Haesun A. Kim - US grants

DESCRIPTION (provided by applicant): The long-term objective of this research is to define molecular mechanisms that are involved in demyelination and re-myelination of the peripheral nervous system (PNS). Demyelination is linked to the loss of all or part of neuronal functions because of the intimate connection between myelinating glial cells and axons. Therefore, the prevention of demyelination or promoting re-myelination is an important therapeutic objective for neurodegenerative diseases of the PNS and the CNS. We and others have shown recently that a function of receptor tyrosine kinase, erbB2, is associated with triggering PNS demyelination. ErbB2 function is also involved in promoting PNS myelination. The mechanisms by which erbB2 elicits two opposing roles in the PNS is unknown. Understanding the receptor signaling mechanisms has important implications for prevention of demyelinating neurodegenerative diseases and regenerative medicines. The broad goal of the present study is to define erbB2 function in initiating injury-induced PNS demyelination. Three specific aims are proposed: 1) determine the mechanism by which erbB2 promotes PNS demyelination, 2) determine whether erbB2 activation is sufficient to induce PNS demyelination and 3) determine the mechanism of signal transduction that is associated with the demyelinating function of erbB2. The study plan exploits a new in vitro compartmentalized Schwann cell-neuron co-culture system that recapitulates Schwann cell nerve injury responses in vivo. The system serves as a powerful tool for studying signal transduction using both pharmacological and genetic intervention. To investigate erbB2 function (Aim 1), we will use an adenoviral delivery system to express dominant-negative erbB2 mutant in adult myelinating Schwann cells, during different stages of PNS demyelination, which will enable us to achieve a conditional erbB2 inhibition both in vivo and in vitro. For the erbB2 gain-of-function study in Aim 2, we will use an innovative regulated erbB2 homo- and hetero-dimerization system. This system allows us to generate ectopic erbB2 signaling within myelinating Schwann cells, independent of the endogenous receptor and the ligand. For the signaling function raised in Aim 3, we will use both biochemical and imaging approaches to define erbB2-signaling mechanism that is associated with the demyelinating function of the receptor. PUBLIC HEALTH RELEVANCE Demyelination is a common pathologic feature in many PNS neurodegenerative diseases including Charcot-Marie-Tooth syndrome, Guillian-Barre syndrome, neuropathies secondary to diabetes and cancer chemotherapy, infectious neuropathy and motor deficits related to glial injury. Demyelination is also linked to the loss of all or part of neuronal functions because of the intimate connection between myelinating glial cells and the axons. Therefore, the prevention of demyelination is an important therapeutic objective for neurodegenerative diseases of the PNS and the CNS. Recent studies suggest that erbB2 activation might play a role in mediating PNS demyelination. Therefore, aberrant activation of the receptor or the associated cytoplasmic effectors may underlie a wide range of demyelinating disorders. Understanding the signal mechanism that initiates demyelination may have implications for the development of therapies that block erbB2 activation or diagnostic tests for detecting early demyelination before further progression of neurodegeneration.

ABSTRACT Acceleration forces induced by impact to the head are the cause of damage to axons throughout the brain. Long axons of white matter tracts are most vulnerable when the tissue is rapidly stretched upon impact. The injury results in significant damage to the myelin. Following mild traumatic brain injury (TBI), oligodendrocyte death is rarely observed. However, myelin abnormalities are nevertheless frequently observed. Ultra-structural analyses have shown myelin loss on intact axons (primary demyelination) as well as excess myelin sheath formation within the lesion, both of which underscore a disruption in myelin homeostasis. We hypothesize that mechanical impact on the brain disrupts myelin homeostasis in the mature oligodendrocyte. As myelin homeostasis is regulated by an array of transcription factors and gene expressions, we hypothesize that TBI initiates active signaling event(s) in oligodendrocytes and that this(these) intrinsic change(s) disrupt(s) myelin stability. In support to this hypothesis, data from our preliminary study show that stretch injury of oligodendrocyte activates the Erk1/2 pathway to induce myelin protein loss. Other injury-associated signals, including Ca2+ increase, glutamate and growth factor stimulation, also induced myelin loss in an Erk1/2-dependent manner. Furthermore, mild TBI on rodent brain induced Erk1/2 activation in white matter oligodendrocytes. Recent studies have shown that aberrant Erk1/2 activation in adult oligodendrocytes disrupts myelin homeostasis. We will test the hypothesis that TBI-induced Erk1/2 activation in oligodendrocytes contributes to myelin abnormalities (Aim 1). For the study, we will combine experimental TBI with the Erk1-/-,Erk2flox/flox:PLP-CreERT mouse line to determine whether inhibiting Erk1/2 activation in mature oligodendrocyte prevents TBI-induced myelin loss or improves myelin stability. Aim 2 will test the hypothesis that myelin abnormalities in TBI results from an active signaling event that involves transcriptional changes in mature oligodendrocytes. We will use in vivo TRAP (translating ribosome affinity purification) to isolate TBI-responsive transcripts specific to mature oligodendrocyte in adult brain. Subsequent RNA-seq analysis will elucidate the TBI-induced oligodendrocyte transcriptome profile associated with myelin dysfunction.

