Xia Yang - US grants

protein structure function; neuromuscular junction; developmental neurobiology; synaptogenesis; nerve /myelin protein; cholinergic receptors; receptor expression; messenger RNA; myofibrils; gene expression; striated muscles; synaptic vesicles; interneurons; motor neurons; RNase protection assay; laboratory mouse; southern blotting; immunocytochemistry; genetically modified animals; polymerase chain reaction; in situ hybridization;

The formation of neuromuscular synapses requires a series of inductive interactions between presynaptic motor nerve terminals and postsynaptic muscle fibers, resulting in the formation of highly specialized pre- and post-synaptic membranes. One aspect of postsynaptic differentiation is the up-regulation of muscle acetylcholine receptor (AChR) expression selectively in synaptic nuclei of myofibers. Neuregulin (NRG), a potential signal for regulating such synapse-specific transcription, and its receptors are localized to neuromuscular synapses. NRG is synthesized both by motor neurons and muscle cells and activates AChR gene expression in cultured muscle cells. Because NRG is synthesized both by motor neurons and muscle cells and activates AChR gene expression in cultured muscle cells. Because NRG is also expressed in endocardial tissues and is required for heart development, mutant mice lacking NRG expression die at embryonic day 10, prior to neuromuscular synapse formation, due to heart malformation. Therefore, the role that NRG may play in synapse formation during development in vivo cannot be evaluated in NRG null- mutant mice. This proposal is devoted to study the role NRG may have in neuromuscular synapse formation. The general approach in this study is to produce mice lacking NRG expression selective in skeletal muscle or in motor neurons, by crossing study is to produce mice lacking NRG expression selectively in skeletal muscle or in motor neurons, by crossing NRG/flox/flox mice with NRG+/-; MLCcre or NRG+/-; HB9cre mice. The formation of neuromuscular synapses will be analyzed by immunohistochemical procedures that will allow me to assess both presynaptic and postsynaptic differentiation. This study will provide information on the function of NRG signaling in neuromuscular synapse formation and may provide insight into signaling mechanisms at interneuronal synapses in the brain.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cadmium (Cd) is a highly toxic pollutant of soils, which inhibits plant growth and yield production, and is frequently accumulated by agriculturally important crops with a significant potential to impair animal and human health. However, in China, up to 20 million hectare has been polluted by this metal. Plants hyperaccumulating Cd are of considerable interest for potential phytoremediation of the contaminated soils. Sedum alfredii has been identified as a Cd hyperaccumulator native to China, and the better understanding of its physiological and molecular mechanisms in hyperaccumulating Cd may contribute to its potential use in phytoremediation. Here, the speciation of metal ions was one of the important aspects in metal hypertolerance of hyperaccumulators. Some ligands (sulfur, oxygen/nitrogen coordinated) was suggested to be involved in the detoxification of metals in the hyperaccumulating plants which exhibited as transport, chelation, compartmentation and cellular restoration. This work is a frontier research on cellular localization and speciation of Cd in the hyperaccumulator S. alfredii by using micro-SXRF and XAS. XAS is an element specific method and, therefore, particularly suited for analyzing the in vivo ligand environment of Cd in plants. Much work has been done by our research group on physiological characterization of uptake, translocation and accumulation of Zn and Cd in S. alfredii, and several experimental evidence has been obtained for the explanation of Zn localization and speciation in the S. alfredii by micro-SXRF and XAS experiments in BSRF, China, and KEK-PF, Japan. Still, it is necessary to further understand the speciation of Cd at cellular levels by using micro-SXRF and XAS, which is not detectable in BSRF or KEK. Thus, we apply for this experiment at SSRL station aiming at explaining the speciation of Cd in S. alfredii and its homeostatic mechanisms of Zn and Cd hyperaccumulation in S. alfredii.

