Molecular and Cellular Neuroscience xxx (2011) xxx–xxx

YMCNE-02599; No of Pages 11

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Molecular and Cellular Neuroscience

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Increased expression of the beta3 subunit of voltage-gated Na+ channels in thespinal cord of the SOD1G93A mouse

Michele Nutini a,c, Alida Spalloni a, Fulvio Florenzano a, Ruth E. Westenbroek b, Claudia Marini a,1,William A. Catterall b, Giorgio Bernardi a,c, Patrizia Longone a,⁎a Molecular Neurobiology Unit, Experimental Neurology, Fondazione Santa Lucia, via del Fosso di Fiorano 64, 00179 Rome, Italyb Department of Pharmacology, University of Washington, Seattle, WA, USAc Department of Neuroscience, University of Rome “Tor Vergata,” Rome, Italy

⁎ Corresponding author. Fax: +39 6 501703302.E-mail address: p.longone@hsantalucia.it (P. Longon

1 Present address: Department of Neurochemistry aInstitute for Neurobiology, Magdeburg, Germany.

1044-7431/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.mcn.2011.03.005

Please cite this article as: Nutini, M., et al., ISOD1G93A mouse, Mol. Cell. Neurosci. (20

a b s t r a c t

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Article history:Received 22 October 2010Revised 14 February 2011Accepted 18 March 2011Available online xxxx

Keywords:Amyotrophic lateral sclerosisCu,Zn superoxide dismutase (SOD1) mouseVoltage-gated Na+ channelBeta3 Na+ channel subunitSpinal motor neuronsLaser-capture microdissection

Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the progressive degeneration ofmotoneurons (MNs). Altered electrical properties have been described in familial and sporadic ALS patients.Cortical and spinal neurons cultured from the mutant Cu,Zn superoxide dismutase 1 (SOD1G93A) mouse, amurine model of ALS, exhibit a marked increase in the persistent Na+ currents. Here, we investigated theeffects of the SOD1G93A mutation on the expression of the voltage-gated Na+ channel alpha subunit SCN8A(Nav1.6) and the beta subunits SCN1B (beta1), SCN2B (beta2), and SCN3B (beta3) inMNs of the spinal cord inpresymptomatic (P75) and symptomatic (P120) mice. We observed a significant increase, within lamina IX,of the beta3 transcript and protein expression. On the other hand, the beta1 transcript was significantlydecreased, in the same area, at the symptomatic stage, while the beta2 transcript levels were unaltered. TheSCN8A transcript was significantly decreased at P120 in the whole spinal cord. These data suggest that theSOD1G93A mutation alters voltage-gated Na+ channel subunit expression. Moreover, the increasedexpression of the beta3 subunit support the hypothesis that altered persistent Na+ currents contribute tothe hyperexcitability observed in the ALS-affected MNs.

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© 2011 Elsevier Inc. All rights reserved.

Introduction

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegen-erative disease characterized by the progressive loss of motoneurons(MNs) in the spinal cord, brain stem and motor cortex (Cleveland andRothstein, 2001). ALS is present in two forms: sporadic (sALS) andfamilial (fALS), which are clinically indistinguishable (Rowland andShneider, 2001). Approximately 20% of all fALS cases result frommutations in the superoxide dismutase 1 gene (SOD1; Rosen et al.,1993) and transgenic mice carrying the human mutation G93A(SOD1G93A) developMN degeneration that closely resembles humanALS (Gurney et al., 1994; Bendotti and Carrì, 2004). Many underlyingmechanisms have been associated with the ALS-related MN degen-eration including mitochondrial dysfunction and astrocyte-mediatedtoxicity (Beal, 2000; Ferri et al., 2004; Di Giorgio et al., 2007; Nagaiet al., 2007), glutamate excitotoxicity via the AMPA/kainate receptors(Van Den Bosch et al., 2000; Carriedo et al., 2000; Spalloni et al.,2004a,b), protein misfolding and accumulation (Munoz et al., 1988;Petrucelli and Dawson, 2004), and endoplasmic reticulum stress

(Atkin et al., 2006, 2008; Saxena et al., 2009). In addition a line ofresearch has looked at an unexpected characteristic of neurons in ALS,hyperexcitability. Cortical and peripheral hyperexcitabilities havebeen described in ALS patients (Bostock et al., 1995; Zanette et al.,2002; Turner et al., 2005; Kanai et al., 2006; Vucic et al., 2008), and inprimary neuronal cultures and slice preparations from neonatalSOD1G93A mice (Pieri et al., 2003; Kuo et al., 2004, 2005; Zonaet al., 2006; Bories et al., 2007; van Zundert et al., 2008; Pieri et al.,2009). These observations indicate an involvement of the Na+ chan-nels in the pathogenesis of ALS; in particular they support a role forthe persistent Na+ currents.

