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An expanded CTG-repeat in the 3′ UTR of theDMPKgene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreasedDMPKsense andSIX5transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability.

The activities of copper, zinc-superoxide dismutase (SOD1), manganese SOD (SOD2), glutathione peroxidase (GPX), glutathione reductase (GSSG-R) and glutathioneS-transferase (GST) were studied in sheep corpora lutea (CL) obtained on days 15, 40, 60, 80 and 128 of pregnancy. Maintained enzymatic activity of SOD1, SOD2, GPX, GSSG-R and GST were found in the sheep CL throughout pregnancy. Enzymatic activity of SOD1, GPX and GST increased significantly from day 15 to day 40 of pregnancy, and thereafter remained constant until day 128. SOD2 and GSSG-R activities were not different between any days of pregnancy examined. Apoptotic luteal cells identified by the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling were very rarely observed, and their incidence (less than 0.5%) was not different between days of pregnancy. These results showed that the activities of antioxidant enzymes in the sheep CL are subject to major changes during early pregnancy, suggesting that the CL of early pregnancy may be rescued from luteolysis through increasing activities of key antioxidant enzymes and inhibition of apoptosis. Maintained levels of antioxidant enzymes in the CL throughout pregnancy may be linked to reactive oxygen species continuously generated in the steroidogenically active luteal cells, and may be involved in the maintenance of luteal steroidogenic activity and cellular integrity.

International audience; Recent work has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Low concentrations of the powerful antioxidant glutathione (GSH) and impaired brain energy metabolism, particularly in the substantia nigra, are key features of Parkinson's disease (PD). The main goal of this study was to better characterize the deleterious effects of brain GSH depletion on mitochondrial function. We depleted GSH in the brains of newborn wild-type (WT) and transgenic (Tg) mice overproducing either human Cu/Zn-superoxide dismutase (h-CuZnSOD) or human Bcl2 (h-Bcl-2), by subcutaneous injection of -buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase. GSH was 97% depleted in brain homogenates and 90% depleted in brain mitochondria for both WT and Tg mice. This depletion of brain GSH led to a decrease in the activity of the GSH-dependent antioxidant enzyme glutathione peroxidase, both in WT and in Tg animals. BSO treatment decreased the activities of respiratory complexes I, II, and IV in the brain homogenates of WT mice. BSO-treated h-CuZnSOD or h-Bcl-2 Tg mice had no respiratory chain deficiencies. Thus, brain GSH depletion leads to the impairment of mitochondrial respiratory chain activity. The protection of mitochondrial respiratory function by overproduction of Bcl-2 may result from a decrease in the generation of reactive oxygen species (ROS) or lipid peroxidation. The protection of mitochondria by overproduction of CuZnSOD is consistent with the involvement of superoxide or superoxide-derived ROS in the mitochondrial dysfunction caused by brain GSH depletion. This study demonstrates that the antioxidant balance is critical for maintenance of brain mitochondrial function, and its disruption may contribute to the pathogenesis of PD.

Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of amyloid beta-peptide (A beta), presumed to play a pathogenic role. However, the precise molecular mechanisms of its neurotoxicity are not fully understood. Recent studies have suggested that it may exert its toxic effect via activation of transcription factors. We investigated A beta-responsive genes in human preneuron NT2 cells, at early stages of A beta (25-35) exposure, by RNA differential display. A beta induced the expression of (i) the growth arrest and DNA damage-inducible gene (gadd45) implicated in the DNA excision-repair process; (ii) a stress-signaling kinase gene encoding the mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1); (iii) a new growth factor-inducible immediate-early gene, CYR61, the product of which functions as an extracellular matrix signaling molecule; (iv) other immediate-early genes, such as c-jun and c-fos; (v) the gene encoding the basic fibroblast growth factor (bFGF); (vi) a gene encoding a constituent of the mitochondrial pyruvate dehydrogenase complex, the dihydrolipoamide dehydrogenase-binding protein (E3-BP); and (vii) an unidentified human gene (KIAA0099). A beta not only activates but also respresses genes: (i) the gene encoding "hinge" protein, a subunit of the mitochondrial cytochrome-c reductase and (ii) the SRp55 gene encoding a splicing factor involved in constitutive pre-mRNA splicing and alternative splice site selection. Our results underscored A beta-responsive genes that play key roles in the response (damage/recovery) of neuron cells to A beta exposure. In particular, the strong upregulation of gadd45, indicating DNA damage, was detected early in A beta cytotoxicity. This suggests that DNA strand breaks occurred rapidly in cells exposed to A beta, which may be a critical event in A beta neurotoxicity.

