Peter Scheiffele - US grants

DESCRIPTION (provided by applicant): The long-term goal of these studies is to understand the molecular mechanism of synapse formation and target recognition in the central nervous system. The aims of the research proposed in this application are: (1) to dissect the structural basis for the synaptogenic activity of neuroligin, a post-synaptic adhesion molecule that can trigger the assembly of pre-synaptic elements in contacting axons, (2) to identify receptors for neuroligin in the pre-synaptic cell, and (3) to analyze the role of neuroligin in vivo. Aim 1 and Aim 2 will be studied with functional cell-based assays in vitro that permit direct manipulation of the pre- and post-synaptic machinery. The neurexin family of proteins will be studied as candidate neuroligin receptors, but at the same time we will perform an unbiased biochemical purification of all molecules that might associate with neuroligin during synapse formation. To analyze neuroligin function in vivo, hammer-head ribozymes will be generated that specifically degrade neuroligin mRNAs. These ribozymes will be expressed in the cerebellum of mice, either by gene transfer into selected cells with a viral vector or in a transgenic animal. Synapse formation is a crucial process in the generation of neuronal circuits in the brain. Aberrant synapse formation and synaptic dysfunction lead to severe nervous system disorders such as epilepsy, schizophrenia, and mental retardation. Neuroligins themselves have been implicated in mental retardation in humans. Besides nervous system disorders, understanding the mechanism of synapse formation will also be most important for controlling or stimulating the regeneration of synaptic circuits after injury, as for example the connectivity in the spinal cord after injuries that lead to paralysis.

[unreadable] DESCRIPTION (provided by applicant): The development and function of the nervous system relies on its ability to form specific neuronal circuits and to alter their properties in response to neuronal activity and signaling. Spatially restricted modification represents one of the key problems in the regulation of neuronal plasticity. That is, mechanisms need to exist that enable the modification and generation of proteins locally, e.g. changes that occur at only one synapse but not at others. The goal of this proposal is to explore whether alternative splicing of mRNAs occurs in the neuronal cytoplasm and can be locally controlled by synaptic signaling mechanisms. To address this question, we will focus on the alternative splicing of a family of neuronal cell surface receptors, called neurexins, which exist in more than 1,000 functionally different splice variants. Neurexin splicing will be analyzed using a combination of RT-PCR, fluorescent in situ hybridization, and splice-form specific antibodies. The aims of this proposal are (1) to characterize cytoplasmic intron-containing neurexin pre-mRNAs, (2) to analyze the splicing machinery that can remove introns from pre-mRNAs in the neuronal cytoplasm, and (3) to investigate the regulation of neurexin splicing in response to neuronal activity and signaling. Findings from this research will provide twofold contributions: First, they will provide novel insights into the mechanisms and regulation of neurexin protein expression and function. Secondly, the principal mechanisms that control cytoplasmic splicing in neuronal cells are likely not restricted to splicing of neurexin proteins but may also control expression of other neuronal proteins. The characterization of local splicing at synapses would support a novel mechanism for regulating neuronal plasticity and function. The findings from these studies have direct relevance for human health. Neurexin splicing is differentially regulated in ischemia. Moreover, splicing of neurexins regulates the interaction of neurexins with two ligands, neuroligins and alpha-dystroglycan. Both ligands have been implicated in nervous system disorders: neuroligins in mental retardation and autism and alpha-dystroglycan in muscular dystrophies. Information obtained in studies on neurexin splicing will therefore also be valuable for understanding aspects of cellular and molecular defects underlying these disorders. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This project investigates the molecular mechanisms that regulate the dendritic morphology of neurons in the central nervous system. The architecture of dendritic arborizations determines the wiring of synaptic circuits and the integration of synaptic inputs. Therefore, control and dynamic regulation of neuronal morphology are crucial for normal nervous system function. This project focuses on one gene named alpha-chimaerin that is likely to be an important regulator of neuronal morphogenesis. Two a-chimaerin isoforms are expressed in the developing nervous system that function as GTPase activating proteins for Rho-GTPases. It is the goal of this proposal to understand the function and regulation of a-chimaerins in the formation and plasticity of neuronal arbors in mice. The project employs a combination of biochemical, cell biological, and anatomical approaches to investigate the function of these proteins in hippocampal and cerebellar neurons. The proposed experiments will first examine the molecular mechanism of a-chimaerin function in regulating the morphology of dendritic arbors (Aim 1). Subsequently, we will investigate how a-chimaerin is regulated by synaptic activity (Aim 2). Finally, we will generate mutant mice lacking individual or multiple a-chimaerin isoforms and analyze the development of dendritic and axonal arbors in vivo (Aim 3). These studies will investigate a molecular mechanism that links neuronal signaling with the dynamic regulation of cell morphology by Rho-GTPases. These mechanisms are likely to be relevant for the normal development of the nervous system but also for the plasticity of neuronal connections in the adult organism. Structural alterations in dendrites are observed after drug abuse. Alpha-chimaerins are good candidate factors to be relevant for such changes since they are functionally coupled to signaling pathways implicated in addiction. Moreover, defects in a-chimaerins have been proposed to be associated with autism and schizophrenia. Understanding the cellular functions of a-chimaerins is therefore highly relevant for human health. [unreadable] [unreadable]