Yoshie Sugiura

Glial Cells Maintain Synaptic Structure and Function and Promote Development of the Neuromuscular Junction In Vivo

To investigate the in vivo role of glial cells in synaptic function, maintenance, and development, we have developed an approach to selectively ablate perisynaptic Schwann cells (PSCs), the glial cells at the neuromuscular junction (NMJ), en masse from live frog muscles. In adults, following acute PSC ablation, synaptic structure and function were not altered. However, 1 week after PSC ablation, presynaptic function decreased by approximately half, while postsynaptic function was unchanged. Retraction of nerve terminals increased over 10-fold at PSC-ablated NMJs. Furthermore, nerve-evoked muscle twitch tension was reduced. In tadpoles, repeated in vivo observations revealed that PSC processes lead nerve terminal growth. In the absence of PSCs, growth and addition of synapses was dramatically reduced, and existing synapses underwent widespread retraction. Our findings provide in vivo evidence that glial cells maintain presynaptic structure and function at adult synapses and are vital for the growth and stability of developing synapses.

Ubiquitin carboxyl-terminal hydrolase L1 is required for maintaining the structure and function of the neuromuscular junction

The enzyme ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is one of the most abundant proteins in the mammalian nervous system. In humans, UCH-L1 is also found in the ubiquitinated inclusion bodies that characterize neurodegenerative diseases in the brain, suggesting its involvement in neurodegeneration. The physiologic role of UCH-L1 in neurons, however, remains to be further elucidated. For example, previous studies have provided evidence both for and against the role of UCH-L1 in synaptic function in the brain. Here, we have characterized a line of knockout mice deficient in the UCH-L1 gene. We found that, in the absence of UCH-L1, synaptic transmission at the neuromuscular junctions (NMJs) is markedly impaired. Both spontaneous and evoked synaptic activity are reduced; paired pulse-facilitation is impaired, and synaptic transmission fails to respond to high-frequency, repetitive stimulation at the NMJs of UCH-L1 knockout mice. Morphologic analyses of the NMJs further revealed profound structural defects—loss of synaptic vesicles and accumulation of tubulovesicular structures at the presynaptic nerve terminals, and denervation of the muscles in UCH-L1 knockout mice. These findings demonstrate that UCH-L1 is required for the maintenance of the structure and function of the NMJ and that the loss of normal UCH-L1 activity may result in neurodegeneration in the peripheral nervous system.

SCG10 expresses growth-associated manner in developing rat brain, but shows a different pattern to p19/stathmin or GAP-43

The gene encoding SCG10 was originally isolated as a neuronal marker from neural crest derivatives, implying that this protein may contribute to fundamental neuronal properties. To examine the developmental change of SCG10 expression in brain, immunoblot analysis and in situ hybridization were performed in embryonic day 15 (E15), E19, postnatal day 0 (P0), P6, P14, P30 and P90 rat brains. The distribution of SCG10 mRNA was compared to those of its homologue, p19/stathmin, and the well-characterized growth-associated protein GAP-43. Overall expression of SCG10 in brain reached a peak at E19 and decreased gradually by P30 to the adult level. The expression pattern of SCG10 in E15 whole body was identical with that of GAP-43; both mRNAs were specifically detected in developing neuronal structures. p19/stathmin mRNA, on the other hand, showed widespread expression throughout the whole body. Expression patterns of the three mRNAs overlapped in many structures in the perinatal brain, yet each showed unique expression during postnatal development. For example, in the developing cerebellum, strong GAP-43 expression was found in the external granule cells, which are presumably extending parallel fibers, while SCG10 strongly hybridized in the internal granule cells which have reached their final position and begun dendrite outgrowth. The unique transient expression of p19/stathmin was found in the subventricular zone in the cortex, the white matter in the cerebellum, the optic nerve layer of the superior colliculus and the inner edge of the dentate granule layer in the hippocampus. Considering the timing, all of these areas are known to produce neurons or glia. This is consistent with the suggestion that p19/stathmin is related to differentiation. SCG10 may be a new member of growth-associated proteins and this protein may contribute to neurite extension in perinatal brain as does GAP-43. However, the differential expression between SCG10 and GAP-43 in later developmental stages suggests their diverse functions, which indicates these proteins may play different roles during postnatal brain development.

A novel omega-conopeptide for the presynaptic localization of calcium channels at the mammalian neuromuscular junction.

SummaryVoltage-sensitive Ca2+ channels are essential to transmitter release at the chemical synapse. To demonstrate the localization of voltage-sensitive Ca2+ channels in relation to the site of transmitter release, mouse neuromuscular junctions were double-labelled with α-bungarotoxin and a novel voltage-sensitive Ca2+ channel probe, SNX-260, a synthetic analog of ω-conopeptide MVIIC. Similar to ω-conopeptide MVIIC, biotinylated SNX-260 blocked nerve-stimulated transmitter release at the mouse neuromuscular junction. Fluorescently-tagged biotinylated SNX-260 labelled the nerve terminal which appeared thinner than and was outlined by acetylcholine receptor clusters as seen inen face view. This SNX-260 labelling was inhibited by preincubation with unconjugated SNX-260. Side-views of the neuromuscular junction indicated that the SNX-260 labelling was on the synaptic side facing the acetylcholine receptor rather than on the nonsynaptic side of the nerve terminal. This presynaptic binding was confirmed by the absence of SNX-260 labelling in denervated muscles following a nerve cut or disjunction after collagenase treatment. Confocal microscopy revealed spots of SNX-260 labelling that may correlate with active zones. The SNX-260 labelling pattern was not affected by preincubation with unconjugated SNX-111 (ω-conopeptide MVIIA), an N-type voltage-sensitive Ca2+ channel blocker. These findings suggest that SNX-260 is a novel probe for localizing non-N type voltage-sensitive Ca2+ channels and that these voltage-sensitive Ca2+ channels are localized near the transmitter release sites at the mammalian motor nerve terminal membrane. The results are consistent with the suggestion that non-N, probably P/Q type voltage-sensitive Ca2+ channels mediate evoked transmitter release at the mammalian neuromuscular junction.

