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The aetiologies of Alzheimer's disease (AD) are complex and multifactorial. Current therapies are largely ineffective, as the pathophysiological pathways are poorly understood. Observations in AD autopsies, as well as in vivo and in vitro observations in transgenic mice, have implicated oxidative stress as pathogenic in AD. This study used the Glutathione Peroxidase-1 knockout mouse (Gpx1--/--) model to investigate the role of antioxidant disparity in neuropathologies. Cultured neurons from control and Gpx1--/-- embryos were treated with AD-related peptides and the degree of cell loss compared. Results show that antioxidant disparity makes Gpx1--/-- cells more susceptible to Abeta toxicity. Surrogate replacement of Gpx1 with the reactive oxygen species scavenger N-acetyl cysteine and the Gpx1 mimetic ebselen, reverses the Gpx1--/-- increased susceptibility to Abeta toxicity. Such results support a role for oxidative stress in AD-related neuronal loss. This study is the first to report such findings using the Gpx1--/-- model, and supports a role for oxidative stress as one of the contributing factors, in development of AD-like pathologies.

We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)–/– mice to neuronal apoptosis following mid-cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H2O2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the phospho-inositide 3-kinase [PI(3)K]–Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI(3)K inhibitor, LY294002. The Gpx1–/– mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1–/– brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia–reperfusion. Our results suggest that the increased susceptibility of Gpx1–/– neurons to H2O2-induced apoptosis and neuronal cell death in vivo following cerebral ischaemia–reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti-apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress-mediated neuropathologies.

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H(2)O(2) accumulation), are presented. Although in most instances accumulation of H(2)O(2) affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three-fold in the glutathione peroxidase-1 (Gpx-1) -/- mouse compared with the wild-type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx-1 -/- mouse compared with the wild-type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase-3 expression is present at an earlier time point in the Gpx-1 -/- mice when compared with the wild-type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx-1 -/- mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.

The RGS proteins comprise a large family of proteins which were recently identified as negative Regulators of G-protein Signaling. They have been shown to act as GTPase Activating Proteins (GAPs) towards the Gα subunits of heterotrimeric G-proteins. In addition to this GAP activity, which has been shown to occur through the RGS domain, RGS proteins are likely to possess other functions due to the existence of other domains in these molecules (De Vries and Farquhar, 1999; Hepler, 1999). Here, we report the molecular characterization of the murine Rgs11 gene. The gene encodes a protein with high homology to human RGS11 (79.9%), containing conserved DEP (Dishevelled/EGL-10/Pleckstrin) and GGL (G protein γ-like) domains. The gene is comprised of at least 13 exons, spanning 8–9 kb. Spliced transcript variants were identified which are co-expressed with 5A3, a transcript that contains the largest ORF. Expression of mouse Rgs11 was found to be restricted to specific tissues with a unique pattern of expression observed in brain.

In this study, we demonstrate that methylation-dependent repression of the Pdha-2core promoter is mediated regionally through a consensus activating transcription factor/cAMP-responsive element-binding site located between nucleotides −54 and −62 upstream of the major transcriptional start site. Targeting of the CpG dinucleotide within thiscis-element significantly disrupts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo. DNase I footprinting experiments indicated that availability of the nuclear factor(s) binding this element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early spermatogenic gene expression. Interestingly, CpG dinucleotides associated with the hypersensitive region flanking the activating transcription factor/cAMP-responsive element-binding site appear to confer some conformational structure on the promoter since mutations at these specific CpG dinucleotides result in elevated basal levels of transcription. This raises the possibility of a potential bifunctional role for CpG dinucleotides in either methylation-dependent or -independent processes. Our data support the notion that hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.

Pyruvate dehydrogenase (PDH) is a multiunit enzymatic complex essential for the process of generating cellular energy. One of the most important of its subunits is the E1 alpha subunit. Perturbations in the expression of this subunit lead to reduced or lost function of the PDH complex as a whole, resulting in a loss of ATP production. The consequence of such perturbations can lead to neurological abnormalities, lactic acidosis, and in males, death. Pdha-2 codes for the mouse testis isoform of the E1 alpha subunit and maps to chromosome 19 (chromosome 4 in humans). This is a fortuitous evolutionary development because the somatic isoform of the E1 alpha subunit is linked to the X-chromosome, which is not only inactivated early in spermatogenesis but is represented in only half of the haploid spermatid population. Consequently, activation of the testis-specific E1 alpha subunit is essential for the progression of spermatogenesis. Despite its importance, the molecular mechanisms governing the tight tissue- and temporal-specific regulation of Pdha-2 have, until recently, remained poorly understood. In this review, we describe our current understanding of the transcriptional regulation of Pdha-2 and propose potential mechanisms that may play a role in this process.

