Elva Diaz - US grants

The human brain comprises ~100 billion neurons precisely organized into networks by specific cell-cell contacts called synapses that give rise to cognitive phenomena such as emotion, learning and memory, and perception. During development, the intricate and precise patterns of neuronal synapses are formed. Synapse formation is initiated by special proteins at the presynaptic and postsynaptic cell surfaces upon cell-cell contact. Dr. Elva Diaz (Principal Investigator) and members of her laboratory have identified a new group of related proteins that are found at the postsynaptic neuronal cell surface. Interestingly, different family members of this new gene family are expressed in specific brain regions, suggesting a role for these molecules in determining synaptic specificity. Their hypothesis is that these proteins are involved in postsynaptic differentiation and transmit signals to the presynaptic neuron to cause recruitment of synaptic vesicles, the first step in the formation of new synapses. In this application, Dr. Diaz proposes experiments using genetic manipulations in mice and methods to identify other molecules with which these proteins interact in order to understand how this interesting class of proteins functions during synapse formation.

The broader impacts of the proposed activities include integration of the research and education activities specifically in the context of broadening the participation of undergraduate students from underrepresented groups (primarily Hispanic/Latino). As an active participant in campus programs aimed to increase the number of underrepresented students pursuing postgraduate degrees in science, Dr. Diaz is in a strong position to recruit such students to her laboratory as members of the research team for this proposal. Undergraduate participation will be directed by Dr. Diaz in conjunction with doctoral students in the laboratory, thereby integrating research, teaching, and community outreach activities for students pursuing graduate studies in the Diaz laboratory.

DESCRIPTION (Provided by the applicant) Abstract: Tumors of the central nervous system (CNS) comprise nearly one quarter of all childhood cancers. Although progress has been made in the treatment of some types of childhood cancer, the outcome for children with primary CNS tumors has remained bleak and little advancement has been made in the last decade. In addition, due to the adverse effects of the tumor on brain development or the treatment required to control its growth, survivors of childhood brain tumors often have severe neurodevelopmental defects that negatively impact their quality of life. Thus, there is a need for better treatments specific for childhood brain tumors. Current models suggest that only a few atypical cells within the cancerous mass are responsible for the initiation, growth and recurrence of brain tumors. These transformed cells have both the defining properties of neural stem cells and the ability to initiate cancer - thus, these cells are referred to as 'brain tumor stem (BTS) cells'. While the isolation of neural stem cells is fairly well established, the isolation of BTS cells remains a difficult and complex issue, suggesting the need for innovative approaches to isolate and characterize these cells. The development of induced pluripotent stem cells (somatic cells that have been reprogrammed to an embryonic-like pluripotent state by retroviral-mediated introduction of specific transcription factors) represents a powerful new approach that might alleviate such confounding issues. Thus, the goals of the proposed project are: 1) to reprogram brain tumor cells towards a more stem-like phenotype;2) to characterize the tumorigenic potential of such reprogrammed tumor stem-like cell lines;and 3) to identify chemical compounds that specifically target the reprogrammed tumor stem-like cells. Completion of these studies will provide a directed strategy for novel therapeutics to specifically target the cellular population responsible for the initiation, growth and recurrence of pediatric brain tumors. Public Health Relevance: Brain tumors are the second leading cause of cancer-related death in children in the United States and medulloblastoma is the most common pediatric brain tumor. An increasing amount of evidence suggests that only a few atypical cells (brain tumor stem cells) within the cancerous mass are responsible for the initiation, growth and recurrence of primary brain tumors. Brain tumor stem cells appear to be resistant to traditional chemo- and radiation therapies compared with the more differentiated cells in the cancerous mass, suggesting a mechanism underlying tumor recurrence after treatment. Thus, the focus of the present grant proposal is to generate stable brain tumor stem cell lines and to identify chemical compounds that specifically target the reprogrammed tumor stem-like cells

Brain cells (neurons) transmit electrical information through the central nervous system and elicit specific behaviors through specialized connections called synapses. During development, the intricate and precise pattern of neuronal synapse connections is formed. The goal of this project is to understand the mechanism by which a novel protein (SynDIG1) that was identified in the Principal Investigator's (PI) laboratory functions in synapse development. This study will test the hypothesis that SynDIG1 anchors AMPA-type glutamate receptors (AMPA-Rs) at the postsynaptic compartment via interaction with scaffolding proteins. The researchers will use a combination of approaches including biochemistry, immunocytochemistry, and electrophysiology to address this possibility. While significant progress has been made in the field regarding presynaptic differentiation such as synaptic vesicle clustering and postsynaptic recruitment of scaffolds and NMDA-type glutamate receptors (NMDA-Rs), less is known about the molecules implicated in AMPA-R recruitment during synapse development. Thus, the results of the proposed experiments will fill a major gap in the field of synapse development and function.

The broader impacts of the proposed studies include integration of the research and education activities of the PI, specifically in the context of broadening the participation of undergraduate students from underrepresented groups (primarily Hispanic/Latino). The PI is an active participant in various campus programs aimed to increase the number of underrepresented students pursuing postgraduate degrees in science; thus, the PI is in a strong position to recruit such students to the laboratory as members of the research team for this project. Undergraduate participation will be directed by the PI in conjunction with graduate students in the laboratory, thereby integrating research, teaching, and scientific community outreach activities for students pursuing graduate studies in the laboratory.