The role of Synaptobrevin1/VAMP1 in Ca2+‐triggered neurotransmitter release at the mouse neuromuscular junction

Non‐technical summary  The neuromuscular junction (NMJ) is the synaptic connection between the nerve and the muscle. The neuromuscular synaptic transmission is highly reliable, as each nerve impulse results in the release of more neurotransmitter than is required for evoking an action potential in the muscle. This feature, often referred as the ‘safety factor’, ensures that a muscle contraction will occur in response to each nerve impulse under normal physiological conditions. Here we show that a small, integral membrane protein of synaptic vesicles, named synaptobrevin (Syb)/vesicle‐associated membrane protein (VAMP), is required for optimum synaptic transmission at the NMJ. A genetic mutation in Syb1/VAMP1 in mice causes marked reduction of neurotransmitter release at the NMJ, suggesting an important role for Syb1/VAMP1 in maintaining the ‘safety factor’ of the NMJ.

Abnormal development of the neuromuscular junction in Nedd4-deficient mice.

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.

Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections

Fic domains can catalyze the addition of adenosine monophosphate to target proteins. To date, the function of Fic domain proteins in eukaryotic physiology remains unknown. We generated genetic models of the single Drosophila Fic domain–containing protein, Fic. Flies lacking Fic were viable and fertile, but blind. Photoreceptor cells depolarized normally following light stimulation, but failed to activate postsynaptic neurons, as indicated by the loss of ON transients in electroretinograms, consistent with a neurotransmission defect. Functional rescue of neurotransmission required expression of enzymatically active Fic on capitate projections of glia cells, but not neurons, supporting a role in the recycling of the visual neurotransmitter histamine. Histamine levels were reduced in the lamina of Fic null flies, and dietary histamine partially restored ON transients. These findings establish a previously unknown regulatory mechanism in visual neurotransmission and provide, to the best of our knowledge, the first evidence for a role of glial capitate projections in neurotransmitter recycling.

APP interacts with LRP4 and agrin to coordinate the development of the neuromuscular junction in mice

ApoE, ApoE receptors and APP cooperate in the pathogenesis of Alzheimer’s disease. Intriguingly, the ApoE receptor LRP4 and APP are also required for normal formation and function of the neuromuscular junction (NMJ). In this study, we show that APP interacts with LRP4, an obligate co-receptor for muscle-specific tyrosine kinase (MuSK). Agrin, a ligand for LRP4, also binds to APP and co-operatively enhances the interaction of APP with LRP4. In cultured myotubes, APP synergistically increases agrin-induced acetylcholine receptor (AChR) clustering. Deletion of the transmembrane domain of LRP4 (LRP4 ECD) results in growth retardation of the NMJ, and these defects are markedly enhanced in APP−/−;LRP4ECD/ECD mice. Double mutant NMJs are significantly reduced in size and number, resulting in perinatal lethality. Our findings reveal novel roles for APP in regulating neuromuscular synapse formation through hetero-oligomeric interaction with LRP4 and agrin and thereby provide new insights into the molecular mechanisms that govern NMJ formation and maintenance. DOI: http://dx.doi.org/10.7554/eLife.00220.001

Β-Catenin stabilization in skeletal muscles, but not in motor neurons, leads to aberrant motor innervation of the muscle during neuromuscular development in mice

β-Catenin, a key component of the Wnt signaling pathway, has been implicated in the development of the neuromuscular junction (NMJ) in mice, but its precise role in this process remains unclear. Here we use a β-catenin gain-of-function mouse model to stabilize β-catenin selectively in either skeletal muscles or motor neurons. We found that β-catenin stabilization in skeletal muscles resulted in increased motor axon number and excessive intramuscular nerve defasciculation and branching. In contrast, β-catenin stabilization in motor neurons had no adverse effect on motor innervation pattern. Furthermore, stabilization of β-catenin, either in skeletal muscles or in motor neurons, had no adverse effect on the formation and function of the NMJ. Our findings demonstrate that β-catenin levels in developing muscles in mice are crucial for proper muscle innervation, rather than specifically affecting synapse formation at the NMJ, and that the regulation of muscle innervation by β-catenin is mediated by a non-cell autonomous mechanism.

Neuron-glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function.

The NMJ (neuromuscular junction) serves as the ultimate output of the motor neurons. The NMJ is composed of a presynaptic nerve terminal, a postsynaptic muscle and perisynaptic glial cells. Emerging evidence has also demonstrated an existence of perisynaptic fibroblast-like cells at the NMJ. In this review, we discuss the importance of Schwann cells, the glial component of the NMJ, in the formation and function of the NMJ. During development, Schwann cells are closely associated with presynaptic nerve terminals and are required for the maintenance of the developing NMJ. After the establishment of the NMJ, Schwann cells actively modulate synaptic activity. Schwann cells also play critical roles in regeneration of the NMJ after nerve injury. Thus, Schwann cells are indispensable for formation and function of the NMJ. Further examination of the interplay among Schwann cells, the nerve and the muscle will provide insights into a better understanding of mechanisms underlying neuromuscular synapse formation and function.