Voltage-gated Na+ channels (VGSC) play a fundamental role inexcitable cells. They transiently increase the Na+ ion permeability ofthe plasma membrane thus transmitting depolarizing impulses andpropagating action potentials (Catterall, 2000). The ion channelconsists of a pore-forming alpha-subunit and one or two auxiliarybeta subunits (Yu and Catterall, 2003). The alpha-subunits form theion-selective pore and are responsible for the voltage-sensitivecharacteristics of the channel. To date 10 different alpha isoformshave been identified, which differ in their voltage dependence,kinetics, tissue-specific expression, and regulation (Catterall et al.,2005). The beta subunits (beta1, beta2, beta3, beta4) are structurallyhomologous and influence current density, channel kinetics, gatingmode and channel cell surface density (Isom, 2001; Qu et al., 2001;

ltage-gated Na+ channels in the spinal cord of the

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Morgan et al., 2000; Yu et al., 2003). VGSC subtypes have charac-teristic regional and cellular distribution that contributes to functionalheterogeneity of neurons in the central (CNS) and the peripheral(PNS) nervous systems. Thus, altered density or biophysical proper-ties of VGSCmay lead to significant alterations in neuronal excitabilityand have important consequences in the development of CNS diseases(Chahine et al., 2008).

These previous results raise the possibility that the VGSC subunitsare differently expressed by the MNs in ALS, and that the alteredexpression would influence their gating characteristics. To addressthis question, we have investigated the expression of four VGSCsubunits: the gene (SCN8A) encoding the NaV1.6α, an essentialsubunit for the postnatal development and function of the spinal MNs(Porter et al., 1996; García et al., 1998), and the genes encoding for thebest-characterized VGSC auxiliary subunits, beta1, beta2 and beta3.This study was undertaken in the spinal cord of SOD1G93A micebefore the onset of disease symptoms (hereafter defined as presymp-tomatic, P75) and near the end-phase of the disease (hereafterdefined as symptomatic, P120). Moreover, since defective alternativesplicing has been well documented in ALS and linked to possiblepathological alterations that might contribute to the disease onset/progression (Anthony and Gallo, 2010; Ticozzi et al., 2010), we haveanalyzed the developmentally regulated SCN8A alternative splicing(Plummer et al., 1998). SCN8A undergoes splicing at exons 5 and 18,resulting in a mature transcript containing either “neonatal” exons(5N, 18N), expressed mainly in fetal tissues, or “adult” exons (5A,18A), becoming dominant after birth (Plummer et al., 1998).

We have found (i) a decrease of the SCN8A transcript in the wholespinal cord of symptomatic mice; (ii) an increase, in the ventral hornarea, of the beta3 transcript at P120 and of the beta3 protein at P75and P120; (iii) a decrease in the ventral horn area of the beta1transcript at P120. Our results indicate that the mutant SOD1 altersVGSC expression which, in turn, could explain some of the electro-physiological abnormalities described in the mutant SOD1 MNs

Results

The SCN8A mRNA decreases in the spinal cord of symptomatic (P120)SOD1G93A mice

Initially we performed real-time PCR analyses of the total spinalcord mRNA to check for temporal changes in the VGSC mRNA expres-

Fig. 1. Laser capture microdissection (LCM) of Nissl stained spinal cord slices. (A) 4x magnifi(bottom). (B to D) The MN-enriched area is shown at 20x magnification in the pre-cut stagebar in A=400 μm, in B, C and D=50 μm.

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

sion in the SOD1G93A mice. At P75, the mRNA expression of SCN8A,SCN1B, SCN2B and SCN3B genes was comparable between WT andSOD1G93Amice (Fig. 2A). At P120, a significant decrease of the SCN8AmRNA expression betweenWT and SOD1G93Amice was found, whilethe mRNA expression of SCN1B, SCN2B and SCN3B showed nosignificant changes between WT and SOD1G93A mice (Fig. 2B). Wehave also analyzed alternative splicing of the SCN8A gene focusing onexon 18, whose neonatal form encodes a truncated, non functionalchannel that appears in fetal tissues and disappears just after birth(Plummer et al., 1997), and found no differences between WT andSOD1G93A from E15 to P120 (Supp. Fig. 3).

