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Research Report

Knockdown of SMN by RNA interference induces apoptosis indifferentiated P19 neural stem cells

Barbara Trülzsch, Catherine Garnett, Kay Davies, Matthew Wood⁎

Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK

A R T I C L E I N F O

⁎ Corresponding author. Fax: +44 1865 272420E-mail address: matthew.wood@dpag.ox.aAbbreviations: AO, acridine orange; EB, ethid

hairpin RNA; siRNA, short interfering RNA; SM

0006-8993/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.09.025

A B S T R A C T

Article history:Accepted 3 September 2007Available online 21 September 2007

Spinal muscular atrophy (SMA) is a common neurodegenerative disease that is caused bymutations in the survival of motor neuron gene (SMN), leading to reduced levels of the SMNprotein in affected individuals. In SMA, motor neurons selectively degenerate, however, themechanism of cell death and the precise role of SMN in this process are not completelyunderstood. In this study, we apply RNA interference (RNAi) to knockdown Smn geneexpression in the murine embryonal carcinoma stem cell line P19, which can bedifferentiated into neuronal cells. A direct effect of Smn loss on apoptotic cell death indifferentiated P19 neuronal cells, and to a lesser extent in undifferentiated cells wasobserved. Apoptosis could be partly reversed by expression of an SMN rescue construct, wasreversible by the addition of the caspase-inhibitor ZVAD-fmk and involved the cytochrome cpathway. This study shows for the first time that knockdown of SMN results in apoptosis inmammalian neuronal cells and has implications for understanding the cause of motorneuron-specific cell loss in SMA, and for identifying novel therapeutic targets for this disease.

© 2007 Elsevier B.V. All rights reserved.

Keywords:RNA interferenceSMNSMAApoptosisP19 cellMotor neuron

1. Introduction

Spinal muscular atrophy (SMA) is one of the most commonautosomal recessive diseases leading to death in infancy andis caused by mutations in the survival of motor neuron gene(SMN) (Lefebvre et al., 1995; Pearn, 1980). The resulting lowSMN protein levels are responsible for the motor neurondegeneration and associated muscle weakness characteristicof SMA. The SMN gene is highly conserved across species andis necessary for survival in all metazoan organisms. Its proteinproduct, SMN, is ubiquitously expressed and localized in boththe cytoplasm and nucleus of most cell types (Liu andDreyfuss, 1996). The SMN protein forms part of the SMN-complex, which also contains the gemin proteins (gemin 2–8),and plays a critical role in small nuclear ribonucleoprotein

.c.uk (M. Wood).ium bromide; MAP2, micrN, survival motor neuron

er B.V. All rights reserved

(snRNP) biogenesis, assembly and pre-mRNA splicing (Buhleret al., 1999; Fischer et al., 1997; Liu et al., 1997; Pellizzoni et al.,1999, 1998, 2002; Yong et al., 2002; Carissimi et al., 2006).Recent studies also point to an involvement of SMN in theregulation of axonal transport, axonal growth and thelocalization of actin mRNA in growth cones of motor neurons(Bechade et al., 1999; Cifuentes-Diaz et al., 2002; McWhorteret al., 2003; Pagliardini et al., 2000; Rossoll et al., 2003).

SMN has thus been implicated in a number of criticalcellular processes; however, its exact function in motorneurons and the reason for the selective motor neurondegeneration seen in SMA are still unknown. Knockout ofthe single murine Smn gene leads to massive apoptotic celldeath and has proved to be embryonic lethal (Jablonka et al.,2000). This has made mouse models of SMA complicated and

otubule-associated protein 2; RNAi, RNA interference; shRNA, shortprotein; ZVAD-fmk, Z-Val-Ala-Asp (Ome)-fluoromethylketone

.

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time-consuming to generate. Consequently, much work intoSMN function has focused on studies in cell culture systems,many of them non-neuronal. Contradictory results haveemerged from such systems regarding the role of SMN inneuronal cell death, with some studies suggesting a pro-apoptotic (Talbot et al., 1998) and others suggesting an anti-apoptotic (Ilangovan et al., 2003; Iwahashi et al., 1997) effect ofSMN. Thus, few studies have investigated the role of SMN inneuronal cell death, and possible apoptotic mechanisms, inmammalian neuronal systems.

