35 results on '"Hirokazu Hirai"'
Search Results
2. Nurr1 overexpression in the primary motor cortex alleviates motor dysfunction induced by intracerebral hemorrhage in the striatum in mice
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Keita Kinoshita, Kensuke Motomura, Keisuke Ushida, Yuma Hirata, Ayumu Konno, Hirokazu Hirai, Shunsuke Kotani, Natsuko Hitora-Imamura, Yuki Kurauchi, Takahiro Seki, and Hiroshi Katsuki
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Hemorrhagic stroke ,Nurr1 ,Motor dysfunction ,Corticospinal tract injury ,Glial cell line-derived neurotrophic factor ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Hemorrhage-induced injury of the corticospinal tract (CST) in the internal capsule (IC) causes severe neurological dysfunction in both human patients and rodent models of intracerebral hemorrhage (ICH). A nuclear receptor Nurr1 (NR4A2) is known to exert anti-inflammatory and neuroprotective effects in several neurological disorders. Previously we showed that Nurr1 ligands prevented CST injury and alleviated neurological deficits after ICH in mice. To prove direct effect of Nurr1 on CST integrity, we examined the effect of Nurr1 overexpression in neurons of the primary motor cortex on pathological consequences of ICH in mice. ICH was induced by intrastriatal injection of collagenase type VII, where hematoma invaded into IC. Neuron-specific overexpression of Nurr1 was induced by microinjection of synapsin I promoter-driven adeno-associated virus (AAV) vector into the primary motor cortex. Nurr1 overexpression significantly alleviated motor dysfunction but showed only modest effect on sensorimotor dysfunction after ICH. Nurr1 overexpression also preserved axonal structures in IC, while having no effect on hematoma-associated inflammatory events, oxidative stress, and neuronal death in the striatum after ICH. Immunostaining revealed that Nurr1 overexpression increased the expression of Ret tyrosine kinase and phosphorylation of Akt and ERK1/2 in neurons in the motor cortex. Moreover, administration of Nurr1 ligands 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane or amodiaquine increased phosphorylation levels of Akt and ERK1/2 as well as expression of glial cell line-derived neurotrophic factor and Ret genes in the cerebral cortex. These results suggest that the therapeutic effect of Nurr1 on striatal ICH is attributable to the preservation of CST by acting on cortical neurons.
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- 2024
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3. Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization
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Hayato Kawabata, Ayumu Konno, Yasunori Matsuzaki, Yumika Sato, Mika Kawachi, Ryo Aoki, Saki Tsutsumi, Shota Togai, Ryosuke Kobayashi, Takuro Horii, Izuho Hatada, and Hirokazu Hirai
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adeno-associated virus ,PHP.eB ,AAV-F ,glial fibrillary acidic protein ,astrocyte ,floxed mouse ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
The production of cell-type– and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)–mediated cell-type– and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier–penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target–flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.
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- 2024
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4. 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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5. Production of Spinocerebellar Ataxia Type 3 Model Mice by Intravenous Injection of AAV-PHP.B Vectors
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Ayumu Konno, Yoichiro Shinohara, and Hirokazu Hirai
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adeno-associated virus ,AAV ,AAV-PHP.B ,Spinocerebellar ataxia type 3 ,SCA3 ,Machado–Joseph disease ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
We aimed to produce a mouse model of spinocerebellar ataxia type 3 (SCA3) using the mouse blood–brain barrier (BBB)-penetrating adeno-associated virus (AAV)-PHP.B. Four-to-five-week-old C57BL/6 mice received injections of high-dose (2.0 × 1011 vg/mouse) or low-dose (5.0 × 1010 vg/mouse) AAV-PHP.B encoding a SCA3 causative gene containing abnormally long 89 CAG repeats [ATXN3(Q89)] under the control of the ubiquitous chicken β-actin hybrid (CBh) promoter. Control mice received high doses of AAV-PHP.B encoding ATXN3 with non-pathogenic 15 CAG repeats [ATXN3(Q15)] or phosphate-buffered saline (PBS) alone. More than half of the mice injected with high doses of AAV-PHP.B encoding ATXN3(Q89) died within 4 weeks after the injection. No mice in other groups died during the 12-week observation period. Mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89) exhibited progressive motor uncoordination starting 4 weeks and a shorter stride in footprint analysis performed at 12 weeks post-AAV injection. Immunohistochemistry showed thinning of the molecular layer and the formation of nuclear inclusions in Purkinje cells from mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89). Moreover, ATXN3(Q89) expression significantly reduced the number of large projection neurons in the cerebellar nuclei to one third of that observed in mice expressing ATXN3(Q15). This AAV-based approach is superior to conventional methods in that the required number of model mice can be created simply by injecting AAV, and the expression levels of the responsible gene can be adjusted by changing the amount of AAV injected. Moreover, this method may be applied to produce SCA3 models in non-human primates.
