Pre- and postsynaptic elements exchange developmentally important signals as synapses form. The simplicity, size and accessibility of the skeletal neuromuscular junction (NMJ) make it an excellent preparation for studying these signals and the intracellular signal transduction mechanisms they use. We and others previously defined a rudimentary pathway for initial steps of postsynaptic differentiation at the NMJ in which the proteoglycan z-agrin is a nerve-derived signal, MuSK is its main receptor, and rapsyn is a critical intracellular effector leading to aggregation of acetylcholine receptors. We will now build on this foundation to analyze the dramatic alterations that transform the postsynaptic apparatus at the embryonic NMJ during early postnatal life. These include topological remodeling of the ovoid plaque to a complex pretzel-shaped array of branches;alterations in molecular architecture;subdivision of the membrane into distinct domains;and changes in biophysical properties and metabolic stability. Using a novel culture system in which the plaque to pretzel transition occurs aneurally, we will analyze ways in which two main nerve-derived signals, agrin and acetylcholine, shape the postsynaptic membrane. Using newly generated conditional mutant mice, we will determine whether agrin is required not only for the formation of NMJs but also for their maturation or maintenance. Using targeted mutagenesis in vivo and time-lapse imaging of cultured myotubes, we will analyze roles of lpha-dystrobrevin and LL5_, two synapse-associated proteins that we have already implicated in postsynaptic maturation. Together, these studies on key transsynaptic signals (agrin and neurotransmitter) and intracellular mediators (alpha-dystrobrevin and LL5_) will provide a framework for understanding how postsynaptic maturation occurs and how it is regulated. Importantly, all of these components are present at central synapses, so results will directly enhance our understanding of synaptic remodeling in the postnatal brain. Such remodeling has been studied intensively, because it underlies plasticity during the critical period and in adults, and because defects in processes that regulate it seem likely to underlie conditions as diverse as autism and addiction. Studies of early events in synapse formation at the NMJ have already informed our understanding of less accessible neuron-neuron synapses, and we believe that studies of synaptic maturation and maintenance will be equally broadly applicable.

Public Health Relevance

Nerve cells communicate with each other at junctions called synapses. Synapses are the sites of information processing and plasticity in the normal nervous system, and the sites of defects believed to underlie many neurological and behavioral disorders. Many of the early steps in synapse formation have been described over the past several years, but less is known about how the initially-formed synapse is remodeled during postnatal maturation. We propose to study these processes at the skeletal neuromuscular junction (NMJ). The simplicity, size and accessibility of this synapse make it an excellent preparation for studying signals that regulate synaptic maturation and the intracellular signal transduction mechanisms they use. Moreover, dramatic alterations transform the postsynaptic apparatus at the embryonic NMJ during early postnatal life. These include topological remodeling of the ovoid plaque to a complex pretzel-shaped array of branches;alterations in molecular architecture;subdivision of the membrane into distinct domains;and changes in biophysical properties and metabolic stability. Specifically, we will use the NMJ to analyze roles of two nerve-derived signals, agrin and acetylcholine, and two intramuscular signaling molecules, alpha-dystrobrevin and LL5_, all of which have already been implicated in postsynaptic development. Using a novel culture system in which the plaque to pretzel transition occurs aneurally, we will analyze how these molecules shape the postsynaptic membrane. Hypotheses derived from these studies will be tested using genetically engineered mutant mice. Importantly, all of these components are present at central synapses, so results will directly enhance our understanding of synaptic remodeling in the postnatal brain. Such remodeling has been studied intensively, because it underlies plasticity during the critical period and in adults, and because defects in processes that regulate it seem likely to underlie conditions as diverse as autism and addiction. Studies of early events in synapse formation at the NMJ have already informed our understanding of less accessible neuron-neuron synapses, and we believe that studies of synaptic maturation and maintenance will be equally broadly applicable.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS059853-05
Application #
8231534
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Gubitz, Amelie
Project Start
2008-03-01
Project End
2013-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
5
Fiscal Year
2012
Total Cost
$288,120
Indirect Cost
$116,620
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Chakkalakal, Joe V; Kuang, Shihuan; Buffelli, Mario et al. (2012) Mouse transgenic lines that selectively label Type I, Type IIA, and Types IIX+B skeletal muscle fibers. Genesis 50:50-8
Chakkalakal, Joe V; Nishimune, Hiroshi; Ruas, Jorge L et al. (2010) Retrograde influence of muscle fibers on their innervation revealed by a novel marker for slow motoneurons. Development 137:3489-99
Latvanlehto, Anne; Fox, Michael A; Sormunen, Raija et al. (2010) Muscle-derived collagen XIII regulates maturation of the skeletal neuromuscular junction. J Neurosci 30:12230-41
Nishimune, Hiroshi; Valdez, Gregorio; Jarad, George et al. (2008) Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction. J Cell Biol 182:1201-15