SCN1B and SCN3B mRNA expression is altered in the Lamina IX of theSOD1G93A mice

ALS is characterized by massive MN loss in the ventral horn,particularly in the L-IX, of the spinal cord. Thus, we wanted toinvestigate the expression of mRNA encoding the VGSC subunits inMNs of this area by taking advantage of the selectivity of LCMtechnique. To this purpose, wemicrodissected the L-IX region from 10(WT) to 15 (SOD1G93A) spinal cord sections obtained from n=4mice per group at P75 and P120 (Fig. 3). Following the LCM, qRT-PCRwas performed. The data indicate that at P75 the mRNAs transcribedfrom the SCN8A, SCN1B and SCN2B genes are comparable in WT andSOD1G93A, while SCN1B is significantly decreased in the SOD1G93Aventral horn at P120 (Fig. 3). On the other hand, this analysis has alsorevealed a significant increase of the SCN3BmRNA expression at P120(Fig. 3). This increase is specific to the ventral horn area, since asimilar LCM real-time RT-PCR analysis of the dorsal horn area at P120did not show any SCN3B mRNA up-regulation in the SOD1G93A micecompared to the WT (Supp. Fig. 4).

Beta3 immunoreactivity in the spinal cord of WT and SOD1G93A mice

Disease progression was histologically verified by the appearanceof intense gliosis at the end-stage.While at P75 (not shown) the tissueappearance is comparable between the WT and the SOD1G93A mice;immunofluorescence analysis of the lumbar spinal cord in the P120SOD1G93A mice shows a massive reactive gliosis as revealed by theintense GFAP staining when compared to the age matched WT mice(Fig. 4). At P75WT and SOD1G93A, ChAT positive MNs were surroun-ded by ChAT-positive boutons, sparsely distributed on soma and

cation of a membrane mounted slice before (top), and after the capture of the cut areas(B), when the laser energy has been just transferred (C), and after the capture (D). Scale

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 2. Expression levels of the voltage-gated Na+ channels subunits at P75 (A) andP120 (B) in the whole spinal cord. At P75 the levels of the transcripts in the twogenotypes are comparable (WT values=1, indicated by the dotted line). At P120 theSCN8A transcript was significantly decreased in the SOD1G93A spinal cord. Averageand standard errors are presented. **pb0.01, n=4.

Fig. 3. Relative quantification of the voltage-gated Na+ channels subunits in the MN-enriched, laser microdissected samples at the P75 (A) and P120 (B) time points. At P120the SCN1B transcript is significantly decreased, while the SCN3B transcript shows astrong increase. *pb0.05, n=4.

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proximal dendrites (Fig. 5 and see also Chang and Martin, 2009). AtP120 the MNs perikarya appeared smaller than the typical alpha-MNswith the loss of the ChAT-positive boutons and the appearance ofcytoplasmic ChAT IR (Fig. 6). This change in the SOD1G93Amice of thepattern of ChAT distribution between the P75 and P120 may beattributed to the neuronal degeneration.

In the spinal cords of WT and SOD1G93A (P75) mice, at lowmagnification, beta3 IR was diffused in the neuropil in the graymatter, while the white matter was characterized by pale stainingwith the exception of some positive bundle of fibers departing fromthe gray matter and directed toward the pial surface of the spinal cord(Fig. 5). In the ventral horn, at higher magnification, the beta3 IR wasprevalent in the neuropil and in some intensely stained puncta which,in some cases, decorated the MNs cellular membranes. These puncta,surrounding MNs, were not ChAT-positive. Beta3 IR was observed inthe cytoplasm of MNs to a lesser degree, in comparison to thesurrounding neuropil, while the nucleus appeared devoid of any beta3IR (Fig. 5). This beta3 expression pattern was substantially conservedin WT mice, at P75 and P120, and in P75 SOD1G93A mice. In P120

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

SOD1G93A mice, an increase in the beta3 IR in the cell bodies of MNswas observed, while in the surrounding neuropil beta3 IR appeareddecreased (Fig. 6). In addition, ChAT IR was also increased in the MNscell bodies showing a diffuse homogeneous intracellular staining.MNs in P120 SOD1G93A mice showed evident signs of degeneration,such as shrunken or irregular shaped cell body, reduced cytoplasmand enlarged cell nucleus (Fig. 6).

In the tissue of the WT and SOD1G93A mice at P75 and P120, inaddition to the appearance of beta3 immunofluorescence in the MNscell bodies, other immunopositive cellular structures resembling glialelements were also evident. On the basis of these observations, weperformed triple-label immunofluorescence to investigate the natureof these cellular structures and their relation to MNs. Confocal micro-scopy analysis showed that many astrocytes in WT and SODG93Aexpressed beta3 IR at both time points (Figs. 7 and 8 and Supp. Fig. 5),which was mainly observed in the cell body and to a much lesserdegree to the astrocytic processes. The distribution of beta3-positiveastrocytes was homogeneous in the tissue and did not show particularrelations to the degenerating MNs, which were outlined by ChATimmunofluorescence (Figs. 7 and 8).

