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Vol. 18, Issue 7, 2755-2767, July 2007
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*Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710 Braga, Portugal; Center for Aging and Developmental Biology, Aab Institute of Biomedical Sciences, and Departments of Biochemistry and Biophysics and Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal
Submitted November 29, 2006;
Revised April 13, 2007;
Accepted April 19, 2007
Monitoring Editor: Thomas Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1- phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1- limit the synthesis of nitric oxide (·NO) in both physiological and oxidative stress conditions. This defect in ·NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1- phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize ·NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The different NCL forms are characterized by massive neuronal death and accumulation of lipopigments in the lysosomes of several cell types (Goebel, 1997). Juvenile NCL (JNCL; Batten or Spielmeyer–Vogt–Sjogren disease; OMIM 204200
[OMIM]
) is the most common form, and it presents a disease course marked by vision loss, psychomotor deterioration, epileptic seizures, and premature death in the mid-20s (Boustany, 1992). JNCL is caused by mutations in CLN3, a gene identified in 1995 (Lerner et al., 1995). Saccharomyces cerevisiae harbors a gene, designated BTN1, that encodes a nonessential protein 39% identical and 59% similar to human Cln3 (Pearce and Sherman, 1997), and deletion of BTN1, btn1- has provided a useful model for JNCL research (Pearce et al., 1999a,b; Kim et al., 2003). In btn1-, it has been shown that pH homeostasis is altered (Pearce et al., 1999c) and that a decrease in vacuolar arginine transport results in a depletion of intracellular arginine levels (Kim et al., 2003). Expression of human Cln3 in btn1- complemented these defects, demonstrating that the function of these proteins is conserved (Pearce et al., 1999c; Kim et al., 2003). These findings allowed the identification of primary biochemical defects in JNCL that were later confirmed in more complex animal models (Golabek et al., 2000; Kim et al., 2003; Pearce et al., 2003; Ramirez-Montealegre and Pearce, 2005). In spite of these and other advances in understanding the disease, an association between the known biochemical alterations and the accumulation of storage bodies or the process of neurodegeneration has not yet been established.
Oxidative stress has been widely reported in neurodegenerative conditions such as amyotrophic lateral sclerosis and Alzheimer's and Parkinson's diseases as a possible cause of cell death and as a possible effector of apoptosis (Dexter et al., 1989; Lassmann et al., 1995; Mochizuki et al., 1996; Beal et al., 1997; Smith et al., 1997; Good et al., 1998; Pedersen et al., 1998; Butterfield et al., 2002), although, in most cases, it remains unclear whether oxidative stress is the primary cause, a mediator, or a downstream consequence of the neurodegenerative process. In NCL, studies presenting increased lipid peroxidation and mitochondrial dysfunction support the hypothesis that oxidative stress might also be implicated in this pathology (Jolly et al., 2002; Guarneri et al., 2004). Nonetheless, no alterations in antioxidant enzymes were detected in several tested tissues from human patients (Marklund et al., 1981; Garg et al., 1982).
We report that btn1- has increased resistance to menadione indicating that a lack of Btn1p results in an increased tolerance to oxidative stress. This phenotype is specifically attributable to the lack of Btn1p, and it can be complemented by the human Cln3. Moreover, we show that this resistance to oxidative stress is a direct cause of decreased L-arginine levels in btn1- limiting the production of nitric oxide (NO), which functions, at high concentrations, as a signal for apoptosis. Our results link a primary biochemical alteration caused by the absence of Btn1p or Cln3 with a possible mechanism of cell death. Because cell lines derived from JNCL patients also have reduced intracellular L-arginine levels (Ramirez-Montealegre et al., 2005), it is possible that the limitation in ·NO production we observe in btn1- might also occur in cells from JNCL patients. Due to the importance of ·NO signaling this defect might have an important role in JNCL pathology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Viability (Colony-forming Ability)
Cells harvested by centrifugation at 4°C (1000 x g for 5 min) were washed, counted, and diluted to a concentration of 3 x 106 cells/ml in 10 ml of YNB medium. Immediately before the addition of the stress agent (t0) and after different times of treatment (tn), 100 µl of cell suspension was diluted 1–10 three times and plated on YEPD plates. Colonies were counted after 48-h incubation at 30°C. Results of survival percentage were obtained using as reference the number of colonies counted in t0 plates versus the number of colonies obtained in tn plates.
