Core Faculty

Louis Reichardt, Ph.D.

Control of Neuronal Development and Synapse Formation

Research Description

Extrecellular 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.

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Current Projects

Neurotrophic Factors and Neural Development

Neurotrophic factors are proteins which function to regulate survival of neurons. In past reports, the laboratory has reported on the essential functions of several of these, including two of the four mammalian neurotrophins BDNF and NT-3, and the TGF-b family member GDNF. Recent work has extended these observations by isolating new mouse mutants and by characterizing in more detail deficits in previously isolated mutant strains.

In the absence of NT-3, mice have much more extensive losses of neurons than observed in the absence of trkC, the the cognate NT-3 receptor, and lose sensory and sympathetic neurons that in postnatal life require NGF or BDNF. In analyses of trigeminal and spinal sensory ganglia development, Isabel Fariñas and George Wilkinson have shown that NT-3 deficiency results in death of newly formed neurons before contact with their final targets. In more recent work, using antibodies specific for the neurotrophin receptors trkA, B, or C, NT-3 has been shown to promote survival of newly formed neurons expressing trkB in addition to trkC in spinal sensory ganglia and of neurons expressing trkA as well as trkC in trigeminal ganglia even though NGF is the cognate ligand for trkA and BDNF and NT-4 the ligands for trkB. Thus, in certain circumstances, NT-3 can activate each of the three trk receptors in vivo and has the properties expected of a survival factor that provides transient support for newly born neurons before their axons contact their final targets. In collaboration with Kevin Jones (University of Colorado), Ardem Patapoutian has shown using a lacZ reporter that BDNF is not expressed adjacent to sensory ganglia during the period of neurogenesis, explaining why NT-3 must be present to prevent apoptosis of trkB expressing neurons. In contrast, using a similar lacZ reporter, NT-3 is strongly expressed during this period in mesenchymal cells adjacent to the ganglia, but expression is lost as the mesenchyme further differentiates. As NT-3 plays a very general, but transient role in sensory ganglia genesis, it is critical to understand its regulation. In the last report, it was suggested that release by neurons of neuregulins was important in regulating the expression of NT-3. More recent work by Ardem Patapoutian indicates that NT-3 is expressed in a normal spatial and temporal pattern in absence of neural innervation and that, instead, interactions between epithelium and mesenchyme, mediated in large part by wnt proteins expressed in the epithelium, regulate the initial induction of NT-3.

Analyses of sensory neuron and precursor pools in the NGF and NT-3 mutants has indicated that loss of neurons results in premature conversion of precursors to neurons, reducing indirectly the numbers of neurons of all types ultimately generated. The neurotrophins do not appear to act directly on the precursors, because none of the trk receptors can be detected in these cells and because neither their proliferation nor apoptosis is altered by neurotrophin absence. Instead neuronal loss reduces interactions of neurons with precursors that sustain the precursor pool. Interactions mediated by notch and notch ligands are leading candidates to explain these observations. In addition, loss of a particular class of neurons appears to result in compensatory changes within sensory ganglia at later stages as reflected in proportions of neurons expressing individual trk receptors.

In efforts to understand the regulatory circuits governing expression of individual trk receptors within sensory neurons, Eric Huang has focused on transcription factors expressed within neuronal precursors. Interestingly, he has shown that trkC-expressing neurons are never observed in the absence of the pou domain-containing transcription factor brn3a. TrkA and trkB populations are born normally and survive initially, but are subsequently lost. Losses of trkA and B proteins from the neurons in vivo and loss of neurotrophin responsiveness in vitro precede apoptosis in vivo, so this transcription factor appears essential for maintenance of trkA and B while it is required for initiation of trkC expression. A novel transcription factor that interacts with brn3a has been identified and its roles in brn3a action and in sensory neuron differentiation are being characterized.

