Renping Zhou - US grants

During the development of the nervous system, neurons undergo a number of processes, including proliferation and differentiation. The purpose of this research is to study the role of a growth factor receptor-like molecule (brain specific kinase, Bsk) in neuronal differentiation. Growth factors and their receptors are known to induce neuronal differentiation and support the survival of neurons, and Bsk has been shown to be associated with neurons in the limbic system of the brain during development. The studies will, first, investigate the timing of the production of Bsk by neurons in the limbic system, and then will attempt to modify the timing of its production by introducing into limbic system neurons an altered Bsk gene. The results of these studies will further our understanding of the development of the limbic system as well as the role of growth factors in neural development.

The long term goal of this study is to elucidate the molecular mechanisms of axonal targeting in the hippocamposeptal system. It has been proposed by Roger Sperry that topographic mapping is accomplished by matching gradients of chemoaffinity labels on the presynaptic and postsynaptic neurons. Consistent with this proposal, the pirncipal investigator has shown that Bsk, an eph family receptor, and its ligand are expressed in complementary gradients in the hippocampus and the target, lateral septum in the adult brain. Furthermore, he has shown one of these ligands, Elf-1, selectively inhibited the growth of topographically incorrect neurites, but allowed the growth of correct hippocampal neurites. These observation sled to the hypothesis that eph family receptors and ligands serve as chemoaffinity labels for the hippocamposeptal topograph mapping. Several predictions can be made based on this hypothesis: 1) the eph family ligands and receptors are expressed in propoer gradients at the time of topographic maping; 2) the ligands inhibit or support the growth of axons from appropriate regions of the hippocampus in vitro; 3) changing the gradients of expression disturbs the topographic projection in vivo. To test these predictions, the investigator proposed to examine the spatial and temporal patterns of expression of eph ligands and receptors in the hippocamposeptal system and study the biological actions of the ligands on the hippocampal neurons in vitro. In addition, as critical tests for the roles of expression gradients of the eph molecules, the applicant plans to take two complementary approaches to alter Bsk expression and function in vivo. First, he will inject a soluble eph ligand, A1-1/RAGS, which serves as an inhibitor for Bsk or related receptors, into developing mouse brain. Second, he will overexpress Bsk using Talphal neuron-specific tublin promoter, which generates high levels of uniform expression, in transgenic mice. The effects of these alterations on the hippocampal topographic projection will be examined using axonal tracing techniques. These studies will help to understand how the output pathways of the hippocampus are organized and may shed light on mechanisms of learning and memory, as well as diseases affecting these processes.

Topographic projection is a general feature of brain architecture, and appears to be critical for appropriate coding and processing of information. The hippocampus and septum, which have been the focus of intense interest, since these structures play a central roles in learning and memory, are interconnected topographically. Although their topographic connections have been known for nearly two decades, and the topography may be critical for cognitive functions, the molecular basis for the topographic projection is unknown. The long term goal of this study is to elucidate the molecular mechanisms of axonal targeting in the hippocamposeptal system. It has been proposed by Roger Sperry that topographic mapping is accomplished by matching gradients of chemoaffinity labels on the pre-synaptic and postsynaptic neurons. Consistent with this proposal, we have shown that Bsk, an eph family receptor, and its ligands are expressed in complementary gradients in the hippocampus and the target, lateral septum in the adult brain. Furthermore, we have shown that, one of the ligands, Elf-1, selectively inhibited the growth of topographically incorrect but allowed the growth of hippocampal neurites. These observations led to the hypothesis that eph family receptors and ligands serve as chemoaffinity labels for the hippocamposeptal topographic mapping. Several predictions can be made based on this hypothesis: 1, the eph family ligands and receptors are expressed in proper gradients at the time of topographic mapping; 2, the ligands inhibit or support the growth of axons from appropriate regions of the hippocampus in vitro; 3. changing the gradients of expression disturbs the topographic projection in vivo. to test these predictions, we propose to examine the spatial and temporal patterns of expression of eph ligands and receptors in the hippocamposeptal system, and study the biological actions of the ligands on the hippocampal neurons in vitro. In additions, as critical tests for the roles of expression gradients of the eph molecules, we plan to take two complementary approaches to alter Bsk expression and function in vivo. First, we will inject a soluble eph ligand, A1-1/rags, which serves as an inhibitor for Bsk or related receptors, into developing mouse brains; Second, we will over-express Bsk using Talpha1 neuron-specific tubulin promoter, which generates high levels of uniform expression, in transgenic mice. The effect of these alterations on the hippocamposeptal topographic projection will be examined using axonal tracing techniques. These studies will help to understand how the output pathways of the hippocampus are organized and may shed light on the mechanism of learning and memory, as well as diseases affecting these processes.

