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Establishment of Neuronal Connectivity
Summary: Alex Kolodkin is investigating how families of invertebrate and vertebrate guidance cues direct the establishment and maintenance of neuronal circuits during development and in the adult nervous system.
The establishment, maintenance, and repair of neuronal connections depend on coordinated neuronal responses to a variety of guidance cues. Our research is focused on understanding how connectivity in the nervous system is initially generated during neural development, how these connections are modified in the adult to provide plasticity, and how these or compensatory connections can be reestablished following neuronal injury or degeneration.
What are the extrinsic and intrinsic factors that mediate repulsive guidance events? Several families of guidance cues and their receptors sculpt developing neural circuits. One of the largest is the phylogenetically conserved semaphorin family of proteins, several members of which are robust neuronal repellents. Our work on the development of neuromuscular connectivity in the fruit fly Drosophila has allowed us to define the role of semaphorin-mediated repulsion in axonal pathfinding, to understand how the output of semaphorin repulsive receptors is modulated, to elucidate intracellular signaling cascades that steer growth cones in response to these guidance cues, and to address how repulsive and attractive guidance cue signaling is coordinated. Our work on rodent models of neural development and regeneration has helped us to define novel semaphorin receptors, to understand the logic by which semaphorin guidance cues establish highly specific neuronal connectivity patterns in a variety of neural systems, to uncover connections between neural and vascular development, and also to address the roles played by repellents in adult neuronal regeneration and synapse function.
To address how neuronal pathways are formed during development, we are investigating how a transmembrane semaphorin called Sema1a and its receptor, plexin A, influence motor axon guidance in Drosophila. This work provides us with experimental paradigms for genetic screens designed to identify intracellular signaling pathways essential for contact-mediated repulsive growth cone steering. For example, our genetic and biochemical approaches raise the novel possibility that repulsive signaling in both invertebrates and vertebrates is regulated by redox signaling.
Using Drosophila as a model system to investigate growth cone guidance mechanisms, we previously showed that a phylogenetically conserved multidomain cytosolic protein called MICAL (molecule that interacts with CasL) is essential for repulsive guidance and may underlie inhibition of neuronal regeneration in the adult mammalian nervous system. We are using Drosophila MICAL to purify components of the Sema1a repulsive signaling complex, and we have generated mouse mutants in two of the three vertebrate MICAL genes. Since our data suggest that compromising MICAL redox functions abrogates semaphorin-mediated repulsive signaling vertebrate neurons, we are using robust pharmacological approaches directed toward enhancing neuronal regeneration through the inactivation of MICAL proteins in mammalian neurons.
Although elucidation of individual guidance cue signaling pathways is essential for understanding how neuronal connectivity is established and maintained, none of these pathways operates in isolation. A crucial question is how crosstalk between individual guidance cue signaling pathways and other more general permissive and inhibitory signaling pathways regulates growth cone steering. We previously found that integrin receptors can mediate responses to semaphorins, and we showed that a membrane-associated semaphorin, Sema7A, can act to promote neuronal outgrowth in vitro and in vivo in the mouse through direct activation of integrin signaling pathways.
Recent collaborative work shows that Sema7A signaling through integrins also mediates cytokine production in monocytes. Our work on the cytosolic MICAL protein points toward intracellular crosstalk between semaphorin and integrin signaling cascades. Vertebrate MICAL interacts with an adapter protein, called CasL, that is an essential intermediate in a major integrin-mediated signaling pathway critical for promoting process outgrowth and cell migration. We are therefore investigating both in Drosophila and in mammals the possibility that Cas proteins lie at an intersection between integrin and semaphorin signaling cascades, acting as a molecular switch controlling attractive and repulsive guidance. These studies identify extracellular and intracellular sites of crosstalk between semaphorin and integrin signaling pathways. Our ongoing work exploits these observations to provide an understanding of how guidance cue signaling from multiple pathways is integrated to produce discrete and directed guidance events in both axons and dendrites during neural development.
