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Molecular Mechanisms Controlling Development and Dysfunction of the Mammalian Brain
Summary: Nathaniel Heintz is interested in the identification of novel pathways that participate in the development, function, and dysfunction of the mammalian brain.
An understanding of brain development and function must rest upon the investigation of molecular mechanisms that contribute to its histological and functional complexities. One avenue toward identification of these mechanisms has been the isolation and analysis of specific genes that mark critical events in the formation of the central nervous system (CNS). Over the past decade, our molecular genetic screens have identified a variety of genes important for the development, function, and dysfunction of the mammalian nervous system. Studies of these genes have led to the discovery of novel components or pathways that are required for normal metabolic function in specific subsets of CNS precursor cells (Blbp, 10-Fdhfd), proliferation of progenitor cells in the cerebellum and peripheral epithelia (Zipro1), modulation of nicotinic receptors in the brain and periphery (Lynx1), and function and degeneration of cerebellar Purkinje neurons (Lurcher [Grid2], nPIST, Beclin1). It has become increasingly apparent that the diversity of cell types present in the CNS and the complexities of their function must reflect the actions of many thousands of genes expressed in the CNS whose products perform myriad, often subtle, roles. Accordingly, our lab has also developed a suite of novel approaches based on the manipulation of bacterial artificial chromosomes for the investigation of genes, cells, and circuits in vivo and in vitro. These methods are the foundation of a large-scale effort (GENSAT) led by our lab and supported by the National Institute of Neurological Disorders and Stroke (NINDS) to provide a molecular map of the mammalian brain.
Blbp, 10-Fdhfd
Brain lipid-binding protein (Blbp, GC9) and 10-formyltetrahydrofolate dehydrogenase (10-Fdhfd, GC44) were first identified in our lab in the context of a large screen for dynamically regulated genes that mark specific cerebellar cell types (collaboration with Mary E. Hatten, Rockefeller University). Subsequent studies confirmed that these genes are coexpressed in developing Bergmann glia and in radial glial cells at many sites in the developing CNS. Radial glial cells function during CNS development as neural progenitors, although their precise contribution to neurogenesis remains controversial. For example, recent work has argued that regional differences may exist in the neurogenic potential of radial glia. We have resolved this controversy by Cre/loxP fate mapping using the BLBP promoter and clonal analysis, demonstrating that the vast majority of neurons in all brain regions derive from radial glia. Thus, radial glial populations within different CNS regions are not heterogeneous with regard to their potential to generate neurons versus glia. Rather, within different regions of the CNS, radial glia pass through their neurogenic stage of development at distinct time points. We believe that further lineage-tracing studies with 10-Fdhfd will precisely define the neurogenic stage of radial glial development and provide insights into the transition from neurogenesis to gliogenesis in the developing brain.
Lynx1, A Mammalian Prototoxin
Lynx1 (GC26) was also discovered in our screen for developmentally regulated, cell-specific cerebellar cDNAs. It is the founding member of a family of mammalian prototoxin genes that share structured motifs with a large family of elapid snake neurotoxins. Evolutionary studies have shown that the members of this family expressed in the CNS are more closely related to the invertebrate genes than those expressed in the immune system, supporting our hypothesis that the ancestral prototoxin genes performed important modulatory functions in the primitive nervous system. In elapid snakes, it is apparent that those functions have been altered through selection to result in the production of the stable, high-affinity inhibitors of specific neurotransmitter receptors that are characteristic of snake venom. Our most recent functional studies (in collaboration with Steve Sine, Mayo Clinic) also support this idea, demonstrating that lynx1 is a powerful modulator of nicotinic acetylcholine receptors (nAchRs), which display characteristically altered channel properties when coexpressed with lynx1. Consistent with these findings, studies of lynx-knockout mice have revealed several behavioral and anatomic phenotypes that reflect perturbations of nAChR function in the CNS and in the periphery. We are investigating additional mammalian prototoxin genes to determine whether they also might function as important CNS modulatory proteins.
Zipro1 and the Control of Cell Proliferation
Cerebellar granule cell precursors divide over extended periods of development to generate the largest population of CNS neurons. Zipro1 is a zinc finger protein we discovered in a screen for transcription factors that might be involved in this process. We developed the methodology for the manipulation of bacterial artificial chromosomes (BACs) by homologous recombination of Escherichia coli to allow the construction of properly controlled gene dosage experiments to complement the already established knockout technology for analysis of mammalian gene function. Application of this strategy to Zipro1 demonstrated a role for this gene proliferation of granule cells and some populations of peripheral epithelial cell precursors. Gene expression profiling of normal mice and mice carrying increased Zipro1 gene dosage identified the Ski proto-oncogene as a potential target of Zipro1 action.