As mice deficient in neurotrophin or trk expression die shortly after birth, Baoji Xu has generated a mouse strain in which trkB can be eliminated in some, but not all tissues by regulated expression of a recombinase. BDNF signaling through trkB appears to regulate development of many classes of CNS neurons, particularly retinal photoreceptors and amacrine cells and inhibitory interneurons in cortex and hippocampus, and to modulate plasticity at several synapses, including LTP in the hippocampus and axon terminal sorting during the "critical period" in the visual cortex. Transgenic strains expressing the recombinase in these cell populations have been generated and are being used to examine functions of trkB within these neurons. Among early findings are: (1) reduction in TrkB protein levels throughout the hippocampus has no discernable effect on hippocampal neuron survival or morphology, but does affect presynaptic function of Schaeffer collateral terminals on CA1 pyramidal neurons. The reduction also reduces the magnitude of long term potentiation at this synapse. (2) complete elimination of TrkB within CA1 pyramidal neurons does not affect their survival, morphology or LTP at the CA3-CA1 synapse, implying a presynaptic locus for modulation of LTP by BDNF. (3) elimination of TrkB within neocortical pyramidal neurons results in cortical degeneration. Initially, dendritic trees of neurons are affected. Eventually, subpopulations of pyramidal neurons are actually lost. For some neurons, loss of TrkB appears to directly affect cell survival. For others, the effects appear to be indirect, possibly reflecting changes in neuronal circuitry.

While expression of an appropriate trk receptor is essential to confer neurotrophin responsiveness, results of a collaboration between George Wilkinson and the laboratory of Ben Barres (Stanford University) indicate that membrane localization of trk receptors is regulated by Ca2+ and other second messengers and suggests that membrane trafficking may be an important determinant of neurotrophin responsiveness. Using antibodies to the extracellular domain of trkB, Retinal Ganglion Cells and motoneurons were shown to express trkB primarily in intracellular compartments in absence of cAMP and to be comparatively unresponsive to BDNF. cAMP application resulted in translocation of trkB to the cell surface and a dramatic increase in responsiveness to BDNF. Regulation of trk surface expression could, in principle, be important in restricting neurotrophin actions and thereby help differentiate electrically active from inactive synapses and neurons.

A past report described the phenotype of mice lacking the trophic factor GDNF. More recently in collaboration with Arnon Rosenthal (Genentech), Isabel Fariñas has characterized the phenotype of mice lacking the GDNF binding subunit GFR-a 1which associates with the c-ret tyrosine kinase to mediate GDNF actions. While mice share major features of the deficiencies observed in the GDNF and c-ret mutants, such as absence of the metanephric kidney and enteric nervous system, some sensory and sympathetic neurons lost in the GDNF mutant persist in the GFR-a 1 mutant, indicating that GDNF must act, in part, via a second binding protein.

Adhesion molecule functions in neurons and other cells

The laboratory continues to characterize functions of integrins expressed in the developing nervous system, particularly those that bind ligands containing an RGD sequence as data suggests that these integrins regulate neural crest migration and synaptic plasticity. Recent laboratory efforts have focused on two of these named a 8b 1 and a Vb 8, both of which are prominently expressed in the nervous system and have been detected at synapses within the hippocampus. Alleles containing a mutated b 8 subunit have been generated and introduced into mice, but the resulting phenotype has not be characterized. Despite its prominent neuronal localization, when the a 8 gene was disrupted, the major phenotype was absence of normal kidney morphogenesis. Ulrich Müller and Sumiko Denda have shown that a 8 is expressed by embryonic mesenchymal cells in response to epithelial cell-derived inducers and that its absence results in deficient signaling from the mesenchyme to the ureteric bud epithelium and vice versa.. The mechanism by which a deficiency in a mesenchymal integrin disrupts the response of epithelial cells which do not express this integrin is not understood. As GDNF signaling via the GFRa -1/c-ret receptor complex mediates mesenchymal induction of ureteric ingrowth, possible deficits in this signaling pathway are being characterized. As integrins are most commonly activated by ligand binding and none of the known ligands of a 8b 1 had an appropriate expression pattern to mediate its interactions in the developing kidney, a screen for novel ligands was initiated using an a 8b 1-alkaline phosphatase chimera. The first ligand to be identified was osteopontin, but this is not the ligand detected on the surface of ureteric epithelial cells using the a 8b 1-alkaline phosphatase chimera. Ralph Brandenberger has recently identified a novel ligand which is bound by a 8b 1 with unusual avidity. This protein is expressed by ureteric epithelial cells during kidney morphogenesis and is therefore a strong candidate to be required for normal kidney morphogenesis.

