Publications by authors named "Takahisa Furukawa"

103 Publications

Post-translational modification enzymes as key regulators of ciliary protein trafficking.

J Biochem 2021 Mar 3. Epub 2021 Mar 3.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan.

Primary cilia are evolutionarily conserved microtubule-based organelles that protrude from the surface of almost all cell types and decode a variety of extracellular stimuli. Ciliary dysfunction causes human diseases named ciliopathies, which span a wide range of symptoms, such as developmental and sensory abnormalities. The assembly, disassembly, maintenance, and function of cilia rely on protein transport systems including intraflagellar transport (IFT) and lipidated protein intraflagellar targeting (LIFT). IFT is coordinated by three multisubunit protein complexes with molecular motors along the ciliary axoneme, while LIFT is mediated by specific chaperones that directly recognize lipid chains. Recently, it has become clear that several post-translational modification enzymes play crucial roles in the regulation of IFT and LIFT. Here we review our current understanding of the roles of these post-translational modification enzymes in the regulation of ciliary protein trafficking as well as their regulatory mechanisms, physiological significance, and involvement in human diseases.
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http://dx.doi.org/10.1093/jb/mvab024DOI Listing
March 2021

Functional analysis of Samd11, a retinal photoreceptor PRC1 component, in establishing rod photoreceptor identity.

Sci Rep 2021 Feb 18;11(1):4180. Epub 2021 Feb 18.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Establishing correct neuronal cell identity is essential to build intricate neural tissue architecture and acquire precise neural function during vertebrate development. While it is known that transcription factors play important roles in retinal cell differentiation, the contribution of epigenetic factors to establishing cell identity during retinal development remains unclear. We previously reported that Samd7, a rod photoreceptor cell-specific sterile alpha motif (SAM) domain protein, functions as a Polycomb repressive complex 1 component (PRC1) that is essential for establishing rod identity. In the current study, we analyzed a functional role of Samd11, another photoreceptor-enriched SAM-domain protein, in photoreceptor differentiation and maturation. We observed that Samd11 interacts with Phc2 and Samd7, suggesting that Samd11 is a component of PRC1 in photoreceptor cells. We generated Samd11-null allele and established Samd7/11 double knock-out (DKO) mouse. The Samd7/11 DKO retina exhibits shortened photoreceptor outer segments by electron microscopy analysis. Microarray analysis revealed that Samd7/11 DKO up-regulated more retinal genes than Samd7 alone, partial functional redundancy of Samd7 and Samd11. Taken together, the current results suggest that Samd7 and Samd11 are PRC1 components and that Samd7 is the major regulator while Samd11 is an accessory factor used for the establishment of precise rod photoreceptor identity.
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http://dx.doi.org/10.1038/s41598-021-83781-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892874PMC
February 2021

Influence of Aging on the Retina and Visual Motion Processing for Optokinetic Responses in Mice.

Front Neurosci 2020 1;14:586013. Epub 2020 Dec 1.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan.

The decline in visual function due to normal aging impacts various aspects of our daily lives. Previous reports suggest that the aging retina exhibits mislocalization of photoreceptor terminals and reduced amplitudes of scotopic and photopic electroretinogram (ERG) responses in mice. These abnormalities are thought to contribute to age-related visual impairment; however, the extent to which visual function is impaired by aging at the organismal level is unclear. In the present study, we focus on the age-related changes of the optokinetic responses (OKRs) in visual processing. Moreover, we investigated the initial and late phases of the OKRs in young adult (2-3 months old) and aging mice (21-24 months old). The initial phase was evaluated by measuring the open-loop eye velocity of OKRs using sinusoidal grating patterns of various spatial frequencies (SFs) and moving at various temporal frequencies (TFs) for 0.5 s. The aging mice exhibited initial OKRs with a spatiotemporal frequency tuning that was slightly different from those in young adult mice. The late-phase OKRs were investigated by measuring the slow-phase velocity of the optokinetic nystagmus evoked by sinusoidal gratings of various spatiotemporal frequencies moving for 30 s. We found that optimal SF and TF in the normal aging mice are both reduced compared with those in young adult mice. In addition, we measured the OKRs of null () mice, in which mislocalization of photoreceptor terminals is observed even at the young adult stage. We found that the late phase OKR was significantly impaired in mice, which exhibit significantly reduced SF and TF compared with control mice. These OKR abnormalities observed in mice resemble the abnormalities found in normal aging mice. This finding suggests that these mice can be useful mouse models for studying the aging of the retinal tissue and declining visual function. Taken together, the current study demonstrates that normal aging deteriorates to visual motion processing for both the initial and late phases of OKRs. Moreover, it implies that the abnormalities of the visual function in the normal aging mice are at least partly due to mislocalization of photoreceptor synapses.
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http://dx.doi.org/10.3389/fnins.2020.586013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736246PMC
December 2020

The potential role of Arhgef33 RhoGEF in foveal development in the zebra finch retina.

