The Journal of Neuroscience, January 1, 1996, 16(1):253-261

Downregulation of Cu/Zn Dismutase Leads to Cell Death v& the Nitric Oxide-Peroxynitrite Pathway

Carol M. Troy,’ Daniele Derossi,* Alain Prochiantz,* Lloyd A. Greene,’ and Michael L. Shelanskil l Department of Pathology, Taub Center for Alzheimer’s Research and Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, New York 10032, and *Developpement et Evolution du Systeme Nerveux, URA 1414, Centre National de la Recherche Scientifique, Ecole Norma/e Superieure, 75230 Paris Cedex 05, France

We previously showed that the downregulation of Cu/Zn su- generators and donors accelerated cell death. N-Acetylcys- peroxide dismutase (SODl) activity in PC12 cells by exposure teine and chlorophenylthiol CAMP, which rescue PC12 cells to an appropriate antisense oligonucleotide causes their apop- and neurons from the withdrawal of nerve growth factor and totic death. In this report, we used this model to examine the other forms of trophic support, did not protect PC12 cells from pathways by which SOD1 downregulation leads to death and to SOD1 downregulation. In contrast, overexpression of bcl-2, compare these pathways with those responsible for death which also rescues these cells from loss of trophic support, caused by withdrawal of trophic support. To improve delivery of was equally effective in saving the cells in the SOD1 downregu- the SOD1 antisense oligonucleotide, we coupled it to a carrier lation paradigm. Taken together with past findings, such ob- “vector” peptide homologous to the third helix of the Drosophila servations suggest that SOD1 downregulation and withdrawal Antennapedia homeodomain. This caused not only efficient of trophic support trigger via distinct initial mecha- cellular uptake even in the presence of serum, but also inhibi- nisms but may utilize a common final pathway to bring about tion of SOD1 activity and promotion of apoptosis at IOO-fold death. Our findings may be relevant to the causes and potential lower concentrations of oligonucleotide. Death induced by amelioration of neuronal degenerative disorders caused by SOD1 downregulation appeared to require the reaction of su- impaired regulation of cellular levels of NO and superoxide. peroxide with nitric oxide (NO) to form peroxynitrite. In support Key words: Cu/Zn (SODI); free radi- of this, inhibitors of NO synthase, the responsible for cals; neuronal cells; PC12 cells; antisense oligonucleotides; NO synthesis, blocked death in our experiments, whereas NO superoxide; peroxynitrite; NO; apoptotic death