With the advent of RNA interference (RNAi), a tool toselectively knockdown genes in cells is now available. Thepresent study takes advantage of the unique properties of themurine P19 stem cells, which can be differentiated intoneuronal cells (Parnas and Linial, 1997; Trulzsch et al., 2004),and uses RNAi to reduce expression of themurine Smn gene. Itshows a relationship between loss of SMN and the induction ofapoptosis in undifferentiated and particularly differentiatedP19 cells, findings that could have important implications forunderstanding disease pathogenesis and for the identificationof new therapeutic targets for SMA.

2. Results

2.1. RNAi reduces SMN expression in undifferentiatedP19 cells

pSilencer plasmids expressing shRNAs targeted against twodifferent regions of themouse Smn transcript (psi-142 and psi-219) were transiently transfected into undifferentiated P19

Fig. 1 – shRNA sequences and SMN knockdown in P19 cells. (A)corresponding mRNA target sequences. (B) Immunoblot analysisP19 cells treatedwith scrambled (Co) and psi-142 (RNAi) shRNA plThe blots were simultaneously probed with the cSrc antibody aswith the psi-142 shRNA or scrambled control (psi-scram) plasmiwith propidium iodide (red), as viewed by confocalmicroscopy (63SMN staining pattern compared to the even SMN staining of the

cells. As a specificity control, pSilencer-expressing a scrambledsequence shRNA was utilized (psi-scram) (Fig. 1A andSupplementary data). Controls, to which only the transfectionreagent (Fugene) had been added, but which had otherwisereceived the same treatment as the plasmid-transfectedsamples, were also included. Transfection efficiency wasoptimized using an eGFP expression plasmid at approximately60% in the undifferentiated P19 cells (see Supplementarydata).

SMN expression in the P19 cells was reduced aftertransfection with both the psi-142 plasmid and separatelywith psi-219 plasmid as determined by quantitative westernblotting, with modest knockdown first visible after 48 h (28%),increasing to (60%) after 72 h (Fig. 1B and Supplementary data).Controls were transfected with the scrambled sequencecontrol. All subsequent SMN knockdown experiments weretherefore carried out using the psi-142 construct with datavalidated using the equally effective psi-219 construct. SMNimmunostaining of psi-142 and psi-scram treated cells furtherconfirmed a reduction in SMN expression. The psi-scram-transfected cells demonstrated a uniform cytoplasmic SMNimmunostaining pattern while the psi-142-transfected cellsshowed greater variability of SMN expression, some cells withvery patchy staining and others with no detectable SMNimmunostaining (Fig. 1C). After 72 h, the density of the psi-142-transfected cells was significantly less compared to thepsi-scram or the Fugene only treated cells as a result of theSMN knockdown (see Supplementary data). Trypan blueexclusion validated the observation that more cells survivedin the untreated or scramble control treated compared withthe psi-142 treated cells.

The hairpin sequences for the psi-142 shRNA and theshowing reduced levels of SMN protein in undifferentiatedasmids. Amaximum inhibition (60%) was observed after 72 h.a loading control. (C) Undifferentiated P19 cells transfected

ds and immunostained for SMN (green) and counterstained×magnification). The psi-142 treated cells displayed a patchyscrambled control (psi-scram) treated cells.

Fig. 2 – RNAi inducesapoptosis inundifferentiatedP19 cells.Acridineorange/ethidiumbromide (AO/EB) stainingwasused toassessapoptosis in live P19 cells and the nuclear DAPI stain in fixed cells that had been transfected with psi-scram (A, C) or psi-142 (B, D)shRNAs. The psi-scram-transfected cells showed predominantly bright green organized chromatin structure characteristic ofhealthy live cells (A) and smooth round, uniform nuclei in the DAPI stain (C). Apoptotic cells detectable with the AO/EB stain andshowing condensed and/or fragmented nuclei are present in the psi-142-transfected P19 cells (arrows in B, D). Scale bar=50 μm.

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2.2. Loss of SMN induces apoptosis in undifferentiatedP19 cells

Having shown that SMN expression could be successfullyknocked down in undifferentiated P19 cells and thatdecreased cell survival was induced predominantly in thecells transfected with the shRNA-expressing plasmid, com-pared to scrambled control or Fugene only, we focused onthe mechanism of cell death and carried out a series ofapoptosis studies.

