Age-related dysfunction of the lacrimal gland and oxidative stress: evidence from the Cu,Zn-superoxide dismutase-1 (Sod1) knockout mice.

An imbalance between free radical generation and radical scavenging antioxidant systems results in oxidative stress, which has been associated with cell injury observed in many age-related diseases. The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase-1 (Sod1) in mice leads to many different phenotypes that resemble accelerated aging. In this study we examined the morphologic features and the secretory functions of the lacrimal glands in Sod1(-/-) mice. Lacrimal glands showed atrophy of acinar units; fibrosis; infiltration with CD4(+) T cells, monocytes, and neutrophils; increased staining with both 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine; increases in apoptotic cells; and the presence of the epithelial-mesenchymal transition in senescent Sod1(-/-) mice. Electron microscopy findings revealed evidence of epithelial-mesenchymal transition, presence of swollen and degenerated mitochondria, and the presence of apoptotic cell death in the lacrimal glands of senescent Sod1(-/-) mice. These alterations were also associated with the accumulation of secretory vesicles in acinar epithelial cells, decreased production of both stimulated and nonstimulated tears, and a decline in total protein secretion from the lacrimal glands. Our results suggest that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated lacrimal gland alterations.

Overexpression of optineurin E50K disrupts Rab8 interaction and leads to a progressive retinal degeneration in mice.

Glaucoma is one of the leading causes of bilateral blindness affecting nearly 8 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure (IOP). However, patients with normal tension glaucoma (NTG), a subtype of primary open-angle glaucoma (POAG), develop the disease without IOP elevation. The molecular pathways leading to the pathology of NTG and POAG are still unclear. Here, we describe the phenotypic characteristics of transgenic mice overexpressing wild-type (Wt) or mutated optineurin (Optn). Mutations E50K, H486R and Optn with a deletion of the first (amino acids 153-174) or second (amino acids 426-461) leucine zipper were used for overexpression. After 16 months, histological abnormalities were exclusively observed in the retina of E50K mutant mice with loss of RGCs and connecting synapses in the peripheral retina leading to a thinning of the nerve fiber layer at the optic nerve head at normal IOP. E50K mice also showed massive apoptosis and degeneration of entire retina, leading to approximately a 28% reduction of the retina thickness. At the molecular level, introduction of the E50K mutation disrupts the interaction between Optn and Rab8 GTPase, a protein involved in the regulation of vesicle transport from Golgi to plasma membrane. Wt Optn and an active GTP-bound form of Rab8 complex were localized at the Golgi complex. These data suggest that alternation of the Optn sequence can initiate significant retinal degeneration in mice.

Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization.

PURPOSE: To investigate the transcriptional factors associated with epithelial-mesenchymal transition (EMT) in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: Paraffin sections of CNV obtained from patients with AMD (n = 12) were stained for transcriptional factors related to EMT, i.e., Snail, Slug, SIP1, and Twist. As a control, postmortem sections of ocular normal tissue were used. Furthermore, using a human retinal pigment epithelial (RPE) cell line (ARPE-19), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence microscopy were performed to explore the cellular localization and expression levels of EMT-associated transcriptional factors upon cytokine stimulation. RESULTS: Of 12 specimens, 11 CNV tissues (91.6%) showed staining for Snail localized in cellular nuclei, particularly in those of RPE cells. Snail was strongly co-localized with α-smooth muscle antigen (SMA) in RPE cells. In contrast, postmortem human retina showed no Snail staining in RPE cells. Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina. In ARPE-19 cells, RT-PCR and immunofluorescence microscopy showed that Snail mRNA was upregulated by transforming growth factor (TGF)-ß and VEGF stimulation. Furthermore, TGF-ß induced relocalization of Snail to the nucleus in RPE cells. CONCLUSIONS: The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.

YPEL5 protein of the YPEL gene family is involved in the cell cycle progression by interacting with two distinct proteins RanBPM and RanBP10.

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.

Retinal dysfunction and progressive retinal cell death in SOD1-deficient mice.