Free radicals represent a classof biologically generated species from spinal cord. In both cases,antioxidants such as vitamin E that posea potential threat to neuronalsurvival. CuiZn superox- prevented the death, which is consistentwith an action of free ide dismutase(SODl) is amongthe key cellular enzymesby which radicals. neuronsand other cells detoxify free radicals and protect them- The present study addressestwo issues.The first regardsthe selves from damage (McCord and Fridovich, 1969; Fridovich, molecular pathway by which SOD1 downregulation and conse- 1986).The observationthat a subsetof casesof familial amyotro- quent increasesin superoxidelead to apoptotic death. Two such phic lateral sclerosis(FALS) is associatedwith mis-sensemuta- pathwayshave been suggested(for review, seeDeby and Goutier, tions of SOD1 has provided the first molecular basisfor involve- 1990;Halliwell and Gutteridge, 1990;Olanow, 1993;Brown, 1995; ment of this enzymein neuronal degenerativedisorders (Rosen et Rowland, 1995). One involves superoxidepurely as a reducing al., 1993). agent for transition metal ions such as Fe3+. In this scheme,the One way to study the role of SOD1 in neuronalmaintenance is reduced metal ion catalyzes the conversionof to assessthe effectsof reducing its activity in living cells.This was to the highly reactive and destructive hydroxyl radical (Halliwell achievedin a past study by treating PC12 rat pheochromocytoma and Gutteridge, 1990).The other pathway invokes the interaction cellswith SOD1 antisenseoligonucleotides under conditions that of superoxidewith nitric oxide (NO), leading to formation of led to a SO-60% decreasein SOD1 activity. This causeddeath, via peroxynitrite. Peroxynitrite then can be protonated and rapidly an apoptotic mechanism,of -50% of the cell populationwithin 24 decomposedto a strong oxidant (Beckman et al., 1990). We hr (Troy and Shelanski,1994). Similar resultswere achieved by present experimentshere designedto distinguishwhether either Rothstein et al. (1994) with motor neurons in explant cultures or both of thesepathways are involved in apoptotic death caused by SOD1 downregulation. Received July 10, 1995; revised Aug. 18, 1995; accepted Sept. 18, 1995. A secondissue addressed here pertains to the relationship, if This study was supported by National Institutes of Health grants (M.L.S., L.A.G., any, between the mechanismsby which SOD1 downregulation C.M.T.), by a Muscular Dystrophy Association grant (C.M.T.), and by the Associa- and trophic factor withdrawal lead to apoptosis.Our initial study tion Francaise contre les Myopathies (A.P., D.D.). D.D. is a fellow of the Association Francaise contre les Myopathies. We thank A. Brunissen for peptide synthesis, and suggestedthat the two causesof death are initially divergent, but Drs. G. Chassaing, Cl. Ferrari, S. Farinelli, and I. Yan for helpful discussions. use a common final pathway to bring about death (Troy and Correspondence should be addressed to Carol M. Troy, Department of Pathology, Shelanski,1994). The present work providesfurther data to sup- College of Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York, NY 10032. port this point of view. Copyright 0 1995 Society for Neuroscience 0270.6474/95/160253-09$05.00/O To carry out our studies,we have chosenthe PC12 cell line as 254 J. Neurosci., January 1, 1996, 76(1):253-261 Troy et al. l Decreased SOD1 Causes Cell Death by NO-Peroxynitrite Pathway