Sci Rep 2020 12 8;10(1):21450. Epub 2020 Dec 8.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

The fovea is a pit formed in the center of the retina that enables high-acuity vision in certain vertebrate species. While formation of the fovea fascinates many researchers, the molecular mechanisms underlying foveal development are poorly understood. In the current study, we histologically investigated foveal development in zebra finch (Taeniopygia guttata) and found that foveal pit formation begins just before post-hatch day 14 (P14). We next performed RNA-seq analysis to compare gene expression profiles between the central (foveal and parafoveal) and peripheral retina in zebra finch at P14. We found that the Arhgef33 expression is enriched in the middle layer of the inner nuclear layer at the parafovea, suggesting that Arhgef33 is dominantly expressed in Müller glial cells in the developing parafovea. We then performed a pull-down assay using Rhotekin-RBD and observed GEF activity of Arhgef33 against RhoA. We found that overexpression of Arhgef33 in HEK293 cells induces cell contraction and that Arhgef33 expression inhibits neurite extension in Neuro 2A cells, which is partially recovered by a Rho-kinase (ROCK) inhibitor. Taken together, we used zebra finch as a model animal to investigate foveal development and identified Arhgef33 as a candidate protein possibly involved in foveal development through modulating RhoA activity.
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http://dx.doi.org/10.1038/s41598-020-78452-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722920PMC
December 2020

Inhibition of HDAC increases BDNF expression and promotes neuronal rewiring and functional recovery after brain injury.

Cell Death Dis 2020 08 18;11(8):655. Epub 2020 Aug 18.

Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka, 565-0871, Japan.

Brain injury causes serious motor, sensory, and cognitive disabilities. Accumulating evidence has demonstrated that histone deacetylase (HDAC) inhibitors exert neuroprotective effects against various insults to the central nervous system (CNS). In this study, we investigated the effects of the HDAC inhibition on the expression of brain-derived neurotrophic factor (BDNF) and functional recovery after traumatic brain injury (TBI) in mice. Administration of class I HDAC inhibitor increased the number of synaptic boutons in rewiring corticospinal fibers and improved the recovery of motor functions after TBI. Immunohistochemistry results showed that HDAC2 is mainly expressed in the neurons of the mouse spinal cord under normal conditions. After TBI, HDAC2 expression was increased in the spinal cord after 35 days, whereas BDNF expression was decreased after 42 days. Administration of CI-994 increased BDNF expression after TBI. Knockdown of HDAC2 elevated H4K5ac enrichment at the BDNF promoter, which was decreased following TBI. Together, our findings suggest that HDAC inhibition increases expression of neurotrophic factors, and promote neuronal rewiring and functional recovery following TBI.
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http://dx.doi.org/10.1038/s41419-020-02897-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434917PMC
August 2020

Anterograde trafficking of ciliary MAP kinase-like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking.

J Biol Chem 2020 09 29;295(38):13363-13376. Epub 2020 Jul 29.

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan. Electronic address:

ICK (also known as CILK1) is a mitogen-activated protein kinase-like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)-B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein-coupled receptors. In addition, we found that in knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.
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http://dx.doi.org/10.1074/jbc.RA120.014142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504932PMC
September 2020

The Genetic Basis of Morphological Diversity in Domesticated Goldfish.

Curr Biol 2020 Jun 8;30(12):2260-2274.e6. Epub 2020 May 8.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.

Although domesticated goldfish strains exhibit highly diversified phenotypes in morphology, the genetic basis underlying these phenotypes is poorly understood. Here, based on analysis of transposable elements in the allotetraploid goldfish genome, we found that its two subgenomes have evolved asymmetrically since a whole-genome duplication event in the ancestor of goldfish and common carp. We conducted whole-genome sequencing of 27 domesticated goldfish strains and wild goldfish. We identified more than 60 million genetic variations and established a population genetic structure of major goldfish strains. Genome-wide association studies and analysis of strain-specific variants revealed genetic loci associated with several goldfish phenotypes, including dorsal fin loss, long-tail, telescope-eye, albinism, and heart-shaped tail. Our results suggest that accumulated mutations in the asymmetrically evolved subgenomes led to generation of diverse phenotypes in the goldfish domestication history. This study is a key resource for understanding the genetic basis of phenotypic diversity among goldfish strains.
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http://dx.doi.org/10.1016/j.cub.2020.04.034DOI Listing
June 2020

Improvement of reduced electroretinographic responses in thymoma-associated retinopathy: a case report and literature review.

Doc Ophthalmol 2020 10 1;141(2):195-204. Epub 2020 Apr 1.

Department of Ophthalmology, The Jikei University School of Medicine, Tokyo, Japan.

Purpose: To report a patient with thymoma-associated retinopathy presenting as having a good visual prognosis.

Methods: Case report and literature review.