Fig. 3 – Percentage of apoptotic cells in RNAi treated undifferentdetermined after transfection with psi-142, psi-scram or Fugene oSignificantly more of the RNAi treated cells were apoptotic afterscrambled (7.6±1.4%) controls. This could be partially reversed band by co-transfection with a rescue plasmid containing a mismThe error bars represent standard deviations calculated from thr

Acridine orange (AO)/ethidium bromide (EB) staining wasused to study apoptosis in live P19 cells, and DAPI stainingused in fixed cells (Fig. 2). The condensed chromatin and cellshrinkage of early apoptosis as well as the broken upchromatin characteristic of late apoptosis can be visualizedby AO staining (Figs. 2A, B). Cells that have lost theirmembrane permeability and are undergoing secondary ne-crosis are stained by EB. DAPI staining was used to identifyapoptotic cells based on their characteristic condensed and/orfragmented nuclei and analysis showed a higher proportion of

iated P19 cells. The percentage of apoptotic cells wasnly, and the number of AO/EB positive cells was determined.72 h (15.6±3.2%) compared to the Fugene only (6.8±0.9%) ory the addition of the caspase inhibitor ZVAD-fmk (11.8±1.8%)atch in the binding region of the shRNA (10.0±1.1%).ee independent experiments (p<0.01).

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apoptotic cells in the RNAi treated cells (Figs. 2C, D; detailedpercentage data not shown). The percentage of apoptotic cellsusing the AO/EB method was determined in undifferentiatedP19 cells at 24 h, 48 h and 72 h after transfection with psi-142,and the psi-scram and Fugene only controls. The RNAi treatedcells displayed greater apoptotic cell death after 72 h (15.6±3.2%) compared to the psi-scram (7.6±1.4%) or Fugene only(6.8±0.9%) treated cells as shown by AO/EB staining (Fig. 3). Foreach set of results, the Student's t-test gave a p value of <0.01,signifying that the association between RNAi treatment andthe induction of apoptosis was not caused by chance alone.

As a functional control, an SMN rescue plasmid containing asilent pointmutation in the binding region of thepsi-142 shRNAwas engineered (see Supplementary data). P19 cells weretransfected with the rescue plasmid and after 24 h additionallytransfectedwith thepsi-142 or the psi-scramplasmids. P19 cellsthat had been transfectedwith the SMN rescue plasmid and thepsi-142 displayed fewer apoptotic cells (10.0±1.1%) than those

Fig. 4 – Morphology and SMN expression in undifferentiated and d(A–C) and differentiated P19 cells (D–F) was confirmed by immunocantibody (green in A, D) and counterstained with PI (red in B, E) anddistributed in a diffuse pattern throughout the cytoplasmof the P19neuronal cells (arrow in F). Differentiated P19 neuronal cells expreswith monoclonal antibody to MAP2 (G), counterstained with DAPI (confocal microscopy at 40× magnification.

treatedwith psi-142 alone (15.6±3.2%) (Fig. 3). That the observedRNAi phenotype was to a large extent reversible with this SMNrescue construct provides evidence that the phenotype was, atleast in part, specifically due to loss of SMN expression.

To investigate the mechanism of the apoptosis observed,i.e. whether the apoptosis induced in the undifferentiated P19cells was caspase-dependent, the broad caspase inhibitorZVAD-fmk was added to cells that had been treated with thepsi-142 plasmid. The effects on apoptosis could partially bereversed by the addition of ZVAD-fmk (11.8±1.8%) confirmingthat the activation of caspases played a role in the apoptosisseen in the RNAi treated cells (Fig. 3).

2.3. RNAi knockdown of SMN induces caspase-dependentapoptosis in differentiated P19 neuronal cells

Having demonstrated a two-fold increase in the number ofapoptotic cells in the undifferentiated P19 cells, the effect of

ifferentiated P19 cells. SMN expression in undifferentiatedytochemistry. Cells were stained with the monoclonal SMNviewed by confocal microscopy at 63× magnification. SMN is

cells and canbe detected in the neurites of the differentiated P19s neuronal markers (G–I). Differentiated P19 cells were stainedH), with the merged image shown in I. Visualization was by

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SMN depletion on differentiated P19 cells was investigated. InSMA, motor neurons selectively degenerate and therefore P19embryonal carcinoma cells were utilized as a model systembecause they can be differentiated into neuronal cells by theaddition of retinoic acid (RA) to the culture medium.