Figure 1. Coupling of Antennapedia peptide to ASODl. A, Schematic illustration of cou- pling of Antennapedia peptide (vector pep- tide) with antisense oligonucleotide. B, PAGE of coupled and free vector peptide. Equimolar concentrations of oligonucleotide and peptide were incubated in deionized wa- ter for 1 hr at 37°C. An aliquot of the free peptide (lane I) or coupled peptide (lane 2) was analyzed by 20% SDS-PAGE. a model system. This line has become widely used as a model for SOD1 levels were determined as described previously (Troy and Shelan- neuronal cell survival and death, and responds both to downregu- ski, 1994) with a modification of the xanthine- system (Beauchamp and Fridovich, 1971), measuring the reduction of nitroblue lation of SOD1 (Troy and Shelanski, 1994) and to withdrawal of tetrazolium- (NBT) at 560 nm in the presence and absence of KCN trophic factor support (Batistatou and Greene, 1991, 1993; (Elrov-Stein et al., 1986). Brieflv. cell extracts or SOD (Siema. St. Louis. Rukenstein et al., 1991). To facilitate our investigations further, MO)-were incubated rh 50 m’M sodium carbonate buff& at pH 10.2 we have exploited a recently developed, highly efficient method to containing 0.1 mM EDTA, 1 X 10e4 M xanthine, 1 mM KCN, 2.5 X 10e5 deliver antisense oligonucleotides. M NBT, and 2.2 X lo-’ M xanthine oxidase in a volume of 1 ml. Reduction of NBT was measured at 560 nm. SOD1 activity was deter- MATERIALS AND METHODS mined from an SOD standard curve and is reported as the KCN-sensitive activity. Peptide synthesis. Peptide synthesis of pAntp,,,, was carried out as described by Derossi et al. (1994). Boc-Cys-(NPyS)OH was coupled RESULTS directly to the peptide as the last (N-terminal) amino acid. Coupling reaction. Oligonucleotides bearing an SH group at their 5’ end Downregulation of SOD1 by peptide-linked and an NH group at their 3’ end were purchased from Appligene (Nice, antisense oligonucleotides France). Oligonucleotides were resuspended in deionized water, an In a previous study, we showed that exposure of PC12 cells to equimolar ratio of NPyS-pAntp,,-,a peptide was added, and the mixture unmodified SOD1 antisense oligonucleotides leads to loss of was incubated at 37°C for 1 hr. The yield of the reaction, estimated by SDS-PAGE followed by Coomassie blue staining, was routinely SOD1 activity and apoptotic death (Troy and Shelanski, 1994). >50%. However, this approach requires a relatively high level of oligo- Cell culture. PC12 cells were grown as previously described (Greene nucleotide, frequent readditions, and the use of serum free me- and Tischler, 1976) on rat-tail collagen-coated dishes in RPM1 1640 dium. To extend our observations, we have used a more efficient medium containing 5% fetal calf serum (FCS) and 10% heat-inactivated horse serum (complete media). Nerve growth factor (NGF)-primed cells method to deliver oligonucleotides to cultured cells. The Anten- were grown for at least 7 d in RPM1 with 10% heat-inactivated horse napedia homeodomain protein translocates across biological serum containing NGF (100 r&ml). For incubation with unmodified membranes (Joliot et al., 1991) and directed mutagenesis has oligonucleotides, cells were washed three times with serum-free RPM1 defined a 16-amino-acid peptide of the third helix (amino acids 1640 and then plated on fresh collagen-coated dishes in RPM1 1640 43-58) defined as the vector peptide, that is responsible for the supplemented with insulin (3 pM) (Troy et al., 1992; Troy and Shelanski, 1994). For incubation with Antennapedia peptide-linked oligonucleo- translocation (Le Roux et al., 1993; Derossi et al., 1994). This tides, cells were replated in complete media or were treated as for smaller sequencedoes not bind to cognate sequencesin potential unmodified oligonucleotides. target promoters, thus avoiding other possible activities inherent Oligonucleotide internalization and visualization. Fluorescent ASODl in the 60-amino-acid homeodomain protein (Derossi et al., 1994; oligonucleotide coupled to pAntpds-ss peptide (penetratin 1) was pur- Allinquant et al., 1995). To facilitate cellular uptake of the SOD1 chased from Appligene. Oligonucleotide was uncoupled by preincubation with 100 mM dithiothreitol (DTT) for 15 min at 37°C. The coupled antisense oligonucleotide (designated ASODl), it was linked to oligonucleotide or the uncoupled oligonucleotide was diluted in complete the vector peptide by a bond (Fig. 1). As illustrated in media, and the volume added to cultured cells was equal to the volume Figure 2, the peptide-linked oligonucleotide (V-ASODl) is taken present in the culture dish. After 2 hr, the cells were washed three times up by PC12 cells grown in serum-containing medium and localizes with complete media, tixed for 5 min at -20°C in ethanol/CH,COOII (9515) dried, and mounted in Moviol before examination. within the nucleus within 2 hr. Uptake is seen in all cells exam- Confocal microscopy. Data were obtained with a confocal scanning ined. Under the same conditions, there is no apparent uptake of laser microscope (Sarastro 2000, Molecular Dynamics, Sunnyvale, CA). uncoupled ASODl. It is likely that once V-ASODl is taken up by Excitation was obtained with an argon laser set at 488 nm for fluorescein the cell, the disulfide bond is reduced to release free ASODl. In isothiocyanate (FITC), and the emitted light was filtered with an appro- support of this, Figure 2 shows an experiment in which incubation priate long-pass filter (510 nm). The sections shown were taken approx- imately at mid-height level of the cells. Photomultiplier gain and laser of V-ASODl with the reducing agent (DTT) causesloss of uptake power were identical within each experiment. of the oligonucleotide in serum-containing media. Uncoupling of Cell viability. Cells were grown in 24-well dishes and lysed in 200 ~1 the vector and oligonucleotide was confirmed by SDS-PAGE of a solution that lyses the cell membrane but leaves the nuclei intact (data not shown). (Soto and Sonnenschein, 1985). The nuclei were counted in a hemocytometer. We next compared the relative potencies of ASODl and V- SOD specific activity. Cells were extracted with 0.5% Nonidet NP-40, ASODl in promoting PC12 cell death and depressing SOD1 and protein was measured by the Bradford method (Bradford, 1976). activity. Figure 3A shows that 50% of the cells die within 24 hr of Troy et al. . Decreased SOD1 Causes Cell Death by NO-Peroxynitnte Pathway J. Neurosci., January 1, 1996, 76(1):253-261 255

