovative because we investigate the spatial transcriptomics of granuloma macrophages, a heretofore- unexamined pathogen niche. Insight into host-pathogen interactions during persistent infection of a mammalian host is impactful as novel biomarkers and treatments of asymptomatic carriers are needed for eradication of this disease reservoir.

NIH Research Projects · FY 2024 · 2014-09

PROJECT SUMMARY The human colon houses a complex community of microbes, known as the gut microbiota, which possesses unmapped metabolic capabilities. Bacterial metabolic pathways process components of diet, like amino acids, and produce an array of ill-defined metabolites. Many of the metabolites produced by this microbial ecosystem are absorbed by the human host, modified by host enzymes, and ultimately excreted by the kidneys. When the kidneys fail, these solutes accumulate and comprise a significant portion of the "uremic" solutes found at very high levels in the plasma of patients maintained on dialysis. These compounds can vary widely between individual patients, yet are relatively stable over time within an individual, potentially reflecting inter-individual differences in gut microbiota composition. A few of these molecules have been investigated and linked to poor health outcomes in renal patients. For most of these compounds, however, neither the biochemical pathways responsible for their formation nor their biological effects on the host have been elucidated. This application is focused on the prevalent high concentration uremic solutes derived from tyrosine, 4- ethylphenylsulfate (4-EPS) and p-cresolsulfate (PCS), as well as 4-hydroxyphenylpropionic acid sulfate, a tyrosine metabolite not associated with uremia but important in understanding the tyrosine-utilization niche within the gut ecosystem. The goals of the research are to (i) determine the genes and species within the gut microbiota responsible for production of the microbial metabolites 4-ethylphenol and p-cresol that serve as precursors to 4-EPS and PCS; (ii) elucidate the effects of these molecules on aspects of host biology relevant to uremic illness; and (iii) investigate two distinct strategies for microbiota reprogramming with a goal of lowering uremic solute levels in a host. Aim 1 employs two approaches to predict microbial metabolic pathways, one using a computational/machine learning approach and a second method using comparative genomics combined with bacterial metabolomic phenotyping. Gene predictions will be genetically validated using gene deletion or heterologous expression. In Aim 2, gnotobiotic mice are used as a platform to investigate the conversion of microbial metabolites into circulating solutes, and how solute levels are affected by diet and other members of the microbiota. Isotopically labeled amino acids are used to trace dietary substrates to uremic solute products. Aim 3 leverages gnotobiotic mice colonized by WT versus mutant bacteria, which differ in the presence or absence of 4-EPS or PCS, to examine the effect of the metabolite on host biology. Changes in arterial thrombosis and cognitive function relevant to uremic illness will be assessed. The focus of Aim 4 is to reprogram the microbiota to reduce production of harmful uremic solutes. Single strain targeted reprogramming or complex consortium-based microbiota reconstitution using a diverse array of culturable bacteria will be tested as complementary strategies for lowering uremic solute levels in mice. Dietary modifications or antibiotic-based ablation of the microbiota will be used to augment the reprogramming therapies, respectively.