In Parkinson's disease (PD), dopaminergic cell death in the substantia nigra was associated with a profound glutathione (GSH) decrease and a mitochondrial dysfunction. The fall in GSH concentration seemed to appear before the mitochondrial impairment and the cellular death, suggesting that a link may exist between these events. The relationships between GSH depletion, reactive oxygen species (ROS) production, mitochondrial dysfunction and the mode of cell death in neuronal cells remain to be resolved and will provide important insights into the etiology of Parkinson's disease. An approach to determine the role of GSH in the mitochondrial function and in neurodegeneration was to create a selective depletion of GSH in a neuronal cell line in culture (NS20Y) by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a nearly complete GSH depletion after 24 hr and induced cellular death via an apoptotic pathway after 5 days of BSO treatment. By using the reactive oxygen species-sensitive probe 2',7'-dichlorofluorescin, we observed that the rapid GSH depletion was accompanied, early in the process, by a strong and transient intracellular increase in reactive oxygen species evidenced after 1 hr with BSO, culminating after 3 hr when the GSH level decreased to 30% of normal. GSH depletion induced a loss of mitochondrial function after 48 hr of BSO treatment. In particular, the activities of complexes I, II and IV of the respiratory chain were decreased by 32, 70 and 65%, respectively as compared to controls. These results showed the crucial role of GSH for maintaining the integrity of mitochondrial function in neuronal cells. Oxidative stress and mitochondrial impairment, preceding DNA fragmentation, could be early events in the apoptotic process induced by GSH depletion. Our data are consistent with the hypothesis that GSH depletion could contribute to neuronal apoptosis in Parkinson's disease through oxidative stress and mitochondrial dysfunction.

The toxicity of expanded transcripts in myotonic dystrophy type 1 (DM1) is mainly mediated by the disruption of alternative splicing. However, the detailed disease mechanisms in the central nervous system (CNS) have not been fully elucidated. In our recent study, we demonstrated that the accumulation of mutant transcripts in the CNS of a mouse model of DM1 disturbs splicing in a region-specific manner. We now discuss that the spatial- and temporal-regulated expression of splicing factors may contribute to the region-specific spliceopathy in DM1 brains. In the search for disease mechanisms operating in the CNS, we found that the expression of expanded CUG-containing RNA affects the expression and phosphorylation of synaptic vesicle proteins, possibly contributing to DM1 neurological phenotypes. Although mediated by splicing regulators with a described role in DM1, the misregulation of synaptic proteins was not associated with missplicing of their coding transcripts, supporting the view that DM1 mechanisms in the CNS have also far-reaching implications beyond the disruption of a splicing program.

International audience; Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Defenses against free radical damage were determined in red blood cells and plasma from 40 patients with dementia of the Alzheimer-type (DAT) and 34 aged control subjects with normal cognitive function. No crude significant difference in erythrocyte copper-zinc superoxide dismutase (E-CuZnSOD), seleno-dependent glutathione peroxidase (E-GSH-Px), glutathione reductase (E-GSSG-RD) activities, and selenium (Se) concentration was found between DAT cases and control subjects. The peroxidation products evaluated in plasma by the thiobarbituric-reactive material (TBARS) were at the same level in the DAT group as compared to controls. In the DAT group, plasma GSH-Px (P-GSH-Px) activity and plasma Se (P-Se) were negatively correlated with age (r = -0.58; p < 0.001 and r = -0.63; p < 0.001 respectively). Moreover, erythrocyte GSH-Px activity and Se were also negatively correlated with age (r = -0.40; p < 0.01 and r = -0.46; p < 0.01, respectively). No significant correlation with age was observed in the controls. When controlling for age, a significant increase for P-GSH-Px activity and P-Se was observed in DAT patients as compared to controls. These significant differences mostly appeared in DAT subjects under 80 years. Some correlations were only observed in the DAT group such as P-GSH-Px and E-GSH-Px (r = +0.68; p < 0.001); P-GSH-Px and E-Se (r = +0.79; p < 0.001). Correlations between P-GSH-Px and P-Se, E-GSH-Px and P-Se, and P-Se with E-Se are greater in the DAT group (r = +0.84; p < 0.001; r = +0.76; p < 0.001 and r = 0.75; p < 0.001) than in the control group (r = 0.54, pI < 0.01; r = 0.43, p < 0.01 and r = +0.34, p < 0.05 respectively). The fact that first -- a significant increase in P-GSH-Px and P-Se, second -- some modifications in the relationships between antioxidant parameters, and third -- age-dependent decreases of glutathione-peroxidase activities and their cofactor, appeared only in the DAT group suggest that DAT is associated with an oxidative stress due to an imbalance between reactive oxygen species and the peripheral antioxidant opposing forces.