? DESCRIPTION (provided by applicant): Contrary to previously expected, high fructose (HF) consumption is emerging as a key contributor to the worldwide epidemic of metabolic syndrome (MetS), posing pressing concerns for the lack of understanding of the mechanisms mediating the effects of HF. Considering the broad actions of fructose in peripheral organs as well as in the brain control of energy homeostasis, nutrigenomic approaches capable of revealing the impact of HF on webs of molecular events and their interactions are essential to capture the whole dimensionality of MetS etiology. We hypothesize that HF induces epigenetic variability to alter the organization of gene networks in tissues underlying the MetS pathology, thereby reprograming metabolism and increasing risks for MetS. The information on gene network organization can be utilized to guide interventions to reverse HF-induced reprogramming, leading to regain of control of metabolic homeostasis. We propose an integrative nutrigenomics study that harnesses the power of high throughput genomic technologies and network modeling approaches, coupled with in vivo experimental studies in mice and genetic studies in humans, to reveal the impact of HF on genomic signatures of MetS etiology. This application represents a unique opportunity to synergize the expertise of two complementary multidisciplinary teams - one specialized in genomics and the other specialized in nutritional research - with the merit to combine nutrigenomic approaches and integrative physiology to advance our understanding of the impact of nutrients on the etiology of MetS. In Aim 1, we will use next-generation sequencing technologies to determine the capacity of HF consumption to promote large- scale changes in the DNA methylome as well as in the transcriptome in selected MetS-related tissues in a mouse model, and identify molecular signatures of MetS pathology. In Aim 2, we will assess how high fructose affects the organization of genes in networks, and will identify key regulatory genes that may be responsible for the shifts in the network dynamic. The novel regulators and gene networks identified will be tested for causal association with MetS by i) targeting novel regulatory genes in genetically modified animal models, and ii) assessing novel regulators/networks for genetic association with metabolic diseases in human genome-wide association studies (GWAS). Lastly, in Aim 3, we will address the therapeutic utility of the gene networks by corroborating the capacity of DHA omega-3 fatty acid to normalize epigenetic variability and gene networks disrupted by HF. Our preliminary data indeed support that fructose induces epigenomic and network-level perturbations, which are reversed using DHA. Completion of the study will provide the much needed integrative and systems-level understanding of the basic molecular processes underlying HF-induced MetS pathology. The mechanistic insights obtained will help guide the selection of novel therapeutic targets and the development of network-based nutritional strategies for alleviating the growing health burden of MetS.

Abstract Given the multifactorial pathophysiological cascades involved in traumatic brain injury (TBI), it is difficult if not impossible to have a thorough view of the molecular mechanisms underlying the complexity of this pathology based on limited information derived from conventional approaches that examine isolated molecular events. This complexity becomes a larger limiting factor for the design of strategies to diagnose and predict the outcome of mild TBI (mTBI) which has a less defined symptomatology. Genomic profiling is emerging as a powerful tool to retrieve fundamental information about gene regulatory mechanisms that govern the mTBI pathology and its diversion towards other neurological disorders. However, genomic studies of mTBI have been primarily based on the use of whole brain regions comprised of heterogeneous cell types that mask information from the most vulnerable cell types which are crucial drivers of the pathogenesis. The unique aspect of our approach is to determine the genomic signatures of mTBI at single-cell resolution as cells are the basic units of biological structure and function. We will use state-of-the-art Drop-seq technology that empowers us to capture the transcriptome of thousands of individual brain cells in parallel to accurately define cell types based on aggregate genomic features and, more importantly, to identify cell-type specific gene markers of mTBI, which are generally hidden to the eyes of conventional approaches. We propose experiments to classify all the cell types in the hippocampus (one of the main action sites of TBI) in an unbiased manner and to assess the vulnerability of each cell type to mTBI using single-cell transcriptome profiles (Aim 1). In addition, we will define cell-type specific signatures of genes that could characterize main events involved in our rodent model of mTBI with varying severity at different time points post-TBI (Aim 2). We expect to retrieve novel cell types defined by the effects of mTBI, and their associated deterministic gene markers in the hippocampus. In doing so, we expect to unravel the fundamental, cell specific gene signatures that can be used as biomarkers of mTBI in a data-driven, unbiased manner. Our findings will derive clues to the underlying molecular events and pathways driving the cellular and functional remodeling. This proposal offers the unique opportunity to synergize efforts by combining the expertise of Dr. Fernando Gomez-Pinilla in TBI and the expertise of Dr. Xia Yang in genomics, bioinformatics, and systems biology of complex disorders. The overall goal of the proposal is to elaborate on an innovative research strategy that can provide a comprehensive understanding of the mTBI pathology at single cell resolution, which can be used to empower patient diagnosis, and to design a new line of strategies to redirect the courses of TBI and overcome subsequent neurological disorders.

PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder which comprises of a spectrum of hepatic abnormalities ranging from simple steatosis to steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Despite significant research efforts, the etiology of this disease is poorly understood; in particular, factors associated with progression from steatosis to NASH are unknown. We have developed mouse models from the Hybrid Mouse Diversity Panel (HMDP) that exhibit the spectrum of NAFLD observed in humans. The overall goal of our proposal is to use population-based approaches in mice to identify pathways and higher order biological networks that contribute to the development and progression of NAFLD. Using Mergeomics, an association-based modeling method we developed, we previously identified and validated several genes associated with steatosis from a cohort of HMDP mice fed a high fat, high sucrose diet. Applying the same strategy to a novel transgenic HMDP mice model of NASH, we have now identified several high confidence NASH candidate genes. In Aim 1, we will perform transcriptomic and metabolomics profiling on resistant and susceptible strains to examine the progression of NASH. We will identify and validate candidate genes for NASH progression using multi-omics approaches and Adeno-Associated Virus (AAV) vectors for rapid screening in mice. We will also identify cell-specific changes in gene expression and cell composition related to liver fibrosis and other NAFLD features. This will allow us to follow functional changes in the major hepatic cell types as well as populations of stellate cells and infiltrated inflammatory cells during NASH progression. In Aim 2, we will examine five prioritized genes contributing to hepatic fibrosis, including one gene, Mgp, that we recently validated using knockout mice. Mechanistic studies will be performed to investigate how these genes affect fibrosis. Additional candidate genes identified in Aim 1 will be examined with a similar strategy. Results from these studies will reveal the underlying genetic mechanisms contributing to NAFLD and may identify potential therapeutic targets.

