Papers - Yuzaki, Michisuke

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Ojima K., Kakegawa W., Yamasaki T., Miura Y., Itoh M., Michibata Y., Kubota R., Doura T., Miura E., Nonaka H., Mizuno S., Takahashi S., Yuzaki M., Hamachi I., Kiyonaka S.

Nature Communications 13 ( 1 )  2022.12

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Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

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The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure

Ito S., Hashimoto H., Yamakawa H., Kusumoto D., Akiba Y., Nakamura T., Momoi M., Komuro J., Katsuki T., Kimura M., Kishino Y., Kashimura S., Kunitomi A., Lachmann M., Shimojima M., Yozu G., Motoda C., Seki T., Yamamoto T., Shinya Y., Hiraide T., Kataoka M., Kawakami T., Suzuki K., Ito K., Yada H., Abe M., Osaka M., Tsuru H., Yoshida M., Sakimura K., Fukumoto Y., Yuzaki M., Fukuda K., Yuasa S.

Nature Communications 13 ( 1 )  2022.12

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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.

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In vivo nanoscopic landscape of neurexin ligands underlying anterograde synapse specification

Nozawa K., Sogabe T., Hayashi A., Motohashi J., Miura E., Arai I., Yuzaki M.

Neuron  2022.10

ISSN  08966273

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Excitatory synapses are formed and matured by the cooperative actions of synaptic organizers, such as neurexins (Nrxns), neuroligins (Nlgns), LRRTMs, and Cbln1. Recent super-resolution nanoscopy developments have revealed that many synaptic organizers, as well as glutamate receptors and glutamate release machinery, exist as nanoclusters within synapses. However, it is unclear how such nanodomains interact with each other to organize excitatory synapses in vivo. By applying X10 expansion microscopy to epitope tag knockin mice, we found that Cbln1, Nlgn1, and LRRTM1, which share Nrxn as a common presynaptic receptor, form overlapping or separate nanodomains depending on Nrxn with or without a sequence encoded by splice site 4. The size and position of glutamate receptor nanodomains of GluD1, NMDA, and AMPA receptors were regulated by Cbln1, Nlgn1, and LRRTM1 nanodomains, respectively. These findings indicate that Nrxns anterogradely regulate the postsynaptic nanoscopic architecture of glutamate receptors through competition and coordination of Nrxn ligands.

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Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

Ojima K., Shiraiwa K., Soga K., Doura T., Takato M., Komatsu K., Yuzaki M., Hamachi I., Kiyonaka S.

Nature Communications 12 ( 1 )  2021.12

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The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

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Subunit-dependent and subunit-independent rules of AMPA receptor trafficking during chemical long-term depression in hippocampal neurons

Matsuda S., Yuzaki M.

Journal of Biological Chemistry 297 ( 2 )  2021.08

ISSN  00219258

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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.

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Destroy the old to build the new: Activity-dependent lysosomal exocytosis in neurons

Ibata K., Yuzaki M.

Neuroscience Research 167   38 - 46 2021.06

ISSN  01680102

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Lysosomes are organelles that support diverse cellular functions such as terminal degradation of macromolecules and nutrient recycling. Additionally, lysosomes can fuse with the plasma membrane, a phenomenon referred to as lysosomal exocytosis, to release their contents, including hydrolytic enzymes and cargo proteins. Recently, neuronal activity has been shown to induce lysosomal exocytosis in dendrites and axons. Secreted lysosomal enzyme cathepsin B induces and stabilizes synaptic structural changes by degrading the local extracellular matrix. Extracellular matrix reorganization could also enhance the lateral diffusion of the co-released synaptic organizer Cbln1 along the surface of axons to facilitate new synapse formation. Similarly, lateral diffusion of dendritic AMPA-type glutamate receptors could be facilitated to enhance functional synaptic plasticity. Therefore, lysosomal exocytosis is a powerful way of building new cellular structures through the coordinated destruction of the old environment. Understanding the mechanisms by which lysosomal exocytosis is regulated in neurons is expected to lead to the development of new therapeutics for neuronal plasticity following spinal cord injury or neurodegenerative disease.

