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Joshua R Sanes

Publications and source records attributed to Joshua R Sanes.

At least 19 recordsLinked to original sources
PubMed2004

PSD93 regulates synaptic stability at neuronal cholinergic synapses.

Neuronal cholinergic synapses play important roles in both the PNS and CNS. However, the mechanisms that regulate the formation, maturation, and stability of neuronal cholinergic synapses are poorly understood. In this study, we use the readily accessible mouse superior cervical ganglion (SCG) and submandibular ganglion (SMG) to examine the assembly of the postsynaptic complex of neuronal cholinergic synapses. We find that novel splicing forms of PSD93 (postsynaptic density 93) are expressed in SCG. By immunostaining, we show that PSD93 proteins precisely colocalize with neuronal nicotinic acetylcholine receptors (nAChRs) at synapses of the SCG and SMG. Subcellular fractionation demonstrates that PSD93 is enriched in the PSD fraction of SCG, and coimmunoprecipitation shows that PSD93 and neuronal nAChRs form a complex in vivo. Furthermore, two additional components of the well characterized glutamatergic postsynaptic complex, GKAP/SAPAP (guanylate kinase domain-associated protein/synapse-associated protein-associated protein) and Shank/ProSAP family proteins, are also present at neuronal cholinergic synapses. To assess the function of this protein complex at neuronal cholinergic synapses in vivo, we examined ganglia in mice that lack PSD93. We find that neuronal cholinergic synapses form properly in PSD93 null mice. After denervation, however, synaptic clusters of nAChRs disassemble much faster in mice lacking PSD93 than those in wild-type mice. These results demonstrate that PSD93 is a key component of the postsynaptic scaffold at neuronal cholinergic synapses and plays an important role in synaptic stability. In addition, these results suggest that the mechanism of postsynaptic scaffolding is conserved between neuronal cholinergic and glutamatergic synapses.

Alternative Splicing↗
PubMed2004

A putative ariadne-like E3 ubiquitin ligase (PAUL) that interacts with the muscle-specific kinase (MuSK).

Formation of the postsynaptic membrane at the skeletal neuromuscular junction (NMJ) requires activation of the muscle-specific receptor tyrosine kinase (MuSK). Few intracellular mediators or modulators of MuSK actions are known. E3 ubiquitin ligases may serve this role, because activities of several receptor tyrosine kinases, G-protein-coupled receptors and channels are modulated by ubiquitination. Here, we report identification of a putative Ariadne-like ubiquitin ligase (PAUL) that binds to the cytoplasmic domain of MuSK. PAUL is expressed in numerous tissues of developing and adult mice, and is present at NMJs in muscle fibers but is not confined to them.

Amino Acid Sequence↗
PubMed2003

Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.

A direct histological assay of axonal regeneration would have many advantages over currently available behavioral, electrophysiological, and radiometric assays. We show that peripheral sensory axons marked with the yellow fluorescent protein in transgenic mice can be viewed transcutaneously in superficial nerves. Degenerating and regenerating axons can be followed in live animals with a dissecting microscope and then, after fixation, studied at high resolution by confocal microscopy. Using this approach, we document differences in regenerative ability after nerve transection, crush injury, and crush injury after a previous "conditioning" lesion. We also show that the chemotherapeutic drug vincristine rapidly but transiently blocks regeneration and that the immunosuppressive drug FK506 modestly enhances regeneration. Moreover, FK506 nearly restores normal regenerative ability in animals treated with submaximal doses of vincristine. Because neuropathy is the major dose-limiting side effect of vincristine, we propose that its efficacy could be enhanced by coadministration of FK506 analogs that are neuroactive but not immunosuppressive.

Animals↗
PubMed2003

Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition.

Synaptic activity drives synaptic rearrangement in the vertebrate nervous system; indeed, this appears to be a main way in which experience shapes neural connectivity. One rearrangement that occurs in many parts of the nervous system during early postnatal life is a competitive process called 'synapse elimination'. At the neuromuscular junction, where synapse elimination has been analysed in detail, muscle fibres are initially innervated by multiple axons, then all but one are withdrawn and the 'winner' enlarges. In support of the idea that synapse elimination is activity dependent, it is slowed or speeded when total neuromuscular activity is decreased or increased, respectively. However, most hypotheses about synaptic rearrangement postulate that change depends less on total activity than on the relative activity of the competitors. Intuitively, it seems that the input best able to excite its postsynaptic target would be most likely to win the competition, but some theories and results make other predictions. Here we use a genetic method to selectively inhibit neurotransmission from one of two inputs to a single target cell. We show that more powerful inputs are strongly favoured competitors during synapse elimination.

Animals↗
PubMed2003

Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease.

