Qin Liu - US grants

The mammalian neocortex is the most complex tissue of the central nervous system. It is subdivided into functionally distinct areas tangentially differing in cytoarchitecture and connectivity. Neurons within different areas underlie our most sophisticated cognitive and perceptual abilities and are thus highly specialized for the analysis of sensory inputs or the generation of motor output. For example, layer V neurons form the major connections between the neocortex and the spinal cord. In the visual cortex, layer V neurons project to the superior colliculus, while in the sensorimotor cortex, layer V neurons send their axons to the spinal cord. In both regions, there is a subset of neurons send axons to the contralateral cortex. This mature projection pattern of layer V neurons is achieved by a combination of selective axon segment elimination and collateral stabilization. Little is known about the molecular mechanism of this process. To try to address this issue and to understand the molecular mechanism of cortical area specification, here I propose to identify and characterize genes that are differentially expressed in neurons of the visual cortex versus the sensorimotor cortex. The long term goal is to unravel the molecular mechanism of cortical area specification and the mechanism of axon pathfinding.

Developmental biologists have long been trying to determine mechanisms underlying the development of vertebrate retina. One of the most fundamental questions to be asked is what are the factors determining or influencing the fate of progenitor cells (e.g. what determines whether the cells become cones instead of rods). Cadherins, in addition to being responsible for adhesion between many types of cells, may also play key roles in cell differentiation, axon pathfinding and synapse formation. Our preliminary results show that both N- and R-cadherins are located in the teleost retina at a time when retinal cells are proliferating and differentiating, and when retinal ganglion cell axons are migrating out of the retina toward their major target in the brain, the optic tectum. The proposed study will first examine the developmental profiles of these two cadherins in the visual system to look for correlations between the distribution of these molecules and the major morphogenetic events of the system. Functional analysis using either cadherin antibodies in an organ culture system or injection of antisense nucleotides to block cadherin mRNA will be employed to assess the role(s) of these cadherin molecules in the development of teleost visual structures.

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this research is to understand molecular mechanisms underlying neuronal differentiation and the formation of specific neuronal connections in the visual system. The objective of the proposed studies is to determine the roles of cadherin molecules in regulating neurite outgrowth and axonal pathfinding of retinal ganglion cells in developing zebrafish. Cadherins are Ca-dependent cell adhesion molecules that have been demonstrated to play critical roles in cell differentiation and tissue formation. Both in vitro and in vivo studies using a variety of vertebrate species have shown that cadherins are involved in retinal axon fasciculation, axonal outgrowth and pathfinding, retinotectalsynaptic formation and stabilization. However, most of the studies to date have focused on N-cadherin, and there is relatively little information on the role(s), and in particular the in vivo functions, of other cadherins, in these processes. Moreover, little is known about relative roles of different cadherin molecules in the development of retinal ganglion cells. I propose to study the expression patterns and functions of both R- and N-cadherins in the visual system of developing zebrafish. This proposal has three specific aims: A) to generate neutralizing antibodies, B) to further characterize the expression patterns of R- and N-cadherins, and C) to assess the relative roles of R- and N-cadherins by application of neutralizing antibodies to the eye and optic pathway, and application of dominant negative constructs to the eye. The proposed studies, designed to uncover mechanisms underlying retinal ganglion cell differentiation, outgrowth and pathfinding of their axons, may provide insights into therapies for injured or congenitally defective human retinal and optic nerve tissues.

DESCRIPTION (provided by applicant): The long-term objective of this research is to determine how coordinated and differential cell adhesion regulates the development of the zebrafish visual system, with special emphasis on the role of cadherin cell adhesion molecules in the development of retinal ganglion cells. Cadherins are important cell adhesion molecules that have been implicated in the development of a variety of tissues and organs including the nervous system. Both in vitro and in vivo studies using a variety of vertebrate species have shown that cadherins are involved in retinal axon fasciculation, axonal outgrowth and pathfinding. However, most of the studies to date have focused on cadherin-2 (N-cadherin), and there is relatively little information on the roles, and in particular the in vivo functions, of other cadherins, in these processes. Moreover, little is known about relative roles of different cadherins in the development of retinal ganglion cells. We have studied expression patterns of both cadherin-2 and cadherin-4 (R-cadherin) in the visual system of developing zebrafish. I propose to study cadherin-2 and cadherin-4 function in zebrafish retinal ganglion cell development. This proposal has two specific aims: A) determine function of cadherin-4 in the development of zebrafish retinal ganglion cells, and B) determine function of cadherin-2 in the development of zebrafish retinal ganglion cells, and compare its function in retinal ganglion cell development with cadherin-4. A variety of techniques (e.g. application of cadherin antibodies, cadherin dominant-negative constructs, examination of cadherin-2 mutant) will be used in the project. The proposed studies, designed to uncover mechanisms underlying retinal ganglion cell development, may provide insights into therapies for injured or congenitally defective human retinal and optic nerve tissues.

