Troy Ghashghaei

It has been found that Shh signaling promotes cortical radial glial cells to switch lineages to generate olfactory bulb interneurons and the two cortical glial cell types at the end of cortical neurogenesis. It is very important to determine when and how these lineages segregate from each other during normal development, especially whether a single cortical progenitor cell can generate all three lineages. Dr. Ghashghaei will collaborate with Dr. Chen to perform lineage analyses of the Fezf2+ cortical radial glial cells and the Ascl1+ cortical intermediate progenitors using the MADM strategy. Dr. Ghashghaei will provide all the guidance and help for Dr. Chen and her students to finish the experiments and data analysis.

The brain critically relies on balanced production of neurons and glia during embryonic and early postnatal development. Recently developed clonal lineage analysis has revealed the behavior of neural stem cells (NSCs) giving rise to neurons in the cerebral cortex with unprecedented single-cell resolution. However, the clonal principles underlying the formation of glia by NSCs remains unclear and has yet to be systematically investigated using these new technologies. Gliogenesis is critical for proper neuronal functions and when disrupted, it can result in various neurological diseases. Reconstructing how glia are generated from individual NSCs and organized in the cortex during development is essential to understand the structure-function relationships and how they can be modulated by clone-specific factors. We have established a genetically-based single-cell lineage tracing technique utilizing MADM (Mosaic Analysis with Double Markers) mice to label NSCs in the developing cortex and begin to address this knowledge gap. Using this method we have found two distinct populations of glia that occupy different territories of the cortex and its related structure the hippocampal formation. The goal of the proposed research is to reconstruct, quantify, and mathematically model the behavior of individually labeled NSCs in vivo in neocortical and paleocortical areas. This effort requires the development of optimized imaging and analytical tools to ensure reliable and repeatable interpretation of quantitative data. To this end we are developing light sheet microscopy and AI-based automated quantification methods to facilitate unbiased and precise imaging and quantification of clonal data in the brain. Successful completion of our study will result in a comprehensive map of single NSCs and their glial progeny in various cortical regions. Our approach will also establish a platform for detailed quantitative and computational analysis of gliogenesis, glial diversity, and their potential for repair and regenerative approaches in the cortex in the context of various neurological disorders and brain injury.

The cerebral cortex critically relies on balanced production of neurons and glia during embryonic and early postnatal development. Recently developed clonal lineage analysis has revealed the behavior of neural stem cells (NSCs) giving rise to neurons in the cerebral cortex with unprecedented single-cell resolution. However, the formation of glia by NSCs remains unclear and has yet to be systematically investigated using these new technologies. Gliogenesis is critical for proper neuronal functions and when disrupted, it can result in various neurological diseases. Reconstructing how glia are generated from individual NSCs and organized in the cortex during development is essential to understand the structure-function relationships and how they can be modulated by clone-specific factors. We have established a genetically-based single-cell lineage tracing technique utilizing MADM (Mosaic Analysis with Double Markers) mice to label NSCs in the developing cortex and begin to address this knowledge gap. The goal of the proposed research is to reconstruct, quantify, and mathematically model the behavior of individually labeled NSCs in vivo. We will use the power of this labeling method to also screen for gene expression of glial clones at single cell resolution, which all together will help us decipher the general principles organizing glial clones in the cortex, and define how clonal siblings interact with each other. Using some of the identified genes that we have already identified, we will test their role in generation of glial clones in the cortex, which will further help define the biological system underlying clonal rules and principles of gliogenesis. Successful completion of our study will result in a comprehensive map of single NSCs and their glial progeny in various cortical regions. Our approach will also establish a platform for detailed quantitative and computational analysis of gliogenesis, glial diversity, and their potential for regenerative approaches in the cortex. Potential for Broader Impact Our approaches to understand how important constituents of the brain, the glial cells, develop have wide implications. Disruption of glial development is the root of a range of pathological conditions in the brain. Therefore, understanding the basic principles and cellular mechanisms that control gliogenesis is critical to appreciate not only how healthy development may be controlled by systematic production of glial cells, but also how abnormalities in gliogenesis may lead to devastating neurodevelopmental disorders.