Apoptosis is a hallmark of motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) [1]. In a widely used mouse model of motoneuron disease (progressive motor neuronopathy or pmn) [2-4], transgenic expression of the anti-apoptotic bcl-2 gene [5] or treatment with glial cell-derived neurotrophic factor [6] prevents the apoptosis of the motoneuron soma; however, they were unable to affect the life span of the animals. The goal of the present work was to determine whether the pmn phenotype could be rescued by means of a gene that inhibits axon degeneration. For this reason, the pmn mice were crossed with mice bearing the dominant Wlds ("slow Wallerian degeneration") mutation, which slows axon degeneration and synapse loss [7-9]. We show here that the Wlds gene product attenuates symptoms, extends life span, prevents axon degeneration, rescues motoneuron number and size, and delays retrograde transport deficits in pmn/pmn mice. These results suggest new pathogenic mechanisms and therapeutic avenues for motoneuron diseases.

Animals↗
PubMed2003

Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions.

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Alternative Splicing↗
PubMed2003

PDAPP; YFP double transgenic mice: a tool to study amyloid-beta associated changes in axonal, dendritic, and synaptic structures.

Neuritic plaques are one of the stereotypical hallmarks of Alzheimer's disease (AD) pathology. These structures are composed of extracellular accumulations of fibrillar forms of the amyloid-beta peptide (Abeta), a variety of other plaque-associated proteins, activated glial cells, and degenerating nerve processes. To study the neuritic toxicity of different structural forms of Abeta in the context of regional connectivity and the entire cell, we crossed PDAPP transgenic (Tg) mice, a model with AD-like pathology, to Tg mice that stably express yellow fluorescent protein (YFP) in a subset of neurons in the brain. In PDAPP; YFP double Tg mice, markedly enlarged YFP-labeled axonal and dendritic varicosities were associated with fibrillar Abeta deposits. These varicosities were absent in areas where there were nonfibrillar Abeta deposits. Interestingly, YFP-labeled varicosities revealed changes that corresponded with changes seen with electron microscopy and the de Olmos silver staining technique. Other silver staining methods and immunohistochemical localization of phosphorylated neurofilaments or phosphorylated tau were unable to detect the majority of these dystrophic neurites. Some but not all synaptic vesicle markers accumulated abnormally in YFP-labeled varicosities associated with neuritic plaques. In addition to the characterization of the effects of Abeta on axonal and dendritic structure, YFP-labeled neurons in Tg mice should prove to be a valuable tool to interpret the localization patterns of other markers and for future studies examining the dynamics of axons and dendrites in a variety of disease conditions in living tissue both in vitro and in vivo.

Alzheimer Disease↗
PubMed2003

Expression of laminin chains by central neurons: analysis with gene and protein trapping techniques.

Laminins exert numerous effects on neurons in vitro, but expression of laminin subunit genes by neurons in vivo remains controversial. To reexamine this issue, we generated mice from ES cells in which the laminin alpha1, alpha5, beta1, and gamma1 genes had been "trapped" by insertion of a histochemically detectable selectable marker, betageo (beta-galactosidase fused to neomycin phosphotransferase). The presence of laminin-betageo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin beta1 and gamma1 genes, but not alpha chain genes, by defined subsets of neurons in brain and retina. We also used the gene traps in a novel way to assay expression of endogenous laminin subunits, which were barely detectable by ordinary immunohistochemical methods. The trapping vector included a transmembrane domain that anchors proteins otherwise destined for secretion. Laminin alpha/beta/gamma heterotrimers are assembled intracellularly, and we show that the trapped laminin gamma1 fusion protein "co-trapped" endogenous beta1 intracellularly. The laminin gamma1 fusion was also able to co-trap transgene-derived alpha chains, but we detected no co-trapped endogenous alpha chains. The co-trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.

Animals↗
PubMed2003

Synaptic adhesion molecules.

Formation, differentiation and plasticity of synapses, the specialized cell-cell contacts through which neurons communicate, all require interactions between pre- and post-synaptic partners. Several synaptically localized adhesion molecules potentially capable of mediating these interactions have been identified recently. Functional studies suggest roles for some of them in target recognition (e.g. SYG-1 and sidekicks), formation and alignment of synaptic specializations (e.g. SynCAM, neuroligin and neurexin), and regulation of synaptic structure and function (e.g. cadherins and syndecan).

Amino Acid Sequence↗
PubMed2003

Synaptic dynamism measured over minutes to months: age-dependent decline in an autonomic ganglion.

Naturally occurring rearrangements of synaptic terminals are common in the nervous systems of young mammals, but little is known about their incidence in adults. Using transgenic mice that express yellow fluorescent protein (YFP) in axons, we repeatedly imaged nerve terminals in the parasympathetic submandibular ganglion. We found that the pattern of synaptic branches underwent significant rearrangements over several weeks in young adult mice. In older mice, rearrangements were less common, and synaptic patterns on individual neurons were recognizable for many months to years. Axonal branches frequently retracted or extended on a time scale of minutes in young adult mice, but seldom in mature animals. These results provide direct evidence for a decrease in plasticity of interneuronal connections as animals make the transition from young adulthood to middle age. The long-term stability of synaptic patterns could provide a structural basis for the persistence of memory in the adult nervous system.

