Biomedical subjects

Yuichiro Takada

Publications and source records attributed to Yuichiro Takada.

8 recordsLinked to original sources

PubMed2006

Retinoschisin expression and localization in rodent and human pineal and consequences of mouse RS1 gene knockout.

PURPOSE: The pineal gland shares a common neuroectoderm origin with the retina, and like the retina, regulates circadian rhythms through melatonin secretion. Recent expressed tag sequence (EST) analysis showed that several gene mutations, including RS1, which cause retinal degeneration, are also expressed in the pineal gland. Mutations in RS1 result in structural delamination of the neural retinal layers, leading to formation of schisis cavities in men affected with "X-linked retinoschisis" (XLRS). In this study, we evaluated RS1 expression in the rat and mouse as well as in human pineal and looked for morphological changes in the pineal of the RS1 knockout (RS1(-/Y)) mouse. METHODS: We analyzed rat and mouse pineal for RS1 expression by Northern blot and in situ hybridization. RS protein, synaptophysin, S-100, and GFAP localization in the pineal of rat and mouse and RS protein in human pineal were evaluated immunohistochemically. Morphological studies were performed using transmission electron microscopy and light microscopy comparing wild-type to the RS1(-/Y) mouse. RESULTS: RS1 expression was detected in RNA isolated from both the pineal and retina as a single major band migrating at about 5.5 kbp in Northern blots. RS1 riboprobe in situ hybridization demonstrated message in rat and mouse pineal, and immunohistochemistry showed RS protein in pinealocytes expressing synaptophysin but not in interstitial GFAP- and S100-positive glial cells. RS protein was present in many pinealocytes in human pineal. In light and electron microscopic examination of the pineal gland from RS1(-/Y) mice none of the structural changes found in the retina, such as cavity formation and loosening of the tissue, were seen. CONCLUSIONS: This study demonstrates that RS protein is expressed in the pinealocytes but not in interstitial glial cells. The lack of structural abnormalities in the RS1(-/Y) mice suggests that RS serves a different function in the pineal gland than in the retina.

Animals↗

PubMed2006

Identification and characterization of two mature isoforms of retinoschisin in murine retina.

Retinoschisin (RS) is a 24 kDa secreted protein expressed in retina and is required for the structural and functional integrity of the retina. RS has been predicted to serve as an adhesive protein but the precise molecular mechanism by which it functions in retina is not yet known. During investigations on structural and functional aspects of RS in murine retina using proteomic tools, we identified two isoforms of RS that differed in mass by 200 Da with no apparent change in charge. Mass spectra and amino acid sequence analysis of the tryptic peptides revealed that these isoforms differed by two amino acids at the N-terminus which suggested processing of RS signal sequence at two cleavage sites by signal peptidase as the basic mechanism underlying the occurrence of two mature RS isoforms in retina. Bioinformatic analysis identified two potential cleavage sites (between amino acids 21-22 and 23-24) in RS signal sequence. The flexibility of the signal peptidase to cleave at two sites is correlated to the amino acid composition of the RS signal sequence. This finding represents a rare example of a naturally occurring signal sequence cleavage at more than one site in vivo.

Amino Acid Sequence↗

PubMed2006

Congenital X-linked retinoschisis classification system.

PURPOSE: To establish a classification system for congenital X-linked retinoschisis (CXLRS) using clinical examination and optical coherence tomography (OCT). METHODS: Thirty-eight eyes of 19 patients who carried a clinical diagnosis of CXLRS were examined with OCT and clinical examination. Eyes were classified into one of four types based on a combination of clinical examination and OCT. RESULTS: All patients had bilateral OCT scanning performed at an average age of 8.64 years (range 2.24-17.4 years). Review of OCT scans revealed that 37 of 38 eyes had foveal schisis (97%) while 31 of 38 (82%) eyes had macular schisis deeper than the nerve fiber layer in areas of ophthalmoscopically normal macular retina. The authors termed this flat schisis phenomenon lamellar schisis. Thirty of 38 (79%) had peripheral bullous schisis cavities present. CONCLUSIONS: OCT examinations of patients with CXLRS reveal lamellar schisis in areas of ophthalmoscopically normal macular retina in 82% of eyes with the clinical diagnosis of CXLRS. Using both clinical examination and OCT, the authors were able to identify foveal, lamellar, and peripheral schisis, lamellar schisis only identifiable by OCT. These findings allow the authors to propose a classification system. The authors hope this classification system will allow a better understanding of the natural history of CXLRS disease and allow testing of therapeutic options.

