Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c

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1 letters Glucose metolism inhiits poptosis in neurons nd cncer cells y redox inctivtion of cytochrome c Allyson E. Vughn 1 nd Mohnish Deshmukh 1,2,3 Neurons nd cncer cells use glucose extensively, yet the precise dvntge of this dpttion remins uncler. These two seemingly disprte cell types lso show n incresed regultion of the poptotic pthwy, which llows for their longterm survivl 1. Here we show tht oth neurons nd cncer cells strictly inhiit cytochrome c-medited poptosis y mechnism dependent on glucose metolism. We report tht the pro-poptotic ctivity of cytochrome c is influenced y its redox stte nd tht increses in rective oxygen species (ROS) following n poptotic insult led to the oxidtion nd ctivtion of cytochrome c. In helthy neurons nd cncer cells, however, cytochrome c is reduced nd held inctive y intrcellulr glutthione (GSH), generted s result of glucose metolism y the pentose phosphte pthwy. These results uncover striking similrity in poptosis regultion etween neurons nd cncer cells nd provide insight into n dptive dvntge offered y the Wrurg effect for cncer cell evsion of poptosis nd for long-term neuronl survivl. Apoptosis is geneticlly regulted process tht is essentil for the development of the orgnism, ut its dysregultion cn led to neurodegenertive disorders s well s cncer 2,3. A crucil event in the poptotic pthwy is the relese of cytochrome c from the mitochondri. In helthy cells, cytochrome c resides in the mitochondril intermemrne spce where it serves s redox crrier for the electron trnsport chin. However, in response to mny poptotic stimuli, cytochrome c is relesed into the cytosol where it cn initite the formtion of the poptosome complex, leding to cspse ctivtion nd susequent cell deth 4. Emerging evidence indictes tht cells, such s postmitotic neurons, tht lst the lifetime of the orgnism, s well s cncer cells, which must overcome cell deth response, inhiit the poptotic pthwy 1. Interestingly, despite the striking morphologicl nd functionl differences etween neurons nd cncer cells, oth extensively metolize glucose 5,6. Here we exmined whether the relince on glucose metolism ws importnt for the incresed resistnce to poptosis in neurons nd cncer cells. We ssessed the ility of endogenous cytochrome c to ctivte poptosis in sympthetic neurons y using truncted form of the pro-poptotic protein Bid (tbid). Full-length Bid is cleved intrcellulrly into tbid in response to certin poptotic stimuli, where it then cts s potent inducer of cytochrome c relese from mitochondri 7. Expression of tbid GFP plsmid DNA in mouse emryonic firolsts (MEFs) resulted in rpid nd complete poptosis, ut sympthetic neurons mintined in nerve growth fctor (NGF) were resistnt to expression of tbid GFP (Fig. 1, Supplementry Informtion, Fig. S1). Sympthetic neurons re known to e resistnt to microinjections of exogenous cytochrome c ecuse of the inhiition of cspses y XIAP. In contrst to wild-type neurons, XIAP-deficient sympthetic neurons re sensitive to microinjection of excess exogenous cytochrome c 8. However, we were surprised to find tht the relese of endogenous cytochrome c with tbid ws incple of inducing poptosis even in XIAP-deficient neurons (Fig. 1). Although it did not induce poptosis, tbid ws completely cple of relesing cytochrome c in these neurons (Fig. 1c; Supplementry Informtion, Fig. S2). Similrly, injection of Bid BH3 peptide in neurons induced the relese of cytochrome c from mitochondri ut this did not result in deth (Supplementry Informtion, Figs S1c, S2). Interestingly, despite hving relesed cytochrome c, these neurons mintin their mitochondril memrne potentil (Supplementry Informtion, Fig. S2c) 9. Thus, in NGF-mintined neurons, the relese of endogenous cytochrome c ws incple of inducing poptosis even in the sence of XIAP (Supplementry Informtion, Fig. S3). To focus on this unexpected XIAP-independent mechnism of post-cytochrome c regultion, we conducted ll following experiments in neurons isolted from XIAP-deficient mice. Despite the resistnce of neurons to deth following cytochrome c relese y tbid, sympthetic neurons redily undergo cytochrome c-medited cell deth in response to multiple poptotic stimuli 1,11. Thus, poptotic stimuli must render endogenous cytochrome c cple of ctivting poptosis in neurons. Indeed, NGF deprivtion, s well s DNA dmge with etoposide, sensitized sympthetic neurons to poptosis induced y tbid-medited cytochrome c relese (Fig. 1d; Supplementry Informtion, Figs S1, c, S4). As tbid my cuse the relese of multiple 1 Deprtment of Cell & Developmentl Biology nd 2 Neuroscience Center, University of North Crolin, Box 725, 115 Mson Frm Rod, Chpel Hill, North Crolin 27599, USA. 3 Correspondence should e ddressed to M.D. (e-mil: mohnish@med.unc.edu) Received 28 July 28; ccepted 22 Septemer 28; pulished online 23 Novemer 28; DOI: 1.138/nc187 nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

