Changes in Gene Expression Profiling and Phenotype in Aged Multidrug Resistance Protein 4-Deficient Mouse Retina

Multidrug resistance protein 4 (MRP4) is an energy-dependent membrane transporter that is responsible for cellular efflux of a broad range of xenobiotics and physiological substrates. In this trial, we aimed to investigate the co-effects of aging and MRP4 deficiency using gene expression microarray and morphological and electrophysiological analyses of the mouse retina. Mrp4knockout (null) mice and wild-type (WT) mice were reared in the same condition to 8–12 wk (young) or 45–55 wk (aged). Microarray analysis identified 186 differently expressed genes from the retinas of aged Mrp4-null mice as compared to that from aged WT mice, and subsequence gene ontology and KEGG pathway analyses showed that differently expressed genes were related to lens, eye development, vision, and transcellular barrier function that are involved in metabolic pathways or viral infection pathways. No significant change in thickness was observed for each retinal layer among young/aged WT mice and young/aged Mrp4-null mice. Moreover, immunohistochemical analyses of retinal cell type did not exhibit an overt change in the cellular morphology or distribution among the 4 age/genotype groups, and the electroretinogram responses showed no significant differences in the amplitude or the latency between aged WT mice and aged Mrp4-null mice. Aging would be an insufficient stress to cause some damage to the retina in the presence of MRP4 deficiency.


Introduction
In general, aging affects all organ systems in individuals via several physiological and pathological processes [1]. Oxidative stress is a major modifier of organ dysfunction and has been lined to age-related diseases, such as cardiovascular diseases, diabetes, chronic kidney diseases, and neurodegenerative diseases [2][3][4][5]. A recent systematic review suggests the association of a long lifespan and less oxidative damage although the authors admitted that the complexity of the etiology and the effects of genetic-environment interaction occurring throughout the lifetime remain unclear [6]. Research has shown that oxidative stress regulates the expression of a subset of efflux ATPbinding cassette (ABC) transporters that are located on the cell membrane that protect cells from against oxidative stress, including P-glycoprotein, multidrug resistance-related proteins, and breast cancer-related protein [7]. The retina is one of the most susceptible tissues to the oxidative damage because it has the highest oxygen consumption rate per kg of the body among body organs, thereby leading to exposure to a large amount of endogenous reactive oxygen species [8]. Oxidative stress RNA microarray and KEGG pathway analysis was performed by Macrogen Inc. (Tokyo, Japan). Onecolor Cy3 RNA labeling and array hybridization to Agilent SurePrint G3 Mouse Gene Expression Arrays 8 x 60 K Ver. 2.0 (Agilent Technologies, Santa Clara, CA, USA) were performed. In brief, labeled cRNA was prepared from 1-5 µg total RNA using the Agilent's Quick Amp Labeling Kit. Following fragmentation, 1.65 µg of cRNA were hybridized to the Agilent expression microarray as per the protocols recommended by the manufacturer. Then, the arrays were scanned using the Agilent Technologies G4900DA SG12494263, and array data export processing and analysis was performed with Agilent Feature Extraction v11.0.1.1. The raw data for same gene was then summarized automatically in the Agilent Feature Extraction protocol to generate raw data text file, providing expression data for each gene that was probed on the array.

Data analyses
Array probes that have Flag A in the samples were filtered out. Selected gProcessed Signal value was transformed by logarithm and normalized using the quantile method. Statistical significance of the expression data was determined using fold change and local pooled error (LPE) test wherein the null hypothesis was that no difference exists between both the groups. Hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Significant counting by the absolute value of fold change ≥ 1.5 and P-value LPE < 0.05 was performed using the processed data. Then, Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using Gene Ontology (GO) (http://geneontology.org) and KEGG (http://kegg.jp). All data analysis and visualization of differentially expressed genes were performed using R 3.3.3 (www.r-project.org). Primary microarray data after significant counting have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number (GSE162376).

Electroretinography recording
Electroretinography (ERG) recording were conducted, as reported previously [27,28] with some modifications. All the animals were dark-adapted overnight before ERG recordings, and all the procedures were performed under dim red light. Mice were anesthetized and positioned on a built-in heating pad that maintains the body temperature at 37°C. After dilating the pupils using 2.5% phenylephrine and 1.0% tropicamide eye drop, (Santen Pharmaceutical Co., Osaka, Japan), a contact lens electrode embedded with gold wire was placed on the cornea as an active electrode (Mayo, Aichi, Japan), and a chloride silver plate was placed in the mouth as a reference electrode. The ERGs were recorded using commercially available equipment with a Ganzfeld bowl (Mayo, Aichi, Japan). Scotopic recordings were obtained from dark-adapted animals at the following increasing light intensities. Responses were amplified 10,000 times, and the band-pass filtered from 0.3 to 500 Hz (PuREC PC-100, Mayo). For the recording of scotopic threshold response (STR), serially increasing luminescence intensities of −6.1, −5.5, −5.1, −4.6, and −4.1 log sc td s were used. Responses were amplified differentially and band-pass filtered at 0.125-50 Hz, and responses from 80 repeated stimuli for each intensity were averaged. Photopic recordings were performed after 5-min light adaptation intervals on a background light intensity of 1.5 log sc td. Responses were amplified differentially and band-pass filtered at 0.3-300 Hz, and the responses from 30 (20 for flicker) repeated stimuli for each intensity were averaged.