Flow Cytometry Analysis
Fluorescence analysis was carried out by flow cytometry with an EPICS XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA) equipped with an argon-ion laser emitting a 488-nm beam at 15 mW. The green and red fluorescence were collected through a 488-nm blocking filter, a 550-nm/long-pass dichroic with a 525-nm/band-pass and a 590-nm/long-pass with a 620-nm/band-pass, respectively. An acquisition protocol was defined to measure forward scatter (FS log), side scatter (SS log), green fluorescence (FL1 log), and red fluorescence (FL3 log) on a four-decade logarithmic scale. Data (20,000 cells/sample) were analyzed with the Multigraph software included in the system II acquisition software for the EPICS XL/XL-MCL, version 1.0.
Assessment of Intracellular Reactive Oxygen Species (ROS) Levels
Flow cytometry quantification of yeast cell populations with high ROS levels was performed after labeling with MitoTracker Red CM-H2XRos (Invitrogen, Carlsbad, CA) as described previously (Ludovico et al., 2002). Briefly, cells were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS), pH 7.4, and incubated for 15 min at 37°C with 50 µg/ml MitoTracker Red CM-H2XRos. Fluorescence intensity was measured by flow cytometry, and cells displaying higher values than a defined threshold of red fluorescence were considered as containing elevated intracellular ROS levels. Epifluorescence microscopy visualization of intracellular ROS was performed after labeling with dihydrorhodamine 123 (DHR123) (Invitrogen). DHR123 was added from a 1-mg/ml stock solution in ethanol, to 5 x 106 cells/ml suspended in PBS, reaching a final concentration of 15 µg/ml. Cells were incubated during 90 min at 30°C in the dark, washed in PBS, and visualized as describe previously (Almeida et al., 2007). Images were acquired in an Olympus BX61 microscope with filter wheels to control excitation and emission wavelengths, equipped with a high-resolution Olympus DP70 digital camera (Olympus, Tokyo, Japan).
Plasma Membrane Integrity
Plasma membrane integrity was determined by examining cellular permeability to propidium iodide (PI) (Invitrogen), as described previously (Delafuente et al., 1992). Cells were incubated with 5 µg/ml PI for 15 min and analyzed by flow cytometry or epifluorescence microscopy. Heat-killed cells were used as a positive control. Cells displaying higher values than a defined threshold of red fluorescence were considered as having compromised plasma membrane integrity.
Assessment of Mitochondrial Function and Integrity
The charged cationic green dye rhodamine 123 (Invitrogen) was used to assess mitochondrial function/integrity as described previously (Ludovico et al., 2001). Cells were harvested by centrifugation and incubated with 25 µg/ml rhodamine 123 for 15 min at 37°C. Fluorescence intensity was measured by flow cytometry, and cells displaying higher values than a defined threshold of green fluorescence were considered has having compromised mitochondrial or functional integrity.
Enzyme Activity Assays
Cells were harvested, washed, and resuspended in 2 ml of 0.05 M phosphate buffer. To disrupt cells, 200 µl of 0.5-mm-diameter glass beads was added, and sonication for 10 times in 30-s intervals interspersed with periods of cooling in ice. Cellular debris was removed by a 15-min centrifugation at 15,000 x g, and the supernatant was collected. Protein concentration was determined using the Bradford method using bovine serum albumin as standard. Superoxide dismutase activity was expressed in units defined as the enzyme amount able to inhibit in 50% the velocity of cytochrome c reduction per minute and milligrams of total protein according to McCord and Fridovich (1969). Glucose-6-phosphate dehydrogenase (G6PDH) activity was based on the increase in absorbance at 340 nm, resulting from NADP reduction, as described previously (Postma et al., 1989).
