Abstract Spinal muscular atrophy (SMA) is a commonmotor neuron degenerative disease caused by mutationsof the survival of motor neuron (SMN) gene. The SMNprotein is expressed ubiquitously as part of a 300-kilo-dalton multi-protein complex, incorporating several pro-teins critically required in pre-mRNA splicing. AlthoughSMN mutations render SMN defective in this role, thespecific α-motor neuron degenerative phenotype seen inthe disease remains unexplained. During the differentia-tion process of spinal motor neurons and cerebellar gran-ule cells, the acquisition of mature electrophysiologicaland molecular properties is linked to the activation of theglutamate receptors of N-methyl-D-aspartate (NMDA)subtype. We have used primary cultures of rat cerebellargranules to study SMN expression during neuronal dif-ferentiation in vitro and in response to the activation ofthe NMDA receptor. We report that the expression ofgems, the nuclear structures where SMN concentrates, isdevelopmentally regulated. The highest expression is as-sociated with the cell clustering phase and expression ofNMDA receptors. Stimulation of the NMDA receptor in-duces an increase in gem number and in SMN transcrip-tion, through activation of its promoter. These results

demonstrate that SMN levels are dependent on synapticactivity, implying that SMN may have important neuron-specific functions downstream of synaptic activation.

Keywords Spinal muscular atrophy · SMN gene · N-methyl-D-aspartate receptor · Cerebellar granule cells

Introduction

Spinal muscular atrophy (SMA) is a common autosomalrecessive disorder characterized by degeneration of theα-motor neurons in the spinal cord. It is the leading ge-netic cause of infant mortality, with a frequency of 1in10,000 live births. In type I SMA, which is the most-se-vere form of the disease, death usually occurs by 2 yearsof age [1]. Loss or mutation of the telomeric survival ofmotor neuron (SMN1) gene, but not its copy the centro-meric survival of motor neuron (SMN2), causes SMA[2]. In humans, the SMN1 and SMN2 genes are located ina large inverted repeat region on 5q13. The two genesare virtually identical, with only one functional differ-ence, a nucleotide change in exon 7 that alters the activi-ty of an exonic splicing enhancer [3, 4]. This results inthe SMN1 gene producing mainly full-length transcripts.The SMN2 gene, however, produces mainly transcriptslacking exon 7. As a consequence, the SMN2 gene pro-duces only a low level of the SMN protein [5, 6].

Rodents possess only one copy of the Smn gene,which is considered to be equivalent to SMN1 [7, 8]. Ahomozygous knockout of the Smn gene in mice results inan embryonic lethal phenotype [9]. Expression of homo-zygous deletion of Smn exon 7 does not rescue the em-bryonic lethality [10]. The introduction of the entireSMN2 gene onto the null Smn-/- background, to mimicthe situation in human SMA patients, does rescue theembryonic lethality, resulting in mice showing a severeSMA-like phenotype [11, 12]. SMN2 copy number andthe level of the SMN protein modulate the severity of thephenotype, as mice carrying eight copies of SMN2 have

C. Andreassi · M.L. Eboli (✉ )Institute of General Pathology, Universita’ Cattolica del Sacro Cuore, Largo F. Vito, 1, 00168 Rome, Italye-mail: mleboli@hotmail.comTel.: +39-06-30154914, Fax: +39-06-3386446

A.L. Patrizi · C. BraheInstitute of Medical Genetics, Universita’ Cattolica del Sacro Cuore, Rome, Italy

U.R. Monani · A.H.M. BurghesDepartment of Molecular and Cellular Biochemistry and Department of Neurology, College of Medicine, Ohio State University, Columbus, Ohio, USA

U.R. Monani · A.H.M. BurghesDepartment of Molecular Genetics, College of Biological Sciences, Ohio State University, Columbus, Ohio, USA

