Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

dog retina that is devoid of cones but has the complete rod cell population. Cone degeneration (cd) is an autosomal recessive disorder that occurs naturally and was originally found in the Alaskan Malamute dog (Aguirre and Rubin, 1974). This disease is caused by a deletion in the cone cyclic nucleotide-gated channel β subunit gene (CNGB3) (Sidjanin et al., 2002).

2 Strategy and Methods

Retinal mRNAs from adult, cone-less cd dogs were subtracted from normal dog retinal mRNAs using two rounds of representational difference analysis (Akhmedov et al., 2002). We then took the output of RDA and shotgun cloned it into a plasmid vector to create a mini-library in a bacterial host. Approximately 2000 cDNA clones generated from the subtracted library were arrayed on microarray chips after amplification of inserts from individual colonies with vector-specific primers. The arrayed target cDNAs were hybridized with the Cy3and Cy5-labeled original amplicons from normal and adult cd dog retinas, and subsequently screened by repetitive probing with mixtures of the inserts that had the brightest signal after hybridization with the initial amplicons (Saghizadeh et al., 2003). This screening created a non-redundant set of clones. Eighty of these clones were differentially expressed. After sequencing, BLAST analyses were performed utilizing the National Center for Biotechnology Database (www.ncbi.nlm.nih.gov/BLAST) and the Institute for Genomic Research (www.tigr.com.).

2.1Microarray Screening Identified Several Potentially Cone-Expressed cDNAs

Out of the 80 sequenced clones, we identified several that have been described as cone-specific (i.e., 3 clones for different regions of cone opsin cDNA, 4 clones for the different subunits of cone transducin and 3 clones for different regions of α’ subunit of cone PDE cDNA), and several that did not correspond to any known gene (8 clones, Table 1) or to genes that had not been described as present in the retina (27 clones, i.e., VPS35 and PTDSR). The rest corresponded to mitochondrial genome fragments. Northern blots and real-time RT-PCR were used to confirm the differential expression of the isolated cDNAs.

3 Preliminary Characterization Of Unknown cDNAs

The 8 differentially expressed clones from Table 1 were used as probes on Northern blots of total RNA from dog retinas. Only three clones (12B7, 21D1 and 4A1) showed detectable signals after one-week exposure (Fig. 1). It is possible that the other 5 clones were less abundant than 12B7, 21D1 and 4A1; at the time we also

Table 1 Unknown dog cDNAs identified as expressed in cone photoreceptors after microarray screening of the RDA-subtracted pool of cDNAs

thought that they could be pseudogenes. 12B7 was expressed specifically in normal retina and was not present in brain, heart, kidney, liver, muscle, or spleen (Fig. 1A). Two transcripts for 12B7 of approximately 7.0 kb and 3.5 kb were observed in normal retinas. The larger transcript and a smaller than 3.5 kb transcript were present in cd dog retinal RNA, (Fig. 1A). 21D1 was preferentially expressed in retina with a transcript approximately 3.0 kb long (Fig. 1B). This transcript was in much lower amount in cd than in normal retina (Fig. 1B). 4A1 was expressed in normal and cd dog retina and also in all dog tissues investigated. The size of the transcript hybridized to the 4A1 cDNA probe was approximately 1.8 kb.

In collaboration with The Institute for Genomic Research (Tigr), human orthologs of both the 21D1 and 15A15 clones were identified as predicted genes. 12B7 and 8H5 were mapped to human chromosome 14 and 13, respectively. The latter was found to encode a known gene, Cullin 4A (GenBankTM accession number AF077188). Similarly, 5E1 mapped to human chromosome X, and was found to encode the known gene, MBTPS2. Clones 13D8 and 4A1 could not be mapped to any human chromosome.

