|Molecular Vision 2006;
Received 6 February 2006 | Accepted 13 September 2006 | Published 26 October 2006
The rat Apg3p/Aut1p homolog is upregulated by ischemic preconditioning in the retina
Bill X. Wu,1
Alix G. Darden,1,2,
Yan Li,1 Craig
E. Crosson,1 E. Starr Hazard
III,1 Jian-xing Ma3
1Department of Ophthalmology, Medical University of South Carolina, 2Department of Biology, The Citadel, Charleston, SC, 3Department of Cell Biology, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK
Correspondence to: Alix G. Darden, PhD., The Citadel, 171 Moultrie Street, Charleston, SC 29409, Phone: (843) 953-7873; FAX: 843-953-7264; email: email@example.com
Purpose: Retinas can be protected from subsequent severe ischemic injury by ischemic preconditioning. Ischemic preconditioning is dependent on gene expression and protein synthesis; however, it is not clear which genes are important in this process. In this study, we have identified and characterized the rat homolog of yeast Apg3p/Aut1p, an important autophagy protein encoded by the autophagy 3-like (APG3L) gene. We have also further characterized the homologous human APG3L gene.
Methods: A fragment of the rat Apg3 cDNA was identified by mRNA differential display from hypoxia-treated E1A-NR3, an immortalized cell line derived from rat retinal cells that manifests phenotypes of retinal neurons. The full length of rat Apg3 (rApg3) cDNA sequence (about 1.4 kb) encoding 341 amino acids was cloned from a rat retinal cDNA library and characterized using Southern and northern blot analysis, and a global GenBank search. Protein expression was determined by western blotting, and immunohistochemistry. Ischemic preconditioning was achieved by ligation of the retinal arteries of the right eye for 5 min followed by 5 h reperfusion. The prolonged retinal ischemia was induced by ligation of the retinal arteries for 45 min followed by 5 h reperfusion. The full-length homologous human APG3L gene was cloned and sequenced from a human genomic DNA library.
Results: The combination of genomic Southern blot analysis and a global GenBank search indicated that rat APG3L is a single copy gene. Rat Apg3 mRNA is expressed in the retina at a high level but is also detected in other tissues. In the process of comparing the rat and human APG3L genes we showed that the organization of the human APG3L gene includes a unique transcriptional start site, a coding region with 12 translated exons and 11 introns and is located on human chromosome 3q13.1. Subcellular localization studies showed that recombinant rat autophagocytosis protein (Apg3p) is a cytosolic protein. Rat Apg3 mRNA level was upregulated by ischemic preconditioning but downregulated by prolonged ischemia.
Conclusions: Our results suggest that the upregulation of rApg3 is a specific response to ischemic preconditioning rather than to retina ischemia, and autophagy may contribute to the neuroprotective effect of ischemic preconditioning in the retina.
Studies of neurons in the central nervous system (CNS) demonstrated that there are substantial differences in gene expression and functional recovery from ischemic stress, depending on the duration of the ischemia or hypoxia [1,2]. Moreover, brief, nondamaging periods of ischemia followed by periods of reperfusion have been shown to protect cells from subsequent, damaging ischemia in cardiomyocytes and the CNS. This has been termed ischemic preconditioning (IPC) or ischemic tolerance [3,4]. The tolerance to ischemia has been demonstrated to be dependent upon gene expression and protein synthesis [5,6]. In IPC, changes in gene expression are different from the gene expression profile seen in programmed cell death . Evidence from histological and functional analyses demonstrated that 5 min of ischemia followed by a period of reperfusion provides complete protection to the retina from the damage caused by subsequent 60 min of sustained ischemia in the rat retina .
It is speculated that IPC induces the expression of protective genes, and their protein products protect cells from the damage caused by subsequent, prolonged ischemia . In cardiomyocytes and CNS neurons, several genes such as NF-κB and certain heat shock proteins have been shown to be induced by IPC, and the increased expression of these genes may contribute to IPC's protective effect [8-10]. In the retina, basic fibroblast growth factor, glial fibrillary acidic protein, and a major anti-apoptosis protein, Bcl-2, were upregulated after IPC [11,12]. Recently, we found that Hsp27 mRNA and protein levels were increased two-fold at 24 h after IPC and returned to control levels after 72 h . Furthermore, HSP27 expression is up-regulated by hypoxia-inducible factor-1 (HIF-1), and over-expression of HSP27 inhibits caspase-3 activation and protects a transformed rat retinal ganglion cell line (RGC5) ganglion cell line from ischemic stress-induced cell death [13,14].