Neurogenetics (2002) 4:29–36DOI 10.1007/s10048-001-0128-y

O R I G I N A L A R T I C L E

Catia Andreassi · Anna Letizia PatriziUmrao R. Monani · A.H.M. BurghesChristina Brahe · Maria Luisa Eboli

Expression of the survival of motor neuron (SMN) gene in primary neurons and increase in SMN levels by activation of the N-methyl-D-aspartate glutamate receptorReceived: 3 August 2001 / Accepted: 16 November 2001 / Published online: 19 January 2002© Springer-Verlag 2002

high protein levels and develop normally [11]. SMN pro-tein levels [5, 6] and SMN2 copy number [13] also corre-late with disease severity in humans. The SMN protein(38 kilodaltons) is expressed ubiquitously at higher lev-els during fetal development [14], except in motor neu-rons, which also show high expression in adult stages[15]. The protein localizes diffusely in the cytoplasm andis concentrated in nuclear structures called gems [16].The number of gems is a strong index of the clinical phe-notype, with type I patients showing very low gem num-bers [5]. SMN is part of a multi-protein complex whoseother identified components are gemin2, gemin3, and ge-min4. In the cytoplasm, the SMN complex appears toplay a critical role in spliceosomal snRNP assembly[17], and in the nucleus it is required for pre-mRNAsplicing [18]. Nuclear SMN is also involved in regulat-ing gene transcription, as it has been shown to interactwith the papillomavirus nuclear transcription activatorE2 [19], with the DNA-binding transactivator proteinFBP [20], and with the putative RNA helicase dp103[21]. These functions would appear to be fundamental tothe survival of any cell and are consistent with the em-bryonic lethal phenotype observed in Smn knock-outmice. However, SMA is not caused by loss of SMN pro-tein, but by insufficient SMN levels that impair the me-tabolism of the motor neurons causing their death. Atpresent there are no data on what function(s) are disrupt-ed by low levels of SMN and how this affects neuronalphysiology.

L-Glutamate is the major excitatory neurotransmitterin the mammalian central nervous system. In addition tofunctioning as a synaptic transmitter, glutamate induceslong-lasting changes in neuronal excitability and in thestructure and function of the synapses. It also affectsneuronal viability and neuronal migration during devel-opment [22]. These effects are produced through the ac-tivation of two general classes of glutamate receptors,the ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors, and the G-protein-coupled metabo-tropic receptors [23]. During the development of the spi-nal cord in both rodents and humans, the NMDA andnon-NMDA receptor subtypes are expressed in a syn-chronized pattern and at high levels [24]. Moreover, dur-ing a circumscribed period in early post-natal life, motorneurons undergo activity dependent development thatleads to the cell acquisition of mature molecular, physio-logical, and anatomical properties. This process requiresthe activation of the NMDA receptor [25]. Prolonged ac-tivation of ionotropic glutamate receptors, however, isresponsible for a particular type of neuronal cell deathcalled excitotoxicity. Excitotoxicity appears to be in-volved in the etiology or progression of several neurode-generative diseases, as well as in brain damage followingstroke [26]. The possible contribution of such a mecha-nism to the pathogenesis of SMA has also been hypothe-sized [24]. These observations underlying the fundamen-tal role played by the NMDA receptor in the pathophysi-ology of the motor neuron cells led us to investigate thepossible relationship between NMDA receptor activity

and SMN expression. We have analyzed SMN proteinlocalization and gene expression during the differentia-tion in vitro of a well-characterized glutamatergic cellmodel, the rat cerebellar granules. We have also studiedthe effect of NMDA receptor stimulation on SMN ex-pression in neurons in primary cultures and in motorneuron cell lines. Our results demonstrate that SMNgene expression is induced by activation of the NMDAreceptor.

