Regulation of sympathetic neuron and neuroblastoma cell death by XIAP and its association with...

Regulation of sympathetic neuron and neuroblastoma cell death byXIAP and its association with proteasomes in neural cells

Li-Ying Yu,a Laura Korhonen,b Rodrigo Martinez,b Eija Jokitalo,c Yuming Chen,b

Urmas Aruma¨e,a and Dan Lindholmb,*a Program of Molecular Neurobiology, Institute of Biotechnology, Biocenter 1, P.O. Box 56 Viikinkaari 9,

University of Helsinki, FIN-00014, Helsinki, Finlandb Department of Neuroscience, Neurobiology, Uppsala University, Biomedical Centre, Box 587, S-751 23 Uppsala, Sweden

c Electron Microscopy Unit, Institute of Biotechnology, Biocenter 1, P.O. Box 56, Viikinkaari 9, University of Helsinki, FIN-00014, Helsinki, Finland

Received 10 July 2002; revised 25 October 2002; accepted 19 November 2002

Abstract

XIAP (X chromosome-linked inhibitor of apoptosis protein) has been shown to inhibit cell death in a variety of cells. XIAP binds toactive caspases, but XIAP also has a carboxy-terminal RING domain that can regulate cell death via protein degradation. Here we havestudied the function of full-length and RING-deleted XIAP in mouse sympathetic neurons microinjected with expression plasmids and inneuroblastoma cells stably overexpressing these proteins. Both full-length and RING-deleted XIAP-protected sympathetic neurons againstdeath induced by nerve growth factor (NGF) withdrawal to about the same extent. However, the two proteins were differentially localizedin transfected neurons, with RING-deleted XIAP present in the cytoplasm and full-length XIAP found mostly in cytoplasmic proteinaggregates, as revealed by transmission electron microscopy. The occurrence of these aggregates was blocked by lactacystin, a proteasomeinhibitor. In neuroblastoma cells, RING-deleted XIAP protected against death induced by staurosporine, thapsigargin, or serum withdrawal,whereas full-length XIAP was without effect. Full-length, but not RING-deleted, XIAP was degraded and ubiquitinated in the neuroblas-toma cells. The results show that the presence of the RING domain differentially affected the neuroprotective ability of XIAP in sensoryneurons and neuroblastoma cells. The RING domain was essentially required for the proteasomal association of XIAP and for itsubiquitination.© 2003 Elsevier Science (USA). All rights reserved.

Introduction

XIAP belongs to the family of inhibitor of apoptosisproteins (IAPs) and has been shown to inhibit apoptosis ina variety of cells. Different IAP family members antagonizecell death on a variety of signals, among others treatmentwith chemotherapeutic agents, Fas activation, and trophicfactor withdrawal (Clem and Duckett, 1997; LaCasse et al.,1998; Silke and Vaux, 2001). XIAP is also involved incellular signaling, as shown for bone morphogenetic protein(BMP)/transforming growth factor� (TGF-�) receptors(Yamaguchi et al., 1999), modulation of the transcriptionfactor NF-�B (Hofer-Warbinek et al., 2000), and induction

of c-Jun N-terminal kinase activity (Sanna et al., 1998). Theamino-terminal region of XIAP contains three baculovirusinhibitory repeat (BIR) domains, which are about 70 aminoacids long, and bind and inhibit caspases that are activatedduring apoptosis (Deveraux et al., 1997; Takahashi et al.,1998). The BIR-2 region and the linker sequence betweenBIR-1 and BIR 2 bind active caspase-3 and -7 at highaffinity, whereas BIR3 domain binds caspase-9 (Huang etal., 2001; Riedl et al., 2001; Sun et al., 2000). XIAP also hasa RING zinc finger domain in the carboxy-terminal region,and a similar domain is found in more than a hundreddifferent proteins. Characteristic of the RING domains aretheir histidine and cysteine residues with zinc-binding ca-pacity which are crucial for activity (Borden, 2000). TheRING domain is found in proteins involved in RNA pro-cessing, organelle transport, and viral replication, among

* Corresponding author. Fax:�46-18-559017.E-mail address: [email protected] (D. Lindholm).