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- 2024
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6. A blood-brain barrier-penetrating AAV2 mutant created by a brain microvasculature endothelial cell-targeted AAV2 variant
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Hayato Kawabata, Ayumu Konno, Yasunori Matsuzaki, and Hirokazu Hirai
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AAV ,PHP.B ,PHP.eB ,BR1 ,blood-brain barrier ,capsid ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Upon systemic administration, adeno-associated virus serotype 9 (AAV9) and the capsid variant PHP.eB show distinct tropism for the central nervous system (CNS), whereas AAV2 and the capsid variant BR1 transduce brain microvascular endothelial cells (BMVECs) with little transcytosis. Here, we show that a single amino acid substitution (from Q to N) in the BR1 capsid at position 587 (designated BR1N) confers a significantly higher blood-brain barrier (BBB) penetration capacity to BR1. Intravenously infused BR1N showed significantly higher CNS tropism than BR1 and AAV9. BR1 and BR1N likely use the same receptor for entry into BMVECs; however, the single amino acid substitution has profound consequences on tropism. This suggests that receptor binding alone does not determine the final outcome in vivo and that further improvements of capsids within predetermined receptor usage are feasible.
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- 2023
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7. Excitatory subtypes of the lateral amygdala neurons are differentially involved in regulation of synaptic plasticity and excitation/inhibition balance in aversive learning in mice
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Mieko Morishima, Sohta Matsumura, Suguru Tohyama, Takashi Nagashima, Ayumu Konno, Hirokazu Hirai, and Ayako M. Watabe
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lateral amygdala (LA) ,medial geniculate nucleus (MGN) ,excitatory cell ,inhibitory cell ,fear conditioning ,experience-dependent plasticity ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The amygdala plays a crucial role in aversive learning. In Pavlovian fear conditioning, sensory information about an emotionally neutral conditioned stimulus (CS) and an innately aversive unconditioned stimulus is associated with the lateral amygdala (LA), and the CS acquires the ability to elicit conditioned responses. Aversive learning induces synaptic plasticity in LA excitatory neurons from CS pathways, such as the medial geniculate nucleus (MGN) of the thalamus. Although LA excitatory cells have traditionally been classified based on their firing patterns, the relationship between the subtypes and functional properties remains largely unknown. In this study, we classified excitatory cells into two subtypes based on whether the after-depolarized potential (ADP) amplitude is expressed in non-ADP cells and ADP cells. Their electrophysiological properties were significantly different. We examined subtype-specific synaptic plasticity in the MGN-LA pathway following aversive learning using optogenetics and found significant experience-dependent plasticity in feed-forward inhibitory responses in fear-conditioned mice compared with control mice. Following aversive learning, the inhibition/excitation (I/E) balance in ADP cells drastically changed, whereas that in non-ADP cells tended to change in the reverse direction. These results suggest that the two LA subtypes are differentially regulated in relation to synaptic plasticity and I/E balance during aversive learning.
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- 2023
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8. State-dependent modulation of positive and negative affective valences by a parabrachial nucleus-to-ventral tegmental area pathway in mice
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Takashi Nagashima, Kaori Mikami, Suguru Tohyama, Ayumu Konno, Hirokazu Hirai, and Ayako M. Watabe
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parabrachial nucleus ,ventral tegmental area ,glutamic acid decarboxylase 65 ,avoidance ,aversive memory ,operant task ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Appropriately responding to various sensory signals in the environment is essential for animal survival. Accordingly, animal behaviors are closely related to external and internal states, which include the positive and negative emotional values of sensory signals triggered by environmental factors. While the lateral parabrachial nucleus (LPB) plays a key role in nociception and supports negative valences, it also transmits signals including positive valences. However, the downstream neuronal mechanisms of positive and negative valences have not been fully explored. In the present study, we investigated the ventral tegmental area (VTA) as a projection target for LPB neurons. Optogenetic activation of LPB-VTA terminals in male mice elicits positive reinforcement in an operant task and induces both avoidance and attraction in a place-conditioning task. Inhibition of glutamic acid decarboxylase (GAD) 65-expressing cells in the VTA promotes avoidance behavior induced by photoactivation of the LPB-VTA pathway. These findings indicate that the LPB-VTA pathway is one of the LPB outputs for the transmission of positive and negative valence signals, at least in part, with GABAergic modification in VTA.