ABSTRACT Cells have a limited capacity to synthesize choline, thus cells depend on protein transporters to import choline. Choline is used to synthesize phosphatidylcholine, from which structural lipid components of myelin are synthesized. Phosphatidylcholine is also metabolized to generate phosphotidylinositols, whose phosphorylated derivatives are important signaling lipids that regulate myelination. Choline is involved in synthesis of the universal methyl donor, S-adenosylmethionine (SAM) for histone and DNA methylation, thus regulating gene expression. Considering the position of choline at the crossroad for the biosynthesis of phospholipids and epigenetic regulation, we have very little to no understanding of the regulation of choline import and choline-dependent metabolism in myelinating glial cells. Choline transporter for Schwann cells has not been identified. We have identified choline-like-transporter 1 (CTL1) as an important regulator of Schwann cell myelination. CTL1 deletion in Schwann cells (CTL1sc-KO) results in early onset of focal hyper-myelination in the PNS. Biochemical analysis revealed an overall decrease in choline-derived   phospholipids in the myelin. Furthermore, CTL1 loss impaired myelin gene expression and exhibited altered DNA modifications in Schwann cells. From these observations, we hypothesize that CTL1 is a Schwann cell choline transporter. We also hypothesize that choline-dependent metabolism feeds into the phospholipid signaling and epigenetic modifications that are important for myelination. To this end, we will investigate three aspects of choline metabolism in Schwann cell myelination. Aim 1 will test the hypothesis that CTL1 is a Schwann cell choline transporter. MALDI-TOF and tandem mass spectrometry will be performed to directly measure choline import into CTL1sc-KO Schwann cells. Impact of CTL1 loss on phosphatidylcholine synthesis will also be analyzed. In Aim 2, we will test the hypothesis that myelin defects in CTL1sc-KO mice results from imbalance in PI(3,5)P2 and PI(3,4,5)P3 synthesis. This is based on the observation that phosphatidylinositol contents are altered in CTL1sc-KO nerve and the myelination defects resemble those seen in mice with dysregulated PI(3,5)P2 and PI(3,4,5)P3 synthesis. Aim 3 will test the hypothesis that CTL1 loss alters gene expression in Schwann cells by modulating histone and DNA methylation. Perturbed lipid metabolism, including choline, is an underlying mechanism in many hereditary diseases associated with PNS myelination defects. Furthermore, dietary supplement of phospholipids has been considered as a potential therapeutic option for treating PNS neuropathies. Therefore, results from this study will provide important insights into understanding the implication of choline metabolism in developing therapeutic strategies to treat PNS neuropathies.

An award is made to Rutgers University – Newark to acquire a Zeiss LSM980 Laser Scanning Confocal Microscope with Airyscan 2. The Zeiss LSM 980 will be integrated into the Advanced Imaging Core Facility (AICF), of the Department of Biological Sciences (DBS), a core university facility that serves Rutgers and institutions of higher education in Newark. Installation of the microscope provides critically needed instrumentation that will have an unquestionable positive impact at Rutgers-Newark as an anchor institution with broad mission to improve and advance STEM education, training, and outreach. Acquisition of the Zeiss LSM 980 will initiate improvement in the undergraduate curriculum in Biology by stimulating development of a six-week long biological imaging laboratory series in a core biology course that enrolls 600 students annually. Using in-person and virtual teaching/learning strategies, students will be able to have an exhilarating first-hand experience at multi-dimensional imaging. DBS will add hands-on laser confocal imaging to a capstone upper division laboratory course in microscopy. To reach a broader undergraduate and graduate student audience, the leadership team will work with NSF-LSAMP, NIH-G-RISE, and Rutgers-NASA Space Grant Consortium programs at Rutgers-Newark to introduce multi-dimensional imaging using the Zeiss LSM 980. It is anticipated that this approach will encourage increased undergraduate participation in research, particularly URM students, and encourage pursuit of STEM careers. STEM faculty at Rutgers-Newark have a long-standing tradition of partnering with American Chemical Society Project SEED and Newark Public Schools to provide urban high school students with hands-on laboratory experience. Microscopy is often a key part of that experience. The leadership team is invested in bringing the wonder of fluorescence imaging to local high school students through the 4-week summer immersion program RUN-IMAGE and the academic year program Aim High NPS-NorthStar Academy. Each of these programs will benefit from both hands-on and virtual imaging relying upon the Zeiss LSM 980 and other microscopes in the AICF. The addition of the Zeiss LSM 980 to the AICF will have societal impacts on multiple fronts. Introducing advanced imaging to students at all levels will improve science literacy and build an understanding of the process of scientific discovery and application. This will have a future positive impact as US citizens are asked to make important societal decisions dependent upon an understanding of science. Further, the Zeiss LSM 980 will strengthen the ability of Rutgers-Newark to hire and retain top-shelf faculty whose research and teaching skills will add to education, training, and outreach at Rutgers-Newark. The LSM 980 will provide new faculty in the biological science with an essential modern-day research instrument that is vital for success in obtaining extramural grant funding and in publishing peer-reviewed work.

