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ABSTRACT: Spinal muscular atrophy (SMA), a motoneuron disease caused by a deficiency of the survival of motor neuron (SMN) protein, is characterized by motoneuron loss and muscle weakness. It remains unclear whether widespread loss of neuromuscular junctions (NMJs) is involved in SMA pathogenesis. We undertook a systematic examination of NMJ innervation patterns in >20 muscles in the SMNΔ7 SMA mouse model. We found that severe denervation (<50% fully innervated endplates) occurs selectively in many vulnerable axial muscles and several appendicular muscles at the disease end stage. Since these vulnerable muscles were located throughout the body and were comprised of varying muscle fiber types, it is unlikely that muscle location or fiber type determines susceptibility to denervation. Furthermore, we found a similar extent of neurofilament accumulation at NMJs in both vulnerable and resistant muscles before the onset of denervation, suggesting that neurofilament accumulation does not predict subsequent NMJ denervation. Since vulnerable muscles were initially innervated, but later denervated, loss of innervation in SMA may be attributed to defects in synapse maintenance. Finally, we found that denervation was amendable by trichostatin A (TSA) treatment, which increased innervation in clinically relevant muscles in TSA-treated SMNΔ7 mice. Our findings suggest that neuromuscular denervation in vulnerable muscles is a widespread pathology in SMA, and can serve as a preparation for elucidating the biological basis of synapse loss, and for evaluating therapeutic efficacy.
Human Molecular Genetics 01/2012; 21(1):185-95. · 7.64 Impact Factor
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Mahru C An,
Weichun Lin,
Jiefei Yang,
Bertha Dominguez,
Daniel Padgett,
Yoshie Sugiura,
Prafulla Aryal,
Thomas W Gould,
Ronald W Oppenheim,
Mark E Hester,
Brian K Kaspar, Chien-Ping Ko,
Kuo-Fen Lee
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ABSTRACT: Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.
Proceedings of the National Academy of Sciences 06/2010; 107(23):10702-7. · 9.68 Impact Factor
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ABSTRACT: Spinal muscular atrophy (SMA) is a major genetic cause of death in childhood characterized by marked muscle weakness. To investigate mechanisms underlying motor impairment in SMA, we examined the spinal and neuromuscular circuitry governing hindlimb ambulatory behavior in SMA model mice (SMNΔ7). In the neuromuscular circuitry, we found that nearly all neuromuscular junctions (NMJs) in hindlimb muscles of SMNΔ7 mice remained fully innervated at the disease end stage and were capable of eliciting muscle contraction, despite a modest reduction in quantal content. In the spinal circuitry, we observed a ∼28% loss of synapses onto spinal motoneurons in the lateral column of lumbar segments 3-5, and a significant reduction in proprioceptive sensory neurons, which may contribute to the 50% reduction in vesicular glutamate transporter 1(VGLUT1)-positive synapses onto SMNΔ7 motoneurons. In addition, there was an increase in the association of activated microglia with SMNΔ7 motoneurons. Together, our results present a novel concept that synaptic defects occur at multiple levels of the spinal and neuromuscular circuitry in SMNΔ7 mice, and that proprioceptive spinal synapses could be a potential target for SMA therapy.
PLoS ONE 01/2010; 5(11):e15457. · 4.09 Impact Factor
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ABSTRACT: Recent studies suggest that glial cells actively participate in the formation, function, maintenance, and repair of the chemical synapse. However, the molecular mechanisms of glia-synapse interactions are largely unknown. We have shown previously that Schwann cell-conditioned medium (SC-CM) promotes synaptogenesis in Xenopus nerve-muscle cocultures. The present study aimed to identify the synaptogenic molecules in SC-CM. Combining biochemical approaches and in vitro bioassays, we found that SC-CM contains transforming growth factor (TGF)-beta1, which is expressed in Schwann cells both in vivo and in vitro. Similar to SC-CM, TGF-beta1 doubled the size of acetylcholine receptor (AChR) clusters at nerve-muscle contacts and significantly increased the percentage of nerve-muscle contacts that show AChR clusters to approximately 60%, compared with approximately 20% seen in control cultures. The synaptogenic effects of SC-CM were abolished if SC-CM was immunodepleted of TGF-beta1 or if the latency-associated protein or a TGF-beta1 receptor kinase inhibitor was added to block the bioactivity of TGF-beta1. Similar to frog SC-CM, mammalian SC-CM also showed synaptogenic effects, which were prevented by immunodepletion of TGF-beta1. TGF-beta1 upregulated agrin expression in spinal neurons, which could explain the increase in AChR clusters in cultures treated with SC-CM. These results suggest that Schwann cells express TGF-beta1, which is both sufficient and necessary for mediating the synapse-promoting effects of Schwann cells at the developing neuromuscular junction. Schwann cell-derived TGF-beta1 thus joins other astrocyte-derived synaptogenic factors in further strengthening the emerging concept that glial cells contribute to synaptogenesis in both the PNS and the CNS.