Materials and methods

Cell cultures and treatments

Primary cerebellar granule cell cultures were prepared from dis-sociated cerebella of 7-day-old Wistar rats according to the proce-dure described by Gallo et al. [27]. Cells (2.5×105/cm2) were plated on poly-L-lysine-precoated plastic dishes or 12-mm glass coverslips and grown in basal Eagle’s medium supplemented with10% fetal calf serum (FCS), 2 mM glutamine, 100 µg/ml gentam-icin sulfate, and 25 mM KCl. If not otherwise indicated, cellswere used for experiments at 2, 4, 6, 8, and 10 days in vitro(DIV).

Downregulation of protein kinase C was achieved by exposingthe cells to 1 µM phorbol 12-myristate-13-acetate (PMA) for 24 h.At 12 h of treatment with PMA, 5 µM glutamate or 100 µM NMDAwas added to the cells. When indicated, 1 µM (+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine (MK-801) (RBI,Natick, Mass., USA), an NMDA receptor antagonist [28], was added to the cells together with NMDA or glutamate.

Mouse motor neuron cell lines EHMN [29] and NSC34 [30]and mouse fibroblasts 3T3 were maintained in Dulbecco modifiedEagle’s medium supplemented with 10% FCS and 2 mM gluta-mine. Differentiation of motor neuron cell lines was performed bytreating the cells with 50 µM retinoic acid for 3 days as previouslydescribed [31]. In all cases, compounds of interest were added di-rectly to the cell culture medium from 50–100× stock solutions.Unless otherwise specified, all the drugs and chemicals were sup-plied by Sigma (St. Louis, Mo., USA).

Immunofluorescence analysis

Immunofluorescence staining of cerebellar granule cells grown onglass coverslips was performed as described by Patrizi et al. [32].Briefly, cells were washed and fixed with 4% paraformaldehyde.Cells were then permeabilized with 0.3% Triton X-100 in phos-phate-buffered saline (PBS) and preincubated in blocking solution[PBS containing 1% bovine serum albumin (BSA)]. The cover-slips were incubated with the anti-SMN monoclonal antibody 2B1[16] for 18 h at 4°C (dilution 1:1,000) and subsequently with thefluorescein isothiocyanate-conjugated goat anti-mouse antibodyfor 30 min at 37°C (dilution 1:50). The coverslips were thenwashed, mounted in PBS containing 40% glycerol and 100 µg/mlp-phenylenediamine, and observed for fluorescence pattern and intensity (Zeiss Axiophot microscope). Digital images were takenusing a CCD camera (CE 200A, Photometrics).

For quantification of gem expression, more than 400 cells percoverslip were counted. Values are expressed as means±SEM. Allexperiments were carried out in duplicate or triplicate on at leasttwo cell preparations. Statistical analysis was performed by one-way ANOVA. A P value <0.05 was considered as significantlydifferent.

Western blot analysis

Cell cultures were lysed in lysis buffer [140 mM β-mercaptoetha-nol, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 50 mM

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TRIS-HCl, pH 6.8] and protein content was determined. Briefly,50 µg of protein was electrophoresed on a 12% SDS-polyacryla-mide gel and subsequently electrotransferred to a PVDF mem-brane (Amersham Pharmacia Biotech, Piscataway, N.J., USA).The membranes were blocked in 5% non-fat dry milk, 0.5% BSAin TBS-0.1% Tween 20 for 2 h at room temperature. Immunode-tection was carried out using the monoclonal antibody 2B1 [15],diluted 1:2,000, or anti-SC35, diluted 1:2,000, followed by use ofthe Enhanced Chemoluminescence (ECL) western blotting detec-tion kit (Amersham Pharmacia Biotech).

