Lack of glutathione peroxidase-1 exacerbates Abeta-mediated neurotoxicity in cortical neurons.

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.

Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury.

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.

Identification and characterization of alternatively spliced murine Rgs11 isoforms: genomic structure and gene analysis.

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 G(alpha) 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 gamma-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.

Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an activating transcription factor/cAMP-responsive element-binding site.

In this study, we demonstrate that methylation-dependent repression of the Pdha-2 core 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 this cis-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.

Oxidative stress and neural dysfunction in Down syndrome.

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.

Review: Pdha-2, past and present.

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.

Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

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.

Cu/Zn-superoxide dismutase and glutathione peroxidase during aging.

During oxidative metabolism harmful reactive oxygen species (ROS) are generated. These species are neutralized by antioxidant enzymes. Firstly, superoxide dismutase (Sod) converts superoxide radicals (.O2-) to hydrogen peroxide (H2O2). Thereafter catalase (Cat) and glutathione peroxidase (Gpx) independently convert this to water. An imbalance in the ratio of Sod to Gpx and Cat results in the accumulation of H2O2 which may participate in the Fenton reaction, resulting in the formation of noxious hydroxyl radicals. These ROS are highly reactive and cause damage to macromolecules such as DNA, protein and lipids. We propose that it is the balance in the activity of the Sod to Gpx plus Cat ratio (Sod/(Gpx plus Cat)) that is an important determinant of cellular aging. This is based on our observation that an altered Cu/Zn-superoxide dismutase (Sod1)/(Gpx1 plus Cat) ratio exists in the brain of aging mice and that this correlates with increased lipid damage. Conversely, aging liver and kidney have an unaffected Sod1/(Gpx1 plus Cat) ratio and lipid damage is not increased with aging. We also examine the Sod1 to Gpx1 ratio in Down syndrome tissue and show that all organs have an altered ratio. This may contribute to the premature aging seen in these individuals. We show that binding of a p50/p65 complex to an NF-kappa B consensus sequence is enhanced by H2O2 treatment in NIH3T3 cells. Thus an altered Sod1/(Gpx1 plus Cat) ratio may also affect gene expression by altering the binding and/or availability of transcription factors to DNA.

Changes in the levels of enzymes which modulate the antioxidant balance occur during aging and correlate with cellular damage.

Oxidative metabolism produces a flux of superoxide anions that must be removed from the cellular environment if the cell is to survive. The levels of antioxidant enzyme involved in the elimination of superoxide anions and/or hydrogen peroxide were investigated in an attempt to correlate any changes in the levels of these enzymes during aging with changes in free radical mediated cellular damage. Cu/Zn superoxide dismutase (Sod1), glutathione peroxidase (Gpx1) and catalase levels were measured in a number of organs during murine aging. Sod1 enzyme activity rose during aging in all organs studied, while the levels of both Gpx1 and catalase showed organ specific profiles. Both organs in which lipid peroxidation damage (which was used as a marker of free radical mediated damage) increased with age, namely the brain and small intestine, also showed a significant increase in the ratio of Sod1 to Gpx1 enzyme activity. In organs where either the ratio of Sod1/Gpx1 activity or Sod1/catalase levels (in the lung only) ratios were maintained during aging, no increased lipid peroxidation damage was detected. In the lung where Sod1/Gpx1 ratio did increase, Sod1/catalase remained constant and this was able to provide protection during aging. Thus our data shows that alterations in the balance between first and second steps of the antioxidant pathway correlate with cellular damage, and that this may contribute to the aging changes seen in some organs.

A model for understanding gene regulation during spermatogenesis: the mouse testis Pdha-2 promoter.

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.

Pdha-2: a model for studying transcriptional regulation in early spermatocytes.

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.

Differential expression of two testis-specific transcripts of the mouse Pdha-2 gene during spermatogenesis.