DESCRIPTION (Applicant's Abstract): The long term goal of this study is to elucidate the molecular mechanisms of axonal targeting during development and plasticity in the adult in the mesencephalic dopaminergic system, which plays key roles in mediating drug dependence and brain reward. The neural structures in the dopamine system are interconnected topographically. Dopaminergic neuron in the ventral tegmental area (VTA) project primarily to the ventromedial striatum (which include the nucleus accumbens, olfactory tubercle and ventromedial portion of the caudate putamen), while neurons in the substantia nigra (SN) project to dorsolateral striatum (which corresponds to dorsolateral caudate putamen). Although the topographic relations have been known for nearly two decades, and appear to be critical for physical and psychological dependence on drugs, the molecular basis for this topographic projection is unknown. It has been proposed that topographic mapping is accomplished by matching gradients of chemoaffinity labels on the presynaptic and postsynaptic neurons. Consistent with this proposal, we have shown Elk, and Eph family receptor, and its ligand, Lerk5, are expressed in complementary patterns in the ventral tegmental area/substantial nigra, and the targets, nucleus accumbens/caudate-putamen. Furthermore, we have shown that Lerk5 is unregulated by cocaine treatment in adult mice. Previous studies conducted in our laboratory and in others have shown that interactions between Eph ligands and receptors expressed in complementary gradients specify the topographic projection maps in the brain. Together, these observations led to the hypothesis that Elk and its ligand regulate axonal targeting and plasticity in the ascending dopaminergic system. To test this hypothesis and examine the roles of Elk and Lerk5 in the ontogeny of the dopamine system, we propose to first examine the spatial and temporal patterns of expression of Elk and its ligand during the development of projection. We will also study the biological effects of Lerk5 on the VTA/SN dopaminergic neurons in vitro using a co-culture assay we have developed. Furthermore, the expression of Elk and Lerk5 expression in vitro will be altered using transgenic technology to examine the effects on the development of the ascending dopamine system. To study whether Elk and Lerk5 also play roles in drug-induced plasticity, we will examine how drug treatment alters their expression in the adult. Understanding the molecular basis governing the axonal connections and modifications may shed new light on the underlying mechanism of drug addiction, as well as diseases involving this system such as Parkinson's disease.

IBN 0095040
Zhou, Renping

Title: Regulation of Contralateral Cortical Projection by the EphA Receptors

The human brain has been considered the most complex object in the universe known to man. Trillions of neurons are wired together precisely through thousands of times more connections. In this immensely complicated network, neurons are interconnected through wire-like processes called axons. How does this complex organ develop? What are the molecular signals that direct the formation of axon pathways and guide the growing axon tip toward proper target? These questions have fascinated scientists for centuries. Recent studies from our laboratory and the laboratories of others indicated that a family of cell surface molecules, termed receptors, that sense extracellular signals, play key roles in guiding axons. This project utilizes several molecular techniques to examine how the Eph receptors and their signaling partners regulate the formation of corpus callosum, an axon fiber that connects the two cerebral hemispheres, and is important for left and right coordination. Experiments examine where these molecules are located, how they affect axon growth and migration, and what are the effects on corpus callosum formation in development when these molecules are inactivated. Results from these studies will reveal the molecular mechanism of axon guidance and help to understand how this extremely complex organ, the brain, is wired together.

Nerve fibers (axons) are guided to make appropriate connections during development of the nervous system. The guidance factors can be either attractive or repulsive to the axons. The Ephrin family molecules and their Eph receptors often function as repulsive guidance cues and induce axon retraction. The molecular mechanisms underlying repulsive axon guidance remain largely unknown. The long-term objective of this project is to elucidate the molecular pathways underlying axon retraction, using Eph receptors as model systems. Eph receptors are tyrosine kinases, and transmit signals through phosphotyrosine (P-Tyr)-dependent or independent coupling of downstream molecules. However, roles of molecular cascades coupled through P-Tyr binding in axon retraction have not been well investigated. In the preliminary studies, a candidate signaling protein, a phosphotyrosine phosphatase, termed Shp2, which contain the Src homology 2-domain known to bind to P-Tyr, has been isolated. It is hypothesized that Shp2, coupled through P-Tyr-binding to activated EphA receptors, initiates critical signaling cascades important for axon retraction induced by the Ephrins. To test this hypothesis, experiments are proposed to (1) identify specific phosphotyrosine residues that are important for Shp2 binding and axon repulsion, using mutagenesis and biochemical analyses; (2) Test whether Shp2 is required for EphA receptor-mediated biochemical and biological responses. To further define functions of Shp2, the roles of Shp2 effector Src in Ephrin-induced repulsive guidance will be examined. Several approaches, including expression of dominant-negative and constitutively active mutants and assaying Ephrin functions in gene-deleted neurons, will be used to test roles of Shp2 in EphA receptor-mediated repulsive axon guidance.

The intellectual merits of proposed experiments lie in addressing an important area of axon guidance mechanisms, namely, functions of the P-Tyr-docked pathways, which have not been well investigated in axon guidance. The proposed studies are likely to identify novel signaling mechanisms mediating Ephrin-induced repulsive axon guidance. Since tyrosine kinases and phosphatases have been shown to play important roles in other axon guidance systems and possibly axon guidance in general, these studies are likely to significantly enhance understanding of how axons are guided, and may provide insights in future interventions to reconnect damaged neural circuits.