2 Survivl (percentge) Survivl (percentge) Survivl (percentge) Survivl (percentge) Nuclei tbid GFP Cyt c letters * MEF Sympthetic neurons GFP tbid GFP tbid GFP GFP tbid GFP + zvad GFP tbid GFP MEF Sympthetic neurons c XIAP +/+ XIAP / GFP tbid GFP Neurons with mitochondril cyt c (percentge) GFP tbid GFP d e NGF NGF GFP tbid GFP tbid GFP + zvad Time (h) +NGF + Rhod + Cyt c NGF + Rhod NGF + Rhod + Cyt c NGF + Rhod + Cyt c + zvad Figure 1 Endogenous cytochrome c relese is incple of inducing poptosis in NGF-mintined sympthetic neurons. () Cultures of MEFs or sympthetic neurons were injected with tbid GFP or GFP plsmid. Cell survivl ws quntified y cell morphology nd expressed s percentge of helthy green cells t 24 h, compred with 5 h post-injection (*no MEF survivl). Representtive photogrphs of MEFs nd neurons re shown; rrows indicte injected cells. () Sympthetic neurons from XIAP / or wild-type (XIAP +/+ ) mice were injected with GFP or tbid GFP, nd cell survivl ws quntified s in. (c) tbid GFP ws injected into sympthetic neurons, nd llowed to e expressed over 24 h. Cells were fixed nd the sttus of cytochrome c nlysed y immunofluorescence fctors from the mitochondri, we exmined whether this oserved effect ws direct result of sensitiztion to cytochrome c. Indeed, NGF-deprived or DNA-dmged neurons injected with cytochrome c (t time-point efore endogenous cytochrome c relese) lso showed incresed sensitivity (Fig. 1e; Supplementry Informtion, Fig. S4). To e poptoticlly ctive, cytochrome c must exist s holoenzyme complete with its hem prosthetic group 12. In vitro studies hve exmined whether the redox stte of cytochrome c ffects its poptotic ctivity. Some studies show tht oxidized cytochrome c is more poptoticlly ctive, ut others suggest tht the reduced form is lso functionl In prticulr, recent study showed tht oxidtion of cytochrome c y cytochrome c oxidse promotes cspse ctivtion, wheres its reduction y tertmethylphenylenedimine locks cspse ctivtion 18. Similrly, oxidized ut not reduced cytochrome c ws found to promote poptosis in permeilized HepG2 cells 19. To determine whether the intrcellulr microscopy. Percentge of neurons with mitochondril cytochrome c ws quntified in XIAP / neurons. (d) XIAP / sympthetic neurons were either mintined in NGF-contining medium, or deprived of NGF for 8 h (with or without 5 μm zvad), followed y injection with GFP or tbid GFP. Cell survivl ws quntified y cell morphology nd expressed s percentge of helthy green cells t 16 h, compred with 5 h post-injection. (e) XIAP / sympthetic neurons were deprived of NGF (with or without 5 μm zvad) for 12 h followed y injection with rhodmine lone, or cytochrome c (2.5 μg μl 1 ) nd rhodmine to lel injected cells. Cell survivl ws ssessed t vrious time points following injection. Dt re men ± s.e.m. of three independent experiments. redox environment ffects the sensitivity of intct neurons to cytochrome c, we treted neurons with either low levels of hydrogen peroxide (H 2 O 2 ) to crete more oxidized environment, or cell-permele reduced glutthione (GSH) to simulte reduced environment. A high deth rte ws oserved for neurons injected with cytochrome c (2.5 μg μl 1 ) only 1 16 h fter injection (Fig. 2, ). However, H 2 O 2 gretly incresed the sensitivity of neurons to injected cytochrome c (Fig. 2), wheres neurons treted with GSH were resistnt (Fig. 2). These results show tht in intct cells, the redox environment hs mrked ffect on the ility of cytochrome c to promote poptosis. As chnges in the redox environment cn hve widespred effects, we determined whether the ility of the reduced cellulr environment to inhiit cytochrome c-medited poptosis is result of the specific inctivtion of cytochrome c. Incution of exogenous cytochrome c (which is predominntly oxidized) with cytochrome 1478 nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

3 letters Survivl (percentge) Time (h) Rhod Rhod + Cyt c H 2 O 2 + Rhod H 2 O 2 + Rhod + Cyt c Survivl (percentge) hrs Rhod Rhod + Cyt c GSH + Rhod + Cyt c c Survivl (percentge) hrs Rhod + Cyt c Cyt c Reductse + Rhod + Cyt c d Survivl (percentge) Oxidized Cyt c Reduced Cyt c e Survivl (percentge) NGF + GFP NGF + tbid GFP NGF + GFP + GSH NGF + tbid + GSH f A * Oxidized Cyt c Reduced Cyt c +NGF lyste NGF lyste +NGF lyste + oxidized Cyt c NGF lyste + oxidized Cyt c Figure 2 Oxidtion of cytochrome c increses its poptotic ctivity. () XIAP / sympthetic neurons were treted with of H 2 O 2 (2 5 μm) for 2 min, then injected with cytochrome c protein nd rhodmine, or rhodmine dye lone. Cell survivl ws ssessed t vrious time points fter injection. () XIAP / sympthetic neurons were treted with GSH ethyl ester (1 mm) for 12 h, then injected cytochrome c nd rhodmine, or rhodmine lone. Cell survivl ws ssessed t 16 h fter injection. (c) XIAP / sympthetic neurons were injected with either cytochrome c or cytochrome c tht hd een pre-incuted with cytochrome c reductse (1 U ml 1 ). Cell survivl ws ssessed 16 h fter injection. (d) XIAP / sympthetic neurons were injected with either cytochrome c, or cytochrome c tht hd c reductse or dithiothreitol rendered this now reduced cytochrome c (Supplementry Informtion, Fig. S7) less effective in promoting poptosis of injected neurons (Fig. 2c, d). Thus, oxidized cytochrome c ws more potent inducer of poptosis thn reduced cytochrome c. In ddition, we generted n N52I mutnt of cytochrome c, which hs incresed stility 2. The redox sttus of this protein in intct cells is uncler; however, injection of the N52I mutnt cytochrome c into sympthetic neurons induced significntly less poptosis thn did wild-type cytochrome c (Supplementry Informtion, Fig. S5). Two predictions my e mde on the sis of the oservtion tht cytosolic cytochrome c requires n oxidized cellulr environment to e pro-poptotic. First, the ility of tbid-induced cytochrome c relese to promote poptosis in MEFs ut not in NGF-mintined neurons my e due to mrked difference in their redox environment. Consistent with this, we found tht the verge intensity of the redox-sensitive dye 5-(nd-6)-chloromethyl-2, 7 -dichlorodihydrofluorescein dicette (CM-H 2 DCFDA) ws 5-fold less in sympthetic neurons, compred with MEFs, indicting reduced levels of rective oxygen species (ROS) in sympthetic neurons (Supplementry Informtion, Fig. S6). Second, NGF deprivtion, which sensitizes neurons to cytochrome c-induced deth, my result in n incresed oxidized cellulr environment. It is known tht the cellulr redox sttus of cells cn chnge during poptosis 21. Indeed, ROS intensity, s mesured y CM-H 2 DCFDA nd dihydrorhodmine (DHR), ws incresed more thn 2.5-fold fter NGF deprivtion in neurons (Supplementry Informtion, Fig. S6, c) 22,23. This increse in ROS following NGF deprivtion ws importnt to permit een pre-incuted with DTT (nd susequently seprted) to reduce this cytochrome c. Percent survivl ws ssessed fter 6 h. (e) XIAP / sympthetic neurons were deprived of NGF for 8 h in the presence of GSH (1 mm), then injected with GFP or tbid GFP constructs. Cell survivl ws quntified y cell morphology nd expressed s percentge of helthy green cells t 16 h, compred with 5 h post-injection. (f) Exogenous cytochrome c (which is primrily oxidized) ws dded t concentrtion of 1 μm to neuronl extrcts, nd sornce (A 55 ) ws mesured fter 15 min incution. Control experiments mesuring sornce of reduced or oxidized cytochrome c re lso shown. Dt re men ± s.e.m. of three independent experiments (*P =.265, pired t-test). cytochrome c-medited poptosis, s ddition of GSH completely inhiited the ility of NGF deprivtion to sensitize neurons to cytochrome c relese y tbid (Fig. 2e). Importntly, we exmined whether there ws difference in the ility of NGF-mintined versus NGF-deprived lystes to ffect the redox stte of cytochrome c. As nticipted, lthough oxidized cytochrome c remined oxidized in NGF-deprived lystes, it ws rpidly reduced on incution with NGF-mintined neuronl lystes (Fig. 2f). Similrly, ddition of reduced cytochrome c resulted in its oxidtion in NGF-deprived, ut not NGF-mintined neuronl lystes (Supplementry Informtion, Fig. S7), indicting tht cytosolic cytochrome c would exist in different redox stte in NGF-mintined, helthy neurons, compred with those undergoing poptosis in response to NGF deprivtion. GSH is one of the most prevlent intrcellulr reducing gents nd mintins redox homeostsis y scvenging ROS. We found tht endogenous GSH is necessry for mintining cytochrome c in n inctive stte in NGF-mintined neurons, s removl of GSH with diethyl mlete (DEM) or inhiition of GSH synthesis y uthionine-sulfoximine (BSO) leds to n increse in ROS 22 nd sensitizes cells to cytosolic cytochrome c (Fig. 3, ; Supplementry Informtion, Fig. S8). Levels of GSH re mintined in cells y reduced nicotinmide denine dinucleotide phosphte (NADPH), which is generted when glucose is metolized y the pentose phosphte pthwy 24. We exmined whether the pentose phosphte pthwy ws importnt for regulting the redox sttus of neurons, nd thus cytochrome c-medited poptosis. Neurons treted with the pentose phosphte pthwy inhiitors dehydroepindrosterone nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