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- 2023
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9. Development of microglia-targeting adeno-associated viral vectors as tools to study microglial behavior in vivo
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Yukihiro Okada, Nobutake Hosoi, Yasunori Matsuzaki, Yuuki Fukai, Akito Hiraga, Junichi Nakai, Keisuke Nitta, Yoichiro Shinohara, Ayumu Konno, and Hirokazu Hirai
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Biology (General) ,QH301-705.5 - Abstract
Microglia-targeting AAV vectors are created which can be utilized to study microglial pathophysiology and microglia-targeted therapies in mice, especially in the regions of striatum and cerebellar cortex.
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- 2022
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10. DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers
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Mingyue Jin, Sakiko Matsumoto, Takashi Ayaki, Hodaka Yamakado, Tomoyuki Taguchi, Natsuko Togawa, Ayumu Konno, Hirokazu Hirai, Hiroshi Nakajima, Shoji Komai, Ryuichi Ishida, Syuhei Chiba, Ryosuke Takahashi, Toshifumi Takao, and Shinji Hirotsune
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Science - Abstract
In this work, the authors show that α-synuclein is posttranslationally dopanized at Tyr136 by tyrosine hydroxylase, which facilitates the formation of oligomers. This modification likely impacts pathogenesis and the selective degeneration of dopaminergic neurons in Parkinson’s disease.
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- 2022
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11. Interaction between Teneurin-2 and microtubules via EB proteins provides a platform for GABAA receptor exocytosis
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Sotaro Ichinose, Yoshihiro Susuki, Nobutake Hosoi, Ryosuke Kaneko, Mizuho Ebihara, Hirokazu Hirai, and Hirohide Iwasaki
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synapse ,microtubules ,adhesion molecule ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Neurons form dense neural circuits by connecting to each other via synapses and exchange information through synaptic receptors to sustain brain activities. Excitatory postsynapses form and mature on spines composed predominantly of actin, while inhibitory synapses are formed directly on the shafts of dendrites where both actin and microtubules (MTs) are present. Thus, it is the accumulation of specific proteins that characterizes inhibitory synapses. In this study, we explored the mechanisms that enable efficient protein accumulation at inhibitory postsynapse. We found that some inhibitory synapses function to recruit the plus end of MTs. One of the synaptic organizers, Teneurin-2 (TEN2), tends to localize to such MT-rich synapses and recruits MTs to inhibitory postsynapses via interaction with MT plus-end tracking proteins EBs. This recruitment mechanism provides a platform for the exocytosis of GABAA receptors. These regulatory mechanisms could lead to a better understanding of the pathogenesis of disorders such as schizophrenia and autism, which are caused by excitatory/inhibitory (E/I) imbalances during synaptogenesis.