Although reactive oxygen species have been proposed to play a major role in the aging process, the exact molecular mechanisms remain elusive. In this study we investigate the effects of a perturbation in the ratio of Cu/Zn-superoxide dismutase activity (Sod1 dismutases.O 2 - to H 2 O 2 ) to glutathione peroxidase activity (Gpx1 catalyses H 2 O 2 conversion to H 2 O) on cell growth and development. Our data demonstrate that Sodl transfected cell lines that have an elevation in the ratio of Sod1 activity to Gpx1 activity produce higher levels of H 2 O 2 and exhibit well characterised markers of cellular senescence viz. slower proliferation and altered morphology. On the contrary, Sod1 transfected cell lines that have an unaltered ratio in the activity of these two enzymes, have unaltered levels of H 2 O 2 and fail to show characteristics of senescence. Furthermore, fibroblasts established from individuals with Down syndrome have an increase in the ratio of Sod1 to Gpx1 activity compared with corresponding controls and senesce earlier. Interestingly, cells treated with H 2 O 2 also show features of senescence and/or senesce earlier. We also show that Cip1 mRNA levels are elevated in Down syndrome cells, Sod1 transfectants with an altered Sod1 to Gpx1 activity ratio and those treated with H 2 O 2 , thus suggesting that the slow proliferation may be mediated by Cip1. Furthermore, our data demonstrate that Cip1 mRNA levels are induced by exposure of cells to H 2 O 2 . These data give valuable insight into possible molecular mechanisms that contribute to cellular senescence and may be useful in the evolution of therapeutic strategies for aging.

Precise temporal and tissue-specific expression of genes during spermatocyte differentiation is crucial for the formation of functional spermatozoa. However, the mechanisms that regulate gene expression during spermatogenesis are poorly understood. One testis-specific gene, Pdha-2, is beginning to emerge as a potentially important model for the study of these events. This review focuses on our current understanding of the expression and regulation of Pdha-2 during spermatogenesis.

The type I interferons (IFNs) are a family of multifunctional cytokines which includes the 15 IFN alpha subtypes and IFN beta. These IFNs compete for binding to cell surface receptors. However, murine cells transfected with a cDNA for a human IFN alpha receptor (IFNAR) developed an antiviral response only to human IFN alpha B, but not to human IFN alpha 2 nor -beta(1). In this study we show, using a panel of CHO-human chromosome 21 hybrid cell lines which all express IFNAR, that only those containing the region 21q22.2 to 21q22.3 transduce signals for IFN responses. Two such hybrid cell lines responded to IFNs alpha 2, -alpha B and -beta by induction of 2‘-5‘ oligoadenylate synthetase and resistance to viral infection. Other hybrid cell lines, that lacked the region 21q22.2-3, failed to transduce signals as above; even though they expressed IFNAR and bound human IFN alpha 2, -alpha B, and -beta. These data demonstrate that a gene(s) located in the region 21q22.2-3 encodes a factor(s) which is necessary for signaling but does not influence ligand binding. This factor is not the cofactor required for IFN gamma signaling which is located in the region 21p to 21q22.1(2).

The increase in Concanavalin A (ConA) binding to sarcolemmal membranes of rat skeletal muscle following denervation has been attributed to conformational changes in membrane glycoproteins resulting in the unmasking of previously cryptic ConA binding sites (Leung et al., 1982). In this study, analysis of lectin binding patterns to alpha-fucosidase- or sialidase-treated sarcolemmal membranes reveals that the fucose moieties of carbohydrate structures may be principally involved in the unmasking process. By contrast, sialic acid has no apparent effect on the availability of the number of ConA binding sites, but plays a significant role in the masking of other lectin recognition sites.

The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic (Sod-1 tg) mouse. To investigate this hypothesis, they crossed the Gpx-1 -/- mouse with the Sod-1 tg and subjected the cross to a mouse model of ischemia/reperfusion. Two hours of focal cerebral ischemia followed by 24 hours of reperfusion was induced via intraluminal suture. The Sod-1 tg/Gpx-1 -/- cross exhibited no neuroprotection when infarct volume was measured; indeed, infarct volume increased in the Sod-1 tg/Gpx-1 -/- cross compared with the wild-type mouse. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to ischemia/reperfusion injury.