NIH Research Projects · FY 2026 · 2014-09

In the previous funding period of this R01, that we are seeking to renew, we have made the remarkable observation that the Swedish mutation in the Alzheimer Precursor Protein (APP), which causes familial Alzheimer's disease (AD), induces an increase in synapse formation in human iPS cell-derived neurons. Importantly, we established that this effect is caused by Amyloid beta (Ab) and not any other APP cleavage product altered due to the more efficient b-secretase cleavage. This finding was surprising since elevated Ab is considered one of the key drivers of neurodegeneration in AD. In the first aim we propose to elucidate the mechanism of how Ab induces synapses. We will test whether the synaptogenic effect is induced by Ab peptides more or less prone to aggregation and whether decreasing aggregated Ab affects synapse formation. After establishing the dynamics of Ab-induced synaptogenesis, we will map out the neuronal signaling pathways that lead to synapse formation in direct or indirect response to Ab. In our second aim, we will establish whether the Ab acts directly on neurons or indirectly via astrocytes or microglia. The latter hypothesis may be attractive since astrocytes promote synapse formation physiologically. Using a newly developed all-human iPS cell-derived neuron, astrocyte, and microglia tri-culture system we will further evaluate whether Ab may be presented differently in a human neuro-glial cell context as opposed to human neurons grown on mouse primary glia which is the context we have studied synaptic biology so far. Human neural organoids consisting of defined proportions of neurons, astrocytes, and microglia will further allow to address the question whether three-dimensional tissue and cell-produced extracellular matrix affects the synaptogenic effects of Ab. To investigate how Ab affects the three different cell types, we will perform single cell and nuclear transcriptional profiling of two-dimensional tri- cultures and organoids from APPSwe and control cells. In our third and final aim, we seek to further investigate the role of Tau pathology and ApoE on neuronal function. In the previous funding period we identified an ApoE- induced synapse formation by activation of ApoE receptors and downstream kinases DLK, MKK7, and ERK1 and phosphorylation of the transcription factor CREB. Intriguingly, ApoE4 stimulated this pathway and induced synapses better than ApoE3, and ApoE3 better than ApoE2. In addition, ApoE variants have been involved in many cell biological aspects. Tau aggregation and its prion-like spreading is another hallmark of AD pathology. We will apply recent breakthrough technologies to model Tau spreading in human neuronal cultures. Other than focusing on the mechanisms of Tau spreading, we here propose to assess its effect on synaptic function, consistent with the overall theme of this project. Here we propose to utilize our improved tri-culture and organoid models which allow more complex cell-cell interactions and our successful conditional gene editing approach that allows the derivation of truly isogenic controls in order to assess DLK/MKK7/ERK-dependent and independent effects of ApoE variants and perturbations on neuronal function.

NIH Research Projects · FY 2025 · 2014-07

Project Abstract Alzheimer’s disease (AD) affects over 5.7 million Americans and is expected to rise to nearly 14 million people by 2060, as the number of people living with this disease doubles every 5 years. AD is a progressive and severely debilitating disease that negatively affects cognitive and memory function and is linked to increased disability in everyday functioning and risk of mortality. Neurodegeneration of focal brain areas in AD progressively impacts large-scale brain circuits, leading to significant cognitive and behavioral impairments. However, little is known regarding aberrant context-dependent dynamic causal interactions between distributed brain regions, and their links to cognitive and memory impairments and neuropathology, across AD clinical stages. Leveraging a productive and high-impact line of research in the current project period, we now propose to address critical gaps in our knowledge of functional circuit mechanisms underlying cognitive dysfunction in AD using innovative computational tools. Our first major goal is to continue to address critical unmet needs in human brain research by developing and validating novel computational tools for identifying context-dependent dynamic causal interactions between distributed brain regions. Building on progress in the current project period, we will further develop novel Multivariate Dynamic Systems Identification-Hamiltonian Monte Carlo techniques taking advantage of recent advances in Bayesian modeling and inferencing. Our computational tools will be validated using optogenetic stimulation with whole-brain fMRI, and stability analysis of normative Human Connectome Project data. Our second major goal is to use MDSI-HMC to investigate aberrancies in dynamic causal circuits underlying cognitive and memory impairment in AD. Our system neuroscience approach will target four key brain systems implicated in AD: default mode network, medial temporal lobe, and two frontal control systems anchored in the frontoparietal and salience networks. To achieve our goals, we will leverage clinical, phenotypic, cognitive, experimental, and state-of-the-art fMRI, and beta amyloid (Aβ) and tau PET, data from multiple NIH-funded AD-specific Human Connectome Projects. Our proposed studies will advance foundational knowledge of cognitive and memory-related circuits across AD clinical stages and their links to neuropathology. More generally, our proposed studies will also contribute novel tools for examining dynamical causal circuits underlying human brain function and dysfunction. The proposed studies are highly relevant to the NIH Focus on AD and PAR- 10-070 which call for innovative characterization of functional brain circuits altered in AD. More broadly, the proposed studies are relevant to the mission of the NIH to encourage development and dissemination of innovative advanced computational tools for clinical neuroscience. We will disseminate our algorithms and software tools to the research community as we have done in the current project period.