Journal of Neuroscience 10/2008; 28(39):9599-609. · 7.11 Impact Factor
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Chien-Ping Ko
The Journal of Physiology 08/2008; 586(13):3021. · 4.72 Impact Factor
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ABSTRACT: The vertebrate neuromuscular junction (NMJ) is a "tripartite" synapse, composed of three cellular elements: the presynaptic nerve terminal, the postsynaptic specialization, and synapse-associated glial cells, called perisynaptic Schwann cells (PSCs; also called terminal Schwann cells). During development, PSCs grow beyond nerve terminals and guide nerve terminal extension. Nerve terminals retract or stop extension after PSC ablation by complement-mediated lysis in vivo, suggesting that PSCs can promote synaptic growth and maintenance at developing NMJs. Schwann cell-conditioned medium (SC-CM), which may be mediated by transforming growth factor-beta1, can promote synapse formation in Xenopus nerve-muscle culture. In addition, SC-CM contains small molecules (within 500-5000 Da), which can enhance spontaneous synaptic activities acutely and potently at developing frog NMJs. In adult muscles, PSCs can detect evoked synaptic activities and are capable of modulating transmitter release. Nerve terminals retract and synaptic efficacy is reduced at 1 week, but not within the first few hours, after PSC ablation. Thus, PSCs are essential for the long-term, but not short-term, maintenance of synaptic structure and function at the adult NMJ. During synaptic remodeling in adult muscles, PSC sprouts lead nerve terminal sprouts. After nerve injury, adult PSCs sprout extensive processes, which guide regenerating nerve terminals. Schwann cells express agrin and neuregulins, which may help the postsynaptic differentiation and synaptic repair. Furthermore, neuregulin-ErbB signaling pathways play an essential role in synapse-glial interactions at the NMJ. These recent findings suggest that PSCs play multiple roles and actively participate in synaptic development, modulation, maintenance, and repair of the vertebrate NMJ.
Annals of the New York Academy of Sciences 07/2008; 1132:19-28. · 3.15 Impact Factor
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ABSTRACT: Glial cells are active participants in the function, formation, and maintenance of the chemical synapse. To investigate the molecular basis of neuron-glia interactions at the peripheral synapse, we examined whether and how Schwann cell-derived factors modulate synaptic function at developing neuromuscular junctions (NMJs). Schwann cell-conditioned medium (SC-CM) from Xenopus Schwann cell cultures was collected and applied to Xenopus nerve-muscle cocultures. We found that SC-CM increased the frequency of spontaneous synaptic currents (SSCs) within 3-15 min by an average of approximately 150-fold at developing neuromuscular synapses. The increase in SSC frequency by SC-CM is a presynaptic effect independent of neuronal excitability and requires the influx of Ca2+. In contrast to its potentiating effect on spontaneous transmitter release, SC-CM suppressed the evoked transmitter release. The SC-CM effect required the presence of motoneuron soma but not protein synthesis. Using molecular weight cutoff filters and dialysis membranes, we found that the molecular weight of functional factor(s) in SC-CM was within 500 and 5000 Da. The SC-CM effect was not attributable to currently known factors that modulate synaptic efficacy, including neurotrophins, glutamate, and ATP. SC-CM also enhanced spontaneous synaptic release at developing NMJs in Xenopus tadpoles in situ. Our results suggest that Schwann cells release small molecules that enhance spontaneous synaptic activities acutely and potently at developing neuromuscular synapses, and the glial cell-enhanced spontaneous neurotransmission may contribute to synaptogenesis.
Journal of Neuroscience 07/2007; 27(25):6712-22. · 7.11 Impact Factor
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ABSTRACT: Emerging studies demonstrate that perisynaptic Schwann cells (PSCs), which are the glia cells juxtaposed to the motor nerve terminal, actively participate in multiple aspects of the neuromuscular junction. During development, PSCs guide and promote synaptic growth. In adult muscles, PSCs can sense nerve stimulation by increasing intracellular calcium and are also capable of modulating transmitter release. Although adult PSCs are not required for acute synaptic maintenance and function, they are indispensable for long-term synaptic maintenance. Furthermore, PSC sprouts lead nerve terminal extension during synaptic remodeling. After nerve injury, PSCs sprout profusely and PSC processes guide regenerating nerve terminals. Future challenges will be to identify the molecular mechanisms by which PSCs interact with the nerve terminal and the muscle fiber.