Adolescent↗

PubMed2006

Ames Waltzer deaf mice have reduced electroretinogram amplitudes and complex alternative splicing of Pcdh15 transcripts.

PURPOSE: Mutations of PCDH15, the gene encoding protocadherin 15, cause either nonsyndromic deafness DFNB23 or Usher syndrome type 1F (USH1F) in humans and deafness with balance problems in Ames waltzer (av) mice. Persons with USH1 usually begin to exhibit signs of retinitis pigmentosa (RP) in early adolescence, but av mice are reported to have functional retinas. In this study, the auditory, visual and molecular biological phenotype of Pcdh15av-5J and Pcdh15av-Jfb mice is characterized, and their usefulness as animal models of USH1 is evaluated. METHODS: Hearing thresholds of mice between 6 and 10 weeks of age were measured by auditory brain stem response (ABR). Immunohistochemistry and histology were used to examine the effect of homozygosity of Pcdh15av-5J on stereocilia bundles of inner ear hair cells and on the photoreceptor cells of the retina. Scotopic and photopic Ganzfeld ERGs were recorded from homozygous Pcdh15av-5J and Pcdh15av-Jfb mice at different ages. Heterozygous littermates served as control subjects. Measurements of the width of the outer nuclear layer (ONL) and the length of rod photoreceptor outer segment (ROS) were made. RESULTS: Homozygous Pcdh15av-5J mice have profound hearing loss and disorganized stereocilia bundles of inner ear hair cells. Compared with heterozygous littermates, homozygous Pcdh15av-5J and Pcdh15av-Jfb mutant mice had scotopic ERG amplitudes consistently reduced by approximately 40% at all light intensities. The b-to-a-wave ratio confirmed that the a- and b-waves were reduced proportionally in homozygous mutant mice. Histologic measurements of retinal sections revealed no significant differences in either the ONL width or the ROS length as a function of genotype. The protocadherin 15 labeling pattern with antisera PB303 in the retina of both heterozygous and homozygous Pcdh15av-5J mice was indistinguishable from the wild type. Wild-type Pcdh15 have many alternatively spliced isoforms. A novel isoform was found in the retina of homozygous Pcdh15av-5J mice, which appears to circumvent the effect of the mutant allele (IVS14-2A-->G), which causes skipping of exon 14, a shift in the translation reading frame and a premature stop codon in exon 15. CONCLUSIONS: Pcdh15(av-5J) and Pcdh15(av-Jfb) mice do not faithfully mimic the RP found in USH1 due to mutations of PCDH15, but have significantly attenuated ERG function in the absence of histologic change. The decline in ERG amplitude with a preserved b-to-a-wave ratio suggests a role for Pcdh15 in retinal function and/or generation of the ERG potentials. Understanding the molecular mechanism by which av mice circumvent degeneration of the retina might offer insights into potential therapies for USH1.

Alternative Splicing↗

PubMed2004

A retinal neuronal developmental wave of retinoschisin expression begins in ganglion cells during layer formation.

PURPOSE: Mutations in the retinoschisin gene, RS-1, cause juvenile X-linked retinoschisis (XLRS), a dystrophy characterized by delamination of the inner retinal layers, leading to visual impairment. Although the retinoschisin protein (RS) is expressed most abundantly in photoreceptors in the outer retina, XLRS disease affects the innermost retinal layers, including the nerve fiber layer that contains retinal ganglion cells (RGCs). Considering the histopathological and electrophysiological characteristics of the clinical disease, the present study was conducted to evaluate the local cellular expression of RS-1 during retinal development. METHODS: RS protein and RS-1 mRNA were localized to specific retinal cell types in embryonic to adult mice by immunohistochemistry, confocal immunofluorescence microscopy, catalyzed reporter deposition in situ hybridization, and laser capture microdissection/RT-PCR. RESULTS: RS-1 mRNA was expressed first in RGCs by postnatal day (P)1, after terminal differentiation. Expression then moved posteriorly through the retina in a spatial and temporal developmental wave, as additional neuronal classes were born and synaptic layers were formed. RS was expressed by bipolar cells at a time when these neurons were establishing functional synapses with photoreceptors, evidenced by the appearance of the electroretinogram b-wave between P12 and P14. CONCLUSIONS: All major classes of adult retinal neurons, with the possible exception of horizontal cells, express RS protein and mRNA, strongly suggesting that retinoschisin in the inner retina is synthesized locally rather than being transported, as earlier proposed, from distal retinal photoreceptors. Continued expression of RS by mature inner-retinal neurons supports the possibility of a therapeutic strategy of protein replacement to treat both infants and adults with XLRS.