Molecular and Cellular Neuroscience 22 (2003) 308–318 www.elsevier.com/locate/ymcne

1044-7431/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S1044-7431(02)00038-6

others (Borden, 2000). The unifying theme for the functionof the different RING domains seems to be that they areinvolved in formation of large macromolar scaffolds. Anexample of this is the activity of RING domain in theautodegradation of IAPs, shown to occur through ubiquiti-nation in thymocytes (Yang et al., 2000). There are also datasuggesting that the RING domain of IAPs from Drosophilamay function as a negative regulator of apoptosis (Hay etal., 1995). Conversely, there are several examples wheredeletion of RING domain has no effect on the antiapoptoticactivity of XIAP (Silke et al., 2002, Yang et al., 2000).Thus, the exact role of the RING domain for the function ofXIAP in different mammalian cells has not been clarified.

XIAP is expressed in the nervous system mainly inneurons, and is regulated by kainic acid, inducing seizures(Korhonen et al., 2001), and by axotomy in spinal motoneu-rons (Perrelet et al., 2002). Available data indicate thatoverexpression of XIAP by adenoviral vectors is neuropro-tective in different types of brain injuries (Eberhardt et al.,2000; Perrelet et al., 2002; Xu et al., 1999). However, therole of XIAP in regulation of death of developing neurons,especially at the single-cell level, is not fully understood.

In the present work, we have studied the function ofXIAP Overexpressed in mouse sympathetic neurons de-prived of nerve growth factor (NGF). We have also ana-lyzed the role of the RING domain of XIAP in proteindegradation and neuroprotection, by comparing effects ofthe RING-deleted protein with those of full-length XIAP.To substantiate data obtained with sympathetic neurons, wehave studied neuroblastoma cells stably overexpressingXIAP. The results show that full-length and RING-deletedXIAP both protected sympathetic neurons against deathinduced by NGF withdrawal. However, in neuroblastomacells only the RING-deleted XIAP protein showed a signif-icant protective effect against cell death induced by thapsi-gargin, staurosporine, or serum withdrawal. This differencein activity between the two XIAP proteins in neuroblastomacells was related to altered protein stability and the ubiq-uitination and association of XIAP with proteasomes (De-Martino and Slaughter, 1999), which required the intactRING domain.

Results

Effect of full-length and RING-deleted XIAP onsympathetic neuron death

Developing superior cervical ganglion (SCG) neuronsobtained from neonatal mice represent a well-establishedsystem in which to study neuronal apoptosis (Deckwerthand Johnson, 1993). These neurons depend on NGF for theirsurvival, and die in the absence of this neurotrophic factor.Expression vectors encoding for full-length and RING-de-leted XIAP were microinjected into SCG neurons, whichwere cultured in the presence and absence of NGF. Figure

1 shows that overexpression of full-length XIAP rescued asignificant number of neurons, which die in the absence ofNGF. Deletion of the C-terminal RING domain slightlyimproved the survival-promoting activity of XIAP in thissystem, but the effect was not statistically significant (Fig.1). The presence of the N-terminally linked GFP moiety hadno significant effect on the antiapoptotic activity of XIAP.In the presence of NGF, XIAP, with or without the RINGdomain, did not affect survival of SCG neurons (Fig. 1). Thesmall decrease in neuron survival observed under theseconditions was comparable to that obtained with emptyvector alone. Thus, XIAP acts as an antiapoptotic protein inSCG neurons, and its effect is independent of the presenceof the RING domain.

Differential distribution of full-length and RING-deletedXIAP in sympathetic neurons

When analyzed by fluorescence microscopy, GFP-XIAPwas localized to distinct inclusions in injected neuronsgrown with NGF (Fig. 2). The nature of these inclusionswas studied by transmission electron microscopy. To un-equivocally identify injected neurons, they were grown oncoverslips with a grid, and low-power phase-contrast andfluorescence images were taken of the injected culturesbefore fixation. The positions of the injected neurons on thephotographs were identified by fluorescence, and localizedin the Epon block according to the grid coordinates. Trans-mission electron microscopic analysis revealed protein ag-gregates of different sizes in the GFP-XIAP-expressing neu-rons (Fig. 3). These were not found in neighboringuninjected neurons. Other aspects of the ultrastructure of

Fig. 1. Overexpression of XIAP in mouse sympathetic neurons. Newbornmouse sympathetic neurons overexpressing XIAP, GFP-XIAP, GFP-XIAPwithout the C-terminal RING domain (GFP-XIAP-�RING), or emptypcDNA3.1 plasmid were grown for 3 days with or without NGF. Livingneurons were counted and expressed as percentages of initial neuronsliving 3 h after injection. Values represent three or six independent exper-iments � SEM. *P � 0.05; ***P � 0.001.