Case Report: A 42-year-old female patient was referred to our hospital for complaints of sudden visual-field defects bilaterally. Decimal corrected visual acuity (VA) was 1.5 and 1.2 in the right (RE) and left eyes (LE), respectively. Fundus autofluorescence revealed hyper-autofluorescence from the posterior pole to mid-peripheral retina in both eyes. Full-field electroretinography (ERG) amplitudes were reduced to 20-50% and 30-50% of our controls for the scotopic and photopic conditions, respectively. A systemic examination revealed the presence of thymoma, and the patient underwent thymectomy and immunosuppression therapies. Immunohistochemical analysis using the patient's serum showed immunolabeling on the photoreceptor inner segment and outer plexiform layer in the monkey retina. Two years later, VA remained at 1.5 and 1.2 in RE and LE. ERG amplitudes improved to 30-60% of the controls for the scotopic conditions. However, photopic ERG showed no remarkable change.

Conclusions: To our knowledge, improvement of reduced rod-mediated ERG responses has not been described in seven previously reported patients with thymoma-associated retinopathy. The good visual prognosis of our patient may be associated with well-timed intervention.
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http://dx.doi.org/10.1007/s10633-020-09764-1DOI Listing
October 2020

Origin and evolution of the Rax homeobox gene by comprehensive evolutionary analysis.

FEBS Open Bio 2020 04 19;10(4):657-673. Epub 2020 Mar 19.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Suita, Japan.

Rax is one of the key transcription factors crucial for vertebrate eye development. In this study, we conducted comprehensive evolutionary analysis of Rax. We found that Bilateria and Cnidaria possess Rax, but Placozoa, Porifera, and Ctenophora do not, implying that the origin of the Rax gene dates back to the common ancestor of Cnidaria and Bilateria. The results of molecular phylogenetic and synteny analyses on Rax loci between jawed and jawless vertebrates indicate that segmental duplication of the Rax locus occurred in an early common ancestor of jawed vertebrates, resulting in two Rax paralogs in jawed vertebrates, Rax and Rax2. By analyzing 86 mammalian genomes from all four major groups of mammals, we found that at least five independent Rax2 gene loss events occurred in mammals. This study may provide novel insights into the evolution of the eye.
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http://dx.doi.org/10.1002/2211-5463.12832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137802PMC
April 2020

GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

Gastroenterology 2020 05 4;158(6):1650-1666.e15. Epub 2020 Feb 4.

Department of Gastroenterology, Graduate school of Medicine, the University of Tokyo, Tokyo, Japan.

Background & Aims: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells.

Methods: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-Kras, LSL-Kras, and LSL-Hras mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition.

Results: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30 chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion.

Conclusions: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.
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http://dx.doi.org/10.1053/j.gastro.2020.01.046DOI Listing
May 2020

Retinal ON and OFF pathways contribute to initial optokinetic responses with different temporal characteristics.

Eur J Neurosci 2020 08 25;52(4):3160-3165. Epub 2020 Feb 25.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan.

Visual information in the retina is processed via two pathways: ON and OFF pathways that originate from ON and OFF bipolar cells. The differences in the receptors that mediate signal transmission from photoreceptors imply that the response speed to light signals differs between ON and OFF pathways. We studied the initial optokinetic responses (OKRs) of mice using two-frame motion stimuli presented with interstimulus intervals (ISIs) to understand functional difference of these pathways. When two successive image frames were presented with an ISI, observers often perceived motion in the opposite direction of the actual shift. This directional reversal results from the biphasic nature of the temporal filters in visual systems whose characteristics can be estimated from the dependence on ISIs. We examined the dependence on ISIs in the OKRs of TRPM1 mice, whose ON bipolar cells are dysfunctional, as well as in those of wild-type control mice. Wild type and TRPM1 mice showed comparable OKRs in the veridical direction when no ISI was present. Both types of mice showed OKRs that decreased and eventually reversed as the ISI increased, but with a directional reversal at a shorter ISI in TRPM1 than wild-type mice. In addition, the temporal filters of TRPM1 mice estimated from dependence on ISIs were tuned for higher frequencies, suggesting that compared with wild-type mice, the visual system of TRPM1 mice responds to light signals with faster dynamics. We conclude that the ON and OFF pathways contribute to initial OKRs by providing visual signals processed with different temporal resolutions.
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http://dx.doi.org/10.1111/ejn.14697DOI Listing
August 2020

Functional and Evolutionary Diversification of Otx2 and Crx in Vertebrate Retinal Photoreceptor and Bipolar Cell Development.