4 letters Survivl (percentge) d 1 Survivl (percentge) c DEM Untreted Time (h) DHEA Untreted CM-H 2 DCFDA CM-H 2 DCFDA Rhod + Cyt c DEM + Rhod DEM + Rhod + Cyt c tbid GFP DHEA + GFP DHEA + tbid GFP 6-AN + GFP 6-AN + tbid GFP Figure 3 Role of the pentose phosphte shunt in cytochrome c-medited poptosis. () Averge ROS levels in sympthetic neurons were oserved y fluorescence intensity of the redox-sensitive dye CM-H 2 DCFDA fter 3 min of GSH depletion with DEM (.1 mm). () XIAP / sympthetic neurons (NGF-mintined) were treted with DEM (.1 mm) for 3 min then injected with cytochrome c nd rhodmine, or rhodmine dye lone. Cell survivl ws ssessed t vrious time-points. (c) Averge ROS levels were oserved s in following inhiition of the Pentose Phosphte Pthwy with 2 µm DHEA for 24 h. (d) XIAP / sympthetic neurons (NGF-mintined) were treted with DHEA (2 μm) for 6 h, 6-AN (.1 mm) for 24 h, or left untreted, then injected with tbid GFP or GFP constructs. Cell survivl ws expressed s percentge of helthy green cells t 16 h, compred with 5 h post-injection. Dt re men ± s.e.m. of t lest three independent experiments. (DHEA) or 6-nicotinmide (6-AN) ccumulted high levels of ROS nd ecme sensitive to endogenous cytochrome c relese with tbid (Fig. 3c, d; Supplementry Informtion, Fig. S8). This effect ws not unique to neurons derived from the superior cervicl gngli, s sensory neurons from the dorsl root gngli (DRG) were lso resistnt to cytochrome c relese y tbid, ut ecme sensitive fter DHEA tretment (Supplementry Informtion, Fig. S9). Together, these dt show tht glucose metolism y the pentose phosphte pthwy promotes neuronl survivl y mintining GSH levels nd directly restricting cytochrome c-medited poptosis. As with neurons, mny cncer cells re known to metolize glucose extensively, phenomenon known s the Wrurg effect 6. To investigte whether incresed glucose metolism in cncer cells directly regultes poptosis t the level of cytochrome c, we first exmined the sensitivity of cncer cells to cytosolic injection of cytochrome c. Cytochrome c ws injected in the presence of Smc to eliminte the ctivity of IAPs tht re known to lock cspse ctivtion in mny cncer cells 25, thus focusing on IAP-independent mechnisms of cytochrome c regultion. Norml cells redily underwent poptosis in response to cytosolic cytochrome c, wheres cncer cells showed n incresed resistnce (Fig. 4; Supplementry Informtion, Fig. S1). The decresed sensitivity to cytochrome c correlted well with the incresed levels of intrcellulr GSH found in these cncer cells (Fig. 4). Interestingly, tretment of MEFs with cell-permele GSH incresed their resistnce to cytosolic cytochrome c (Fig. 4c). In ddition, lthough incution of oxidized cytochrome c with MEF lyste did not significntly ffect the redox stte of oxidized cytochrome c, oxidized cytochrome c ws rpidly reduced on incution with lystes generted from cncer cells (Fig. 4d). We next exmined whether enhnced glucose metolism through the pentose phosphte pthwy in HeL nd JM2 cncer cells contriutes to cellulr redox homeostsis, nd thus resistnce to cytochrome c. Indeed, ddition of the pentose phosphte inhiitor DHEA incresed ROS levels nd specificlly rendered these cncer cells sensitive to cytosolic cytochrome c (Fig. 4e, f; Supplementry Informtion, Fig. S1). Importntly, glucose deprivtion lso resulted in similr increse in sensitivity to cytochrome c (Fig. 4g). Together, these results show tht the ility of cytochrome c to induce poptosis is strictly regulted y its redox stte nd tht glucose flux is criticl regultor of cytochrome c-medited poptosis. By coupling the pro-poptotic ctivity of cytochrome c to the pentose phosphte pthwy, cells in which glucose metolism is incresed, such s neurons nd cncer cells, re le to effectively mintin restrictive environment for cytochrome c-medited poptosis. Such regultion would e physiologiclly importnt for neurons tht hve limited regenertive potentil nd survive long-term, nd dvntgeous to cncer cells in evding poptosis. Flux through the pentose phosphte pthwy is likely to inhiit poptosis under vrious conditions. For exmple, upregultion of TIGAR following p53 ctivtion y DNA dmge ws recently shown to engge the pentose phosphte pthwy nd promote cell survivl 26. Overexpression of glucose-6-phosphte dehydrogense ws lso shown to promote tumour formtion y incresing GSH through pentose phosphte pthwy 27. Our results suggest tht specific mechnism y which this could inhiit poptosis is through direct inctivtion of cytochrome c. Although mintennce of redox homeostsis is primry function of the pentose phosphte pthwy, flux through this pthwy my lso inhiit poptosis t other levels. For exmple, NADPH production through the pentose phosphte pthwy mintins cspse-2 in n inctive stte in Xenopus levis egg extrcts y mechnism independent of GSH synthesis 28. Neurons undergoing cytochrome c-medited poptosis must overcome multiple locks to undergo cell deth. We hve shown previously 148 nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