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3′UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro–RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes., Author Summary Myotonic dystrophy type 1 (DM1) is caused by the abnormal expansion of a CTG repeat located in the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form toxic nuclear foci that affect other RNAs. DM1 involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro–RNA metabolism. We previously generated transgenic mice carrying the human DM1 locus and very large expansions >1,000 CTG (DMSXL mice). Here we described for the first time, the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. We also demonstrate that DMPK antisense transcripts are expressed in various tissues from DMSXL mice and human. Both sense and antisense transcripts form nuclear foci. DMSXL mice showed molecular DM1 features such as foci and mild splicing defects as well as muscles defects, reduced muscle strength, and lower motor performances. These mice recapitulate some molecular features of DM1 leading to physiological abnormalities. DMSXL are not only a tool to decipher various mechanisms involved in DM1 but also to test the preclinical impact of systemic therapeutic strategies.

Myotonic dystrophy is an RNA gain-of-function disease caused by expanded CUG or CCUG repeats, which sequester the RNA binding protein MBNL1. Here we describe a newly discovered function for MBNL1 as a regulator of pre-miR-1 biogenesis and find that miR-1 processing is altered in heart samples from people with myotonic dystrophy. MBNL1 binds to a UGC motif located within the loop of pre-miR-1 and competes for the binding of LIN28, which promotes pre-miR-1 uridylation by ZCCHC11 (TUT4) and blocks Dicer processing. As a consequence of miR-1 loss, expression of GJA1 (connexin 43) and CACNA1C (Cav1.2), which are targets of miR-1, is increased in both DM1- and DM2-affected hearts. CACNA1C and GJA1 encode the main calcium- and gap-junction channels in heart, respectively, and we propose that their misregulation may contribute to the cardiac dysfunctions observed in affected persons.

The copper-zinc-dependent superoxide dismutase messenger RNA expression was studied at cellular level by in situ hybridization, using a 35S-labelled complementary DNA probe homologous to human copper-zinc-dependent superoxide dismutase messenger RNA, in the dopaminergic neuron-containing areas of the human mesencephalon (the substantia nigra pars compacta, ventral tegmental area, central gray substance and peri- and retrorubral region corresponding to catecholaminergic cell group A8). The autoradiographic labelling signal was localized in neurons. No detectable hybridization signal could be found in the glial cells. Copper-zinc-dependent superoxide dismutase messenger RNA was detected in melanin-containing neurons as well as in non-melanized neurons. Quantification at cellular level, taking the autoradiographic silver grain density as an index of the abundance of copper-zinc-dependent superoxide dismutase messenger RNA, indicated that hybridization level was higher in the melanized than in the non-melanized neurons within a region. Among melanized neurons, cellular copper-zinc-dependent superoxide dismutase messenger RNA content was lowest in the neurons of the substantia nigra. No significant difference in levels of transcripts was evidenced between the groups of non-melanized neurons. The data suggest that the abundance of copper-zinc-dependent superoxide dismutase messenger RNA is higher in the mesencephalic neurons containing neuromelanin compared to other neurons. Thus, the melanized neurons have a particular defence system against oxygen toxicity, which may represent a basis for their preferential vulnerability to Parkinson's disease.

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid perxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47); P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 ± 0.71 μmole MDA/g tissue) thatn at 2 months (5.69 ± 0.90) and significantly lower than 28 months (5.13 ± 0.33) than at 12 months. Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.