Statistical analyses
Statistical analyses were performed using MedCalc software version 11.3 (MedCalc Software, Mariakerke, Belgium). All the descriptive statistics are presented as mean ± standard deviation values (all the experiments were performed at least in triplicate). Comparison between the groups and among > 3 groups was analyzed using Student's t-test and analysis of variance (ANOVA), respectively. Student-Newman-Keuls test for all pairwise comparisons were conducted when the F ratio was significant in ANOVA. A P-value < 0.05 was considered to indicate statistical significance. Microarray data analyses were conducted, as described above.

Altered gene expression profile in aged Mrp4-null mouse retina
In order to examine comprehensive transcriptome information on aged Mrp4-null mouse retina, we performed oligonucleotide-based microarray analysis with Agilent SurePrint G3 Mouse Gene Expression Arrays 8 x 60 K Ver. 2.0 that covers the content source from RefSeq, Ensembl, RIKEN, UniGene, and GenBank databases to provide full coverage of the mouse transcriptome: 27,112 Entrez Genes, 4,578 lncRNAs, 39,430 Entrez Gene RNAs, and 16,251 lincRNAs. As compared to age-matched WT mice, 186 differently expressed genes were identified from the retinas of aged (about 50 wk old) Mrp4-null mice with the absolute value of fold change ≥ 1.5 and P-value of LPE < 0.05 (Table S1). Top 10 up-or down-regulated genes have been listed in Table 1.  (Figure 2, b-d). Subsequent KEGG pathway analysis revealed that differently expressed genes were mainly involved in the "metabolic pathway," "glycerophospholipid metabolism," "herpes simplex virus 1 infection," and "Kaposi sarcoma-associated herpes virus infection" ( Figure S1).

Thickness changes in each retinal layer
We hypothesized that differently expressed genes in aged Mrp4-null mouse retina have some associations with the retinal architecture. Therefore, we then assessed the thickness changes in each retinal layer: nerve fiber layer (NFL)/ ganglion cell layer (GCL) complex, inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and photoreceptor layer (PhR). Figure 3 shows the results of H&E staining. No significant change in the thickness was observed for each retinal layer among young (8-12 wk) WT mice, aged (45-55 wk) WT mice, young Mrp4-null mice, and aged Mrp4-null mice. Quantitative comparison of thickness in each retinal layer. No significant difference was observed (n = 6). WT, wild-type mice; Mrp4KO, Mrp4-null mice; NFL, nerve fiber layer (NFL); GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PhR, photoreceptor layer.

Morphology and distribution of each retinal cell type
In response to the results of H&E staining, we checked the morphology and distribution of each retinal cell type because the changes related to aging and Mrp4 deficiency might be detected at the cellular level. Section immunohistochemical staining demonstrated no apparent changes in the morphology and distribution among young/aged WT mice and young/aged Mrp4-null mice for vascular endothelial cells (CD31), astrocytes (GFAP), Muller cells (glutamine synthetase), horizontal and amacrine cells (Calbindin), bipolar cells (Chx10), or photoreceptors (peanut agglutinin) (Figure 4). Retinal whole mount immunostaining was performed for the two-dimensional evaluation of retinal vessels and astrocyte network that could not be used for performing section immunohistochemistry. Further, the results showed no overt change in young/aged WT mice and young/aged Mrp4-null mice in terms of the retinal vasculature or astrocyte network although some layers showed a small but significant difference in the vascular area and/or total vascular length ( Figures 5 and 6).

Electrophysiological function of the retina
In order to test whether the retina of aged Mrp4-null mice has the same retinal function as that of WT mice, we conducted electrophysiological analyses. Electroretinography (ERG) performed for aged Mrp4-null mice showed no significant difference from aged WT mice in terms of the amplitude and latency of a-wave, b-wave, and the amplitude of positive STR (Figure 7 and Figure S2).