309L-Y. Yu et al. / Molecular and Cellular Neuroscience 22 (2003) 308–318

GFP-XIAP-overexpressing neurons were normal (Fig. 3).Immunostaining showed that the surfaces of the aggregateswere positive for XIAP whereas the interior of the aggre-gates was not stained due to inability of the antibodies topenetrate the densely packed proteins (Figs. 3C, 3D).

Formation of the fluorescent aggregates was partiallyblocked by lactacystin (Fig. 2), which is a proteasomeinhibitor (Fenteany and Schreiber, 1998). The lactacystin-treated cultures deteriorated within 3 days. Therefore theinhibition of aggregate formation was not complete in someneurons, whereas in some, the aggregates did not form at all.Both examples are in Fig. 2. Partial inhibition of the aggre-gate by lactacystin suggests, but does not formally prove,that proteasome activity is necessary for their formation.Further biochemical studies of proteasome inhibition inprimary cultured neurons are hard due to scarcity of thematerial. Attempts to stain the GFP-XIAP-expressing neu-rons with anti-ubiquitin antibodies failed, due to high non-specific background of these antibodies in primary neurons(data not shown). Deletion of the RING domain in XIAP

resulted in a diffuse localization of the fluorescent proteinthroughout the neuron, showing that the formation of ag-gregates was mediated by the presence of the RING domain.The data show that full-length and RING-deleted GFP-XIAP had different distributions in injected sympatheticneurons grown in the presence of NGF. A similar situationwas observed for these neurons in the absence of NGF andkept alive by overexpression of full-length or RING-deletedXIAP. Figure 2 shows an example of this for GFP-XIAP-injected SCG neurons, which had many fluorescent aggre-gates in the cytoplasm in the absence of NGF.

Comparison of full-length and RING-deleted XIAP ondeath of neuroblastoma cells

Sympathetic neurons are postmitotic cells and the stateof cell proliferation may possibly affect the function ofXIAP. Also, the small amount of material does not allowbiochemical analysis of primary neurons. We therefore gen-erated mouse neuroblastoma N2A cells stably overexpress-

Fig. 2. Localization of GFP-XIAP in microinjected sympathetic neurons. Representative pairs of phase-contrast and fluorescence microscope images ofsympathetic neurons expressing GFP-XIAP or GFP-XIAP without the C-terminal RING domain (GFP-XIAP-�RING) taken 24 h after injection from neuronsgrown with or without NGF. Shown are also GFP-XIAP-expressing neurons grown in the presence of 1 �M lactacystin for 24 or 72 h. Note the disappearanceof fluorescent aggregates in the presence of lactacystin. Neurons treated with lactacystin appeared less healthy at 72 h, but not yet so at 24 h after treatment.The images were captured with a 63� objective. Bar � 20 �m.

310 L-Y. Yu et al. / Molecular and Cellular Neuroscience 22 (2003) 308–318

ing full-length or RING-deleted XIAP. As shown in West-ern blots, transfected N2A cells exhibited an equalexpression level of XIAP protein, the RING-deleted proteinband migrating slightly faster, as expected (Fig. 4B). XIAP-overexpressing neuroblastoma cells were then challengedwith different agents to induce death, and cell viability wasdetermined by the MTT assay. Figure 4A shows that over-expression of RING-deleted XIAP afforded significant pro-

tection of N2A cells against death induced by thapsigargin(calcium overload) and serum withdrawal (trophic factordeprivation). RING-deleted XIAP-expressing cells alsoseemed to be more protected against staurosporine-induceddeath, compared with control N2A cells, but only at higherdrug concentrations (Fig. 4). In contrast, neuroblastomacells overexpressing full-length XIAP were not significantlymore protected compared with control for any of the treat-