Analysis of the expression of the testis-specific isoform of the mouse pyruvate dehydrogenase E1 alpha subunit gene (Pdha-2) during various stages of spermatogenesis has shown that a 2.0-kb Pdha-2 mRNA is initially transcribed in meiotic prophase. The initial appearance of Pdha-2 mRNA precedes that of Pgk-2 and corresponds to the appearance of Ldh-3 mRNA. A second Pdha-2 1.7-kb transcript is present in post-meiotic round spermatids. Polysomal analysis of purified spermatogenic cell populations demonstrates that the 2.0-kb mRNA species is translated in diploid, pachytene spermatocytes and the 1.7-kb mRNA species is translated in round spermatids, although a large proportion is present on the nonpolysomal fraction and may be stored for use in later stages of spermiogenesis.

Mouse testis Pdha-2 promoter upstream sequences confer tissue-and temporal-specific activity in transgenic mice.

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.

Temporal and tissue-specific interactions involving novel transcription factors and the proximal promoter of the mouse Pdha-2 gene.

We have determined by deletion analysis that the most proximal region of the Pdha-2 promoter between nucleotide position -187 to +22 harbors a transcriptionally active core. This "core" promoter directs high levels of CAT (chloramphenicol acetyltransferase) reporter gene transcription in HeLa cells. DNase I footprinting of the proximal promoter revealed four regions of protection. One of these contains the consensus sequence for the Sp1 binding site and another the ATF/CREB binding site. The cis-sequences of the remaining two protected regions (designated MEP-2 and MEP-3; Mouse E1 alpha Promoter site) show no apparent consensus homology with cis-elements of other known transcription factors. Results of electrophoretic mobility shift assays confirm that the ATF/CREB and MEP binding sites interact in a characteristic and specific manner with factors present in nuclei of both testis and somatic tissue. The factor which recognizes the MEP-3 motif appears to be ubiquitous, whereas the MEP-2-protein complexes were tissue-specific. Interestingly, formation of a complex involving MEP-2 and a putative testis-specific binding factor (tau-MEP-2BF) is first observed in the testis of 2-week-old mice, this correlates with the expression of Pdha-2. In contrast, the formation of complexes between the MEP-2 binding site and a somatic variant of MEP-2BF (sigma-MEP-2BF) decreases in the testis as spermatogenesis proceeds. Our results suggest that 1) the MEP-2 binding factors are temporally regulated during spermatogenesis, and 2) interactions involving these factors with the MEP-2 cis-element may be important for modulating Pdha-2 expression.

Transcriptional expression of a testis-specific variant of the mouse pyruvate dehydrogenase E1 alpha subunit.

An isogene of the E1 alpha subunit of the mouse pyruvate dehydrogenase complex was shown in a previous study to map to chromosome 19. Here we demonstrate using Northern blot analysis that this gene is expressed in a testis-specific fashion. Two testis-specific E1 alpha transcripts were detected: (1) a 2.0-kb transcript that is abundant in pachytene cells but also detectable during earlier stages of spermatogenesis, and (2) a shorter 1.7-kb transcript detectable only in round spermatids. Analysis of both transcripts following RNase H digestion revealed that the smaller message is not derived from the shortening of the poly(A) tail and most likely results from the alternate use of two polyadenylation signals. Finally, analysis of polysomes isolated from 20- and 80-day-old mouse testes demonstrated that the 2.0-kb transcript is associated with the polysomal fraction, suggesting that this message is transcribed and actively translated at the same time. We conclude from these results that expression of the testis-specific E1 alpha is initially expressed during the meiotic prophase stage of spermatogenesis and not under any apparent post-transcriptional regulation.

Denervation-induced changes in lectin binding to sarcolemmal glycoproteins: exposure of cryptic recognition sites.

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.

Cardiac troponin T gene expression in muscle.