Cell-to-cell communication in the nervous system is mediated by small chemicals called neurotransmitters that are released from one cell and bind to receptor proteins present on the membrane of the adjacent cell in a process called synaptic neurotransmission. The amino-acid neurotransmitter glutamate and its conjugate glutamate receptors are responsible for the majority of the excitatory neurotransmission in the mammalian brain. Glutamate receptors are protein complexes of four subunits that assemble in various combinations, creating membrane-embedded ion-channels of different types and properties. In recent years, it has become increasingly evident that glutamate receptors are acted on by various auxiliary factors that regulate channel properties and localization at synapses. Therefore, elucidating the structure and function of glutamate receptors in general, and how these receptors are modulated by association with accessory proteins in particular, is central to understanding the basic principles of brain physiology. This grant will concentrate on the interaction of the AMPA-type glutamate receptors with the SynDIG protein family. A combination of molecular biology, biochemistry and electrophysiology experiments will be undertaken to test the hypothesis that SynDIG proteins influence the delivery of AMPA-type glutamate receptors to the cell surface of the postsynaptic membrane as well as alter the properties of ion flow through the glutamate receptor channel critical for synaptic neurotransmission.

The broader impacts of the proposed studies include integration of the research and education activities of the PI, specifically in the context of broadening the participation of undergraduate students from underrepresented groups (primarily Hispanic/Latino) pursuing postgraduate degrees in science. Undergraduate participation will be directed by the PI in conjunction with graduate students in the laboratory, thereby integrating research, teaching, and scientific community outreach activities for students pursuing graduate studies in the laboratory. Additionally undergraduate and graduate students will participate in international exchanges as part of the collaboration with the Israeli PI for this project which is jointly supported by NSF and United States-Israel Binational Science Foundation.

We are interested in how synapses form in the brain. The objective of this proposal is to investigate the mechanism underlying the novel finding that Brain-Specific Angiogenesis Inhibitor 2 (BAI2) increases synapse number when overexpressed in cultured neurons. BAI2 is a member of adhesion G-protein coupled receptor (GPCR) family, comprised of a large N- terminal extracellular (adhesive) domain followed by a seven transmembrane GPCR domain. This novel finding extends our published work that identified a functional mutation in a critical extracellular region of BAI2 underlying a forward mutagenesis hyperactivity screen in mice that reduced surface expression of BAI2 in heterologous cells. Our hypothesis is that the intracellular C-terminus of BAI2 is indispensable for the increase in synapse number that we observed. Specifically, there are two motifs?a domain that binds a key regulator of the actin cytoskeleton and a PDZ binding sequence?that may be critical mediators of early synapse formation. We will employ a molecular biology approach coupled with super-resolution microscopy in dissociated hippocampal neurons in the presence or absence of BAI2 to test this hypothesis. Intriguingly, a de novo C-terminal mutation of BAI2 that leads to constitutive receptor signaling was identified in a human patient suffering from progressive spastic paraparesis and other symptoms. Thus, results of these studies will provide insight into BAI2 function in synapse development as well as dysfunction that leads to human disease.

Activity-dependent variation in synaptic AMPA receptor (AMPAR) content, referred to as ?synaptic plasticity?, is a mechanism whereby information is stored in neural networks that give rise to higher order cognitive skills such as learning and memory. During long-term potentiation (LTP), a widely studied form of synaptic plasticity, extrasynaptic AMPARs are recruited from nearby reserve pools, including perisynaptic regions on the cell surface and intracellular compartments, and subsequent anchored with the postsynaptic density (PSD). A large body of evidence spanning decades of investigation has established mechanisms by which AMPARs are anchored within the PSD. In contrast, the molecular mechanisms that govern AMPAR synaptic targeting to establish reserve pools of extrasynaptic receptors are largely unknown. Given that recruitment of reserve pools of extrasynaptic AMPARs underlies the rapid strengthening of synapses that occurs during LTP, the molecular mechanisms that establish such reserve pools are critical to our understanding of synaptic plasticity and represent a major gap in our knowledge. SynDIG (Synapse Differentiation Induced Gene) defines a family of four genes (SynDIG1-4) that encode brain- specific transmembrane proteins. Here we will determine the function of SynDIG4 (SD4), also known as Prrt1 (Proline-rich transmembrane protein 1) in the regulation of the reserve pool of AMPARs. Proteomic studies indicate that SD4 is a component of AMPAR complexes; however, SD4 is not enriched in the PSD, but instead colocalizes with GluA1-containing AMPARs at non-synaptic sites. Remarkably, tetanus-induced LTP, which is dependent on GluA1, is abolished in acute hippocampal slices from SD4 knockout (KO) while theta-burst stimulation LTP (TBS-LTP), which is independent of GluA1, is not impaired. Furthermore, SD4 KO mice exhibit profound deficits in two independent cognitive assays (Morris water maze, novel object recognition), demonstrating a critical role for SD4 in hippocampal-dependent learning and memory. Moreover, extrasynaptic AMPARs are reduced in SD4 KO compared with wild-type (WT) neurons. Given that reserve pools of extrasynaptic AMPARs are critical for synaptic plasticity, we hypothesize that SD4 maintains such reserve pools of extrasynaptic GluA1-containing AMPARs that are deployed during tetanus-induced LTP. In this collaborative dual-PI application we propose a comprehensive multidisciplinary approach to investigate SD4-dependent regulation of extrasynaptic GluA1-containing AMPARs with molecular, cellular, and electrophysiological methods. In Aim 1 we will define the mechanism by which SD4 maintains extrasynaptic GluA1-containing AMPARs. In Aim 2 we will determine whether synaptic targeting of reserve pools of GluA1- containing AMPARs during plasticity requires SD4. In Aim 3 we will test whether homomeric GluA1-dependent synapse plasticity mechanisms require SD4 ex vivo at multiple ages. The results of these studies will provide molecular insight into fundamental mechanisms that govern establishment and maintenance of the reserve pools of extrasynaptic AMPARs critical for synaptic plasticity and address a major gap in our knowledge.