The proposed studies will also have a broader impact. These studies will help generate viral vectors carrying various signaling proteins, which will be made available to other investigators. In addition, the proposed studies will train students and postdoctoral fellows. The PI also participates in the American Chemical Society SEED Program. This program provides training opportunities for minority high school students. Three minority high school students have performed summer research in the PI's laboratory in the past three years. The proposed studies will allow continued exposure for minority students to research experience in a laboratory environment.

Axons travel frequently in bundles to reach their target. Upon arriving at the target, axon terminals defasciculate, migrate to topographicaJly defined positions, and form synapses with appropriate target neurons. Molecular signals regulating these processes are not well understood. Our previous studies indicate that members of the Eph family tyrosine kinase receptors, and their ligands, the ephdns, play critical roles in regulating hippocampat projections. The Eph receptors consist of EphA and EphB subfamilies. Our studies show that the EphA receptors and ligands are expressed in opposing gradients in the hippocampus and its major subcortical target, the lateral septum, respectively, and interactions between the receptor and the ligand gradients define topographic positions of hippocampal axon terminals. In contrast, our preliminary studies showed that a member of the EphB-type receptors, EphB2, is expressed uniformly in the hippocampus, and a ligand, ephrin-B3, is transcribed uniformly in the septal target. Exposure of ephrin-B3 leads to the dispersion of hippocampal neurons and defasciculation of hippocampal axons. Mice missing the EphB2 receptor show abnormal axon bundling in the septum. These observations tend to the hypothesis that EphA and EphB receptors and their cognate ligands are functionally distinct in regulating hippocampal axon targeting to the lateral septum, with the A-type as topographic mapping tags and the B-type as modulators of axon defasciculation. To test this hypothesis, we propose to (1) Elucidate the spatial and temporal expression of EphB receptors and ligands. (2) Delineate effects of B-ephdns on hippocampal axons in vitro. (3) Analyze effects of inactivation of EphB receptors and ligands on hippocampal axon defasciculation in vivo. (4) Examine the molecular mechanisms of Eph receptor functions. The proposed studies will provide new insights into the molecular mechanisms of axon guidance, which may facilitate development of strategies to regenerate neural circuits after injuries or in diseases such as Alzheimer's and Parkinson's diseases.

DESCRIPTION (provided by applicant): Lens transparency is made possible by a combination of the highly ordered organization of the lens fiber cells, their unique refractive index, and the lack of organelles in the fiber cells. The highly ordered arrangement of the lens fiber cells is critical for proper light transmission, and disruption of this structure by alterations of cell-cell interactions is likely to lead to cataracts. However, signals that regulate lens fiber cell interaction remain largely unknown. Our preliminary studies have identified a new class of molecules, the Eph tyrosine kinase receptor family that regulates lens cell organization. Inactivation of ephrin-A5, a ligand of the Eph receptors, leads to the disruption of N-cadherin localization, change in lens fiber cell shape, disorganization of lens cells, and the development of cataracts. We hypothesize that ephrin-A5, interacting with its receptor(s), regulates N-cadherin-mediated fiber cell adhesion to maintain proper lens cell organization. To test this hypothesis, we will: (1) Examine the spatial and temporal characteristics of the ephrin-A5-null lens, determine when and where during development the lens defects first occur, and whether the loss of ephrin-A5 results in disruptions of lens fiber cell differentiation. The morphology of the lens at different developmental stages will be analyzed using both light and electron microscope techniques. Antibodies against markers of lens cell differentiation will be used in immunohistochemical experiments to study the expression of differentiation markers. (2) Elucidate receptor mechanisms of ephrin-A5 in lens development by examining which specific Eph receptors are expressed in the developing lens and where they are expressed, using Real-Time PCR, in situ hybridization, and immunohistochemistry. Since the interaction between Eph receptors and ligands leads to bidirectional signaling, we plan to analyze whether receptor- mediated signaling, the ligand-mediated reverse signaling or both are required for lens development using selective inactivation of different receptor domains. (3) Study the molecular alterations that lead to cataracts in ephrin-A5-null mice. Preliminary studies have revealed a disruption of N-cadherin distribution in the lens fiber cells. We will determine whether ephrin-A5 receptors interact physically with adherens junction molecules, and analyze effects of the ligand on N-cadherin functions. To critically evaluate roles of N-cadherin in mediating ephrin-A5 function and lens cell adhesion, we plan also to examine expression of a N-cadherin-2-catenin fusion protein in a phenotypic rescue experiment. The proposed studies will establish roles of a previously unsuspected family of molecules in lens development and reveal novel regulations of N-cadherin functions. These studies will enhance our understanding of how lens cell interaction is regulated to ensure lens transparency and provide insights into the mechanisms of cataractogenesis. PUBLIC HEALTH RELEVANCE: Cataracts are a leading cause of blindness. The molecular mechanisms underlie cataractogenesis are incompletely understood. The proposed studies will elucidate molecular mechanisms by which defects in ephrin-A5 signaling lead to cataracts and provide insights into future prevention and treatment of human cataracts.