5 Annexin V-positive cells (percentge) Annexin V letters c Untreted 5 mm GSH Survivl (percentge) Norml Cncer Norml Cncer HuMec MEF HDF Sk-Mel 13 HeL JM2 GSH (ng µg 1 of protein) 6 5 HuMec MEF HeL JM MEFs Cyt c + Rhod GSH + Cyt c + Rhod Rhod d A Lyste Lyste + Oxidized Cyt c g Oxidized Cyt c Reduced Cyt c MEF Sk-Mel 13 HeL JM2 e Untreted DHEA CM-H 2 DCFDA f Survivl (percentge) Sk-Mel 13 Hel JM2 Cyt c + Rhod DHEA + Rhod DHEA + Cyt c + Rhod Survivl (percentge) HeL JM2 + Glucose + Cyt c + Rhod Glucose + Rhod Glucose + Cyt c + Rhod +Glucose + Cyt c Glucose + Cyt c Figure 4 Glucose metolism protects cncer cells from cytochrome c-medited poptosis. () Norml cells (humn mmmry epithelil cells (HuMECs)), MEFs, humn derml firolsts (HDFs) or cncer cell lines (Sk-Mel 13, HeL, JM2) were injected with cytochrome c nd Smc protein, nd cell survivl ws ssessed fter 3 min. () Totl GSH ws mesured in norml mitotic cells s well s cncer cell lines, nd expressed s concentrtion of GSH to totl cellulr protein. (c) MEFs were treted with 5 mm GSH ethyl ester for 15 min, followed y injection with cytochrome c. After 1 h, injected cells were ssessed for nnexin V positivity. Photogrphs show representtive nnexin V-FITC stining of cytochrome c injected cells (rrows). (d) Exogenous cytochrome c (which is primrily tht endogenous XIAP is potent inhiitor of cytochrome c-medited poptosis in neurons 8, nd here, tht endogenous cytochrome c itself is incple of inducing poptosis in NGF-mintined neurons. In response to developmentl poptosis fter trophic fctor deprivtion, these neurons not only showed degrdtion of XIAP to llow for cspse ctivtion, ut lso mrked decrese in glucose uptke 29 nd increse in ROS (Supplementry Informtion, Fig. S6, c) 8,22,23, which our results show is essentil for the ctivtion of cytochrome c, nd thus, poptosis. Increses in intrcellulr ROS s well s mitochondril dmge re commonly seen during geing nd re ssocited with poptotic cell deth in mny neurodegenertive diseses, including Prkinson s disese 3,31. Our results provide mechnistic insight into how even modest increse in ROS cn prime neurons to undergo poptosis in response to otherwise potentilly non-lethl events of mitochondril dmge nd cytochrome c relese. oxidized) ws dded t concentrtion of 1 μm to norml or cncer cell extrct, nd sornce ws mesured fter 15 min incution. (e) Averge ROS levels in HeL cells mesured y fluorescence of CM-H 2 DCFDA in the sence or presence of the pentose phosphte pthwy inhiitor, DHEA (2 μm) for 6 h. (f) The pentose phosphte pthwy ws inhiited in vrious cncer cell lines for 6 h y ddition of DHEA (2 µm), followed y injection of cytochrome c nd Smc protein. Cell survivl ws ssessed t 3 h. (g) JM2 nd HeL cells were deprived of glucose for 16 h followed y injection with cytochrome c nd Smc, nd ssessed for survivl t vrious time points. Imges re representtive of JM2 cells t 3 h fter cytochrome c injection. Dt re men ± s.e.m. of t lest three independent experiments. Neurons nd cncer cells re indeed strikingly distinct y most criteri. Different cncer cells themselves pper to inhiit the poptotic pthwy t different points. However, these results ring into focus the possiility tht multiple mechnisms evolved y neurons to restrict poptosis would e similr to those dpted y some mitotic cells during their progression to ecoming cncerous. METHODS Regents. All regents were purchsed from Sigm or Fisher Scientific, unless otherwise stted. Collgense nd trypsin were purchsed from Worthington Biochemicl Corportion. The GSH:GSSG kit ws purchsed from Cliochem. Horse cytochrome c ws purchsed from Sigm. zvadfmk ws purchsed from Enzyme Systems. Annexin V-FITC ws purchsed from R&D Systems. Recominnt Smc protein ws purified from cteri s descried previously 8 nd used t concentrtion of 1.5 mg ml 1. MEFs were gift from Dougls Green (St. Jude s Children s Reserch Hospitl, Memphis, TN). HDFs nd HeL cells were otined from UNC Tissue Culture Fcility, nd JM2 cells were gift from Ekhson Holmuhmedov (UNC, Chpel Hill, nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

