Details of a Researcher - Yuzaki, Michisuke

Papers - Yuzaki, Michisuke

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Dynein- and activity-dependent retrograde transport of autophagosomes in neuronal axons

Katsumata Kiyoshi, Nishiyama Jun, Inoue Takafumi, Mizushima Noboru, Takeda Junzo, Yuzaki Michisuke

Autophagy 6 ( 3 ) 378 - 385 2010.04

ISSN 1554-8627

The accumulation of autophagosomes within axons is often observed in axonopathies associated with various neurological disorders, including those following excitotoxic insults. Nevertheless, the life cycle of autophagosomes in axons is not well understood. In the present study, we used microexplant cultures of cerebellar granule cells from GFP-LC3 transgenic mice to perform time-lapse imaging of LC3-positive dots in identified axons. Since these GFP-LC3 dots were never observed in granule cells on an Atg5-null background, they were considered to represent autophagosomes. Under physiological conditions, the autophagosomes showed bidirectional and saltatory movement with a bias towards one direction. Such vectorial movement was largely blocked by the dynein motor inhibitor EHNA (erythro-9-[3-(2-hydroxynonyl)] adenine), suggesting that the autophagosomes moved towards the soma, where most lysosomes are located. Interestingly, the application of the glutamate analog N-methyl-D-aspartic acid (NMDA) as an excitotoxin increased the number of autophagosomes in axons, while it did not significantly change its movement characteristics. These results suggest that autophagosomes play important roles in axons and are dynamically regulated under physiological and pathological conditions. © 2010 Landes Bioscience.

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Reevaluation of neurodegeneration in lurcher mice

Nishiyama Jun, Matsuda Keiko, Kakegawa Wataru, Yamada Nobuaki, Motohashi Junko, Mizushima Noboru, Yuzaki Michisuke

Journal of Neuroscience 30 ( 6 ) 2177 - 2187 2010.02

ISSN 0270-6474

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 (GluD2^Lc) 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 GluD2^Lc in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2^Lc or by removal of extracellular Na⁺. In addition, although GluD2^Lc 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 GluD2^Lc 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 GluD2^Lc-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.

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Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells

Tao Osamu, Shimazaki Takuya, Okada Yohei, Naka Hayato, Kohda Kazuhisa, Yuzaki Michisuke, Mizusawa Hidehiro, Okano Hideyuki

Journal of Neuroscience Research 88 ( 2 ) 234 - 247 2010.02

ISSN 0360-4012

Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a .oating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6-8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na⁺ or Ca2 ⁺ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley-Liss, Inc.

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Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Sugimoto Mayumi, Sasaki Shinji, Watanabe Toshio, Nishimura Shota, Ideta Atsushi, Yamazaki Maya, Matsuda Keiko, Yuzaki Michisuke, Sakimura Kenji, Aoyagi Yoshito, Sugimoto Yoshikazu

PLoS ONE 5 ( 11 ) 2010

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/ isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1^Asn has a weaker affinity to glutamate than GRIA1^Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1^Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1^Ser. In addition, cows with GRIA1^Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1^Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy. © 2010 Sugimoto et al.

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Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins

Iijima Takatoshi, Miura Eriko, Watanabe Masahiko, Yuzaki Michisuke

European Journal of Neuroscience 31 ( 9 ) 1606 - 1615 2010

ISSN 0953-816X

Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system. © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

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New (but old) molecules regulating synapse integrity and plasticity

Yuzaki M.