[unreadable] DESCRIPTION (provided by applicant): The long-term objectives of this project are to determine the roles of cadherin molecules in the development of vertebrate visual system, with special emphasis on the retina. Cadherins are important cell adhesion molecules that have been implicated in the development of a variety of tissues and organs including the visual system. Usher syndrome type 1F in humans (visual and auditory defects) is due to mutations in a cadherin gene. There is extensive information on expression and function of type I classic cadherins in the vertebrate visual system, but there is no published report on type II cadherins function in retinal development. Using zebrafish as our model organism, we recently examined several type II cadherins expression in developing visual system and have begun to study the role of cadherin6, a member of the type II cadherin subfamily, in zebrafish retinal development. The specific aims of the current proposal are: 1. to test the hypothesis that differentiation of zebrafish retinal ganglion and amacrine cells require cadherin6 function; and 2. To test the hypothesis that cadherin10 is involved in zebrafish retinal ganglion and amacrine cells development. A variety of techniques (e.g. morpholino antisense oligonucleotides technique, application of dominant negative construct, mosaic analysis) will be employed in the project to study cadherin function at both the retinal cell and tissue levels. The proposed studies, designed to uncover mechanisms underlying vertebrate retinal cell development, may provide insights into therapies for injured or congenitally defective human retinal and optic nerve tissues. The proposed research will take place in a department that has a demonstrated commitment to development of undergraduate researchers. In the last 5 years, QL (PI) and RLL (Co- investigator) have mentored a total of 35 undergraduate research projects. Here we propose to strengthen and deepen the undergraduate research experience for selected students (by direct involvement in the proposed experiments), and to broaden the exposure to their research experience to hundreds of students (by presenting the students' research data to students in large Introductory Biology classes). [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objectives of this project are to determine the roles of cadherin molecules in the development of vertebrate visual system, with special emphasis on the retina. Cadherins are important cell adhesion molecules that have been implicated in the development of a variety of tissues and organs including the visual system. Mutations in protocadherins (pcdhs) cause humans diseases including epilepsy, mental retardation and Usher syndrome (visual and/or auditory defects). There is extensive information on function of classic cadherins (e.g. N-cadherin and R- cadherin, also called cadherin-2 and cadherin-4, respectively) in the vertebrate visual system, but little is known about other cadherins including the pcdhs function in retinal development, and there is no published reports on pcdh17 and pcdh9 function in the development of the vertebrate visual system. Using zebrafish as our model organism, we recently examined pcdh17 and pcdh9 expression in developing visual system and have begun to study the role of pcdh17 in zebrafish retinal development. The specific aims of the current proposal are: 1. to test the hypothesis that differentiation of retinal cells requires pcdh17 function;and 2. to test the hypothesis that pcdh9 is involved in RGCs differentiation and pathfinding. A variety of techniques including morpholino antisense oligonucleotides technique, pcdh overexpression, proteomics, DNA microarray, will be employed in the project to study pcdhs function in retinal development. The proposed studies, designed to uncover mechanisms underlying vertebrate retinal cell development, may provide insights into therapies for injured or congenitally defective human retinal and optic nerve tissues. The proposed research will take place in a department that has a demonstrated commitment to the development of undergraduate researchers. We propose to strengthen and deepen the undergraduate research experience and to broaden the exposure to their research experience. We will significantly enhance hands-on research experience for students enrolled in the laboratory component of Cell Physiology, Developmental Biology, Neurobiology and Vertebrate Embryology (through their participating in aspects of the proposed experiments). We will also solicit proposals from students to apply for summer fellowships, where they can pursue their original research (centered on the themes in this proposal). These students will then present their research to other undergraduates at local or regional undergraduate and graduate students'research symposiums. PUBLIC HEALTH RELEVANCE: The proposed studies, designed to uncover mechanisms underlying vertebrate retinal cells differentiation, may provide insights into therapies for injured or congenitally defective human retinal and optic nerve tissues.

