1. The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. 2. Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p). 3. Administration of CaM, but not heat-inactivated CaM, encapsulated in liposomes (1000 units ml-1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM. 4. Administration of antiCaM, but not heat-inactivated antiCaM, in liposomes (50 μl ml-1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. 5. Bath application of the membrane-permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. 6. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ.

A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites. At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis. Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18483号 / 生博第312号 / 新制||生||41(附属図書館) / 31361 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Autoimmunity is a failure to tolerate circulating or cell surface expressed self antigens,leading to activation of the immune system and attack of self tissues. Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a disease caused by antibodies to MuSK and hallmarked by fatigable muscle weakness. MuSK is a tyrosine kinase that interacts with low-density lipoprotein receptor-related protein 4 (LRP4), resulting in maintenance of the high density of acetylcholine receptors (AChRs) at the neuromuscular junction; this high density is essential for efficient transmission of signals from nerve to muscle, and MuSK antibodies impair this transmission. MuSK antibodies are predominantly IgG4, a subclass that does not induce immunological damage. Thus how these antibodies cause neuromuscular junction dysfunction is not clear. Potential mechanisms of the MuSK antibodies were explored in in vitro experiments. Plasmas from fourteen MuSK-MG patients were studied. IgG antibodies and IgG subclass profiles were measured with flow cytometry. Total IgG, Fabs, IgG4 and IgG1-3 subclass antibodies were prepared and purified; these were used to investigate the effects on MuSK surface expression, binding of LRP4 to MuSK, and agrin-LRP4-MuSK-induced AChR clustering in C2C12 mouse myotubes. No evidence for MuSK endocytosis by MuSK IgG, IgG1-3 or IgG4 antibodies was found. The predominant IgG4 subclass, and the monovalent IgG Fabs, blocked binding between LRP4 and MuSK but both IgG4 and IgG1-3 subclass antibodies were equally able to disperse pre-formed and newly-induced AChR clusters in C2C12 myotubes. The block of LRP4-MuSK interaction by IgG4 antibodies is likely to be a major pathogenic mechanism in MuSK-MG, which may lead to disrupted signal transduction, reduced AChR aggregation and neuromuscular transmission failure at the neuromuscular junction. In addition, MuSK IgG1-3, until now described as nonpathogenic, may also contribute to the reduced AChR density and neuromuscular dysfunction in MuSK-MG. These results provide new evidence concerning the pathogenic antibodies and their mechanisms in MuSK-MG.

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.