To obtain a comprehensive profile of the erythrocyte antioxidant defense potential during aging, we investigated copper-zinc superoxide dismutase (CuZn-SOD), seleno-dependent glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-RD), and glutathione-S-transferase (GSH-S-T) activities in human erythrocytes from 167 apparently healthy subjects, ages one month to 63 years (102 females, 65 males). We found a negative correlation between age and activities of CuZn-SOD (r = 0.362, P less than 0.001), GSSG-RD (r = 0.549, P less than 0.001), and GSH-S-T (r = 0.575, P less than 0.001). In contrast, we found a positive correlation between age and GSH-Px activity (r = 0.401, P less than 0.001). To evaluate aging changes, we divided the subjects into five groups: Group 1 (newborn-age one year), Group 2 (1-11 years), Group 3 (11-25 years), Group 4 (25-40 years), and Group 5 (40-63 years). Significant age-related modifications in erythrocyte enzyme activities appeared in Group 3 for CuZn-SOD, GSSG-RD, and GSH-Px activity, whereas for GSH-S-T activity age-related modifications appeared in Group 2. We found no sex-related differences in erythrocyte CuZn-SOD, GSSG-RD, GSH-Px, and GSH-S-T activities.

Superoxide dismutase CuZn (SOD1) enzymatic activity was measured in five orangutans (Pongo pygmaeus, PPY) and compared to that of man, chimpanzee, and gorilla. It was found to be increased by a factor of two in one orangutan (Ralfina) and by a factor of 1.5 in the four others. In situ hybridization of the SOD1 cDNA human probe showed a heterozygous intra-chromosomal rearrangement of pair PPY XXI, possibly an insertion, in Ralfina. Southern blotting showed that the SOD1 gene is modified in the three orangutans that were investigated and that a further modification of the 5'-end of the gene had occurred in Ralfina. The evolutionary implications of these observations are discussed.

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions (“big jumps”), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG•CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis., Author Summary Many neurological and/or neuromuscular diseases, such as myotonic dystrophy, Huntington disease, and fragile X mental retardation are caused by an increase in the size of a repeated DNA sequence within a specific gene. These repetitive DNA sequences are prone to expansion, increasing in size when transmitted from one generation to the next, which results in more severe symptoms and earlier age of onset. In myotonic dystrophy, the DNA repeat can undergo very large increments of several hundred units (frequently called “big jumps”), usually associated with the most severe clinical picture. Until now, big jumps have not been observed in mice carrying the disease mutation, leading to questions about the adequacy of mice to fully model DNA repeat instability. We now report that these large increments in the size of DNA repeats can occur in transgenic mice, resulting in animals that carry extremely large repeated sequences. These mice are remarkably small and display abnormalities in the metabolism of multiple messenger RNAs, notably in brain and muscle. Our findings strongly support the use of transgenic mice to resolve the complex dynamics of simple repetitive DNA sequences associated with human inherited diseases, and to investigate the molecular events that underlie the development of disease symptoms.

Background: Transient neonatal diabetes (TND) is a rare form of diabetes usually present in the first few days after birth that resolves within 1 year but that has a tendency to recur later in life. It can be associated with chromosome 6 paternal uniparental disomy (UPD), paternal duplications or loss of maternal methylation at the 6q24 imprinted locus. Objective: To report on a cohort of 13 sporadic TND cases, including five with birth defects (congenital abnormalities of heart, brain and bone) and eight without. Results: The hallmarks of diabetes were similar in patients with or without 6q24 defects. The chromosome 6 abnormalities in our patients (n = 13) included 2 of 13 (approximately 15.4%) cases of paternal UPD6, 2 of 11 (approximately 18%) cases of complete and 3 of 11 (approximately 27%) cases of partial loss of the maternal methylation signature upstream of ZAC1-HYMAI imprinted genes in non-UPD cases, and 1 of 13 (approximately 7.7%) cases of hemizygotic deletion. Conclusion: The deletion was found in a patient with severe congenital abnormalities. This genetic lesion was not reported previously. The hypothesis of an effect on regulatory elements critical for imprinting and tissue-specific gene expression in early development by the deletion is raised. The data presented here may contribute to the diagnosis and the understanding of imprinting in the region.