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- 2023
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12. Astrocytic GPCR-Induced Ca2+ Signaling Is Not Causally Related to Local Cerebral Blood Flow Changes
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Katsuya Ozawa, Masaki Nagao, Ayumu Konno, Youichi Iwai, Marta Vittani, Peter Kusk, Tsuneko Mishima, Hirokazu Hirai, Maiken Nedergaard, and Hajime Hirase
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astrocytes ,optogenetic GPCR ,OptoA1AR ,Ca2+ elevation ,blood flow ,cortex ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Activation of Gq-type G protein-coupled receptors (GPCRs) gives rise to large cytosolic Ca2+ elevations in astrocytes. Previous in vitro and in vivo studies have indicated that astrocytic Ca2+ elevations are closely associated with diameter changes in the nearby blood vessels, which astrocytes enwrap with their endfeet. However, the causal relationship between astrocytic Ca2+ elevations and blood vessel diameter changes has been questioned, as mice with diminished astrocytic Ca2+ signaling show normal sensory hyperemia. We addressed this controversy by imaging cortical vasculature while optogenetically elevating astrocyte Ca2+ in a novel transgenic mouse line, expressing Opto-Gq-type GPCR Optoα1AR (Astro-Optoα1AR) in astrocytes. Blue light illumination on the surface of the somatosensory cortex induced Ca2+ elevations in cortical astrocytes and their endfeet in mice under anesthesia. Blood vessel diameter did not change significantly with Optoα1AR-induced Ca2+ elevations in astrocytes, while it was increased by forelimb stimulation. Next, we labeled blood plasma with red fluorescence using AAV8-P3-Alb-mScarlet in Astro-Optoα1AR mice. We were able to identify arterioles that display diameter changes in superficial areas of the somatosensory cortex through the thinned skull. Photo-stimulation of astrocytes in the cortical area did not result in noticeable changes in the arteriole diameters compared with their background strain C57BL/6. Together, compelling evidence for astrocytic Gq pathway-induced vasodiameter changes was not observed. Our results support the notion that short-term (2+ signaling.
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- 2023
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13. A cortical cell ensemble in the posterior parietal cortex controls past experience-dependent memory updating
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Akinobu Suzuki, Sakurako Kosugi, Emi Murayama, Eri Sasakawa, Noriaki Ohkawa, Ayumu Konno, Hirokazu Hirai, and Kaoru Inokuchi
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Science - Abstract
Animals refer to related past experiences when processing sensory inputs. The authors show that a cellular ensemble in the posterior parietal cortex that is activated during past experience mediates an interaction between past and current information to update memory through a circuit including the anterior cingulate cortex.
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- 2022
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14. Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice.
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Kasane Yaguchi, Mitsue Hagihara, Ayumu Konno, Hirokazu Hirai, Hiroko Yukinaga, and Kazunari Miyamichi
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Medicine ,Science - Abstract
Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother-pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother-pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions.
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- 2023
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15. Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation
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Takeshi Tokuyama, Hideki Uosaki, Ayumu Sugiura, Gen Nishitai, Keisuke Takeda, Shun Nagashima, Isshin Shiiba, Naoki Ito, Taku Amo, Satoshi Mohri, Akiyuki Nishimura, Motohiro Nishida, Ayumu Konno, Hirokazu Hirai, Satoshi Ishido, Takahiro Yoshizawa, Takayuki Shindo, Shingo Takada, Shintaro Kinugawa, Ryoko Inatome, and Shigeru Yanagi
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Physiology ,Cellular physiology ,Molecular biology ,Developmental biology ,Science - Abstract
Summary: Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.
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- 2022
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16. Intestinal immunomodulatory activity of indigestible glucan in mice and its utilization by intestinal bacteria in vitro
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Ayumu Horinouchi, Hirokazu Hirai, Rika Hirano, Shin Kurihara, Hiroki Takagi, and Kenji Matsumoto
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Indigestible glucan ,Intestinal IgA ,Mucin ,Animal experiment ,Intestinal bacteria ,Prebiotics ,Nutrition. Foods and food supply ,TX341-641 - Abstract
The functional properties of indigestible glucan (IG), a newly developed water-soluble glucose polymer, on intestinal health were investigated. Of high amylose cornstarch, fructooligosaccharides, and IG, IG showed the strongest intestinal IgA induction during the 5-week experiment with BALB/cA mice (3% per diet). Next, separated IG samples, degree of polymerization (DP) ≥ 5 (High-IG) and DP ≤ 5 (Low-IG) fractions, were fed to BALB/cA mice for 9 weeks (3% per diet). High-IG consumption immediately increased the fecal IgA, whereas Low-IG consumption gradually increased it. Fecal mucin was significantly increased in the High-IG group but not in the Low-IG group. To identify utilization of High-IG and Low-IG by intestinal bacteria, in vitro assays were performed using 57 human gut bacterial strains. Few bacteria utilized High-IG, whereas more bacteria, particularly Bifidobacterium strains, utilized Low-IG. These results indicate that IG ingestion benefits the intestinal environment via strengthening the intestinal barrier function and prebiotic effects.
