Overview
We study the molecular events of synapse formation and maintenance in the nervous system. At the neuromuscular junction, the point of contact between spinal motor neurons and skeletal muscle fibers, the protein agrin provides an essential signal for the differentiation of cells on the muscle side of the junction. We are continuing to identify new functions of agrin, both at the neuromuscular junction and in other sites such as the vasculature of the brain, where agrin may contribute to the blood-brain-barrier and influence the accumulation of beta-amyloid, a pathological marker of Alzheimer's Disease. We are also examining the requirements of maintaining neuromuscular junctions in a number or genetic models of motor neuron diseases (ALS), and peripheral neuropathies (Charcot-Marie-Tooth). Finally, we are working to find mechanisms in central nervous system development that parallel those we have studied at the neuromuscular junction. We are using the retina as our experimental system, and have identified Dscam as an adhesion molecule that provides an essential label for cells to recognize one another as neuronal circuits in the retina form.
Research details
Genetics of Synapse Formation and Maintenance
Synapse formation
The primary focus of my research has been on the molecular cues that direct neuronal synapse formation and neurodevelopment. At the vertebrate neuromuscular junction (NMJ), the extracelllular matrix molecule agrin is an essential signal from motor neurons to muscle fibers. If agrin is genetically deleted from mice, NMJ formation fails completely, causing neonatal lethality. Although agrin is an excellent candidate to also be involved in central nervous system (CNS) synapse formation, its function in the brain remains unclear. We are currently addressing the roles of agrin in various disease models using a combination of knockout and transgenic strategies, and in the retina as a model for development of the CNS. In addition, we are investigating new mutations that affect the maturation and stability of the NMJ and retina.
Agrin knockout studies
Loss-of-function mutations in agrin demonstrate that it is essential for embryonic NMJ formation. However, the NMJ phenotype of these mutants results in neonatal lethality, making it difficult to determine if agrin is also required for the postnatal maintenance of the NMJ and to study its role in CNS development and function. To circumvent this problem, we generated an isoform-specific deletion of agrin that removes the predominant form of the protein expressed in the CNS without affecting NMJ formation, and a loxP-flanked conditional allele of agrin. The loxP allele allows us to remove all agrin isoforms from specific neuronal populations in the brain to study agrin's function in the CNS, while leaving it intact in motor neurons so that NMJs will form normally. In addition, we can delete the gene from motor neurons after NMJs have formed to determine if agrin signaling is required to maintain postsynaptic differentiation in the muscle in adult animals. These studies are also aided by a newly identified point mutation in agrin that causes a milder phenotype with a postnatal onset. This mutation suggests that agrin signaling is indeed required postnatally, and identifies a previously uncharacterized domain of agrin as important for the maintenance of postsynaptic differentiation.
Agrin transgenic studies
We have generated transgenic mice that overexpress agrin tagged with either Cyan or Yellow Fluorescent Proteins (agrin-CFP or -YFP) (Fuerst et al., 2006) to allow the protein to be more easily visualized. These transgenic mice produce fully functional agrin and are being used for studies of agrin's localization in the brain. These mice also allow studies on the effects of agrin's overexpression. The agrin-CFP transgenic mice show no overt phenotype except in the line with the highest transgenic expression, which has severe defects in eye development. The defects are directly attributable to agrin overexpression, and may be the result of the mispresentation of growth factors and signaling molecules during development, or the result of aberrantly strong interactions between the extracellular matrix and cell adhesion molecules. Furthermore, the eye phenotype in the agrin-CFP transgenic mice is strain-dependent, appearing with complete penetrance in a C57BL/6 inbred background, which has allowed us to map three loci in C57BL/6 that predispose this strain to eye defects.
Agrin and disease models
Our work on agrin in the maintenance of NMJs indicates that postnatal deficiencies in agrin lead to synaptic dysfunction and eventually death from a severe myasthenia. This finding has relevance to human disease because both autoimmunity and mutations affecting MuSK (Muscle Specific Kinase)-the receptor that transduces agrin's signal-cause a myasthenia in humans. While such a disease has not yet been directly attributed to agrin in the human population, our results suggest that it is a formal possibility and indeed likely. In addition to its role at the NMJ, agrin is proposed to be involved in the formation of amyloid plaques in the brains of Alzheimer's disease patients. Agrin is a heparan sulfate proteoglycan (HSPG), a class of proteins invariably present in the amyloid plaques that form in Alzheimer's disease. Agrin is the core protein most often associated with HSPG in human Alzheimer's samples. However, whether agrin plays a causative role in plaque formation is unknown. We are currently using our agrin transgenic and knockout mice, in combination with an amyloid plaque-forming transgenic model, to test agrin's role in plaque formation. Our results to date indicate that agrin associated with the microvasculature of the brain, but not with the cell surface of neurons, influences amyloid deposition in the brain. This is an intriguing result that suggests a key role for the blood-brain barrier in the progression of amyloid plaque pathology.
