Age-related macular degeneration (AMD) is a major cause of blindness which lead to the deterioration of the center of the retina, among the elderly period. Nearly 40% of people over 75 years old, have some pathologic signs of AMD . Many lines of evidence suggest that mitochondrial disorders in eyes contribute to AMD, and mitochondrial disorders are common in other neurodegeneration diseases, such as Parkinson’s disease and Alzheimer disease [2,3]. Mitochondrial disorders are clinical phenotypes associated with mitochondrial dysfunction, and abnormalities of OXPHOS.
OXPHOS is responsible for 90% of ATP production in a respiring cell. It has five multi-subunit complexes, the respiratory chain complexes, and two additional electron carriers. It is controlled on the genetic level by two distinct genomes: the mitochondrial genome and the nuclear genome. The mitochondrial genome of nearly 16.6 kb, encodes 13 subunits of complex I, III, IV and V .
Deficiencies of OXPHOS are able to lead direct and indirect changes in metabolic homeostasis, which includes concentrations of ROS, Ca2+, and ratios of ADP/ATP, and NAD/NADH . Transcriptional response of OXPHOS genes, both nDNA and mtDNA, is often used to adapt these metabolic changes. Besides, OXPHOS genes have a feature of co-expression. And it is evident from studies of transcriptome analysis across different species. Particularly, in mice, co-expression of OXPHOS genes are studied among various tissues . What is more, it is found that OXPHOS genes are likely to have co-regulations. There may be the likely core promoters for most OXPHOS genes and assembly factors .
Complex I deficiency is the most common cause of the mitochondrial disease. NDUFS2 is a constitutive component of the core of complex I, considered to be essential for electron transfers. It is encoded by nDNA and has thirteen exons. The NDUFS2 mutation may lead to dysfunction of OXPHOS complex I where the mutation caused a catalytic defect .This mutation may have relation with Leigh syndrome . Leigh syndrome is a severe neurological disorder disease with features of progressive degeneration of mental and movement abilities.
Cytochrome c oxidase(COX), embedded in the inner mitochondrial membrane, is in the terminal enzyme of electron transport chain. It catalyzes transfer of electrons from cytochrome c to oxygen . Deficiency of COX is one of the most common metabolic reasons associated with respiratory chain defects and it is associated with various mitochondrial and neurodegenerative diseases [10,11]. COX10, one of subunits of COX, is heme-O-farnesyl transferase and also an assembly factor for COX in complex IV. It is encoded by nDNA and has seven exons. It is reported that mice lacking COX10 in skeletal muscle exhibited a progressive mitochondrial disease phenotype .
Materials and Methods
Mice were provided by Finnemann’s lab, Department of Biological Sciences, Fordham University.Young, middle-aged mice were three and six months old, respectively. Mice were sacrificed by CO2 and eyeballs were isolated immediately and immersed in Davidson's fixative. Retina and eyecups were separated and then were grind.
RNA was extracted from young mice retina, eyecups and middle-aged mice retina and eyecups using RNeasy® Plus Mini Kit (QIAGEN), according to the manufacturer’s instructions.
Seven pairs of primers were designed for this experiment. 4 pairs of primers were used to cover gene ndufs2 and the other primers were used to cover gene cox10. Most expected size of product is about 500 bp. The Tm of most of the primers is near 60°C.
Reverse Transcriptase-PCR (RT-PCR)
RT-PCR was performed using QIAGEN® One-Step RT-PCR Kit following instructions.Ten nanograms of RNA was amplified in 20 ul RT-PCRs. Temperature cycles as follow: one cycle of 50°C for 30min and 95°C for 15min, 94°C for 30 s, 57°C for 30s, and 72°C for 30s, and a final extension of 72°C for 10 min followed by a final hold at 4°C. Cycle number was 50.
5 ul of loading dye was added to each RT-PCR product. 5 ul of each product was then added to a 1% agarose gel, and electrophoresis was performed at 160 V. Band intensities were visualized by ethidium bromide in a UV trans-illuminator (BioRad). 100 bp marker was used to to measure the size of bands.
Gel extraction and sequencing
The target products was extracted by QIAquick Gel Extraction Kit(QIAGEN) following the manufacturer's instructions and subsequently sequenced by GENEWIZ® in order to identify PCR products.
I. No alternative variants were found in NDUFS2
In order to detect alternative splicing of NDUFS2, four pairs of primers were used for ndfus2, from exon1 to exon3 and from exon3 to exon7, from exon6 to exon10, and from exon9 to exon13. They overlapped all exons of ndufs2. If alternative splicing existed, two or more bands were observed from electrophoresis gel. But no alternative splicing was observed in ndufs2 (Fig. 1). Only expected bands were obtained. And expression level of ndufs2 were also constant during aging (Fig. S1). The result indicated that alternative splicing of NDUFS2 might not be affected by aging.
II. Spliced variants of COX10 were observed
In order to detect alternative splicing of COX10, three pairs of primers were used for cox10, from exon1 to exon3 and from exon2 to exon4, and from exon3 to exon7. No alternative splicing were detected with first two pairs of primers(Fig. S2). However, it was surprising that three bands in young mice and two bands in middle-age mice were detected within primers from exon3 to exon7 (Fig. 2).Variant C was only found to be expressed strongly in young mice. Three bands were purified and sent out for sequencing and were confirmed that they were three variants of COX10 by blast (Fig. 3). The sequences of three bands were aligned and analyzed by blast and clustalW. It was found that variant A was expected results, in which no exons were spliced out and variant B resulted from part of exon6, 45 bp was spliced out. No reading frame shift happened. Conserved region for alternative splicing acceptor site were also analyzed and compared with variant A (Fig. 4). Polypyrimidine tract, T and C rich region, and 3’ end AG were necessary for acceptor site, and they both found spliced end region on variant B. In variant C, exon6 was entirely spliced out, without read frame shift. Variant B and variant C were not reported in mouse database, but variant C matched a homologous variant in human database. Variant B has not been reported in any database.
This project found expressions of three variants of COX10, in mice retina and eyecups, changed with aging. Transmembrane domains were predicted by TMHMM Server. Variant A has nine putative transmembrane domains.The spliced region in variant B is located in two transmembrane domains; and variant C lacks four transmembrane domains. It is surprising that variant C is expressed strongly in young mice eyes. The biological meaning of these spliced variants is not understood clearly. Variant B and C are not found in mouse genomic plus transcript database in NCBI. Variant C matched a homologous variant in the human database, variant B, however, has not been reported in any database. Western blot of variant B and variant C are still required, because those two variants may not be translated into proteins.
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