·NO Measurements
Kinetics of ·NO production and decay was measured using an ·NO-selective carbon fiber microelectrode coated with Nafion and o-phenylenediamine as described previously (Ledo et al., 2002; Ferreira et al., 2005). Early log cells (9 x 108) were washed, resuspended in 2 ml of 10 mM Tris-HCl, pH 7.4, and transferred to a recording cell with agitation under aerobic conditions. Amperometric currents originated from the oxidation of ·NO at the electrode surface were recorded by differential pulse amperometry (between +0.7 and +0.9 V, the range that includes the oxidation peak of ·NO for the experimental conditions used) using on a PGSAT 12 potentiostat (EcoChimie, Utrecht, The Netherlands) with low current module installed and controlled by GPES software, version 4.9 (EcoChimie), by using a three-electrode system with a platinum wire auxiliary electrode, a silver/silver chloride (3 M) reference electrode, and the ·NO-selective carbon fiber electrode as the working electrode. The electrodes were calibrated by a single stream flow injection analysis system using ·NO solutions prepared from ·NO donor (diethylenetriamine-NO) or from ·NO gas, as described previously (Ferreira et al., 2005). The intracellular levels of ·NO were assessed using the ·NO-selective probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) (Invitrogen). Cells were harvested, washed, and resuspended in PBS, pH 7.4, and incubated for 15 min at 37°C with 5 µg/ml DAF-FM. The mean fluorescence intensity of 20,000 cells per sample was measured by flow cytometry.
Quantification of Intracellular Levels of L-Arginine
A standard procedure for extraction of total amino acids was used (Ohsumi et al., 1988). In summary, cells at OD600 of 0.6 were harvested, washed twice in distilled water, resuspended in AA buffer (2.5 mM potassium phosphate buffer, pH 6.0, 0.6 M sorbitol, 10 mM glucose, and 0.2 M CuCl2) and incubated for 10 min at 30°C. The cell suspension was collected by filtration on 0.45-µm membrane filters (Millipore, Billerica, MA) and washed four times with AA buffer lacking 0.2 M CuCl2, representing the cytosolic fraction (Ohsumi et al., 1988). Cells retained on the filters were resuspended in water, boiled for 15 min, and then centrifuged at 5000 x g for 5 min. The supernatant was collected as the vacuolar fraction. Both fractions were combined, and homoserine was added as an internal control. Arginine concentration was then measured using high-performance liquid chromatography following the manufacturer's protocol (ESA, Boston, MA).
Nucleic Markers of Programmed Cell Death
Study of nuclear morphological alterations was performed by staining with 4',6-diaminilo-2-phenylindol dihydrochloride (DAPI). Cells were harvested, washed, and then resuspended in PBS, pH 7.4, and incubated for 10 min at 37°C with 0.5 µg/ml DAPI. Stained cells were washed twice with PBS and visualized by epifluorescence microscopy (Auxioskop 2 Plus; Carl Zeiss, Jena, Germany). DNA strand breaks were demonstrated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay with the In Situ Cell Death Detection kit, fluorescein (Roche Diagnostics, Indianapolis, IN).
Assessment of Caspase-like or ASPase Activity
Study of yeast caspase-like or other ASPase activity was performed using CaspSCREEN Flow Cytometric Analysis kit (Chemicon International, Temecula, CA). Briefly, cells were incubated with the nonfluorescent substrate (Asp)2-rhodamine 110 (D2R) at 37°C for 45 min, and then they were analyzed by flow cytometry. Cells displaying higher values than a defined threshold of fluorescence were considered has presenting caspase-like or other ASPase activity (Vachova et al., 2005; Almeida et al., 2007).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Has Increased Resistance to Oxidative Stress cells sensitivity to oxidative stress induced by several prooxidant agents. Strains deleted in BTN1 (for details, see Table 1) presented increased resistance to menadione (Figure 1A), diamide, and hydrogen peroxide (see Supplemental Material) compared with wild-type cells. The most marked differences were obtained for menadione treatment, in which considerable loss of btn1- cells viability was only obtained for concentrations >0.1 mM, whereas wild-type cells were severely affected by lower menadione concentrations, presenting <10% of viability when treated with 0.1 mM.