The high probability of breakdown in the functioning of the central nervous system (CNS) during late stages of aging, as in Alzheimer������������������s disease and various dementias is a major concern for the elderly. Triggers that initiate age-associated diseases and neurological conditions are for the most part unknown. A key to these associations could be the population of ependymal cells in the brain. Ependymal cells form a monolayer that functions as a barrier between the cerebrospinal fluid (CSF) and the overlying cellular compartments of the brain. As such, they regulate CSF production, circulation, and filtering, and thus ependymal cells are a key component of the newly described ���������������glymphtic-lymphatic������������������ system. This system is purported to control CSF-vascular interactions in the brain parenchyma and thus contribute to the overall clearance of the brain of toxicants and metabolic byproducts and allow entry of immune cells into the brain. The ependymal layer appears damaged in the aged brain, yet whether the damage is caused by malfunctioning signals in the overlying CNS tissue, or if ependymal damage causes defects in neurons and glia in the CNS remain unknown. We have developed several genetic mouse models which suggest the ependymal layer may be the root of many problems in the brain interstitium related to various neurodegenerative diseases and during normal aging in the CNS. We will use these models to study this novel concept. Studies in our mouse models have revealed a previously unknown expression and clearance of mucins by ependymal cells in the CNS. Since mucins function to protect against inflammation and infectious diseases in other tissues, our results have led to the central hypothesis that mucin secretion by ependymal cells is required for maintenance and functional integrity of homeostasis in the forebrain during aging, and that disruption of mucin secretion can lead to aberrant function and disease in the CNS. Our project uses a variety of genetic mice, together with cellular, molecular, and biochemical approaches to test our hypothesis. Potential for Broader Impact: Our approaches to understand how aging affects the brain through its monolayer of ependymal cells have wide implications. Disruption of filtration and protective functions of ependymal cells may be the root of a range of pathological conditions that emerge during late stages of aging. Therefore, undertaking the basic cellular mechanisms that control aging of the brain is critical to understanding not only how healthy aging may be controlled by ependymal cells, but also how abnormalities in ependymal aging may lead to devastating diseases such as Alzheimer������������������s. Moreover, the mechanisms we study can be harnessed to develop novel aging therapeutics by targeting ependymal functions selectively.

The objective of this proposal is to obtain funding from the North Carolina Biotechnology Center to purchase a unique, automated, and high throughput slide scanning imager manufactured by Olympus. The new equipment allows for rapid acquisition of highly detailed images of cells and tissues. In biological research, often the generation of large amounts of samples from experiments is desired to tackle important research questions. Yet, sample generation often is not the limiting factor. Rather the imaging and data analysis of large numbers of samples is usually prohibitive and limits the types and impact of research questions that can be asked. As described in this proposal, the Olympus imaging system would unlock a wide range of important biomedical questions in diverse systems from zebrafish to human, bone marrow to brain, by providing an over 10-fold improvement in sample imaging throughput and analysis. The instrument will be installed within an NCSU Shared Core Research Facility, the Cellular and Molecular Imaging Facility (CMIF), which lacks the proposed technology currently, and is staffed by professional staff that will manage, maintain, and operate the proposed equipment. Acquisition of the Olympus slide scanner and installation within CMIF will increase the rate of data acquisition by research groups at NCSU and, in so doing, ensure their continued competitiveness for research funding.