The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase (SOD1) in mice leads to many different phenotypes that resemble accelerated aging. The purpose of this study was to examine the morphology and physiology of the sensory retina in Sod1(-/-) mice. The amplitudes of the a- and b-waves of electroretinograms elicited by stimuli of different intensity were reduced in senescent Sod1(-/-) mice, and this reduction in amplitude was more pronounced with increasing age. Retinal morphometric analyses showed a reduced number of nuclei in both the inner nuclear cell layer and outer nuclear cell layer. Electron microscopy revealed swollen cells and degenerated mitochondria in the inner nuclear cell and outer nuclear cell layer of senescent Sod1(-/-) mice indicating necrotic cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed no significant differences in the number of apoptotic cells between Sod1(-/-) and wild-type mice, and activated caspase-3 could not be detected in the retina of Sod1(-/-) mice. In addition to the age-related macular degeneration-like phenotypes previously reported, Sod1(-/-) mice also present progressive retinal degeneration. Our results indicate that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated retinal degeneration.

The role of mitochondrial superoxide anion (O2(-)) on physiological aging in C57BL/6J mice.

Much attention has been focused on the mitochondrial superoxide anion (O2(-)), which is also a critical free radial produced by ionizing radiation. The specific role of the mitochondrial O2(-) on physiological aging in mammals is still unclear despite wide-spread evidence that oxidative stress is involved in aging and age-related diseases. The major endogenous source of O2(-) is generated as a byproduct of energy metabolism from mitochondria. In order to better understand how O2(-)relates to metazoan aging, we have comprehensively examined age-related changes in the levels of oxidative damage, mitochondrial O2(-) production, mitochondrial antioxidant enzyme activity and apoptosis induction in key organs of an inbred mouse strain (C57BL/6J). Oxidative damage accumulated and excess apoptosis occurred in the brain, oculus and kidney with aging, but comparatively little occurred in the heart and muscle. These rates are correlated with O2(-) levels. Mitochondrial O2(-) production levels increased with aging in the brain, oculus and kidney, and did not significantly increased in the heart and muscle. In contrast to O2(-) production, mitochondrial SOD activities increased in heart and muscle, and remained unchanged in the brain, oculus and kidney with aging. These results suggest that O2(-) production has high organ specificity, and oxidative damage by O2(-) from mitochondria mediated apoptosis can lead to organ atrophy and physiological dysfunction. In addition, O2(-) from mitochondria plays a core role in physiological aging.

Expression of the mRNA and protein of serine racemase in primary cultures of rat neurons.

Real-time quantitative PCR, Western blot and in situ hybridization techniques were employed to clarify the presence of serine racemase in the primary cultures of rat neurons. We have detected both serine racemase mRNA and protein in the cultured neurons. Both the mRNA and the protein levels in the neurons are higher than those in the astrocytes. Sequential detection of serine racemase mRNA and MAP2 immunoreactivity also revealed that serine racemase and MAP2 are co-localized in the cultured neurons. These data are the first to demonstrate that a substantial amount of serine racemase exists in the cultured neurons.

Characterization of AOC2 gene encoding a copper-binding amine oxidase expressed specifically in retina.

We have previously cloned a human, retina-specific, amine oxidase gene (RAO, gene symbol: AOC2), a member of the copper-binding amine oxidase super family. AOC2 shares sequence identity with the human kidney amine oxidase gene (KAO, gene symbol: AOC1) and the vascular adhesion protein-1 gene (VAP-1, gene symbol: AOC3). For further analysis of AOC2, the sequences surrounding the human AOC2 and the complete mouse and partial rat homologue of AOC2 were cloned for characterization. Real-time quantitative PCR, in situ hybridization, and immunohistochemistry were performed to determine the specific expression of AOC2 in the mouse retina and especially in the retinal ganglion cells. Our results demonstrated that the copper-binding motif and the enzyme active site of AOC1 and AOC3 were both conserved in mouse AOC2. The human and mouse AOC2 was flanked by two genes, the Psme3 gene for PA-28 gamma subunit and, surprisingly, the AOC3 gene. Rat AOC2 contained a stop codon that terminated the peptide length to 127 amino acids. The presence of human and rat AOC pseudogene in this region, in addition to the tandemly positioned two AOC genes, indicates the possibility of successful AOC3 replication to retina-specific AOC2 for human and mouse but unsuccessful for rat.

Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head.

PURPOSE: To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS: Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O(2)) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS: The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS: The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.

Distributions of Actin, Vinculin and Fibronectin in the Duodenum of Developing Chick Embryos: Immunohistochemical Studies at the Light Microscope Level: (chick embryo/duodenal morphogenesis/immunofluorescent staining/actin/vinculin).

Microscopic studies were made on the localizations of three different cytoskeletal proteins, actin, vinculin and fibronectin, in the duodenum of developing chick embryos and chicks by an indirect immuno-fluorescent staining method with specific antibodies. Topographical changes in the distributions of these three proteins seemed to be related to stages in morphogenesis of the duodenum. In early stages of embryonic development, findings suggested interaction between actin and vinculin in the apical region of epithelial cells and between actin and fibronectin in the basal region of these cells. From this stage, vinculin and fibronectin seemed to be of importance in determination and continuity of the polarity of the duodenal epithelium, and in control of the intracellular arrangement of actin. This relation between actin and vinculin seemed to continue throughout embryogenesis. The main role of actin in epithelial cells seemed to change on day 12 from that of forming constricting bundles for morphogenesis of previllous ridges to that of microfilaments in the microvilli and the terminal web.

The serine racemase mRNA is expressed in both neurons and glial cells of the rat retina.

D-Serine, an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in mammals, is synthesized from L-serine by serine racemase. Serine racemase and D-serine have long been believed to occur predominantly in astrocytes, according to immunohistochemical studies. Recent studies have demonstrated, however, that both the mRNA and protein levels of serine racemase are considerably higher in neurons than in astrocytes in primary cultures of the rat brain and that the mRNA level of serine racemase predominates in neurons of the adult rat brain. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat retina and optic nerve head. The localization of serine racemase mRNA could be demonstrated in ganglion cells, amacrine cells, bipolar cells, horizontal cells, and Müller cells of the retina as well as in the astrocytes of the optic nerve head and the lamina cribrosa. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level in the retina and the optic nerve head. These results suggest that both the neuron- and glia-derived D-serine could modulate neurotransmission via the glycine site of the N-methyl-D-aspartate receptors in the retina.

Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins.

We identified 11 proteins that are associated with DGCR8 by immunoprecipitation assay and mass spectrometry. These proteins included Nucleolin, ILF3 and others, most of which appeared to be involved in the RNA processing or RNA transportation. We detected at least four kinds of protein complex, such as DROSHA/DGCR8, DGCR8/Nucleolin, DGCR8/ILF3 and ILF3/XPO5, by co-immunoprecipitation. The complex formation of DGCR8 with Nucleolin was dependent on RNA. Subcellular localization analysis by the immunofluorescent microscopy and immunoelectron microscopy indicated that DGCR8 locates at the nucleolus and small foci adjacent to splicing speckles in the nucleoplasm. Furthermore, the localization of DGCR8 at the nucleolus was changed by the inhibition of RNA transcription. Thus, our studies provided additional new evidence for the involvement of various protein complexes in the molecular mechanisms of apparently complex innate RNA interference machinery.

The serine racemase mRNA is predominantly expressed in rat brain neurons.

D-serine is an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in the mammalian brain. D-serine is synthesized from L-serine by serine racemase; immunohistochemical studies have long been believed to indicate that serine racemase and D-serine occur predominantly in astrocytes. However, we have recently demonstrated in the primary cultures that both the mRNA and protein levels of serine racemase are higher in neurons than in astrocytes. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat brain. Serine racemase mRNA could be demonstrated in a large number of neurons throughout the brain, especially in the forebrain such as the cerebral cortex, striatum, and hippocampus. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level. These results suggest that neuron-derived D-serine could modulate neurotransmission via the glycine site of N-methyl-D-aspartate receptors.

Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: a model of age-related macular degeneration.

