76 results on '"Hirokazu Hirai"'
Search Results
2. Protein kinase Cγ negatively regulates the intrinsic excitability in zebrin-negative cerebellar Purkinje cells
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Masashi Watanave, Mika Kawachi, Ayumu Konno, Ryo Aoki, Yuuki Fukai, Yasunori Matsuzaki, Ryosuke Kaneko, and Hirokazu Hirai
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protein kinase ,cerebellum ,action potential ,Purkinje cells ,aldolase C ,zebrin ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Protein kinase C γ (PKCγ), a neuronal isoform present exclusively in the central nervous system, is most abundantly expressed in cerebellar Purkinje cells (PCs). Targeted deletion of PKCγ causes a climbing fiber synapse elimination in developing PCs and motor deficit. However, physiological roles of PKCγ in adult mouse PCs are little understood. In this study, we aimed to unravel the roles of PKCγ in mature mouse PCs by deleting PKCγ from adult mouse PCs of PKCγfl/fl mice via cerebellar injection of adeno-associated virus (AAV) vectors expressing Cre recombinase under the control of the PC-specific L7-6 promoter. Whole cell patch-clamp recording of PCs showed higher intrinsic excitability in PCs virally lacking PKCγ [PKCγ-conditional knockout (PKCγ-cKO) PCs] than in wild-type (WT) mouse PCs in the zebrin-negative module, but not in the zebrin-positive module. AAV-mediated PKCγ re-expression in PKCγ-deficient mouse PCs in the zebrin-negative module restored the enhanced intrinsic excitability to a level comparable to that of wild-type mouse PCs. In parallel with higher intrinsic excitability, we found larger hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents in PKCγ-cKO PCs located in the zebrin-negative module, compared with those in WT mouse PCs in the same region. However, pharmacological inhibition of the HCN currents did not restore the enhanced intrinsic excitability in PKCγ-cKO PCs in the zebrin-negative module. These results suggested that PKCγ suppresses the intrinsic excitability in zebrin-negative PCs, which is likely independent of the HCN current inhibition.
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- 2024
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3. The Ser19Stop single nucleotide polymorphism (SNP) of human PHYHIPL affects the cerebellum in mice
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Hisako Sugimoto, Takuro Horii, Jun-Na Hirota, Yoshitake Sano, Yo Shinoda, Ayumu Konno, Hirokazu Hirai, Yasuki Ishizaki, Hajime Hirase, Izuho Hatada, Teiichi Furuichi, and Tetsushi Sadakata
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PHYHIPL ,PHYHIP ,dbSNP ,HapMap Project ,Cerebellum ,Purkinje cell ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases.
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- 2021
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4. Chronic optogenetic stimulation of Bergman glia leads to dysfunction of EAAT1 and Purkinje cell death, mimicking the events caused by expression of pathogenic ataxin-1
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Anton N. Shuvaev, Olga S. Belozor, Oleg Mozhei, Dariya A. Yakovleva, Ilya V. Potapenko, Andrey N. Shuvaev, Marina V. Smolnikova, Vladimir V. Salmin, Alla B. Salmina, Hirokazu Hirai, Anja G. Teschemacher, and Sergey Kasparov
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Bergmann glia ,Excitatory amino acid transporter type 1 ,Spinocerebellar ataxia type 1 ,Cerebellum ,Purkinje cell ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Bergmann glia (BG) are highly specialized radial astrocytes of the cerebellar cortex, which play a key role in the uptake of synaptic glutamate via the excitatory amino acid transporter EAAT1. Multiple lines of evidence suggest that in cerebellar neurodegenerative diseases reactive BG has a negative impact on neuronal function and survival through compromised EAAT activity. A family of such diseases are those caused by expansion of CAG repeats in genes of the ataxin family, resulting in spinocerebellar ataxias (SCA).We investigated the contribution of BG to the pathogenesis of cerebellar neurodegeneration in a model of SCA1, which was induced by expression of a polyglutamine mutant of ataxin-1 (ATXN1[Q85]) in BG specifically. We compared the outcomes with a novel model where we triggered excitotoxicity by a chronic optogenetic activation of BG with channelrhodopsin-2 (ChR2). In both cases we detected evidence of reduced glutamate uptake manifested by prolongation of excitatory postsynaptic currents in Purkinje cells which is consistent with documented reduction of expression and/or function of EAAT1. In both models we detected astroglyosis and Purkinje cells atrophy. Finally, the same pattern was detected in a knock-in mouse which expresses a polyglutamine mutant ataxin-1 ATXN1[Q154] in a non-cell-selective manner.Our results suggest that ATXN1[Q85] and ChR2-induced insult targeted to BG closely mimics SCA1 pathology, where excessive glutamate signaling appears to be a common feature likely being an important contributor to cerebellar neurodegeneration.
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- 2021
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5. Pharmacological enhancement of retinoid-related orphan receptor α function mitigates spinocerebellar ataxia type 3 pathology
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Masashi Watanave, Chiaki Hoshino, Ayumu Konno, Yumi Fukuzaki, Yasunori Matsuzaki, Tohru Ishitani, and Hirokazu Hirai
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Cerebellum ,Purkinje cell ,Spinocerebellar ataxia type 3 ,Machado–Joseph disease ,AAV vector ,Retinoid-related orphan receptor alpha ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado–Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.
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- 2019
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6. Minimal Purkinje Cell-Specific PCP2/L7 Promoter Virally Available for Rodents and Non-human Primates
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Keisuke Nitta, Yasunori Matsuzaki, Ayumu Konno, and Hirokazu Hirai
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Purkinje cell ,L7 ,PCP2 ,cerebellum ,lentivirus ,adeno-associated virus ,viral vector ,cell type-specific promoter ,marmoset ,non-human primate ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Cell-type-specific promoters in combination with viral vectors and gene-editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood-brain barrier confirmed the Purkinje cell specificity of the L7-6 in the CNS. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.
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- 2017
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7. Contribution of Thyrotropin-Releasing Hormone to Cerebellar Long-Term Depression and Motor Learning
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Masashi Watanave, Yasunori Matsuzaki, Yasuyo Nakajima, Atsushi Ozawa, Masanobu Yamada, and Hirokazu Hirai
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thyrotropin-releasing hormone ,motor learning ,cerebellum ,LTD ,NO ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Thyrotropin-releasing hormone (TRH) regulates various physiological activities through activation of receptors expressed in a broad range of cells in the central nervous system. The cerebellum expresses TRH receptors in granule cells and molecular layer interneurons. However, the function of TRH in the cerebellum remains to be clarified. Here, using TRH knockout (KO) mice we studied the role of TRH in the cerebellum. Immunohistochemistry showed no gross morphological differences between KO mice and wild-type (WT) littermates in the cerebellum. In the rotarod test, the initial performance of KO mice was comparable to that of WT littermates, but the learning speed of KO mice was significantly lower than that of WT littermates, suggesting impaired motor learning. The motor learning deficit in KO mice was rescued by intraperitoneal injection of TRH. Electrophysiology revealed absence of long-term depression (LTD) at parallel fiber-Purkinje cell synapses in KO mice, which was rescued by bath-application of TRH. TRH was shown to increase cyclic guanosine monophosphate (cGMP) content in the cerebellum. Since nitric oxide (NO) stimulates cGMP synthesis in the cerebellum, we examined whether NO-cGMP pathway was involved in TRH-mediated LTD rescue in KO mice. Pharmacological blockade of NO synthase and subsequent cGMP production prevented TRH-induced LTD expression in KO mice, whereas increase in cGMP signal in Purkinje cells by 8-bromoguanosine cyclic 3’,5’-monophosphate, a membrane-permeable cGMP analog, restored LTD without TRH application. These results suggest that TRH is involved in cerebellar LTD presumably by upregulating the basal cGMP level in Purkinje cells, and, consequently, in motor learning.
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- 2018
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8. Inhibition gates supralinear Ca2+ signaling in Purkinje cell dendrites during practiced movements
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Michael A Gaffield, Matthew J M Rowan, Samantha B Amat, Hirokazu Hirai, and Jason M Christie
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cerebellum ,inhibition ,calcium ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.
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- 2018
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9. Contribution of Thyrotropin-Releasing Hormone to Cerebellar Long-Term Depression and Motor Learning.
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Masashi Watanave, Yasunori Matsuzaki, Yasuyo Nakajima, Atsushi Ozawa, Masanobu Yamada, and Hirokazu Hirai
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THYROTROPIN releasing factor ,CYCLIC guanylic acid ,GRANULE cells ,CENTRAL nervous system ,MOTOR learning - Abstract
Thyrotropin-releasing hormone (TRH) regulates various physiological activities through activation of receptors expressed in a broad range of cells in the central nervous system. The cerebellum expresses TRH receptors in granule cells and molecular layer interneurons. However, the function of TRH in the cerebellum remains to be clarified. Here, using TRH knockout (KO) mice we studied the role of TRH in the cerebellum. Immunohistochemistry showed no gross morphological differences between KO mice and wild-type (WT) littermates in the cerebellum. In the rotarod test, the initial performance of KO mice was comparable to that of WT littermates, but the learning speed of KO mice was significantly lower than that of WT littermates, suggesting impaired motor learning. The motor learning deficit in KO mice was rescued by intraperitoneal injection of TRH. Electrophysiology revealed absence of longterm depression (LTD) at parallel fiber-Purkinje cell synapses in KO mice, which was rescued by bath-application of TRH. TRH was shown to increase cyclic guanosine monophosphate (cGMP) content in the cerebellum. Since nitric oxide (NO) stimulates cGMP synthesis in the cerebellum, we examined whether NO-cGMP pathway was involved in TRH-mediated LTD rescue in KO mice. Pharmacological blockade of NO synthase and subsequent cGMP production prevented TRH-induced LTD expression in KO mice, whereas increase in cGMP signal in Purkinje cells by 8-bromoguanosine cyclic 3',5'-monophosphate, a membrane-permeable cGMP analog, restored LTD without TRH application. These results suggest that TRH is involved in cerebellar LTD presumably by upregulating the basal cGMP level in Purkinje cells, and, consequently, in motor learning. [ABSTRACT FROM AUTHOR]
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- 2024
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10. Synaptic pruning through glial synapse engulfment upon motor learning
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Yosuke M. Morizawa, Mami Matsumoto, Yuka Nakashima, Narumi Endo, Tomomi Aida, Hiroshi Ishikane, Kaoru Beppu, Satoru Moritoh, Hitoshi Inada, Noriko Osumi, Eiji Shigetomi, Schuichi Koizumi, Guang Yang, Hirokazu Hirai, Kohichi Tanaka, Kenji F. Tanaka, Nobuhiko Ohno, Yugo Fukazawa, and Ko Matsui
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Neurons ,Mice ,Neuronal Plasticity ,Cerebellum ,General Neuroscience ,Synapses ,Animals ,Neuroglia - Abstract
Synaptic pruning is a fundamental process of neuronal circuit refinement in learning and memory. Accumulating evidence suggests that glia participates in sculpting the neuronal circuits through synapse engulfment. However, whether glial involvement in synaptic pruning has a role in memory formation remains elusive. Using newly developed phagocytosis reporter mice and three-dimensional ultrastructural characterization, we found that synaptic engulfment by cerebellar Bergmann glia (BG) frequently occurred upon cerebellum-dependent motor learning in mice. We observed increases in pre- and postsynaptic nibbling by BG along with a reduction in spine volume after learning. Pharmacological blockade of engulfment with Annexin V inhibited both the spine volume reduction and overnight improvement of motor adaptation. These results indicate that BG contribute to the refinement of the mature cerebellar cortical circuit through synaptic engulfment during motor learning.