Cellular homeostasis depends on a balance between anabolic and catabolic processes [15,16]. Autophagy is defined as the bulk degradation of the cell's organelles by the lysosomal system for recycling of essential molecules or removing extra as well as hazardous molecules by the autophagosome or autophagic vacuole, a double/multiple membrane structure. This physiological process is important for human health [17,18]. There is evidence that autophagy is associated with neurodegeneration and may play a role in neuroprotection by removing aggregated proteins [19-22].
Although autophagy is an important process in maintaining the homeostasis of nondividing neural cells , the metabolism of Apg proteins in the retina has not been widely investigated. In our present study, we have further characterized the human APG3L gene. Additionally, we have identified a mammalian homolog of a yeast autophagocytosis protein (Apg3p), which is induced by retinal IPC in a rat model, suggesting that the autophagy system may play a role in the protective effects of IPC.
Cloning of the rat and human APG3 cDNAs
A fragment of the rat Apg3 cDNA was first identified by mRNA differential display from hypoxia-treated E1A-NR3, an immortalized cell line derived from rat retinal cells and that manifests phenotypes of retinal neurons [23,24]. An oligonucleotide (5'-GGT CAA TGG TCA CAT CTA TG-3') was synthesized based on the partial APG3 cDNA and utilized as a probe to isolate the full-length cDNA from a rat retinal cDNA library (Stratagene, La Jolla, CA) .
The human APG3 cDNA was amplified from human retinal RNA using reverse transcription-polymerase chain reaction (RT-PCR). The PCR primers 5'-GCC GCT ACT CCG GCC CCA GG-3' and 5'-AAC ATA TCA GTC CCA TTA TTA GAG-3', which span the full-length coding region, were used, and PCR was performed using the Titan One Tube RT-PCR System (Roche, Indianapolis, IN) following the manufacturer's protocol. The PCR products were cloned into the pCRII vector (Invitrogen, Gaithersburg, MD) and selected by colony hybridization as described in the literature .
Genomic structure characterization and chromosomal localization of human APG3L
A lambda phage human genomic DNA library was screened using a 32P-labeled probe containing the entire human APG3P coding sequence. After hybridization, the filters were rinsed twice at room temperature with a 2x SSC, 0.1% SDS solution for 15 min. High stringency washes with 0.1x SSC and 0.1% SDS at 50 °C were performed for two rounds of 30 min each. Filters were exposed to X-ray film using an intensifying screen at -80 °C. The DNA sequence from positive clones were subcloned followed by DNA sequence analysis using an ABI (Foster City, CA) Model 377 automated DNA sequencer. Exonic fragments were determined by comparison of the obtained genomic sequences with the published human APG3 cDNA sequence .
Purified APG3L genomic DNA fragment was labeled with digoxigenin dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to metaphase chromosomes derived from PHA-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2x SSC. Specific hybridization signal was detected by incubating the hybridized slides in fluoresceinated anti-digoxigenin antibodies followed by counterstaining the nucleus with DAPI. A second experiment was conducted in which a genomic clone which had been previously mapped to 3q26 was cohybridized with the purified APG3L genomic DNA fragment. A total of 80 metaphase cells were analyzed with 45 exhibiting specific labeling.
Genomic Southern blot analysis
Ten micrograms of rat genomic DNA per assay was digested with the restriction enzymes, BamHI, BglII, EcoRI, HaeI, HindIII, NdeI, PstI, and XbaI, respectively. Digested DNAs were electrophoresed in a 0.8% agarose gel at low voltage (20 V) overnight and transferred to Hybond nylon membranes (Amersham; Piscataway, NJ). The membrane was hybridized using a full-length rat Apg3 cDNA as a probe labeled with 32P-dCTP. We hybridized the membrane at 42 °C overnight in Ultrahyb solution (Ambion; Austin, TX) with 100 μg/ml denatured salmon sperm DNA. The membrane was washed twice at 42 °C with 2x SSC/0.1% SDS and followed by two more rinses using 0.1x SSC/0.1% SDS (15 min each time). The membrane was then exposed to Kodak (Rochester, NY) Biomax film.
Northern blot analysis
Total RNA was isolated from rat retina and other tissues. The same amount of RNA (20 μg) from each tissue was loaded for northern blot analysis. A 1.4-kb rat Apg3 cDNA was labeled with 32P-α-dCTP using a Nick Translation kit (Invitrogen, Gaithersburg, MD). Ultrahyb hybridization solution (Ambion, Austin, TX) was used according to the manufacturer's protocol. After hybridization with the rat Apg3 probe, the RNA blots were stripped and reprobed with an oligonucleotide specific to the 18S ribosomal RNA (for tissue distribution) or GADPH cDNA probe (for IPC and ischemia experiments).