New genetic models affecting NMJ maintenance
By screening mice with induced and spontaneous mutations for overt neuromuscular dysfunction, we are identifying new genetic models that affect the maintenance of synaptic connectivity in the peripheral nervous system. One such model, which we recently identified by positional cloning, is a dominant mutation in Gars, the gene encoding glycyl tRNA synthetase (GlyRS) (Seburn et al., 2006). Mutations in the human GARS gene cause the peripheral neuropathy Charcot-Marie-Tooth disease, type 2D (CMT2D). Our studies on the mouse model have indicated that these mutations can affect sensory as well as motor axons, a point that was ambiguous based on the analysis of human CMT2D patients. They have also indicated that the disease mechanism is complex, requiring the expression of mutant forms of GlyRS and not simply resulting from a loss of GlyRS activity. Analyses of additional mutations affecting both the neuromuscular junction and the development of the retina are also ongoing.
Lab staff
Principal Investigator: Robert W. Burgess, Ph.D.
Postdoctoral Fellows: Peter G. Fuerst, Ph.D., Laurent P. Bogdanik, Ph.D.
Research Assistant III: Steven M. Rauch, M.D., M.S.,
Research Assistant II: Kathy Miers
Laboratory Technician IV: Christine Rosales
Research Administrative Assistant: Ashley Stanton
Publication listings
Fuerst PG, Koizuma A, Masland R, Burgess RW. 2008. Neurite arborization and mosaic spacing in the mouse retina requires DSCAM. Nature, (in press).Misgeld T, Kerschensteiner M, Bareyre FM, Burgess RW, Lichtman JW. 2007. Imaging axonal transport of mitochondria in vivo. Nat Methods Jul;4(7):559-61.
Harvey SJ, Jarad G, Cunningham J, Rops AL, van der Vlag J, Berden JH, Moeller MJ, Holzman LB, Burgess RW, Miner JH. 2007. Disruption of Glomerular Basement Membrane Charge through Podocyte-Specific Mutation of Agrin Does Not Alter Glomerular Permselectivity. Am J Pathol Jul;171(1):139-52.
Fuerst PG, Rauch SM, and Burgess RW. 2007. Defects in eye development in transgenic mice overexpressing the heparan sulfate proteoglycan agrin. Dev Biol 303(12):165-180.
Seburn KL, Nangle LA, Cox GA, Schimmel P, and Burgess RW. 2006. An active dominant mutation of Glycyl-tRNA synthetase causes neuropathy in a Charcot Marie Tooth 2D mouse model. Neuron 51(6):715-26.
Burgess RW, Jucius TJ, Ackerman SL. 2006. Motor axon guidance of the mammalian trochlear and phrenic nerves: dependence on the netrin receptor Unc5c and modifier loci. J Neurosci 26:5756-5766.
Stacy RC, Demas J, Burgess RW, Sanes JR, Wong RO. 2005. Disruption and recovery of patterned retinal activity in the absence of acetylcholine. J Neurosci 25(41:9347-9357.
Burgess RW, Peterson KA, Johnson MJ, Roix JJ, Welsh IC, O'Brien TP. 2004. Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice. Mol Cell Biol 24(3):1096-1105.
Buffelli M, Burgess RW, Feng G, Lobe C, Lichtman JW, Sanes JR. 2003. Genetic evidence that relative synaptic activity biases the outcome of synaptic competition. Nature 424:430-434.
Bhattacharya S, Stewart BA, Niemeyer BA, Burgess RW, McCabe BD, Lin P, Boulianne
G, O'Kane CJ, Schwarz TL. 2002. Members of the Synaptobrevin/Vesicle Associated
Membrane Protein (VAMP) Family in Drosophila Are Functionally Interchangeable In
Vivo for Neurotransmitter Release and Cell Viability. Proc Nat Acad Sci, 99:13867
13872.
Burgess RW, Dickman DK, Nunez L, Glass DJ, Sanes JR. 2002. Mapping Sites
Responsible for Interactions of Agrin With Neurons. J. Neurochem 83:271-284.
Misgeld T, Burgess RW, Lewis RM, Cunningham JM, Lichtman JW, Sanes JR.
2002. Roles of Neurotransmitter in Synapse Formation: Development of Neuromuscular Junctions Lacking Choline Acetyltransferase. Neuron 36:271-84.
Books, Book Chapters and Reviews:
Burgess, RW. 2006. The Formation of the Vertebrate Neuromuscular Junction: Roles for the Extracellular Matrix in Synaptogenesis, Chapter 1, in Molecular Mechanisms of Synaptogenesis, El-Husseini, A. and Dityatev, A., editors. Springer Verlag, New York.
Patton B, Burgess RW. 2005. Synaptogenesis in Developmental Neurobiology, 4th ed., edited by Rao MS, Jacobson M. Published by Kluwer Academic/Plenum Publishers, NY.
Sanes JR, Apel ED, Burgess RW, Emereson RB, Feng G, Gautam M, Glass D, Grady RM, Krejci E, Lichtman JW, Lu JT, Massoulie J, Miner JH, Moscoso LM, Nguyen Q, Nichol M, Noakes PG, Patton BL, Son YJ, Yancopoulos GD, Zhou H. 1998 Development of the neuromuscular junction: genetic anaylsis in mice. J Physiol Paris 92:167-172.