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mutants expressing either gene. Expression of BTN1 or CLN3 in btn1- strain (denoted pBTN1 and pCLN3, respectively) decreased the viability after menadione treatment of these strains to the wild-type level (Figure 1B), indicating the dependence of the resistance phenotype on the lack of BTN1/CLN3. The first report showing a Btn1p-specific phenotype in yeast was the increased btn1- resistance to D-(–)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), a breakdown product of chloramphenicol with pH-dependent toxicity (Pearce and Sherman, 1998), allowing the finding of altered pH homeostasis in btn1-. This study also establishes parallels between fundamental biological processes in yeast and characteristics in the human disease by correlating the increased resistance of btn1- to ANP and the severity of JNCL. To further evaluate the relationship between higher resistance to menadione and the function of Btn1p, we tested the menadione sensitivity of several btn1- strains expressing Btn1p with the following amino acid replacements: R293H, L44P, L112P, E243K, V289F, and R293C. These mutations are the counterparts of a spectrum of Cln3 mutations known to occur in the human disease heterozygously with the most frequent 1.02-kb deletion (GenBank X99832). The degree of btn1- menadione resistance is decreased by the expression of mutated Btn1p, and for the majority of these mutations, the degree of menadione resistance can be correlated with JNCL severity (Table 2).
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; Croopnick et al., 1998). To test whether btn1- increased menadione resistance was specific to btn1- and not an effect attributable to general vacuolar dysfunction, we determined the viability of strains harboring deletions in other vacuolar proteins, namely, Cup5p (a subunit of the vacuolar H+-ATPase) and Pep4p (a vacuolar aspartyl protease). Both cup5- and pep4- strains display low resistance to menadione similar to the wild-type control (Figure 1B).
Increased btn1- Menadione Resistance Is Associated with Lower ROS Levels
Treatment with menadione induces the catalytic reduction of oxygen to superoxide and other ROS, triggering mild-to-severe cellular damage and ultimately cell death (Thor et al., 1982; Morrison et al., 1984). For additional characterization of the btn1- phenotype, we performed the analysis of intracellular ROS levels, cell death, plasma membrane integrity, and mitochondrial function/integrity along 200-min treatment with 0.1 mM menadione (Figure 2, A–D). A slow increase in the percentage of btn1- cells with elevated endogenous ROS, resulting in a mean value of 45.2% of cells with a positive response at the end of the treatment, was observed. In contrast, the wild-type strain showed an abrupt increase in cells with elevated intracellular ROS with a mean value of 74.7% after 60-min treatment and 97.3% after 120 min (Figure 2A). These data correlate with the kinetic analysis of cell death in which the slow death rate of btn1- cells was markedly different from the obtained for the wild-type strain where <10% survival was observed after 120-min treatment (Figure 2B). At this time point, the entire wild-type population presented compromised plasma membrane integrity, whereas only 44.9% of btn1- cell population had lost membrane integrity (Figure 2C). Rh123 cell staining showed that btn1- strain displayed reduced percentages of cells with defective mitochondrial function/integrity at the later stages of treatment compared with the wild-type strain (Figure 2D). To assess whether the differences in intracellular ROS levels between btn1- and wild-type cells preceded loss of cellular integrity and cell death, we visualized cells colabeled with DHR123 and PI after 30 min of menadione treatment (
70% viability in wild-type cells). The results show that live wild-type cells with preserved membrane integrity present increased intracellular ROS levels when compared with btn1- cells (Figure 2E), suggesting the presence of a different ROS metabolism in both strains. Therefore, to evaluate whether the lower levels of ROS in btn1- cells were due to an increased detoxification capacity, we have performed an evaluation of antioxidant defenses. No significant alterations in the activity of superoxide dismutases were detected when comparing btn1- and wild-type cells (Table 3). The activity of catalase was also not altered in btn1- cells, even though it could only be detected in stationary phase (data not shown). Additionally, we studied whether differences in glutathione synthesis and redox balance were involved in the btn1- phenotype. The viability of btn1- and wild-type strains was largely reduced when cells with inhibited glutathione biosynthesis were treated with 0.1 mM menadione (see Supplemental Material). However, when using lower menadione concentrations, the differences in resistance between both strains were maintained (see Supplemental Material), indicating that glutathione synthesis is not accountable for btn1- phenotype. In addition, the activity of glucose-6-phosphate dehydrogenase, the major source of NADPH necessary for the redox balance of glutathione in yeast (Nogae and Johnston, 1990) was similar in both strains (Table 3). These results are in accordance with the data from DNA microarray analysis of btn1- strain that shows no differential expression of genes encoding for enzymes involved in ROS detoxification (Pearce et al., 1999a).