Guidance cue signaling involves the intrinsic and extrinsic modulation of receptor output. Our genetic and biochemical screens in Drosophila identified critical, phylogenetically conserved, semaphorin signaling components, including an A kinase–anchoring protein (AKAP) and a receptor guanylate cyclase (RGC). We have also observed in the mammalian central nervous system that chondroitin sulfate proteoglycans (CSPGs), well-known and extremely potent inhibitors of adult neuronal regeneration, can act as extrinsic regulators that determine whether a vertebrate transmembrane semaphorin, called Sema5A, acts as a repellent or as an attractant for rodent CNS axons. Recent analyses of Sema5A and Sema5B mouse mutants also show that these cues play critical roles in the regulation of cortical neuronal migration events. Our current studies on CSPG and heparin sulfate proteoglycan (HSPG) modulation of semaphorin signaling will define the molecular bases for proteoglycan modulation of Sema5A and Sema5B guidance and migration events and may assess the degree to which CSPG-semaphorin interactions contribute to the inhibition of neuronal regeneration following injury.
To understand how axon guidance cues sculpt neuronal connections, we identified some time ago, in collaboration with David Ginty (HHMI, Johns Hopkins University), the first cell surface semaphorin receptor, neuropilin-1 (Npn-1). We showed that Npn-1 and the related protein Npn-2 are vertebrate secreted semaphorin receptors, conclusions reached independently by Marc Tessier-Lavigne (Genentech, formerly HHMI). Over the past several years we have shown in vivo how vertebrate secreted semaphorins, through their interactions with their neuropilin receptors, direct the organization of select sensory, motor, sympathetic, and central neuronal connectivity throughout neural development and into adulthood.
In collaboration with David Ginty, Thomas Jessell (HHMI, Columbia University College of Physicians and Surgeons), and Christopher Henderson (Columbia University College of Physicians and Surgeons), we addressed how timing of axonal growth and dynamic expression of axonal guidance cues and their receptors are coordinated to allow for the establishment of functional spinal motor circuitry. We found that the two neuropilin receptors control distinct aspects of motor axon pathfinding, including regulation of axon growth into the developing limb, fasciculation of motor axons in the plexus, and dorsoventral pathfinding choices within the limb. These results show that regulation of semaphorin guidance cue distribution and receptor expression play critical roles in the establishment of spinal motor axon connectivity. Our current work is directed toward elucidation of how secreted semaphorins and their receptors, those known and those yet to be defined, function in the elaboration of spinal sensory and motor circuits. Given our previous observations (also in collaboration with Ginty, Jessell, and Henderson) that secreted Sema3E binds directly to the divergent plexin receptor plexin D1 independently of neuropilins to pattern the developing somitic vasculature, we are pursuing preliminary results suggesting the existence of novel neuronal secreted semaphorin receptors.
Do these same ligand receptor signaling systems function in the adult nervous system? Recently, in collaboration with David Ginty and Richard Huganir (HHMI, Johns Hopkins University), we found that a secreted semaphorin called Sema3F and its Npn-2 receptor are localized to hippocampal synapses. Moreover, Sema3F signaling at mature synapses alters basal synaptic transmission, and mice with a null mutation at the sema3F locus are prone to seizures. These results showed an unexpected and unprecedented role for semaphorins in synaptic physiology. We are investigating the roles played by secreted semaphorins and their neuropilin receptors in the regulation of adult cortical and hippocampal neuronal morphology and synapse assembly. We have observed that secreted semaphorin-neuropilin signaling does have a profound effect on hippocampal and cortical neuronal morphology and synaptic development and that that Npn-2 and Npn-1 function independently in vivo to regulate dendritic spine morphology and dendritic complexity. We are exploiting a variety of genetic reagents to investigate these effects in vivo, and we anticipate principles of guidance cue function will inform our analyses of their function in the adult nervous system.
This research is also supported by grants from the National Institutes of Health.
Last updated October 22, 2008
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