Aging↗

PubMed2004

RS-1 Gene Delivery to an Adult Rs1h Knockout Mouse Model Restores ERG b-Wave with Reversal of the Electronegative Waveform of X-Linked Retinoschisis.

PURPOSE: To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the retinoschisin protein by gene delivery can reverse the abnormal "electronegative" electroretinogram (ERG) retinal response. METHODS: An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functional analysis was performed. RESULTS: No RS protein was detected by Western blot analysis or immunohistochemistry in the Rs1h-KO mouse. Dark-adapted ERG responses showed an electronegative configuration, with b-wave reduction in both Rs1h(-/Y) and Rs1h-/- mice, typical of XLRS in humans. Histologic examination of Rs1h-KO mice showed disorganization of multiple retinal layers, including duplication and mislocalization of ganglion cells, laminar dissection through the inner plexiform layer, disorganization of the outer plexiform layer, loss of regularity of the outer nuclear layer, and shortening of the inner/outer segments with mislocalization of photoreceptor nuclei into this layer. After intraocular administration of AAV(2/2)-CMV-Rs1h, immunohistochemistry showed retinoschisin expression in all retinal layers of Rs1h(-/Y) mice, and ERG recordings showed reversal of the electronegative waveform and restoration of the normal positive b-wave. CONCLUSIONS: The RS-KO mouse mimics structural features of human X-linked juvenile retinoschisis with dissection through, and disorganization of, multiple retinal layers. The Rs1h-KO functional deficit results in an electronegative ERG waveform that is characteristic of human retinoschisis disease and that implicates a synaptic transmission deficit in the absence of retinoschisin protein. Replacement therapy by supplementing normal Rs1h protein in the adult Rs1h-KO mouse restored the normal ERG configuration. This indicates that gene therapy is a viable strategy of therapeutic intervention even in the postdevelopmental adult stage of XLRS disease.

Animals↗

PubMed2003

Early changes in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays.

The liver, skin, and gastrointestinal tract are major target organs of acute graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT). In order to gain a better understanding of acute GVHD in the liver, we compared the gene expression profiles of livers after experimental allogeneic and syngeneic BMT using oligonucleotide microarray. At 35 days after allogeneic BMT when hepatic GVHD was histologically evident, genes related to cellular effectors and acute-phase proteins were up-regulated, whereas genes largely related to metabolism and endocrine function were down-regulated. At day 7 after BMT before the development of histologic changes in the liver, interferon gamma (IFN-gamma)-inducible genes, major histocompatibility (MHC) class II molecules, and genes related to leukocyte trafficking had been up-regulated. Immunohistochemistry demonstrated that expression of IFN-gamma protein itself was increased in the spleen but not in hepatic tissue. These results suggest that the increased expression of genes associated with the attraction and activation of donor T cells induced by IFN-gamma early after BMT is important in the initiation of hepatic GVHD in this model and provide new potential molecular targets for early detection and intervention of acute GVHD.

Acute-Phase Proteins↗

PubMed2003

Molecular cloning of ELOVL4 gene from cynomolgus monkey (Macaca fascicularis).

ELOVL4, elongation factor of very long chain fatty acids-4, is known to be responsible for autosomal dominant macular degeneration and Stargardt-like macular degeneration. In this study, we cloned the monkey homologue of ELOVL4 and determined the cellular and tissue distribution of the gene product. Sequence analysis of the monkey ELOVL4 gene revealed a high degree of homology between human and monkey. The cloned full-length cDNA of monkey ELOVL4 encoded 314 amino acids, the same length as human and two amino acids longer than mouse. The monkey ELOVL4 conserved the characteristics typical of the super family of ELO enzymes involved in the metabolism of membrane-bound fatty acid elongation. Real-time quantitative PCR demonstrated that the monkey ELOVL4 gene was highly expressed in restricted tissue-specific fashion, not only in the retina but also in the skin (90% of retina) and thymus (111% of retina). Immunohistochemical analysis detected signals predominantly in the photoreceptor layer of the monkey retina.

Amino Acid Sequence↗