Current Opinion in Pharmacology 06/2007; 7(3):316-24. · 6.86 Impact Factor
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CNS Drug Reviews 06/2006; 4(4):380 - 383. · 4.92 Impact Factor
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ABSTRACT: Glial cells are widely distributed throughout the nervous system, including at the chemical synapse. However, our knowledge of the role of glial cells at the synapse is rudimentary. Recent studies using a model synapse, the vertebrate neuromuscular junction (NMJ), have demonstrated that perisynaptic Schwann cells (PSCs), which are the glia juxtaposed to the nerve terminal at the NMJ, play active and essential roles in synaptic function, maintenance, and development. PSCs can respond to nerve activity by increasing intracellular calcium and are capable of modulating synaptic function in response to pharmacological manipulations. Studies using PSC ablation in vivo have shown that PSCs are essential for the long-term maintenance of synaptic structure and function at the adult NMJ. In vivo observations have also shown that PSCs guide presynaptic nerve terminal extension and dictate the pattern of innervation during synaptic regeneration and remodeling at adult NMJs. PSCs may also induce postsynaptic acetylcholine receptor aggregation. Furthermore, PSCs play an essential role in synaptic growth and maintenance during development of NMJs in vivo, and Schwann cell-derived factors can promote synaptogenesis and enhance synaptic transmission in tissue culture. These recent findings advance the emerging concept that glial cells help make bigger, stronger, and more stable synapses.
The Neuroscientist 11/2005; 11(5):503-13. · 4.57 Impact Factor
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ABSTRACT: The process by which excess axons are pruned during development has remained unclear. In this issue of Neuron, Bishop et al. use time-lapse imaging and serial electron microscopy of developing neuromuscular junctions to describe a novel cellular mechanism in which retracting axon branches shed fragments rich in normal synaptic organelles. These "axosomes" are engulfed by adjacent Schwann cells and may be assimilated into the glial cytoplasm. Shedding of axosomes and glial engulfment may represent a widespread mechanism of synapse elimination.
Neuron 11/2004; 44(4):578-80. · 14.74 Impact Factor
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Journal of Neuroscience 11/2004; 24(42):9250-60. · 7.11 Impact Factor
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ABSTRACT: 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.
Neuron 11/2003; 40(3):563-80. · 14.74 Impact Factor
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ABSTRACT: Recent studies have shown that the survival of mammalian motoneurons in vitro is promoted by neurotrophins (NTs) and cAMP. There is also evidence that neurotrophins enhance transmitter release. We thus investigated whether these agents also promote synaptogenesis. Cultured Xenopus spinal cord neurons were treated with a mixture of BDNF, glia-derived neurotrophic factor, NT-3, and NT-4, in addition to forskolin and IBMX or the cell-permeant form of cAMP, to elevate the cAMP level. The outgrowth and survival of neurons were dramatically increased by this trophic stimulation. However, when these neurons were cocultured with muscle cells, the trophic agents resulted in a failure of synaptogenesis. Specifically, the induction of ACh receptor (AChR) clustering in cultured muscle cells was inhibited at nerve-muscle contacts, in sharp contrast to control, untreated cocultures. Because AChR clustering induced by agrin or growth factor-coated beads in muscle cells was unaffected by trophic stimulation, its effect on synaptogenesis is presynaptic in origin. In the control, agrin was deposited along the neurite and at nerve-muscle contacts. This was significantly downregulated in cultures treated with trophic stimuli. Reverse transcriptase-PCR analyses showed that this decrease in agrin deposition was caused by an inhibition of agrin synthesis by trophic stimuli. Both agrin synthesis and induction of AChR clustering were restored under trophic stimulation when Schwann cell-conditioned medium was introduced. These results suggest that trophic stimulation maintains spinal neurons in the growth state, and Schwann cell-derived factors allow them to switch to the synaptogenic state.