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the extracellular deposition of amyloid beta-peptide (Abeta) in the brain. Abeta is involved in the pathogenesis of AD but the molecular mechanisms of its neurotoxicity are unknown. Here, we report that Abeta exposure on human preneuronal NT2 cells provoked a strong and early up-regulation of growth arrest and DNA damage inducible gene (Gadd45 mRNA), an indicator of DNA damage and DNA excision-repair processes, strongly suggesting that Abeta causes an early DNA strand breakage leading to a cellular DNA repair response. Comet assay clearly demonstrated that both full-length Abeta (1-42), and its minimal cytotoxic fragment Abeta (25-35), caused DNA breakage as early as 3h after the start of Abeta exposure. This extensive DNA damage provoked by Abeta constitutes an early event in the pathogenic cascade leading to neuronal death which could contribute to the neuropathogenesis of AD.

The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid beta-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer's disease victims. In this study, we have compared the toxicity of amyloid beta-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper-zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper-zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid beta-peptide(25-35) [the minimal cytotoxic fragment of amyloid beta-peptide(1-42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid beta-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper-zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid beta-peptide(25-35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid beta-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid beta-peptide(25-35) and amyloid beta-peptide(1-42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer's disease.

International audience; If permanent focal ischemia is induced by middle cerebral artery occlusion (MCAO), neurons within the infarcted territory die by necrosis and apoptosis (or programmed cell death). We have previously shown, using a mouse strain transgenic (tg) for the nerve growth factor (NGF) gene, that tg mice have consistently smaller infarcted areas than wild-type (wt) animals, correlated with upregulated NGF synthesis and impaired apoptotic cell death. We studied, in wt and tg mice subjected to MCAO, the activities of several antioxidant enzymes and the synthesis of the proteins of the Bcl-2 family. Our results show that the antiapoptotic Bcl-2 protein and glutathione peroxidase are recruited after MCAO. NGF-tg mice also had an intrinsic resistance to oxidative stress because their basal copper zinc superoxide dismutase (SOD) and glutathione transferase activities were high. Additionally, manganese SOD activity increased in NGF-tg mice after MCAO, correlating strongly with the resistance of these mice to apoptosis.

International audience; After an ischemic episode induced by the electrocoagulation of the left middle cerebral artery (MCA) in mouse, neurons within the damaged territory die either by an apoptotic or by a necrotic process. Most of the cortical neurons within the ischemic area display both morphological and biochemical signs of programmed cell death: nuclear condensation, DNA degradation, formation of apoptotic bodies, and glutathione depletion. In fact, apoptosis essentially contributes to the expansion of the ischemic lesion and the maximum of damaged territory is reached 24 h postocclusion. Several potentially neuroprotective pathways have been evidenced in different experimental models of ischemia including the activation of antioxidant enzyme activities and/or the recruitment of neurotrophic as well as antiapoptotic factors. In our model of permanent focal ischemia induced by MCA occlusion, we measured the temporal synthesis of nerve growth factor (NGF) and examined the status of antioxidant enzymes as well as Bcl-2 antiapoptotic product. We detected in both cortices a transient increase of NGF which peaks at 6 h. Moreover, we reported that glutathione peroxidase is recruited with a time course which parallels NGF synthesis. Finally, we observed the induction of Bcl-2 in safe neurons; this may represent a self-protective response against ischemia-induced apoptosis. We provide evidence that in a model of permanent focal ischemia, several neuroprotective pathways could be coactivated.

Summary Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease

Oxidative stress may play an important role in the pathogenesis of brain injuries associated with aging and neurodegenerative disorders such as Down’s syndrome, Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Brain contain several antioxidant enzymes, most importantly, superoxide dismutases (MnSOD and CuZnSOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) and catalase. The tripeptide reduced glutathione (GSH) serves as one of the major endogenous antioxidants in protecting mammalian cells against oxidative stress. GSH has a role not only as a substrate for GSH-Px to break down hydrogen peroxide and lipid peroxides but also as free radical scavenger. The individual contribution of these antioxidants in neuronal protection remains unknown and requires elucidation. A first approach to this question was to establish a comprehensive profile of the brain antioxidant defense potential during aging in control mice. A second approach was to use genetic manipulations to construct transgenic mice that overexpress CuZnSOD. These constructed transgenic mice which exhibit increased CuZnSOD activity in the brain would provide a suitable model to further investigate the role of an increased CuZnSOD activity in neuronal aging and Alzheimer-like neuropathology in Down syndrome. A third strategy in assessing the role of a particular antioxidant in brain fonction was to use metabolic manipulations to deplete brain GSH levels with electrophiles that conjugate with GSH in the presence of glutathione S-transferases, such as diethylmaleate (DEM), and to correlate this depletion with brain injury or susceptibility to oxidative stress. Obtention of such a variation of a particular antioxidant in the brain and neurobiological consequences will be evaluated and discussed.

