Kinase- and rapsyn-independent activities of the muscle-specific kinase (MuSK).

The muscle-specific receptor tyrosine kinase (MuSK) is co-localized with nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the skeletal neuromuscular junction, and is required for all known aspects of postsynaptic differentiation. Studies in vitro have shown that Z(+)-agrin, a nerve-derived proteoglycan, activates MuSK's kinase activity to promote clustering of AChRs and MuSK itself with a cytoplasmic, receptor-associated protein, rapsyn. These studies, however, have used soluble forms of agrin, whereas agrin is cell- or matrix-attached in vivo. We show here that immobilized (particle- or cell-attached) agrin but not soluble agrin is able to aggregate MuSK in the absence of rapsyn and that this aggregation does not require MuSK's kinase activity but does require MuSK's cytoplasmic domain. Moreover, immobilized agrin can promote clustering of AChRs by a mechanism that requires MuSK and rapsyn but does not require MuSK's kinase activity. These results imply that rapsyn and signaling components activated by MuSK kinase may be dispensable for some early aspects of postsynaptic differentiation.

Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin alpha2-deficient mice.

To optimize and evaluate treatments for muscular dystrophy, it is important to know the natural history of the disease in the absence of therapeutic intervention. Here we characterized disease progression of three mutant mouse strains of muscular dystrophy: mdx mice, which lack dystrophin; mdx:utrn-/- mice, which also lack utrophin; and dy/dy mice, which are deficient in laminin alpha2. Normal mice show a marked increase in forelimb strength over the first 10 weeks of life and little fatigue (<5%) over five consecutive strength trials. Mdx and mdx:utrn-/- mice demonstrate less strength then normal mice and approximately 40% fatigue at each age. Mdx mice become obese but mdx:utrn-/- mice do not. Dy/dy mice remain small and are much weaker than mdx and mdx:utrn-/- mice at all ages even when normalized to weight; however, they show only minimal fatigue (10%). This work demonstrates a distinct pattern of disease progression in each model and provides a foundation for assessing strategies for improving strength in each model.

Glial cell line-derived neurotrophic factor administration in postnatal life results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) in embryonic muscle fibers causes dramatic hyperinnervation of neuromuscular junctions. However, it is not known whether GDNF induces the extra innervation by regulation of axonal branching and/or synaptic maintenance. To address this issue, high levels of circulating GDNF were established by administering subcutaneous injections starting either at birth or later and continuing for up to 40 d. Treatment with exogenous GDNF beginning in the first week, but not later, increased the number of axons converging at neuromuscular junctions. The effect of GDNF on the branching pattern of individual motor axons was determined by reconstructing labeled axonal arbors from transgenic mice expressing yellow fluorescent protein in subsets of motor neurons. Whereas, at postnatal day 8 (P8) individual axons in control animals branched to sporadically innervate junctions within circumscribed regions of the muscle, motor units from GDNF injected animals had significantly more axonal branches and exhibited a high degree of localized arborization such that adjacent muscle fibers were often innervated by the same axon. Administration beginning at P0 and continuing through P40 prolonged multiple innervation of most fibers throughout the period of injection. Between P30 and P40 there was no net change in multiple innervation, although there was evidence of retraction bulbs, suggesting that axon extension and retraction were in equilibrium. We conclude that GDNF has a developmentally regulated effect on presynaptic branching and that sustained administration of GDNF induces a state of continuous synaptic remodeling.

Asynchronous synapse elimination in neonatal motor units: studies using GFP transgenic mice.

In developing muscle, synapse elimination reduces the number of motor axons that innervate each postsynaptic cell. This loss of connections is thought to be a consequence of axon branch trimming. However, branch retraction has not been observed directly, and many questions remain, such as: do all motor axons retract branches, are eliminated branches withdrawn synchronously, and are withdrawing branches localized to particular regions? To address these questions, we used transgenic mice that express fluorescent proteins in small subsets of motor axons, providing a unique opportunity to reconstruct complete axonal arbors and identify all the postsynaptic targets. We found that, during early postnatal development, each motor axon loses terminal branches, but retracting branches withdraw asynchronously and without obvious spatial bias, suggesting that local interactions at each neuromuscular junction regulate synapse elimination.

Gephyrin-independent clustering of postsynaptic GABA(A) receptor subtypes.

Gephyrin has been shown to be essential for the synaptic localization of the inhibitory glycine receptor and major GABA(A) receptor (GABA(A)R) subtypes. However, in retina certain GABA(A)R subunits are found at synaptic sites in the absence of gephyrin. Here, we quantitatively analyzed GABA(A)R alpha1, alpha2, alpha3, alpha5, beta2/3, and gamma2 subunit immunoreactivities in spinal cord sections derived from wild-type and gephyrin-deficient (geph -/-) mice. The punctate staining of GABA(A)R alpha1 and alpha5 subunits was unaltered in geph -/- mice, whereas the numbers of alpha2-, alpha3-, beta2/3-, and gamma2-subunit-immunoreactive synaptic sites were significantly or even strikingly reduced in the mutant animals. Immunostaining with an antibody specific for the vesicular inhibitory amino acid transporter revealed that the number of inhibitory presynaptic terminals is unaltered upon gephyrin deficiency. These data show that in addition to gephyrin other clustering proteins must exist that mediate the synaptic localization of selected GABA(A)R subtypes.