Journal of Neuroscience 07/2003; 23(12):5050-60. · 7.11 Impact Factor
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Eugene P Brandon,
Weichun Lin,
Kevin A D'Amour,
Donald P Pizzo,
Bertha Dominguez,
Yoshie Sugiura,
Silke Thode, Chien-Ping Ko,
Leon J Thal,
Fred H Gage,
Kuo-Fen Lee
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ABSTRACT: In this study we examined the developmental roles of acetylcholine (ACh) by establishing and analyzing mice lacking choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh. As predicted, ChAT-deficient embryos lack both spontaneous and nerve-evoked postsynaptic potentials in muscle and die at birth. In mutant embryos, abnormally increased nerve branching occurs on contact with muscle, and hyperinnervation continues throughout subsequent prenatal development. Postsynaptically, ACh receptor clusters are markedly increased in number and occupy a broader muscle territory in the mutants. Concomitantly, the mutants have significantly more motor neurons than normal. At an ultrastructural level, nerve terminals are smaller in mutant neuromuscular junctions, and they make fewer synaptic contacts to the postsynaptic muscle membrane, although all of the typical synaptic components are present in the mutant. These results indicate that ChAT is uniquely essential for the patterning and formation of mammalian neuromuscular synapses.
Journal of Neuroscience 02/2003; 23(2):539-49. · 7.11 Impact Factor
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ABSTRACT: Voltage-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 -bungarotoxin and a novel voltage-sensitive Ca2+ channel probe, SNX-260, a synthetic analog of -conopeptide MVIIC. Similar to -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 (-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.
Journal of Neurocytology 12/1994; 24(1):15-27. · 1.94 Impact Factor
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ABSTRACT: To search for ultrastructural correlates of differences in synaptic safety factor and neurotransmitter release, neuromuscular junctions from the cutaneous pectoris and cutaneous dorsi muscles of the grass frogRana pipiens were freeze fractured. Synaptic efficacy in these muscles was determined by the extent to which isometric twitch tension could be blocked by lowering [Ca2+] in the bathing solution. We found that junctions in the cutaneous pectoris were significantly more effective than those of the cutaneous dorsi. Morphometric analysis of 16 junctions from each type of muscle showed significant differences in some aspects of active zone structure. Cutaneous pectoris terminals had longer active zone segments and active zones spaced more closely together. This resulted in 20% more active zone length per unit terminal length in the cutaneous pectoris. Cutaneous dorsi terminals had active zones that were more often segmented into two or more sections at a single junctional fold. Mean active zone length per junctional fold and the number of active zone particles per micrometre of active zone length were not significantly different. As a result of the somewhat larger terminal width in the cutaneous dorsi, the percentage of terminal width occupied by active zone was greater in the cutaneous pectoris. As an attempt to indirectly estimate active zone spacing with the light microscope, we applied rhodamine-conjugated alpha bungarotoxin to neuromuscular junctions from the cutaneous pectoris and cutaneous dorsi. No significant difference in the spacing of fluorescently labelled acetylcholine receptor bands was found between the two types of junctions. Our results indicated that the greater active zone length per unit terminal length in the cutaneous pectoris was associated with its increased synaptic efficacy. In addition the continuity and particle organization of active zones may have contributed to the observed differences in synaptic safety factor at frog neuromuscular junctions.
Journal of Neurocytology 07/1986; 15(4):525-534. · 1.94 Impact Factor
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ABSTRACT: Like other vertebrate synapses, the neuromuscular junction (NMJ) has glial cells that are closely associated with the pre- and post-synaptic components. These "perisynaptic Schwann cells" (PSCs) cover nerve terminals and are in close proximity to the synapse, yet their role at the NMJ has remained mysterious for decades. In this review we explore historical perspectives on PSCs and highlight key developments in recent years that have provided novel insight into PSC functions at the NMJ. First among these developments is the generation of specific antibody probes for PSCs. Using one such antibody and the principle of complement-mediated cell lysis, we have developed a novel technique to selectively ablate PSCs en masse from frog NMJs in vivo. Applying this approach, we have shown that PSCs are essential for the long-term maintenance of synaptic structure and function. In addition, PSCs are essential for the growth and maintenance of NMJs during development. Probes for PSCs also allow us to observe in vivo that processes extended by PSCs guide nerve terminals during synapse development, remodeling, and regeneration. PSCs may therefore dictate the pattern of innervation at the NMJ. Finally, PSCs may also induce postsynaptic acetylcholine receptor expression and aggregation. This wealth of recent findings about PSCs suggests that these synapse-associated glial cells are a more integral and essential component of the NMJ than previously appreciated. New approaches currently being applied at the NMJ may further support the emerging view that glial cells help make bigger, stronger, and more stable synapses.
Journal of Neurocytology 32(5-8):987-1002. · 1.94 Impact Factor