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- 2021
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17. Indirect Negative Effect of Mutant Ataxin-1 on Short- and Long-Term Synaptic Plasticity in Mouse Models of Spinocerebellar Ataxia Type 1
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Anton N. Shuvaev, Olga S. Belozor, Oleg I. Mozhei, Andrey N. Shuvaev, Yana V. Fritsler, Elena D. Khilazheva, Angelina I. Mosyagina, Hirokazu Hirai, Anja G. Teschemacher, and Sergey Kasparov
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spinocerebellar ataxia type 1 ,PPF ,PPD ,DSE ,LTD ,synaptic plasticity ,Cytology ,QH573-671 - Abstract
Spinocerebellar ataxia type 1 (SCA1) is an intractable progressive neurodegenerative disease that leads to a range of movement and motor defects and is eventually lethal. Purkinje cells (PC) are typically the first to show signs of degeneration. SCA1 is caused by an expansion of the polyglutamine tract in the ATXN1 gene and the subsequent buildup of mutant Ataxin-1 protein. In addition to its toxicity, mutant Ataxin-1 protein interferes with gene expression and signal transduction in cells. Recently, it is evident that ATXN1 is not only expressed in neurons but also in glia, however, it is unclear the extent to which either contributes to the overall pathology of SCA1. There are various ways to model SCA1 in mice. Here, functional deficits at cerebellar synapses were investigated in two mouse models of SCA1 in which mutant ATXN1 is either nonspecifically expressed in all cell types of the cerebellum (SCA1 knock-in (KI)), or specifically in Bergmann glia with lentiviral vectors expressing mutant ATXN1 under the control of the astrocyte-specific GFAP promoter. We report impairment of motor performance in both SCA1 models. In both cases, prominent signs of astrocytosis were found using immunohistochemistry. Electrophysiological experiments revealed alteration of presynaptic plasticity at synapses between parallel fibers and PCs, and climbing fibers and PCs in SCA1 KI mice, which is not observed in animals expressing mutant ATXN1 solely in Bergmann glia. In contrast, short- and long-term synaptic plasticity was affected in both SCA1 KI mice and glia-targeted SCA1 mice. Thus, non-neuronal mechanisms may underlie some aspects of SCA1 pathology in the cerebellum. By combining the outcomes of our current work with our previous data from the B05 SCA1 model, we further our understanding of the mechanisms of SCA1.
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- 2022
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18. D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation
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Erika Ueda, Tomoko Ohta, Ayumu Konno, Hirokazu Hirai, Yuki Kurauchi, Hiroshi Katsuki, and Takahiro Seki
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aromatic-turmerone ,dopaminergic neurons ,microglia ,Nrf2 ,Cytology ,QH573-671 - Abstract
Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.
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- 2022
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19. 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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20. Development of Superconducting Cable and Coated Conductors for Electric Propulsion System of Airplane
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Teruo Izumi, Kei Shiohara, Takato Machi, Akira Ibi, Koichi Nakaoka, Hishiro Hirose, Kazuhisa Adachi, Tatsuhisa Nakanishi, Michio Sato, Yuji Aoki, Yasuo Takahashi, Masashi Miura, Hirokazu Hirai, Msayuki Konno, and Masataka Iwakuma
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Electrical and Electronic Engineering ,Condensed Matter Physics ,Electronic, Optical and Magnetic Materials - Published
- 2023
21. 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.
- Published
- 2022
22. Electrophysiological and Imaging Analysis of GFP-Tagged Protein Kinase C γ Translocation in Cerebellar Purkinje Cells
- Author
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Hirokazu Hirai, Yuuki Fukai, Ayumu Konno, and Nobutake Hosoi
- Subjects
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
- Published
- 2022
23. Development and Optimization of a Multilayer Rat Purkinje Neuron Culture
- Author
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Ida Margrethe Uggerud, Torbjørn Kråkenes, Hirokazu Hirai, Christian Alexander Vedeler, and Manja Schubert
- Subjects
Neurology ,Neurology (clinical) - Abstract
Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.
- Published
- 2023
24. Long-term depression–inductive stimulation causes long-term potentiation in mouse Purkinje cells with a mutant thyroid hormone receptor
- Author
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Ayane Ninomiya, Izuki Amano, Michifumi Kokubo, Yusuke Takatsuru, Sumiyasu Ishii, Hirokazu Hirai, Nobutake Hosoi, and Noriyuki Koibuchi
- Subjects
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.