V-ASODI

Figure 2. Nuclear accumulation of ASODl in serum-containing media requires coupling to Antennapedia peptide. Analysis by confocal microscopy of internalization of FITC-labeled ASODl (100 nM) coupled (lefi and middle) or uncoupled (right) to the vector peptide. Incubation with the antisense was for 2 hr in RPMI, 5% FCS, and 10% horse serum. Uncoupling was achieved by preincubating V-ASODl with 100 mM DTT for 15 min at 37°C. exposure to ASODl and V-ASODI concentrations of 4 PM and We also evaluated responses to two additional NOS inhibitors, 50 nM, respectively. In contrast, incubation with vector alone or N-monomethyl-L- (MMA) and N-nitro-L-arginine (NA). with the vector linked to the sense sequence of ASODl did not Figure 4, C and D, shows that MMA (30-100 PM) and NA (100 alter cell viability. Our previous work (Troy and Shelanski, 1994) PM) fully prevent death caused by exposure to V-ASODl. showed that the action of ASODl is sequence-specific with nei- ther a scrambled oligonucleotide nor a sense oligonucleotide NO generators potentiate and accelerate affecting either SOD1 enzymatic activity or the intensity of cellu- cell death lar SOD1 staining. Figure 3B shows the results of an experiment If the formation of peroxynitrite from superoxide and NO is a in which PC12 cells were exposed to 4 JLLM ASODl and 50 nM major pathway by which V-ASODl induces cell death, then in- V-ASODl for various times up to 6 hr (time period before cell creasing the available NO should alter the rate and/or extent of death was evident) and then evaluated for SOD1 activity. At these this process. The data in Figure 5A show that the NO generators concentrations, there was a similar rate of loss of activity down to S-nitroso penicillamine (SNAP) and sodium nitroprusside (SNP) a level of about one-third that in control cells. Thus, V-ASODl both increase cell death achieved in the presence of V-ASODl. effectively decreases SOD1 activity, but does so at concentrations This was observed for primed and for naive cultures, although approximately two orders of magnitude lower than ASODl. primed cells showed greater sensitivity and fewer surviving cells. V-ASODl was equally efficacious in serum-containing medium The NO generators increase the rate of V-ASODl-induced cell (data not shown) and in serum-free medium supplemented with death when cell survival at 5-6 and 24 hr of treatment in the insulin (Fig. 3). presence and absence of SNP, SNAP, and the NO adduct NO synthase (NOS) inhibitors protect cells from DETA-NO (all at 100 pM) is compared. At 5-6 hr there was no V-ASODI -induced death loss of viability in the presence of V-ASODI alone, whereas the Recent findings indicate that PC12 cells contain low, but detect- generators plus V-ASODl resulted in significant losses. Only by able, levels of noninducible NOS (Peunova and Enikolopov, 24 hr did viability in the cultures treated with V-ASODI alone 1995). One potential mechanism by which may lead decrease to the levels in cultures treated with the combination of to death is by interaction with cellular NO to form peroxynitrite antisense construct plus NO generator. These data indicate that (Beckman et al., 1990; Oury et al., 1993). To test this possibility, NO generators increase both the rate and extent of cell death PC12 cells were incubated with V-ASODl in the presence of NOS induced by downregulation of SODl. inhibitors and cell viability was evaluated. Figure 4A shows that N-nitro-L-arginine methyl ester (L-NAME) completely protected Free radical scavengers/transition metal chelators the cells from death at a concentration of 10 pM. This protective inhibit V-ASODl-induced death effect was eliminated in the presence of excess L-arginine (100 PM) We next examined the capacities of several iron chelators/free (data not shown). Similar results were found with PC12 cells that radical scavengers for their effect on V-ASODl-induced death had been neuronally differentiated (primed) by NGF. However, in (Fig. 6). Desferrioxamine, an iron chelator that also scavenges this case full protection required 30 FM L-NAME (Fig. 4B). This free radicals, including peroxynitrite (Beckman et al., 1990), pro- difference in L-NAME sensitivity may reflect the higher levels of tected the cells completely at a concentration of 10 FM (Fig. 6A). NOS in primed cells (Peunova and Enikilopov, 1995). Some Mimosine, another iron chelator that scavenges free radicals and toxicity of L-NAME became apparent in both control and that has antimitotic activity (Lalande, 1990), provided complete V-ASODl-treated cultures at 20 PM for naive cells and at 100 PM rescue at doses of 10 and 100 PM. However, at 400 pM this drug for the primed cells. However, in both instances, L-NAME main- was without protective effect (Fig. 6B). A third iron chelator, tains viability equal to that seen in control cells without V-ASODl diethylenetriaminepentaacetic acid (DTPA), gave complete pro- treatment. tection from death at 10 and 30 PM, but not at 100 FM (Fig. 6C). 256 J. Neurosci., January 1, 1996, 76(1):253-261 Troy et al. l Decreased SOD1 Causes Cell Death by NO-Peroxynitrite Pathway