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 in vitro and in animals. In the last report, results of experiments in which disruption of linkages between integrins and the actin cytoskeleton prevented stabilization, but not initiation of motile process formation were described. In more recent work, agents that inhibit activity of the cytoplasmic tyrosine kinase named pp125FAK, which is regulated by integrins, have been shown to inhibit initiation of motile process formation. Thus integrins appear to regulate more than one pathway required for normal cell motility.

In past work, the laboratory showed that the cytoplasmic domains of cadherins are important for mediating cadherin -dependent neurite outgrowth and that trkA, the major signal-mediating receptor for NGF, phosphorylates a cytoplasmic protein named b -catenin that links cadherins to the actin cytoskeleton. The laboratory also identified a phosphatase whose association with the cadherin-catenin complex is regulated by trkA which dephosphorylates b -catenin. More recent work by Ravinder Sehgal has shown that regulated association of a -catenin with b -catenin is crucial for normal early development and for morphogenetic interactions mediated by the wnt family of differentiation factors. As cadherins have recently been implicated in synaptogenesis in addition to neurite outgrowth, and the association between b -catenin and a -catenin appears to regulate cadherin function, consequences of preventing normal regulation of this interaction are currently under investigation for effects on both growth cone motility and synapse formation and stability.

Synapse Formation and Function

Baerbel Rohrer has been examining development of innervation of the optic tectum by terminals of retinal ganglion cells by characterizing distributions during development of chimeric proteins targeted to organelles concentrated within the synapse. Using confocal videomicroscopy she is determining effects of neurotrophic factors and electrical activity on development of these synaptic terminals. Robin Kleiman is examining pathways activated by neurotrophins which modulate synaptic efficiency at the neuromuscular junction by introducing into Xenopus embryos mutations in receptors or signal-transducing molecules that interfere with intracellular pathways known to be activated by trk receptors.

List of Abbreviations
BDNF, brain-derived neurotrophic factor
FAK, focal-adhesion kinase
GDNF, glial-derived neurotrophic factor
GFR, glial-derived neurotrophic factor family receptor
LTP, long term potentiation
NGF, nerve growth factor
NT-3, neurotrophin-3 NT-4, neurotrophin-4
RGD, arginine-glycine-aspartic acid
trk, tropomyosin-related kinase

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Selected Publications

Xu, B., Goulding, E., Zang, K., Cepoi, D., Cone, R.D., Jones, K.R., Tecott, L.H., and Reichardt, L.F. 2003. Brain-derived Neurotrophic Factor is a Component of the Melanocortin-4 Receptor Pathway in the Regulation of Energy Balance. Nature Neuroscience 6: 736-742.

Beggs, H.E., Schahin-Reed, D., Sretavan, D., Zang, K., Jones, K.R. Goebbels, S., Nave, K.-A., and Reichardt, L.F. 2003. FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies. Neuron 40: 501-514.

Bamji, S.X., Shimazu, K., Kimes, N., Huelsken, J., Birchmeier, W., Lu, B., and Reichardt, L.F. 2003. Role of b -catenin in synaptic vesicle localization and presynaptic assembly. Neuron 40: 719-731.

Rico, B., Beggs, H.E., Schahin-Reed, D., Kimes, N., Schmidt, A. and Reichardt. 2004. Control of axonal branching and synapse formation by focal adhesion kinase. Nature Neuroscience 7: 1059-1069.

Niewmierzycka, A., Mills, J., St.-Arnaud, R., Dedhar, S., and Reichardt, L.F. 2005. Integrin linked kinase (ILK) deletion from mouse cortex results in cortical lamination defects resembling cobblestone lissencephaly. J. Neurosci. 25: 7022-31.

Huang, Z., Zang, K., and Reichardt, L.F. 2005. Dendrite and dendritic spine development regulated by the origin recognition core complex. J. Cell Biol. 170: 527-35.

Proctor,J.M., Zang, K., Wang, D., Wang, R., and Reichardt, L.F. 2005. Vascular Development of the Brain Requires b 8 Integrin Expression in the Neuroepithelium. J. Neuroscience 25: 9940-48.

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Contact Information

Email: louis.reichardt@ucsf.edu
Phone: 415-476-8503
Mailind Address:
Genetics, Development & Behavioral Sciences
1550 4th Street
Box 2611
San Francisco, CA 94143-2611
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