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- 2022
11. Electrophysiological and Imaging Analysis of GFP-Tagged Protein Kinase C γ Translocation in Cerebellar Purkinje Cells
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Hirokazu Hirai, Yuuki Fukai, Ayumu Konno, and Nobutake Hosoi
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Purkinje Cells ,Neurology ,Cerebellum ,Synapses ,Dendrites ,Neurology (clinical) ,Protein Kinase C - Abstract
The cerebellum contains the highest density of protein kinase C (PKC) in the central nervous system. PKCγ, the major isotype accounting for over half of the PKCs in the cerebellum, is expressed exclusively in Purkinje cells (PCs). Inactivated PKCγ, which is localized in the cytoplasm of PC dendrites and soma, begins to translocate to the cell membrane upon activation. However, the physiological conditions that induce PKCγ translocation in PC remain largely unknown. Here, we virally expressed PKCγ-GFP in PCs and examined the conditions that induced its translocation to PC dendrites by whole-cell patch clamp analysis combined with confocal GFP fluorescence imaging. A single or repetitive (150 pulses at 5 Hz for 30 s) electrical stimulation to a climbing fiber (CF), which produced a complex spike(s) in PC, failed to induce translocation of PKCγ-GFP to the dendritic shaft of PCs. Direct current injection (+ 2 nA for 3 s) to PC also did not induce the translocation, although PCs generated simple spikes continuously at high rates. In contrast, high-frequency parallel fiber (PF) stimulation (50 pulses at 50 Hz for 1 s), which triggered action potentials followed by sustained depolarization (known as mGluR1-mediated slow depolarization), caused translocation of cytoplasmic PKCγ-GFP to the membrane. Low-frequency PF stimulation (150 pulses at 5 Hz for 30 s) induced continuous simple spike firing but did not induce translocation. These results suggest that CF-triggered depolarization, which causes Ca
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- 2022
12. Long-term depression–inductive stimulation causes long-term potentiation in mouse Purkinje cells with a mutant thyroid hormone receptor
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Ayane Ninomiya, Izuki Amano, Michifumi Kokubo, Yusuke Takatsuru, Sumiyasu Ishii, Hirokazu Hirai, Nobutake Hosoi, and Noriyuki Koibuchi
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Mice ,Purkinje Cells ,Receptors, Thyroid Hormone ,Multidisciplinary ,Depression ,Long-Term Synaptic Depression ,Cerebellum ,Long-Term Potentiation ,Synapses ,Congenital Hypothyroidism ,Animals ,Calcium - Abstract
Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)–inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)–PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF–PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca 2+ condition. Confocal calcium imaging revealed that dendritic Ca 2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP 3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism–induced dysfunctions of central nervous system and cerebellum.
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- 2022
13. Consensus Paper: Strengths and Weaknesses of Animal Models of Spinocerebellar Ataxias and Their Clinical Implications
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Mario Manto, Michael Strupp, Mandi Gandelman, Hirokazu Hirai, Stefan M. Pulst, Jan Cendelin, Harry T. Orr, Filip Tichanek, Jan Tuma, and Marija Cvetanovic
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medicine.medical_specialty ,Consensus ,Neurology ,Cerebellar ataxia ,business.industry ,Cognition ,medicine.disease ,Article ,Clinical trial ,Mice ,Quality of life (healthcare) ,Animal model ,Cerebellum ,Models, Animal ,Quality of Life ,Spinocerebellar ataxia ,Animals ,Spinocerebellar Ataxias ,Medicine ,Neurology (clinical) ,medicine.symptom ,business ,Neuroscience ,Strengths and weaknesses - Abstract
Spinocerebellar ataxias (SCAs) represent a large group of hereditary degenerative diseases of the nervous system, in particular the cerebellum, and other systems that manifest with a variety of progressive motor, cognitive, and behavioral deficits with the leading symptom of cerebellar ataxia. SCAs often lead to severe impairments of the patient's functioning, quality of life, and life expectancy. For SCAs, there are no proven effective pharmacotherapies that improve the symptoms or substantially delay disease progress, i.e., disease-modifying therapies. To study SCA pathogenesis and potential therapies, animal models have been widely used and are an essential part of pre-clinical research. They mainly include mice, but also other vertebrates and invertebrates. Each animal model has its strengths and weaknesses arising from model animal species, type of genetic manipulation, and similarity to human diseases. The types of murine and non-murine models of SCAs, their contribution to the investigation of SCA pathogenesis, pathological phenotype, and therapeutic approaches including their advantages and disadvantages are reviewed in this paper. There is a consensus among the panel of experts that (1) animal models represent valuable tools to improve our understanding of SCAs and discover and assess novel therapies for this group of neurological disorders characterized by diverse mechanisms and differential degenerative progressions, (2) thorough phenotypic assessment of individual animal models is required for studies addressing therapeutic approaches, (3) comparative studies are needed to bring pre-clinical research closer to clinical trials, and (4) mouse models complement cellular and invertebrate models which remain limited in terms of clinical translation for complex neurological disorders such as SCAs.
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- 2021
14. Global Knockdown of Retinoid-related Orphan Receptor α in Mature Purkinje Cells Reveals Aberrant Cerebellar Phenotypes of Spinocerebellar Ataxia
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Hirokazu Hirai, Hiroyuki Yasui, Ayumu Konno, and Yasunori Matsuzaki
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0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Cerebellum ,Purkinje cell ,Biology ,Mice ,Purkinje Cells ,Retinoids ,03 medical and health sciences ,0302 clinical medicine ,microRNA ,medicine ,Animals ,Humans ,Spinocerebellar Ataxias ,Transcription factor ,Mice, Knockout ,Orphan receptor ,Gene knockdown ,Cerebellar ataxia ,General Neuroscience ,Dendrites ,medicine.disease ,Cell biology ,Phenotype ,030104 developmental biology ,medicine.anatomical_structure ,Spinocerebellar ataxia ,medicine.symptom ,030217 neurology & neurosurgery - Abstract
Retinoid-related orphan receptor α (RORα) is a transcription factor expressed in a variety of tissues throughout the body. Knockout of RORα leads to various impairments, including defects in cerebellar development, circadian rhythm, lipid metabolism, immune function, and bone development. Previous studies have shown significant reduction of RORα expression in Purkinje cells (PCs) of spinocerebellar ataxia (SCA) type 1 and type 3/MJD (Machado-Joseph disease) model mice. However, it remains unclear to what extent the RORα reduction in PCs is involved in the disease pathology. Here, RORα expression was downregulated specifically in mature mouse PCs by intravenous infusion of blood-brain barrier-permeable adeno-associated virus (AAV), expressing a microRNA against RORα (miR-RORα) under the control of the PC-specific L7-6 promoter. The systemic AAV infusion led to extensive transduction of PCs. The RORα knock-down caused degeneration of PCs including disruption of the PC monolayer alignment and dendrite atrophy. In behavioral experiments, mice expressing miR-RORα showed motor learning deficits, and later, overt cerebellar ataxia. Thus, RORα in mature PCs plays pivotal roles in maintenance of PC dendrites and the monolayer alignment, and consequently, motor learning and motor function. Decrease in RORα expression in PCs could be a primary etiology of the cerebellar symptoms in patients with SCA1 and SCA3/MJD.
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- 2021
15. Masao Ito—A Visionary Neuroscientist with a Passion for the Cerebellum
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Michisuke Yuzaki, Hirokazu Hirai, Masanobu Kano, and Soichi Nagao
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Cerebellum ,General Neuroscience ,Philosophy ,media_common.quotation_subject ,Emotions ,Neurosciences ,Art history ,Passion ,Introductory Journal Article ,Neuroscientist ,media_common - Published
- 2021
16. Ataxic phenotype and neurodegeneration are triggered by the impairment of chaperone‐mediated autophagy in cerebellar neurons
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Hirokazu Hirai, Takahiro Seki, Yuri Morikawa, Hiroshi Katsuki, Masahiro Sato, Akinori Hisatsune, Tomoko Ohta, Ayumu Konno, and Yuki Kurauchi
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0301 basic medicine ,Cerebellum ,Synapsin I ,Histology ,Cerebellar Ataxia ,Chaperone-Mediated Autophagy ,Biology ,Protein degradation ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Chaperone-mediated autophagy ,Lysosomal-Associated Membrane Protein 2 ,Physiology (medical) ,medicine ,Animals ,Neurons ,Mice, Inbred ICR ,Gene knockdown ,Neurodegeneration ,medicine.disease ,Astrogliosis ,Cell biology ,Phenotype ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,Neurology ,Gliosis ,Nerve Degeneration ,Neurology (clinical) ,medicine.symptom ,030217 neurology & neurosurgery - Abstract
Aims Chaperone-mediated autophagy (CMA) is a pathway involved in the autophagy lysosome protein degradation system. CMA has attracted attention as a contributing factor to neurodegenerative diseases since it participates in the degradation of disease-causing proteins. We previously showed that CMA is generally impaired in cells expressing the proteins causing spinocerebellar ataxias (SCAs). Therefore, we investigated the effect of CMA impairment on motor function and the neural survival of cerebellar neurons using the micro RNA (miRNA)-mediated knockdown of lysosome-associated protein 2A (LAMP2A), a CMA-related protein. Methods We injected adeno-associated virus serotype 9 vectors, which express green fluorescent protein (GFP) and miRNA (negative control miRNA or LAMP2A miRNA) under neuron-specific synapsin I promoter, into cerebellar parenchyma of 4-week-old ICR mice. Motor function of mice was evaluated by beam walking and footprint tests. Immunofluorescence experiments of cerebellar slices were conducted to evaluate histological changes in cerebella. Results GFP and miRNA were expressed in interneurons (satellite cells and basket cells) in molecular layers and granule cells in the cerebellar cortices, but not in cerebellar Purkinje cells. LAMP2A knockdown in cerebellar neurons triggered progressive motor impairment, prominent loss of cerebellar Purkinje cells, interneurons, granule cells at the late stage, and astrogliosis and microgliosis from the early stage. Conclusions CMA impairment in cerebellar interneurons and granule cells triggers the progressive ataxic phenotype, gliosis and the subsequent degeneration of cerebellar neurons, including Purkinje cells. Our present findings strongly suggest that CMA impairment is related to the pathogenesis of various SCAs.
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- 2020
17. Targeting inhibitory cerebellar circuitry to alleviate behavioral deficits in a mouse model for studying idiopathic autism
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Hirokazu Hirai, Kevin Wickman, Lisa A. Duvick, Yi Mei Yang, Owen Y. Chao, Ezequiel Marron Fernandez de Velasco, Salil Saurav Pathak, Harry T. Orr, Swati Maitra, and Hao Zhang
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Male ,Brachyury ,Cerebellum ,Autism Spectrum Disorder ,Inhibitory postsynaptic potential ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Postsynaptic potential ,mental disorders ,medicine ,Animals ,Autistic Disorder ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Excitability ,business.industry ,Autism spectrum disorders ,medicine.disease ,Potassium channel ,Mice, Inbred C57BL ,Disease Models, Animal ,Psychiatry and Mental health ,medicine.anatomical_structure ,nervous system ,Autism spectrum disorder ,Autism ,business ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Autism spectrum disorder (ASD) encompasses wide-ranging neuropsychiatric symptoms with unclear etiology. Although the cerebellum is a key region implicated in ASD, it remains elusive how the cerebellar circuitry is altered and whether the cerebellum can serve as a therapeutic target to rectify the phenotype of idiopathic ASD with polygenic abnormalities. Using a syndromic ASD model, e.g., Black and Tan BRachyury T+Itpr3tf/J (BTBR) mice, we revealed that increased excitability of presynaptic interneurons (INs) and decreased intrinsic excitability of postsynaptic Purkinje neurons (PNs) resulted in low PN firing rates in the cerebellum. Knowing that downregulation of Kv1.2 potassium channel in the IN nerve terminals likely augmented their excitability and GABA release, we applied a positive Kv1.2 modulator to mitigate the presynaptic over-inhibition and social impairment of BTBR mice. Selective restoration of the PN activity by a new chemogenetic approach alleviated core ASD-like behaviors of the BTBR strain. These findings highlight complex mechanisms converging onto the cerebellar dysfunction in the phenotypic model and provide effective strategies for potential therapies of ASD.