Metabolism is tightly regulated and disruptions in metabolic homeostasis could lead to metabolic diseases such as type 2 diabetes. Inflammation is the prominent disrupter of metabolic homeostasis, which is associated with hyperglycemia, hypercholesterolemia, hypertriglyceridemia and obesity. Thus, understanding of molecular pathways of inflammation-induced metabolic disorders is critical to combat metabolic diseases. Inflammatory signaling molecules such as c-Jun N-terminal kinase (JNK) and NF-��������B have been identified as mediators of metabolic disruption. JNK and NF-kB modulate and inhibit insulin receptor substrate 1 (IRS1), which is clearly responsible for insulin resistance in several tissues including liver and adipose tissues. However, inflammation is associated with many other metabolic disorders including impairment of hypothalamic neurons, which regulate appetite and glucose homeostasis. Many of the mechanisms by which inflammation regulates metabolism still remain to be identified. We found that neuron-specific deletion of a protein kinase TAK1 blocks inflammation-associated obesity. TAK1 belongs to the mitogen-activated protein kinase kinase kinase (MAP3K) family., and a intermediate of inflammatory signaling pathway. TAK1 can activate JNK or NF-��������B; however, we found that activity of JNK and NF-��������B is unchanged in Tak1 deletion in neurons. Thus, TAK1 modulate neurons and systemic metabolism through a mechanism independently of JNK or NF-��������B. In the effort to determine new downstream pathways of TAK1, we have identified lipogenesis is increased by Tak1 deletion. TAK1 modulates lipid metabolism, which may alter overall metabolic state in the cells. We hypothesize that inflammation-induced TAK1 activation changes cellular metabolism through modulating lipogenesis, which is one of the pathway of inflammation-induced metabolic disorder. In this project: 1) we will delineate the TAK1-dependent mechanism for metabolic regulation at the cellular level: 2) we will determine how the TAK1-mediated cellular level metabolic change modulates systemic metabolic homeostasis such as weight control. Outcomes of this project will reveal a new mechanistic link between inflammation and metabolism, and could provide therapeutic targets in inflammation-induced metabolic diseases.

Neurons and glia, the operating units of the mature brain, are derived from neural stem cells (NSCs) largely during embryonic development. NSCs that give rise to neurons and glia in the cerebral cortex are particularly important to mammals as they ultimately generate the tissue that allows us to perform high-order cognitive tasks. Many neurodevelopmental disorders are caused by abnormalities in molecular and cellular machinery involved in various NSC functions. For example severe disruptions in generation and migration of new neurons can cause microcephaly and anencephaly, whereas milder developmental defects may result in imperfections in connectivity of neurons such as those becoming apparent in Autism spectrum and schizophrenia. The developmental timing of molecular and cellular signals that regulate cortical development are particularly important as temporally distinct insults may impact the cortex, activity in the brain, and behavior differentially. A number of defects associated with mechanisms that impact cytokinesis in NSCs underlie distinct diseases. Therefore understanding how stem cells divide, and what governs changes in their division during the course of brain development and NSC maturation is critical to understanding neurodevelopmental disorders. In the course of cortical development NSCs must maintain an extremely important balance in their cellular divisions. They must first expand their own pool through symmetric divisions, after which they must switch how they divide so that they can generate neurons and glia through asymmetric divisions. The current understanding of cellular and molecular mechanisms that regulate these important divisions remains fragmented and much remains to be discovered regarding master regulators of this process. We recently discovered a novel regulator of this process belongs to a family of zinc-finger specificity protein transcription factors, called Sp2. We found accumulation of stem cells at the expense of neurogenesis when we deleted the Sp2 gene only in NSCs of the developing cerebral cortex. In contrast overexpression of Sp2 rapidly pushes stem cells to delaminate from their epithelial home in the ventricular surface of the developing cortex, and precociously generate cortical neurons. We have discovered a number of intriguing cell biological themes that underlie the potent effects of Sp2 on NSCs, which we present in our preliminary data. With these findings, we propose to use a combination of state-of-the-art genetic mouse strains, cell and slice culture assays, live imaging protocols, biochemical assays, and mapping of RNA and protein landscapes that are Sp2-depenent to test the central hypothesis that Sp2-dependent transcription regulates the correct balance of proliferation and differentiation by regulating symmetric and asymmetric divisions of NSCs in the developing cerebral cortices. We provide preliminary evidence that Sp2 may carry out this critical function in NSCs through its interactions with known mechanisms and pathways of cell division. Thus, our study proposes to explore a novel mechanistic model that links molecular machineries that drive cytokinesis with asymmetric division of NSCs for production of neurons in the cerebral cortices. Potential for Broader Impact: Our approaches to understand how cortical stem cells divide symmetrically or asymmetrically have wide implications. Symmetric and asymmetric decisions in various stem cells are key to tissue development and regeneration throughout the body. Disruption of this balance in division of stem cells can lead to a range of pathological conditions from developmental retardation of tissues to oncogenesis. Therefore, undertaking the basic cellular mechanisms that control this key neural stem cell function is critical to understanding not only how appropriate divisions are controlled in stem cells during normal development, but also how their abnormal divisions in pathological conditions lead to devastating diseases such as cancer.

