Lu Chen, Ph.D. - US grants

DESCRIPTION (provided by applicant): The application's long-term objective is to delineate the molecular mechanisms underlying synaptic differentiation and modulation. Regulated targeting of ionotropic glutamate receptors to the synapse is a critical step in synaptogenesis and synaptic plasticity. Recent studies indicate that stargazin and its family members are essential for glutamate receptor targeting. Two distinct mechanisms are involved: interactions between stargazin and AMPA receptor subunits are required for surface delivery of the receptors;and interactions between stargazin and PSD-95 family proteins is required for targeting AMPA receptors to the synapse. Many questions remain regarding this novel regulation of AMPA receptor trafficking: 1) In which steps of the AMPA receptor targeting processes is stargazin involved? 2) Does stargazin interact with AMPA receptors directly or through other intermediate proteins? 3) If they interact directly, where are the functional domains in stargazin that interact with AMPA receptors, transport them to the plasma membrane and target them to the synapse? 4) Where are the functional domains in AMPA receptors that interact with stargazin? I propose to address these questions by focusing on the following aims. The first aim of this application is to investigate the roles of stargazin in different stages of AMPA receptor targeting. Stargazin may potentially control AMPA receptor targeting at different stages along its transport pathway-i.e. ER ->Golgi apparatus ->membrane fusion machinery ->internalization machinery. We will selectively block some of these steps and examine its impact on AMPA receptor trafficking. The second aim is to map the functional domains in stargazin and the AMPA receptor that mediate their interactions and control their targeting. There are potentially two types of functional domains in stargazin and the AMPA receptor, one responsible for bringing AMPA receptor and stargazin into same protein complex (the interaction domains), the other for bring the whole complex to plasma membrane (the trafficking domains). We will use Co-IP and FRET methods to map the interaction domains, and surface biotinylation and electrophysiological approaches to determine the trafficking domains. A large number of brain disorders are related to abnormal brain development and plasticity. The proposed studies will advance our understanding on molecular mechanisms of synaptogenesis and plasticity, thereby providing insights in these brain diseases, and their remediation.

DESCRIPTION (provided by applicant): Lymphatic research represents an explosive field of new discovery owning to the recent identification of several lymphatic specific markers. The cornea provides an ideal tissue for lymphatic studies due to its accessible location, transparent nature, and lymphatic-free and -inducible character. Additionally, corneal transplantation offers an excellent model for lymphatic research because it allows for (i) functional lymphatic cell trafficking studies;and (ii) identification of cellular contributions (donor or recipient source) to the process of lymphangiogenesis (LG). Studies on corneal LG bear direct applications to high risk (HR) transplantation where grafting is performed on lymphatic-rich host beds and the rejection rate can be as high as 90%, a category that many patients blind from corneal diseases fall into after inflammatory, infectious, traumatic, or toxic insults. In this setting, the trafficking of antigen presenting cells (ARC) afforded by lymphatics is greatly enhanced. To date, there is no effective management for this situation. My long-term goal is to elucidate the molecular mechanisms of LG using in vitro and corneal models, a necessary prerequisite to the development of new therapeutic protocols. My hypothesis is that corneal LG can be manipulated through specific lymphatic factors or cell populations (such as the macrophages), and combined blockade of several factors optimizes the outcomes of HR graft survival. This hypothesis is based on my preliminary data that 1) vascular endothelial growth factor receptor-3 (VEGFR-3) is expressed on both APCs and newly developed lymphatics, and its blockade leads to improved graft survival in uninflamed beds;2) a1 integrin (very late antigen-1, VLA-1) deficiency leads to diminished corneal LG, and universal graft survival in uninflamed beds;and 3) lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) is expressed on normal conjunctival cells of macrophage lineage and these cells may supply functional corneal LG during inflammation. The specific aims of this proposal are to: 1) define the role of VEGFR-3. I will study the role of VEGFR-3 in both development and maintenance of LG, and modifications of HR host beds and graft survival at both early and late stages;2) determine the role of VLA-1.1 will identify the expressional patterns of VLA-1 during corneal inflammation, and the role of VLA-1 in lymphatic endothelial cell functions in vitro, and corneal LG, lymphatic trafficking, modifications of HR host beds and graft survival in vivo;and 3) investigate the interaction between VEGFR-3, VLA-1 and LYVE-1.1 will investigate the role of conjunctival macrophages in corneal LG, the interplay between these three factors, and the effect of the concurrent blockade on modifications of HR host beds and graft survival. Research on corneal LG will have broader clinical implications beyond the treatment of ocular diseases alone, since lymphatic dysfunctions have been found in a diverse array of diseases including cancer metastasis, lymphedema, diabetics, arthritis, and AIDS, among many others.