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- 2020
18. The neurotoxic effect of lactational PFOS exposure on cerebellar functional development in male mice
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Ayane Ninomiya, Abdallah Mshaty, Asahi Haijima, Hiroyuki Yajima, Michifumi Kokubo, Miski Aghnia Khairinisa, Winda Ariyani, Yuki Fujiwara, Sumiyasu Ishii, Nobutake Hosoi, Hirokazu Hirai, Izuki Amano, and Noriyuki Koibuchi
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Male ,Fluorocarbons ,Behavior, Animal ,Neurotoxins ,General Medicine ,Anxiety ,Toxicology ,Dietary Exposure ,Mice ,Alkanesulfonic Acids ,Maternal Exposure ,Cerebellum ,Animals ,Lactation ,Female ,Psychomotor Performance ,Food Science - Abstract
Recent studies showed a possible association between perfluorooctane sulfonate (PFOS) and developmental disabilities. We previously found the specific effects of PFOS exposure on learning and memory, however, its effect on the other developmental disabilities such as motor and social deficits remains unclear. We examined the effect of early lactational PFOS exposure on motor coordination, social activity, and anxiety in male mice. We orally administered a PFOS solution to dams from postnatal day 1-14. At 10 weeks old, we conducted a behavior test battery to evaluate motor performance, social activity, and anxiety, followed by electrophysiology and Western blot analysis. PFOS-exposed mice displayed impaired motor coordination. Whole-cell patch-clamp recordings from Purkinje cells revealed that the short-term and long-term plasticity at parallel fiber-Purkinje cell synapses are affected by PFOS exposure. Western blot analysis indicated that PFOS exposure increased syntaxin binding protein 1 (Munc18-1) and glutamate metabotropic receptor 1 (mGluR1) protein levels, which may be associated with the change in neurotransmitter release from parallel fibers and the level of long-term depression, respectively. The present study demonstrates that lactational PFOS exposure may have disrupted the pre- and postsynaptic plasticity at parallel fiber-Purkinje cell synapses, causing profound, long-lasting abnormal effects on the cerebellar function.
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- 2021
19. Pharmacological enhancement of retinoid-related orphan receptor α function mitigates spinocerebellar ataxia type 3 pathology
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Hirokazu Hirai, Yumi Fukuzaki, Tohru Ishitani, Chiaki Hoshino, Yasunori Matsuzaki, Masashi Watanave, and Ayumu Konno
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Pathology ,medicine.medical_specialty ,Cerebellum ,congenital, hereditary, and neonatal diseases and abnormalities ,Purkinje cell ,Biology ,Receptors, Metabotropic Glutamate ,Protein Aggregation, Pathological ,lcsh:RC321-571 ,Purkinje Cells ,Spinocerebellar ataxia type 3 ,medicine ,Animals ,Humans ,Ataxin-3 ,Retinoid-related orphan receptor alpha ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Orphan receptor ,Nuclear Receptor Subfamily 1, Group F, Member 1 ,Dendrites ,Machado-Joseph Disease ,Polyglutamine tract ,medicine.disease ,Mice, Inbred C57BL ,Repressor Proteins ,AAV vector ,medicine.anatomical_structure ,Neurology ,Cerebellar cortex ,Spinocerebellar ataxia ,Metabotropic glutamate receptor 1 ,Peptides ,Machado–Joseph disease ,Signal Transduction - Abstract
Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.
- Published
- 2019
20. The Ser19Stop single nucleotide polymorphism (SNP) of human PHYHIPL affects the cerebellum in mice
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Yasuki Ishizaki, Hajime Hirase, Ayumu Konno, Hisako Sugimoto, Hirokazu Hirai, Yoshitake Sano, Tetsushi Sadakata, Izuho Hatada, Yo Shinoda, Jun Na Hirota, Teiichi Furuichi, and Takuro Horii
- Subjects
0301 basic medicine ,Cerebellum ,dbSNP ,Purkinje cell ,Single-nucleotide polymorphism ,Mice, Transgenic ,HapMap Project ,Biology ,Motor Activity ,PHYHIP ,Polymorphism, Single Nucleotide ,lcsh:RC346-429 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Mice ,Purkinje Cells ,0302 clinical medicine ,Nerve Fibers ,Interneurons ,Gene expression ,medicine ,SNP ,Animals ,Humans ,Learning ,Amino Acid Sequence ,Gene Knock-In Techniques ,International HapMap Project ,PHYHIPL ,Molecular Biology ,Gene ,Cell Shape ,lcsh:Neurology. Diseases of the nervous system ,Genetics ,Sequence Homology, Amino Acid ,Research ,Intracellular Signaling Peptides and Proteins ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Rotarod Performance Test ,Codon, Terminator ,Female ,CRISPR-Cas Systems ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00766-x.
- Published
- 2021
21. Chronic optogenetic stimulation of Bergman glia leads to dysfunction of EAAT1 and Purkinje cell death, mimicking the events caused by expression of pathogenic ataxin-1
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Anja G. Teschemacher, M. V. Smolnikova, A. N. Shuvaev, Ilya V. Potapenko, Hirokazu Hirai, Dariya A. Yakovleva, Olga S Belozor, Oleg Mozhei, Anton N. Shuvaev, Vladimir V. Salmin, Alla B. Salmina, and Sergey Kasparov
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0301 basic medicine ,Cerebellum ,Bergmann glia ,Purkinje cell ,Excitotoxicity ,Ataxin 1 ,Gene Expression ,Neurosciences. Biological psychiatry. Neuropsychiatry ,Mice, Transgenic ,Biology ,medicine.disease_cause ,03 medical and health sciences ,Mice ,Purkinje Cells ,0302 clinical medicine ,medicine ,Animals ,Spinocerebellar ataxia type 1 ,Ataxin-1 ,Excitatory amino acid transporter type 1 ,Cell Death ,Neurodegeneration ,Glutamate receptor ,medicine.disease ,Excitatory Amino Acid Transporter 1 ,Mice, Inbred C57BL ,Optogenetics ,030104 developmental biology ,medicine.anatomical_structure ,Neurology ,nervous system ,Ataxin ,Cerebellar cortex ,biology.protein ,Neuroscience ,Neuroglia ,030217 neurology & neurosurgery ,Photic Stimulation ,RC321-571 - Abstract
Bergmann glia (BG) are highly specialized radial astrocytes of the cerebellar cortex, which play a key role in the uptake of synaptic glutamate via the excitatory amino acid transporter EAAT1. Multiple lines of evidence suggest that in cerebellar neurodegenerative diseases reactive BG has a negative impact on neuronal function and survival through compromised EAAT activity. A family of such diseases are those caused by expansion of CAG repeats in genes of the ataxin family, resulting in spinocerebellar ataxias (SCA). We investigated the contribution of BG to the pathogenesis of cerebellar neurodegeneration in a model of SCA1, which was induced by expression of a polyglutamine mutant of ataxin-1 (ATXN1[Q85]) in BG specifically. We compared the outcomes with a novel model where we triggered excitotoxicity by a chronic optogenetic activation of BG with channelrhodopsin-2 (ChR2). In both cases we detected evidence of reduced glutamate uptake manifested by prolongation of excitatory postsynaptic currents in Purkinje cells which is consistent with documented reduction of expression and/or function of EAAT1. In both models we detected astroglyosis and Purkinje cells atrophy. Finally, the same pattern was detected in a knock-in mouse which expresses a polyglutamine mutant ataxin-1 ATXN1[Q154] in a non-cell-selective manner. Our results suggest that ATXN1[Q85] and ChR2-induced insult targeted to BG closely mimics SCA1 pathology, where excessive glutamate signaling appears to be a common feature likely being an important contributor to cerebellar neurodegeneration.
- Published
- 2020
22. Therapeutic potential of d-cysteine against in vitro and in vivo models of spinocerebellar ataxia
- Author
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Hirokazu Hirai, Akinori Hisatsune, Takahiro Seki, Ayumu Konno, Yuki Kurauchi, Yuri Morikawa, Masahiro Sato, Hiroshi Katsuki, and Tomoko Ohta
- Subjects
Male ,0301 basic medicine ,Ataxia ,Mutant ,Mice, Transgenic ,Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Developmental Neuroscience ,Pregnancy ,In vivo ,Cerebellum ,medicine ,Animals ,Spinocerebellar Ataxias ,Cysteine ,Rats, Wistar ,Ataxin-1 ,Cells, Cultured ,Mice, Inbred ICR ,medicine.disease ,In vitro ,Rats ,Cell biology ,030104 developmental biology ,Neurology ,Gliosis ,Spinocerebellar ataxia ,Metabotropic glutamate receptor 1 ,Female ,medicine.symptom ,030217 neurology & neurosurgery ,Immunostaining - Abstract
Spinocerebellar ataxia (SCA) is a group of autosomal-dominantly inherited ataxia and is classified into SCA1-48 by the difference of causal genes. Several SCA-causing proteins commonly impair dendritic development in primary cultured Purkinje cells (PCs). We assume that primary cultured PCs expressing SCA-causing proteins are available as in vitro SCA models and that chemicals that improve the impaired dendritic development would be effective for various SCAs. We have recently revealed that D-cysteine enhances the dendritic growth of primary cultured PCs via hydrogen sulfide production. In the present study, we first investigated whether D-cysteine is effective for in vitro SCA models. We expressed SCA1-, SCA3-, and SCA21-causing mutant proteins to primary cultured PCs using adeno-associated viral serotype 9 (AAV9) vectors. D-Cysteine (0.2 mM) significantly ameliorated the impaired dendritic development commonly observed in primary cultured PCs expressing these three SCA-causing proteins. Next, we investigated the therapeutic effect of long-term treatment with D-cysteine on an in vivo SCA model. SCA1 model mice were established by the cerebellar injection of AAV9 vectors, which express SCA1-causing mutant ataxin-1, to ICR mice. Long-term treatment with D-cysteine (100 mg/kg/day) significantly inhibited the progression of motor dysfunction in SCA1 model mice. Immunostaining experiments revealed that D-cysteine prevented the reduction of mGluR1 and glial activation at the early stage after the onset of motor dysfunction in SCA1 model mice. These findings strongly suggest that D-cysteine has therapeutic potential against in vitro and in vivo SCA models and may be a novel therapeutic agent for various SCAs.