Northern blot analysis

Poly(A)+ RNA from granule cells was prepared using the Oli-gotex Direct mRNA kit (Qiagen, Germantown, Md., USA).Poly(A)+ RNA (2 µg) was electrophoresed on a 1% agarose gelcontaining 10% MOPS 10× (200 mM 3-N-morpholinopropanesulfonic acid, 50 mM sodium acetate, 10 mM EDTA, pH 7) and2.2 M paraformaldehyde, transferred overnight to a positivelycharged membrane in 20× SSPE (3.6 M NaCl, 200 mMNa2HPO4, 20 mM EDTA, pH 7.4) and baked for 2 h at 80°C. Thefilters were prehybridized using ExpressHyb hybridization solu-tion (Clontech, Palo Alto, Calif., USA) at 68°C for 1 h and hy-bridized with [32P]-labeled rat SMN cDNA [8] at 68°C for 2 h.Blots were stripped and reprobed with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Washing was per-formed following the manufacturer’s instructions, and the blots,hybridized with SMN or GAPDH probes, were exposed for7 days or 5–36 h, respectively, at –80°C. Northern blot quantifi-cation was performed by scanning the autoradiographs with a dual wave-length scanner (CS9000U, Shimadzu) to determineSMN/GAPDH ratios.

RT-PCR amplification

Total RNA was extracted from EHMN, NSC-34, and 3T3 cellsand mouse brain tissue using TRIzol reagent (Gibco-BRL, GrandIsland, N.Y., USA) and first-strand cDNA synthesis was per-formed using Superscript reverse transcriptase (Gibco-BRL), ac-cording to the manufacturer’s instructions. The NMDA R1 Fwd(5’-AAGCAGCCCAGCGTGTTTGTGA-3’) and NMDA R1 Rev(5’-GACTCGTTCTTGCCGTTGATTA-3’) primers were used forthe amplification of a 529-bp fragment from the mouse NMDA re-ceptor subunit 1 transcript. The βactin Fwd (5’-GTGGGCCGC-CCTAGGCACCAG-3’) and βactin Rev (5’-CTCTTTAATGTCA-CGCACGATTTC-3’) primers were used to generate a 539-bpfragment from the mouse βactin transcript. PCR conditions wereas follows: after a denaturation step of 3 min at 94°C, the amplifi-cation was carried out at 94°C for 30 s, 50°C for 30 s, and 72°Cfor 1 min in a reaction mixture containing 20 ng of cDNA and4 mM magnesium. We used 35 and 28 cycles for the amplificationof NMDA R1 and βactin, respectively. PCR products were thenresolved on a 2% agarose gel.

Transient transfections

Undifferentiated or retinoic acid-differentiated EHMN and NSC34cells were transiently transfected using Lipofectamine reagent (Gibco-BRL) as previously described [33]. Briefly, 1 µg of p3.4kb-luciferase or p0.75kb-luciferase constructs was co-transfectedalong with 0.5 µg of cytomegalovirus (CMV) β-galactosidase re-porter plasmid that serves as a control for transfection efficiency.Beginning 24 h after transfection, cells were treated for the indicat-ed times with 100 µM NMDA and reporter activities measured42 h after transfection. Cell lysates (40 µg) were assayed for luci-ferase activity using Luciferase Assay System (Promega, Madison,Wis., USA) and galactosidase activity using Galacton substrate(Tropix, Bedford, Mass., USA), according to the manufacturer’s in-structions.