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4.

Precise apposition of pre- to postsynaptic specializations is required for optimal function of chemical synapses, but little is known about how it is achieved. At the skeletal neuromuscular junction, active zones (transmitter release sites) in the nerve terminal lie directly opposite junctional folds in the postsynaptic membrane. Few active zones or junctional folds form in mice lacking the laminin beta2 chain, which is normally concentrated in the synaptic cleft. beta2 and the broadly expressed gamma1 chain form heterotrimers with alpha chains, three of which, alpha2, alpha4 and alpha5, are present in the synaptic cleft. Thus, alpha2beta2gamma1, alpha4beta2gamma1 and alpha5beta2gamma1 heterotrimers are all lost in beta2 mutants. In mice lacking laminin alpha4, active zones and junctional folds form in normal numbers, but are not precisely apposed to each other. Thus, formation and localization of synaptic specializations are regulated separately, and alpha4beta2gamma1 (called laminin-9) is critical in the latter process.

Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos.

Approximately half of the motoneurons produced during development die before birth or shortly after birth. Although it is believed that survival depends on a restricted supply of a trophic sustenance produced by the synaptic target tissue (i.e., muscle), it is unclear whether synapse formation per se is involved in motoneuron survival. To address this issue, we counted cranial motoneurons in a set of mutant mice in which formation of neuromuscular junctions is dramatically impaired (i.e., null mutants for agrin, nerve-derived agrin, rapsyn, and MuSK). We demonstrate that in the absence of synaptogenesis, there is an 18-34% increase in motoneuron survival in the facial, trochlear, trigeminal motor, and hypoglossal nuclei; the highest survival occurred in the MuSK-deficient animals in which synapse formation is most severely compromised. There was no change in the size of the mutant motoneurons as compared with control animals, and the morphology of the mutant motoneurons appeared normal. We postulate that the increased axonal branching observed in these mutants leads to a facilitated "access" of the motoneurons to muscle-derived trophic factors at sites other than synapses or that inactivity increases the production of such factors. Finally, we examined motoneurons in double mutants of CNTFRalpha(-/-) (in which there is a partial loss of motoneurons) and MuSK(-/-) (in which there is an increased survival of motoneurons). The motoneuron numbers in the double mutants parallel those of the single MuSK-deficient mice, indicating that synapse disruption can even overcome the deleterious effect of CNTFRalpha ablation.

Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse.

The development of chemical synapses is regulated by interactions between pre- and postsynaptic cells. At the vertebrate skeletal neuromuscular junction, the organization of an acetylcholine receptor (AChR)-rich postsynaptic apparatus has been well studied. Much evidence suggests that the nerve-derived protein agrin activates muscle-specific kinase (MuSK) to cluster AChRs through the synapse-specific cytoplasmic protein rapsyn. But how postsynaptic differentiation is initiated, or why most synapses are restricted to an 'end-plate band' in the middle of the muscle remains unknown. Here we have used genetic methods to address these issues. We report that the initial steps in postsynaptic differentiation and formation of an end-plate band require MuSK and rapsyn, but are not dependent on agrin or the presence of motor axons. In contrast, the subsequent stages of synaptic growth and maintenance require nerve-derived agrin, and a second nerve-derived signal that disperses ectopic postsynaptic apparatus.

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

Development. The decade of the developing brain.

Our understanding of neural development has advanced dramatically over the past decade. Significant insights have now been obtained into seven fundamental developmental processes: first, induction of the neural plate; second, regionalization of the neural tube along the dorsoventral and anteroposterior axes; third, generation of neurons and glia from multipotential precursors; fourth, apoptotic cell death; fifth, migration of neurons; sixth, guidance of axons to their targets; and seventh, formation of synapses.

Agrin isoforms with distinct amino termini: differential expression, localization, and function.

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

Nerve terminals form but fail to mature when postsynaptic differentiation is blocked: in vivo analysis using mammalian nerve-muscle chimeras.