Abstract Concussive injury or mild/moderate TBI (mTBI) accounts for a large majority of the brain injuries in USA and compromises neuronal function and cognitive abilities that can last for years. Neurons that survive the initial insult show a decline in function, and one of the most intriguing aspects of mTBI is that many patients become vulnerable to secondary injury or neurological disorders, which underlying instructions are hiding in alterations of gene programs. The lack of information how TBI alters gene regulatory programs that govern pathogenesis has precluded major advances in strategies to guide TBI therapeutics. Traditional medicine relies on manifestations of symptoms and phenotypes rather than causative factors of the pathology. Instead, alterations in the program of genes are likely causative factors of the pathology and can reveal therapeutic targets that can support precision medicine initiatives. We have recently implemented the use of single-cell genomic analysis to elucidate the impact of TBI on cell types, genes, pathways, and cell-cell interactions that can help inform on novel targets for therapy. Our results from single-cell genomic analysis point to cell metabolism as a driver of mTBI pathogenesis at the cell level and has helped us to prioritize thyroid hormone (important metabolic modulator) as a potential therapeutic agent. The underlying hypothesis is that treatment with thyroid hormone T4 can activate gene regulatory mechanisms that control functionality of circuits in brain regions important for processing of higher order information. Leveraging the expertise of Dr. Xia Yang in genomics and systems biology, and Dr. Fernando Gomez-Pinilla in TBI, we will utilize state-of-the-art parallel single cell sequencing (drop-seq) to assess changes in gene expression in cells forming circuits in brain regions related to cognitive processing. A unique aspect of our project is the implementation of highly sophisticated genomic procedures to understand unsolved questions in the field of neural repair and plasticity and to monitor the efficacy of treatments, using basic concepts of precision medicine. Astrocytes supply energy used by neurons, and they play a crucial role in the incorporation of thyroid hormone from blood into neuronal cells, and according to our preliminary data, astrocytes are highly vulnerable to TBI. We will modulate astrocyte activities to probe the role of astrocytes on circuit reorganization after TBI and on the effects of thyroid hormone. Our studies have the promise to open new avenues to mitigate mTBI pathology based on cell-specific functional aspects of gene regulation, which is also a main premise for precision medicine initiatives.

Abstract Traumatic brain injury (TBI) has a complex neuropathology involving progressive alterations in brain centers that process cognitive and emotional behaviors and consist of heterogeneous cell populations. The complex spatiotemporal cell and molecular circuits underlying progressive TBI pathologies that can evolve into other disorders such as chronic traumatic encephalopathy and posttraumatic stress disorder remain to be understood. A comprehensive understanding of the molecular mechanisms underlying the complexity of TBI has been hindered by the lack of effective approaches to examine molecular events in individual brain cells that drive the overall pathology. We recently conducted a single cell resolution study of the hippocampus at the acute phase (24hr) of TBI using single cell RNA sequencing (scRNAseq) and revealed cell-type specific pathways and regulators of TBI. In particular, we found that depression of cell metabolism to be a key pathogenic component in the hippocampus at the acute phase of TBI. This finding suggests that tracking metabolic state of cells can be used to address key knowledge gaps on the spatial and time dependent progression of key pathologic drivers of TBI. Here we propose to test the hypothesis that cell metabolic regulators determine dynamic and spatial pathogenic pathways of TBI by harnessing the power of modern high-throughput technologies. We propose a highly integrative team approach to profit from recent advances in single cell RNA sequencing (scRNAseq) and multiplexed error robust fluorescent in situ hybridization (MERFISH) along with advanced gene-gene and cell-cell network modeling to inform on targets for intervention at specific time points or brain sites, a fundamental unsolved question in the TBI field. In Aim 1, we propose to utilize a unique combination of scRNAseq, MERFISH, and network modeling approaches to assess and validate the spatial and temporal vulnerability of each cell type to TBI in multiple brain regions at multiple time points in a data-driven, unbiased manner, which can inform us about hidden regulators of TBI pathogenesis. We will focus on the spatial and temporal changes in cellular metabolic pathways during TBI progression. Our preliminary results support that mt-Rnr2, encoding a mitochondrial peptide humanin and involved in cell metabolism, is a major site- and time-dependent driver of TBI. In Aim 2, we will functionally assess whether modulating mt-Rnr2 (humanin) has therapeutic potential to mitigate TBI pathology and prevent progression. We will also explore the cell-type specific mechanisms, especially the role of metabolism, underlying the actions of humanin. The overall goal of the proposal is to elaborate on an innovative strategy that can offer a comprehensive mechanistic understanding of the spatiotemporal cell substrates of TBI pathology and uncover novel targets and mechanisms to redirect the courses of TBI to overcome subsequent neurological disorders.