Fig. 1 Northern blots of dog RNAs hybridized with cDNA fragments corresponding to three isolated clones. 30 μg of total RNA from multiple normal dog tissues and cd dog retina were electrophoresed on a 1.2% agarose gel containing formaldehyde, transferred to Hybond N+ and hybridized at 68◦ c with radiolabeled cDNA probes. (A) Expression of 12B7 mRNA. (B) The same blot was stripped and probed with a radiolabeled 21D1 cDNA fragment. (c) Different blot showing the expression of 4A1 mRNA. Lanes: 1, brain; 2, heart; 3, liver; 4,kidney; 5, lung; 6, testis; 7, normal retina; 8, cd retina

Therefore, preliminary characterization of unknown cDNAs and computer analysis of their primary sequences led us to choose the cDNA candidates for further investigation.

4 Characterization of Mouse 21D1

4.1 Molecular Cloning

The clone originally obtained was a part of the 5’ dog archive sequence, intronic to the cDNA sequence, and it had 94% homology to a human sequence and 95% homology to a mouse sequence. 21D1 was mapped to human chromosome 9 and mouse chromosome 2. The complete 21D1 mouse cDNA, including the coding region and 3’ and 5’ UTRs, was obtained by 5’ and 3’ RACE using oligo-dT-primed mouse retinal first-strand cDNA and gene-specific sequences. The 2970–bp cDNA comprises an open reading frame of 1506 bp, a 367-bp 5’-UTR, and a 1097-bp 3’-UTR.

4.2Northern Blot Analysis and Developmental Studies of Mouse 21D1 mRNA

Using RT-PCR, a 700 bp probe from the coding region of the 21D1 mouse sequence was subcloned. Northern blots of mRNAs from different mouse tissues hybridized to this 21D1 cDNA probe showed a transcript of the same size of dog 21D1 mRNA, approximately 3.0 kb long in retina and larger than 3.0 kb in other tissues. This mRNA was abundantly expressed in retina and brain, in lesser amounts in liver and testis and was present at low levels in kidney, lung, and muscle and barely detected in heart (Fig. 2A). Northern blots of retinal mRNAs from mice at different times during postnatal development showed that 21D1mRNA expression increased from birth and peaked at 15-21 day (Fig. 2B). Interestingly, the developmental increase in 21D1 mRNA expression correlates with the time of differentiation, growth and elongation of outer segments of photoreceptor cells. This observation suggests that 21D1 may be expressed in visual cells.

4.3 Computer Analysis of the 21D1 Primary Sequence

Search of the protein database with the deduced amino acid sequence of the original dog 21D1 clone revealed its homology with the human KIAA1896 protein and the mouse peroxisomal Ca2+-dependent solute carrier-like protein. However, after cloning the full-length mouse cDNA from retina, we found that the first 73 amino acids of the predicted protein corresponding to the 21D1 cDNA had no homology with other proteins in the database. The amino-terminal-half of the 21D1 predicted

MCSC-c (Mouse Ortholog) (Mitochondrial Ca2+ dependent Solute Carrier, isoform c)

Fig. 3 Human mitochondrial carrier protein family and two corresponding mouse orthologs

MCSC

MCSC-c

Fig. 4 Genomic organization of MCSC and the MCSC-c variant. Dark gray boxes are the 5’ or 3’-UTRs and light gray boxes are the 10 exons that form the mRAN coding region

codon of these two variants is present in exon 1, the mRNA 5’ UTRs as well as the N-terminal sequences of both resulting proteins are different.

5 Characterization of the Human 15A15 Clone

5.1 Computer Analysis of the 15A15 Primary Sequence

The sequence of the 15A15 dog clone that we originally obtained (260 bp) showed 89% homology to the coding region of a human predicted gene, ZBED4. This predicted gene is located on human chromosome 22 and mouse chromosome 15, is 34.5 kb long in human and 29.8 kb in mouse and has 2 exons in both species. The ZBED4 cDNA sequence predicts a protein formed by 1171-amino acids that contains four BED type zinc finger domains and a hATC type dimerization domain near the C terminus.

5.2 Molecular Cloning of ZBED4

Using RT-PCR, a 713-bp probe from the 3’ coding region of the mouse sequence was obtained. This fragment was subcloned and used for Northern blot hybridization. In addition, the complete coding region of ZBED4 was amplified from mouse retina mRNA using the appropriate primers, and the isolated cDNA was subcloned into an expression vector for subcellular localization studies.