Results

SMN expression during in vitro differentiation of glutamatergic neurons

To evaluate SMN expression during neuronal differentia-tion in vitro, we used primary cultures of rat cerebellargranule cells, a model widely used to study survival anddifferentiation of glutamatergic neurons. The cultures areprepared from 7-day-old animals and are approximately95% pure. As many neuronal cell types, cerebellar gran-ule cells in primary culture are post-mitotic and do notproliferate in vitro. During a 10-day culture period, thecells undergo a maturation process characterized by bio-chemical and morphological changes, including neuriteoutgrowth (2–3 days in vitro, DIV), subsequent forma-tion of cell aggregates (4–6 DIV), and neurite fascicula-tion (7–9 DIV) [34]. We have studied SMN expressionand localization during these differentiation phases. Im-munofluorescence analysis showed typically one, rarelytwo, discrete intensely stained dots (gems) in the nucleusand a fine granular staining in the cell cytoplasm(Fig. 1A). The appearance of gems was developmentallyregulated (with a maximum of 80% of total nuclei withgems between 4 and 6 DIV), whereas the cytoplasmicstaining did not appreciably change during the time peri-od investigated (Fig. 1B). SMN expression at later stagesof differentiation was not examined, as a process of cul-ture aging and death begins at 12 DIV [34]. Western blotanalysis of SMN levels in granule cells at different stages showed no alteration during the culture period(Fig. 1C). SMN gene expression was analyzed by northern blots of poly(A+) RNA extracted from cerebel-lar granule cells at different stages (Fig. 1D). Densito-metric analysis of the SMN hybridization signal, relativeto that of GAPDH used as a control, did not show anysignificant difference in SMN mRNA levels during the2–10 DIV period examined. Thus, in differentiating cer-ebellar granule cells, the appearance of nuclear gems isdevelopmentally regulated and the variations in gemnumber are not due to modifications in total protein or inSMN transcription rate.

SMN expression in response to NMDA receptor stimulation

During the in vivo and in vitro differentiation process,cerebellar granule cells express various subtypes of glu-tamate receptors. The metabotropic class of receptors istransiently expressed during the first days of culture,contributing significantly to the first differentiationevents (i.e., neurite outgrowth and growth cone motility).Conversely, the ionotropic class of receptors, principallyof NMDA subtype, develops later (4–6 DIV) and is in-volved in critical aspects of mature neuronal function,including plasticity responses and sensitivity to excito-toxic insult [35]. The observation that many gems are ex-pressed at the 6 DIV stage prompted us to investigate

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whether SMN expression in granule cells is affected bystimulation of the NMDA-sensitive glutamate receptors.Neuronal cultures at different stages (2, 4, 6, and 8 DIV)were treated with 100 µM NMDA and the effect onSMN expression was monitored by immunofluorescencewithin 24 h. The percentage of cells expressing gems increased at all stages, except for 2 DIV when NMDAreceptor expression was low (Fig. 2). At all stages themaximum effect of NMDA occurred after 12 h of treat-ment. This response was transient and decayed withinthe following 12 h (data not shown). Cell stimulationwith physiological concentrations (5 µM) of glutamateelicited a similar effect (data not shown). The responseto NMDA was mediated by the receptor activity, asshown by the complete inhibition obtained by co-admin-istration of 1 µM MK-801, a selective NMDA receptorantagonist, [28] or by downregulation (1 µM PMA for 24 h) of protein kinase C, a major signaling compo-nent acting downstream of the NMDA receptor [36],(Table 1).

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Fig. 1 A–D. SMN expression during in vitro development of cere-bellar granule cells. A Immunofluorescence analysis of SMN ex-pression in cells at 6 days in vitro (DIV). Monoclonal anti-SMNantibody 2B1 [16] stained both the cell cytoplasm and the nucleargems (arrows). The cell nucleus is counterstained by the nucleardye DAPI. Magnification 1,250×. B Graph summarizing the resultsof gem count analysis over the 9-day developmental period. Valuesare expressed as percentage (mean±SEM) of positive cells.*P<0.005 DIV vs. the preceding DIV. C Western blot analysis ofthe SMN protein in cerebellar granule cells at different stages.Whole-cell lysates were separated on a 12% polyacrylamide geland blotted. The 2B1 antibody detects two bands of approximately36 and 34 kilodaltons. Lower panel, the same blot after strippingoff the antibody was reprobed with anti SC-35 antibody, a nuclearmarker, to control for protein amounts in individual lanes. D North-ern blot analysis of SMN mRNA content in granule cells. Poly(A)+

RNA was extracted from cells at different stages (2–10 DIV). Theblot was hybridized with rat SMN cDNA [8] and subsequently(lower panel) reprobed with human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) cDNA as loading control