DESCRIPTION (provided by applicant): Ocular itch is a refractory symptom of many ocular conditions, and severely affect the quality of life and productivity. Ocular itch is thought to be mediated by a group of primary sensory neurons residing in the trigeminal ganglia. These neurons detect endogenous itch-inducing mediators (pruritogens) via their peripheral axons in the conjunctiva, and transmit signals to the brainstem via their central axons. However, the molecular identification of these itch-sensing neurons remains elusive. Previously, we identified a novel itch receptor, called MrgprA3. We found that MrgprA3 marks a highly restricted population of primary sensory neurons that mediates acute and chronic itch in the skin. Interestingly, our latest results revealed that MrgprA3-expressing sensory neurons also project to the conjunctiva but not to other mucosal membrane tissues examined. However, the function of MrgprA3-expressing neurons in ocular itch remains to be determined. This proposal aims to uncover the neural mechanisms of ocular itch. Aim 1 will characterize the innervation pattern and physiological properties of MrgprA3-expressing sensory fibers in the conjunctiva. Using genetic labeling tools, we will perform detailed anatomical analysis of the innervation of MrgprA3-expressing sensory fibers in the conjunctiva during development and in adulthood. In addition, we will test whether MrgprA3-expressing sensory fibers in the conjunctiva can be activated by various pruritogens. These studies will provide important information about the potential role of MrgprA3-expressing sensory fibers in ocular itch. Aim 2 will investigate whether MrgprA3-expressing neurons mediate acute ocular itch. We will determine whether ablation of MrgprA3-expressing neurons alleviates the ocular itch produced by various pruritogen. Furthermore, we will examine the behavioral consequence of selective activation of MrgprA3-expressing sensory fibers in the conjunctiva. These loss-of-function and gain-of-function studies will firmly establish the role of MrgprA3- expressing neurons in ocular itch, which will, for the frst time, unravel the neural mechanism of ocular itch at the peripheral level. In Aim 3, we seek to understand the interaction between MrgprA3-expressing sensory fibers and mast cells in allergic conjunctivitis and determine whether MrgprA3-expressing fibers mediate related ocular itch. Based on our preliminary data, we hypothesize that mast cells release endogenous pruritogens upon allergen-induced degranulation and excite MrgprA3-expressing sensory fibers to induce itch. Using a novel imaging tool combined with molecular and behavioral analysis, we will investigate this hypothesis. These studies will reveal the neural basis underlying ocular itch that occurs in perennial and seasonal allergic conjunctivitis and will have a significant impact on both the study of ocular itch pathogenesis and the clinical treatment of chronic itch.

Itch is a primary symptom of many allergic conditions, and severely affects the quality of life and productivity. Primary sensory neurons residing in the trigeminal ganglia and dorsal root ganglia (DRG) play an essential role in the generation of allergic itch. These neurons detect endogenous itch-inducing molecules (pruritogens) via a group of itch receptors on their peripheral axons in the skin or mucosal membrane tissues, and transmit signals to the spinal cord via their central axons. In contrast to the well-studied histamine pathway, histamine-independent itch pathways in allergic itch remain poorly defined. Previously, we identified several novel itch receptors within a large family of G-protein?coupled receptors called Mrgprs. We found that several Mrgprs recognize distinct pruritogens, and mediate histamine-independent itch. Our latest results indicate that Mrgprs are required for mast cell-mediated allergic itch. This proposal aims to uncover the underlying mechanisms. We will test whether Mrgprs mediate the interaction between mast cells and primary sensory fibers in allergic itch using molecular, genetic and imaging approaches in Aim 1. Furthermore, we will identify the endogenous Mrgpr ligands released by mast cells upon degranulation in Aim 2. In Aim 3, we seek to translate our discoveries from mice to humans. Human Mrgprs do not form clear orthologous pairs with mouse Mrgprs. Their role in itch is therefore unclear. Based on our preliminary data, we hypothesize that human MrgprX1 mediates neuronal and behavioral response to mast cell-mediated allergy, which will be tested in Aim 3. These studies will reveal the neural basis underlying allergic itch and will have a significant impact on both the study of itch pathogenesis and the clinical treatment of itch.