- Published
- 2022
25. Liver-secreted fluorescent blood plasma markers enable chronic imaging of microcirculation
- Author
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Xiaowen Wang, Christine Delle, Antonis Asiminas, Sonam Akther, Marta Vittani, Peter Brøgger, Peter Kusk, Camilla Trang Vo, Ayumu Konno, Hirokazu Hirai, Masahiro Fukuda, Pia Weikop, Steven A Goldman, Maiken Nedergaard, and Hajime Hirase
- Abstract
Studying blood microcirculation is vital for gaining insights into vascular diseases. Acute administration of fluorescent tracers is currently used for deep tissue blood flow imaging. This is invasive, and the plasma fluorescence decreases within an hour of administration. We report a novel approach for the longitudinal study of vasculature. Using a single systemic administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to label the blood in mice. All segments of the vasculature in brain and peripheral tissue are observable by two-photon microscopy within two weeks of vector administration. This approach allows for observation of circulation without the need for repeated administration for several months. We demonstrate the chronic assessment of vascular functions at micro-and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease.
- Published
- 2022
26. Development and optimization of a rat Purkinje neuron culture to study paraneoplastic cerebellar degeneration
- Author
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Ida Margrethe Uggerud, Torbjorn Krakenes, Hirokazu Hirai, Christian Alexander Vedeler, and Manja Schubert
- Subjects
nervous system - Abstract
Background: Elucidation of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that replicate in vivo features. Methods: We developed a refined protocol for generation of age-dependent, well-developed, and synaptically active rat Purkinje neurons in a multilayer cell network culture that can be used to study neurodegenerative diseases such as paraneoplastic cerebellar degeneration (PCD). Result: Independent of the age of the tissue from which the culture was driven, the properties of the generated rat Purkinje neuron culture are complex and robust, allowing great experimental flexibility. Using our model, we found that the application of Yo antibodies, which are associated with PCD, alters the structure of the dendritic arbour of cultured Purkinje neurons within 48h. The numbers of dendrites per branch-order, the branch-order in itself, and the dendritic length were reduced by treatment with anti-Yo. Conclusion: The here presented in vitro model is flexible and can be used to investigate disease mechanisms that disturb Purkinje neuron function and communication in a multilayer network. It proved that anti-Yo has a functional role in the pathogenesis of PCD.
- Published
- 2022
27. Protein kinase Cγ in cerebellar Purkinje cells regulates Ca 2+ -activated large-conductance K + channels and motor coordination
- Author
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Masashi Watanave, Nobutaka Takahashi, Nobutake Hosoi, Ayumu Konno, Hikaru Yamamoto, Hiroyuki Yasui, Mika Kawachi, Takuro Horii, Yasunori Matsuzaki, Izuho Hatada, and Hirokazu Hirai
- Subjects
Multidisciplinary - Abstract
Significance The cerebellum, the site where protein kinase C (PKC) was discovered, contains the highest amount of PKCγ in the central nervous system. PKCγ in the cerebellum is exclusively confined to Purkinje cells (PCs), sole outputs from the cerebellar cortex. Systemic PKCγ-knockout mice show impaired motor coordination; however, the cause of motor defects remains unknown. Here we show that activation of PKCγ suppresses the Ca 2+ -activated large-conductance K + (BK) channels located along the PC dendrites. A consequential increase in the membrane resistance attenuates electrical signal decay during propagation, resulting in an altered complex spike waveform. Our results suggest that synaptically activated PKCγ in PCs plays a critical role in motor coordination by negative modulation of BK currents.
- Published
- 2022
28. Development and Optimization of A High-Throughput 3D Rat Purkinje Neuron Culture to Study Paraneoplastic Cerebellar Degeneration
- Author
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Ida Margrethe Uggerud, Torbjorn Krakenes, Hirokazu Hirai, Christian Alexander Vedeler, and Manja Schubert
- Abstract
Improved understanding of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that faithfully replicate in vivo features. Here, we present a refined protocol for generating age-dependent, well-developed and synaptically active rat Purkinje neurons in a 3D cell network culture which are responsive to a disease inducer. Using our model, we found that the application of autoantibody Yo, a paraneoplastic cerebellar degeneration (PCD) inducer, alters the structure of the dendritic arbour of cultured Purkinje neurons. The numbers of dendrites per branch-order, the branch-order in itself and the dendritic length were reduced by anti-Yo, proving a functional role for anti-Yo in the pathogenesis of PCD. Our new ex-vivo model is flexible and can be used to investigate disease mechanisms that disturb Purkinje neuron function and communication in 3D. Since it is possible to use the approach in a multi-well format, this method also has high-throughput screening potential.