To better understand the role of the postsynaptic cell in the differentiation of presynaptic terminals, we transplanted muscles that lacked postsynaptic differentiation from mutant mice into normal adult immunocompatible hosts and attached the host nerve to the grafts. Host motor axons innervated wild-type grafted muscle fibers and established normal appearing chimeric neuromuscular junctions. By repeated in vivo imaging, we found that these synapses were stably maintained. Results were different when nerves entered transplanted muscles derived from mice lacking muscle-specific receptor tyrosine kinase (MuSK) or rapsyn, muscle-specific components required for postsynaptic differentiation. Initial steps in presynaptic differentiation (e.g., formation of rudimentary arbors and vesicle clustering at terminals) occurred when wild-type neurites contacted MuSK- or rapsyn deficient muscle fibers, either in vivo or in vitro. However, wild-type terminals contacting MuSK or rapsyn mutant muscle fibers were unable to mature, even when the chimeras were maintained for up to 7 months. Moreover, in contrast to the stability of wild-type synapses, wild-type nerve terminals in mutant muscles underwent continuous remodeling. These results suggest that postsynaptic cells supply two types of signals to motor axons: ones that initiate presynaptic differentiation and others that stabilize the immature contacts so that they can mature. Normal postsynaptic differentiation appears to be dispensable for initial stages of presynaptic differentiation but required for presynaptic maturation.

Identification and expression of mouse netrin-4.

Netrins are secreted proteins that serve as potent axon guidance molecules in vertebrates and invertebrates. We report the identification of a novel mammalian member of this family. Netrin-4 is similar in predicted size and secondary structure to the other three netrins; all contain, in order, an amino-terminal signal sequence, a laminin-type globular domain of the 'VI' type, three laminin-type epidermal growth factor (EGF) repeats, and a carboxyl-terminal 'netrin module'. In terms of primary sequence, however, netrin-4 is a distant relative of netrins-1-3, and its globular domain is more closely related to those of laminins than to those of other netrins. Netrin-4 is broadly expressed in both neural and non-neural tissues of embryonic and adult mice. In embryonic spinal cord, it is selectively expressed by cells at the lateral margins of the floor plate. In postnatal brain, it is selectively expressed in subsets of neurons, including cerebellar granule and hippocampal pyramidal cells.

Syne-1, a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction.

We describe a novel protein, Syne-1, that is associated with nuclear envelopes in skeletal, cardiac, and smooth muscle cells. Syne-1 contains multiple spectrin repeats similar to those found in dystrophin and utrophin, as well as a domain homologous to the carboxyl-terminal of Klarsicht, a protein associated with nuclei and required for a subset of nuclear migrations in Drosophila. In adult skeletal muscle fibers, levels of Syne-1 are highest in the nuclei that lie beneath the postsynaptic membrane at the neuromuscular junction. These nuclei are transcriptionally specialized, expressing genes for synaptic components at higher levels than extrasynaptic nuclei in the same cytoplasm. Syne-1 is the first protein found to be selectively associated with synaptic nuclei. Syne-1 becomes concentrated in synaptic nuclei postnatally. It remains synaptically enriched following denervation or degeneration/regeneration, and is also present at high levels in the central nuclei of dystrophic myotubes. The location and structure of Syne-1 suggest that it may participate in the migration of myonuclei in myotubes and/or their anchoring at the postsynaptic apparatus. Finally, we identify a homologous gene, syne-2, that is expressed in an overlapping but distinct pattern.

Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho.

We demonstrate that within the intact and spontaneously active retina, dendritic processes of ganglion cells exhibit rapid and extensive movements during the period of synaptogenesis. Marked restructuring occurs in seconds, but structural changes are relatively balanced across the dendritic arbor, maintaining overall arbor size and complexity over hours. Dendritic motility is regulated by spontaneous glutamatergic transmission. Both the rate and extent of the movements are decreased by antagonists to NMDA and non-NMDA glutamate receptors but are unaffected by tetrodotoxin, a sodium channel blocker. The dendritic movements are actin dependent and are controlled by the Rho family of small GTPases. Transfection of dominant-negative and constitutively active mutants into ganglion cells showed that Rac and Rho exert reciprocal effects on motility. We suggest that the Rho family of small GTPases could integrate activity-dependent and -independent signals from afferents, thereby adjusting target motility and maximizing the chance for initial contact and subsequent synaptogenesis.

Maturation and maintenance of the neuromuscular synapse: genetic evidence for roles of the dystrophin--glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) links the cytoskeleton of muscle fibers to their extracellular matrix. Using knockout mice, we show that a cytoplasmic DGC component, alpha-dystrobrevin (alpha-DB), is dispensable for formation of the neuromuscular junction (NMJ) but required for maturation of its postsynaptic apparatus. We also analyzed double and triple mutants lacking other cytoskeletal DGC components (utrophin and dystrophin) and myotubes lacking a alpha-DB or a transmembrane DGC component (dystroglycan). Our results suggest that alpha-DB acts via its linkage to the DGC to enhance the stability of postsynaptic specializations following their DGC-independent formation; dystroglycan may play additional roles in assembling synaptic basal lamina. Together, these results demonstrate involvement of distinct protein complexes in the formation and maintenance of the synapse and implicate the DGC in the latter process.

Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers.

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.