Fig. 2 Effect of N-methyl-D-aspartate (NMDA) receptor stimula-tion on nuclear SMN in cerebellar granules at different stages. Thecells were exposed to 100 µM NMDA for 12 h and then assayedby immunofluorescence. Values are expressed as percentage ofcells with gems (mean±SEM). Note that control values refer to2.5, 4.5, 6.5, and 8.5 DIV (compare also with Fig. 1B). *P<0.05NMDA-treated cultures vs. respective controls

Table 1 Effects of different drugs on N-methyl-D-aspartate(NMDA)-induced increase in gem positive cell number (PMAphorbol 12-myristate-13-acetate)a

Treatment Percentage of positive cells

–NMDA +NMDA

None 100.0±1.1 147.5±1.5*MK-801 (1 µM) 98.2±1.3 93.6±1.2**PMA (1 µM for 24 h) 89.6±1.1 105.8±0.9**

*P<0.005 NMDA treatment vs. control; ** P<0.005 drug treat-ment vs. NMDA treatmenta 6-day in vitro cultures were incubated for 12 h in the presence or absence of 100 µM NMDA and then assayed by immunofluo-rescence. Drugs were added either 12 h before (PMA) or with(MK-801) NMDA and maintained throughout the entire incuba-tion. The percentage of positive cells was determined. Data are ex-pressed as mean±SEM percentage of values of untreated controlcultures

To investigate whether NMDA altered SMN mRNAlevels, northern blot analysis was performed on 6 DIVcerebellar granule cells treated with the agonist for dif-ferent time periods. A significant increase in SMNmRNA level was observed after 6 h of treatment(Fig. 3A) and peaked after 10 h (3.4-fold increase inSMN mRNA levels compared with untreated cells)(Fig. 3B). Thus, the activation of the NMDA receptor incerebellar granule cells modulates the expression of nu-clear gems and induces an increase in transcription of theSMN gene.

Effect of NMDA receptor activation on SMN promoter activity

To determine whether the increase in SMN gene expres-sion following NMDA receptor stimulation could be as-cribed to promoter activation, we investigated the effectof NMDA administration on the motor neuron cell linesEHMN [29] and NSC34 [30] transfected with luciferasereporter constructs driven by the human SMN promoter.We first verified whether the cells expressed highNMDA receptor levels when induced to differentiate byretinoic acid. Treatment with 50 µM retinoic acid for

3 days significantly increased the expression of the func-tional subunit (R1) of the NMDA receptor (Fig. 4A).Then undifferentiated or differentiated cells were tran-siently co-transfected with the luciferase constructs andCMV β-galactosidase reporter plasmid. The CMV β-ga-lactosidase reporter serves as an internal control fortransformation efficiency. We used two different con-structs, one (p3.4 kb) containing a 3.4-kb fragment of thehuman SMN gene, which includes a negative regulatoryelement, and another (p0.75 kb) containing a smallerfragment of 750 bp, which lacks the repressor activity[33]. Beginning 24 h post transfection, the cultures werestimulated with 100 µM NMDA for the indicated peri-ods of time and reporter activities assayed 42 h aftertransfection. As shown in Fig. 4B, the activity of the

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Fig. 3A, B Time course of SMN mRNA induction in cerebellargranule cells stimulated with NMDA. A 6 DIV cultures were ex-posed to NMDA for the indicated times. Poly(A)+ mRNA was ex-tracted and analyzed by northern blot technique. Lower panel, fil-ters were consecutively hybridized with rat SMN and internal con-trol GAPDH probes. B Quantification of relative levels of SMNmRNA by densitometry of the autoradiographs. Each value ismean±SEM of at least three different experiments conducted ondifferent cell preparations

