Published papers

© 2018, The Author(s). Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1–4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion.

© 2018 by Annual Reviews. All rights reserved. Research in the last two decades has identified many synaptic organizers in the central nervous system that directly regulate the assembly of pre- and/or postsynaptic molecules, such as synaptic vesicles, active zone proteins, and neurotransmitter receptors. They are classified into secreted factors and cell adhesion molecules, such as neurexins and neuroligins. Certain secreted factors are termed extracellular scaffolding proteins (ESPs) because they are components of the synaptic extracellular matrix and serve as a scaffold at the synaptic cleft. These include Lgi1, Cbln1, neuronal pentraxins, Hevin, thrombospondins, and glypicans. Diffusible secreted factors, such as Wnts, fibroblast growth factors, and semaphorins, tend to act from a distance. In contrast, ESPs remain at the synaptic cleft and often help synaptic adhesion and/or accumulation of postsynaptic receptors. Many fundamental questions remain about when, how, and why various synaptic organizers establish and modify the vast numbers of connections during development and throughout life.

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+–NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.

Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity–induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit–independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.

Mutations in the gene encoding the chromatin remodeler chromodomain helicase DNA-binding protein 8 (CHD8) are a highly penetrant risk factor for autism spectrum disorder (ASD). Although cerebellar abnormalities have long been thought to be related to ASD pathogenesis, it has remained largely unknown whether dysfunction of CHD8 in the cerebellum contributes to ASD phenotypes. We here show that cerebellar granule neuron progenitor (GNP)-specific deletion of Chd8 in mice impairs the proliferation and differentiation of these cells as well as gives rise to cerebellar hypoplasia and a motor coordination defect, but not to ASD-like behavioral abnormalities. CHD8 is found to regulate the expression of neuronal genes in GNPs. It also binds preferentially to promoter regions and modulates local chromatin accessibility of transcriptionally active genes in these cells. Our results have thus uncovered a key role for CHD8 in cerebellar development, with important implications for understanding the contribution of this brain region to ASD pathogenesis.

Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

The localization of certain mRNAs to dendrites and their local translation in synaptic regions are proposed to be involved in certain aspects of synaptic plasticity. A cis-acting element within the 3′ untranslated region (3′ UTR) of the targeted mRNAs, which is bound by a trans-acting RNA-binding protein, controls the dendritic mRNA localization. Here, we identified hematopoietic zinc finger (Hzf) as a trans-acting factor that regulates the dendritic mRNA localization of the type 1 inositol 1,4,5-trisphosphate receptor (IP3RI), a dendritically localized mRNA in cerebellar Purkinje cells, via binding to the 3′ UTR. In Hzf-deficient mice, the dendritic localization of IP3RI mRNA and brain-derived neurotrophic factor-induced IP3RI protein synthesis in the cerebellum were impaired. These findings suggest that Hzf is an RNA-binding protein that controls the dendritic mRNA localization and activity-dependent translation of IP3RI, and may be involved in some aspects of synaptic plasticity. © 2005 by The National Academy of Sciences of the USA.

The fate of grafted neuroepithelial stem cells in the normal mature brain environment was assessed both morphologically and electrophysiologically to confirm their feasibility in the functional repair of damaged neural circuitry. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of transgenic green fluorescence protein-carrying rat embryos, and implanted into the normal adult rat striatum. The short- and long-term differentiation pattern of donor-derived cells was precisely monitored immunohistochemically. The functional abilities of the donor-derived cells and communication between them and the host were investigated using host-rat brain slices incorporating the graft with whole-cell patch-clamp recording. Vigorous differentiation of the neuroepithelial stem cells into mostly neurons was noted in the short-term with positive staining for tyrosine hydroxylase, suggesting that the donor-derived cells were exclusively following their genetically programmed fate, together with gamma-aminobutyric acid (GABA) and glutamate expression. In the long-term, the large number of donor-derived neurons was sustained, but the staining pattern showed expression of dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein 32, suggesting that some neurons were following environmental cues, together with the appearance of some cholinergic neurons. Some donor-derived astrocytes were also seen in the graft. Many action potentials indicating the presence of both dopaminergic and non-dopaminergic patterns could be elicited and recorded in the donor-derived neurons in addition to spontaneous glutamatergic and GABAergic post-synaptic currents which were strongly shown to be of host origin. Neuroepithelial stem cells are therefore an attractive candidate as a source of donor material for intracerebral grafting in functional repair. © 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GIuR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical synapses allow neurons to perform complex computations and regulate other systems of the body. At a chemical synapse, pre- and postsynaptic sites are separated by a small space (the synaptic cleft) and surrounded by astrocytes. The basement membrane (BM), a sheetlike, specialized extracellular matrix (ECM), is found ubiquitously in the PNS. It has become clear that the ECMs not only play a structural role but also serve as barriers and filters in the PNS and CNS. Moreover, proteoglycans and tenascin family proteins in the ECM regulate synapse formation and synaptic plasticity. Although CNS synapses lack the BMs, recent results indicate that the BM-associated collagens are also present in the CNS synaptic cleft and affect synaptogenesis in both the CNS and the PNS. The C1q domain-containing family proteins are important components of the CNS synaptic cleft in regulating synapse formation, maintenance, and the pruning process. The ECM is regarded as a crucial component of the tetrapartite synapse, consisting of pre- and postsynaptic neurons, astrocyte, and ECM.