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The autism-associated protein CHD8 is required for cerebellar development and motor function

Kawamura A., Katayama Y., Kakegawa W., Ino D., Nishiyama M., Yuzaki M., Nakayama K.I.

Cell Reports 35 ( 1 )  2021.04

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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.

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Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent

Thimaradka V., Hoon Oh J., Heroven C., Radu Aricescu A., Yuzaki M., Tamura T., Hamachi I.

Bioorganic and Medicinal Chemistry 30 2021.01

ISSN  09680896

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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.

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PIP <inf>3</inf> -Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover

Xie M., Ishikawa Y., Yagi H., Iguchi T., Oka Y., Kuroda K., Iwata K., Kiyonari H., Matsuda S., Matsuzaki H., Yuzaki M., Fukazawa Y., Sato M.

Scientific Reports (Scientific Reports)  9 ( 1 ) 4305 2019.12

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© 2019, The Author(s). The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP 3 , functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP 3 -responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2 −/− mice.

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Hyaluronan synthesis supports glutamate transporter activity.

Hayashi MK, Nishioka T, Shimizu H, Takahashi K, Kakegawa W, Mikami T, Hirayama Y, Koizumi S, Yoshida S, Yuzaki M, Tammi M, Sekino Y, Kaibuchi K, Shigemoto-Mogami Y, Yasui M, Sato K

Journal of neurochemistry 150 ( 3 ) 249 - 263 2019.08

ISSN  0022-3042

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Activity-Dependent Secretion of Synaptic Organizer Cbln1 from Lysosomes in Granule Cell Axons.

Ibata K, Kono M, Narumi S, Motohashi J, Kakegawa W, Kohda K, Yuzaki M

Neuron 102 ( 6 ) 1184 - 1198.e10 2019.06

ISSN  0896-6273

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Interneuronal NMDA receptors regulate long-term depression and motor learning in the cerebellum

Kono M., Kakegawa W., Yoshida K., Yuzaki M.

Journal of Physiology (Journal of Physiology)  597 ( 3 ) 903 - 920 2019.02

ISSN  00223751

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© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society Key points: NMDA receptors (NMDARs) are required for long-term depression (LTD) at parallel fibre–Purkinje cell synapses, but their cellular localization and physiological functions in vivo are unclear. NMDARs in molecular-layer interneurons (MLIs), but not granule cells or Purkinje cells, are required for LTD, but not long-term potentiation induced by low-frequency stimulation of parallel fibres. Nitric oxide produced by NMDAR activation in MLIs probably mediates LTD induction. NMDARs in granule cells or Purkinje cells are dispensable for motor learning during adaptation of horizontal optokinetic responses. Abstract: Long-term potentiation (LTP) and depression (LTD), which serve as cellular synaptic plasticity models for learning and memory, are crucially regulated by N-methyl-d-aspartate receptors (NMDARs) in various brain regions. In the cerebellum, LTP and LTD at parallel fibre (PF)–Purkinje cell (PC) synapses are thought to mediate certain forms of motor learning. However, while NMDARs are essential for LTD in vitro, their cellular localization remains controversial. In addition, whether and how NMDARs mediate motor learning in vivo remains unclear. Here, we examined the contribution of NMDARs expressed in granule cells (GCs), PCs and molecular-layer interneurons (MLIs) to LTD/LTP and motor learning by generating GC-, PC- and MLI/PC-specific knockouts of Grin1, a gene encoding an obligatory GluN1 subunit of NMDARs. While robust LTD and LTP were induced at PF–PC synapses in GC- and PC-specific Grin1 (GC-Grin1 and PC-Grin1, respectively) conditional knockout (cKO) mice, only LTD was impaired in MLI/PC-specific Grin1 (MLI/PC-Grin1) cKO mice. Application of diethylamine nitric oxide (NO) sodium, a potent NO donor, to the cerebellar slices restored LTD in MLI/PC-Grin1 cKO mice, suggesting that NO is probably downstream to NMDARs. Furthermore, the adaptation of horizontal optokinetic responses (hOKR), a cerebellar motor learning task, was normally observed in GC-Grin1 cKO and PC-Grin1 cKO mice, but not in MLI/PC-Grin1 cKO mice. These results indicate that it is the NMDARs expressed in MLIs, but not in PCs or GCs, that play important roles in LTD in vitro and motor learning in vivo.