6 letters NC). Sk-Mel 13 cells were gift from Chnning Der (UNC, Chpel Hill, NC). XIAP / mice were gift from Crig Thompson (University of Pennsylvni, Phildelphi, PA). Sympthetic neuronl cultures. Primry sympthetic neurons were dissected from the superior cervicl gngli of postntl dy 1 mice (wild-type ICR or XIAP-deficient C57BL/6 mice) nd mintined in culture s descried previously 8. Cells were plted on collgen-coted dishes t density of 1, cells per well. Neurons were grown for 4 5 dys in NGF-contining medium efore sujecting them to experimentl conditions. For NGF deprivtion, cultures were rinsed three times with medium lcking NGF, followed y the ddition of got nti-ngf neutrlizing ntiody. Cells were treted with vrious compound: etoposide (2 μm); DHEA (2 μm), 6-AN (.1 mm); DEM (.1 mm); GSHethyl ester (1 mm); BSO (2 μm). Microinjection nd quntifiction of cell survivl. Cells were injected with horse cytochrome c protein (2.5 μg μl 1 ) or Bid BH3 peptide (8 mm) (AnSpec) nd rhodmine dextrn (4 μg μl 1 ) in microinjection uffer contining 1 mm KCl nd 1 mm KPi, ph 7.4 s descried previously 8. Immeditely fter injections, the numer of rhodmine-positive cells were counted. At vrious times fter injections, the numer of vile injected cells remining ws determined using the sme counting criteri nd expressed s percentge of the originl numer of microinjected cells. This method of ssessing survivl correltes well with other cell-survivl ssys, such s trypn lue exclusion nd stining with clcein AM 8. In experiments involving microinjection of DNAs, 1 ng μl 1 of the tbid expressing plsmid, or EGFP vector in microinjection uffer ws injected into the nucleus of neurons or MEFs. To quntify survivl fter injection, GFP-positive cells were counted 5 nd 24 h fter injection. Cell survivl is expressed s rtio of vile GFP-positive cells t 24 h post-injection to GFPpositive cells t 5 h post-injection. As tbid rpidly induces deth (under certin conditions), the window etween detectle expression of tbid GFP in cells nd poptosis is nrrow. Therefore, we chose n erly time-point for time zero, to void excluding cells tht my die soon fter this. Under conditions where injection of tbid GFP or GFP lone does not kill cells, this numer ecomes greter thn 1% s few dditionl cells egin to express visile GFP fter the initil count. Under conditions where cell deth occurred, deth ws confirmed s cspse-dependent s it could e inhiited with zvad-fmk (5 μm). Cell survivl ws lso ssessed y nnexin-v positivity, ccording to the mnufcturer s protocol. Immunofluorescence microscopy for ssessing cytochrome c relese. The sttus of cytochrome c (whether intct in the mitochondri or relesed) ws exmined y immunofluorescence microscopy. Sympthetic neurons show punctte mitochondril stining with nti-cytochrome c ntiody, which is lost s cytochrome c is relesed from the mitochondri. Briefly, cultured sympthetic neurons were fixed in 4% prformldehyde nd incuted overnight with nti-cytochrome c (1:1; , BD Biosciences), or nti-gfp (1:1; Cell Signling) primry ntiody followed y 2 h incution with nti-mouse Cy3 (1:4), nti-mouse Alex 488 (1:1) or nti-chicken Alex 488 (1:1) secondry ntiody (Jckson Ls). Nuclei were stined with Hoechst (Moleculr Proes). ROS mesurement. The redox-sensitive dye CM-H 2 DCFDA or dihydrorhodmine (DHR) (Moleculr Proes) ws used to mesure ROS levels in sympthetic neuronl cultures nd cell lines using the protocol descried previously 22. CM-H 2 DCFDA is non-fluorescent when reduced nd fter cell permeiliztion, is cleved y esterses nd trpped in the cell where oxidtion converts it to fluorescent form. After tretment of cells with vrious experimentl conditions, cells were incuted with 1 μm CM-H 2 DCFDA or 1 μm DHR for 2 min then rinsed three times in PBS. Fluorescent intensity of cells ws represented s n imge, nd/or quntified y mesuring pixel intensity in 5 μm 2 re within individul som using Metmorph softwre or y mesuring the fluorescence intensity using Fluoroskn (ThermoLSystems) plte reder. Prepring reduced cytochrome c, nd ssessing cytochrome c redox sttus. Cytochrome c ws reduced y incuting it with 1 U ml 1 of cytochrome c reductse (NADPH) followed y injection into neurons. Alterntively, cytochrome c ws reduced with.5 mm DTT, then seprted on PD-1 columns (GE Helthcre). The redox sttus of cytochrome c ws ssessed y monitoring its sornce t 55 nm on Nnodrop spectrophotometer. For experiments involving nlysis of redox ctivity of cytochrome c fter NGF deprivtion, sympthetic neurons were plted t density of 1, cells nd mintined in NGF or deprived of NGF for 12 h. Neuronl lyste ws collected nd incuted with oxidized cytochrome c (1 μm) for 15 min efore nlysis, s descried ove. For mitotic cells, oxidized cytochrome c (1 μm) ws incuted with cell lystes (2 μg) for 15 min efore mesurement t 55 nm. Reduced glutthione mesurements. Mesurements of GSH:GSSG were crried out using the GSH:GSSG rtio kit ccording to modified version 32 of the mnufcturer s. Briefly, cells were lysed in 5% metphosphoric cid for 15 min, centrifuged nd the GSH content of the superntnt ws mesured y the rte of colorimetric chnge of 5,5 -dithiois(nitroenzoic cid) t 412 nm in the presence of glutthione reductse nd NADPH. The cid pellet ws resuspended in 1 N NOH nd the soluilized protein concentrtion mesured y the Lowry method (BioRd). Totl GSH is represented s ng of GSH per µg of cellulr protein. Imge cquisition nd processing. All imges were cquired y Hmmtsu ORCA-ER digitl B/W CCD cmer mounted on Leic inverted fluorescence microscope (DMIRE 2). The imge cquisition softwre ws Metmorph version 5. (Universl Imging Corportion). Imges were scled down nd cropped in Adoe Photoshop to prepre the finl figures. Note: Supplementry Informtion is ville on the Nture Cell Biology wesite. Acknowledgements We thnk Jeffery Rthmell, Gry Pielk, Eugene Johnson nd memers of the Deshmukh L for helpful discussions nd criticl review of this mnuscript. This work ws supported y NIH grnts NS42197 nd GM78366 (to M.D.), NS55486 (to A.E.V.) nd y the UNC Cncer Reserch Fund. Author contriutions A.E.V. performed ll experiments; A.E.V. nd M.D. plnned the project nd nlysed the dt. Competing finncil interests The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Wright, K. M. & Deshmukh, M. Restricting poptosis for postmitotic cell survivl nd its relevnce to cncer. Cell Cycle 5, (26). 2. Yun, J. & Ynkner, B. A. Apoptosis in the nervous system. Nture 47, (2). 3. Green, D. R. & Evn, G. I. A mtter of life nd deth. Cncer Cell 1, 19 3 (22). 4. Wng, X. The expnding role of mitochondri in poptosis. Genes Dev. 15, (21). 5. Schuert, D. Glucose metolism nd Alzheimer s disese. Ageing Res. Rev. 4, (25). 6. Wrurg, O. On the origin of cncer cells. Science 123, (1956). 7. Esposti, M. D. The roles of Bid. Apoptosis 7, (22). 8. Potts, P. R., Singh, S., Knezek, M., Thompson, C. B. & Deshmukh, M. Criticl function of endogenous XIAP in regulting cspse ctivtion during sympthetic neuronl poptosis. J. Cell Biol. 163, (23). 9. Deshmukh, M., Kuid, K. & Johnson, E. M., Jr. Cspse inhiition extends the commitment to neuronl deth eyond cytochrome c relese to the point of mitochondril depolriztion. J. Cell Biol. 15, (2). 1. Neme, S. J., Ruin, L. L. & Philpott, K. L. Blocking cytochrome c ctivity within intct neurons inhiits poptosis. J. Cell Biol. 142, (1998). 11. Vughn, A. E. & Deshmukh, M. Essentil postmitochondril function of p53 uncovered in DNA dmge-induced poptosis in neurons. Cell Deth Differ. 14, (27). 12. Yng, J. et l. Prevention of poptosis y Bcl-2: relese of cytochrome c from mitochondri locked. Science 275, (1997). 13. Hncock, J. T., Desikn, R. & Neill, S. J. Does the redox sttus of cytochrome C ct s fil-sfe mechnism in the regultion of progrmmed cell deth? Free Rdic. Biol. Med. 31, (21). 14. Pn, Z., Voehringer, D. W. & Meyn, R. E. Anlysis of redox regultion of cytochrome c-induced poptosis in cell-free system. Cell Deth Differ. 6, (1999). 15. Suto, D., Sto, K., Oh, Y., Yoshimur, T. & Fujii, J. Suppression of the pro-poptotic function of cytochrome c y singlet oxygen vi hem redox stte-independent mechnism. Biochem. J. 392, (25) nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