- Published
- 2021
23. Viral Vector-Based Evaluation of Regulatory Regions in the Neuron-Specific Enolase (NSE) Promoter in Mouse Cerebellum In Vivo
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Ayumu Konno, Toshinori Ohtani, Yoichiro Shinohara, and Hirokazu Hirai
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0301 basic medicine ,Cerebellum ,Interneuron ,Genetic Vectors ,Green Fluorescent Proteins ,Purkinje cell ,Enolase ,Biology ,Green fluorescent protein ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,medicine ,Animals ,Promoter Regions, Genetic ,Neurons ,Promoter ,Dependovirus ,Immunohistochemistry ,Molecular biology ,Rats ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Neurology ,Regulatory sequence ,Phosphopyruvate Hydratase ,Neurology (clinical) ,030217 neurology & neurosurgery - Abstract
We investigated the neuron-specific enolase (NSE) promoter in terms of its promoter strength and neuronal specificity in the cerebellum in vivo. The 1.8 kb rat NSE promoter was divided into three regions, A (0.8 kb), B (0.7 kb), and C (0.3 kb), starting from the 5' side. Then, we made various deletion constructs and assessed them by virally expressing GFP under the control of one of the deleted promoters. Removing region A reduced GFP expression to ~6% of that of the original 1.8 kb promoter. Further deletion of region B (presence of region C alone) did not influence the promoter strength, but removing region B from the original 1.8 kb promoter reduced the GFP expression to ~6% of the original level, similar to the level observed after deletion of region A. Immunohistochemistry showed robust GFP expression in Purkinje cells and modest expression in interneurons by the original promoter. Removing region A and/or region B abolished the GFP expression in Purkinje cells in most cerebellar lobules, with the expression in interneurons almost unchanged. These results suggest that region C, which is a proximal 0.3 kb sequence, contains cis-acting elements that drive transcription predominantly in interneurons. The addition of either region A or B onto region C does not alter the promoter properties; however, the addition of both regions A and B to region C drastically enhanced the promoter activity in Purkinje cells, suggesting the synergistic action of cis-acting regulatory elements in regions A and B for strong activation in Purkinje cells.
- Published
- 2017
24. Task Force Paper On Cerebellar Transplantation: Are We Ready to Treat Cerebellar Disorders with Cell Therapy?
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Mario Manto, Lorenzo Magrassi, Frantisek Vozeh, Annalisa Buffo, Hiroshi Mitoma, Rachel M. Sherrard, Jan Cendelin, and Hirokazu Hirai
- Subjects
medicine.medical_specialty ,Cerebellum ,Neurology ,Central nervous system ,Cell- and Tissue-Based Therapy ,Disease ,Stem cells ,050105 experimental psychology ,Cell therapy ,03 medical and health sciences ,0302 clinical medicine ,Cerebellar Diseases ,medicine ,Animals ,0501 psychology and cognitive sciences ,Cerebellar disorder ,Neurotransplantation ,Cerebellar reserve ,Ataxias ,Task force ,business.industry ,05 social sciences ,Transplantation ,medicine.anatomical_structure ,nervous system ,Neurology (clinical) ,business ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Restoration of damaged central nervous system structures, functional recovery, and prevention of neuronal loss during neurodegenerative diseases are major objectives in cerebellar research. The highly organized anatomical structure of the cerebellum with numerous inputs/outputs, the complexity of cerebellar functions, and the large spectrum of cerebellar ataxias render therapies of cerebellar disorders highly challenging. There are currently several therapeutic approaches including motor rehabilitation, neuroprotective drugs, non-invasive cerebellar stimulation, molecularly based therapy targeting pathogenesis of the disease, and neurotransplantation. We discuss the goals and possible beneficial mechanisms of transplantation therapy for cerebellar damage and its limitations and factors determining outcome.
- Published
- 2019
25. Loss-of-function mutation of c-Ret causes cerebellar hypoplasia in mice with Hirschsprung disease and Down's syndrome
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Hirokazu Hirai, Mayumi Jijiwa, Ichiro Yajima, Masahide Takahashi, Toyonori Tsuzuki, Nobutaka Ohgami, Naoya Asai, Akira Iizuka, Masashi Kato, Machiko Iida, and Atsuyoshi Shimada
- Subjects
0301 basic medicine ,Cerebellum ,KI, knock-in ,endocrine system diseases ,DS, Down's syndrome ,Developmental Disabilities ,Proto-Oncogene Mas ,Biochemistry ,Mice ,Purkinje Cells ,Loss of Function Mutation ,EGL, external germinal layer ,Glial cell line-derived neurotrophic factor ,Gene Knock-In Techniques ,Phosphorylation ,neurological disease ,Sonic hedgehog ,Mice, Knockout ,biology ,Hypoplasia ,HSCR, Hirschsprung disease ,GDNF, glial cell line–derived neurotrophic factor ,medicine.anatomical_structure ,IGL, internal granular layer ,Shh, sonic hedgehog ,Neuroglia ,GCPs, granule neuron progenitors ,Research Article ,endocrine system ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,neurite outgrowth ,cerebellum ,Neurite ,Mice, Transgenic ,GFRα1, GDNF family receptor α1 ,Nervous System Malformations ,PCs, Purkinje cells ,03 medical and health sciences ,Internal medicine ,Gene knockin ,medicine ,Animals ,Hedgehog Proteins ,Hirschsprung Disease ,GCs, granule cells ,neoplasms ,Molecular Biology ,sonic hedgehog (Shh) ,ENS, enteric nervous system ,030102 biochemistry & molecular biology ,Proto-Oncogene Proteins c-ret ,Cell Biology ,phosphotyrosine ,medicine.disease ,EDNRB, endothelin receptor B ,Disease Models, Animal ,030104 developmental biology ,Endocrinology ,receptor tyrosine kinase ,biology.protein ,Cerebellar hypoplasia (non-human) ,Down Syndrome ,Smoothened - Abstract
The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.
- Published
- 2021
26. Progressive impairment of cerebellar mGluR signalling and its therapeutic potential for cerebellar ataxia in spinocerebellar ataxia type 1 model mice
- Author
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Anton N. Shuvaev, Dai Yanagihara, Nobutake Hosoi, Yamato Sato, and Hirokazu Hirai
- Subjects
0301 basic medicine ,Spinocerebellar Ataxia Type 1 ,Cerebellum ,Cerebellar ataxia ,Physiology ,musculoskeletal, neural, and ocular physiology ,Purkinje cell ,medicine.disease ,Motor coordination ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,nervous system ,Metabotropic glutamate receptor ,Synaptic plasticity ,medicine ,Spinocerebellar ataxia ,medicine.symptom ,Psychology ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease that presents with cerebellar ataxia and motor learning defects. Previous studies have indicated that the pathology of SCA1, as well as other ataxic diseases, is related to signalling pathways mediated by the metabotropic glutamate receptor type 1 (mGluR1), which is indispensable for proper motor coordination and learning. However, the functional contribution of mGluR signalling to SCA1 pathology is unclear. In the present study, we show that SCA1 model mice develop a functional impairment of mGluR signalling which mediates slow synaptic responses, dendritic Ca2+ signals and short- and long-term synaptic plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in a progressive manner from the early disease stage (5 postnatal weeks) prior to PC death. Notably, impairment of mGluR-mediated dendritic Ca2+ signals linearly correlated with reduction of PC capacitance (cell surface area) in the disease progression. Enhancement of mGluR signalling by baclofen, a clinically available GABAB receptor agonist, led to an improvement of motor performance in SCA1 mice and the improvement lasted ∼1 week after a single application of baclofen. Moreover, the restoration of motor performance in baclofen-treated SCA1 mice matched the functional recovery of mGluR-mediated slow synaptic currents and mGluR-dependent short- and long-term synaptic plasticity. These results suggest that impairment of synaptic mGluR cascades is one of the important contributing factors to cerebellar ataxia in early and middle stages of SCA1 pathology, and that modulation of mGluR signalling by baclofen or other clinical interventions may be therapeutic targets to treat SCA1. This article is protected by copyright. All rights reserved
- Published
- 2016
27. Safety profile of the intravenous administration of brain-targeted stable nucleic acid lipid particles
- Author
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Pedro M. Costa, Clévio Nóbrega, João Nuno Moreira, Hirokazu Hirai, Luís Pereira de Almeida, N. Manjunath, Maria C. Pedroso de Lima, Célia Gomes, Mariana Conceição, and Liliana Mendonça
- Subjects
0301 basic medicine ,Cerebellum ,Pathology ,medicine.medical_specialty ,Mouse tissue ,02 engineering and technology ,Pharmacology ,lcsh:Computer applications to medicine. Medical informatics ,03 medical and health sciences ,Immune system ,medicine ,Research article ,lcsh:Science (General) ,Data Article ,Multidisciplinary ,business.industry ,Immunogenicity ,021001 nanoscience & nanotechnology ,3. Good health ,Safety profile ,030104 developmental biology ,medicine.anatomical_structure ,Nucleic acid ,lcsh:R858-859.7 ,0210 nano-technology ,business ,lcsh:Q1-390 - Abstract
In a clinical setting, where multiple administrations of the therapeutic agent are usually required to improve the therapeutic outcome, it is crucial to assess the immunogenicity of the administered nanoparticles. In this data work, we investigated the safety profile of the repeated intravenous administration of brain-targeted stable nucleic acid lipid particles (RVG-9r-targeted SNALPs). To evaluate local activation of the immune system, we performed analysis of mouse tissue homogenates and sections from cerebellum. To investigate peripheral activation of the immune system, we used serum of mice that were intravenously injected with RVG-9r-targeted SNALPs. These data are related and were discussed in the accompanying research article entitled “Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado–Joseph disease neurological phenotype” (Conceição et al., in press) [1].
- Published
- 2016
28. Inhibition gates supralinear Ca2+ signaling in Purkinje cell dendrites during practiced movements
- Author
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Hirokazu Hirai, Michael A. Gaffield, Samantha B. Amat, Jason M. Christie, and Matthew J. M. Rowan
- Subjects
0301 basic medicine ,Cerebellum ,cerebellum ,QH301-705.5 ,Science ,Purkinje cell ,Parallel fiber ,Inhibitory postsynaptic potential ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,medicine ,Biology (General) ,calcium ,General Immunology and Microbiology ,Chemistry ,General Neuroscience ,General Medicine ,Climbing fiber ,inhibition ,030104 developmental biology ,medicine.anatomical_structure ,Cerebellar cortex ,Climbing ,Medicine ,Motor learning ,Neuroscience ,human activities - Abstract
Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.
- Published
- 2018
29. d-Cysteine promotes dendritic development in primary cultured cerebellar Purkinje cells via hydrogen sulfide production
- Author
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Yuki Kurauchi, Akinori Hisatsune, Ayumu Konno, Masahiro Sato, Hiroshi Katsuki, Takahiro Seki, and Hirokazu Hirai
- Subjects
0301 basic medicine ,Cerebellum ,Sulfide ,Hydrogen sulfide ,Neurogenesis ,D-amino acid oxidase ,chemistry.chemical_element ,Oxidative phosphorylation ,Biology ,Antioxidants ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Purkinje Cells ,0302 clinical medicine ,medicine ,Animals ,Cysteine ,Hydrogen Sulfide ,Molecular Biology ,chemistry.chemical_classification ,Oxidase test ,Brain ,Cell Biology ,Sulfur ,Cell biology ,Rats ,Oxidative Stress ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,030217 neurology & neurosurgery - Abstract
Hydrogen sulfide and reactive sulfur species are regulators of physiological functions, have antioxidant effects against oxidative stresses, and are endogenously generated from l-cysteine. Recently, a novel pathway that generates hydrogen sulfide and reactive sulfur species from d-cysteine has been identified. d-Amino acid oxidase (DAO) is involved in this pathway and, among the various brain regions, is especially abundant in the cerebellum. d-Cysteine has been found to be a better substrate in the generation of hydrogen sulfide in the cerebellum than l-cysteine. Therefore, d-cysteine might be a novel neuroprotectant against cerebellar diseases such as spinocerebellar ataxia (SCA). However, it remains unknown if d-cysteine affects cerebellar Purkinje cells (PCs), which are important for cerebellar functions and are frequently degenerated in SCA patients. In the present study, we investigated whether the production of hydrogen sulfide from d-cysteine affects the dendritic development of cultured PCs. d-Cysteine was found to enhance the dendritic development of PCs significantly, while l-cysteine impaired it. The effect of d-cysteine was inhibited by simultaneous treatment with DAO inhibitors and was reproduced by treatment with 3-mercaptopyruvate, a metabolite of d-cysteine produced by the action of DAO, and disodium sulfide, a donor of hydrogen sulfide. In addition, hydrogen sulfide was immediately produced in cerebellar primary cultures after treatment with d-cysteine and 3-mercaptopyruvate. These findings suggest that d-cysteine enhances the dendritic development of primary cultured PCs via the generation of hydrogen sulfide.