Fig. 3. Transmission electron microscopic analysis of GFP-XIAP-overexpressing sympathetic neuron. (A) Low-power micrograph of a section through theneuron, the epifluorescence image of which is shown in the inset, showing normal ultrastructure and three inclusion bodies (arrows). Note that the inclusionbodies locate at different levels in the round neuron; therefore on any given ultrathin section, only some inclusion bodies are included, although all are visiblein the epifluorescence image. Secondary lysosomes (dark structures) and lipid droplets (asterisks) are the normal structures of sympathetic neurons. (B)Higher-power micrograph of an inclusion body from the same neuron. The amorphous structure and lack of surrounding membrane are indicative of theprotein aggregate. Note normal ultrastructure of mitochondria and rough endoplasmic reticulum. (C, D) Aggregates found in the GFP-XIAP-injected neuronswere stained with anti-XIAP antibodies. Note the strong staining of the surface and the absence of staining of the interior due to the inability of antibodiesto penetrate the densely packed protein aggregate. Note the normal ultrastructure and only background labeling of the mitochondria (M), Golgi complex (G),and endosomes (E). Bars � (A) 2 �m, (B) 200 nm, (C,D) 500 nm.


ments (Fig. 4). Thus, the RING domain is required for XIAPto protect neuroblastoma cells against apoptotic stimuli usedin this study. The results indicate that the antiapoptoticfunction of XIAP is different in dividing neuroblastomacells and postmitotic primary sympathetic neurons.

Stability and ubiquitination of XIAP in neuroblastomacells

The differential effects of full-length and RING-deletedXIAP on the survival of neuroblastoma cells prompted us tostudy the stability of XIAP in N2A cells. As shown in Fig.5, full-length XIAP protein is downregulated in staurospo-rine-treated N2A cells. This degradation was abolished bythe presence of lactacystin, which alone also slightly ele-vated XIAP levels in these cells. In contrast, neither lacta-cystin nor staurosporine affected the levels of RING-deletedXIAP in transfected N2A cells (Fig. 5). Thus, full-lengthand RING-deleted XIAP have different half-lives in thetransfected neuroblastoma cells which most probably resultsfrom proteasome-mediated degradation of RING domain-containing XIAP. Similar results were obtained usingMG132, another proteasome inhibitor (data not shown). Asshown by fluorescence microscopy, full-length but notRING-deleted GFP-XIAP is localized in cytoplasmic aggre-gates in transfected N2A cells (Fig. 6). Thus, the RINGdomain induces an aggregation of XIAP both in neurons

and in neuroblastoma cells. Immunostaining using an anti-body against ubiquitin showed that these aggregates werealso positive for ubiquitin (Fig. 7a). This indicates that thefull-length XIAP is ubiquitinated after overexpression, andthat it is present in aggregates, presumably for proteasome-mediated degradation. To substantiate this and to directlydemonstrate ubiquitination of XIAP in apoptotic neuroblas-toma cells, wild-type N2A cells were incubated with stau-

Fig. 4. Effect of full-length and RING-deleted XIAP on cell death of neuroblastoma cells. (A) N2A neuroblastoma cell lines stably expressing full-length(gray bars) or RING-deleted XIAP (white bars) were treated for 24 h with different death-inducing agents or deprived of serum for 48 h. Mock-transfectedcells were used as controls (black bars). Cell viability was determined by MTT assay as described under Experimental Methods. TG, thapsigargin, 05 �M;STS, staurosporine, 50 or 300 nM. Values represent means of three independent experiments � SEM. RING-deleted but not full-length XIAP protected N2Acells against cell death and the difference between the activities of the two constructs was statistically significant. (B) Western blot analysis of cell linesoverexpressing EGFP-linked full-length XIAP (X) or EGFP-linked RING-deleted XIAP (X-R). Note the difference in molecular weight between the twoproteins due to deletion of the RING domain.

Fig. 5. Stability of full-length and RING-deleted XIAP in transfected N2Acells. N2A cells overexpressing EGFP-linked full-length XIAP (XIAP) orEGFP-linked RING-deleted XIAP (XIAP-R) were cultured in the absenceor presence of 50 nM staurosporine (STS). Lactacystin 10 �M was addedto half of the cultures to inhibit proteasomes. Cells were incubated for 16 hand the levels of XIAP were determined by Western blots as describedunder Experimental Methods. Note altered levels of EGFP-XIAP but not ofRING-deleted XIAP. The endogenous XIAP at 57 kDa did not significantlychange in these experiments.


rosporine in the presence and absence of MG132. XIAP wasthen immunoprecipitated and the Western blot probed usingthe ubiquitin antibody. Figure 7b shows that blocking of theproteasomes by MG132 dramatically increases the amountof ubiquitinated XIAP in the N2A cells, indicating thatXIAP is constantly degraded via a ubiquitin-mediated path-way in the N2A cells. Staurosporine decreased the levels ofendogenous XIAP in the N2A cells and further increasedthe amount of ubiquitinated XIAP in MG132-treated cells(Fig. 7B).