7 letters 16. Hmpton, M. B., Zhivotovsky, B., Slter, A. F., Burgess, D. H. & Orrenius, S. Importnce of the redox stte of cytochrome c during cspse ctivtion in cytosolic extrcts. Biochem. J. 329, (1998). 17. Kluck, R. M. et l. Cytochrome c ctivtion of CPP32-like proteolysis plys criticl role in Xenopus cell-free poptosis system. EMBO J. 16, (1997). 18. Borutite, V. & Brown, G. C. Mitochondril regultion of cspse ctivtion y cytochrome oxidse nd tetrmethylphenylenedimine vi cytosolic cytochrome c redox stte. J. Biol. Chem. 282, (27). 19. Li, M., Wng, A. J. & Xu, J. X. Redox stte of cytochrome c regultes cellulr ROS nd cspse cscde in permelized cell model. Protein Pept. Lett. 15, 2 25 (28). 2. Doyle, D. F. et l. Chnging the trnsition stte for protein (Un) folding. Biochemistry 35, (1996). 21. Ci, J. & Jones, D. P. Mitochondril redox signling during poptosis. J. Bioenerg. Biomemr. 31, (1999). 22. Kirklnd, R. A. & Frnklin, J. L. Evidence for redox regultion of cytochrome C relese during progrmmed neuronl deth: ntioxidnt effects of protein synthesis nd cspse inhiition. J. Neurosci. 21, (21). 23. Greenlund, L. J., Deckwerth, T. L. & Johnson, E. M., Jr. Superoxide dismutse delys neuronl poptosis: role for rective oxygen species in progrmmed neuronl deth. Neuron 14, (1995). 24. Kplowitz, N., Aw, T. Y. & Ookhtens, M. The regultion of heptic glutthione. Annu Rev. Phrmcol. Toxicol..25, (1985). 25. Verhgen, A. M. & Vux, D. L. Cell deth regultion y the mmmlin IAP ntgonist Dilo/Smc. Apoptosis 7, (22). 26. Bensd, K. et l. TIGAR, p53-inducile regultor of glycolysis nd poptosis. Cell 126, (26). 27. Kuo, W., Lin, J. & Tng, T. K. Humn glucose-6-phosphte dehydrogense (G6PD) gene trnsforms NIH 3T3 cells nd induces tumors in nude mice. Int. J. Cncer 85, (2). 28. Nutt, L. K. et l. Metolic regultion of oocyte cell deth through the CMKII-medited phosphoryltion of cspse-2. Cell 123, (25). 29. Deckwerth, T. L. & Johnson, E. M., Jr. Temporl nlysis of events ssocited with progrmmed cell deth (poptosis) of sympthetic neurons deprived of nerve growth fctor. J Cell Biol. 123, (1993). 3. Crney, J. M., Smith, C. D., Crney, A. M. & Butterfield, D. A. Aging- nd oxygeninduced modifictions in rin iochemistry nd ehvior. Ann. NY Acd. Sci. 738, (1994). 31. Rego, A. C. & Oliveir, C. R. Mitochondril dysfunction nd rective oxygen species in excitotoxicity nd poptosis: implictions for the pthogenesis of neurodegenertive diseses. Neurochem. Res. 28, (23). 32. Tietze, F. Enzymic method for quntittive determintion of nnogrm mounts of totl nd oxidized glutthione: pplictions to mmmlin lood nd other tissues. Anl. Biochem. 27, (1969). nture cell iology volume 1 numer 12 DECEMBER Mcmilln Pulishers Limited. All rights reserved.