NIH Research Projects · FY 2026 · 2014-06

PROJECT SUMMARY Humans rapidly comprehend the continuous visual input - perceiving who are the people, what are their actions, and where are they. These percepts emerge from computations across ventral, lateral, dorsal streams, respectively. However, it is unknown how the interplay between brain structure, function, and computation supports these visual behaviors. Leveraging advancements from the prior funding period, we propose a unique multimodal and computational approach to answer this question. The research will focus on ventral and lateral streams, as the ventral is the most understood and the lateral is the least understood. Aim 1 will elucidate the interplay between function, white matter connections, and cytoarchitecture using functional, anatomical, and diffusion MRI, as well as cytoarchitectonic data. We will quantitatively measure the relation between functional and structural data, develop computational models linking these metrics, and evaluate their predictivity in left out data. Aim 2 will determine the nature of basic spatiotemporal computations in the visual system and how they affect visual capacity. Aim 2a, will use fMRI and novel spatiotemporal population receptive field (ST-pRF) modeling to determine how basic spatiotemporal computations vary with stimuli and attention. Aim 2b, will use fMRI and behavioral measurements to measure neural and visual capacity, respectively, to multiple stimuli presented sequentially or simultaneously under different attention conditions. Using ST-pRFs, we will determine what spatiotemporal computations predict neural capacity, and then develop a linking model predicting from brain responses visual capacity. Aim 3 will use exciting new topographic deep neural networks (TDANNs) together with massive fMRI datasets of brain responses to visual stimuli to determine how behavioral goals and structural constraints affect the function and spatial organization of the human visual system. This will not only shed important light into the utility of implementational factors, but also generate a single model linking structure, function, and visual behavior. The research will (i) significantly advance understanding of the interplay between visual function, structure, and computations in multiple visual streams filling in longstanding empirical and theoretical gaps, (ii) generate new computational and theoretical understanding how basic spatiotemporal computations and structural constraints affect visual processing, and (iii) generate innovative open source empirical and computational methodologies. This research has important health ramifications for developing noninvasive diagnostics for clinical conditions associated with malfunction of high-level visual cortex including prosopagnosia, action agnosia, and dyslexia, and will provide a new computational testing bed for developing cortical visual prosthetics.

NIH Research Projects · FY 2026 · 2014-05

Project Abstract This application requests funding to continue the highly successful T32 program called Stanford Training Program in Aging Research (TPAR). The principal mission of TPAR is to provide state-of-the-art, mentored, multidisciplinary research career development for outstanding junior investigators (TPAR Fellows) who will enter careers in aging research and improve the health and wellbeing of older Americans. The success of the TPAR training program in the past ten years is evidenced by its strong track record of attracting exceptional candidates, successfully training them and consistently launching our graduates into prestigious and highly competitive positions in aging research. For this renewal, we have convened thirty-seven Faculty Mentors (representing 3 Schools, 16 Departments and 10 Institutes and centers at Stanford). Our interdisciplinary faculty and leadership team is well equipped to help the TPAR Fellows acquire the skills and experiences needed to transition them into productive, independent research careers that advances longevity and healthy aging. We propose a postdoctoral (PhD, MD/PhD and MD), interdisciplinary fellowship program that has six specialized tracks (1) Stem Cells and Aging; (2) Genetics of Aging and Longevity; (3) Neurobiology of Aging; (4) Genomic Stability and Age-Related Cancer; (5) Immunology and Immunosenescence and (6) Translational Research and team Science. The program will support 6 postdoctoral trainees per year, providing 2 years of full-time support for each trainee. The training includes required and elective coursework, guided research experiences, and the development of an individualized, integrated research project. Fellows will participate in seminars and gain critical professional development skills, including training in grant writing and manuscript preparation. This intentionally structured training pathway, with a personalized curriculum that aligns with each fellow’s individual skills, competencies, and growth areas, combined with immersive, hands-on research mentorship will foster the development of TPAR fellows into independent researchers. An Internal Advisory Board and an External Advisory Board will provide guidance and oversight to maximize programmatic impact. Embedding the TPAR program within Stanford’s renowned culture of innovation and interdisciplinary collaboration creates a uniquely empowering environment—one that is exceptionally well-suited to cultivate the next generation of independent investigators poised to drive transformative advances in longevity and healthy aging for all.