5.3 Northern Blot Analysis

Northern blots of mouse retinal and brain mRNAs hybridized to the corresponding ZBED4 cDNA probe showed a major transcript of 5.3 kb, which is consistent with the molecular size estimated for ZBED4 mRNA (Fig. 5).

5.4 Localization of ZBED4 to Human Cone Photoreceptor Cells

Immunohistochemistry studies using a polyclonal ZBED4 antibody and human retinal sections localized ZBED4 to the nuclei and inner segments of cone photoreceptors (Fig. 6). These results were confirmed with double immunostaining of human sections with anti-ZBED antibody and rhodamine-conjugated peanut agglutinin, which binds to the matrix surrounding cone photoreceptors, but not to that surrounding rods (data not shown).

5.5 Subcellular Localization of ZBED4

Immunostaining studies using Y79 retinoblastoma cells and anti-ZBED4 antibody showed the nuclear localization of ZBED4 (Fig. 7). This localization was also seen when Y79 retinoblastoma cells transfected with an expression vector containing the

Brain Retina

5.3 kb

Fig. 5 Northern blots of mouse mRNAs from retina and brain probed with a radiolabeled 15A15 (ZBED4) cDNA fragment. Each lane contains 2 μg of mRNA

Fig. 6 Localization of the ZBED4 protein in human retina. Human retinal sections were incubated with a polyclonal antibody to a ZBED4 peptide (that we generated in rabbit), followed by reaction with FITC-conjugated goat anti-rabbit antibody. Arrow shows nuclei and arrowhead shows cone inner segment staining. Magnification 400X

Fig. 7 Subcellular localization of the ZBED4 protein in Y79 retinoblastoma cells. Y79 retinoblastoma cells incubated with anti-ZBED4 antibody show the enogenous localization of ZBED4 to nuclei of these cells A. DAPI staining of nuclei. B. Anti-ZBED4 antibody staining

complete coding region of ZBED4 attached to the Xpress epitope were reacted with anti-Xpress antibody: the conjugated ZBED4-Xpress protein was detected in the nuclei (data not shown).

6 Discussion

Microarray screening of our subtracted cDNA pool (retinal cDNAs from cd dog subtracted from those of normal dog retina) helped us to identify several uncharacterized cDNAs expressed in cone photoreceptors. After preliminary studies on several of these cDNAs and computer analysis of their primary sequences, we chose a couple cDNAs for further investigation.

We report here the identification in retina of a new variant of mouse mitochondrial carrier solute carrier, MCSC-c. This protein belongs to the Ca2+-dependent mitochondrial transporter subfamily and is encoded by an mRNA that has a different exon 1 than the liver MCSC mRNA (see Fig. 4). The N-terminal of the MCSC-c protein has no homology to that of MCSC. These variants may have different functional characteristics or sensitivity to Ca+2, which could explain their tissue or cell specificity.

Several studies have established that 60–65% of retinal mitochondria are located in the inner segments of photoreceptors (Hoang et al., 2002; Kageyama and Wong-Riley, 1984). These cells have 2- to 3-fold greater oxygen consumption and higher cytochrome c oxidase (CO) activity than the cells of the inner retina (Chen et al., 1989). It has also been shown that rod and cone mitochondria have fundamental substructural and functional differences (Perkins et al., 2003). Cone inner segments contain 2-fold more mitochondria and create more CO activity than rod inner segments. Therefore, cones utilize complementary mechanisms to compensate for their differences with rods in bioenergetic processes. These include: increased number of mitochondria, increased cristae surface membrane area and probably, the presence of specific proteins or variants of functional proteins in the outer or inner membrane of their mitochondria with different functional sensitivities.