Transfection, expression, and immunocytochemistry
The rat Apg3 cDNA was cloned into the pCDNA3/V5/His vector (Invitrogen) in frame with the V5 epitope and His-tag. The expression construct and pCDNA3/V5/His control vector were transfected into COS-7 and Chinese hamster ovary (CHO) cells using lipofectamin (Invitrogen). The cells were treated, 48 h after transfection, with DRAQ5TM (Alexis) for 10 min for nucleic staining and then fixed, permeabilized, and stained with the anti-V5 antibody followed by an FITC-conjugated secondary antibody as described previously .
Expression, subcellular fractionation, and western blot analysis
The transfected CHO cells were lysed. The insoluble fraction was pelleted by centrifugation at 30,000 μg for 15 min. The supernatant was then centrifuged at 100,000 xg for 30 min to separate microsomes from cytosolic proteins . The cytosolic supernatant was centrifuged again to remove any contaminant microsomes. Both the microsomal and insoluble pellets were washed twice and then solubilized in 1% SDS. Equal amounts of protein (6 μg) from each fraction were subjected to SDS-PAGE and western blot analysis, using antibody specific to the His-tag as described previously .
Retinal ischemic preconditioning and ischemia
All animal procedures and interventions followed the guidelines of the Association for Research in Vision and Ophthalmology. Retinal ischemic preconditioning and ischemia was performed as previously described . Briefly, to induce retinal ischemia, rats were anesthetized with ketamine and xylazine (50 mg/kg and 6 mg/kg, intraperitoneally). The cornea was then anesthetized with 1 drop of 0.5% proparacaine (Alcon, Fort Worth, TX). IPC was achieved by ligation of the retinal arteries of the right eye for 5 min followed by 5 h reperfusion. Retinal ischemia and reperfusion were monitored by fundus examination by direct ophthalmoscopy. The prolonged retinal ischemia was induced by ligation of the retinal arteries for 45 min followed by 5 h reperfusion. The left eye of each animal was sham operated and used as the control. At the end of the reperfusion periods, animals were sacrificed by an overdose of pentobarbital, and the eyes were enucleated and the retinas dissected. Five retinas from each group were pooled for RNA preparation.
Cloning and sequence analysis of the rat Apg3 cDNA
The full-length sequence of the rat cDNA consists of 1,425 bp, including 285 bp of the 5' untranslated region (UTR), 945 bp of the coding region and 195 bp of the 3' UTR (GenBank accession AF175224). The 3' UTR contains a polyadenylation signal AATAAA and a poly-A tail. The open reading frame (ORF) encodes a protein of 314 amino acid residues (Figure 1). The deduced protein has a calculated molecular mass of 35,822 and a pI of 4.49.
The cloned sequence is not identical to any known genes but shares 90% and 97% sequence identity to the human Apg3 at the nucleotide and amino acid levels, respectively. Thus, we conclude that the identified sequence is rat Apg3 (rAPG3). rApg3 also showed sequence homology to two yeast autophagocytosis proteins. It shares 47.7% and 53.0% sequence similarity to AUT1_YEAST, a yeast autophagocytosis protein from Saccharomyces cerevisiae at the nucleotide and amino acid levels, respectively. It also shares 51.8% nucleotide sequence similarity and 50.5% amino acid sequence similarity to AUT_YEASTSp, another autophagocytosis protein from Schizosaccharomyces pombe (GenBank Accession AL022070).
Genomic Southern blot analysis
Genomic Southern blot analysis with multiple enzymes all produced a simple banding pattern, suggesting that the APG3L gene is unlikely to belong to a multigene family (Figure 2A). Genomic Southern blot analysis is a commonly used method to estimate if a new gene belongs to a gene family . Although it is not an accurate measure, this method can provide a fairly reliable estimate based on the complexity of the banding pattern [31-33]. In our Southern blot, the Apg3L gene showed several bands after restriction digestion. Given the length of the full-length gene of Apg3L and the sizes of the positive bands, we cannot conclude that these bands are from multiple genes, after we add the all the bands together. In other words, the multiple bands in each lane can be ascribed to a single gene, based on their sizes. A genomic Southern blot banding pattern of a gene in a gene family "would be" more complex than this  and might have larger sized bands. We have also performed a global GenBank search based on sequence homology, using several databases . No search identified any genes with high sequence homology with Apg3L further supporting the results of the genomic Southern blot analysis, suggesting that Apg3L is a single copy gene.