from apoptotic death causedby lossof trophic support (Ferrari et al., 1995).NAC increasesintracellular levelsof glutathione (Meis- ter and Anderson, 1983; Isselset al., 1988) which, in turn, is V-ASOD1 involved in destruction of cellular hydrogen peroxide. Despite MOD1 these activities, NAC (0.1-60 mM) did not prevent V-ASODl- induced cell death (Fig. 7), even at concentrations that rescue serum- and NGF-deprived sympathetic neurons (Ferrari et al., 1995). Permeant AMP analogs, such as CPT-CAMP, protect PC12 cells and cultured sympathetic neuronsfrom withdrawal of sup- port by trophic factors (Rydel and Greene, 1988; Rukenstein et al., 1991). However, 100 pM CPT-CAMP, which protects PC12 cells from apoptosisinduced by serumand NGF withdrawal, did not rescuethe cells from death causedby exposureto V-ASODl (data not shown).These resultswith NAC and with CAMP ana- logs point to distinct differencesin the pathwaysby which SOD1 downregulationand trophic factor withdrawal lead to cell death. 10 160 loo0 10066 They point awayfrom a role for hydroxyl radical and glutathione- mediateddeath. oligonucleotide (nM) bcl-2 protects PC12 cells from V-ASODl -induced cell death The proto-oncogenebcl-2 inhibits cellular apoptotic death under a variety of circumstances(Korsmeyer, 1992a,b;Hockenbery et -i)- V-ASODl treated al., 1993; Kane et al., 1993). For instance,PC12 cells and sympa- thetic neuronsoverexpressing bcl-2 showresistance to death after withdrawal of trophic support (Garcia et al., 1992; Batistatou et al., 1993).To determinewhether bcl-2 alsooffers protection from lossof SODl, we testedthe effect of V-ASODl on a line of PC12 cells that overexpressthis . Figure 8 showsthat, although bcl-2 expressiondoes not affect the ability of V-ASODl to reduce SOD1 activity, it protects the cells from death at V-ASODl concentrationsof up to 480 nM. This finding indicates that bcl-2 can protect cells from apoptotic death triggered by either lossof trophic support or diminution of SOD1 activity.