Oxidative stress has long been linked to the pathogenesis of neurodegenerative diseases; however, whether it is a cause or merely a consequence of the degenerative process is still unknown. We show that mice deficient in Cu, Zn-superoxide dismutase (SOD1) have features typical of age-related macular degeneration in humans. Investigations of senescent Sod1(-/-) mice of different ages showed that the older animals had drusen, thickened Bruch's membrane, and choroidal neovascularization. The number of drusen increased with age, and exposure of young Sod1(-/-) mice to excess light induced drusen. The retinal pigment epithelial cells of Sod1(-/-) mice showed oxidative damage, and their beta-catenin-mediated cellular integrity was disrupted, suggesting that oxidative stress may affect the junctional proteins necessary for the barrier integrity of the retinal pigment epithelium. These observations strongly suggest that oxidative stress may play a causative role in age-related retinal degeneration, and our findings provide evidence for the free radical theory of aging. In addition, these results demonstrate that the Sod1(-/-) mouse is a valuable animal model to study human age-related macular degeneration.

Characterization of human and mouse TRPM2 genes: identification of a novel N-terminal truncated protein specifically expressed in human striatum.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable cation channel activated by ADP-ribose or reactive oxygen species. In human, a major transcript of 6.5 kb is expressed in various tissues, whereas a minor transcript of 5.5 kb is detected only in striatum (caudate nucleus and putamen). We found that the 5.5-kb shorter transcript is transcribed from the intron 4 of the TRPM2 gene and encodes the striatum short form protein (SSF-TRPM2) with 1289 amino acid residues as compared to the long form protein (LF-TRPM2), in which the N-terminal 214 amino acid residues are removed. The SSF-TRPM2 protein still maintained H2(O2)-induced Ca2+ influx activity. In addition, we found that the major transcripts in human and mouse start from a novel 5' non-coding exon; however, we could not detect any striatum short transcript in mouse brain. These new findings are invaluable to further study the regulation of TRPM2 gene expression and to examine the possible involvement of the TRPM2 gene in the pathophysiology of bipolar disorder.

Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region.

We have identified and cloned a novel gene (DGCR8) from the human chromosome 22q11.2. This gene is located in the DiGeorge syndrome chromosomal region (DGCR). It consists of 14 exons spanning over 35kb and produces transcripts with ORF of 2322bp, encoding a protein of 773 amino acids. We also isolated a mouse ortholog Dgcr8 and found it has 95.3% identity with human DGCR8 at the amino acid sequence level. Northern blot analysis of human and mouse tissues from adult and fetus showed rather ubiquitous expression. However, the in situ hybridization of mouse embryos revealed that mouse Dgcr8 transcripts are localized in neuroepithelium of primary brain, limb bud, vessels, thymus, and around the palate during the developmental stages of embryos. The expression profile of Dgcr8 in developing mouse embryos is consistent with the clinical phenotypes including congenital heart defects and palate clefts associated with DiGeorge syndrome (DGS)/conotruncal anomaly face syndrome (CAFS)/velocardiofacial syndrome (VCFS), which are caused by monoallelic microdeletion of chromosome 22q11.2.

Expression of slow skeletal myosin heavy chain 2 gene in Purkinje fiber cells in chick heart.

In avian, there are three slow skeletal myosin heavy chain (MHC) isoforms, slow skeletal MHC 1, 2, and 3. While slow skeletal MHC 3 has been characterized, slow skeletal MHC 1 and 2 are not yet fully studied. To determine the complete sequence of slow skeletal MHC 2, we isolated six overlapping cDNA clones, each encoding a portion of chick slow skeletal MHC 2, using the reverse transcription polymerase chain reaction (RT-PCR). The entire slow skeletal MHC 2 cDNA consisted of 5927 nucleotides including a 104 bp 3'-untranslated region and encoded 1941 amino acids. Using one of the cDNA clones, we made a probe for in situ hybridization. We also used immunohistochemistry to localize slow skeletal MHC 2 in skeletal and cardiac tissues. These studies showed that in addition to its expected expression in the adult chicken slow skeletal muscle, slow skeletal MHC 2 was expressed in the subendocardial cluster of cells and around the blood vessels within the ventricle of late embryos and adults. This isoform was not expressed in the myocardium throughout the life of the chicken. Based on morphological criteria as well as rich desmin expression, we concluded that the subendocardial cluster of cells were Purkinje cells. Although the physiological significance of the slow skeletal MHC expression remains elusive at this time, this MHC isoform may be used as a specific marker for Purkinje cells.