Beta3 protein expression and immunoreactivity are increased in theventral horn region and in the MNs cell bodies of the SOD1G93A mice

In order to establish whether the increased beta3 mRNA ex-pression was accompanied by a parallel increase in beta3 proteinexpression we performed Western blot analyses and immunohisto-chemistry. Western blotting, performed on homogenates preparedfrom sections of the ventral horn region confirmed that the beta3protein was significantly increased at P75 and P120 (Fig. 9). In

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 4. Confocal images showing an intense astrogliosis in the P120 SOD1G93A mouse. Double immunofluorescence performed with anti-GFAP (green, astrocytes) and anti-ChAT(red, choline acetyltransferase) antibodies. The ChAT channel is merged with the DAPI channel (gray, nuclei). The anti-ChAT antibody was used to identify the MNs cell body in theventral horn of the spinal cord. Scale bar=40 μm.

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addition, the quantitative analysis of the intensity levels of the beta3immunoreactivity, performed on MN cell bodies, showed a significantincrease between WT and SOD1G93A groups at both time points.At P75, the beta3 immunofluorescence intensity in the MNs ofSOD1G93A mice was significantly increased (~1.5-fold) compared toage-matched WT littermates (Fig. 7). At P120, the beta3 immunoflu-orescence intensity in the MNs of SOD1G93A mice was furtherincreased to more than two-fold above that of the age-matched WTlittermate (Fig. 8). Overall, these results demonstrate a significantincrease in expression of beta3 in MNs of SOD1G93Amice at both P75and P120.

Discussion

In the present work, we analyzed the expression of the genesencoding SCN8A, SCN1B, SCN2B and SCN3B VGSC subunits in thespinal cord of pre-symptomatic and symptomatic SOD1G93A miceand compared with age-matched WT controls. Our major finding isthe significant increase of the SCN3B mRNA and protein in the L-IX ofthe SOD1G93Amouse spinal cord compared to theWT. The incrementof the beta3 mRNA was unmasked when the L-IX was analyzedseparately from the whole spinal cord and was even more evident forthe beta3 protein, which we have found up-regulated in the ventralhorn area and in the MN cell bodies at the pre-symptomatic andsymptomatic stages.

The beta3 subunit is expressed in neural tissues including thespinal cord. Previous studies have found its transcript in the layers I/IIand X of the spinal cord (Shah et al., 2000). Qu et al. (2001) using “insitu” hybridization have found the beta3 transcript in the superficiallaminae of the dorsal horn, in lamina V, in the preganglionic sym-pathetic neurons and around the central canal, whereas expression inthe MNs was not detected. Our analyses focused on the lamina IX,which is the MN-enriched region in the spinal cord and is the anato-

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

mical region that is most affected in ALS. Our data show that the beta3transcript and protein are present at very low levels in L-IX and in thecell bodies of MNs in bothWT and mutant mice. At P75, the transcriptlevels were comparable between WT and mutant, while the beta3protein was significantly higher in the mutant mice. In contrast, insymptomatic mice at P120, both beta3 mRNA and protein wereincreased. These data suggest that over-expression of SOD1G93Aregulates the beta3 subunit at some post-translational level thatenhances folding and assembly of the protein and/or decreases itsturnover at P75 in presymptomatic mice. The significant increase, atP75, of the beta3 protein in the ventral horn area, and specifically inthe MN cell bodies, indicates that this increment is peculiar for theMNs, and will in turn modify their gating characteristics.

Among the VGSC auxiliary subunits, the beta3 subunit has beenmost consistently related to persistent sodium currents, which play animportant role in the control of neuronal excitability near firingthreshold (Crill, 1996). Qu et al. (2001) demonstrated that the beta3subunit increases persistent sodium currents conducted by VGSCexpressed transiently in tsA-201 cells and that its co-expression withthe beta2 subunit further enhances these persistent currents. In linewith these data, Aman et al. (2009) concluded that beta3, as well asbeta2 and beta4, all promote persistent currents conducted byrecombinant VGSC expressed in HEK-293 cells. On the other hand,beta1 consistently accelerates inactivation and decreases persistentcurrents. The beta1 and beta3 subunits have similar molecularcharacteristics and are thought to have similar noncovalent associa-tionwith VGSCs (Qu et al., 2001). Thus, although the ability of beta3 tomodulate persistent currents depends on the alpha subunit present(Vijayaragavan et al., 2004), our findings indicate that increasedexpression of beta3 in MNs relative to beta1 might contributesignificantly to their increased excitability in ALS.