? DESCRIPTION (provided by applicant): The National lung Screening Trial has demonstrated that a 20% reduction in lung cancer mortality is associated with routine LDCT screening of older individuals with a heavy smoking history, but of the patients that had a positive screen for lung cancer based on lung nodules detected, approximately 96% proved to be false positives. These statistics highlight two unmet medical needs required to maximize the diagnostic potential of LDCT: 1) the development of diagnostic platforms that will distinguish malignant from benign nodules identified by routine LDCT, and 2) the development of inexpensive, non-invasive methods that can identify at risk individuals who would benefit from follow up with LDCT. The proposed research in Project 1 capitalizes on technical advances for assaying gene expression and abundant prior evidence that tumors are highly interactive with the immune system. Our previous studies demonstrated that it is possible to diagnose early-stage lung cancer with 90% sensitivity and 80% specificity using gene expression signatures from PBMC. The proposed research translates the PBMC diagnostic to a more clinically viable sample collection platform with the additional goal of increasing accuracy and assessing immunological processes affected by the presence or removal of a lung tumor. We present preliminary studies that support the hypothesis that this can be done. We have enriched the signature development process by assessing both mRNA and miRNA expression profiles to assess complimentary mechanisms for regulating gene expression and will also integrate Natural Killer cell and Myeloid cell markers associated with prognosis. We also introduce in Project 2 an assay for tumor associated antigens, the cancer testis antigens (CTAs) also associated with circulating tumor cells, cancer cell derived exosomes or other potentially important cells such as cancer stem cells. We provide strong preliminary evidence that detection of the mRNA for the CTA AKAP4 in PBMC derived RNA is possible and that detection is very highly correlated with the verified presence of a lung tumor. Strong preliminary results are presented for both projects. We also propose to integrate and expand the signatures from these 2 studies and assess accuracy on a single reliable platform that can assess both mRNA and miRNA expression, and is already FDA approved for a breast cancer prognosis signature, the nCounter from Nanostring.

Project Summary ? Core D    The  primary  objective  of  the  Biostatistics  Core  in  this  P01  is  to  facilitate  the  understanding  of  the  biology  of  melanomas  and  the  development  of  novel  strategies  for  melanoma  treatment  based  on  the  most  stringent  and  highest  quality  of  statistical  data  analyses  and  inferences.  The  Biostatistics  Core  is  well  organized  and  equipped  to  achieve  this  goal.  This  Core  will  provide  expertise  on  experimental  design  and  critical  analytical  services  to  Investigators that are  tailored  to  each project  and provided  in a  timely and  cost-­effective  manner.  The  biostatisticians  in  this  Core  will  meet  regularly  with  project  investigators  to  develop analytic  strategies,  assess  the  statistical  needs  for  each  particular  project  on  an  ongoing  basis,  and  to  modify  analysis  plans  as  the  research  evolves.  Since  many  elements  of  the  statistical  analysis  plans  will  be  specific  to  a  particular  study, each project  will benefit from the  centralized  resource,  including  individuals  who  can provide expertise  in  study  design,  biostatistics  methodology  and  specialized  data  analysis  techniques,  and  who  have  had  significant  experience  in  solving  statistical  issues  in  biological  cancer  research.  This  level  of  collaboration  between  Core  biostatisticians  and  project  Investigators  will  ensure  that  the  quality  and  validity  of  each  research  project  will  be  of  the  highest  degree.  In  addition  to  the  statistical  services  provided  to  each  project,  this  Core  is  the  essential  centralized  resource  to  integrate  the  data  across  all  projects  in  this  program  and  synthesize  study  findings.  Using  state-­of-­the-­art  statistical  techniques  combined  with  high-­throughput  data  garnered  from  each  project,  the  Core  will  provide  a  clear  and  complete  view  of  the  associations  between  studied  inhibitors  and  genotypes,  and  provide  quantitative  assessment  regarding  the  development  of  novel  strategies of melanoma treatment in this P01.   