Cell Rep 2020 01;30(3):658-671.e5

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Otx family homeoproteins Otx2 and Crx are expressed in photoreceptor precursor cells and bind to the common DNA-binding consensus sequence, but these two proteins have distinct functions in retinal development. To examine the functional substitutability of Otx2 and Crx, we generate knockin mouse lines in which Crx is replaced by Otx2 and vice versa. We find that Otx2 and Crx cannot be substituted in photoreceptor development. Subsequently, we investigate the function of Otx2 in photoreceptor and bipolar cell development. High Otx2 levels induce photoreceptor cell fate but not bipolar cell fate, whereas reduced Otx2 expression impairs bipolar cell maturation and survival. Furthermore, we identify Otx2 and Crx in the lamprey genome by using synteny analysis, suggesting that the last common ancestor of vertebrates possesses both Otx2 and Crx. We find that the retinal Otx2 expression pattern is different between lampreys and mice, suggesting that neofunctionalization of Otx2 occurred in the jawed vertebrate lineage.
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http://dx.doi.org/10.1016/j.celrep.2019.12.072DOI Listing
January 2020

Cul3-Klhl18 ubiquitin ligase modulates rod transducin translocation during light-dark adaptation.

EMBO J 2019 12 7;38(23):e101409. Epub 2019 Nov 7.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan.

Adaptation is a general feature of sensory systems. In rod photoreceptors, light-dependent transducin translocation and Ca homeostasis are involved in light/dark adaptation and prevention of cell damage by light. However, the underlying regulatory mechanisms remain unclear. Here, we identify mammalian Cul3-Klhl18 ubiquitin ligase as a transducin translocation modulator during light/dark adaptation. Under dark conditions, Klhl18 mice exhibited decreased rod light responses and subcellular localization of the transducin α-subunit (Tα), similar to that observed in light-adapted Klhl18 mice. Cul3-Klhl18 promoted ubiquitination and degradation of Unc119, a rod Tα-interacting protein. Unc119 overexpression phenocopied Tα mislocalization observed in Klhl18 mice. Klhl18 weakly recognized casein kinase-2-phosphorylated Unc119 protein, which is dephosphorylated by Ca -dependent phosphatase calcineurin. Calcineurin inhibition increased Unc119 expression and Tα mislocalization in rods. These results suggest that Cul3-Klhl18 modulates rod Tα translocation during light/dark adaptation through Unc119 ubiquitination, which is affected by phosphorylation. Notably, inactivation of the Cul3-Klhl18 ligase and calcineurin inhibitors FK506 and cyclosporine A that are known immunosuppressant drugs repressed light-induced photoreceptor damage, suggesting potential therapeutic targets.
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http://dx.doi.org/10.15252/embj.2018101409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885741PMC
December 2019

Molecular mechanisms underlying selective synapse formation of vertebrate retinal photoreceptor cells.

Cell Mol Life Sci 2020 Apr 4;77(7):1251-1266. Epub 2019 Oct 4.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

In vertebrate central nervous systems (CNSs), highly diverse neurons are selectively connected via synapses, which are essential for building an intricate neural network. The vertebrate retina is part of the CNS and is comprised of a distinct laminar organization, which serves as a good model system to study developmental synapse formation mechanisms. In the retina outer plexiform layer, rods and cones, two types of photoreceptor cells, elaborate selective synaptic contacts with ON- and/or OFF-bipolar cell terminals as well as with horizontal cell terminals. In the mouse retina, three photoreceptor subtypes and at least 15 bipolar subtypes exist. Previous and recent studies have significantly progressed our understanding of how selective synapse formation, between specific subtypes of photoreceptor and bipolar cells, is designed at the molecular level. In the ON pathway, photoreceptor-derived secreted and transmembrane proteins directly interact in trans with the GRM6 (mGluR6) complex, which is localized to ON-bipolar cell dendritic terminals, leading to selective synapse formation. Here, we review our current understanding of the key factors and mechanisms underlying selective synapse formation of photoreceptor cells with bipolar and horizontal cells in the retina. In addition, we describe how defects/mutations of the molecules involved in photoreceptor synapse formation are associated with human retinal diseases and visual disorders.
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http://dx.doi.org/10.1007/s00018-019-03324-wDOI Listing
April 2020

miR-124 dosage regulates prefrontal cortex function by dopaminergic modulation.

Sci Rep 2019 03 5;9(1):3445. Epub 2019 Mar 5.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

MicroRNA-124 (miR-124) is evolutionarily highly conserved among species and one of the most abundantly expressed miRNAs in the developing and mature central nervous system (CNS). Previous studies reported that miR-124 plays a role in CNS development, such as neuronal differentiation, maturation, and survival. However, the role of miR-124 in normal brain function has not yet been revealed. Here, we subjected miR-124-1 mice, to a comprehensive behavioral battery. We found that miR-124-1 mice showed impaired prepulse inhibition (PPI), methamphetamine-induced hyperactivity, and social deficits. Whole cell recordings using prefrontal cortex (PFC) slices showed enhanced synaptic transmission in layer 5 pyramidal cells in the miR-124-1 PFC. Based on the results of behavioral and electrophysiological analysis, we focused on genes involved in the dopaminergic system and identified a significant increase of Drd2 expression level in the miR-124-1 PFC. Overexpression or knockdown of Drd2 in the control or miR-124-1 PFC demonstrates that aberrant Drd2 signaling leads to impaired PPI. Furthermore, we identified that expression of glucocorticoid receptor gene Nr3c1, which enhances Drd2 expression, increased in the miR-124-1 PFC. Taken together, the current study suggests that miR-124 dosage modulates PFC function through repressing the Drd2 pathway, suggesting a critical role of miR-124 in normal PFC function.
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http://dx.doi.org/10.1038/s41598-019-38910-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401137PMC
March 2019