DESCRIPTION (provided by applicant): Lymphatic research represents an explosive field of new discovery in recent years. The cornea provides an ideal tissue for lymphatic studies due to its accessible location, transparent nature, and lymphatic-free and - inducible features. Additionally, corneal transplantation offers an excellent model for lymphatic research because it allows for (i) functional lymphatic cell trafficking studies; and (ii) the identification of cellulr contributions (donor or recipient source) to the process of lymphangiogenesis (LG). Studies on corneal LG bears direct implication to high-risk (HR) transplantation where grafting is performed on lymphatic-rich host beds and the rejection rate can be as high as 90%. Many patients who are blind from corneal diseases fall into this category after inflammatory, infectious, traumatic, r chemical insults. To date, there is no effective management for this situation. Our long-term goal is to elucidate the molecular and cellular mechanisms of LG using both in vitro cell culture and in vivo corneal models, a necessary prerequisite to the development of new therapeutic protocols. Our hypothesis is that corneal LG can be manipulated through specific lymphatic factors or cell populations. Molecular blockade of the lymphatic factors promotes HR graft survival. This proposal is based on the most recent advances in technology and lymphatic research, and a large amount of preliminary data we have generated during the past few years. The specific aims are: 1) define the role of integrin alpha 9 (Itga-9) in corneal lymphatic valve formation and function using neutralizing antibodies and knockout mice; 2) determine the role of Angiopoietin-2 (Ang-2) in lymphatic endothelial cell functions in vitro, and LG and transplant rejection in vivo using small interference RNAs, neutralizing antibodies, and knockout mice; and 3) investigate the cell origins of lymphatic vessels using cell type-specific gene-manipulated mice and live imaging techniques. Research on corneal LG will have broader clinical implications beyond the treatment of ocular diseases alone since lymphatic dysfunction is associated with a diverse array of diseases and conditions, which include but are not limited to cancer metastasis, inflammation, infections, diabetes, obesity, hypertension and lymphedema.

Project Summary Glaucoma is a leading cause of blindness and affects over 60 million people worldwide. The mechanisms underlying glaucoma have been studied extensively but are not yet fully understood. We recently reported the first evidence that Schlemm?s canal (SC) expresses Prox-1, the master control gene for lymphatic development. The endothelial characteristics of SC has been debated for a long time. Regardless of its being considered as a blood, lymphatic, or intermediate vascular type, in this proposal, we will apply our knowledge and experience with lymphatic and blood vascular research to investigate SC, a critical structure of the conventional outflow pathway of aqueous humor that regulates intraocular pressure (IOP). IOP elevation is the primary and most important risk factor of glaucoma. Our long-term goal is to elucidate the underlying mechanisms governing SC function using various in vivo ocular hypertension models and in vitro SC cell culture systems, a necessary prerequisite to the development of new therapeutic protocols. Our main hypothesis is that Wnt pathway plays a critical role in SC, and its intervention can modulate IOP and treat glaucoma. This proposal is based on a large amount of preliminary data and the most recent advances in technology. Our specific aims are: 1) investigate the specific role of Wnt pathway in SC cells in vitro using human SC cell culture system and small interfering RNAs (siRNAs); 2) explore the specific role of Wnt pathway in SC in vivo using conditional knockout mice, ocular hypertension model, and advanced live imaging technology; and 3) assess the effect of Wnt pathway intervention on ocular hypertension and glaucoma using ocular hypertension models and optical coherence tomography (OCT). Our study promises for revealing novel mechanisms and therapeutic strategies for ocular hypertension and glaucoma, and possibly other vascular-related diseases in the body.