Neuroscience 162 ( 3 ) 633 - 643 2009.09

ISSN 0306-4522

The δ2 glutamate receptor (GluRδ2) is predominantly expressed in cerebellar Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2-null mice display ataxia and impaired motor learning. Interestingly, the contact state of synapses between parallel fibers (PFs) and Purkinje cells is specifically and severely affected, and the number of normal PF synapses is markedly reduced in GluRδ2-null Purkinje cells. Furthermore, long-term depression at PF-Purkinje cell synapses is abrogated. Cbln1, a member of the C1q/tumor necrosis factor (TNF) superfamily, is predominantly expressed and released from cerebellar granule cells. Unexpectedly, the behavioral, physiological and anatomical phenotypes of cbln1-null mice precisely mimic those of GluRδ2-null mice. Thus, we propose that Cbln1, which is released from granule cells, and GluRδ2, which is predominantly expressed in Purkinje cells, are involved in a common signaling pathway crucial for synapse formation/maintenance and plasticity in the cerebellum. Since molecules related to Cbln1 are expressed in various brain regions other than the cerebellum, other C1q/TNF superfamily proteins may also regulate various aspects of synapses in the CNS. Therefore, an understanding of the signaling mechanisms underlying Cbln1 and GluRδ2 in the cerebellum will provide new insights into the roles of C1q/TNF superfamily proteins as new cytokines that regulate normal and abnormal brain functions. © 2009 IBRO.

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Polarized sorting of AMPA receptors to the somatodendritic domain is regulated by adaptor protein AP-4

Matsuda Shinji, Yuzaki Michisuke

Neuroscience Research 65 ( 1 ) 1 - 5 2009.09

ISSN 0168-0102

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.

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The N-terminal domain of GluD2 (GluRδ2) recruits presynaptic terminals and regulates synaptogenesis in the cerebellum in vivo

Kakegawa Wataru, Miyazaki Taisuke, Kohda Kazuhisa, Matsuda Keiko, Emi Kyoichi, Motohashi Junko, Watanabe Masahiko, Yuzaki Michisuke

Journal of Neuroscience 29 ( 18 ) 5738 - 5748 2009.05

ISSN 0270-6474

The δ2 glutamate receptor (GluRδ2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2^wt) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2^wt, but not of a mutant GluD2 lacking the NTD (GluD2^ΔNTD), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2 ^NTD-GluK2) did. Similarly, GluD2^wt and GluD2 ^NTD-GluK2, but not GluD2^ΔNTD, induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF-Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively. Copyright © 2009 Society for Neuroscience.

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Activity-dependent repression of Cbln1 expression

Iijima Takatoshi, Emi Kyoichi, Yuzaki Michisuke

Journal of Neuroscience 29 ( 17 ) 5425 - 5434 2009.04

ISSN 0270-6474

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is released from cerebellar granule cells and plays a crucial role in forming and maintaining excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells not only during development but also in the adult cerebellum. Although neuronal activity is known to cause morphological changes at synapses, how Cbln1 signaling is affected by neuronal activity remains unclear. Here, we show that chronic stimulation of neuronal activity by elevating extracellular K⁺ levels or by adding kainate decreased the expression of cbln1 mRNA within several hours in mature granule cells in a manner dependent on L-type voltage-dependent Ca²⁺ channels and calcineurin. Chronic activity also reduced Cbln1 protein levels within a few days, during which time the number of excitatory synapses on Purkinje cell dendrites was reduced; this activity-induced reduction of synapses was prevented by the addition of exogenous Cbln1 to the culture medium. Therefore, the activity-dependent downregulation of cbln1 may serve as a new presynaptic mechanism by which PF-Purkinje cell synapses adapt to chronically elevated activity, thereby maintaining homeostasis. In addition, the expression of cbln1 mRNA was prevented when immature granule cells were maintained in high-K ⁺ medium. Since immature granule cells are chronically depolarized before migrating to the internal granule layer, this depolarization-dependent regulation of cbln1 mRNA expression may also serve as a developmental switch to facilitate PF synapse formation in mature granule cells in the internal granule layer. Copyright © 2009 Society for Neuroscience.

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Cbln1 accumulates and colocalizes with Cbln3 and GluRδ2 at parallel fiber-Purkinje cell synapses in the mouse cerebellum

Miura Eriko, Matsuda Keiko, Morgan James I., Yuzaki Michisuke, Watanabe Masahiko

European Journal of Neuroscience 29 ( 4 ) 693 - 706 2009.02

ISSN 0953-816X

Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluRδ2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluRδ2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluRδ2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum. © The Authors (2009).