Two-frame apparent motion presented with an inter-stimulus interval reverses optokinetic responses in mice.

Sci Rep 2018 12 13;8(1):17816. Epub 2018 Dec 13.

Department of Integrative Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Two successive image frames presented with a blank inter-stimulus interval (ISI) induce reversals of perceived motion in humans. This illusory effect is a manifestation of the temporal properties of image filters embedded in the visual processing pathway. In the present study, ISI experiments were performed to identify the temporal characteristics of vision underlying optokinetic responses (OKRs) in mice. These responses are thought to be mediated by subcortical visual processing. OKRs of C57BL/6 J mice, induced by a 1/4-wavelength shift of a square-wave grating presented with and without an ISI were recorded. When a 1/4-wavelength shift was presented without, or with shorter ISIs (≤106.7 ms), OKRs were induced in the direction of the shift, with progressively decreasing amplitude as the ISI increased. However, when ISIs were 213.3 ms or longer, OKR direction reversed. Similar dependence on ISIs was also obtained using a sinusoidal grating. We subsequently quantitatively estimated temporal filters based on the ISI effects. We found that filters with biphasic impulse response functions could reproduce the ISI and temporal frequency dependence of the mouse OKR. Comparison with human psychophysics and behaviors suggests that mouse vision has more sluggish response dynamics to light signals than that of humans.
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http://dx.doi.org/10.1038/s41598-018-36260-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292883PMC
December 2018

Compound heterozygous splice site variants in the SCLT1 gene highlight an additional candidate locus for Senior-Løken syndrome.

Sci Rep 2018 11 13;8(1):16733. Epub 2018 Nov 13.

Department of Ophthalmology, The Jikei University School of Medicine, Tokyo, Japan.

Senior Løken syndrome (SLS) is a heterogeneous disorder characterized by severe retinal degenerations and juvenile-onset nephronophthisis. Genetic variants in ten different genes have been reported as the causes of SLS. Clinical evaluation of a patient with SLS and her unaffected parents revealed that the patient had infantile-onset retinal dystrophy and juvenile-onset nephronophthisis. Other systemic abnormalities included hepatic dysfunction, megacystis, mild learning disability, autism, obesity, and hyperinsulinemia. Whole-exome sequencing identified compound heterozygous SCLT1 variants (c.1218 + 3insT and c.1631A > G) in the patient. The unaffected parents were heterozygous for each variant. Transcript analysis using reverse transcription PCR demonstrated that the c.1218 + 3insT variant leads to exon 14 skipping (p.V383_M406del), while the other variant (c.1631A > G) primarily leads to exon 17 skipping (p.D480EfsX11) as well as minor amounts of two transcripts (6 bps deletion in the last of exon 17 [p.V543_K544del] and exons 17 and 18 skipping [p.D480E, S481_K610del]). Immunohistochemical analysis demonstrated that the Sclt1 protein was localized to the distal appendage of the photoreceptor basal body, indicating a ciliary protein. In conclusion, we identified compound heterozygous splice site variants of SCLT1 in a patient with a new form of ciliopathies that exhibits clinical features of SLS.
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http://dx.doi.org/10.1038/s41598-018-35152-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233217PMC
November 2018

Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors.

Cell Rep 2018 10;25(1):130-145.e5

Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA. Electronic address:

Establishing synaptic contacts between neurons is paramount for nervous system function. This process involves transsynaptic interactions between a host of cell adhesion molecules that act in cooperation with the proteins of the extracellular matrix to specify unique physiological properties of individual synaptic connections. However, understanding of the molecular mechanisms that generate functional diversity in an input-specific fashion is limited. In this study, we identify that major components of the extracellular matrix proteins present in the synaptic cleft-members of the heparan sulfate proteoglycan (HSPG) family-associate with the GPR158/179 group of orphan receptors. Using the mammalian retina as a model system, we demonstrate that the HSPG member Pikachurin, released by photoreceptors, recruits a key post-synaptic signaling complex of downstream ON-bipolar neurons in coordination with the pre-synaptic dystroglycan glycoprotein complex. We further demonstrate that this transsynaptic assembly plays an essential role in synaptic transmission of photoreceptor signals.
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http://dx.doi.org/10.1016/j.celrep.2018.08.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203450PMC
October 2018

Enriched expression of the ciliopathy gene Ick in cell proliferating regions of adult mice.