We have been analyzing the regulatory regions of the cardiac troponin T gene promoter as a mean toward understanding the mechanisms that govern the transcription of genes which are cross-expressed in cardiac and skeletal muscles during development. By analyzing the activities of mutant cardiac troponin T gene promoter by transient transfection of primary embryonic muscle cells, we showed that both common and distinct elements are required for activity of the cardiac troponin T promoter in these embryonic muscle cells. In skeletal muscle the minimal promoter sufficient to direct activity of the cardiac troponin T promoter is only 99 nucleotides upstream from the transcription initiation site. Within the distal half of this promoter are two tandem copies of a conserved hexanucleotide sequence (5'-CATTCCT-3') we termed the 'M-CAT motif'. Since mutation of either one of the M-CAT motifs abolishes promoter activity, we concluded that both M-CAT motifs are essential for activity of the promoter. The above minimal promoter is insufficient to confer promoter activity in embryonic cardiocytes. In these cells an additional 48-nucleotide region approximately 100 nucleotides upstream of the minimal promoter is needed for efficient promoter activity. We have named this region the 'cardiac element'. This element contains a conserved sequence motif found in other muscle gene promoter. The cardiac element can also act irrespective of orientation and is relatively independent of position, characteristics that are like transcriptional enhancers. This element alone, however, is insufficient to direct cardiac promoter activity. Activity of the 48-nucleotide cardiac element is dependent on either direct or indirect interaction with the downstream M-CAT motifs because mutation of either M-CAT motif also abolishes promoter activity in cardiac cells. The third regulatory region (nucleotide position -550 to -268) is not essential for promoter activity but can enhance activity of the cTNT promoter or a heterologous promoter in both cardiac and skeletal muscle cells by three to five folds. Within this region are sequences which show similarity to motifs found in other muscle gene enhancers. In vitro DNA-protein binding studies showed that the above three regulatory regions interact with nuclear factors. A direct correlation exists between promoter activity and sequence specific binding of a nuclear factor we termed the 'M-CAT binding factor' to the M-CAT motifs. Similar interaction of nuclear regulatory molecules with sequences within the cardiac element and the upstream enhancer region is likely to be the mechanisms which control the action of these regulatory regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a promoter element required for transcription in myocardial cells.

Transcription of the chicken cardiac troponin T (cTNT) gene in myocardial cells requires upstream sequences not required for expression of this gene in embryonic skeletal muscle cells. Deletion analysis shows that the segment between nucleotides -247 and -201 (numbered relative to the transcription initiation site at +1) is capable of conferring cardiac specific expression to a "minimal" cTNT promoter. The cardiac element within this segment contains at least two essential subregions: one residing upstream of position -215, which bears no homologies to known transcription elements, and an A/T-rich segment residing between positions -215 and -201. Conserved M-CAT motifs within the cTNT minimal promoter which are required for activity in skeletal muscle cells are also required for activity in myocardial cells, suggesting an interaction between the upstream cardiac element and proximal promoter elements. Gel-shift experiments demonstrate interaction between the upstream portion of the cardiac element and factor(s) present in the nuclei of cardiac and non-cardiac tissues. Thus, additional cis and trans factors are required for transcription of the cTNT gene in myocardial cells which are not required for expression in skeletal muscle cells.

Glycoproteins of rat skeletal muscle sarcolemma: characterization by two-dimensional gel electrophoresis and effect of denervation.

Sarcolemmal membrane glycoproteins from rat mixed, fast, and slow muscles were characterized by concanavalin A (ConA) binding following two-dimensional polyacrylamide gel electrophoresis (PAGE). Analysis of electrophoretic profiles revealed that although sarcolemmal membranes prepared from these muscle types contained common glycoprotein species, each had a distinct glycoprotein pattern. In sarcolemma from mixed muscle, three major classes of ConA binding glycoproteins could be distinguished: (i) an acidic species of 110,000-120,000 Da, pI 5.0 to 5.3 (CG-1, ConA binding glycoproteins-1); (ii) a group of highly charged isomers, ranging from 75,000 to 80,000 Da pI 5.2 to 8.2 (CG-2); and (iii) a group of charged isomers of predominantly acidic nature of approximately 50,000 Da pI 5.2 to 5.8 (CG-3). ConA bound exclusively to CG-1 in sarcolemma from a fast muscle (extensor digitorum longus muscle, EDL). In soleus muscle sarcolemma (slow fiber type) both CG-1 and CG-3 were readily detected but CG-2 was markedly diminished. ConA binding to slow muscle sarcolemma revealed as well a glycoprotein species of 66,000-70,000 Da, pI 4.3-5.1 (CG-4), which was unique to this fiber type and as such may be a specific marker for slow fiber type. Denervation had no significant effect on the properties of ConA binding to mixed or slow muscle sarcolemma but dramatically altered the ConA binding to fast muscle sarcolemma, specifically increasing binding to CG-2. These findings demonstrate that denervation differentially affects the metabolism of ConA binding glycoproteins in these muscle types.