8 Percent Survivl supplementry informtion DOI: 1.138/nc187 % Cells Annexin positive MEFs GFP tbid-gfp tbid-gfp + zvad % Cells Annexin positive Sympthetic neurons +NGF + tbid-gfp -NGF + GFP -NGF + tbid-gfp -NGF + tbid-gfp + zvad c Sympthetic neurons +NGF + Bid BH3 + Rhod -NGF + Rhod -NGF + Bid BH3+ Rhod -NGF + Bid BH3 + zvad + Rhod Figure S1 tbid induces n poptotic cell deth in MEFs nd NGF-deprived neurons. ) MEFs were injected with tbid-gfp in the presence or sence of zvad. After 5 hrs, cells were nlyzed for Annexin V-Cy3 positivity. ) XIAP-/- sympthetic neurons were either mintined in, or deprived of NGF for 8 hrs, followed y injection with tbid-gfp in the presence or sence of zvad. Cells were then nlyzed for Annexin V positivity. c) NGF deprivtion sensitizes neurons to cytochrome c relese y Bid BH3. XIAP-/- neurons were either mintined in medi contining NGF, or deprived of NGF for 1 hrs, followed y injection with Bid BH3 peptide long with rhodmine to mrk the injected cells. zvad ws dded to the indicted condition t the time of injection. Cell survivl ws ssessed 6 hrs fter injection. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

9 % Neurons with mitochondril cyt c % Neurons with mitochondril cyt c supplementry informtion tbid Bid BH3 Nuclei Nuclei * GFP tbid-gfp tbid Cytochrome c Overly Rhod Bid BH3 + Rhod Rhodmine Cytochrome c c Bid BH3 Phse 1 % Neurons which hve relesed cyt c Mitotrcker Mintined Mitotrcker stining Lost Mitotrcker stining Cytochrome c Nuclei Figure S2 tbid induces cytochrome c relese ut not poptosis in neurons. ) XIAP-/- sympthetic neurons were injected with plsmids encoding GFP, tbid-gfp or n untgged tbid construct, nd fter 24 hrs, the sttus of cytochrome c in injected neurons ws ssessed nd quntified y immunofluorescence. * represents 1% cytochrome c relese. Photogrphs re representtive of cytochrome c stining fter 24 hrs of tbid expression. Arrow points to cell injected with tbid. ) Neurons were injected with Bid BH3 peptide long with rhodmine to mrk the injected cells. Cytochrome c sttus ws ssessed y immunofluorescence 8 hrs fter injection. Arrow points to cell injected with the Bid BH3 peptide. c) tbid expressing neurons relese cytochrome c, ut mintin mitochondril memrne potentil. Neurons were injected with Bid BH3 peptide. After 3 hrs, cells were scored either s mintining memrne potentil (Mitotrcker stining mintined) or lost memrne potentil (Mitotrcker stining lost) using Mitotrcker Ornge. Cells were then fixed nd cytochrome c relese ssessed y immunofluorescence. Dt is represented s percentge Bid BH3 injected cells which hve relesed cytochrome c, tht hve either mintined or lost their memrne potentil. Arrows represent cell which hs een injected with the Bid BH3 peptide. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

10 supplementry informtion Percent Survivl tbid-gfp tbid-gfp + Smc Figure S3 Neuronl resistnce to cytochrome c relese is not medited y IAPs. XIAP-/- neurons were injected with tbid-gfp, or tbid-gfp + Smc protein. Cell survivl ws ssessed y morphology y quntifying the numer of GFP positive cells t 16 hrs compred to 5 hrs post-injection. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

11 Percent Survivl Percent Survivl supplementry informtion NGF GFP tbid-gfp +NGF + Etop Time (hrs) Rhod + Cyt c Etop + Rhod Etop + Rhod + Cyt c Figure S4 DNA dmge sensitizes neurons to cytochrome c-medited poptosis. ) XIAP-/- sympthetic neurons were treted with 2 µm etoposide for 12 hrs, followed y injection of tbid-gfp or GFP plsmids. Cell survivl ws ssessed y morphology y quntifying the numer of vile GFP positive cells t 16 hrs compred to 5 hrs post-injection. ) XIAP-/- sympthetic neurons were treted with 2 µm etoposide for 12 hrs followed y injection with cytochrome c nd rhodmine, or rhodmine lone. Cell survivl ws ssessed t multiple time points. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

12 supplementry informtion Percent Survivl t 4 hrs Sympthetic Neurons WT Cyt c N52I Cyt c Figure S5 Cytochrome c N52I hs reduced potency in inducing poptosis in XIAP-/- intct neurons. XIAP-/- sympthetic neurons were injected with recominnt wildtype (WT) or N52I cytochrome c (4 µg/ul) nd ssessed for survivl fter 4 hrs. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

13 Fold DHR Intensity Fold CM-H 2 DCFDA Intensity DHR Fold CM-H 2 DCFDA Intensity supplementry informtion Sympthetic neurons MEFs + NGF -NGF c + NGF -NGF + NGF -NGF Figure S6 ROS levels in MEFs, nd sympthetic neurons fter NGF deprivtion. ) MEFs were co-cultured with sympthetic neurons, followed y incution with 1 µm CM-H 2 DCFDA redox-sensitive dye. Fluorescent intensity ws mesured in 5 µm 2 re within individul som nd quntified using Metmorph softwre. ) XIAP-/- sympthetic neurons were mintined in NGF, or deprived of NGF for 12 hrs, followed y incution with CM-H 2 DCFDA. Fluorescent intensity ws quntified s in (). c) Sympthetic neurons were mintined in NGF, or deprived of NGF for 12 hrs, followed y incution with 1 um Dihydrorhodmine (DHR). Fluorescent intensity ws quntified s in (). Representtive photogrphs re shown Mcmilln Pulishers Limited. All rights reserved.