Gene Expr Patterns 2018 09 7;29:18-23. Epub 2018 Apr 7.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address:

Cilia are essential for sensory and motile functions across species. In humans, ciliary dysfunction causes "ciliopathies", which show severe developmental abnormalities in various tissues. Several missense mutations in intestinal cell kinase (ICK) gene lead to endocrine-cerebro-osteodysplasia syndrome or short rib-polydactyly syndrome, lethal recessive developmental ciliopathies. We and others previously reported that Ick-deficient mice exhibit neonatal lethality with developmental defects. Mechanistically, Ick regulates intraflagellar transport and cilia length at ciliary tips. Although Ick plays important roles during mammalian development, roles of Ick at the adult stage are poorly understood. In the current study, we investigated the Ick gene expression in adult mouse tissues. RT-PCR analysis showed that Ick is ubiquitously expressed, with enrichment in the retina, brain, lung, intestine, and reproductive system. In the adult brain, we found that Ick expression is enriched in the walls of the lateral ventricle, in the rostral migratory stream of the olfactory bulb, and in the subgranular zone of the hippocampal dentate gyrus by in situ hybridization analysis. We also observed that Ick staining pattern is similar to pachytene spermatocyte to spermatid markers in the mature testis and to an intestinal stem cell marker in the adult small intestine. These results suggest that Ick is expressed in proliferating regions in the adult mouse brain, testis, and intestine.
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http://dx.doi.org/10.1016/j.gep.2018.04.005DOI Listing
September 2018

Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity.

Cell Rep 2018 03;22(13):3548-3561

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function.
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http://dx.doi.org/10.1016/j.celrep.2018.03.007DOI Listing
March 2018

Metabolic shift induced by systemic activation of T cells in PD-1-deficient mice perturbs brain monoamines and emotional behavior.

Nat Immunol 2017 Dec 23;18(12):1342-1352. Epub 2017 Oct 23.

Laboratory for Mucosal Immunity, Center for Integrative Medical Sciences, RIKEN Yokohama Institute, Yokohama, Japan.

T cells reorganize their metabolic profiles after being activated, but the systemic metabolic effect of sustained activation of the immune system has remained unexplored. Here we report that augmented T cell responses in Pdcd1 mice, which lack the inhibitory receptor PD-1, induced a metabolic serum signature characterized by depletion of amino acids. We found that the depletion of amino acids in serum was due to the accumulation of amino acids in activated Pdcd1 T cells in the lymph nodes. A systemic decrease in tryptophan and tyrosine led to substantial deficiency in the neurotransmitters serotonin and dopamine in the brain, which resulted in behavioral changes dominated by anxiety-like behavior and exacerbated fear responses. Together these data indicate that excessive activation of T cells causes a systemic metabolomic shift with consequences that extend beyond the immune system.
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http://dx.doi.org/10.1038/ni.3867DOI Listing
December 2017

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity.

Proc Natl Acad Sci U S A 2017 09 12;114(39):E8264-E8273. Epub 2017 Sep 12.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan;

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the -deficient retina, showing that deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity.
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http://dx.doi.org/10.1073/pnas.1707021114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625914PMC
September 2017

The TRPM1 Channel Is Required for Development of the Rod ON Bipolar Cell-AII Amacrine Cell Pathway in the Retinal Circuit.

J Neurosci 2017 10 12;37(41):9889-9900. Epub 2017 Sep 12.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University,

Neurotransmission plays an essential role in neural circuit formation in the central nervous system (CNS). Although neurotransmission has been recently clarified as a key modulator of retinal circuit development, the roles of individual synaptic transmissions are not yet fully understood. In the current study, we investigated the role of neurotransmission from photoreceptor cells to ON bipolar cells in development using mutant mouse lines of both sexes in which this transmission is abrogated. We found that deletion of the ON bipolar cation channel TRPM1 results in the abnormal contraction of rod bipolar terminals and a decreased number of their synaptic connections with amacrine cells. In contrast, these histological alterations were not caused by a disruption of total glutamate transmission due to loss of the ON bipolar glutamate receptor mGluR6 or the photoreceptor glutamate transporter VGluT1. In addition, deficiency led to the reduction of total dendritic length, branch numbers, and cell body size in AII amacrine cells. Activated Goα, known to close the TRPM1 channel, interacted with TRPM1 and induced the contraction of rod bipolar terminals. Furthermore, overexpression of Channelrhodopsin-2 partially rescued rod bipolar cell development in the retina, whereas the rescue effect by a constitutively closed form of TRPM1 was lower than that by the native form. Our results suggest that TRPM1 channel opening is essential for rod bipolar pathway establishment in development. Neurotransmission has been recognized recently as a key modulator of retinal circuit development in the CNS. However, the roles of individual synaptic transmissions are not yet fully understood. In the current study, we focused on neurotransmission between rod photoreceptor cells and rod bipolar cells in the retina. We used genetically modified mouse models which abrogate each step of neurotransmission: presynaptic glutamate release, postsynaptic glutamate reception, or transduction channel function. We found that the TRPM1 transduction channel is required for the development of rod bipolar cells and their synaptic formation with subsequent neurons, independently of glutamate transmission. This study advances our understanding of neurotransmission-mediated retinal circuit refinement.
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http://dx.doi.org/10.1523/JNEUROSCI.0824-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596597PMC
October 2017

Versatile functional roles of horizontal cells in the retinal circuit.