Our recent studies have confirmed that Ski is a direct target of Zipro1 action, and led to the discovery that Zipro1 is itself regulated in response to the Sdf1/Cxcr4 chemokine signaling pathway. Further analyses of Zipro1-knockout mice have revealed misregulation of Ski expression and alterations of cell proliferation in peripheral epithelia. These studies provide a framework in which to place Zipro1 in the control of cell proliferation in vivo.
Lurcher, Cell Death, and Synaptic Plasticity
The phenotypes of strains carrying spontaneous mouse neurological mutations identify the affected genes as essential for either normal cerebellar development or for its maintenance in mature animals. Lurcher (Lc) is a spontaneous, semidominant mouse neurological mutation. Heterozygous lurcher mice (Lc/+) display ataxia due to the selective, cell-autonomous, and apoptotic death of 90 percent of cerebellar Purkinje cells during postnatal development. The death of homozygous lurcher mice (Lc/Lc) shortly after birth is due to massive loss of mid- and hindbrain neurons during late embryogenesis. By positional cloning, we identified the mutations responsible for neurodegeneration in two independent Lc alleles as identical G-to-A transitions that change a highly conserved alanine to a threonine residue in transmembrane domain III of the mouse δ2 glutamate receptor gene (GluRδ2), resulting in a large, constitutive inward current.
Our recent studies have revealed that cell death in lurcher mice involves multiple pathways. We have recently demonstrated that one key pathway activated in Lc/+ Purkinje cells is autophagy. Autophagy is a pathway for bulk degradation of subcellular constituents that is hyperactivated in many neurodegenerative conditions. It is sometimes considered a second form of programmed cell death. We have identified protein interactions between GluRδ2, a novel isoform of a PDZ domain-containing protein (nPIST) that binds to this receptor and Beclin1. nPIST and Beclin1 can synergize to induce autophagy. GluRδLc, but not GluRδ2wt, can also induce autophagy. Furthermore, dying lurcher Purkinje cells contain morphological hallmarks of autophagic death in vivo. These results provide strong evidence that a direct link exists between the GluRδ2Lc receptor and stimulation of the autophagic pathway in dying lurcher Purkinje cells. They also provide the first insights into molecular mechanisms regulating this critical pathway in neurodegenerative disease.
The Use of BAC-Mediated Transgenesis to Analyze Mammalian CNS Gene Expression and Function
The anatomical complexity of the mammalian CNS presents special problems for the analysis of CNS gene expression and function. The most difficult challenge is that there are thousands of functionally and morphologically defined cell types in the CNS. Given this complexity, the interpretation of CNS phenotypes is often problematic. The preparation of transgenic mice carrying marked BACs provides an important avenue for improving our understanding of CNS-expressed genes and phenotypes. This approach can allow efficient analysis of patterns of gene expression, subcellular localization of their encoded gene products, and mapping of neuronal projection patterns.
BAC transgenic mice can also provide access to information relevant to gene function based on phenotypes arising from increased gene dosage or expression of activating and dominant-negative alleles. Our present studies are aimed at developing combinatorial methods for restricting gene expression to unique cell types in the CNS, at developing additional methodology for manipulation of activities and pathways important for CNS function, and at identifying useful affinity tags for the biochemical characterization of protein complexes from genetically modified animals.
A Large-Scale Screen for Novel CNS-Expressed Genes
The increased precision with which one can collect accurate gene expression data using BAC transgenic mice carrying marked genes suggested to us that this approach could be used to analyze a large number of CNS genes. In collaboration with Mary Beth Hatten and Alexandra Joyner (HHMI, New York University), we have developed the GENSAT Project, a large-scale screen to map gene expression in the mouse brain (funded by the NINDS). This project is composed of an in situ hybridization prescreen to localize the expression of ~1,000 genes in the developing and adult brain (in a collaboration with Tom Curran, St. Jude Children's Research Hospital), followed by BAC transgenic analysis of the most interesting 25 percent of these genes. The data from this project are entered into a publicly available database being developed at the National Institutes of Health. Although the immediate aim of this project is to provide precise information concerning gene expression in CNS cell types, the BAC vectors and BAC transgenic animals generated as part of GENSAT are made available to the neuroscience community for additional physiologic and genetic studies.
Last updated: September 14, 2006
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