DISCUSSION ” I I 1 I Use of Antennapedia peptide for delivery of 0 2 4 6 antisense oligonucleotides time (hours) We have shownthat linking ASODl to an Antennapedia peptide enhancedits potency by loo-fold and facilitated its uptake, caus- Antennapedia peptide-linked ASODl is more efficient than ing nuclear localization within 2 hr. Moreover, in contrast to our unmoditied ASODl. A, Dose-response with V-ASODl or ASODl. experiencewith unmodified oligonucleotides(Troy et al., 1992; Naive PC12 cells were washed and plated in serum free RPM1 1640 Troy and Shelanski, 1994), the peptide-linked oligonucleotide supplemented with 3 pM insulin and then incubated with indicated concentration of the different oligonucleotides. Cells were lysed at 24 could be used effectively in serum-containingmedium. This ap- hr treatment, and nuclei were counted. The number of surviving cells proach has the potential for enabling the use of antisenseoligo- is expressed relative to the number present without oligonucleotide nucleotides in a broader array of cell culture conditions and (designated as 100). B, SOD1 specific activity over 6 hr with V-ASODl potentially in intact organisms.Several other approacheshave or ASODl. Naive PC12 cells were incubated with 50 nM V-ASODl or with 4 p.~ ASODl. Cells were extracted with 0.5% NP-40, and protein been usedto modify oligonucleotidesto increasetheir utility (for was measured by the Bradford method. SOD1 levels were determined review, see Colman, 1990); the most widely usedis the phospho- with the xanthine-xanthine oxidase system, with measurement of the rothioate (P-S) modification of the backbone. The P-S oligonu- reduction of nitroblue tetrazolium at 560 nm in the presence and cleotides are nuclease-resistant,but the modification alters the absence of KCN, at the indicated times. SOD1 activity was determined from a SOD standard curve and is reported as the KCN-sensitive chirality of the moleculeswhich can result in suboptimalbinding activity. to mRNA. In contrast, for the peptide delivery systemused here, interaction with mRNA is unimpeded, because the oligonucleo- DTPA hasbeen reported to have no interaction with peroxynitrite tide is releasedin an unmodified form by cytoplasmicreducing (Beckman et al., 1990). agents. N-Acetylcysteine (NAC) and CAMP derivatives do not Pathways of ASODl-induced cell death inhibit V-ASODl -induced death Our experimentsexplored the mechanismby which SOD1 down- NAC is an antioxidant compoundthat is reported to protect cells regulation leadsto apoptotic cell death. Past observations,that from oxidative stressand that rescuesPC12 cells and neurons antioxidants protect cells from SOD1 loss,pointed to oxidative Troy et al. l Decreased SOD1 Causes Cell Death by NO-Peroxynitrite Pathway J. Neurosci., January 1, 1996, 76(1):253-261 257

control

40 I I I I 0 10 20 30 -1 I 0 2'6 5'0 7'6 A0 NAME (PM) NAME (PM) C D

Yi .-5 T ? 100’ --mm_------______ii f

= 60- ,* control ti p” I 60 -- o- - V-ASODl treated $ ,I

0 25 60 75 100

MMA (PM)

Figure 4. NOS inhibitors protect cells from death induced by downregulation of SODl. A, Protection of naive cells by L-NAME. Naive PC12 ceils were incubated with the indicated concentrations of L-NAME in the presence or absence of V-ASODl (50 nM). B, Protection of NGF-primed cells by L-NAME. NGF-primed PC12 cells were incubated with the indicated concentrations of L-NAME in the presence or absence of V-ASODl (50 nM). C, Protection of PC12 cells by MMA. Naive PC12 cells were incubated with the indicated concentrations of MMA in the presence or absence of V-ASODl (50 nM). D, Protection of cells by other NOS inhibitors. Naive PC12 cells were incubated with MMA or NA (100 pM) in the presence or absence of V-ASOD (50 nM). Relative cell survival was determined as described in Figure 3. damage as the initiating cause of death, presumably via excess In pathway 1, superoxidepromotes hydroxyl radical production accumulation of superoxides(Rothstein et al., 1994; Troy and by the iron-mediatedHaber-Weiss reaction, wherein superoxide Shelanski,1994; Greenlund et al., 199.5).The presentfindings with reducesFe+‘, which then reacts with hydrogenperoxide to form metal chelators/freeradical scavengersfurther support this possi- hydroxyl radicals(Halliwell and Gutteridge, 1990).The releaseof bility. As illustrated below, we have examined two alternative reduced iron from ferritin by superoxidecan also lead directly to pathways that have been proposed to account for the death lipid peroxidation (Thomaset al., 1985;Deby and Goutier, 1990; promoting effectsof superoxide. Halliwell and Gutteridge, 1990). The protective actions of metal HzOz chelatorsthat we observedpoint to a possiblerole for Fe and/or other transition metals. However, the chelators may have multiple , Fe3+ *Fe’+ L OH a\ / actions and may also scavengefree radicals or interact directly Pathway 1 ’ Apoptotic with peroxynitrite (Beckman et al., 1990; Radi et al., 1991). The 1 SOD1 -+ t 0; cell death \ 7 inability of NAC to protect the cells from SOD1 downregulation NO. + ON00 also arguesagainst a primary role for the reduction pathway. Pathway 2 Although NAC does not scavengesuperoxide, it doesscavenge 256 J. Neurosci.. January 1, 1996, 76(1):253-261 Troy et al. l Decreased SOD1 Causes Cell Death by NO-Peroxynitrite Pathway