The College of Veterinary Medicine at North Carolina State University (NCSU) is requesting funds to acquire an X-RAD 320XL X-Ray irradiator from Precision X-Ray Inc. for cell and small animal research. Using an x-ray tube with a homogenic beam designed for clinical orthovoltage radiation and powered by a 320 kV high frequency ultra-stable x-ray generator, the X-RAD 320XL is capable of precise, repeatable irradiations, and features a chamber able to accommodate specimens as large as mini-pigs. Although irradiation studies are fundamental to the research of a considerable number of investigators at NCSU, there is currently no equipment on campus that offers easy and affordable access to radiation for either cells or small animals. The acquisition of the X-RAD 320XL will hence meet a significant demand from many NSCU investigators whose research spans a broad variety of fields including regenerative medicine, neurobiology, immunology/cancerology, material science and bioengineering, and who represent all departments of the Colleges of Veterinary Medicine and Engineering, as well as faculty colleagues from the joint UNC/NCSU Department of Biomedical Engineering. In the same vein, implementing easy and affordable access to a safe and easy-to-use irradiator on our campus will authorize and stimulate the development of a broad variety of innovative research projects that have been hampered until now. While a significant percentage of the users are already federally funded investigators (NIH, NSF, Army Research Office, USDA), the acquisition of the X-RAD320XL will undoubtedly increase competitiveness for additional federal funding. The high level of qualification of the persons involved, the space availability, the simplicity and safety of use of the X-RAD 320XL, and the strong support from our institution further guarantee the success of the X-RAD 320XL implementation, which will undoubtedly have positive and measureable repercussions for the development of innovative research at NCSU.

Stem cells are the basic units of developing tissues, and they are employed in regenerative medicine, cellular reprogramming, and cancer biology. Mechanisms of how stem cells communicate with themselves and their offspring is important for basic and applied studies, but are not well understood. An emerging question is how ���������������clonally������������������ related offspring (cells derived from division of a common ���������������mother������������������ stem cell) speak to each other and their mother stem cell, and how this communication dictates their fate. We have established cutting-edge genetic, imaging, and electrophysiological technologies to track the number, fate, and mode of communication among clonal cells in vivo, using the developing brain as a model. Our focus is on a critical period when neuronal production (neurogenesis) switches to production of glia (gliogenesis), a transition vital for normal brain development. Our results are revealing exciting novel concepts in stem cell biology. We propose to mathematically model complex datasets to uncover underlying rules and principles that govern clonal stem cell behaviors. Data obtained from proposed experiments will be used for an application to NIH.

The adult central nervous system (CNS) lacks endogenous mechanisms for replacing its damaged or diseased tissue, such as the neural circuits susceptible to epilepsy. Use of embryonic neural stem cells for replacement of CNS tissue poses a number of ethical and methodological limitations. Alternatively, the discovery of adult neural stem cells has raised hope for utilization of endogenous mechanisms for cell replacement in the CNS. This endeavor requires a comprehensive understanding of molecular mechanisms that distinguish adult stem cells from their embryonic counterparts. We have identified a Fork head transcription factor, FoxJ1, that is specifically expressed by cells forming the adult stem cell niche. Moreover, we recently discovered that FoxJ1 expression is ?turned on? when embryonic neural stem cells initiate their transformation into their adult form. This proposal is designed to test our hypothesis that FoxJ1 expression and activity is essential for the transformation of embryonic neural stem cells into their adult form in the CNS.