Discussion

In the present article we show that XIAP can protectdeveloping mouse neonatal sympathetic neurons in cultureagainst death induced by NGF withdrawal. These neuronsdepend on NGF during development, and express TrkAreceptors, mediating the effect of NGF. NGF deprivationactivates the mitochondrial pathway of cell death, includingtranslocation of Bax to mitochondria (Deckwerth et al.,1996; Putcha et al., 1999), transcriptional induction of BH3-

Fig. 6. Distribution of full-length and RING-deleted XIAP in transfected N2A cells. Transfection and distribution of control EGFP (A, B), EGFP-linkedfull-length XIAP (XIAP) (C, D), and EGFP-linked RING-deleted XIAP (E, F) in neuroblastoma cells. (A, C, E) Phase-contrast images; (B, D, F) fluorescenceimages. Note the difference in distribution of full-length and RING-deleted XIAP proteins. Size Bars � (A,B,E,F) 45 �m; (C,D) 30 �m.


only proteins Bim (Putcha et al., 2001; Whitfield et al.,2001) and DP5/harakiri (Harris and Johnson, 2001;Imaizumi et al., 1997), as well as the release of cytochrome

c from the mitochondria to cytosol (Deshmukh and John-son, 1998; Neame et al., 1998). As death of sympatheticneurons induced by NGF deprivation can be blocked by

Fig. 7. Ubiquitination and proteasome aggregation of XIAP in N2A cells. (a) Neuroblastoma cells were transiently transfected with control EGFP (A–C) andEGFP-linked full-length XIAP (D–F). Cells were visualized under the fluorescence microscope (A,D), before immunocytochemistry was made using thespecific antiubiquitin antibody (B,E), as described under Experimental Methods. (C, F) Overlay of EGFP and ubiquitin labeling. Note that the XIAP-containing aggregates were also positive for ubiquitin (arrows). Bar � 15 �m. (b) N2A neuroblastoma cells were cultured in the absence or presence of 100or 300 nM staurosporine (STS). MG132 3 �M was added to half of the cultures to inhibit proteasomes. Cells were cultured for 24 h and XIAP wasimmunoprecipitated. The amount of ubiquitin in XIAP was determined by Western blotting using a specific anti-ubiquitin antibody. Upper plate: Note theincrease in ubiquitination of XIAP by MG132 in control cells and further increase in the presence of both MG132 and staurosporine. Lower plate: Levelsof XIAP proteins in cell lysates shown by Western blots.


pan-caspase inhibitors, overexpression of XIAP most prob-ably inhibits critical caspases activated in microinjectedSCG neurons. Caspase-9 and caspase-3 are activated down-stream of the mitochondria, and can be bound by XIAP(Deveraux et al., 1997; Takahashi et al., 1998). However,results of studies on the roles played by these and othercaspases in NGF-deprived sympathetic neurons are notcompletely in agreement. SCG neurons obtained fromcaspase-3- and caspase-9-deficient mice do not die in theabsence of NGF, although cytochrome c is released from themitochondria (Deshmukh et al., 2000). On the other hand,acute inhibition of caspase-2, but not of caspase-3 orcaspase-9, rescued sympathetic neurons from death inducedby NGF withdrawal (Stefanis et al., 1998; Troy et al., 2001).The possible inhibition of caspase-2 activity by XIAP hasnot yet been demonstrated. Thus, the identity of caspasesblocked by XIAP in microinjected SCG neurons remains tobe studied. However, our results demonstrate that XIAPmay be one critical checkpoint in NGF deprivation-induceddeath. Moreover, the significant neuroprotection affordedby XIAP in sympathetic neurons strengthens the view thatthis protein could be a target to counteract neuronal death inthe peripheral nervous system.