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In vivo Two-Photon Imaging of Anesthesia-Specific Alterations in Microglial Surveillance and Photodamage-Directed Motility in Mouse Cortex.

Sun W, Suzuki K, Toptunov D, Stoyanov S, Yuzaki M, Khiroug L, Dityatev A

Frontiers in neuroscience 13   421 2019

ISSN  1662-4548

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PhotonSABER: new tool shedding light on endocytosis and learning mechanisms in vivo.

Matsuda S, Kakegawa W, Yuzaki M

Communicative & integrative biology 12 ( 1 ) 34 - 37 2019

ISSN  1942-0889

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Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions.

Saegusa S, Fukaya M, Kakegawa W, Tanaka M, Katsumata O, Sugawara T, Hara Y, Itakura M, Okubo T, Sato T, Yuzaki M, Sakagami H

PloS one 14 ( 5 ) e0216960 2019

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Spatiotemporal regulation of the GPCR activity of BAI3 by C1qL4 and Stabilin-2 controls myoblast fusion

Hamoud N., Tran V., Aimi T., Kakegawa W., Lahaie S., Thibault M., Pelletier A., Wong G., Kim I., Kania A., Yuzaki M., Bouvier M., Côté J.

Nature Communications (Nature Communications)  9 ( 1 ) 4470 2018.12

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© 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.

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Improvement of cerebellar ataxic gait by injecting Cbln1 into the cerebellum of cbln1-null mice

Takeuchi E., Ito-Ishida A., Yuzaki M., Yanagihara D.

Scientific Reports (Scientific Reports)  8 ( 1 ) 6184 2018.12

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© 2018 The Author(s). Patients and rodents with cerebellar damage display ataxic gaits characterized by impaired coordination of limb movements. Here, gait ataxia in mice with a null mutation of the gene for the cerebellin 1 precursor protein (cbln1-null mice) was investigated by kinematic analysis of hindlimb movements during locomotion. The Cbln1 protein is predominately produced and secreted from cerebellar granule cells. The cerebellum of cbln1-null mice is characterized by an 80% reduction in the number of parallel fiber-Purkinje cell synapses compared with wild-Type mice. Our analyses identified prominent differences in the temporal parameters of locomotion between cbln1-null and wild-Type mice. The cbln1-null mice displayed abnormal hindlimb movements that were characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles and knees. When recombinant Cbln1 protein was injected into the cerebellum of cbln1-null mice, the step cycle and stance phase durations increased toward those of wild-Type mice, and the angular excursions of the knee during a cycle period showed a much closer agreement with those of wild-Type mice. These findings suggest that dysfunction of the parallel fiber-Purkinje cell synapses might underlie the impairment of hindlimb movements during locomotion in cbln1-null mice.

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Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum

Nozawa K., Hayashi A., Motohashi J., Takeo Y., Matsuda K., Yuzaki M.

Cerebellum (Cerebellum)  17 ( 6 ) 709 - 721 2018.12

ISSN  14734222

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© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.

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Caveolin-1 Promotes Early Neuronal Maturation via Caveolae-Independent Trafficking of N-Cadherin and L1.

Shikanai M, Nishimura YV, Sakurai M, Nabeshima YI, Yuzaki M, Kawauchi T

iScience 7   53 - 67 2018.09

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La Dolce Vita of Neurexin: Synaptic Partnerships through Glycosaminoglycans.

Suzuki K, Yuzaki M

Cell 174 ( 6 ) 1337 - 1338 2018.09

ISSN  0092-8674

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