- Published
- 2022
29. Liver-secreted fluorescent blood plasma markers enable chronic imaging of the microcirculation
- Author
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Xiaowen Wang, Christine Delle, Antonis Asiminas, Sonam Akther, Marta Vittani, Peter Brøgger, Peter Kusk, Camilla Trang Vo, Tessa Radovanovic, Ayumu Konno, Hirokazu Hirai, Masahiro Fukuda, Pia Weikop, Steven A. Goldman, Maiken Nedergaard, and Hajime Hirase
- Subjects
Genetics ,Radiology, Nuclear Medicine and imaging ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,Computer Science Applications ,Biotechnology - Abstract
Studying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.
- Published
- 2022
30. Protein kinase Cγ in cerebellar Purkinje cells regulates Ca
- Author
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Masashi, Watanave, Nobutaka, Takahashi, Nobutake, Hosoi, Ayumu, Konno, Hikaru, Yamamoto, Hiroyuki, Yasui, Mika, Kawachi, Takuro, Horii, Yasunori, Matsuzaki, Izuho, Hatada, and Hirokazu, Hirai
- Subjects
Mice, Knockout ,Mice ,Potassium Channels, Calcium-Activated ,Purkinje Cells ,Animals ,Calcium Signaling ,Genetic Therapy ,Motor Activity ,Synaptic Potentials ,Gene Deletion ,Protein Kinase C - Abstract
The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature
- Published
- 2021
31. Abstract 10460: Urinary Fatty Acid Binding Protein 1 is a Biomarker for Impaired Proximal Tubular Protein Reabsorption and is Synergistically Enhanced by Concurrent Liver Injury
- Author
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Ryo Kawakami, Miki Matsui, Ayumu Konno, Ryosuke Kaneko, Hiroaki Sunaga, Hirofumi Hanaoka, Masaru Obokata, Hiroki Matsui, Norimichi Koitabashi, Hirokazu Hirai, Masahiko Kurabayashi, and Tatsuya Iso
- Subjects
Physiology (medical) ,Cardiology and Cardiovascular Medicine - Abstract
Introduction: Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Methods and Results: Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or PTEC-specific manner, or both. Upon diphtheria toxin (DT) administration, massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Conclusions: Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function.
- Published
- 2021
32. Cell-type-specific transduction using blood-brain-barrier-penetrating AAV vector
- Author
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Ayumu Konno and Hirokazu Hirai
- Subjects
Applied Mathematics ,General Mathematics - Published
- 2022
33. Establishment of a mouse model of spinocerebellar ataxia type 34
- Author
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Morikawa Yuri, Takahiro Seki, Ayumu Konno, Hirokazu Hirai, Yuki Kurauchi, and Hiroshi Katsuki
- Subjects
Applied Mathematics ,General Mathematics - Published
- 2022
34. Impaired dendritic development is a common phenotype observed in primary cultured Purkinje cells expressing various SCA-causing proteins
- Author
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Ueda Erika, Ayumu Konno, Hirokazu Hirai, Yuki Kurauchi, Hiroshi Katsuki, and Takahiro Seki
- Subjects
Applied Mathematics ,General Mathematics - Published
- 2022
35. Protein kinase Cγ in cerebellar Purkinje cells regulates Ca2+-activated large-conductance K+ channels and motor coordination.
- Author
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Masashi Watanave, Nobutaka Takahashi, Nobutake Hosoi, Ayumu Konno, Hikaru Yamamoto, Hiroyuki Yasui, Mika Kawachi, Takuro Horii, Yasunori Matsuzaki, Izuho Hatada, and Hirokazu Hirai
- Subjects
MOTOR ability ,PURKINJE cells ,PROTEIN kinases ,PROTEIN kinase C ,CENTRAL nervous system ,SOYBEAN meal ,COMMERCIAL products - Abstract
The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ- knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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