- Published
- 2018
30. Protein Kinase C in the Cerebellum: Its Significance and Remaining Conundrums
- Author
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Hirokazu Hirai
- Subjects
0301 basic medicine ,Gene isoform ,Cerebellum ,Kinase ,Cell ,Purkinje cell ,Climbing fiber ,Biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Neurology ,medicine ,Animals ,Humans ,Neurology (clinical) ,Neuroscience ,030217 neurology & neurosurgery ,Protein kinase C ,Protein Kinase C ,Diacylglycerol kinase - Abstract
Protein kinase C (PKC), a family of serine/threonine protein kinases, mediates a myriad of patho-physiological cellular events in various tissues. The originally discovered PKC (conventional) requires the binding of diacylglycerol and Ca2+ for full activation. The conventional PKC consists of four isoforms, PKCα, PKCβI/βII, and PKCγ. PKCα and PKCβI/βII are expressed in the cells of various tissues including the brain, while PKCγ is present specifically in neurons of the brain and spinal cord. The cerebellum expresses the largest amount of PKC with all its four isoforms. Purkinje cells express PKCα and PKCγ. Previous studies have shown that PKCα is involved in the induction of long-term depression (LTD) at parallel fiber-Purkinje cell synapses. On the other hand, analysis of PKCγ-deficient mice has revealed that PKCγ plays a critical role in eliminating supernumerary climbing fiber synapses from developing Purkinje cells. Although why PKCα has no compensatory action in climbing fiber pruning in PKCγ-deficient Purkinje cells had so far remained unclear, we have recently demonstrated that PKCα is also capable of pruning supernumerary climbing fiber synapses, but the expression levels of PKCα are too low to achieve pruning in PKCγ-null Purkinje cells. Notably, although PKCγ is most abundant in Purkinje cells, its physiological role in mature Purkinje cells remained totally unknown. In addition to a concise review of the physiological and pathological roles of conventional PKCs in Purkinje cells, this report postulates a contribution of PKCα in developing Purkinje cells and a possible involvement of PKCγ in motor coordination in the mature cerebellum.
- Published
- 2017
31. Plasticity of the developmentally arrested staggerer cerebellum in response to exogenous RORα
- Author
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Hirokazu Hirai, Yasunori Matsuzaki, Ayumu Konno, and Akira Iizuka
- Subjects
0301 basic medicine ,Cerebellum ,Histology ,Internal granular layer ,Genetic Vectors ,Purkinje cell ,Synaptogenesis ,Parallel fiber ,Biology ,Receptors, Metabotropic Glutamate ,Membrane Potentials ,Mice ,Mice, Neurologic Mutants ,Purkinje Cells ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Transcription factor ,Orphan receptor ,Neuronal Plasticity ,General Neuroscience ,Lentivirus ,Excitatory Postsynaptic Potentials ,Nuclear Receptor Subfamily 1, Group F, Member 1 ,Dendrites ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Metabotropic glutamate receptor ,Synapses ,Anatomy ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Retinoid-related orphan receptor α (RORα) is a critical master transcription factor that governs postnatal cerebellar development. An RORα-deficient cerebellum has a persistent external granular layer (EGL), rudimentary Purkinje cell (PC) dendrites, grossly reduced numbers of immature parallel fiber (PF)-PC synapses, and multiple climbing fibers (CF) innervating PCs in mice after 3 weeks of age when these features have disappeared in wild-type mice. Functionally, metabotropic glutamate receptor (mGluR)-mediated signaling in PCs is completely abrogated. Here we examined whether these defects could be corrected by lentivirally providing the RORα gene to 3-week-old PCs of RORα-deficient homozygous staggerer (sg/sg) mice. RORα expression in sg/sg PCs significantly increased the numbers of PF–PC synapses, spines on PC dendritic branchlets, and internal granule cells, concomitant with regression of the EGL, suggesting enhanced proliferation in the EGL and migration of post-mitotic progeny into the internal granular layer with augmented synaptogenesis between PFs and PC dendrites. However, the primary dendritic stems were only slightly extended, and mGluR signaling and the loss of redundant CF synapses in sg/sg PCs remained unrestored. These results suggest that the mitogenic and migratory potential of external granule cells in response to RORα was preserved in the >3-week-old sg/sg mouse cerebellum. Moreover, sg/sg PCs sprouted spines and formed synapses with PFs. However, lengthening of the primary dendritic stems, establishment of mGluR signaling, and removal of CF synapses in sg/sg PCs were regressed by 3 weeks of age.
- Published
- 2015
32. Long-term oral administration of the NMDA receptor antagonist memantine extends life span in spinocerebellar ataxia type 1 knock-in mice
- Author
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Hirokazu Hirai, Akira Iizuka, and Kazuhiro Nakamura
- Subjects
medicine.medical_specialty ,Cerebellum ,Spinocerebellar Ataxia Type 1 ,Chromosomal Proteins, Non-Histone ,Longevity ,Administration, Oral ,Ataxin 1 ,Receptors, N-Methyl-D-Aspartate ,Memantine ,Gene knockin ,Internal medicine ,Animals ,Spinocerebellar Ataxias ,Medicine ,Gene Knock-In Techniques ,Neurons ,Cell Death ,biology ,business.industry ,General Neuroscience ,Antagonist ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Dorsal motor nucleus ,Endocrinology ,nervous system ,biology.protein ,NMDA receptor ,business ,Neuroscience ,medicine.drug - Abstract
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by extension of a CAG repeat in the Sca1gene. Although the mechanisms underlying the symptoms of SCA1 have not been determined, aberrant neuronal activation potentially contributes to the neuronal cell death characteristic of the disease. Here we examined the potential involvement of extrasynaptic N-methyl-d-aspartate receptor (NMDAR) activation in the pathogenesis of SCA1 by administering memantine, a low-affinity noncompetitive NMDAR antagonist, in SCA1 knock-in (KI) mice. In KI mice, the exon in the ataxin 1 gene is replaced with abnormally expanded 154CAG repeats. Memantine was administered orally to the SCA1 KI mice from 4 weeks of age until death. The treatment significantly attenuated body-weight loss and prolonged the life span of SCA1 KI mice. Furthermore, memantine significantly suppressed the loss of Purkinje cells in the cerebellum and motor neurons in the dorsal motor nucleus of the vagus, which are critical for motor function and parasympathetic function, respectively. These findings support the contribution of aberrant activation of extrasynaptic NMDARs to neuronal cell death in SCA1 KI mice and suggest that memantine may also have therapeutic benefits in human SCA1 patients.
- Published
- 2015
33. Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado-Joseph disease mice
- Author
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Hirokazu Hirai, Liliana Mendonça, Clévio Nóbrega, Luís Pereira de Almeida, and Brian K. Kaspar
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Cerebellum ,Pathology ,medicine.medical_specialty ,Purkinje cell ,Mice, Transgenic ,Cell Separation ,Neuropathology ,Mice ,Neural Stem Cells ,Neuritis ,Neurotrophic factors ,medicine ,Animals ,Cells, Cultured ,business.industry ,Brain-Derived Neurotrophic Factor ,Cell Differentiation ,Machado-Joseph Disease ,Neural stem cell ,Receptors, Neurotransmitter ,Motor coordination ,Mice, Inbred C57BL ,body regions ,Transplantation ,Neuroepithelial cell ,medicine.anatomical_structure ,Ataxia ,Neurology (clinical) ,business ,Neuroscience ,Psychomotor Performance - Abstract
Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.
- Published
- 2014
34. Type 1 metabotropic glutamate receptor and its signaling molecules as therapeutic targets for the treatment of cerebellar disorders
- Author
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Hirokazu Hirai and Masanobu Kano
- Subjects
0301 basic medicine ,Cerebellum ,Cell signaling ,Purkinje cell ,Cerebellar Purkinje cell ,Receptors, Metabotropic Glutamate ,03 medical and health sciences ,Purkinje Cells ,0302 clinical medicine ,Cerebellar Diseases ,Drug Discovery ,Medicine ,Animals ,Humans ,Cerebellar disorder ,Pharmacology ,business.industry ,030104 developmental biology ,medicine.anatomical_structure ,Metabotropic glutamate receptor ,Metabotropic glutamate receptor 1 ,Signal transduction ,business ,Neuroscience ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Neurodegenerative diseases such as spinocerebellar ataxias and autoantibody-associated disorders of the central nervous system often affect the cerebellum, resulting in motor deficits. Recent studies have revealed that most of these disorders impair type 1 metabotropic glutamate receptor (mGluR1) and/or the closely associated signaling molecules in cerebellar Purkinje cell. Since the signaling pathway triggered by mGluR1 activation in Purkinje cell plays a pivotal role in coordinated movements and motor learning, pharmacological repair of aberrant mGluR1 signaling in Purkinje cell is critical for mitigation of cerebellar symptoms. Here we review recently identified pathophysiology underlying the neurodegenerative and autoimmune diseases affecting mGluR1 signaling in Purkinje cell and possible therapeutic interventions.