P19 cells were differentiated by the addition of RA (day −5)over a period of 4 days and subsequently plated on cell culturedishes for a further 3–4 days (see timeline, Fig. 5A). Undiffer-entiated P19 cells and differentiated P19 neurons stronglyexpressed SMN as identified by immunocytochemistry usingthe SMN antibody (Figs. 4A–C and D–F, respectively). Cells ofneuronal morphology could be identified and neurite out-growths could be detected in the differentiated P19 cells, theneuronal nature of these cells being confirmed by immuno-cytochemistry for neuronal lineage markers (e.g. MAP2,nestin) (Figs. 4G–I).

The effect of SMN knockdown on the differentiated P19cells that had undergone four days of RA treatment beforebeing transfected with the shRNA-expressing plasmids wasdetermined (Fig. 5). Transfection of the differentiated P19cells was optimized as for the undifferentiated cells and asimilar efficiency (approximately 60%) was obtained. Western

Fig. 5 – SMN knockdown by RNAi and induction of apoptosis inwere transfected on day 1 with the psi-142 plasmid and SMN pro(representative results for day 4 are shown in B). SMN protein in tthe psi-scram sample. SYPRO staining of the blot was carried outof differentiated P19 neuronal cells under varying conditions, Fufollowing addition of ZVAD caspase inhibitor. Scale bar=50 μm.under varying conditions. The percentage of apoptotic cells was sscrambled control treated cells (12.8±1.6%). Samples treated withapoptotic cells than the RNAi treated cells, 26.5±2.3% and 21.5±

blot analysis and quantification of SMN expression indicateda 60% reduction in SMN levels in the RNAi treated samples72 h after transfection with psi-142 (Fig. 5B). The proportionof MAP2 positive neuronal cells was not significantlydifferent between the psi-142 (13.5±2.6%), psi-scram (14.8±3.2%) and Fugene only (13.9±3.2%) treated cells. Similarlythere was no difference in the total number of cells betweenthese three groups.

Clear differences in the proportions of AO/EB staineddifferentiated P19 cells were observed under different exper-imental conditions (Fig. 5C). Quantification of these differ-ences in the AO/EB stained cells (Fig. 5D) showed a dramaticincrease in the percentage of apoptotic cells in the psi-142treated sample with a maximum occurring at 72 h aftertransfection (39.5±2.1%), compared to Fugene only (10.0±1.9%) and scrambled controls (12.8±1.6%), which was ofstatistical significance (p<0.01). This result was confirmedwith the psi-219 construct (see Supplementary data). Cells thathad been transfected with the SMN rescue plasmid on day 0displayed significantly fewer apoptotic cells, again indicatingthat the RNAi effect was SMN specific andmediated by the psi-142 plasmid.

differentiated P19 neuronal cells. (A) Differentiated P19 cellstein expression was determined on day 4 by immunoblottinghe psi-142-transfected cells was reduced by 60% compared toto confirm equal loading and even transfer. (C) AO/EB staininggene only, psi-scram (scramble), psi-142 (RNAi) and(D) Quantification of AO/EB positive differentiated P19 cellsignificantly higher in the RNAi treated (39.5±2.1%) versus theZVAD-fmk or the SMN rescue plasmid displayed less

2.2%, respectively (p<0.01).

Fig. 6 – Cytochrome c is released in differentiated P19 cells treated with RNAi. Cytochrome c immunocytochemistry ofRNAi (A green: cytochrome c, B blue: DAPI, C merge) and scrambled control treated differentiated P19 neuronal cells (D–F). TheRNAi treated cells display the patchy, clumped cytochrome c staining pattern and the condensed small nuclei characteristic ofapoptotic cells. Scale bar=10 μm.

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The caspase-dependency of apoptosis in differentiated P19cells was further investigated. The pan-caspase inhibitorZVAD-fmk was added to samples that had been treated withthe psi-142 or psi-scram plasmids. Treatment with ZVAD-fmkled to a reduction in the percentage of apoptotic cells in theRNAi treated cells (26.5±2.3%) compared with those nottreated with ZVAD-fmk (39.5±2.1%) (Figs. 5C, D), indicatingthat the apoptosis observed had occurred by (although notexclusively) caspase-dependent pathways.