The copper-zinc superoxide dismutase (SOD-1) gene, located on chromosome 21 and triplicated in Down's syndrome (DS), is suspected to be involved in the neuropathology observed in Alzheimer's disease (AD), DS and physiological aging. In order to explore the effect of an overproduction of SOD-1 in the mouse hippocampus, we investigated the Timm-stained mossy fiber (MF) innervation in the hippocampus of transgenic mice for the human SOD-1 gene (hSOD-1 mice). The results showed a decrease of the MF projection area in the hSOD-1 mice overexpressing the SOD-1 protein. These findings suggest that free radicals could play a role in this particular synaptic loss.

Down’s Syndrome (DS), the most frequent of congenital birth defects, results from the trisomy of the chromosome numbered 21 in all cells of affected patients. This disease is characterized by developmental anomalies, mental retardation and features of rapid aging, particularly in the brain where the occurrence of Alzheimer’s disease (AD) is observed in all trisomy 21 patients over the age of 35. Elucidation of the biological mechanisms leading to brain aging in DS might provide new insight into the understanding of brain aging and AD in normal people.

It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Down's syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammon's horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Down's syndrome patients.

It was suggested that increased Cu-Zn superoxide dismutase (SOD-1) might be involved in the various biological abnormalities found in Down's syndrome (DS) such as premature aging and Alzheimer-type neurological lesions. As a model system for testing this hypothesis we have developed two strains of transgenic mice carrying only one copy of the human SOD-1 gene. In the first strain (TG1), no expression has been found by northern blot analysis. The second strain (TG2) exhibited human SOD-1 mRNA and increased SOD-1 activity in the brain (1.93 fold), in the heart (1.69 fold), thymus (1.49 fold) and to a lesser extent in muscle (1.25 fold), liver (1.19 fold), kidney (1.18 fold), spleen (1.35 fold), lung (1.26 fold) and erythrocytes (1.09 fold). In this strain, increased SOD-1 activity in the brain did not induce modifications in the seleno-dependent glutathione peroxidase, glutathione reductase and glutathione S-transferase activities. In brain homogenates, we have focused our studies on Tau proteins which are known to be the major antigenic components of paired helical filaments (PHF), both in DS and Alzheimer's disease. Our results suggested that, in our experimental conditions, the overexpression of SOD-1 did not induce the modifications of Tau proteins similar to those seen during neurofibrillary degeneration.

The distribution of cells containing copper-zinc superoxide dismutase (CuZn SOD) protein and mRNA was studied in hippocampi from normal humans and patients with Alzheimer's disease (AD) by using immunohistochemistry and in situ hybridization. Using antisera against native and denatured CuZn SOD protein, we have determined that immunostaining was intense in pyramidal neurons of the cornu ammonis, in granule cells of the dentate gyrus and very weak in other cells. In the hippocampus of an Alzheimer's patient, successive immunostaining of the same tissue section by antiCuZn SOD and antipaired helical filaments antisera show that both normal and degenerating cells were labeled by the antiCuZn SOD antiserum. Thus, large pyramidal neurons which are susceptible to degenerative processes in AD have the property to contain high amount of CuZn SOD protein. In situ hybridization was performed on paraformaldehyde-fixed hippocampus sections of normal human brains and AD brains with a 35 S labeled DNA probe homologous to human CuZn SOD mRNA. Our results show that CuZn SOD transcripts are present at high abundance in pyramidal neurons of the CA1-CA4 fields, subiculum, and in granule cells of the dentate gyrus. This cellular distribution is similar to that obtained with the antiCuZn SOD antiserum. This might indicate that biochemical pathways leading to superoxide radicals generation are specially active in these neurons, requiring an active transcription of CuZn-SOD gene.