- Published
- 2017
35. Beclin 1 mitigates motor and neuropathological deficits in genetic mouse models of Machado-Joseph disease
- Author
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Hirokazu Hirai, Clévio Nóbrega, David Albuquerque, Nicole Déglon, Isabel Onofre, Célia A. Aveleira, Luís Pereira de Almeida, Ana Vasconcelos-Ferreira, and Isabel Nascimento-Ferreira
- Subjects
Male ,Cerebellum ,Dopamine and cAMP-Regulated Phosphoprotein 32 ,congenital, hereditary, and neonatal diseases and abnormalities ,Purkinje cell ,Green Fluorescent Proteins ,Mice, Transgenic ,Nerve Tissue Proteins ,Biology ,Motor Activity ,Transfection ,Mice ,medicine ,Autophagy ,Animals ,Humans ,Ataxin-3 ,Postural Balance ,Cells, Cultured ,Analysis of Variance ,Cerebellar ataxia ,Neurodegeneration ,Age Factors ,Membrane Proteins ,Nuclear Proteins ,Machado-Joseph Disease ,medicine.disease ,Motor coordination ,Mice, Inbred C57BL ,Repressor Proteins ,Disease Models, Animal ,medicine.anatomical_structure ,Animals, Newborn ,Gene Expression Regulation ,Ataxin ,Nerve Degeneration ,Sensation Disorders ,Spinocerebellar ataxia ,Beclin-1 ,Female ,Neurology (clinical) ,medicine.symptom ,Apoptosis Regulatory Proteins ,Peptides ,Machado–Joseph disease ,Neuroscience ,Psychomotor Performance - Abstract
Machado-Joseph disease or spinocerebellar ataxia type 3, the most common dominantly-inherited spinocerebellar ataxia, results from translation of the polyglutamine-expanded and aggregation prone ataxin 3 protein. Clinical manifestations include cerebellar ataxia and pyramidal signs and there is no therapy to delay disease progression. Beclin 1, an autophagy-related protein and essential gene for cell survival, is decreased in several neurodegenerative disorders. This study aimed at evaluating if lentiviral-mediated beclin 1 overexpression would rescue motor and neuropathological impairments when administered to pre- and post-symptomatic lentiviral-based and transgenic mouse models of Machado-Joseph disease. Beclin 1-mediated significant improvements in motor coordination, balance and gait with beclin 1-treated mice equilibrating longer periods in the Rotarod and presenting longer and narrower footprints. Furthermore, in agreement with the improvements observed in motor function beclin 1 overexpression prevented neuronal dysfunction and neurodegeneration, decreasing formation of polyglutamine-expanded aggregates, preserving Purkinje cell arborization and immunoreactivity for neuronal markers. These data show that overexpression of beclin 1 in the mouse cerebellum is able to rescue and hinder the progression of motor deficits when administered to pre- and post-symptomatic stages of the disease. * Abbreviation : SCA : spinocerebellar ataxia
- Published
- 2017
36. Regulatory connection between the expression level of classical protein kinase C and pruning of climbing fibers from cerebellar Purkinje cells
- Author
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Anton N. Shuvaev, Hirokazu Hirai, Ayumu Konno, Masashi Watanave, Nobutaka Takahashi, and Yasunori Matsuzaki
- Subjects
0301 basic medicine ,Male ,Cerebellum ,Purkinje cell ,Biology ,behavioral disciplines and activities ,Biochemistry ,Synapse ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Mice ,Purkinje Cells ,0302 clinical medicine ,Nerve Fibers ,medicine ,Animals ,Protein Isoforms ,Protein kinase C ,Protein Kinase C ,Mice, Knockout ,Messenger RNA ,Climbing fiber ,humanities ,Cell biology ,Reverse transcription polymerase chain reaction ,Blot ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Animals, Newborn ,Female ,Transcriptome ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Cerebellar Purkinje cells (PCs) express two members of the classical protein kinase C (cPKC) subfamily, namely, PKCα and PKCγ. Previous studies on PKCγ knockout (KO) mice have revealed a critical role of PKCγ in the pruning of climbing fibers (CFs) from PCs during development. The question remains as to why only PKCγ and not PKCα is involved in CF synapse elimination from PCs. To address this question, we assessed the expression levels of PKCγ and PKCα in wild-type (WT) and PKCγ KO PCs using PC-specific quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical analysis. The results revealed that the vast majority of cPKCs in PCs were PKCγ, whereas PKCα accounted for the remaining minimal fraction. The amount of PKCα was not up-regulated in PKCγ KO PCs. Lentiviral expression of PKCα in PKCγ KO PCs resulted in a 10-times increase in the amount of PKCα mRNA in the PKCγ KO PCs, compared to that in WT PCs. Our quantification showed that the expression levels of cPKC mRNA in PKCγ KO PCs increased roughly from 1% to 22% of that in WT PCs solely through PKCα expression. The up-regulation of PKCα in PKCγ KO PCs significantly rescued the impaired CF synapse elimination. Although both PKCα and PKCγ are capable of pruning supernumerary CF synapses from developing PCs, these results suggest that the expression levels of cPKCs in PKCγ KO PCs are too low for CF pruning.
- Published
- 2017
37. One-year follow-up of transgene expression by integrase-defective lentiviral vectors and their therapeutic potential in spinocerebellar ataxia model mice
- Author
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H Saida, Yasunori Matsuzaki, Shigeru Yanagi, Hirokazu Hirai, Ayumu Konno, Akira Iizuka, and Kiyohiko Takayama
- Subjects
Transgene ,Genetic Vectors ,Mutant ,Vectors in gene therapy ,GTP Phosphohydrolases ,Insertional mutagenesis ,Mice ,In vivo ,Cerebellum ,Genetics ,medicine ,Animals ,Humans ,Spinocerebellar Ataxias ,Transgenes ,Molecular Biology ,Gene ,Integrases ,biology ,Lentivirus ,medicine.disease ,Molecular biology ,Integrase ,Disease Models, Animal ,HEK293 Cells ,Rotarod Performance Test ,Mutation ,biology.protein ,Spinocerebellar ataxia ,Molecular Medicine ,Follow-Up Studies ,HeLa Cells ,Signal Transduction - Abstract
We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.
- Published
- 2014
38. Mesenchymal Stem Cells Ameliorate Cerebellar Pathology in a Mouse Model of Spinocerebellar Ataxia Type 1
- Author
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Serina Matsuura, Anton N. Shuvaev, Hirokazu Hirai, Kazuhiro Nakamura, and Akira Iizuka
- Subjects
Genetically modified mouse ,Spinocerebellar Ataxia Type 1 ,Pathology ,medicine.medical_specialty ,Ataxia ,Transgene ,Mice, Transgenic ,Biology ,Mesenchymal Stem Cell Transplantation ,Mice ,Purkinje Cells ,Cerebellum ,medicine ,Animals ,Spinocerebellar Ataxias ,Neurons ,Cerebellar ataxia ,Mesenchymal stem cell ,Mesenchymal Stem Cells ,Neurodegenerative Diseases ,Dendrites ,Polyglutamine tract ,Transplantation ,Disease Models, Animal ,Neurology ,Neurology (clinical) ,medicine.symptom - Abstract
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder caused by the expansion of a polyglutamine tract in the ataxin-1 protein. To date, no fundamental treatments for SCA1 have been elucidated. However, some studies have shown that mesenchymal stem cells (MSCs) are partially effective in other genetic mouse models of cerebellar ataxia. In this study, we tested the efficacy of the intrathecal injection of MSCs in the treatment of SCA1 in transgenic (SCA1-Tg) mice. We found that intrathecal injection of only 3 × 10(3) MSCs greatly mitigated the cerebellar neuronal disorganization observed in SCA1 transgenic mice (SCA1-Tg mice). Although the Purkinje cells (PCs) of 24-week-old nontreated SCA1-Tg mice displayed a multilayer arrangement, SCA1-Tg mice at a similar age injected with MSCs displayed monolayer PCs. Furthermore, intrathecal injection of MSCs suppressed the atrophy of PC dendrites in SCA1-Tg mice. Finally, behavioral tests demonstrated that MSCs normalized deficits in motor coordination in SCA1-Tg mice. Future studies should be performed to develop optimal protocols for intrathecal transplantation of MSCs in SCA1 model primates with the aim of developing applications for SCA1 patients.
- Published
- 2013
39. Postural dysfunction in a transgenic mouse model of spinocerebellar ataxia type 3
- Author
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Hirokazu Hirai, Dai Yanagihara, and Hiroshi Yamaura
- Subjects
Genetically modified mouse ,Cerebellum ,Movement ,Posture ,Purkinje cell ,Mice, Transgenic ,Hindlimb ,Electromyography ,Mice ,medicine ,Cerebellar Degeneration ,Animals ,Postural Balance ,medicine.diagnostic_test ,General Neuroscience ,Machado-Joseph Disease ,medicine.disease ,Muscle atrophy ,Biomechanical Phenomena ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Spinocerebellar ataxia ,medicine.symptom ,Psychology ,Neuroscience ,psychological phenomena and processes - Abstract
During voluntary limb movements, humans exert anticipatory postural adjustments (APAs) to prevent any upcoming equilibrium disturbance that might be provoked by limb movements. Dysfunction in generation or control of APAs is associated with postural deficits in some human patients with cerebellar damage. To examine the role of the cerebellum in APAs, we investigated a conditional transgenic mouse of spinocerebellar ataxia type 3 (SCA3Tg) that has defective cerebellar Purkinje cells. Kinematic analyses and monitoring of electromyographic activities during quadrupedal standing showed that SCA3Tg mice exhibited greater hindlimb instability than wild-type (WT) mice. This instability increased during a reaching task that required postural adjustments associated with voluntary neck movements. Normally, the activities of the hindlimb muscles are synchronized with those in the neck that are the agonists for movement of the head in this reaching task; however, in SCA3Tg mice, activities in the hindlimbs were markedly delayed compared to the neck. These observations cannot simply be explained as a secondary outcome of the muscle atrophy that occurs in SCA3Tg mice. In WT mice with muscle atrophy induced by immobilization of the hindlimbs, we did not find impairment of APAs. These findings suggest that the deficits in APAs during the reaching task in SCA3Tg mice were not due to muscle atrophy in the hindlimbs, but were mainly caused by cerebellar degeneration. Therefore, we conclude that the cerebellum is critically involved in APAs.
- Published
- 2013
40. Arc/Arg3.1 Is a Postsynaptic Mediator of Activity-Dependent Synapse Elimination in the Developing Cerebellum
- Author
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Hiroyuki Okuno, Takayasu Mikuni, Karl Deisseroth, Masanobu Kano, Haruhiko Bito, Naofumi Uesaka, and Hirokazu Hirai
- Subjects
Cerebellum ,Neuroscience(all) ,Purkinje cell ,Mice, Transgenic ,Nerve Tissue Proteins ,Biology ,Article ,Photostimulation ,Rats, Sprague-Dawley ,Synapse ,Mice ,Purkinje Cells ,Postsynaptic potential ,medicine ,Biological neural network ,Animals ,Arc (protein) ,General Neuroscience ,Climbing fiber ,Synaptic Potentials ,Coculture Techniques ,Rats ,Cell biology ,Mice, Inbred C57BL ,Cytoskeletal Proteins ,medicine.anatomical_structure ,Animals, Newborn ,Neuroscience - Abstract
SummaryNeural circuits are shaped by activity-dependent elimination of redundant synapses during postnatal development. In many systems, postsynaptic activity is known to be crucial, but the precise mechanisms remain elusive. Here, we report that the immediate early gene Arc/Arg3.1 mediates elimination of surplus climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum. CF synapse elimination was accelerated when activity of channelrhodopsin-2-expressing PCs was elevated by 2-day photostimulation. This acceleration was suppressed by PC-specific knockdown of either the P/Q-type voltage-dependent Ca2+ channels (VDCCs) or Arc. PC-specific Arc knockdown had no appreciable effect until around postnatal day 11 but significantly impaired CF synapse elimination thereafter, leaving redundant CF terminals on PC somata. The effect of Arc knockdown was occluded by simultaneous knockdown of P/Q-type VDCCs in PCs. We conclude that Arc mediates the final stage of CF synapse elimination downstream of P/Q-type VDCCs by removing CF synapses from PC somata.
