Inducible and tissue-specific gene inactivation in mice has become a powerful tool to bypass embryonic and postnatal lethality of knockout mice. The most frequently used inducible system is based on Cre recombinase fused to either one or two mutated estrogen receptor ligand binding domains, thus rendering Cre function tamoxifen-dependent. To achieve Cre-mediated inactivation of a given gene, 4-OH tamoxifen (4-OHT) dissolved either in alcohol and/or oil is usually administered by repeated intraperitoneal (i.p.) injections. Since this procedure imposes considerable stress on mice, we compared the effect of tamoxifen citrate, mixed into a standard mouse diet at different concentrations, with that of i.p. administration of 4-OHT on Cre-mediated, heart-specific inactivation of thioredoxin reductase 2. Here we show that tamoxifen citrate in the chow was equally effective as 4-OHT given i.p. Oral tamoxifen administration is thus a convenient and cost-saving way for gene induction, and, most importantly, it reduces stress and avoids adverse effects in mice.

Background— Excessive formation of reactive oxygen species contributes to tissue injury and functional deterioration after myocardial ischemia/reperfusion. Especially, mitochondrial reactive oxygen species are capable of opening the mitochondrial permeability transition pore, a harmful event in cardiac ischemia/reperfusion. Thioredoxins are key players in the cardiac defense against oxidative stress. Mutations in the mitochondrial thioredoxin reductase (thioredoxin reductase-2, Txnrd2) gene have been recently identified to cause dilated cardiomyopathy in patients. Here, we investigated whether mitochondrial thioredoxin reductase is protective against myocardial ischemia/reperfusion injury. Methods and Results— In mice, α-MHC-restricted Cre-mediated Txnrd2 deficiency, induced by tamoxifen ( Txnrd2-/-ic ), aggravated systolic dysfunction and cardiomyocyte cell death after ischemia (90 minutes) and reperfusion (24 hours). Txnrd2-/-ic was accompanied by a loss of mitochondrial integrity and function, which was resolved on pretreatment with the reactive oxygen species scavenger N-acetylcysteine and the mitochondrial permeability transition pore blocker cyclosporin A. Likewise, Txnrd2 deletion in embryonic endothelial precursor cells and embryonic stem cell-derived cardiomyocytes, as well as introduction of Txnrd2-shRNA into adult HL-1 cardiomyocytes, increased cell death on hypoxia and reoxygenation, unless N-acetylcysteine was coadministered. Conclusions— We report that Txnrd2 exerts a crucial function during postischemic reperfusion via thiol regeneration. The efficacy of cyclosporin A in cardiac Txnrd2 deficiency may indicate a role for Txnrd2 in reducing mitochondrial reactive oxygen species, thereby preventing opening of the mitochondrial permeability transition pore.

Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons. INTRODUCTIONForward and reverse genetics has been successfully used in Drosophila to identify new genes involved in neuronal degeneration and in the study of human genes linked to neurodegenerative diseases. Recently, a number of articles have corroborated the use of Drosophila as a powerful model organism to investigate the process of age-related neuronal cell death (reviewed in Fortini and Bonini, 2000). It is well known that neurons need a constant supply of growth factors for their survival, and in cell culture, the withdrawal of these factors or blocking their signal transduction pathways leads to cell death Raff et al., 1993;Xia et al., 1995;Le-Niculescu et al., 1999). A variety of factors including fibroblast growth factor (FGF) and epidermal growth factor (EGF) promote cell survival by binding and activating receptor tyrosine kinases (RTKs), which stimulate the activation of the Ras proto-oncogene products (Gardner and Johnson, 1996;Yamada et al., 1997). The involvement of Ras-dependent pathways in the process of neuronal cell survival has been studied in cell culture (Bonni et al., 1999;Mazzoni et al., 1999) and in vivo using Drosophila. Perhaps the most interesting data linking Ras activation with cell survival arise from the studies in photoreceptor apoptosis in Drosophila: Ras promotes cell survival in the eye by downregulating the expression of the apoptotic gene hid during development (Bergmann et al., 1998;Kurada and White, 1998). On the other hand, the overexpression of argos, an inhibitor of the Egfr/Ras signaling pathway, causes extensive cell death in developing Drosophila eyes (Sawamoto et al., 1998).Conversely, some reports exist that associate an activated Ras cascade with enhanced cell death. The ectopic expression, for instance, of an active form of Ras leads to hyperplastic growth and induces widespread cell death in Drosophila imaginal discs (Karim and Rubin, 1998), and the expression of oncogenic mutated Ras in human cancer cells leads to cell death that shares features of autophagic degeneration (cell death type 2) (Chi et al., 1999;Kitanaka and Kuchino, 1999). The expression of oncogenic Ras has been also implicated in senescence in cultured human fibroblasts. In primary cells, Ras is initially mitogenic but eventual...

BackgroundUbiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined.Methods and ResultsThe tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated.ConclusionsThese results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.

Thioredoxin reductases (Txnrds) are a group of selenoenzymes participating in cellular redox regulation. Three Txnrd isoforms are known, each of which exhibits distinct cellular localisation and tissue-specific expression pattern. Txnrd1 is found in the cytoplasm, expression of Txnrd2 is restricted to mitochondria and Txnrd3 shows testis-specific expression. Recently, it was shown that Txnrd2 strongly affects the development of blood cells, since mouse embryos deficient for Txnrd2 are severely anaemic, show increased apoptosis in foetal liver and possess haematopoietic liver stem cells of reduced capacity to proliferate in vitro. However, because Txnrd2-deficient mice die at embryonic day 13.5, it was not known how this enzyme affects blood cell function in the adult animal. In the present study we show that conditional Txnrd2 knockouts generated using CD4- and CD19Cre transgenic mice lack Txnrd2 expression in CD4-- and CD19-positive T- and B-lymphocytes, respectively. However, the development and differentiation of both cell types in thymus and bone marrow was not significantly impaired. In addition, B-cell proliferation and activation in response to CD40 and IL-4 was unaltered in Txnrd2-deficient B-cells.