Previous studies conducted by Zona and co-workers (1998) (Pieriet al., 2003; Zona et al., 2006; Pieri et al., 2009) and Heckman and

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 5. Distribution of beta3 at postnatal day 75 (P75). A) Double immunofluorescence performed with anti-beta3 (green, voltage-gated Na+ channels subunit) and anti-ChAT (red,choline acetyltransferase) antibodies. The ChAT channel is merged with the DAPI channel (gray, nuclei). The anti-ChAT antibody was used to identify the MN cell bodies in theventral horn of the spinal cord, which were selected for subsequent analysis. B) Intensity level analysis of beta3 immunofluorescence showing a significant increase of beta3 in theSOD1G93A mouse at the pre-symptomatic stage (P75) compared with the age matched WT littermate. Average and standard errors are presented. P-value *pb0.05. Scalebar=40 μm.

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co-workers (Kuo et al., 2004, 2005, 2006; Elbasiouny et al., 2010) haveshown that hyperexcitability and increased persistent sodiumcurrents are characteristics of spinal MNs and cortical neuronscultured from mutant SOD1 mice. Van Zundert et al. (2008) havedescribed an early onset (P4–P10) of increased intrinsic excitabilityand persistent Na+ current in hypoglossal MNs of SOD1G93A mice.Although our molecular data are on the adult mouse while thefunctional analyses have been performed on primary cultures andpostnatal mice, both findings are indicative of an abnormal neuralactivity of the VGSC expressed by the ALS-affected MNs. Moreover,the functional data combined with ours support the hypothesis thathyperexcitability is an early and intrinsic characteristic of the MNs inALS, leading to an increased excitotoxic vulnerability throughoutlife. Consistent with the experimental data are the findings on theneuronal hyperexcitability in ALS patients (Vucic and Kiernan, 2006a,b; Kanai et al., 2006; Vucic et al., 2007; Vucic et al., 2008), which haverevealed changes indicative of widespread dysfunction in axonalmembrane ion channels, including increased persistent VGSC con-ductances, in the spinal cord and in the cerebral cortex as well. In a

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

recent study conducted on 13 asymptomatic SOD-1 mutation carriers,6 clinically defined familial ALS patients carrying SOD1 mutations,and 45 sporadic ALS patients, Vucic and Kiernan (2010) concludedthat persistent sodium conductances were up-regulated in thefamilial ALS population (the mutant SOD1 carriers) and were linkedto axonal degeneration and cortical hyperexcitability. Moreover,Urbani and Belluzzi (2000) demonstrated that riluzole, a neuropro-tective agent and the only drug currently approved for the treatmentof ALS, is able to produce a dose-dependent inhibition of the per-sistent sodium currents in rat brain cortical slices at therapeutic con-centrations. Therefore, we hypothesize that the mutant SOD1 altersthe beta3 subunit expression, which in turn enhances the excitabilityof MNs and could contribute to excitotoxicity and cell degeneration inALS.

Our analyses also revealed a significant decrease, at end stage(P120), of the beta1 subunit mRNA expression in the ventral horn ofthe mutant mice, while the beta2 mRNA levels remain unchanged.Although beta1 is expressed by reactive astrocytes (Gorter et al.,2002), which at this disease stage have completely invaded the

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 6. Distribution of beta3 at postnatal day 120 (P120). A) Double immunofluorescence performed with anti-beta3 (green, voltage-gated Na+ channels subunit) and anti-ChAT(red, choline acetyltransferase) antibodies. The ChAT channel is merged with the DAPI channel (gray, nuclei). The anti-ChAT antibody was used to identify the MN cell bodies in theventral horn of the spinal cord, which were selected for subsequent analysis. B) Intensity levels analysis of beta3 immunofluorescence showing a significant increase of beta3 in theSOD1G93A mouse at the symptomatic stage (P120) compared with the age matched WT littermate. Average and standard deviations are presented. P-value *pb0.05. Scalebar=40 μm.

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ventral horn (Almer et al., 1999; Jaarsma et al., 2008; presentmanuscript), our analyses suggest a concurrent decrease of the beta1transcript in the MNs. The beta1 subunit accelerates fast inactiva-tion (Isom et al., 1992, 1995; Chen and Cannon 1995). Thus, we mayhypothesize a scenario in which the MNs in mutant mice containVGSC complexes with an increased complement of beta3 subunitsduring life and a reduced complement of beta1 subunits towardthe end-stage. These characteristics might lead to VGSCs withincreased excitability in the presymptomatic stage, which is furtherenhanced by the reduced beta1 expression at the manifestation of thesymptoms.