Cytochrome c immunostaining was carried out to gainfurther insight into the apoptosis mechanism. Cytochrome cis a member of the mitochondrial respiratory electrontransport chain and also a mediator of the caspase cascade.When cytochrome c is released from the mitochondria intothe cytoplasm it binds to Apaf-1 to form the apoptosomewhich activates caspase 9, an upstream initiator of apopto-sis. In normal cells, cytochrome c can be visualized in apredominately punctate distribution in the cytoplasm whileapoptotic cells show a patchy disorganized staining pattern.The scrambled control treated cells show large smoothnuclei with uniformly distributed, punctate cytoplasmiccytochrome c staining (Figs. 6D–F). In contrast, the psi-142treated cells displayed patchy clumped cytochrome c accu-mulation in the cytoplasm and nucleus of the cells as well asthe condensed shrunken nuclei characteristic of apoptosis(Figs. 6A–C). This indicates that the mitochondrial pathwaycould be activated in response to the RNAi treatment inthese cells.

3. Discussion

It is well recognized that mutations of the SMN gene are thecausative factors in SMA. However, as yet, the precise cellularfunction of SMN and the reasons for the motor neuronspecificity of the disease remain unclear. This study demon-strates that as a consequence of SMN knockdown by RNAi,differentiated neuronal cells and to a lesser degree undifferen-tiated P19 stem cells die due to a predominantly caspase-dependent apoptosis. Overexpression of an SMN rescue con-struct, with a silentmutation, partially reversed the effect of theRNAi treatment, indicating that the observed effects werespecifically due to loss of SMN protein. Drawing these findingstogether, it seems reasonable to hypothesize that SMN plays ananti-apoptotic role in the cell, and that themotor neuron deathcharacteristic of SMA may result from loss of this neuro-protective effect, hence the abnormal induction of apoptosis.

Crucial to performing successful RNAi experiments is theuse of a range of controls. Scrambled control shRNAs andFugene only controls were carried out in each experiment andall sequences used in this study were BLASTed against theNCBI database ensuring that no homologous sequences weretargeted. Datawere replicatedwith a second functional shRNAtargeted at a separate region of the Smn mRNA sequence (psi-219). Furthermore, a functional control in which an SMN-expressing plasmid harbored a silent point mutation in theshRNA binding site was included. The rescue effect of this

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plasmid was partial suggesting that the shRNA may not havediscriminated fully between the wild-type endogenous SmnmRNA sequence and the transfected plasmid expressing thepoint mutant Smn. Recent allele-specific gene silencingstudies indicate that the location of the nucleotide mismatchis an important factor in determining to what degree siRNAscan discriminate between wild-type and mutant transcripts(Abdelgany et al., 2003; Schwarz et al., 2006). However, suchdiscrimination is at best partial, although centrally placednucleotide mismatches in general confer efficient selectivity.

The striking observation in the present study is thedramatically enhanced vulnerability to apoptotic cell deathin differentiated neurons in which SMN was knocked down,compared with undifferentiated P19 stem cells. Apoptosis is aform of programmed cell death, known to play an integral partin nervous system development. It is mediated by the acti-vation of a family of proteases known as caspases (cysteine-dependent, aspartate-specific proteases) which can be trig-gered by the activation of “cell death receptors” such as theTNFα receptor, or by the intrinsic mitochondrial pathway,which may be induced by diverse intracellular stresses andregulated by a variety of proteins, including Bcl-2, Bcl-c, Baxand the tumor suppressor p53 (Hengartner, 2000). Involvementof the caspase-dependent pathway was confirmed in thisstudy due to reversibility of apoptosis after addition of thecaspase inhibitor ZVAD-fmk. Furthermore, the finding thatknockdown of SMN induces cytochrome c release from mito-chondria in differentiated P19 cells suggests activation of theintrinsic mitochondrial apoptosis pathway in these neurons.Our data therefore suggest that there is a greater requirementfor SMN as a neuro-protective or anti-apoptotic factor in the72 h immediately after neuronal differentiation of P19 stemcells compared with undifferentiated cells. We hypothesizethat this finding may be related to role that SMN plays inaxonal and dendritic transport (Bechade et al., 1999; Cifuentes-Diaz et al., 2002; Pagliardini et al., 2000), and its localization inmotile granules in neurites and at growth cones (Zhang et al.,2003), given that these differentiated P19 neurons are activelygrowing and extending new neurites at this time.