- naive cell control (SNAP)

- naive cell control (SNP)

- primed cell control (SNP)

- naive cell SNAP + V-ASODl - naive cell SNP + V-ASODl

primed cell SNP + V-ASOD’

NO generator (PM)

q V-ASODl

0 + DETA*NO

q + SNAP

5 - 6 hours 24 hours

time of treatment

Figure 5. Enhancement and acceleration of cell death by NO generators. A, Naive or NGF-primed PC12 cells were incubated with the indicated concentrations of the NO generators in the presence or absence of V-ASODl (50 nM). B, PC12 cells were incubated with the indicated NO generators (all at 100 NM) in the presence of V-ASODl (50 nM). Surviving cells were counted at the times noted. Relative cell survival was determined ‘as described in Figure 3. hydroxyl radicals (Aruoma et al., 1989). Furthermore, this com- 1995).Thus, our findingsare consistentwith the notion that diminu- pound increasesintracellular levels of glutathione, which should tion of cellular SOD1 activity permits the formation of death-pro- enhanceclearance of hydrogen peroxide (Isselset al., 1988). In moting reaction productsbetween NO and superoxide.One some- contrast to our observations,Rothstein et al. (1994) reported that what surprisingoutcome of our pastwork wasthat NGF-pretreated NAC prevented death inducedby inhibition of SOD1 in organo- PC12 cellsshow enhanced sensitivity to ASODl (Troy and Shelan- typic spinal cord cultures. The presenceof multiple cell types in ski, 1994).The recent demonstrationthat NGF treatment substan- the primary cultures may account for this apparent discrepancy in tially increasesPC12 cell levels of constitutivelyactive NOS (Peunova results. and Enikilopov, 1995)might accountfor this phenomenon.Thus, the In pathway 2, superoxideinteracts with NO to form peroxynitrite. increasedsensitivity of NGF-primed cells to ASODl could be a The protective effectsof NOS inhibitors and the accelerationand resultof their enhancedcapacity to synthesizeNO and, therefore, to increaseof V-ASODl-promoted death by NO generatorsstrongly generateincreased amounts of peroxynitrite. This is supportedby suggestthat damage from downregulation of SOD1 involves NO. our finding that primedcells require higher dosesof NOS inhibitors Recentfindings have demonstratedthat PC12cells possess a detect- for full protection againstV-ASODl-induced death and are more able level of constitutively active NOS (Peunova and Enikilopov, sensitiveto the effectsof NO generators. Troy et al. l Decreased SOD1 Causes Cell Death by NO-Peroxynitrite Pathway J. Neurosci., January 1, 1996, 16(1):253-261 259

100 1

‘0 60- : , I

11’ --O- control I --s 25 60=- z V-ASODl treated ?J;-T&“’ --*-- T

11 1 nn -1 1 0 0.01 0.1 1 10 100 0 ; 1’0 1’5 2’0 N-acetylcysteine (mM) desferrioxamine (PM)

Figure Z NAC does not protect the cells. PC12 cells were incubated in q the presence of 50 nivt V-ASOD and the indicated concentrations of control NAC. At 24 hr, cells were lysed and nuclei were counted. The number of surviving cells is expressed relative to the number present without oligo- hp V-ASODI treated nucleotide and without NAC (designated as 100).