The Zeiss LSM 980 with Airyscan 2 provides a state-of-the-art microscopy workstation that provides multi-dimensional fluorescence imaging as well as super-resolution research capacity. The microscope will support fundamental research examining the mechanisms of cell signaling and membrane-protein sorting in neural, intestinal epithelial, and bacterial cells. The Zeiss LSM 980 will enable researchers will use novel fluorescent-reporter proteins in conjunction with innovative genetic animal and in vitro cell/organoid models to follow the dynamics and regulatory function of small GTPases and transcription factors in regulating cell and tissue differentiation and maintenance. Super-resolution fluorescence imaging and photo-manipulation will be used to discover novel protein-membrane and protein-protein interactions in the assembly of macromolecular complexes that regulate lipid transport, assembly of cell surface receptors, and intracellular sorting of signaling complexes in bacterial membranes, during myelin formation in peripheral nervous system, and embryonic development in mammalian central. The Zeiss LSM980 will be used in state-of-the-art optogenetic experimentation on the important role of cytoskeleton-chromatin interactions on gene expression and epigenetics. Additionally, the microscope will be used to provide multi-dimensional fluorescence imaging of cell signaling and remodeling pathways activated in response to tissue and cell injury from trauma including, impact, stretch, radiation, and chemical insult. All the research projects are fully integrated with STEM education/training of high school, undergraduate, graduate students and postdoctoral students serving as the foundation for improved STEM literacy and future leadership.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This award is jointly supported by the Major Research Instrumentation and the Chemistry Research Instrumentation Programs. Rutgers University – Newark is acquiring a high-resolution mass spectrometer (HRMS) equipped with electrospray Ionization (ESI), atmospheric pressure chemical ionization (APCI), and Direct Analysis in Real Time (DART) to support the research of Professor Frieder Jaekle and colleagues Elena Galoppini, Haesun Kim, Michal Szostak, and Colin Kinz-Thompson. This instrument facilitates research in the areas of organic chemistry, catalysis, semiconductors, inorganic chemistry, biochemistry, and biological sciences. In general, mass spectrometry (MS) is one of the key analytical methods used to identify and characterize small quantities of chemical species embedded in complex samples. In a typical experiment, the components are heated and flow into a mass spectrometer where they are ionized. The ions' masses are measured very accurately. The capabilities of the mass spectrometer instrument are augmented by complementary ESI and ambient pressure APCI and DART sources and will serve a wide host of research needs. The acquisition strengthens the research infrastructure at the University and regional area. This instrument enhances the educational, research, and teaching efforts of students at all levels in many departments as well as provides accessibility for use at nearby institutions. The instrument gives students experience using vital instrumentation that they carry with them into their careers. The research groups using the instrument are also actively participate in multiple successful programs to aid recruiting from underrepresented groups. <br/><br/>The award of this mass spectrometer is aimed at enhancing research and education at all levels. The new instrument will serve as an important characterization tool for research that spans areas ranging from synthetic chemistry, catalysis, materials science to biophysical and biological sciences. research will be enabled in amide bond activation, cross-coupling and catalysis, organocatalysis and green synthesis, molecular electrocatalysts for energy conversion, chromophore-semiconductor interfaces, organoboranes in materials science and catalysis, bacterial chemosensing, choline transport in myelin-forming glial cells, lipid metabolism in brain health and disease, lysozyme-mediated bacterial cell-wall-processing, and biological sciences.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.