Rod-derived cone viability factor promotes cone survival by stimulating aerobic glycolysis.

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication.

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1-BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.

Therapeutic strategy for handling inherited retinal degenerations in a gene-independent manner using rod-derived cone viability factors.

The most common hereditary retinal degeneration, retinitis pigmentosa (RP), leads to blindness by degeneration of cone photoreceptors. Meanwhile, genetic studies have shown that a significant proportion of RP genes is expressed only by rods, which raises the question of the mechanism leading to the degeneration of cones. Following the concept of sustainability factor cones, rods secrete survival factors that are necessary to maintain the cones, named Rod-derived Cone Viability Factors (RdCVFs). In patients suffering from RP, loss of rods results in the loss of RdCVFs expression and followed by cone degeneration. We have identified the bifunctional genes nucleoredoxin-like 1 and 2 that encode for, by differential splicing, a thioredoxin enzyme and a cone survival factor, respectively RdCVF and RdCVF2. The administration of these survival factors would maintain cones and central vision in most patients suffering from RP.

The thioredoxin-like protein rod-derived cone viability factor (RdCVFL) interacts with TAU and inhibits its phosphorylation in the retina.

Rod-derived cone viability factor (RdCVF) is produced by the Nxnl1 gene that codes for a second polypeptide, RdCVFL, by alternative splicing. Although the role of RdCVF in promoting cone survival has been described, the implication of RdCVFL, a putative thioredoxin enzyme, in the protection of photoreceptors is presently unknown. Using a proteomics approach we identified 90 proteins interacting with RdCVFL including the microtubule-binding protein TAU. We demonstrate that the level of phosphorylation of TAU is increased in the retina of the Nxnl1(-/-) mice as it is hyperphosphorylated in the brain of patients suffering from Alzheimer disease, presumably in some cases through oxidative stress. Using a cell-based assay, we show that RdCVFL inhibits TAU phosphorylation. In vitro, RdCVFL protects TAU from oxidative damage. Photooxidative stress is implicated in retinal degeneration, particularly in retinitis pigmentosa, where it is considered to be a contributor to secondary cone death. The functional interaction between RdCVFL and TAU described here is the first characterization of the RdCVFL signaling pathway involved in neuronal cell death mediated by oxidative stress.

Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes.

Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.

The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcgammaRIIIb and cytochrome b558.

Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. In the current study, we demonstrate the presence of PR3, FcgammaRIIIb, and cytochrome b558 of the NADPH oxidase in neutrophil lipid rafts. Activation of neutrophils with PMA, fmet-leu-phe, or TNFalpha known to increase the membrane expression of PR3 did not affect the amount of PR3 in rafts. Unexpectedly, the cytosolic subunits of the NADPH oxidase, p67phox and p47phox, the recruitment of which to the membrane requires cell stimulation, were detected in the rafts of unstimulated neutrophils. Treatment of neutrophils with the cholesterol-sequestering agent methyl-beta-cyclodextrin (MbetaCD) reduced raft p22phox and PR3. MbetaCD diminished membrane FcgammaRIIIb upregulating membrane PR3 (mPR3) and CD11b/CD18. In addition, MbetaCD significantly reduced PMA-induced activity of the NADPH oxidase without altering fmet-leu-phe-elicited activity. Antibody-mediated cross-linking of membrane PR3 caused activation of ERK and JNK kinases and their translocation to rafts. Confocal analysis revealed colocalization of mPR3, FcgammaRIIIb, and p22phox in the membrane, confirmed by their coimmunoprecipitation. Cleavage of neutrophil GPI-anchors by PI-PLC reduced mPR3 and FcgammaRIIIb, implicating a GPI-protein, possibly FcgammaRIIIb, in the attachment of PR3 to the membrane.