The mechanismby which peroxynitrite may lead to cell death is presently unclear and requires further study. Death could be triggered by nonenzymaticdecomposition of peroxynitrite to hy- droxyl radical and nitrogen dioxide (Beckman et al., 1990). How- ever, the ineffectivenessof NAC in our V-ASODl experiments 60 arguesagainst a major role for the hydroxyl radical.An alternative possibility worthy of further considerationis that peroxynitrite could lead to cell death by a mechanisminvolving nitrosylation of critical tyrosine residues(Stammler, 1994). Taken together, our resultspoint to the NO-peroxynitrite path- way as a major contributor to the cell death causedby SOD1 0 10 100 downregulation.Our data,however, do not rule out entirely a role mimosine (PM) for the pathway involving reduction of iron. Thus, both pathways may participate in triggering cell death, and inhibition of either may be sufficient to promote survival. q control Comparison of apoptotic death induced by q V-ASODI treated SOD1 downregulation and by serum or trophic factor deprivation One advantageof the PC12 cell systemis that it hasbeen usedto model death causedboth by SOD1 downregulationand by with- 76 drawal of trophic support. This has permitted comparisonof the mechanismsinvolved in the two events. The present observations support and extend the view that death induced by these two causesinvolves initially independentpathways. This was suggested in part by the observation that, although insulin and long-term NGF treatment promote PC12 cell survival, they did not protect them from SOD1 downregulation (Troy and Shelanski, 1994). Also consistentwith this was the conversefinding that, although vitamin E protects PC12 cellsfrom SOD1 downregulation,it does 0 10 30 100 not prevent their death when it is brought about by withdrawal of NGF or other trophic support (Ferrari et al., 199.5).Furthermore, DTPA (PM) as shownhere, CPT-CAMP and NAC, two agentsthat effectively replace NGF as a survival-promoting factor for neurons and Iron chelators protect from V-ASODl-induced cell death. serum-deprivedPC12 cells, are ineffective in preventing death in Cells were incubated with the indicated concentrations of the respec- tive chelators, with and without V-ASODl (50 nkt). A, Desferrioxam- the SOD1 downregulationparadigm. Finally, our findingspoint to ine; B, mimosine; C, DPTA. Relative cell survival was determined as an oxidative mechanismfor initiation of death by downregulation described in Figure 3. of SODl, whereasit has been arguedthat death causedby NGF 260 J. Neurosci., January 1, 1996, 76(1):253-261 Troy et al. l Decreased SOD1 Causes Cell Death by NO-Peroxynitrite Pathway

downregulation of SODl. These observations thus provide further support for the existence of a common final pathway. T ,o,T T T SOD1 and neuronal degeneration 100 1 o-o- ,o---,-- T -4 -__-___------o Our findings indicate that interfering with the destruction of IL 1 1 superoxide and increasing NO levels accelerates death of neuro- nal cells. It is presently controversial whether the SOD1 defects

--O-- associated with certain cases of FALS lead to death by a mecha- 75 bcl-2 transfected PC12 nism associated with loss or gain of function (Brown, 1995; Row- land, 1995). If the former occurs, then our findings are relevant to wild-type PC12 cells this disorder. In this case, diminution of SOD1 may enhance susceptibility of motor neurons to the formation of peroxynitrite, 50 T T T thereby leading to degeneration and death. There is evidence that A 1 1 some forms of amyotrophic lateral sclerosis may involve impaired 111 lb = uptake of glutamate (Choi, 1988; Rothstein et al., 1992). Excess I I I I r glutamate in turn can lead to enhanced levels of intracellular 0 100 200 300 400 Cazt which, in turn, can activate Ca’+-sensitive forms of NOS and increase NO synthesis (Coyle and Puttfarcken, 1993). Thus, V-AS001 (nM) there may be a critical threshold for levels of NO and superoxide in neurons that, if exceeded for either species, leads to excessive peroxynitrite formation and, ultimately, apoptosis. Our findings suggest several agents that may be effective in blocking this damage. * wild-type PC12 cells

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