Discussion
In the current study, we performed a comprehensive transcriptome analysis and identified 186 differently expressed genes in aged Mrp4-null mice as compared to that in aged WT mice and obtained the information on significant GO terms and KEGG pathways using the differently expressed gene. However, no overt retinal phenotype related to aging or Mrp4 deficiency was observed in the retinal architecture, morphology, and distribution of each retinal cell type or electrophysiological function.
A total of 186 differently expressed genes would reflect the effects of the blood-retinal barrier (BRB) dysfunction on the neural retina because (1) MRP4 is almost exclusively expressed in vascular endothelial cells in the retina [23,24], (2) endothelial cells are major components of the BRB [29,30], (2) MRP4 restricts transcellular transport [22], and (4) endothelial cells account for only a small cell population (0.1%) in the retina [31]. The GO terms significantly classified using 186 differently expressed genes involved those related to the lens (lens development in camera-type eye, lens fiber cell differentiation, and lens fiber cell development), eye development (camera-type eye development, visual system development, sensory system development, eye development, and sensory organ development), and vision (visual perception and sensory perception of light stimulus) for biological process. We confirmed that the extracted GO terms in our study varied from those in other studies that investigated the effect of ocular inflammation, ultraviolet radiation, or strain [32][33][34]. It surprised us that differently expressed genes were enriched within lens-related GO because the retina and lens are being recognized as two entirely different tissue types. However, this result might convey an important message to us. For example, crystallins are primarily found as major structural proteins of the ocular lens; however, it was then disclosed that αB-crystallin acts as a regulator of retinal angiogenesis [35]. The significant GO terms for molecular function or cellular components, such as structural molecule activity, intrinsic component of vacuolar membrane, and integral component of synaptic vesicle membrane might be associated to the MRP4 transcellular barrier function in the CNS [36]. Specific signaling pathway affected by Mrp4 deficiency in aged mouse retina included "metabolic pathway," "glycerophospholipid metabolism," "herpes simplex virus 1 infection," and "Kaposi sarcomaassociated herpes virus infection." Although distinct relationships between the former 2 pathways and MRP4 remain unknown, the latter 2 pathways might be associated with MRP4 function as an efflux transporter because antiviral agents are MRP4 substrates [22,37].
Before focusing on some key genes that were significantly up-regulated or down-regulated in aged Mrp4-null mice in the list, we checked the severity of the impact on the retina aging and Mrp4 deficiency. Thickness change in the specific retinal layer is likely to be a good marker of retinal degeneration [38]; therefore, we measured the thickness of retinal layers in young/aged WT mice and young/aged Mrp4null mice and performed a comparison. Unexpectedly, although > 100 of the genes are differently expressed in aged Mrp4-null mouse retinas, changes in the thickness of each retinal layer did not show any significant differences. We checked some retinal sections from WT and Mrp4-null mice aged 2 y and found no overt retinal phenotype (data not shown). Moreover, immunohistochemical analyses of the retinal cell type did not exhibit any obvious change in the cellular morphology or distribution among the 4 age/genotype mouse groups. Inconspicuous vascular phenotypes observed in the intermediate and/or deep retinal layer might be attributed to Mrp4 deficiency, aging, or both because we previously disclosed that Mrp4 deficiency led to retinal vascular phenotypes after forskolin administration in neonatal mice [17]. An extensive electrophysiological analysis confirmed these histological results, showing no significant ERG responses in the amplitude or the latency between the aged WT mice and aged Mrp4-null mice. Based on these experimental results, we concluded that aging is an insufficient stress to cause some damage to the retina in Mrp4-null mice. However, we are currently unable to explain the reasons why aged Mrp4-null mice show no evident retinal phenotype in spite of the presence of many differently expressed genes in the retina. We made the following speculations: (1) as the mice had been housed under well-controlled and specific-pathogen-free condition, accumulated endogenous and exogenous stresses were beneath the threshold that caused retinal damage, (2) other efflux transporter family proteins expressed in the retina compensated for the lack of MRP4, and (3) the pleiotropic effects of the loss of MRP4 function canceled the molecular pathways causing retinal dysfunction. In any case, the point is that co-effects of MRP4 deficiency and aging unlikely exert serious damage to the retina. It might be a boon to patients who continue taking drugs with MRP4 inhibitory activity; however, further studies should be performed to find a resolution of this issue because they have systemic diseases, such as hyperlipidemia, hypertension, and cancer, that may impose another stress in addition to aging [11,21,22]. This study has certain limitations. First, microarray analysis was performed at only one time point. If microarray data was acquired at many different time points, the co-effects of MRP4 and chronological aging on retinal phenotype could be further understood. Second, we used whole retinas to investigate the differences in gene expression. Although the major component of the retina is the neuron, the retina has several types of neurons as well as glial cells and vascular endothelial cells. Therefore, we observed the sum of gene expressions derived from different cell types in our microarray data. Third, although technically difficult, we did not evaluate cell behaviors that would facilitate brain researchers because the retina is an ideal tissue for in vivo imaging, and the cellular events that occurred in the retina were linked to those in the brain.
In conclusion, MRP4 deficiency in aged mice caused different gene expression than in WT mice. The analyzed gene profiles suggest that aging and MRP4 deficiency affects the gene expressions in the retina associated with the lens, eye development, vision, and transcellular barrier function that are involved in metabolic pathways or viral infection pathways. However, the magnitude of the impact of gene expression changes observed in aged Mrp4-null mice on the retinal morphology and function would be small. Aging is insufficient as a stress to cause some damage to the retina in Mrp4-null mice, and further studies should be performed to investigate the co-effects of Mrp4 deficiency and other types of stress, such as hyperglycemia and inflammation.