Background: The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets. Results: Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state. Conclusions: Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets. © 2014 Zhou et al.; licensee BioMed Central Ltd.

The delay eyeblink conditioning (EBC) is a cerebellum-dependent type of associative motor learning. However, the exact roles played by the various cerebellar synapses, as well as the underlying molecular mechanisms, remain to be determined. It is also unclear whether long-term potentiation (LTP) or long-term depression (LTD) at parallel fiber (PF)-Purkinje cell (PC) synapses is involved in EBC. In this study, to clarify the role of PF synapses in the delay EBC, we used mice in which a gene encoding Cbln1 was disrupted (cbln1-/- mice), which display severe reduction of PF-PC synapses. We showed that delay EBC was impaired in cbln1-/- mice. Although PF-LTD was impaired, PF-LTP was normally induced in cbln1-/- mice. A single recombinant Cbln1 injection to the cerebellar cortex in vivo completely, though transiently, restored the morphology and function of PF-PC synapses and delay EBC in cbln1-/- mice. Interestingly, the cbln1-/- mice retained the memory for at least 30 days, after the Cbln1 injection's effect on PF synapses had abated. Furthermore, delay EBC memory could be extinguished even after the Cbln1 injection's effect were lost.These results indicate that intact PF-PC synapses and PF-LTD, not PF-LTP are necessary to acquire delay EBC in mice. In contrast, extracerebellar structures or remaining PF-PC synapses in cbln1-/- mice may be sufficient for the expression, maintenance, and extinction of its memory trace. © 2013 Emi, Kakegawa, Miura, Ito-Ishida, Kohda and Yuzaki.

Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells. A new in utero electroporation (IUE) protocol has been developed to deliver genes preferentially into cerebellar Purkinje cells. IUE did not alter the physiological characteristics or normal synaptic plasticity of Purkinje cells. IUE allowed selective, effective, and temporally regulated expression of multiple foreign genes in Purkinje cells in vivo. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding δ2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2Lc) triggers cell death has remained elusive. Here, taking advantage of recent knowledge about the domain structure of GluD2, we reinvestigated Lc-mediated cell death, focusing on the "autophagic cell death" hypothesis. Although autophagy and cell death were induced by the expression of GluD2Lc in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2Lc or by removal of extracellular Na+. In addition, although GluD2Lc is reported to directly activate autophagy, mutant channels that are not associated with n-PIST (neuronal isoform of protein-interacting specifically with TC10)-Beclin1 still caused autophagy and cell death. Furthermore, cells expressing GluD2Lc showed decreased ATP levels and increased AMP-activated protein kinase(AMPK)activities in a manner dependent on extracellular Na+. Thus, constitutive currents were likely necessary and sufficient to induce autophagy via AMPK activation, regardless of the n-PIST-Beclin1 pathway in vitro. Interestingly, the expression of dominant-negative AMPK suppressed GluD2Lc-induced autophagy but did not prevent cell death in heterologous cells. Similarly, the disruption of Atg5, a gene crucial for autophagy, did not prevent but rather aggravated Purkinje-cell death in Lc mice. Furthermore, calpains were specifically activated in Lc Purkinje cells. Together, these results suggest that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro. Copyright © 2010 the authors.

Neurons are highly polarized cells comprising somatodendritic and axonal domains. For proper neuronal function, such as neurotransmission and synaptic plasticity, membrane proteins must be transported to precise positions. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPA receptors) are membrane proteins localized in the somatodendritic domain. AMPA receptors mediate excitatory synaptic transmission; thus, regulation of the intracellular trafficking of AMPA receptors has a critical role in synaptic plasticity. An understanding of the molecular mechanisms that regulate AMPA receptor trafficking is essential for gaining further insight into neuronal function. Despite its importance, however, how neurons selectively transport AMPA receptors to the somatodendritic domain is largely unknown. In this Update Article, we discuss recent progress in studies of the mechanisms underlying the somatodendritic targeting of AMPA receptors in neurons. © 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society.