Finally, we have observed decreased expression, at the symptom-atic stage, of the SCN8A transcript encoding Nav 1.6 channels notrestricted to the ventral horn area but observed in the whole spinalcord, which may have resulted from the massive neuronal lossaffecting MNs as well as other spinal cord neurons at the end stage

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

(Pardo et al., 1995; Martin et al., 2007). Besides having beenfundamental in MNs development, the Nav 1.6 channels are one ofthe highest expressed channels in the adult CNS (Catterall et al.,2005). They are more effective then other channels in generatingpersistent Na+ currents and have a key role in sustaining repetitivefiring (Chen et al., 2008; Osorio et al., 2010). Functionally, thesefindings might indicate a generalized alteration of the systemicneuronal excitability in the spinal cord. Interestingly, Gunasekaranet al. (2009) reported a decreased expression of Nav1.6 following theexposure to cerebrospinal fluid from sporadic ALS patients, indicatingthat the expression of this subunit, and of the VGSC subunits ingeneral, is affected in ALS. Our data, along with the intrinsic MNhyperexcitability induced by the SOD1 mutation and observed in theALS patients, support the idea that an abnormal neuronal function andan altered VGSC expression is a unique and functionally importantcharacteristic of MNs in ALS.

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 7. Beta 3 is expressed by astrocytes in spinal cord sections of a SOD1G93A presymptomatic mouse (P75). Confocal images of triple immunofluorescence performed with anti-ChAT (cyan, choline acetyltransferase), anti-beta3 (green, voltage-gated Na+ channels subunit) and anti-GFAP (red, astrocytes). The ChAT channel is merged with the DAPI channel(gray, nuclei). Beta3 immunofluorescence colocalizes with GFAP in cell bodies and in the processes (white arrows). Scale bar=40 μm.

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Experimental methods

Animals

B6SJL-TgN(SOD1-G93A)1Gur mice expressing the human G93ACu/Zn superoxide dismutasemutationwere obtained from the JacksonLaboratories (Bar Harbor,ME, USA). Selective breedingmaintained thetransgene in the hemizygous state on an F1 hybrid C57BL6×SJLgenetic background (Gurney et al., 1994). Screening for the presence ofthe human transgene was performed on tail tips of adult mice asdescribed (Spalloni et al., 2006). Transgenic mice (SOD1G93A) andwild-type (WT) non-transgenic littermates were housed in a temper-ature controlled room (22 °C) with a light–dark 12:12 cycle (lights on07:00–19:00 h). Food and water were given ad libitum.

This SOD1G93A fALS mouse model with a high copy number ofmutant allele reveals the first phenotypical signs of the ongoingneurodegenerative disease around 90 days, when they are no longerable to couple and their bodyweight stops to increase in comparison totheWTmice. These first signs progress tomotor impairments between100 and 115 days. At 120 days themice showclearmotor impairmentswith at least one hindlimb paralyzed. This time point (P120) has beenselected as the symptomatic stage. As a pre-symptomatic time point,we selected 75 days of age (P75). Although some molecular andmorphological changes have been described in embryonic prepara-tions and in mice younger than 75 days of age (Tortarolo et al., 2003;Spalloni et al., 2004a,b; Van Zundert et al., 2008), these animalspresent no clear phenotypic signs of growthormotor impairments andno loss of MNs number or decrease in the density of cholineacetyltransferase-positive boutons (Chang and Martin, 2009). Exper-iments were conducted in compliance with the European CouncilDirective (86/609/EEC) for the use and care of laboratory animals.Every effort was made to minimize animal suffering. All experiments

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

were carried out in SOD1G93Amale mice, controls were age-matchednon-transgenic littermates of the mutants. Independent groups ofmice were used for the whole tissue mRNA and the Western blotanalyses, while for the laser microcapture analyses and the immuno-fluorescence staining the same groups of mice were used.

Whole tissue: RNA isolation and cDNA synthesis

Animals under deep anesthesia (chloral hydrate, 400 mg/kg) weredecapitated and the whole spinal cords were ejected from thevertebral column by means of sterile 0.1 M phosphate-buffered saline(PBS, pH 7.2) injection in the vertebral column. Tissues were imme-diately frozen on dry ice and stored at−80 °C until use. Total RNAwasthen extracted using Trizol Reagent (Invitrogen) following manufac-turer's protocol and dissolved in 0.1% DEPC (Sigma) water. RNAquantity and quality were assayed with the NanoDrop ND-1000spectrophotometer (Thermo Scientific).