Aging↗
PubMed2002

Gamma protocadherins are required for survival of spinal interneurons.

The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.

Animals↗
PubMed2002

Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase.

Activity-dependent and -independent signals collaborate to regulate synaptogenesis, but their relative contributions are unclear. Here, we describe the formation of neuromuscular synapses at which neurotransmission is completely and specifically blocked by mutation of the neurotransmitter-synthesizing enzyme choline acetyltransferase. Nerve terminals differentiate extensively in the absence of neurotransmitter, but neurotransmission plays multiple roles in synaptic differentiation. These include influences on the numbers of pre- and postsynaptic partners, the distribution of synapses in the target field, the number of synaptic sites per target cell, and the number of axons per synaptic site. Neurotransmission also regulates the formation or stability of transient acetylcholine receptor-rich processes (myopodia) that may initiate nerve-muscle contact. At subsequent stages, neurotransmission delays some steps in synaptic maturation but accelerates others. Thus, neurotransmission affects synaptogenesis from early stages and coordinates rather than drives synaptic maturation.

Acetylcholine↗
PubMed2002

Sidekicks: synaptic adhesion molecules that promote lamina-specific connectivity in the retina.

A major determinant of specific connectivity in the central nervous system is that synapses made by distinct afferent populations are restricted to particular laminae in their target area. We identify Sidekick (Sdk)-1 and -2, homologous transmembrane immunoglobulin superfamily molecules that mediate homophilic adhesion in vitro and direct laminar targeting of neurites in vivo. sdk-1 and -2 are expressed by nonoverlapping subsets of retinal neurons; each sdk is expressed by presynaptic (amacrine and bipolar) and postsynaptic (ganglion) cells that project to common inner plexiform (synaptic) sublaminae. Sdk proteins are concentrated at synaptic sites, and Sdk-positive synapses are restricted to the 2 (of > or =10) sublaminae to which sdk-expressing cells project. Ectopic expression of Sdk in Sdk-negative cells redirects their processes to a Sdk-positive sublamina. These results implicate Sdks as determinants of lamina-specific synaptic connectivity.

Amacrine Cells↗
PubMed2002

Neurotransmitter receptor dynamics studied in vivo by reversible photo-unbinding of fluorescent ligands.

We show that fluorescently tagged ligands with high affinity for their targets can be reversibly unbound by focused laser excitation. By sequential unbinding and relabeling with different colors of alpha-bungarotoxin, we selectively labeled adjacent pools of acetylcholine receptors (AChRs) at neuromuscular junctions of adult mice. Timelapse imaging in vivo revealed that synaptic AChRs completely intermingle over approximately 4 days and many extrasynaptic AChRs are incorporated into the synapse each day. In mice that lacked alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex, rates of AChR turnover, and intermingling were increased approximately 4- to 5-fold. These results demonstrate remarkable molecular dynamism underlying macroscopic stability of the postsynaptic membrane, and establish alpha-dystrobrevin as a key control point for regulation of mobility and turnover.

Animals↗
PubMed2002

Dystroglycan is selectively associated with inhibitory GABAergic synapses but is dispensable for their differentiation.

The dystrophin glycoprotein complex (DGC) is a multimolecular complex that links the extracellular matrix to the cytoskeleton. The DGC is present at the skeletal neuromuscular junction and required for its maturation and maintenance. Members of the DGC are also expressed in brain. We used cultured hippocampal neurons to analyze the distribution, regulation, and role in synaptogenesis of the major transmembrane component of the DGC, dystroglycan; one of its extracellular ligands, agrin; and one of its cytoskeletal binding partners, dystrophin. alpha-Dystroglycan, beta-dystroglycan, and dystrophin clustered at a subset of inhibitory synapses containing GABA(A)R subunits alpha1, alpha2, and gamma2, and the inhibitory receptor anchoring protein gephyrin. DGC components were not detected at excitatory glutamatergic synapses. Dystroglycan is the first identified adhesive macromolecule at mature GABA synapses. Developmentally, dystroglycan clustered at synaptic loci after synaptic vesicles, GABA(A)R, and gephyrin, the latter being closely associated with GABA(A)R at all stages of synaptogenesis analyzed. Analysis of gephyrin -/-, agrin -/-, and mdx mouse hippocampal neurons in culture indicated that synaptic clustering of dystroglycan occurs independently of gephyrin, agrin, and dystrophin. In dystroglycan-deficient neurons, cultured from a conditional mutant strain, GABAergic synapses differentiated with clusters of gephyrin and GABA(A)R apposed to synaptic terminals, but these synapses did not contain detectable dystrophin. Thus the DGC is not essential for GABAergic synaptogenesis but is likely to function in modulating inhibitory synapses or conferring specialized properties on a subset of them.

Agrin↗