- Published
- 2013
41. Fusion of Human Fetal Mesenchymal Stem Cells with 'Degenerating' Cerebellar Neurons in Spinocerebellar Ataxia Type 1 Model Mice
- Author
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Hirokazu Hirai, Fathul Huda, Yiping Fan, Jerry Kok Yen Chan, Yasunori Matsuzaki, Mamiko Suzuki, Nobutaka Takahashi, and Ayumu Konno
- Subjects
0301 basic medicine ,Cerebellum ,Pathology ,Molecular biology ,Epidemiology ,Cellular differentiation ,lcsh:Medicine ,Disease Vectors ,Cell Fusion ,Purkinje Cells ,Mice ,Animal Cells ,Medicine and Health Sciences ,Fetal Stem Cells ,lcsh:Science ,Ataxin-1 ,Neurons ,Cerebral Cortex ,Multidisciplinary ,biology ,Stem Cells ,Transdifferentiation ,Brain ,Cell Differentiation ,Cell biology ,medicine.anatomical_structure ,Cellular Types ,Anatomy ,Viral Vectors ,Research Article ,Cell Physiology ,medicine.medical_specialty ,Ataxin 1 ,Cre recombinase ,Mice, Transgenic ,DNA construction ,Mesenchymal Stem Cell Transplantation ,Microbiology ,03 medical and health sciences ,Fetus ,Interneurons ,Virology ,medicine ,Animals ,Humans ,Mesenchymal stem cell ,lcsh:R ,Biology and Life Sciences ,Mesenchymal Stem Cells ,Cell Biology ,Research and analysis methods ,Transplantation ,Disease Models, Animal ,Molecular biology techniques ,030104 developmental biology ,Cellular Neuroscience ,Plasmid Construction ,Cell Transdifferentiation ,biology.protein ,lcsh:Q ,Viral Transmission and Infection ,Neuroscience - Abstract
Mesenchymal stem cells (MSCs) migrate to damaged tissues, where they participate in tissue repair. Human fetal MSCs (hfMSCs), compared with adult MSCs, have higher proliferation rates, a greater differentiation capacity and longer telomeres with reduced senescence. Therefore, transplantation of quality controlled hfMSCs is a promising therapeutic intervention. Previous studies have shown that intravenous or intracortical injections of MSCs result in the emergence of binucleated cerebellar Purkinje cells (PCs) containing an MSC-derived marker protein in mice, thus suggesting a fusion event. However, transdifferentiation of MSCs into PCs or transfer of a marker protein from an MSC to a PC cannot be ruled out. In this study, we unequivocally demonstrated the fusion of hfMSCs with murine PCs through a tetracycline-regulated (Tet-off) system with or without a Cre-dependent genetic inversion switch (flip-excision; FLEx). In the FLEx-Tet system, we performed intra-cerebellar injection of viral vectors expressing tetracycline transactivator (tTA) and Cre recombinase into either non-symptomatic (4-week-old) or clearly symptomatic (6-8-month-old) spinocerebellar ataxia type 1 (SCA1) mice. Then, the mice received an injection of 50,000 genetically engineered hfMSCs that expressed GFP only in the presence of Cre recombinase and tTA. We observed a significant emergence of GFP-expressing PCs and interneurons in symptomatic, but not non-symptomatic, SCA1 mice 2 weeks after the MSC injection. These results, together with the results obtained using age-matched wild-type mice, led us to conclude that hfMSCs have the potential to preferentially fuse with degenerating PCs and interneurons but not with healthy neurons.
- Published
- 2016
42. Mutant PKCγ in Spinocerebellar Ataxia Type 14 Disrupts Synapse Elimination and Long-Term Depression in Purkinje CellsIn Vivo
- Author
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Takahiro Seki, Hanna Goenawan, Akira Iizuka, Tomohiko Irie, Hajime Horiuchi, Anton N. Shuvaev, Hirokazu Hirai, and Norio Sakai
- Subjects
Male ,Protein Kinase C-alpha ,Parallel fiber ,Neurotransmission ,Biology ,Synapse ,Mice ,Purkinje Cells ,Organ Culture Techniques ,TRPC3 ,Cerebellum ,medicine ,Animals ,Humans ,Spinocerebellar Ataxias ,Long-term depression ,Cells, Cultured ,Protein Kinase C ,Spinocerebellar Degenerations ,Mice, Knockout ,Long-Term Synaptic Depression ,General Neuroscience ,Cell Membrane ,Articles ,Climbing fiber ,medicine.disease ,Cell biology ,Isoenzymes ,Mice, Inbred C57BL ,Protein Transport ,medicine.anatomical_structure ,Mutation ,Synapses ,Synaptic plasticity ,Spinocerebellar ataxia ,Female ,Neuroscience - Abstract
Cerebellar Purkinje cells (PCs) express a large amount of the gamma isoform of protein kinase C (PKC gamma) and a modest level of PKC alpha. The PKC gamma is involved in the pruning of climbing fiber (CF) synapses from developing PCs, and PKC alpha plays a critical role in long-term depression (LTD) at parallel fiber (PF)-PC synapses. Moreover, the PKC signaling in PCs negatively modulates the nonselective transient receptor potential cation channel type 3 (TRPC3), the opening of which elicits slow EPSCs at PF-PC synapses. Autosomal dominant spinocerebellar ataxia type 14 (SCA14) is caused by mutations in PKC gamma. To clarify the pathology of this disorder, mutant (S119P) PKC gamma tagged with GFP was lentivirally expressed in developing and mature mouse PCs in vivo, and the effects were assessed 3 weeks after the injection. Mutant PKC gamma-GFP aggregated in PCs without signs of degeneration. Electrophysiology results showed impaired pruning of CF synapses from developing PCs, failure of LTD expression, and increases in slow EPSC amplitude. We also found that mutant PKC gamma colocalized with wild-type PKC gamma, which suggests that mutant PKC gamma acts in a dominant-negative manner on wild-type PKC gamma. In contrast, PKC alpha did not colocalize with mutant PKC gamma. The membrane residence time of PKC alpha after depolarization-induced translocation, however, was significantly decreased when it was present with the mutant PKC gamma construct. These results suggest that mutant PKC gamma in PCs of SCA14 patients could differentially impair the membrane translocation kinetics of wild-type gamma and alpha PKCs, which would disrupt synapse pruning, synaptic plasticity, and synaptic transmission.
- Published
- 2011
43. Disruption of metabotropic glutamate receptor signalling is a major defect at cerebellar parallel fibre-Purkinje cell synapses instaggerermutant mice
- Author
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Hirokazu Hirai, Kazuhiro Mitsumura, Nobuhiko Furuya, and Nobutake Hosoi
- Subjects
Cerebellum ,Spinocerebellar Ataxia Type 1 ,Physiology ,musculoskeletal, neural, and ocular physiology ,Purkinje cell ,Neural facilitation ,Biology ,Neurotransmission ,Cell biology ,medicine.anatomical_structure ,TRPC3 ,nervous system ,Metabotropic glutamate receptor ,medicine ,Metabotropic glutamate receptor 1 ,Neuroscience - Abstract
Non-technical summary Homozygous staggerer mutant (sg/sg) mice exhibit cerebellar atrophy and congenital ataxia, and serve as an important extreme mouse model of the hereditary spinocerebellar ataxia type 1 (SCA1), since the staggerer mutation is closely related to SCA1 pathology. However, we know little about synaptic abnormalities at cerebellar parallel fibre (PF)–Purkinje cell (PC) synapses in sg/sg mice, which could underlie SCA1 pathology. In this study, we report that PFs still make reasonably functional fast synapses onto PCs in sg/sg mice despite reduction in the number of PF–PC synapses. In contrast, sg/sg mice lack metabotropic glutamate receptor (mGluR)-mediated slow synaptic transmission completely. Synaptic modulation caused by mGluR-mediated endocannabinoid release is also abolished at sg/sg PF–PC synapses. Our results indicate that major synaptic abnormality is disruption of cerebellar mGluR signalling in SCA1-related sg/sg mice, and that mGluR signalling can be one of the key factors to SCA1 pathology. Abstract Staggerer mutant mice have functional loss of a transcription factor, retinoid-related orphan receptor α (RORα), which is abundantly expressed in Purkinje cells (PCs) of the cerebellum. Homozygous staggerer (sg/sg) mice show cerebellar hypoplasia and congenital ataxia. Sg/sg mice serve as an important extreme mouse model of the hereditary spinocerebellar ataxia type 1 (SCA1), since it has been shown that RORα dysfunction is strongly correlated with SCA1 pathogenesis. However, synaptic abnormalities, especially at parallel fibre (PF)–PC synapses, in SCA1-related sg/sg mice have not been examined in detail electrophysiologically. In this study, we report that PFs can still establish functional synapses onto PCs in sg/sg mice in spite of reduction in the number of PF–PC synapses. Compared with PF-evoked EPSCs in the wild-type or heterozygotes, the success rate of the EPSC recordings in sg/sg was quite low (∼40%) and the EPSCs showed faster kinetics and slightly decreased paired pulse facilitation at short intervals. The prominent synaptic dysfunction is that sg/sg mice lack metabotropic glutamate receptor (mGluR)-mediated slow EPSCs completely. Neither intense PF stimulation nor an exogenously applied mGluR agonist, DHPG, could elicit mGluR-mediated responses. Western blot analysis in the sg/sg cerebellum revealed low-level expression of mGluR1 and TRPC3, both of which underlie mGluR-mediated slow currents in PCs. Immunohistochemical data demonstrated marked mislocalization of mGluR1 on sg/sg PCs. We found that mGluR-mediated retrograde suppression of PF–PC EPSCs by endocannabinoid is also impaired completely in sg/sg mice. These results suggest that disruption of mGluR signalling at PF–PC synapses is one of the major synaptic defects in sg/sg mice and may manifest itself in SCA1 pathology.
- Published
- 2011
44. Inhibitory effects of the antiepileptic drug ethosuximide on G protein-activated inwardly rectifying K+ channels
- Author
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Toru Kobayashi, Hirokazu Hirai, Kazuo Washiyama, Ichiro Fuse, Kazutaka Ikeda, Masae Iino, Shinya Kasai, and Kazuhiro Mitsumura
- Subjects
Patch-Clamp Techniques ,Microinjections ,Charybdotoxin ,Xenopus ,Barium Compounds ,In Vitro Techniques ,Pharmacology ,Inhibitory postsynaptic potential ,Membrane Potentials ,Mice ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Chlorides ,Cerebellum ,medicine ,Animals ,Channel blocker ,Patch clamp ,G protein-coupled inwardly-rectifying potassium channel ,Neurons ,Membrane potential ,Dose-Response Relationship, Drug ,Ethanol ,urogenital system ,Chemistry ,Central Nervous System Depressants ,Neural Inhibition ,Carbamazepine ,Electric Stimulation ,Adenosine Diphosphate ,Mice, Inbred C57BL ,Ethosuximide ,Animals, Newborn ,G Protein-Coupled Inwardly-Rectifying Potassium Channels ,Anticonvulsants ,medicine.drug - Abstract
Antiepileptic drugs protect against seizures by modulating neuronal excitability. Ethosuximide is selectively used for the treatment of absence epilepsy, and has also been shown to have the potential for treating several other neuropsychiatric disorders in addition to several antiepileptic drugs. Although ethosuximide inhibits T-type Ca(2+), noninactivating Na(+), and Ca(2+)-activated K(+) channels, the molecular mechanisms underlying the effects of ethosuximide have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) channels (GIRK, or Kir3) play an important role in regulating neuronal excitability, heart rate and platelet aggregation. In the present study, the effects of various antiepileptic drugs on GIRK channels were examined first by using the Xenopus oocyte expression assay. Ethosuximide at clinically relevant concentrations inhibited GIRK channels expressed in Xenopus oocytes. The inhibition was concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. However, the other antiepileptic drugs tested: phenytoin, valproic acid, carbamazepine, phenobarbital, gabapentin, topiramate and zonisamide, had no significant effects on GIRK channels even at toxic concentrations. In contrast, Kir1.1 and Kir2.1 channels were insensitive to all of the drugs tested. Ethosuximide also attenuated ethanol-induced GIRK currents. These inhibitory effects of ethosuximide were not observed when ethosuximide was applied intracellularly. In granule cells of cerebellar slices, ethosuximide inhibited GTPgammaS-activated GIRK currents. Moreover, ADP- and epinephrine-induced platelet aggregation was inhibited by ethosuximide, but not by charybdotoxin, a platelet Ca(2+)-activated K(+) channel blocker. These results suggest that the inhibitory effects of ethosuximide on GIRK channels may affect some of brain, heart and platelet functions.