We have analysed the DNA of 2 patients with many manifestations of Down syndrome and partial duplication of distinct regions of chromosome 21, respectively, q11.205----q22.300 and q22.300----qter (Rahmani et al.: Proceedings of the National Academy of Sciences of the United States of America 86:5958-5962, 1989). Assessment of the copy number of five chromosome 21 sequences (SOD1, D21S17, D21S55, ETS2, and D21S15) has shown that D21S55 was duplicated in both cases. The size of the common duplicated region can be estimated between 400 and 3,000 Kb, after the results of pulsed-field gel analysis and from the knowledge of regional mapping of the probes D21S17, D21S55, and ETS2. This region, located on the proximal part of 21q22.3, is postulated to contain genes the overexpression of which plays a major role in the pathogenesis of Down syndrome.

The human CuZn superoxide dismutase (superoxide dismutase 1) a key enzyme in the metabolism of oxygen free-radicals, is encoded by a gene located on chromosome 21 in the region 21 q 22.1 known to be involved in Down's syndrome. A gene dosage effect for this enzyme has been reported in trisomy 21. To assess the biological consequences of superoxide dismutase 1 overproduction within cells, the human superoxide dismutase 1 gene and a human superoxide dismutase 1 cDNA were introduced into mouse L cells and NS20Y neuroblastoma cells. Both cell types expressed elevated levels (up to 3-fold) of enzymatically active human superoxide dismutase 1. These human superoxide dismutase 1 overproducers, especially neuronal cell lines, showed an increased activity in the selenodependent glutathione peroxidase. These data are consistent with the possibility that gene dosage of superoxide dismutase 1 contributes to oxygen metabolism modifications previously described in Down's syndrome.

A patient with the phenotype of trisomy 21 (Down syndrome) was found to have a normal karyotype in blood lymphocytes and fibroblasts. Assessment of the chromosome 21 markers SOD1, CBS, ETS2, D21S11, and BCEI showed partial trisomy by duplication of a chromosome segment carrying the SOD1, CBS, and ETS2 loci and flanked by the BCEI and D21S11 loci, which are not duplicated. This submicroscopic duplication at the interface of 21q21 and 21q22.1 reduces to about 2000-3000 kb the critical segment the trisomy of which is responsible for the phenotype of trisomy 21.

The distribution of cells containing CuZn superoxide dismutase (CuZn SOD) was determined in hippocampi and associative cortex from normal and Alzheimer's individuals by using antisera against native and denatured CuZn SOD proteins. Immunostaining was intense in large pyramidal neurons, moderate in hippocampal granule cells and very weak in other cells. In the hippocampus of an Alzheimer's patient, successive immunostaining of the same tissue section by anti CuZn SOD and anti paired helical filaments antisera show that both normal and degenerating cells are labelled by the anti CuZn SOD antiserum. Thus, large pyramidal neurons which are potentially susceptible to degenerative processes in AD have the property to contain higher amounts of CuZn SOD than other brain cells.

The molecular cloning and nucleotide sequence of a cDNA clone (pR SOD) for rat CuZn superoxide dismutase (CuZnSOD) is reported. Nucleotide sequence homology with human superoxide dismutase is 86% for the coding region and 71% for the 3' untranslated region. The deduced amino acid sequence is given and the homologies with the sequences reported for other species are presented. Northern blot analysis of total RNA from various rat and mouse tissues and from two mouse cell lines show that pR SOD hybridizes with one mRNA species of about 0.7 kb. The amount of CuZnSOD mRNA in each tissue, measured by densitometry of the Northern blot autoradiograms, correlates with the enzymatic activity based on protein content. These results indicate that the control of CuZnSOD activity in mammalian tissues is largely dependent on the regulation of CuZnSOD mRNA levels. In human liver, fibroblasts and FG2 hepatoma cells, two CuZnSOD mRNAs (0.7 kb and 0.9 kb) are observed. The level of CuZnSOD mRNA in FG2 is 25% that of the liver and four times more abundant than in fibroblasts.

With the recently cloned complementary DNA probe, lambda Am4 for the chromosome 21 gene encoding brain amyloid polypeptide (beta amyloid protein) of Alzheimer's disease, leukocyte DNA from three patients with sporadic Alzheimer's disease and two patients with karyotypically normal Down syndrome was found to contain three copies of this gene. Because a small region of chromosome 21 containing the ets-2 gene is duplicated in patients with Alzheimer's disease, as well as in karyotypically normal Down syndrome, duplication of a subsection of the critical segment of chromosome 21 that is duplicated in Down syndrome may be the genetic defect in Alzheimer's disease.