ABSTRACT Dry eyes and contact lens wear often cause foreign body sensation. This abnormal mechanosensation is associated with ?Lid Wiper Epitheliopathy? (LWE), a condition induced by the mechanical damage to the marginal palpebral conjunctiva (also known as ?lid wiper?). However, the underlying neurosensory mechanism remains elusive. We recently found that a population of primary sensory neurons defined by the expression of MrgprD selectively innervates the lid wiper and is sensitive to shear force. This proposal aims to determine whether MrgprD-expressing sensory fibers mediate ocular mechanosensation and regulate lacrimation. In Aim 1, we will characterize the innervations and mechanosensitivity of MrgprD-expressing sensory fibers in the lid wiper. Using genetic labeling and axonal tracing approaches, we will perform detailed anatomical analysis of organization and terminal ultrastructure of MrgprD-expressing sensory fibers in the lid wiper. In addition, we will test whether MrgprD-expressing sensory fibers in the lid wiper can be activated by shear force by conducting ex vivo calcium imaging. These studies will shed light on the role of MrgprD-expressing sensory fibers in ocular mechanosensation. In Aim 2, we will further determine whether MrgprD-expressing sensory fibers sense shear force during eye movements. We will determine whether genetic ablation or pharmacological silencing of MrgprD-expressing neurons alleviates the ocular mechanosensation induced by enhanced shear force. This study will provide insight on the neurosensory mechanism of the lid wiper mechanosensation. In Aim 3, we will determine whether the lid wiper mechanosensation regulates lacrimation to maintain the lubrication of the ocular surface. Specifically, we will examine whether ablation of MrgprD-expressing sensory fibers affects basal lacrimation and mechanically-induced lacrimation. Furthermore, we will test whether chemogenetic activation of MrgprD-expressing sensory fibers in the lid wiper promotes lacrimation. Finally, we will determine whether pharmacological activation of MrgprD-expressing sensory fibers is a potential therapeutic strategy for promoting lacrimation under dry eye conditions. These studies will reveal the neural basis of ocular mechanosensation associated with physiological tear evaporations and pathological dryness of the ocular surface, which will have a significant impact in our understanding of ocular mechanosensation as a protective mechanism and its clinical implication in dry eye treatments.

Abstract Allergic rhinitis is the most common mucosal allergy. Its cardinal symptoms include excessive sneezing and rhinorrhea, which severely impact our life quality and productivity. Although antihistamines effectively relieved sneezing induced by intermittent mild allergic rhinitis, they are ineffective against persistent moderate/severe allergic rhinitis. The development of new drugs for alleviating allergic sneezing is hindered by a lack of information about the principal nasal sensory neurons that mediate sneezing and their interactions with immune cells. In this proposal, we hypothesize that a highly restricted population of nasal sensory neurons defined by the expression of MrgprC11 detect mast cell mediators in allergic rhinitis and trigger the sneezing reflex. In Aim 1, we will characterize the innervation pattern of MrgprC11-expressing fibers in the nasal mucosa and examine their pathological changes under allergic rhinitis using genetic labeling and axonal tracing approaches. Furthermore, we will determine their physiological responses to a variety of sneeze-inducing molecules using a novel ex vivo calcium-imaging tool. These studies will provide important information on the initial detection of nasal irritants and transduction of sneezing signals. In Aim 2, we will define the role of MrgprC11+ fibers in acute sneezing. We will determine whether ablation of MrgprC11+ neurons attenuates sneezing responses to a variety of nasal irritants and whether selective activation of MrgprC11+ sensory fibers in the nasal mucosa evokes sneezing. These studies will establish whether MrgprC11+ sensory fibers are required for sneezing induced by different sensory stimuli. In Aim 3, we will investigate the neuro-immune interactions between MrgprC11+ nasal sensory fibers and mast cells in allergic rhinitis. We will test whether degranulated mast cells activate MrgprC11+ nasal sensory fibers to induce sneezing in allergic rhinitis. Furthermore, we will determine whether pharmacological silencing of MrgprC11+ sensory fibers is a feasible therapeutic strategy to control sneezing associated with allergic rhinitis. These studies will not only advance our understanding of the neuro-immune interactions that trigger sneezing, but also provide a novel neuronal target for controlling nasal allergic symptoms.