EXTRACELLULAR FACTORS AFFECTING NEURON DEVELOPMENT
My laboratory studies several interrelated topics in developmental neurobiology: neuronal survival, axon growth and pathfinding, and synapse formation. Trophic factors regulate each of these processes. Adhesion-promoting molecules in the extracellular matrix and on the surfaces of cells regulate axonal growth and differentiation at synapses. Some of these proteins are also important for maintaining the structure and function of the mature nervous system. Several may affect the progression of neurodegenerative diseases.
Neurotrophic Factor Regulation of Neuronal Development and Function
Neurons typically require contact with targets to survive during development and target organs have been shown to synthesize trophic factors that promote their survival. Focusing on a family of four factors named neurotrophins, we have shown that these proteins are essential for normal neural development of many populations of sensory neurons. Laboratory members have identified several transcription factors expressed in neurons that control expression of the neurotrophin receptors of the trk receptor tyrosine kinase family. Understanding the regulatory circuits that induce different neurons to express different receptors and thereby acquire responsiveness to different neurotrophins is central to understanding how interactions between neurons and their targets results in a normal nervous system.
Neurotrophins and their receptors continue to be expressed in the mature brain. Our observations indicate that neurotrophin signaling is essential in the adult brain to prevent neuronal degeneration, suggesting that deficiencies in neurotrophin signaling may contribute to disease-induced neurodegeneration. Examining mice with reduced levels of neurotrophin-mediated signaling, we have observed multiple behavioral abnormalities, including aggressiveness, hyperactivity, anxiety, and obesity. These observations indicate that neurotrophin signaling is essential for normal function of the brain circuits controlling these behaviors and the laboratory is trying to identify the loci within the brain where neurotrophin signaling controls each of these behaviors. As one example, reductions in TrkB-mediated signaling result in a robust obesity phenotype where mice are not normally satiated during meals. The locus of this phenotype has been traced to a subnucleus of the hypothalamus previously implicated in control of obesity. Neurons in this subnucleus express BDNF, a ligand for TrkB, and expression of BDNF in this nucleus is regulated by perturbations of feeding as well as by activators of the melanocortin-4 receptor. BDNF in turn suppresses some of the abnormal feeding responses observed in mice lacking melanocortin-4 receptor signaling. In summary, the phenotypes of these animals indicate that neurotrophins are important for normal function and animal behavior.
Neurotrophins also mediate the efficiency of synaptic communication. Dr. Beatriz Rico has utilized the mice lacking trkB in some, but not all cells to examine the functions of neurotrophins in regulating synapse formation in the cerebellum. She has shown that absence of TrkB-mediated signaling results in severe deficits in inhibitory interneuron function. These neurons have deficiencies in GABA synthesis and transport and form many fewer synapses than in normal mice. Similar studies in the hippocampus and retina have also revealed that neurotrophins have important modulatory roles in assuring appropriate synaptic interactions within the mature as well as developing brain.
Integrin-dependent Signaling Pathways in Neuronal Development
Our laboratory has devoted much effort to identifying cues that regulate and direct axonal growth, and has focused recently on signaling events activated by cell adhesion molecules that affect the movements of growth cones. Members of a particularly interesting family of adhesion-promoting receptors important in development of many types of neurons are named integrins. These are heterodimeric receptors with ligands including both extracellular matrix and cell surface-associated glycoproteins. Dr. Zhen Huang has collaborated with the laboratory of Dr. Ulrich Müller (Scripps Institute) to analyse the functions of b1integrins in cortical development. Loss of beta1 integrins results in severed deficits in migration of neurons, resulting in a phenotype resembling lissencephaly. In collaboration with the laboratories of Drs. Shoukhat Dedhar Kevin Jones, and Klaus-Armin Nave (Universities of British Columbia, Colorado and Heidelberg), Drs. Agnieszka Niewmierzycka and Hilary Beggs have shown that absence of either of two tyrosine kinases associated with integrins named Integrin-linked Kinase (ILK) and Focal Adhesion Kinase (FAK) result in very similar phenotypes. In each case, the defect results from deficient basal lamina assembly on the surface of the developing brain and appears to reflect a requirement for each protein to regulate rearrangements of the actin cytoskeleton within cells that in turn is essential for normal basal lamina assembly. Thus this work represents a step forward in characterizing integrin signaling pathways essential for normal brain development. In collaborative work with the laboratory of Michael Stryker, the absence of FAK has been shown to result in severe perturbations of visual representations in the cerebral cortex. This suggests that FAK is involved in the signaling pathways controlling formation of the axon projections that establish these representations of visual space. In collaboration with the laboratory of Dr. Yi Rao (Northwestern University) we have shown that FAK function is required for normal axon growth and guidance responses to netrin, one of the axon guidance molecules important within the brain. The laboratory is trying to determine more precisely where misrouting of axons occurs and to identify additional pathways involved in growth cone motility that are perturbed by absence of FAK signaling.