Fig. 4A, B Effect of NMDA receptor activation on the transcrip-tion of a luciferase reporter gene driven by the human SMN promoter in differentiated EHMN cells. A Effect of retinoic acid treatment on the NMDA receptor subunit R1 (NMDA R1) ex-pression. Cells were treated with 50 µM retinoic acid for 3 days.RNA was isolated, reverse transcribed, and amplified by RT-PCR.Lane 1, undifferentiated cells; lane 2, 50 µM retinoic acid treatedcells; lane 3, mouse brain tissue as positive control; lane 4, 3T3mouse fibroblasts as negative control. Lower panel, controls forsimilar amounts of template cDNA were performed with primersfor β-actin. B Effect of NMDA treatment on luciferase activity intransfected EHMN cells. Undifferentiated or differentiated EHMNmotor neurons were transiently transfected with luciferase reporterplasmids driven by a human 3.4-kb SMN promoter fragment (p 3.4 Kb) or a smaller fragment of 750 bp (p 0.75 Kb). After24 h, the cells were stimulated with 100 µM NMDA for the indi-cated times and assayed for luciferase activity. The SMN promoterresponse is indicated as the ratio of luciferase activity in stimulat-ed to that in unstimulated cells. Luciferase activity was normal-ized to β-galactosidase activity in each sample. Values are mean±SEM of four independent experiments

entire SMN promoter (p3.4 kb) was modestly influencedby NMDA receptor activation in differentiated EHMNcells (approximately twofold after 12 h of treatment).Conversely, the activity of the 750-bp promoter fragment(p0.75 kb) was greatly enhanced (approximately five-fold) by the stimulation of the NMDA receptor. Similarresults (data not shown) were obtained under the sameexperimental conditions using the NSC-34 cells, anothermouse cell line whose motor neuron-like properties andsensitivity to glutamate toxicity have already been de-scribed [30]. Thus, we conclude that the SMN promoteris activated by the stimulation of the NMDA receptor inmotor neuron cell lines.

Discussion

Despite the numerous studies indicating that SMN1 is theSMA-determining gene [37] and advances in the knowl-edge of the biochemical role of the SMN protein, the cellular process leading to motor neuron degeneration in SMA are still obscure, and the link between reducedlevels of SMN protein and motor neuron loss remainselusive. Various classes of glutamate receptors, classifiedas ionotropic, NMDA and non-NMDA, and metabotrop-ic receptors, mediate most of the excitatory neurotrans-mission in the mammalian central nervous system [23].During the differentiation of both spinal cord motor neu-rons [24] and cerebellar granule cells [35], the expres-sion profile of the ionotropic types of glutamate recep-tors changes, reflecting developmental modifications.These alterations in the excitable properties of the cellsare believed to modulate their activity dependent devel-opment [25]. Therefore, on the basis of the similar roleplayed by the NMDA receptors in the development ofspinal motor neurons and cerebellar granule cells, wehave chosen the latter to study SMN expression duringin vitro neuronal development. This model has allowedus to investigate for the first time SMN expression dur-ing specific neuronal differentiation events and in re-sponse to the activation of a neurotransmitter receptor.

Our results show that SMN gene expression and pro-tein levels are constant during in vitro differentiation ofglutamatergic cells. However, the appearance of gems,the nuclear structures where SMN appears to concen-trate, is developmentally regulated. In particular, thegreat majority of cerebellar neurons are positive for nuclear gems within a narrow developmental window,corresponding to a late stage of differentiation (typicallybetween 4 and 6 days of culture). To our knowledge, thisis the first demonstration of a stage-specific expressionof gems in developing neurons. Although this findingcould be related to the particular cellular model exam-ined, it seems to suggest that, in the same cell type, vari-ation of nuclear gem number is not an exclusive featureof a pathological condition, as seen in SMA cell linescompared with normal [5, 32]. Instead the presence ofnuclear gems can also be affected by physiological cuesinvolved in the differentiation process. This is distinct

from and must not be confused with factors affecting thecell cycle, as cerebellar granule cells do not proliferate inculture [34]. The cell stage associated with the highestlevel gems is the cell-clustering phase. An associationbetween appearance of gems and neuronal cell body con-tacts is indirectly confirmed by our previous findingsthat nuclear gems were never observed in granule cellscultured under conditions that allow cell survival butprevent proper neural differentiation, including forma-tion of cell aggregates [38]. At present the functionalsignificance of this stage-specific expression of gems isnot clear. However, it is interesting to note that late dif-ferentiation stages of rat hippocampus pyramidal neu-rons are associated with changes in nuclear organization,including the proliferation of coiled bodies, the subnu-clear organelles structurally and functionally related togems [39]. It remains to be determined whether a devel-opmental specific reorganization of nuclear structures isa phenomenon common to other neurons and to in vivoneuronal differentiation.