In contrast to other cell types, the expression and func-tions of IAPs in the nervous system have been less studied.These proteins are present in the central nervous system,and their levels change by brain injury (Belluardo et al.,2002; Korhonen et al., 2001), and after axotomy of spinalmotoneurons (Perrelet et al., 2002). The relative levels ofthe different IAPs in the sympathetic neurons are so far notknown. However, preliminary data indicate that peripheralrodent neurons express both XIAP and RIAP-2, the homo-logue of human cIAP2 (unpublished). Overexpression ofITA, the chick homologue of the human cIAP2 protein, hasbeen shown to protect chick sensory neurons against NGFdeprivation, whereas its downregulation led to death ofNGF-maintained sensory neurons (Wiese et al., 1999). Itremains to be studied how the levels of the different IAPs inthe sympathetic neurons change during development, andwhether they are affected by neurotrophic factors.

A striking finding in the present study relates to the roleof the RING domain in the neuroprotective effects of XIAP.In the sympathetic neurons, both full-length and RING-deleted XIAP proteins were about equally effective in in-hibiting death after NGF withdrawal. As described for othercells (Silke et al., 2002; Yang et al., 2000), the RINGdomain may mediate the proteasome-dependent self-degra-dation of XIAP in sympathetic neurons. Overexpression ofXIAP may saturate the proteasome machinery, whereas thesurplus may still be able to protect the neurons from apop-tosis. Deletion of the RING domain would abolish theself-degradation of XIAP, but does not affect caspase bind-ing and inactivation. However, it should be noted that in ourstudies on proteasomal activity in sympathetic neurons wehave used mainly lactacystin, a known proteasome inhibi-tor, which may also have other effects as shown by its

interference with cell survival at longer time points. There-fore the exact contribution of proteasomes to XIAP degra-dation and its protective ability in the sympathetic neuronsremain to be studied in more detail.

In N2A neuroblastoma cells, the situation with the twoXIAP proteins was different. Death in these cells was in-duced by thapsigargin, which elevates intracellular calciumlevels, blocking uptake into intracellular stores (Wei et al.,1998); by withdrawal of serum; or by blocking of differentprotein kinases by staurosporine. In all cases, only theRING-deleted XIAP protein was antiapoptotic, whereasfull-length XIAP had no significant effect. Why the pres-ence of the RING domain is necessary to protect some cells,but not others, is not immediately obvious. It could berelated to the proliferation state of the cells, the sympatheticneurons being postmitotic cells and the N2A neuroblastomacells exhibiting high rates of cell proliferation. On the otherhand, the stability and/or posttranslational modifications ofXIAP could differ between the two neural cell types. Thesmall number of neurons does not allow biochemical anal-ysis of XIAP in primary neurons. However, in staurospo-rine-treated N2A cells the stability of the RING-deletedprotein was greater, compared with full-length XIAP. Thedecreased stability of full-length XIAP protein was corre-lated to its higher degree of ubiquitination. It should benoted that the effects of ubiquitination of XIAP on cellularactivity are not always easy to predict, since the autodeg-radation of XIAP tends to lower the effective concentrationof the protein in cells, while the stimulation of degradationof active caspases by XIAP via proteasomes (Suzuki et al.,2001) would lead to an effective removal of these proteins,hindering cell death. The biological outcome of overexpres-sion of XIAP may therefore vary significantly between celltypes, as shown here for sympathetic neurons and neuro-blastoma cells. XIAP could also have caspase-independentactivities in different cell types; e.g., it has been shown thatXIAP can interfere with signaling from different receptors,including those for tumor necrosis factor and BMP/TGF-�(Li et al., 2002; Rothe et al., 1995; Yamaguchi et al., 1999).More studies are required to understand the reasons for thedifferent actions of full-length XIAP in neurons and neuro-blastoma cells. However, in both cell types the presence ofthe carboxy-terminal RING domain led to formation oflarge protein aggregates that were blocked by proteasomeinhibitors. In N2A cells, these aggregates could also bestained by ubiquitin antibodies, which was technically im-possible to do in the primary neurons. The protein aggre-gates are most probably the sites of active degradation inproteasomes, and are formed by the XIAP RING domain.As shown for other proteins, such a RING domain is knownto be able to assemble large macromolecular scaffolds forcomplex enzymatic processes (Borden, 2000). Proteasomeinhibition had earlier been shown to induce cell death, instudies using inhibitors, such as lactacystin and MG132(Drexler, 1997; Pasquini et al., 2000; Qiu et al., 2000;Sadoul et al., 1996; Wagenknecht et al., 2000). However,