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Cbln1 binds to specific postsynaptic sites at parallel fiber-Purkinje cell synapses in the cerebellum

Matsuda Keiko, Kondo Tetsuro, Iijima Takatoshi, Matsuda Shinji, Watanabe Masahiko, Yuzaki Michisuke

European Journal of Neuroscience 29 ( 4 ) 707 - 717 2009.02

ISSN 0953-816X

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis. © The Authors (2009).

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[How polarized sorting to neuronal dendrites is achieved

Matsuda Shinji, Yuzaki Michisuke

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 16 Suppl ) 2214 - 2219 2008.12

ISSN 0039-9450

AP-4

Matsuda Shinji, Yuzaki Michisuke

Autophagy 4 ( 6 ) 815 - 816 2008.08

ISSN 1554-8627

Neurons are highly polarized cells composed of two distinct domains, the axon and the somatodendritic domain. Although AMPA-type glutamate receptors, which mediate fast excitatory neurotransmission in the vertebrate CNS, are preferentially expressed in the somatodendritic domain, the molecular mechanisms underlying such polarized distribution have remained elusive. We recently demonstrated that adaptor protein complex-4 (AP-4) binds to transmembrane AMPA receptor regulatory proteins (TARPs), thereby mediating the selective trafficking of AMPA receptors to the somatodendritic domain; generic disruption of AP-4 (AP-4β^-/-), results in the mislocalization of TARPs and AMPA receptors in the axons. Similarly, low-density lipoprotein receptors and δ2 glutamate receptors are mislocalized in axons, while other cargos, such as NMDA receptors and metabotropic glutamate receptors, are properly excluded from AP-4β^-/- axons. These findings indicate that there exist AP-4-dependent and -independent sorting mechanisms. Unexpectedly, mislocalized AMPA receptors do not reach the cell surface and accumulate in autophagosomes in the bulging portions of AP-4β^-/- axons. Several lines of evidence indicate that mislocalized AMPA receptors activate the autophagic pathway. Since increased autophagy and axonal swelling are suggested to occur in various neuronal disorders, further studies using AP-4β^-/- mice are warranted to understand the mechanisms regulating autophagy in axons. ©2008 Landes Bioscience.

AP-4: autophagy-four mislocalized proteins in axons.

Matsuda S, Yuzaki M

Autophagy 4 ( 6 ) 815 - 6 2008.08

ISSN 1554-8627

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Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar purkinje cells in vitro and in vivo

Ito-Ishida Aya, Miura Eriko, Emi Kyoichi, Matsuda Keiko, Iijima Takatoshi, Kondo Tetsuro, Kohda Kazuhisa, Watanabe Masahiko, Yuzaki Michisuke

Journal of Neuroscience 28 ( 23 ) 5920 - 5930 2008.06

ISSN 0270-6474

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders. Copyright © 2008 Society for Neuroscience.

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Phosphorylation of delta2 glutamate receptors at serine 945 is not required for cerebellar long-term depression

Nakagami Ryoichi, Kohda Kazuhisa, Kakegawa Wataru, Kondo Tetsuro, Kato Nobumasa, Yuzaki Michisuke