Organization and chromosomal localization of the human APG3L gene
By screening a human genomic DNA library, we identified clones containing APG3L. Sequencing results show that the putative human APG3L gene covers approximately 30 kb and contains 12 exons and 11 introns (Figure 2B). The sizes of exons vary from 35 to more than 400 bp, and the lengths of introns are from 255 bp to more than 5 kb (Table 1). Exon 1 contains one putative start codon ATG, and exon 12 the translation termination codon TAA.
A human APG3L genomic DNA clone labeled with dUTP was used to perform fluorescent in situ hybridization FISH on human lymphocyte metaphase chromosomes (Figure 2C). Initial results based on size, morphology, and banding pattern of the labeled chromosomes indicated that the probe specifically hybridized to the long arm of chromosome 3. Subsequent cohybridization of this APG3L clone with a genomic clone previously mapped to 3q26, and measurements of ten of these specifically hybridized chromosomes demonstrated that the human APG3L gene is located at a position that is 19% of the distance from the centromere to the telomere of chromosome arm 3q, an area that corresponds to locus 3q13.1 (Figure 2D) .
Tissue distribution of the Apg3 mRNA
As shown by northern blot analysis using 20 μg of total RNA from each rat tissue and the rApg3 cDNA as a probe, the Apg3 transcript was identified as a single band of 1.4 kb, which matches the length of the cloned cDNA. The Apg3 mRNA was detected in most of the rat tissues analyzed (Figure 3). After normalization with the 18S ribosomal RNA, the highest level of the Apg3 mRNA was found in the retina.
Expression and subcellular localization of Apg3 protein
As shown by immunocytochemistry, an antibody against the V5 tag, which is fused to the recombinant Apg3, detected Apg3p in the transfected CHO and COS-7 cells (Figure 4A,B). The cytoplasm was evenly stained without any detectable punctate signal, suggesting a cytosolic localization in these cells under the assay conditions. The antibody did not stain the nucleus or plasma membrane (Figure 4A,B). The same staining pattern was observed with the anti-His-tag antibody (data not shown). Neither antibody showed detectable signals in untransfected cells.
Subcellular fractionation by differential centrifugation followed by western blot analysis demonstrated that Apg3p is present as a single band only in the cytosolic fraction but not in microsomal, or insoluble fractions containing the nucleus, mitochondria, or plasma membrane (Figure 4C). The apparent molecular weight of the recombinant protein is approximately 47 kDa which is substantially higher than the calculated molecular weight 40.5 kDa based on the sequence of the fusion protein (Apg3p fused with the V5 epitope, His-tag, and linker sequence from the vector), suggesting significant posttranslational modifications of Apg3p.
Induced expression of Apg3 by retinal IPC but not by prolonged ischemia
In the retina after IPC, Apg3 mRNA levels were elevated compared to the retinas of sham controls. In contrast, retinal mRNA levels of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were not changed by IPC when the same RNA blot was stripped and probed again by the GAPDH cDNA (Figure 5A). Semiquantification by densitometry showed a 1.7 fold increase in Apg3 mRNA levels in the IPC-treated retinas over the sham controls after normalization with the GAPDH levels (Figure 5B).
Apg3 mRNA levels were also determined in the retina after 45 min of sustained ischemia followed by 5 h reperfusion. In contrast to the effect of IPC, the sustained ischemia decreased Apg3 mRNA levels by approximately two fold compared to respective sham controls after normalization by GAPDH mRNA levels, suggesting that the increased Apg3 expression is a specific response to IPC but not to ischemia in general (Figure 5C,D).
IPC has been shown to protect the retina from subsequent damaging ischemic events . It is speculated that the protective action results from the induction of endogenous protective factors or reduction in endogenous harmful proteins. This process is complex and likely involves multiple, yet to be identified genes. Apg3p, a mammalian homolog of Apg3/Aut1p yeast autophagocytosis protein , is one of the genes induced by IPC but not by sustained retinal ischemia in a rat model, suggesting that it is a candidate for IPC-induced protective factors.
Autophagy is a degradative pathway by which cells sequester bulk cytosol into autophagosomes, double-membrane vesicles and deliver them to the vacuole or lysosome for recycling (for recent review articles see [15,16,18,37-42]). In yeast, autophagocytosis is a stress-induced process [36,43]. This process plays an important role in the adaptation of cells to conditions of nutrient limitation and is critical for cell survival under certain stress conditions. In multicellular organisms, cellular autophagy has been identified as a process for regulating cellular protein turnover in cells [44,45]. Under starvation conditions autophagy has also been identified as a strategy for eukaryotic cells to survive. When nutrition is rich, the autophagy system is also utilized to transport and recycle via the cytoplasm to vacuole targeting (Cvt) pathway. The formation process of autophagosomes is conserved between yeast and multicellular organisms. In all systems, the induction of autophagy requires the membrane binding of Aut7/Apg8 protein and a conjugation process mediated by Apg3p/Aut1p [27,46].