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Has Reduced ·NO Production, Impairing Oxidative Signaling; Packer et al., 1996). Moreover, L-arginine, the substrate of the ·NO synthesis, is decreased in btn1- cells (Kim et al., 2003), leading to the hypothesis that the reduced ROS levels of btn1- are associated to an impairment in ·NO synthesis. Because the synthesis of ·NO by yeast cells is still poorly studied, we first assessed whether nitric-oxide synthase (NOS)-like activity could be detected in S. cerevisiae. Using a standard NOS activity assay, we were able to detect conversion of arginine to citrulline in yeast protein extract, partially inhibited by 1 mM nitro-L-arginine methyl ester (L-NAME) (see Supplemental Material). Additionally, using an ·NO-selective electrode, we measured that yeast cells produce ·NO in response to menadione, and we found that ·NO production was enhanced by L-arginine (and not by its inactive enantiomer D-arginine; data not shown) and that it was inhibited by L-NAME (Figure 3A). Together, these findings suggest that an enzyme with NOS-like activity may exist in yeast. We then studied the levels of ·NO production in btn1- cells. Our results indicate that the production of ·NO in response to menadione treatment is decreased in btn1-. Importantly, this defective ·NO production in btn1- can be overcome by the addition of L-arginine, strongly suggesting that the impairment in L-arginine homeostasis in btn1- underlies this signaling defect. This result was corroborated by the use of a fluorescent probe selective for ·NO, DAF-FM (Figure 3B). In addition, this methodology allowed detecting significant lower ·NO levels under physiological conditions when comparing btn1- to wild-type cells (Figure 3B). Furthermore, the analysis of btn1- and btn1- strains expressing mutated Btn1p showed different ·NO levels that could be directly correlated with the different intracellular arginine levels present in each strain (Figure 4). Collectively, these data imply that the defective arginine levels caused by the lack of Btn1p function disrupt endogenous ·NO production in yeast.
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, that has <90–95% of the total wild-type superoxide dismutase activity; and for this reason, it is highly sensitive to redox cycling compounds (Gralla, 1997), including menadione (Figure 5B). Most interestingly, the deletion of BTN1 within sod1- background resulted in a marked survival increase after menadione treatment to values similar to btn1- cells (Figure 5B). This increased resistance of sod1- btn1- cells was lost when treatment was performed in the presence of L-arginine (Figure 5B). Deletion of BTN1 in strains lacking other ROS detoxifying enzymes, namely, sod2- and ctt1- also resulted in increased survival rates to menadione treatment that were reverted by L-arginine addition (data not shown). Collectively, these results suggest that the deletion of BTN1 causes a high decrease in the production of intracellular ROS after menadione treatment to levels that are tolerated even in the absence of Sod1p. This phenotype was reverted by L-arginine, suggesting that ·NO was mediating a burst of intracellular ROS production after menadione stimulus. To study the relevance of ·NO signaling in the response to menadione, we tested the viability after menadione treatment of btn1- and wild-type cells in the presence of L-NAME or L-arginine. Interestingly, wild-type cells showed a sevenfold increase in viability when preincubated with L-NAME, reaching values similar to btn1- (Figure 6). In contrast, incubation with L-arginine before menadione treatment resulted in a marked decrease in btn1- survival to values similar to those obtained for menadione treated wild-type cells (Figure 6). No effect in the growth rate of either strain was obtained in media supplemented with this concentration of L-arginine (data not shown). Additionally, to test the specificity of L-arginine effect on menadione-induced apoptosis, viability of both strains was assayed after menadione treatment in the presence of similar concentrations of L-alanine or L-lysine. These amino acids had no effect on survival rates (Figure 6), suggesting that the observed phenomenon is specific of L-arginine.