Keio Journal of Medicine 57 ( 2 ) 105 - 110 2008.06

ISSN 0022-9717

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell synapses is thought to regulate motor learning and memory formation in the cerebellum. Neuronal activity-evoked protein kinase C (PKC) activation is required for the induction of LTD. In addition, the δ2 glutamate receptor (GluRδ2), which is predominantly expressed at PF-Purkinje cell synapses, is indispensable for the induction of LTD; however, the mechanisms by which GluRδ2 regulates LTD and its relationship with PKC activation remain elusive. Interestingly, GluRδ2 is phosphorylated by PKC on serine 945 (Ser945) near its C-terminus and a postsynaptic protein S-SCAM, which could potentially regulate glutamate receptor trafficking and synaptic plasticity, binds to the extreme C-terminus of GluRδ2 in a phosphorylation-dependent manner on Ser945. Here, using a Sindbis-based virus expression approach, we show that a mutant GluRδ2, in which alanine replaced Ser945 and did not undergo PKC phosphorylation, was normally localized at the postsynaptic sites of PF-Purkinje cell synapses. In addition, like wild-type GluRδ2, the phosphorylation-disrupted GluRδ2 successfully rescued abrogated LTD in GluRδ2-null Purkinje cells. These results indicate that Ser945, a major PKC phosphorylation site of of GluRδ2, may not play a crucial role in induction of LTD in the cerebellum.

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Cbln and C1q family proteins - New transneuronal cytokines

Yuzaki M.

Cellular and Molecular Life Sciences 65 ( 11 ) 1698 - 1705 2008.06

ISSN 1420-682X

The C1q family is characterized by a C-terminal conserved global C1q domain, which is structurally very similar to the tumor necrosis factor homology domain. Although some C1q family members are expressed in the central nervous system, their functions have not been well characterized. Cbln1, a member of the Cbln subfamily of the C1q family, is predominantly expressed in cerebellar granule cells. Interestingly, Cbln1 was recently shown to play two unique roles at excitatory synapses formed between cerebellar granule cells and Purkinje cells: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytosis pathway. Since other Cbln subfamily members, Cbln2-Cbln4, are expressed in various regions of developing and mature brains, Cbln subfamily proteins may generally serve as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions. © 2008 Birkhaueser.

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Accumulation of AMPA Receptors in Autophagosomes in Neuronal Axons Lacking Adaptor Protein AP-4

Matsuda Shinji, Miura Eriko, Matsuda Keiko, Kakegawa Wataru, Kohda Kazuhisa, Watanabe Masahiko, Yuzaki Michisuke

Neuron 57 ( 5 ) 730 - 745 2008.03

ISSN 0896-6273

AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the β subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons. © 2008 Elsevier Inc. All rights reserved.

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Differential regulation of synaptic plasticity and cerebellar motor learning by the C-terminal PDZ-binding motif of GluRδ2

Kakegawa Wataru, Miyazaki Taisuke, Emi Kyoichi, Matsuda Keiko, Kohda Kazuhisa, Motohashi Junko, Mishina Masayoshi, Kawahara Shigenori, Watanabe Masahiko, Yuzaki Michisuke

Journal of Neuroscience 28 ( 6 ) 1460 - 1468 2008.02

ISSN 0270-6474

The δ2 glutamate receptor (GluRδ2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2^-/- mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluRδ2^-/- Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluRδ2^-/- Purkinje cells by the virus-mediated expression of the wild-type GluRδ2 transgene (Tg_wt) but not by that of mutant GluRδ2 lacking the C-terminal seven residues to which several PDZ proteins bind (Tg _ΔCT7). These results indicated that the C terminus of GluRδ2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluRδ2^-/- cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluRδ2 in vivo, we generated transgenic mice that expressed Tg_ΔCT7 on a GluRδ2^-/- background. Interestingly, although Tg_ΔCT7 restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluRδ2^-/- Purkinje cells. Furthermore, although the gross motor discoordination of GluRδ2^-/- mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluRδ2, whereas other functions may be differentially regulated by other regions of GluRδ2. Copyright © 2008 Society for Neuroscience.

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Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death

Nishiyama Jun, Miura Eriko, Mizushima Noboru, Watanabe Masahiko, Yuzaki Michisuke

Autophagy 3 ( 6 ) 591 - 596 2007.11

ISSN 1554-8627

Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes [Atg) have been identified in yeast; a ubiquitin-like Atg12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5 ^flox/flox with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell-specific promoter, pcp2. In Atg5 ^flox/flox; pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons. ©2007 Landes Bioscience.

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