Variations in the N-terminal half of the human SCaMC-2 splice variants result in different number of EF-hand domains in the proteins. It has been reported that the splice isoforms of UCP5 and phosphate carrier proteins (another carrier protein family) differ in their functional characteristics (Fiermonte et al., 1998). It is possible that absence of a specific EF-hand motif in different variants of SCaMC-2 or in MCSC and MCSC-c provides a mechanism for Ca2+ signaling diversification. Furthermore, different N-terminal variants arising from tissue or cell-specific promoter usage may also provide additional mechanisms to modulate sensitivity to Ca2+.

Another cDNA that we identified after substractive hybridization and microarray screening of normal and cone-less mRNAs is 15A15. This cDNA encodes a novel 1171-amino acid protein, ZBED4. Primary sequence analysis of ZBED4 revealed characteristic features of a nuclear regulatory protein. It contains four zinc finger BED domains that have the Cx2CxnHx3−5[H/C] signature in the amino-terminal- half, and a hATC dimerization domain in the carboxyl-terminal-half. Interestingly, two nuclear receptor-interacting modules (LXXLL) are present in the ZBED4 amino acid sequence, suggesting its possible direct or indirect interaction with nuclear hormone receptors. The hATC dimerization domain of ZBED4 also makes this protein a member of the human hAT transposase family. It has been reported that the hATC domain is very conserved among proteins of this family and functions in self-association, an essential feature required for nuclear accumulation and DNA binding (Yamashita et al., 2007).

Expression of ZBED4 is limited to cone photoreceptors and completely absent from rods in human retina, as determined by immunocytochemistry (Fig. 6). Furthermore, ZBED4 is present in cell nuclei, as shown by co-localization of transfected ZBED4 with the nuclear marker DAPI. Considering the specific features of the ZBED4 primary sequence: that ZBED fingers bind to DNA, that hATC domains

usually facilitate accumulation of proteins in nuclei and that LXXLL motifs bind to nuclear hormone receptors, we hypothesize that ZBED4 may be directly or indirectly involved as a co-activator or co-repressor in the regulation of transcription of cone-specific genes.

Acknowledgments This work was supported by NIH R01 grant EY 08285 to DBF, NIH training grant EY07062 (MS) and a grant from the Foundation Fighting Blindness (DBF). We thank Dr. Alex Yuan for his help in the preparation of this chapter. Mehrnoosh Saghizadeh was the recipient of an RD2006 Young Investigator Award.

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Clinical and Genetic Characterization of a Chinese Family with CSNB1

Ruifang Sui, Fengrong Li, Jialiang Zhao, and Ruxin Jiang

1 Introduction

Human congenital stationary night blindness (CSNB) is a group of non-progressive retinal dystrophies characterized by night blindness from birth and other symptoms such as myopia, hyperopia, reduced visual acuity and occasionally accompanied by nystagmus, and optic disc hypoplasia (Heonand and Musarella, 1994). CSNB can be inherited on autosomal dominant, autosomal recessive and X-linked recessive mode. According to the clinical and genetic studies, X-linked recessive CSNB (XLCSNB) can be divided into two subtypes: complete (CSNB1; MIM 310500) and incomplete (CSNB2; MIM 300710). The distinction between the two subtypes of XLCSNB is in electroretinogram (ERG) and in genetic basis. CSNB1 is characterized by the complete absence of the rod b-wave, but almost normal cone amplitudes, while CSNB2 is associated with a reduced rod activity and a significantly abnormal cone ERG. The disease gene responsible for CSNB2 (CACNA1F) at Xp11.23 encodes a retina-specific L-type calcium channel α-subunit. CSNB1 results from mutations in the NYX (nyctalopin on chromosome X) gene at Xp11.4 (Miyake et al., 1986; Bech-Hansen et al., 1998). We describe a Chinese family with multiple individuals affected with reduced vision and high myopia, which has been misdiagnosed as “pathologic myopia and amblyopia”. Clinical and genetic investigation indicated a phenotype of CSNB1. Linkage analysis for the family mapped this phenotype to Xp11.4. Sequencing of NYX identified one novel mutation.

R. Sui

Department of Ophthalmology, Peking Union Medical College Hospital, Beijing 100730, China, Tel: 86-1065296358, Fax: 86-1065296565

e-mail: hrfsui@yahoo.com