First-strand cDNA was synthesized from 1 μg of RNA by reversetranscription using the Superscript III enzyme (Invitrogen) primedwith 2.5 μM random hexamers (Amersham). cDNA synthesis wasperformed using the following program: 25 °C for 5 min, 50 °C for60 min, 70 °C for 15 min.

The SCN8A splicing was analyzed by agarose gel electrophoresisafter classical RT-PCR amplification using specific primers (forward:5′-AAATGGACAGCCTATGGCTTC-3′, reverse: 5′-TCACCTCGTCGATTTC-GAACCG-3′; Plummer et al., 1997).

Tissue preparation for laser capture microdissection (LCM) andimmunofluorescence procedures

Adult WT and SOD1G93A mice (P75 or P120) were anesthetized(chloral hydrate, 400 mg/kg) and subjected to intracardial perfusion

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 8. Beta 3 is expressed by astrocytes in spinal cord sections of a SOD1G93A symptomatic mouse (P120). Confocal images of triple immunofluorescence performed with anti-ChAT(cyan, choline acetyltransferase), anti-beta3 (green, voltage-gated Na+ channels subunit) and anti-GFAP (red, astrocytes). The ChAT channel is merged with the DAPI channel (gray,nuclei). Beta3 immunofluorescence colocalizes with GFAP in many cell bodies (white arrows) and in some processes. Scale bar=40 μm.

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of ice-cold 0.9% NaCl followed by 4% paraformaldehyde (PAF) inphosphate buffer (PB, 0.1 M, pH 7.2). After fixation, vertebral columnswere dissected and immersed in ice-cold PAF for 16 h then spinalcords were dissected and immersed in filtered 30% sucrose PB forcryoprotection. The lumbar tract was cut on a cryostat (Leica,Germany) to a thickness of 40 μm and L3–L6 transverse sectionswere selected for immunofluorescence or LCM procedures. Foridentification of MNs during LCM procedure a Nissl staining wasperformed. Sections were kept in 100% ethanol for 1 h and re-hydrated in descending alcohols (95% ethanol 5 min, 70% ethanol5 min, 50% ethanol 1 min) and water (3 min). The specimens werethen incubated in a 0.02% cresyl violet acetate buffer solution(45 min) and the excessive stain was removed by washes in waterand 50% ethanol. Sections were finally sampled on membranemounted metal frame slides (Molecular Machines and Industries)and dried in a fume hood at room temperature until LCM wasperformed. All the solutions were prepared in 0.1% DEPC water, andthe cryostat blade was rinsed in ethanol and chloroform before use toensure a complete removal of DNAses and RNAses.

RNA isolation and cDNA synthesis for LCM

Spinal cord sections were examined with an inverted Nikonmicroscope (Nikon Eclipse TE2000-S) coupled with an SL MicrotestUV laser control unit microdissector and controlling software (SLMicrotest, GmbH). Histological slides, stained with cresyl violet werevisualized with 4× and 20× objectives. Neurons located in Lamina IX(L-IX) and showing a maximum diameter exceeding 20 μm wereconsidered as MNs and regions of L-IX enriched in MNs were selected,hereinafter defined as MN-enriched region. These regions were cutwith laser pulses, and an RNase free tube with adhesive lid (MolecularMachines and Industries) was used to capture the samples. LCM

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

samples from the same animal were pooled together, and about 200MNs were collected from each mouse and tubes were stored at−80 °C until RNA extraction (Fig. 1).

The Absolutely® RNA FFPE kit (Stratagene) was used to isolateRNA from LCM samples, following manufacturer's protocol forformalin fixed tissues, excluding any deparaffinizing procedure. Theeluted, DNase treated RNA was further precipitated with glycogen(Invitrogen), 0.1 volume of sodium acetate 3 M (pH=5) and 2.5volumes of absolute ethanol. After an over-night incubation at−20 °C, the sample was centrifuged, the pellet subjected to 3 roundsof 75% ethanol wash/centrifugation and finally dissolved in 10 μl ofDEPC water.

The RT reaction was performed immediately after, using 5 μl ofsolubilised RNA in a final volume of 20 μl, under the above-mentionedexperimental conditions.