Studies on human [5, 6] and rodent [40] tissues have shown that SMN is constitutively expressed in all tissues. Our data show that the activation of the NMDAreceptor promotes the expression of gems, and this effectis accompanied by an increase in SMN mRNA. Thus,basal SMN levels can be upregulated in response to spe-cific signals, and this inducible expression could repres-ent a particular aspect of SMN neuronal expression. Theexpression of the NMDA receptor is developmentallyregulated in human [24] and rodent spinal cords [41].Autoradiography studies have revealed that NMDA re-ceptors are transiently expressed at high levels in the de-veloping ventral horns over the first weeks of life. It hasbeen shown that during this period the activation of theNMDA receptor provides a growth-promoting or differ-entiation signal to developing motor neurons [42]. In theabsence of this signal, the proper development of themotor neuron dendritic arbor is severely compromised.The proliferation of spinal cell precursors appears not tobe impaired in SMA, as shown by the results obtained inanimal models of SMA [11]. Therefore, our results couldsuggest that SMN plays a role in the NMDA signaling indeveloping motor neurons, an activity that would be lim-ited or suppressed by the reduction of SMN levels. Stud-ies of markers of motor neuron differentiation in animalmodels of SMA [43] could provide valuable informationabout the influence of SMN expression on the motorneuron development.

We show that SMN is constitutively expressed duringneuronal development but both the endogenous gene, incerebellar granule cells, and the human SMN promoterin a reporter expression system, in motor neuron celllines, can be upregulated by the activation of the NMDAreceptor. As expected, this effect is not observed whenthe receptor expression levels are low, i.e., in 2-day-oldgranule cultures and in undifferentiated motor neuroncell lines. Very recently Baron-Delage et al. [44] haveshown that SMN can be induced by interferon-β and -γ,possibly through the activation of an interferon response

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element present in the SMN promoter. Our data showthat the NMDA-responsive elements are contained in aregion 750 bp upstream of the initiation site. Previousstudies have shown that the activity of this fragment ishigher than that of the entire promoter in motor neurons,but not in other cell lines [33], suggesting that this regionmay contain enhancer or transcription factor bindingsites regulating SMN expression in motor neuron cells.Our present findings indicate that transcription factorsinduced by activation of the NMDA receptors, such asAP-1, could be responsible for the higher levels of SMNexpression in motor neurons.

The implications of our observations remain to be explored. Further studies aimed at the identification ofthe role played by SMN in the developmental eventscontrolled by the NMDA receptor would not only repres-ent a major advance in our knowledge of the pathophysi-ology of SMA, but could also provide the basis of a ther-apeutic approach for this disease.

Acknowledgements We thank Dr. G. Dreyfuss for kindly provid-ing the 2B1 monoclonal antibodies, Dr. G. Battaglia for the ratSMN cDNA, and Dr. N. Cashman for the NSC34 cells. We aregrateful to Drs. S. Mozzetti and B. Bedogni for assistance in den-sitometric analysis and to Dr. A. Riccio for critical reading of themanuscript. C.A. is the recipient of a postdoctoral fellowship fromthe Families of SMA. This work has been supported by a grant“Ministero Universita’ e Ricerca Scientifica” to MLE, Telethongrant 828 to C.B. and NIH grant NS38650 to A.H.M.B. All the ex-perimental procedures described in this study are approved by theItalian Government and are in compliance with US federal laws.

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