inhibition of proteasome activity, in some cells, such asthymocytes, can decrease cell death (Stefaneli et al., 1998).The mechanism for the different effects of proteasome in-hibition is not known but may be related to differences indegradation of specific proteins. We have, in the presentwork, studied the role of full-length and RING-deletedXIAP in neuroblastoma cells, and showed that the RINGdomain renders XIAP susceptible to ubiquitination and deg-radation. In view of this, only the RING-deleted, but not thefull-length, XIAP protein was protective against death of theN2A cells. In contrast to our neuroblastoma cells, overex-pression of XIAP in glioma cells has been shown to affordsignificant protection against apoptosis (Wagenknecht et al.,1999). This indicates a differential effect of full-lengthXIAP in various brain tumors depending on the cell type.The functional roles of various domains of XIAP in regu-lation of survival and growth of different neural cells remainto be studied in more detail.

Experimental methods

Vectors and cloning

Full-length human XIAP cDNA (Mercer et al., 2000) corres-ponding to amino acids (aa) 1–497 was subcloned in-frameinto the expression vectors pCDNA3.1 and pEGFPC1. PCRwas used to obtain a construct where the last 48 aa, consti-tuting the RING domain, were deleted. The RING-deletedXIAP construct was subcloned in-frame into pEGFPC1 forexpression studies. All constructs were verified by auto-mated sequencing, and protein expression studies by tran-sient expression in COS-7 cells followed by Western blot-ting.

Microinjections

Culture and microinjection of sympathetic neurons wereperformed essentially as described (Hamner et al., 2001,Sun et al., 2001). Briefly, SCG neurons from newbornNMRI strain mice were grown for 5–6 days with 30 ng/ml2.5S NGF (Promega). Lactacystin (Calbiochem) was addedto the culture medium at 1 �M concentration, which wasshown previously to be optimal for sympathetic neurons(Sadoul et al., 1996). Neurons were microinjected withexpression plasmids at 50 ng/�l, together with plasmidencoding for EGFP (10 ng/�l), and grown further in thepresence or absence of NGF after medium change. In thelatter case function-blocking NGF antibodies (Roche) werealso added to neutralize possible remaining NGF. The initialneurons surviving the injection procedure were counted 3 hafter injection. Three days after injection, the living fluo-rescent neurons were counted according to the map drawnbefore injection, and expressed as a percentage of initialneurons. Counting was blind, performed by a person un-aware of the plasmids used in a given dish. The statistical

significance of the differences between means was calcu-lated by one-way ANOVA using Tukey’s honest significantdifference test for an equal or unequal n at a significant levelof 0.05. For microscopic examination, the neurons weregrown in two-well chambered coverglasses (Nunc, Den-mark).

Electron microscopic analysis

Neurons were cultured on CellLocate coverslips (Eppen-dorf) for 4–5 days and microinjected with plasmid encodingfor GFP-XIAP. Two days later phase-contrast and fluores-cence microphotographs were taken of the injected cultures.The neurons were then fixed with 2% glutaraldehyde for 30min at room temperature, postfixed with 1% reduced os-mium tetroxide for 1 h, and processed for Epon embedding,as described (Seemann et al., 2000). Injected fluorescentneurons were localized according to the coordinates of Cell-Locate coverslips using microphotographs taken before fix-ation. Sections were cut from the injected neurons parallelto the coverslip, picked up on single-slot copper grids,poststained with uranyl acetate and lead citrate, and exam-ined using a Jeol JEM-1200EX II transmission electronmicroscope at 80 kV.

For immunolabeling, cells were fixed with PLP fixative(2% formaldehyde, 0.01 M periodate, 0.075 M lysine–HClin 0.075 M phosphate buffer, pH 7.4) for 2 h at roomtemperature. Cells were permeabilized with 0.01 % saponin(Sigma), and immunolabeled using affinity-purified rabbitanti-human/mouse XIAP antiserum (R&D Systems, Ger-many) and 1.4-nm gold particle-conjugated Fab’ fragmentsagainst rabbit IgG (Nanoprobes, USA). Nano-gold was sil-ver-enhanced using the HQ Silver kit (Nanoprobes) for 2min, and gold-toned with 0.05% gold chloride (Arai et al.,1992). After being washed the cells were processed forEpon embedding as above.