14 supplementry informtion 55 nm Cyt c Cyt c + Cyt c Reductse Cyt c + GSH Cyt c + DTT 55 nm Oxidized Cyt c Reduced Cyt c + NGF Lyste - NGF Lyste + NGF Lyste + Reduced Cyt c - NGF Lyste + Reduced Cyt c Figure S7 Neurons undergoing poptosis re le to oxidize cytochrome c. ) 4 µm horse cytochrome c ws incuted with either 1 units/ ml cytochrome c reductse, 1 mm glutthione ethyl ester (GSH), or.5 mm DTT for 15 minutes, followed y mesurement of As 55. ) Sympthetic neurons were mintined in NGF or deprived of NGF for 12 hrs. Lystes were then incuted with 4 µm cytochrome c which hd een reduced with DTT (nd susequently seprted from) followed y mesurement of As 55. Control experiments mesuring As 55 of reduced or oxidized cytochrome c re lso shown. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

15 Percent survivl supplementry informtion * Percent survivl * Cyt c + Rhod BSO + Cyt c + Rhod BSO + Rhod tbid 6-AN + tbid 6-AN + tbid + zvad Figure S8 Inhiition of GSH synthesis or the pentose phosphte pthwy sensitizes neurons to poptosis y cytochrome c. ) XIAP-/- sympthetic neurons were treted with 2 µm BSO for 16 hrs, followed y injection with cytochrome c. Survivl ws ssessed t 3 hrs post-injection. * sttisticl significnce of n=3 with p=.14 compred to BSO + Rhod. ) XIAP-/- neurons were treted with 6-AN for 24 hrs, followed y injection with tbid long with GFP to mrk injected cells. zvad ws dded t time of injection. Survivl ws ssessed y quntifying the numer of GFP positive cells t 9 hrs vs. 3 hrs post-injection. *sttisticl significnce of n=3 with p=.17 s compred to 6-AN + tbid Mcmilln Pulishers Limited. All rights reserved.

16 Percent Survivl supplementry informtion DRG Neurons Bid BH3 DHEA + Bid BH3 Bid BH3+ Rhodmine DHEA + Rhodmine DHEA + Bid BH3+ Rhodmine Figure S9 DRG sensory neurons re resistnt to cytochrome c relese, ut ecome sensitive upon inhiition of the pentose phosphte pthwy. Dorsl Root Gnglion neurons were treted with 1 um DHEA for 12 hrs, followed y injection with 2 mm Bid BH3 peptide long with Rhodmine dextrn. Cell survivl ws ssessed 6 hrs fter injection. Representtive photogrphs re shown. Arrows point to the injected cells. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

17 supplementry informtion % Annexin V positive cells HDF HuMec MEF HeL Sk-Mel 13 Cyt c + Rhod Cyt c + Rhod + zvad Fold Chnge in DHR Intensity HeL Sk-Mel 13 Untreted HeL DHEA Untreted DHEA Figure S1 Cytochrome c induces poptosis in norml mitotic cells, nd inhiition of the pentose phosphte pthwy induces ROS in cncer cells. ) Norml mitotic cells were either left untreted, or treted with zvad, nd these cells, in ddition to cncer cell lines, were injected with cytochrome c (nd Smc) long with rhodmine to mrk injected cells. One hour following injections, rhodmine positive cells were ssessed for Annexin V-FITC positivity. ) HeL cells nd Sk-Mel 13 cells were treted with DHEA for 6 hrs, followed y incution with 1 um Dihydrorhodmine (DHR). Fluorescent intensity of DHR ws quntitted on Fluoroskn plte reder. Representtive photogrphs of HeL cells re shown. Error rs represent ±SEM of n> Mcmilln Pulishers Limited. All rights reserved.

18 Supplementry Methods: Determintion of mitochondril memrne potentil. Prior to fixing to ssess cytochrome sttus, the mitochondril memrne potentil in individul cells ws ssessed with Mitotrcker Ornge. Neurons were incuted with 1 µm Mitotrcker (prepred freshly in DMSO) in cell culture medium for 3 min t 37ºC in the drk. Neurons were then wshed 3 times nd individul cells were scored s either highly stining with Mitotrcker (retined memrne potentil) or wekly stining for Mitotrcker (lost memrne potentil). DRG neuronl cultures. Dorsl Root Gngli were dissected from postntl dy, XIAP-deficient C57BL/6 mice nd mintined in culture with medi contining DMEM, 1% FBS, supplemented with 5 ng/ml NGF. Cells were plted on collgen coted dishes, nd grown for 4 dys in NGF contining medi efore treting them with experimentl conditions. Genertion nd expression of wildtype nd N52I Cytochrome c. The plsmid pbtr(hcc) (kindly provided y Dr. Gry Pielk, UNC) expresses oth the horse cytochrome c nd yest heme lyse. A 6 histidine tg ws inserted t the N-terminus of pbtr(hcc), nd site directed mutgenesis ws used to crete the N52I mutnt. The 6XHis wildtype (WT Cyt c) nd N52I mutnt plsmids were trnsformed into BL21 (DE3) to express recominnt protein descried previously 1. The cteril pellet ws lysed y soniction, nd Ni-NTA grose eds (QIAGEN) were used to purify the His- 28 Mcmilln Pulishers Limited. All rights reserved.

19 tgged protein ccording to the mnufcturer s protocol. The protein ws dilyzed overnight, nd concentrted with Amicon Ultr 1, MWC tues. 1. Ptel, C.N., Lind, M.C. & Pielk, G.J. Chrcteriztion of horse cytochrome c expressed in Escherichi coli. Protein Expr Purif 22, (21). 28 Mcmilln Pulishers Limited. All rights reserved.