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; Gerasimenko et al., 2002; Chen et al., 2003). The increased resistance of btn1- cells and the reduced levels of ·NO and ROS found in this strain after menadione treatment led us to question whether menadione-induced apoptosis was impaired in btn1- cells. Menadione-treated cells were examined for nuclear markers of apoptosis such as DNA fragmentation and alterations in nuclear morphology. No DNA fragmentation could be observed in btn1- cells by TUNEL assay, whereas several wild-type cells presented a TUNEL-positive phenotype, indicative of the presence of fragmented DNA (Figure 7A). Furthermore, no nuclear morphological alterations were detected in btn1- cells after menadione treatment, whereas wild-type cells showed typical apoptotic nuclear alterations (Figure 7B), suggesting that menadione-induced apoptosis is largely impaired in btn1- cells. We also tested the involvement of yeast metacaspase activation (Madeo et al., 2002) in menadione-induced cell death. We measured ASPase activity in btn1- and wild-type cells by flow cytometric analysis of cells labeled with the substrate D2R, which was shown to become fluorescent only after cleavage by ASPase activity in yeast cells (Vachova et al., 2005; Almeida et al., 2007). Menadione treatment induced a subpopulation of 
39% of wild-type cells with high fluorescence, indicative of ASPase activity, whereas no significant activity was detected in btn1- cells (Figure 8A). Additionally, ASPase activity seems to be dependent on ·NO production, because the treatment with menadione in the presence of L-arginine resulted in an increase in percentage of wild-type and btn1- cells with positive staining, whereas L-NAME decreased the positive subpopulation. Furthermore, the survival to menadione treatment of cells deleted in yeast metacaspase (yca1-) is significantly increased compared with the survival of wild-type cells (Figure 8B). These results suggest that yeast metacaspase is involved in menadione-induced apoptosis and that btn1- cells are unable to activate yeast metacaspase, possibly due to the ·NO production defect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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results in an increased resistance to oxidative stress induced by several prooxidant agents, including menadione. These data describe a new phenotype specifically attributable to the lack of BTN1, because it is complemented by the expression of Btn1p. Phenotypic complementation also results from expression of the human Cln3, representing additional evidence of high functional conservation between Btn1p and Cln3. Similarly to a previous report on increased resistance of btn1- to ANP (Pearce et al., 1998), a correlation between JNCL severity and menadione resistance was obtained for most of the corresponding Btn1p/Cln3 mutations. The potential of this type of analysis is largely affected by the difficulties in evaluating the severity of the disease and by the reduced number of JNCL patients that are compound heterozygous. Nonetheless, these data constitute important validation of the model.
Menadione, is a quinone that induces toxicity mainly by redox cycling that consists in the catalytic reduction of oxygen to superoxide anion and other reactive oxygen species (Thor et al., 1982). Interestingly, following menadione treatment btn1- cells show decreased levels of intracellular ROS, while presenting no significant increases in the major ROS detoxification systems compared with wild-type cells. Because L-arginine, the substrate in ·NO synthesis pathway, is decreased in btn1- cells (Kim et al., 2003) and ·NO may have a role in enhancing ROS generation within the cell (Zamzami et al., 1995; Packer et al., 1996; Boveris et al., 2000), it was questioned whether ·NO was being produced in response to menadione treatment and whether it was involved in btn1- menadione-resistance phenotype. We have shown that yeast cells produce ·NO in response to oxidative stress generated by menadione by the use of both a selective electrode and an ·NO-sensitive fluorescent probe. This finding is consistent with studies on the function of yeast flavohemoglobin (Yhb1p) that imply the production of ·NO in response to oxidative stress (Cassanova et al., 2005). Our results also show that the production of ·NO is enhanced by L-arginine and inhibited by L-NAME, and we were able to detect conversion of radioactive-labeled arginine to citrulline in yeast protein extracts. Together, these results suggest enzymatic production of ·NO from L-arginine. Previous studies suggest that S. cerevisiae produces ·NO by a mechanism similar to a classical NOS (Kanadia et al., 1998; Domitrovic et al., 2003). However, bioinformatic analysis of the S. cerevisiae genome fails to identify sequences that are homologous to known NOS (data not shown). Thus, it is likely that S. cerevisiae has an unknown enzyme with NOS-like activity that is structurally unrelated to classical NOS, similarly to what has been observed in plants (Guo et al., 2003). A recent study shows that yeast cells generate ·NO from nitrite in hypoxic conditions in a manner dependent from mitochondrial respiratory chain that is consequently abolished by cyanide (Castello et al., 2006). Contrarily, the blockage of mitochondrial respiratory chain by cyanide did not alter ·NO production and cell survival after menadione treatment of wild-type and btn1- cells (data not shown), indicating an ·NO production process distinct from that described by Castello and coworkers. Menadione-induced ·NO production seems to be an essential upstream cell death signal, because the inhibition of ·NO production by L-NAME treatment decreases the intracellular ROS levels and caspase-like activity and largely increases wild-type cell resistance to menadione. Although metacaspases can be kept inactive through S-nitrosylation, a recent publication showed that after activation, a second Cys residue that is conserved in all metacaspases can rescue the first S-nitrosylated site, rendering metacaspases even more active in a high ·NO environment (Belenghi et al., 2007). Our results are consistent with this mechanism, because we show that high ·NO production triggers ROS production that has been described to mediate metacaspase activation (Madeo et al., 1999, 2002).