In related experiments, the laboratory has also characterized the role of the integrin alphaVbeta8 in controlling development of the CNS. This integrin is expressed in the developing neuroepithelium and its absence results in massive hemorrhage from the developing CNS vasculature. John Proctor has demonstrated that this integrin functions in the neuroepithelium to control vascular development through use of cell-specific deletion of the integrin using a neuroepithelial cre line. He has also observed later degenerative responses in the CNS of mice that survive perinatal hemorrhage. This appears to reflect a distinct requirement for this integrin in maintenance of neuronal health and viability. The laboratory is attempting to characterize the signaling pathways through which this integrin controls vascular development and neuronal viability.
Cadherin-dependent Signaling Pathways in Neuronal Development
A second family of of adhesion-promoting receptors is named cadherins. A very large number of different cadherins are expressed in the brain where they have been postulated to regulate axon and dendrite growth and synapse formation. Dr. Tamira Elul has interfered with cadherin function in Xenopus embryos by expressing fragments of an essential linker between cadherins and the cytoskeleton named b-catenin. Disruption of this linkage does not affect axon growth from the retina to the optic tectum, but severely affects the guidance and branching of these axons within the tectum, thereby interfering with synapse formation. In collaboration with the laboratories of Drs. Walter Birchmeier (Berlin) and Bai Lu (N.I.H.), Dr. Shernaz Bamji has examined effects of genetic deletion of b-catenin in the hippocampus and has observed severe perturbations in formation of synapses. b-catenin functions as a scaffold anchored by cadherins that recruits synaptic vesicles and other proteins essential for normal synaptic function. A second protein associated with cadherins is named p120ctn. p120ctn stabilizes cadherins on the surfaces of cells and also controls the localization and activities of a tyrosine kinase Fer and of small G proteins of the Rho family that regulate the cytoskeleton. Dr. Lisa Elia has demonstrated that the presence of this protein is essential for formation of synaptic spines and synapses on dendrites in vivo and in vitro. In its absence, the forebrain contains less than half the normal density of synapses. Absence of p120ctn results in reduced cadherin levels, elevated levels of active rho and reduced levels of active rac. The reduction in spine density appears to reflect alterations in rho activity, while deficits in spine maturation may be caused by changes in cadherin levels. Our studies on b-catenin and p120ctn focus on identification of the signaling pathways through which they control differentiation and synapse formation by neurons.
Integrin signaling in metanephric kidney development
Several years ago, we observed surprisingly that deletion of an RGD-family integrin named alpha8beta1 that my laboratory had characterized as an integrin with prominent expression in the CNS resulted in deficits in formation of the metanephric kidney. The laboratory demonstrated that this integrin is expressed in the metanephric mesenchyme, but affects initial ingrowth of the ureteric bud, suggesting that it controls aspects of signaling from the mesenchyme to the bud. The laboratory identified a novel ligand for this integrin named nephronectin that is expressed by the ureteric bud and is secreted into the basal lamina between the bud and mesenchyme. We have been pursuing genetic studies to understand the role of this ligand in metanephric kidney development. We have also be characterizing pathways through which the integrin alpha8beta1 controls signaling from the metanephric mesenchyme to the ureteric bud.