Cystic fibrosis (CF) has recently been linked to the group of human diseases in which cultured fibroblasts express premature aging. As the deleterious effect of oxygen derivatives in the cell is one of the numerous pathways associated with cell aging, the activity of the enzymatic defense system, superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR), was examined in the erythrocytes of 12 CF children and was compared to age-matched normal controls. No significant differences were found in CF children when compared to the controls. Glutathione-S-transferase was also assayed, but the significant difference found between CF children and normal controls is probably not specific of CF as it is found in other pathologic situations such as hyperbilirubinemia or renal insufficiency.

The activity of manganese superoxide dismutase (MnSOD) revealed by specific staining after gel electrophoresis of cell extracts, is decreased in human fibroblasts transformed by SV 40. The decrease in enzyme activity is attributable to decreased amount of enzyme protein as determined by radial immunodiffusion. Total fibroblast RNAs were translated in the presence of (35S) methionine in a cell-free translation system and the neo synthesized proteins submitted to immunoprecipitation with an anti MnSOD antiserum. Gel electrophoresis of the immunoprecipitated material followed by fluorography shows that MnSOD is translated as a peptide which is 2000 daltons larger than the mature enzyme subunit. This precursor (pre-MnSOD) is processed in vitro to mature MnSOD by the action of an isolated mitochondrial preparation. Levels of translatable MnSOD mRNA in normal and SV 40 transformed cells were compared in terms of the radioactivities incorporated into pre MnSOD bands. The results indicate that the decreased amount of MnSOD in SV 40 transformed fibroblasts is due to a decreased level of translatable mRNA for MnSOD.

Down’s syndrome is defined by a characteristic phenotype, mental retardation and associated pathological manifestations such as rapid aging and Alzheimer’s disease. In most cases, Down’s syndrome results from the presence of an extra chromosome 21 in all cells. Genes which in triplicate participate in Down’s syndrome pathology have yet to be found. Studies of partial trisomies have shown that they are in the distal part (21q21→q22) of chromosome 21. We are currently investigating several patients with most of the features of Down’s syndrome but an apparently normal karyotype. Enzymes coded by chromosome 21, namely superoxide dismutase (SOD) and cystathionine beta synthase, have been assessed, as well as gene copy number for the amyloid protein precursor, the protooncogene ETS2 and various anonymous DNA sequences. Results indicate that duplication of two regions might be important in Down’s syndrome pathogeny: one containing the SOD gene, the other the protooncogene ETS2.

The possibility of a genetic link between Alzheimer’s disease (AD) and Down’s syndrome (DS) is supported by the similarity of the neuropathological and neurochemical changes found in the brain of AD patients and middle-aged DS patients. To test this hypothesis we performed gene dosage experiments with DNA sequences that we found duplicated in DS patients with partial trisomy. Various parameters which could modify the results of gene dosage have been tested. Among these parameters, RNase treatment was shown to introduce significant variations: in fibroblasts from one AD patient that we have studied extensively, dosage of the (β-amyloid gene and the protooncogene ETS2 gave the same values for RNase-untreated DNA and increased copy number for RNase-treated DNA from fibroblasts. DS models for AD suggest that overexpression of one or a few chromosome 21 genes leads to AD pathology. Thus it is potentially interesting to study chromosome 21 gene expression in AD. RNA quantification experiments were run on fibroblast cultures from five AD patients, three age-matched controls and two DS patients: mRNA for CuZn superoxide dismutase was significantly increased in four AD patients and two DS patients as compared to actin RNA. A t-test common to the five blots showed a significant increase (p < 0.001). For ETS2 sequences qualitative results were obtained: the level of ETS2 RNA was higher in AD patients and DS patients than in controls. For AD patients these results could be explained by variations of the regulation of gene expression, either at the transcriptional level or through the control of the stability of RNA. However, the observed increase is similar in DS and AD patients. Furthermore, in one AD patient gene dosage and RNA level quantification were performed on the same fibroblasts; increased values were found for both ETS2 DNA sequences and ETS2 RNA sequences. Therefore these results are also compatible with duplication of some chromosome 21 sequences in some AD patients.