Sci Rep 2017 07 17;7(1):5540. Epub 2017 Jul 17.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, JST, CREST, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

In the retinal circuit, environmental light signals are converted into electrical signals that can be decoded properly by the brain. At the first synapse of the visual system, information flow from photoreceptors to bipolar cells is modulated by horizontal cells (HCs), however, their functional contribution to retinal output and individual visual function is not fully understood. In the current study, we investigated functional roles for HCs in retinal ganglion cell (RGC) response properties and optokinetic responses by establishing a HC-depleted mouse line. We observed that HC depletion impairs the antagonistic center-surround receptive field formation of RGCs, supporting a previously reported HC function revealed by pharmacological approaches. In addition, we found that HC loss reduces both the ON and OFF response diversities of RGCs, impairs adjustment of the sensitivity to ambient light at the retinal output level, and alters spatial frequency tuning at an individual level. Taken together, our current study suggests multiple functional aspects of HCs crucial for visual processing.
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http://dx.doi.org/10.1038/s41598-017-05543-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514144PMC
July 2017

A modulatory role of the Rax homeobox gene in mature pineal gland function: Investigating the photoneuroendocrine circadian system of a Rax conditional knockout mouse.

J Neurochem 2017 10 31;143(1):100-111. Epub 2017 Aug 31.

Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The retinal and anterior neural fold homeobox gene (Rax) controls development of the eye and the forebrain. Postnatal expression of Rax in the brain is restricted to the pineal gland, a forebrain structure devoted to melatonin synthesis. The role of Rax in pineal function is unknown. In order to investigate the role of Rax in pineal function while circumventing forebrain abnormalities of the global Rax knockout, we generated an eye and pineal-specific Rax conditional knockout mouse. Deletion of Rax in the pineal gland did not affect morphology of the gland, suggesting that Rax is not essential for pineal gland development. In contrast, deletion of Rax in the eye generated an anophthalmic phenotype. In addition to the loss of central visual pathways, the suprachiasmatic nucleus of the hypothalamus housing the circadian clock was absent, indicating that the retinohypothalamic tract is required for the nucleus to develop. Telemetric analyses confirmed the lack of a functional circadian clock. Arylalkylamine N-acetyltransferase (Aanat) transcripts, encoding the melatonin rhythm-generating enzyme, were undetectable in the pineal gland of the Rax conditional knockout under normal conditions, whereas the paired box 6 homeobox gene, known to regulate pineal development, was up-regulated. By injecting isoproterenol, which mimics a nocturnal situation in the pineal gland, we were able to induce pineal expression of Aanat in the Rax conditional knockout mouse, but Aanat transcript levels were significantly lower than those of Rax-proficient mice. Our data suggest that Rax controls pineal gene expression and via Aanat may modulate melatonin synthesis.
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http://dx.doi.org/10.1111/jnc.14120DOI Listing
October 2017

Regulation of bone mass through pineal-derived melatonin-MT2 receptor pathway.

J Pineal Res 2017 Sep 20;63(2). Epub 2017 Jun 20.

Systems Biology of Bone, Department of Mouse and Zebrafish Genetics, Wellcome Trust Sanger Institute, Cambridge, UK.

Tryptophan, an essential amino acid through a series of enzymatic reactions gives rise to various metabolites, viz. serotonin and melatonin, that regulate distinct biological functions. We show here that tryptophan metabolism in the pineal gland favors bone mass accrual through production of melatonin, a pineal-derived neurohormone. Pineal gland-specific deletion of Tph1, the enzyme that catalyzes the first step in the melatonin biosynthesis lead to a decrease in melatonin levels and a low bone mass due to an isolated decrease in bone formation while bone resorption parameters remained unaffected. Skeletal analysis of the mice deficient in MT1 or MT2 melatonin receptors showed a low bone mass in MT2-/- mice while MT1-/- mice had a normal bone mass compared to the WT mice. This low bone mass in the MT2-/- mice was due to an isolated decrease in osteoblast numbers and bone formation. In vitro assays of the osteoblast cultures derived from the MT1-/- and MT2-/- mice showed a cell intrinsic defect in the proliferation, differentiation and mineralization abilities of MT2-/- osteoblasts compared to WT counterparts, and the mutant cells did not respond to melatonin addition. Finally, we demonstrate that daily oral administration of melatonin can increase bone accrual during growth and can cure ovariectomy-induced structural and functional degeneration of bone by specifically increasing bone formation. By identifying pineal-derived melatonin as a regulator of bone mass through MT2 receptors, this study expands the role played by tryptophan derivatives in the regulation of bone mass and underscores its therapeutic relevance in postmenopausal osteoporosis.
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http://dx.doi.org/10.1111/jpi.12423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575491PMC
September 2017