- Published
- 2009
45. The scaffold protein JSAP1 regulates proliferation and differentiation of cerebellar granule cell precursors by modulating JNK signaling
- Author
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Takashi Torashima, Hirokazu Hirai, Katsuji Yoshioka, Masahide Asano, Tokiharu Sato, and Kazushi Sugihara
- Subjects
Scaffold protein ,animal structures ,Recombinant Fusion Proteins ,Mutant ,Nerve Tissue Proteins ,Signal transduction ,Biology ,Mice ,Cellular and Molecular Neuroscience ,Cerebellum ,medicine ,Animals ,Humans ,Hedgehog Proteins ,Sonic hedgehog ,Molecular Biology ,Cells, Cultured ,Adaptor Proteins, Signal Transducing ,Cell Proliferation ,Gene knockdown ,Stem Cells ,Cell Cycle ,JNK Mitogen-Activated Protein Kinases ,Cell Differentiation ,Cell Biology ,Cell cycle ,Granule cell ,Cell biology ,Ki-67 Antigen ,medicine.anatomical_structure ,Mitogen-activated protein kinase ,embryonic structures ,biology.protein ,MAP kinase ,Granule cell precursor ,Cyclin-Dependent Kinase Inhibitor p27 - Abstract
金沢大学がん研究所がん分子細胞制御, Cerebellar granule cell precursors (GCPs) proliferate in the outer part of the external granular layer (EGL). They begin their differentiation by exiting the cell cycle and migrating into the inner part of the EGL. Here we report that JSAP1, a scaffold protein for JNK signaling pathways, is expressed predominantly in the post-mitotic GCPs of the inner EGL. JSAP1 knockdown or treatment with a JNK inhibitor enhances the proliferation of cultured GCPs, but the overexpression of wild-type JSAP1 leads to increased proportions of p27Kip1- and NeuN-positive cells, even with saturating concentrations of Sonic hedgehog (Shh), a potent GCP mitogen. However, these differentiation-promoting effects on GCPs are attenuated significantly in cells overexpressing a mutant JSAP1 that lacks the JNK-binding domain. Together, these data suggest that JSAP1 antagonizes the mitogenic effect of Shh on GCPs and promotes their exit from the cell cycle and differentiation, by modulating JNK activity. © 2008 Elsevier Inc. All rights reserved.
- Published
- 2008
46. Purkinje-cell-preferential transduction by lentiviral vectors with the murine stem cell virus promoter
- Author
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Takashi Torashima, Hirokazu Hirai, Kiyohiko Takayama, and Hajime Horiuchi
- Subjects
Genetic Vectors ,Green Fluorescent Proteins ,Purkinje cell ,Gene Expression ,Gene delivery ,Viral vector ,Mice ,Purkinje Cells ,Viral Proteins ,Transduction (genetics) ,Transduction, Genetic ,Cerebellum ,medicine ,Animals ,Humans ,RNA, Messenger ,Promoter Regions, Genetic ,Cell Line, Transformed ,Rous sarcoma virus ,biology ,Stem Cells ,General Neuroscience ,Lentivirus ,Promoter ,biology.organism_classification ,Molecular biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Cell culture ,Stem cell - Abstract
Viral-vector-mediated gene delivery into Purkinje cells is a promising method for exploring the pathophysiology of the cerebellum; however, it is generally difficult to achieve sufficiently high levels of gene expression in Purkinje cells using viral vectors with a cell-type-specific promoter because of the weakness of transcriptional activity. In this study, we prepared lentiviral vectors that express GFP under the control of various ubiquitous promoters derived from murine stem cell virus (MSCV), cytomegalovirus (CMV), CMV early enhancer/chicken beta actin (CAG), and Rous sarcoma virus (RSV) and compared their potential to transduce Purkinje cells. Mice were sacrificed 7 days after lentiviral injection and the ratios of GFP(+) Purkinje cells to all transduced cells were determined. The highest transduction ratio was observed when we used lentivectors containing the MSCV promoter: approximately 70% of GFP(+) cells were Purkinje cells, the next highest ratio was for the CMV promoter (approximately 40%), then the CAG promoter (approximately 35%), and the lowest ratio was for the RSV promoter (approximately 10%). Moreover, the highest levels of GFP expression were also caused by the MSCV promoter. Thus, among the ubiquitous promoters examined, the MSCV promoter was the best for the expression of a foreign gene in Purkinje cells in vivo.
- Published
- 2008
47. Progress in transduction of cerebellar Purkinje cells in vivo using viral vectors
- Author
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Hirokazu Hirai
- Subjects
Cerebellum ,viruses ,Purkinje cell ,Genetic Vectors ,adeno-associated virus ,medicine.disease_cause ,Viral vector ,Transduction (genetics) ,Mice ,Purkinje Cells ,Viral Envelope Proteins ,lentivirus ,medicine ,Animals ,Adeno-associated virus ,Tropism ,biology ,viral vector ,Dependovirus ,biology.organism_classification ,Virology ,gene therapy ,Cell biology ,medicine.anatomical_structure ,Neurology ,Vesicular stomatitis virus ,Tissue tropism ,Neurology (clinical) ,Signal Transduction - Abstract
金沢大学大学院医学系研究科, Expression of a foreign gene in cerebellar Purkinje cells in vivo is a powerful method for exploring the pathophysiology of the cerebellum. Although using developmental engineering many gene-modified mice have been generated, this approach is time-consuming and requires a lot of effort for crossing different lines of mice, genotyping and maintenance of animals. If a gene of interest can be transferred to and efficiently expressed in Purkinje cells of developing and mature animals, it saves much time, effort and money. Recent advances in viral vectors have markedly contributed to selective and efficient gene transfer to Purkinje cells in vivo. There are two approaches for selective gene expression in Purkinje cells: one is to take advantage of the viral tropism for Purkinje cells, which includes the tropism of adeno-associated virus and the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentivirus. Another method, which might be used in combination with the first one, is utilization of a Purkinje-cell-specific promoter. Focusing mainly on these points, recent progress in viralvector-mediated transduction of Purkinje cells in vivo is reviewed. © 2008 Springer Science+Business Media, LLC., This article has not been published yet.
- Published
- 2008
48. Lentivector‐mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia
- Author
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Haruyasu Yamaguchi, Kiyohiko Takayama, Kazuhiro Mitsumura, Chiho Koyama, Hirokazu Hirai, Takashi Torashima, Akira Iizuka, Miho Oue, and Shigeru Yanagi
- Subjects
Cerebellum ,Ataxia ,Transgene ,Genetic enhancement ,Genetic Vectors ,Scientific Report ,Purkinje cell ,Mice, Transgenic ,Biology ,Biochemistry ,GTP Phosphohydrolases ,Mice ,Purkinje Cells ,Genetics ,medicine ,Animals ,Spinocerebellar Ataxias ,Molecular Biology ,Cerebellar ataxia ,Reverse Transcriptase Polymerase Chain Reaction ,Lentivirus ,Dendrites ,Genetic Therapy ,medicine.disease ,Immunohistochemistry ,Molecular biology ,Cell biology ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Spinocerebellar ataxia ,Triphosphatase ,medicine.symptom ,Peptides - Abstract
Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin–proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases.
- Published
- 2008
49. A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance
- Author
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Maya Yamazaki, Hisako Nakayama, Tomoki Nishioka, Kouichi Hashimoto, Hirokazu Hirai, Kozo Kaibuchi, Kohtarou Konno, Manabu Abe, Natsumi Ageta-Ishihara, Kenji Sakimura, Fathul Huda, Tsuyoshi Miyakawa, Masahiko Watanabe, Satoko Hattori, Kenji Hashimoto, Kohichi Tanaka, and Makoto Kinoshita
- Subjects
Male ,rho GTP-Binding Proteins ,Cerebellum ,Dendritic spine ,General Physics and Astronomy ,Glutamic Acid ,macromolecular substances ,Biology ,Septin ,General Biochemistry, Genetics and Molecular Biology ,Article ,Mice ,GTP-Binding Protein Regulators ,medicine ,Animals ,Mice, Knockout ,Neurons ,Multidisciplinary ,GTPase-Activating Proteins ,Glutamate receptor ,RNA-Binding Proteins ,General Chemistry ,Glutamic acid ,Cell biology ,Excitatory Amino Acid Transporter 1 ,Cytoskeletal Proteins ,medicine.anatomical_structure ,Gene Expression Regulation ,Excitatory postsynaptic potential ,Neuroglia ,Septins ,Septin cytoskeleton - Abstract
The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4−/− mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1–5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance., Glutamate transporters mediate neurotransmitter reuptake at glutamatergic synapses. Here the authors show that CDC42 effector protein CDC42EP4 supports efficient glutamate clearance by promoting the tethering of a glutamate transporter GLAST to perisynaptic clusters of septins in Bergmann glia.
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- 2015
50. Transduction Profile of the Marmoset Central Nervous System Using Adeno-Associated Virus Serotype 9 Vectors
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Yasunori Matsuzaki, Ayumu Konno, Hirokazu Hirai, Ryo Mukai, Fumiaki Honda, Yuhei Yoshimoto, and Masafumi Hirato
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0301 basic medicine ,Central Nervous System ,Male ,Cerebellum ,Central nervous system ,Genetic Vectors ,Neuroscience (miscellaneous) ,medicine.disease_cause ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Transduction, Genetic ,biology.animal ,Cisterna Magna ,Inferior olivary nucleus ,medicine ,Animals ,Hepatitis B Virus, Woodchuck ,Adeno-associated virus ,biology ,Pontine nuclei ,Marmoset ,Callithrix ,Dependovirus ,Spinal cord ,Rats ,030104 developmental biology ,medicine.anatomical_structure ,Neurology ,Cerebellar cortex ,Marmota ,Female ,Neuroscience - Abstract
The common marmoset is a small New World primate that has attracted remarkable attention as a potential experimental animal link between rodents and humans. Adeno-associated virus (AAV) vector-mediated expression of a disease-causing gene or a potential therapeutic gene in the brain may allow the construction of a marmoset model of a brain disorder or an exploration of the possibility of gene therapy. To gain more insights into AAV vector-mediated transduction profiles in the marmoset central nervous system (CNS), we delivered AAV serotype 9 (AAV9) vectors expressing GFP to the cisterna magna or the cerebellar cortex. Intracisternally injected AAV9 vectors expanded in the CNS according to the cerebrospinal fluid (CSF) flow, by retrograde transport through neuronal axons or via intermediary transcytosis, resulting in diffuse and global transduction within the CNS. In contrast, cerebellar parenchymal injection intensely transduced a more limited area, including the cerebellar cortex and cerebellar afferents, such as neurons of the pontine nuclei, vestibular nucleus and inferior olivary nucleus. In the spinal cord, both administration routes resulted in labeling of the dorsal column and spinocerebellar tracts, presumably by retrograde transport from the medulla oblongata and cerebellum, respectively. Motor neurons and dorsal root ganglia were also transduced, possibly by diffusion of the vector down the subarachnoid space along the cord. Thus, these two administration routes led to distinct transduction patterns in the marmoset CNS, which could be utilized to generate different disease animal models and to deliver therapeutic genes for the treatment of diseases affecting distinct brain areas.
- Published
- 2015
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