Cell culture

Neuro-2a (N2A) mouse neuroblastoma cells, obtainedfrom ATCC, were cultured in DMEM supplemented with10% fetal bovine serum, and transfected with differentXIAP expression vectors as previously described (Mercer etal., 2000). Stable clones were selected using G418 (600�g/ml, Life Technologies). The levels of full-length andRING-deleted XIAP fusion proteins were determined byWestern blot analysis. To study cell survival control, mock-transfected and XIAP stably expressing Neuro-2a cell lineswere treated for 24 h with different cell death-inducingagents: thapsigargin or staurosporine (Sigma, Sweden). Inserum withdrawal experiments, the time of incubation was48 h. Cell viability was determined using the 3-(4,5-di-methylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide (MTT,Sigma) assay (Korhonen et al., 2001), which is based on thereduction of MTT by mitochondrial hydrogenases. Statisti-cal analysis was performed using ANOVA and a two-tailed


Student t test. P values less than 0.05 were consideredstatistically significant. To study the role of ubiquitinationin XIAP degradation, cells were incubated in the presenceof proteasome inhibitors, lactacystin (Sigma) or MG132(Sigma). Following the treatments, cell lysates were ana-lyzed by Western blotting using antibodies against XIAP(Transduction Laboratories, USA), and ubiquitin (Dako,Denmark).

Immunocytochemistry

For immunocytochemistry, transiently transfected cellswere fixed for 15 min with 4% paraformaldehyde. Afterfixation, cells were washed with Tris-buffered saline, andblocked with TNB-blocking reagent (NEN). Primary anti-ubiquitin antibody (Dako, Denmark) was added at the dilu-tion of 1:50 and incubated overnight at �4°C. After exten-sive washing, secondary biotinylated antibody (1:200;Vector laboratories,) was added. Specific signal was visu-alized using avidin–Texas red (NEN).

Western blot analysis

Wild-type and transfected cells were lysed after 24 hwith ice-cold RIPA buffer (150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris,pH 8.0) supplemented with protease inhibitor cocktail(Roche). Protein concentrations were determined by proteinassay DC (Bio-Rad) and equal amounts of proteins wereloaded onto a 12% SDS–gel for Western blotting (Bellu-ardo et al., 2002; Korhonen et al., 2001). The proteins weretransferred onto a PVDE membrane (PallGel) and incubatedwith the anti-XIAP antibody (Transduction Laboratories).After being washed the membrane was incubated withhorseradish peroxidase-conjugated secondary anti-mouseantibody (Dako, Denmark) followed by detection with theECL method (Pierce, USA).

Immunoprecipitation

N2a cells were treated for 12 h with staurosporine (100–300 nM; Sigma) and/or protease inhibitor MG132 (20 �M;Calbiochem), and lysed in ice cold RIPA buffer supple-mented with protease inhibitors (Roche, Germany). Lysateswere precleared for 1 h at �4°C with protein G–Sepharose(Pharmacia). Anti-XIAP antibody (0.5 �l/400 �l lysate;Transduction Laboratories) was incubated with lysatesovernight at �4°C. Complexes formed were then immuno-precipitated using protein G–Sepharose for 1 h at 4°C, andwashed three times with the lysis buffer. The Sepharosebeads were boiled in SDS–PAGE sample buffer, and thesamples were resolved by SDS–PAGE followed by transferto PVDF membranes (PallGell). Western blot analysis wasperformed as described above using anti-ubiquitin (1:100;Dako) and anti-XIAP (1:2500; Transduction Laboratories)as primary antibodies.

Acknowledgments

This study was supported by the Swedish Cancer Foun-dation (Cancerfonden); Barncancerfonden, Academy ofFinland (Program 44896, Finnish Center of Excellence Pro-gram 2000-2006); Sigrid Juselius Foundation, University ofHelsinki, and Uppsala University. L.K. received a grantfrom the Swedish Society for Medical Research, and R.M.received a Marie Curie Individual Fellowship. We thankHelena Wensman for contribution to neuroblastoma work,and Mervi Lindman for excellent technical assistance.

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