Most interestingly, ·NO production in btn1- cells is decreased both in physiological conditions and in response to menadione treatment. This ·NO synthesis defect in btn1- cells is partially overcome by the addition of high L-arginine concentrations to the media. Furthermore, caspase-like activity can be increased and the resistance phenotype specifically reversed when btn1- cells are treated with menadione in the presence of L-arginine. Because btn1- cells are able to take up arginine from the media (Vitiello et al., 2007), this suggests that defective ·NO production in btn1- cells is due to substrate limitation and that when btn1- intracellular L-arginine levels are increased menadione-induced cell death is restored. Supporting the importance of Btn1p in menadione-induced cell death, we have observed a large viability increase when deleting BTN1 in strains that are highly sensitive to menadione, such as sod1- cells. As summarized in Figure 9, collectively these data indicate that the endogenous production of ·NO by S. cerevisiae cells seems to be required for oxidative stress induced active cell death. In particular, ·NO seems to be synthesized from L-arginine, increasing the production of ROS within the cell and inducing the activation of a cell death cascade. Furthermore, ·NO synthesis might be limited in btn1- cells by low L-arginine levels, possibly blocking ROS production and metacaspase activation, causing an impairment of menadione-induced apoptosis.
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) alters coupling of the V-ATPase and proton-pumping across the vacuolar membrane (Padilla-Lopez and Pearce, 2006). This disruption in the electrochemical gradient between the cytosol and the vacuole likely underlies the decreased vacuolar transport of arginine observed in btn1- strains. It is possible that Btn1p/CLN3 has a role in regulating intracellular levels of arginine through active regulation of vacuolar content. Importantly, these defects have been confirmed in human cell lines from JNCL patients (Ramirez-Montealegre et al., 2005) where it has been demonstrated that the absence of functional Cln3 results in defective lysosomal transport of L-arginine and in reduced intracellular L-arginine levels. By the use of the yeast model for JNCL, we have shown for the first time that mutations in BTN1/CLN3 cause an L-arginine–related limitation in ·NO synthesis both in physiological and oxidative stress conditions. This could be a relevant advance in our understanding of JNCL, because a defect in ·NO synthesis may contribute to the extensive neuronal death observed in the patients. Among other protective functions (for review, see Kang et al., 2004), ·NO synthesis was shown to inhibit the generation of the proapoptotic sphingolipid ceramide (De Nadai et al., 2000) that Puranam et al. (1997) found to be increased in the brains of JNCL patients. Therefore, defective ·NO synthesis might be the event in JNCL pathology that is causing ceramide accumulation and neuronal apoptosis. However, proving this hypothesis requires further studies targeted at in detail evaluation of ·NO signaling in more complex in vitro and in vivo JNCL model systems.
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ACKNOWLEDGMENTS |
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ão para a Ciência e a Tecnologia (FCT) grant POCI/BIA-BCM/57364/2004, PTDC/SAU-NEU/70161/2006, and National Institutes of Health grant R01 NS-36610. N.S.O., A.C., and A.J.A. are supported by scholarships from the FCT. |
Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: Fernando Rodrigues (frodrigues{at}ecsaude.uminho.pt)
Abbreviations used: ANP, D-(–)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol; JNCL, juvenile neuronal ceroid lipofuscinose; L-NAME, nitro-L-arginine methyl ester; NCL, neuronal ceroid lipofuscinose; ·NO, nitric oxide; NOS, nitric-oxide synthase; ROS, reactive oxygen species.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.001 versus wild-type, t test; n = 5.
3.