The mechanisms leading to neurodegeneration are complex and multifactorial. Oxidative stress has been identified as an important constituent in this process and the use of transgenic and knockout mice has allowed the role of key components of the antioxidant pathway to be evaluated. In this study, we have used mice lacking the glutathione peroxidase-1 gene in order to determine the consequences of a reduced capacity to neutralize hydrogen peroxide toward the pathological outcomes following cold-induced brain injury. Analysis of brain cryosections using TUNEL staining revealed a significant increase in brain cell death in knockout mice compared to that seen in wild-type mice. Interestingly, cell death appeared to be uncoupled to a neuro-inflammatory response which was observed in both knockout and wild-type mice but which proceeded in an accelerated manner in glutathione peroxidase-1 knockout mice at 24 h, rapidly diminishing by 96 h postinjury. Our data suggest an important role for glutathione peroxidase-1 in modulating molecular pathways involved in both the level of cell death and inflammatory cascades in brain through its antioxidant capacity in regulating levels of oxygen species such as hydrogen peroxide.

Total or partial trisomy of chromosome 21 occurs with relatively high frequency and is responsible for the occurrence of Down syndrome. Phenotypically, individuals with Down syndrome display characteristic morphological features and a variety of clinical disorders. One of the challenges for researchers in this field has been to ascertain and understand the relationship between the Down syndrome phenotype with the gene dosage effect resulting from trisomy of chromosome 21. Much attention therefore, has been given towards investigating the consequences of overexpressing chromosome 21-linked genes. In particular, an extensive analysis of SOD1 and APP have provided important insights as to how perturbations in the expression of their respective genes may contribute to the Down syndrome phenotype. In this review we will highlight studies which support a key role for SOD1 and APP in the pathogenesis of neural abnormalities observed in individuals with Down syndrome. Central to this relationship is how the redox state of the cell is affected and its consequences to neural function and integrity.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

Publisher Summary In addition to mental retardation, individuals with Down syndrome suffer from congenital heart defects, in utero growth retardation, increased susceptibility to infection; and a higher incidence of leukemia. These individuals show abnormalities of the viscerocranium those result in the characteristic facial features of Down syndrome; and features of premature aging. All individuals develop Alzheimer-type neuronal pathology. The involvement of reactive oxygen species (ROS) in some of the Down syndrome pathologies was initiated after assignment of the gene coding for Cu/Zn-superoxide dismutase (also known as Sodl) to chromosome21 in humans. All individuals with Down syndrome are either trisomic for the entire chromosome or part thereof. Increased gene dosage for Sodl has been proposed to contribute to the premature aging and/or mental retardation that occur as part of the syndrome. In gaining an understanding of how elevated Sodl levels may contribute to various pathologies in Down syndrome, this chapter delineates the physiological role for Sodl within cells. Elevated Sodl activity in Down syndrome could result in the accumulation of H202, which through the Fenton reaction could lead to a loss of cellular function through damage to macromolecules. The chapter discusses in detail the Sodl and Gpx1 expression levels in Down syndrome, premature aging of Down syndrome individuals, and apoptosis related to Down syndrome—some of the neurodegenerative symptoms and thymic destruction in Down syndrome may be because of the oxidative stress caused by the imbalance in the Sod/Gpx1 ratio, which in turn triggers apoptosis directly or confers an increased susceptibility of cells to apoptotic stimuli. Down syndrome is a situation where the antioxidant balance is affected because of gene dosage. Damage to biologically important macromolecules resulted because of the inability to prevent oxidative interactions. This accumulated macromolecular damage may be responsible for the abnormalities that are seen as part of the syndrome.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. How these genes are regulated during spermatogenesis is poorly understood. However, the elucidation of these mechanisms has significant implications for both medicine and the primary livestock industry. The delineation of this process is of particular interest and, in this study, Pdha-2, a gene which codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex, has been used as a model. This review focuses on current knowledge about its expression and regulation during spermatogenesis.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex. To elucidate the mechanisms regulating its expression in vivo, we have begun to investigate the Pdha-2 promoter in transgenic mice. In this paper, a construct containing 3.0 kb of promoter and upstream sequences is reported to be sufficient for directing the testis-specific expression of a CAT reporter gene in mice harbouring the transgene. Similarly to the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. However, the 3.0-kb Pdha-2 promoter is not active in somatic tissue suggesting that repressor elements may be present within these sequences.