Critical involvement of ZEB2 in collagen fibrillogenesis: the molecular similarity between Mowat-Wilson syndrome and Ehlers-Danlos syndrome.

Sci Rep 2017 04 19;7:46565. Epub 2017 Apr 19.

Department of Dermatology, Kochi Medical School, Nankoku 783-8505, Japan.

Mowat-Wilson syndrome (MOWS) is a congenital disease caused by de novo heterozygous loss of function mutations or deletions of the ZEB2 gene. MOWS patients show multiple anomalies including intellectual disability, a distinctive facial appearance, microcephaly, congenital heart defects and Hirschsprung disease. However, the skin manifestation(s) of patients with MOWS has not been documented in detail. Here, we recognized that MOWS patients exhibit many Ehlers-Danlos syndrome (EDS)-like symptoms, such as skin hyperextensibility, atrophic scars and joint hypermobility. MOWS patients showed a thinner dermal thickness and electron microscopy revealed miniaturized collagen fibrils. Notably, mice with a mesoderm-specific deletion of the Zeb2 gene (Zeb2-cKO) demonstrated redundant skin, dermal hypoplasia and miniaturized collagen fibrils similar to those of MOWS patients. Dermal fibroblasts derived from Zeb2-cKO mice showed a decreased expression of extracellular matrix (ECM) molecules, such as collagens, whereas molecules involved in degradation of the ECM, such as matrix metalloproteinases (MMPs), were up-regulated. Furthermore, bleomycin-induced skin fibrosis was attenuated in Zeb2-cKO mice. We conclude that MOWS patients exhibit an EDS-like skin phenotype through alterations of collagen fibrillogenesis due to ZEB2 mutations or deletions.
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http://dx.doi.org/10.1038/srep46565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396187PMC
April 2017

Ick Ciliary Kinase Is Essential for Planar Cell Polarity Formation in Inner Ear Hair Cells and Hearing Function.

J Neurosci 2017 02 23;37(8):2073-2085. Epub 2017 Jan 23.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan,

Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling. The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport regulation with PCP formation in HCs and hearing function.
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http://dx.doi.org/10.1523/JNEUROSCI.3067-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705687PMC
February 2017

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport.

J Biol Chem 2016 Nov 28;291(47):24465-24474. Epub 2016 Sep 28.

From the Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, and; PRESTO, Japan Science and Technology Agency (JST), 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan,. Electronic address:

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases.
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http://dx.doi.org/10.1074/jbc.M116.738658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114401PMC
November 2016

Temporal Properties of Cone ERGs of Pikachurin Null Mutant Mouse.

Invest Ophthalmol Vis Sci 2016 Mar;57(3):1264-9

Department of Ophthalmology Nagoya University Graduate School of Medicine, Nagoya, Japan.

Purpose: Pikachurin is an extracellular matrix-like protein located in the synaptic cleft of photoreceptors. Pikachurin null mutant (Pika-/-) mice have abnormal ON bipolar cell function. This study aimed to determine the contribution of the ON bipolar cell pathway (ON pathway) of Pika-/- mice to the flicker ERG response and, using vector analysis, identify how the contribution varies with the stimulus frequency.

Methods: Flicker ERGs were recorded from wild-type (WT), Pika-/-, and mGluR6 null mutant (mGluR6-/-) mice. The frequency of stimulation was 3.906 to 31.250 Hz. The amplitude and phase of the fundamental components were obtained by harmonic and vector model analysis. The mGluR6-/- mice were used as a model wherein the ON pathway is known to be absent.

Results: The amplitudes of the fundamental components of the Pika-/- mice were significantly smaller than those of WT mice for stimulation frequencies between 3.906 and 17.578 Hz. The phase of the fundamental components of the Pika-/- mice was between those of the WT and mGluR6-/- mice. Vector analyses showed that the functioning of the ON pathway of Pika-/- mice was 12% to 25% of that of the WT mice at low frequencies (i.e., <15.625 Hz); however, it was reduced to noise level at frequencies >17.578 Hz.

Conclusions: Vector model analysis can determine the degree of contribution of the ON pathway of Pika-/- mice to flicker ERG response and may be useful for determining the retinal function in mice models with abnormalities of the ON pathway.
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http://dx.doi.org/10.1167/iovs.15-17708DOI Listing
March 2016