In recent years, it has been suggested that the pathologicalpersistence or reactivation of normally occurring apoptosismay be responsible for the supra-physiological motor neurondeath seen in SMA patients. Indeed, a substantial number ofmotor neurons in children with SMA type 1 are thought to dieby apoptosis (Simic et al., 2000) and fetal SMA spinal cordshows a significant increase in apoptotic cells, with a markeddecrease in expression of the anti-apoptotic proteins Bcl-2 andBcl-x in affected motor neurons (Soler-Botija et al., 2002).Undoubtedly, interest in the role of apoptosis in SMA patho-logy is growing. However, investigations into the precise role ofSMN in this process continue to produce highly contradictoryresults. In 1998, Talbot et al. demonstrated that dose-depen-dent apoptosis could be induced in HeLa cells by overexpres-sion of the SMN-related protein, implying a role for SMN as apro-apoptotic factor (Talbot et al., 1998). In contrast, wild-type(but not mutated) SMN was shown to synergize with the antiapoptotic protein Bcl-2 to inhibit Bax- or Fas-mediated celldeath, suggesting that decreased anti-apoptotic activity ofSMN in concert with Bcl-2 may underlie SMA pathogenesis(Iwahashi et al., 1997). This interaction. however, remains

controversial as it has not been reproduced in independentlaboratories. More concrete, perhaps, is the interaction of SMNwith the tumor suppressor protein, p53, a powerful inducer ofapoptosis. In 2002, it could be shown that pathogenic muta-tions in SMN disrupt p53 binding in a manner correlated withdisease severity, implying a functional interaction betweenSMN and p53 and the potential for apoptosis when this inter-action is impaired (Young et al., 2002). Interestingly, abnor-mally high p53 levels have also been associatedwith increasedmotor neuron apoptosis in the neurodegenerative disease,amyotrophic lateral sclerosis (Martin et al., 2001). Althoughp53distribution in SMN-depleted motor neurons is yet to be in-vestigated, this may also emerge as an important mechanismof cell death in SMA.

Subsequent overexpression studies of SMN in differentsystems have found SMN to be anti-apoptotic in primaryneurons and differentiated neuron-like stem cells (Kerr et al.,2000) but a similar effect could not be shown inmotor neurons(Cisterni et al., 2001). Notably, these studies utilized not onlydifferent cell types but also different inducers of apoptosis.The majority of investigations into apoptosis have involvedSMN overexpression, a situation far removed form the in vivostatus of SMA patients. However, a recent study in DrosophilaS2 cells also demonstrated a relationship between loss of SMNby RNA interference and apoptotic cell death (Ilangovan et al.,2003), which supports the conclusions of this paper, namelythat reduced levels of SMN lead to enhanced apoptosis inmammalian neurons, compared with undifferentiated cells,in the phase immediately post differentiation. Further studiesin motor neurons and a detailed dissection of the apoptoticpathways involved could be a starting point for a better un-derstanding of SMA pathology and the emergence of new SMAtherapeutics.

4. Experimental procedures

4.1. Short hairpin RNA (shRNA) and SMN rescue construct

Smn specific short hairpin RNAs (shRNAs) (for sequences seeFig. 1 and Supplementary data) and scrambled sequencecontrols were expressed from the pSilencer plasmid (Ambion).pSilencer expresses hairpin sequences utilizing the H1 RNApol III promoter. For each target, the sense and antisensestrands are separated by a loop comprising 9 nucleotides(5′ TTCAAGAGA 3′) and a polythymidine tract to terminatetranscription. As a functional control, a rescue plasmid (PVM6,generous gift of Nick Owen, Department of Physiology, Ana-tomy and Genetics) harboring the full-length Smn sequencewith a silent mutation in the center of the shRNA bindingregion was employed (see Supplementary data). The Smnpoint mutation in the rescue plasmid was generated with theQuikChange Site-Directed Mutagenesis Kit (Stratagene) andefficient expression was confirmed by Western analysis (datanot shown).

4.2. P19 cell culture and differentiation

P19 embryonal carcinoma cells were obtained from Dr. RuthBrown (Dept ofGenetics, University ofOxford) and cultured inα-

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minimum essential medium (α-MEM) supplemented with 10%fetal bovine serum (Sigma), 100 unit penicillin/streptomycin(Sigma), 2 mM L-glutamine (Sigma) at 37 °C and 5% CO2. Cells(5×105)were induced to differentiate in 10 cmPetri dishes by theaddition of 1 μM retinoic acid (RA; Sigma) and allowed to formaggregates for 2 days after which fresh RA was added and thecells were allowed to differentiate for a further 2 days accordingto the protocol by Bain (http://thalamus.wustl.edu/gottlieblab/gottlieb_lab_3.html). Aggregates were dispersed with 0.125%trypsin and plated on 24-well dishes (1×105 cells per well) andgrown for up to 4 days in α-MEM with supplements.