The Apg3p/Aut1p proteins play critical roles in autophagy in both yeast and multicellular organisms. In an AUT1 gene-deficient yeast strain, the yeast was viable under normal growth conditions but showed a significantly decreased survival rate during starvation compared to wild-type strains, suggesting that AUT1 has a protective effect under stress conditions . In 2002, the human APG3P/AUT1P gene was cloned, identified, and the interactions between hApg3p and hApg7p, three human Apg8p homologues (GATE-16, GABARAP, and MAP-LC3) were demonstrated . We have added to the information about the human Apg3/Aut1p gene by mapping the exons and introns, identifying the intron splice sites and locating the gene to the long arm of human chromosome 3. Because the human APG3P/AUT1P has been identified, it is interesting to compare the similarity and difference between human and rat APG3L gene. In addition, the novel information for the human APG3L gene structure and chromosomal localization provides the basis for future mutation screening to determine if it is associated with genetic diseases in the eye or other tissues. In Drosophila, DrAut1, the homolog of yeast Aut1, was also identified and shown to be an important autophagy protein . Knockdown DrAut1 expression level not only blocked the induction of autophagy in fat body cells of wandering larvae but also shortened the lifespan of adult Drosophila . The DrAut1 protein was also located in the cytosol. This result is consistent with the hAPG3p (Figure 4), confirming that both human and Drosophila Apg3p homologues play a role during autophagy in the cytosol.
The roles of autophagy in diseases are still controversial; autophagy may act as "a double-edged sword" (for detailed review see [17,19,39]). On the one hand, autophagy is known as another type of programmed cell death (type II) in vertebrates. It is a pathway to eliminate damaged cells after cytotoxic injury [48,49]. The levels of beclin 1, an autophagy protein, are low in MCF7 carcinoma cells  and the incidence of tumorogenesis increased in Beclin1 heterozygous knockout mice . On the other hand, autophagy serves as a mechanism by which cells eliminate damaged proteins that are necessary for normal growth but harmful under certain stressful conditions. In an early response to stress, cells produce molecular chaperones, such as heat shock proteins, to repair damaged proteins. When these damaged proteins are beyond rescue by molecular chaperones, they are transported into autophagosomes for degradation to prevent potential cytotoxicity from the damaged proteins [36,43]. Autophagy is therefore suggested to play a protective role against stress at the subcellular levels . Recently, it has been reported that autophagy could protect cancer cells against radiation damage . It has also been shown that the induction of autophagocytosis in an insulinoma cell line, NIT, (having the non obese diabetic-NOD, genotype and transgenic for SV40 large T antigen) through starvation leads to an increased resistance to oxidative stress (H2O2) and to protection from apoptotic cell death . The proposed mechanism of this protective effect in mammalian cells is an autophagic transport of cytoplasmic ferritin into lysosomes, thereby reducing the lysosomal membrane damage through binding of reactive oxygen species (e.g., iron) [52,53]. Autophagic transport of ferritin into lysosomes might be responsible for reduction of lysosomal reactive oxygen species and prevention of cell damage through autolysis [52,53]. These observations suggest a mechanism for autophagic cell protection, which might be applicable to our model of IPC. It will be interesting to see if the induction of Apg3 contributes to the protective effect of IPC or associates with the autophagic cell death pathway.
Our present study showed that Apg3 is induced by the nondamaging IPC procedure but not by 45 min of ischemia which induces significant cell death in the retina. These results support the hypothesis that the upregulation of Apg3 may play neuroprotective roles during IPC in the retina. Autophagy may play a role in eliminating damaged proteins from retinal neurons and reducing their cytotoxicity as well as decreasing reactive oxygen species during ischemia, and thus, contributes to the protective effect of IPC. This is noteworthy, because the ligation of the retinal arteries could compress the retinal ganglion cell axons. It will be interesting to investigate the effects of nerve cell insults on the regulation of autophagy in the future.
In summary, these findings suggest a potential association between the autophagy process and the protective effects of IPC in the retina, and will contribute to a better understanding of the mechanisms of IPC protection as well as the autophagy pathway in multicellular organisms.
The authors would like to thank Dr. Gail Siegel of the University of Rochester for providing E1A-NR3 cells. This study was supported by grants from National Institutes of Health, EY015650 and EY012231, research awards from American Diabetes Association, Juvenile Diabetes Research Foundation, and a South Carolina Research Initiative Grant.
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