Diane C. Darland - US grants

A grant has been awarded to the University of North Dakota under the direction of Dr. Van Doze to acquire a stereology microscope system for quantitative microscopic analysis of nerve cells. The instrument will allow researchers to make detailed observations of cell structure to study nerve cell development, plasticity, and cellular interactions in the nervous system. Using fluorescent probes and imaging montage software, the investigators will be able to construct three-dimensional images on computers at several workstations. The equipment will be used in courses and in student research, including several programs aimed at rural students and students from Native American tribes.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of the present work is to characterize the cell and molecular mechanisms that regulate neural and vascular cell interactions during development. The central objective of this work is to determine the role of vascular endothelial growth factor (VEGF) in regulation of neurogenesis. Results from the proposed study will impact our understanding of the development of the central nervous system (CNS) as well as the plasticity of the CNS in response to mechanical injury or pathologic stress. Neural and vascular systems develop in concert and several factors have been identified with overlapping function in the two systems. An emerging model of neural and vascular system interdependence includes the potent angiogenesis factor, VEGF, and its dual role as a neural regulator. The angiogenesis-inducing effects of VEGF are largely mediated via activation of the VEGF receptor-2 (VEGFR2) homodimer that can use the co-receptor, neuropilin 1. Neuropilin 1 plays a critical role in mediating axon guidance cues, but can also contribute to angiogenesis via its association with VEGFR2. VEGF is expressed predominantly as three isoforms in the mouse, VEGF120, VEGF164 and VEGF188. Results from homologous recombination studies in mice have led to the suggestion of distinct roles for the different VEGF isoforms. The isoforms differ in their ability to bind to heparan sulfate proteoglycans in the matrix and on the cell surface and to interact with the neuropilin 1 co-receptor. Only VEGF164 has been shown to bind to and activate the VEGFR2/neuropilin 1 complex. VEGF164 is the predominant isoform in the brain. Although a number of studies have suggested a role for VEGF in the nervous system, little is known about the direct role that VEGF plays in neurogenesis. The hypothesis of this proposal is that VEGF regulates developmental neurogenesis via the VEGFR2-neuropilin pathway. The hypothesis will be tested with the following aims: 1) to characterize the cell-type specificity and expression patterns for VEGF, VEGFR2 and activated VEGFR2 during developmental neurogenesis in the CNS and 2) to test the role of VEGF-neuropilin signaling in developmental neurogenesis in mice lacking VEGF-neuropilin signaling. [unreadable] Project Narrative: The results from experiments described in this proposal are directly relevant to human health in the areas of brain development, function, and repair. Neuronal stem cells are the source of all brain neurons and have recently been found in the adult. Understanding how neural stem cells arise, differentiate, divide and die may be the key to finding out how to restore brain function after debilitating CNS injuries such as stroke, lesion, and neurodegenerative disease. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY/ABSTRACT. PROJECT 2, D. DARLAND. Functional integration between neural stem cells (NSC) and vascular cells is critical for neural-glia formation during cortical development and glioma progression. Vascular cells can act as epigenetic drivers to induce NSC specification and tumor transformation. However, these epigenetic factors remain poorly defined. We will identify epigenetic mechanisms that regulate NSC fate decisions in response to vascular investment. We have established a unique NSC-vascular coculture system in which NSC adopt a glial fate in response to vascular cues have used a transcriptome-level, unbiased screening approach that has identified Class IIa Hdacs as potential candidates for the epigenetic drivers. Of the histone deacetylases (Hdacs) expressed, only the Class IIa Hdacs expressed in the brain (Hdac 4, Hdac 5, Hdac 7) were upregulated in NSC in vascular coculture, suggesting that they play a role in mediating NSC transition to glial cells. Based on results from the Cancer Genome Atlas (TCGA), elevated levels of Class IIa Hdacs are also associated with human glioma tumor grades, particularly Hdac 4 and Hdac 5. The Class IIa Hdacs have the unique ability to move from the cytoplasm to the nucleus, recruiting protein partners such as Hdac3 and Mecp2 to facilitate deacetylation reactions. This property makes them attractive candidates to transduce microenvironmental cues. We have developed a coculture system that models NSC interactions with vascular cells (endothelial cells and pericytes) and will use this to address the question of NSC fate decisions in response to vascular environmental cues in early cortical development and in a glioma-vascular model. Here we test the hypothesis that changes in Class IIa Hdacs are critical for gliogenesis in response to vascular cell developmental cues and in glioblastoma during cancer progression. In Aim 1 we will test if Class IIa Hdacs are required for gliogenesis in a neural-vascular coculture model. The working hypothesis is that increasing expression of Class IIa Hdacs expression precedes the cell fate transition from NSC to glia under the influence of vascular cell-derived Lif derived. In Aim 2 we will test if Class IIa Hdacs are required for glioma progression in a vascular coculture model. Since Class IIa Hdac are upregulated in human glioblastoma, the working hypothesis is that Class IIa Hdacs are required for the glioblastoma stage transition that occurs in a highly vascular microenvironment. We predict that the Class IIa Hdacs regulate changes in chromatin structure by decreasing transcription of proliferation and stem-specific genes and inducing gliogenesis during development or promoting glioma progression in response to vascular investment. The proposed studies address a critical need to understand how microenvironment-induced, acetylation-based modifications to chromatin influence cortical gliogenesis during development and in glioma tumor progression. The model systems established will also provide a valuable resource for testing potential Class IIa Hdac-based targets for preventing tumor transition in a glioblastoma-vascular cell culture model.