4.3. Transfection

200 ng of the appropriate shRNA-expressing plasmid wastransfected into P19 cells using Fugene 6 (Roche) transfectionreagent according to the manufacturer's instructions. Cellswere seeded into 24 well plates at a density of 1×104 cells perwell and transfected either with empty plasmid or the SMNrescue plasmid on the day of plating, and 24 h later again withthe shRNA-expressing plasmids.

4.4. Immunocytochemistry

P19 cells were rinsedwith phosphate-buffered saline (PBS) andfixed for 5 min at room temperature with 4% (w/v) parafor-maldehyde in PBS, followedby three 5minwashes in PBS.Non-specific binding siteswere blocked by incubationwith 1% (w/v)bovine serum albumin (BSA) and 10% goat serum for 1 h atroom temperature. Samples were incubated for 2 h at roomtemperature with the primary antibodies, and washed fourtimes for 5 min. Primary antibodies used were mousemonoclonal antibody against SMN (Transduction laborato-ries); the mouse monoclonal Nestin (Chemicon International,Inc., CA); rabbit polyclonal microtubule-associated protein 2(MAP2; Chemicon International, Inc., CA) and mouse mono-clonal cytochrome c (Pharmingen). Secondary antibodies wereapplied for 1 h at room temperature, followed by four 5 minwashes in PBSandwere either goat anti-mouse IgG (Alexa fluor488) or goat anti-rabbit IgG (Alexa fluor 594) (Molecular Probes,Oregon). Cell nuclei were counterstained with 50nM DAPI inPBS for 1 min, followed by four 5 min washes in PBS. Ascontrols, the first antibody was omitted and stained withsecondary antibody alone. Cellswere viewed either by invertedfluorescence microscopy (Zeiss) or confocal microscopy(Leica). For confocal microscopy, cells were grown on cover-slips, fixed, stained with the appropriate antibodies andcounterstained with propidium iodide (PI).

4.5. Western blotting

Protein from P19 cells was resolved by SDS-PAGE (10% poly-acrylamide) and transferred to a PVDF membrane (Amersham,UK) by electroblotting. Blotswere probedwith theSMNantibody(Transduction Laboratories, USA) and, to ensure equal proteinloading, with the cSrc IgG antibody (Santa Cruz Biotechnology)or by SYPRO ruby red (Invitrogen) staining according to themanufacturer's instructions. Bands were detected using theenhanced chemiluminescence (ECL) system (Roche, Switzer-land) and quantified using Typhoon Scanner and software.

4.6. Apoptosis assays

Acridine orange/ethidium bromide (AO/EB) was used toassess apoptosis in live cells and to differentiate necroticfrom apoptotic cells (McGahon et al., 1995). Live cells werestained for 5 min in AO (5 μg/ml)/EB (5 μg/ml) in 500 μlmedium in 24-well plates. The cells were briefly rinsed withfresh medium and were evaluated immediately by fluores-cence microscopy. Apoptosis was also evaluated by DAPIstaining. Cells were fixed in 5% paraformaldehyde andpermeabilized with 0.1% Triton-X-100 in PBS, and werestained for 10 min with DAPI (1μg/ml) and analyzed byfluorescence microscopy. For both methods, the number ofapoptotic cells in each condition was expressed as apercentage of the total cell number with a minimum of 300cells counted in triplicate. The pan-caspase inhibitor Z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone)(Alexis Biochemicals) was added at a final concentration of 5μM where appropriate. Cytochrome c immunocytochemistrywas carried out to determine cytochrome c release and togain further insight into the apoptosis mechanism. TheStudent's t-test, which is used to determine the actualdisparity between two means in relation to the variation ofthe data, was used to determine statistical significance of theresults.

Acknowledgments

The authors would like to thank the Marie Curie Associationfor financial support of BT; Ruth Brown for generouslyproviding the P19 cells; Allison Potter for valuable cell cultureadvice; Nick Owen for helpful discussion; and Aviva Tolkovskyfor the generous gift of the cytochrome c antibody.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.brainres.2007.09.025.

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