Real-time PCR on microdissected samples and whole tissue

Real-time PCR was performed with the IQ™ 5 Real-Time PCRDetection System (Bio-Rad). Reactions were performed in a 20 μlvolumemixture containing 1× IQ™SYBR®Green Supermix (Bio-Rad),and 50 ng of the retrotranscribed RNA. Primer pairs were designedwith the Primer3 program to obtain short amplicons (150 bp or less)that work better in real-time PCR (Table 1). All oligos, but the SCN2B,were designed to span between adjacent exons and were tested foramplification efficiencies by 1:5 serial dilution standard curves. Ameltcurve was run to determine specific amplification (Supp. Fig. 1) usingcDNA from both total spinal cord and LCM samples, further confirmedby sequencing of the PCR products and by verifying the presence ofDNA of the appropriate size on a agarose gel visualized by ethidiumbromide, and by the absence of amplified DNA in the no templatecontrol. Oligos concentrations were 150 nM for glyceraldehydes-3-

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

Fig. 9. Western blot analysis of beta3 expression in lumbar, ventral horn spinal cord ofSOD1G93A and WT mice at P75 (A) and P120 (B). Upper panels show representativebeta3 immunoblots and relative GAPDH as loading control, on 3.5 μg of protein from thetotal homogenates. The graphs below show an increased expression of the beta3subunit both at P75 and at P120. Values are expressed relative to WT expression (=1)±SEM. P-value pb0.05.

9M. Nutini et al. / Molecular and Cellular Neuroscience xxx (2011) xxx–xxx

phosphatase dehydrogenase (GAPDH) as housekeeping gene, and500 nM for SCN8A, SCN1B, SCN2B and SCN3B (GenBank accessionnumbers: GAPDH—BC096440; SCN8A—AF049617; SCN1B—NM011322; SCN2B—XM134787; SCN3B—NM153522). Three repli-cates from each individual cDNA sample were processed.

Table 1Primer pairs used in real-time RT-PCR.

Gene name Forward primer

GADPH 5′-TGCGACTTCAACAGCAACTC-3′SCN8A 5′-TCATGAATCAGTCGCTGCTC-3′SCN1B 5′-TATGGCATCCATCGTGTCAG-3′SCN2B 5′-CATAGCCACTGCTTCCACAA-3′SCN3B 5′-TCTACTGGGTCAGAGTCTGCTTC-3′

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

Fold expression was determined using the 2−ΔΔCt method forrelative quantification (User Bulletin #2 and Applied Biosystems,1997; Johnson et al., 2000; Livak and Schmittgen, 2001; Bookout andMangelsdorf, 2003). The results of the real-time PCR are presented as2−ΔΔCt values, where Ct is the threshold cycle, defined as the point ofthe amplification reaction at which the fluorescence rises significantlyabove the background; ΔCt is the difference in the Ct values derivedfrom the specific gene being assayed and the GAPDH internal control,while the ΔΔCt represents the difference between the paired tissuesamples, as calculated by the formula ΔΔCt=ΔCt of SOD1G93A—ΔCtof WT sample. The N-fold differential expression in a specific VGSCtranscript of a SOD1G93A sample compared to the WT counterpartwas expressed as 2−ΔΔCt.

The GAPDH mRNA levels were similar across age groups andgenotypes (LCM samples Ct values: WT P75: 26.8±1.1; WT P120:26.2±1.6; SOD1G93A P75:26.9±1.5; SOD1G93A P120: 25.8±1.2).

Lumbar spinal cord dissection and Western blot analysis

Whole spinal cords were isolated from P75 and P120 animalsfollowing the procedure described above. The ejected spinal cord wascross cut at 5 mm, rostrally, from the L2 level, in order to discard thecervical and thoracic tracts. The sacral tract was also excluded bymeans of a second cross cut at the L6–S1 level. The isolated lumbartract was divided into two halves by a longitudinal incision on themedial line (sulcus medianus dorsalis) of the tissue. Each half, themedial side facing upward, was further sectioned following thecentral canal for the entire length of the tissue to obtain the dorsal andventral horn samples. The ventral samples were homogenized in asolution containing sucrose 0.32 M and Hepes 5 mM (pH=7.4) plus10 μl/ml of protease inhibitor cocktail (Sigma-Life Sciences). Aftertotal protein isolation, 3.5 μg were loaded onto 10% SDS–PAGE andtransferred to PVDF membrane (Amersham). The membrane wasblocked with 5% milk in PBS 0.1% Tween 20, followed by incubationwith primary antibodies, rabbit anti-beta3 (1:50) and mouse anti-GAPDH (Calbiochem, 1:5000). Immunoreactivity was detected usinganti-rabbit (beta3, 1:5000) and anti-mouse (GAPDH, 1:5000) conju-gated to HRP (Promega) and visualized with enhanced chemilumi-nescence (ECL Plus, Amersham). The bands were visualized using theStorm Scanner and analyzed using the Image Quant TL-1D software(Amersham). The assays were conducted under conditions in whichdensitometric signal increased linearly with the loaded quantity ofprotein, as determined by preliminary experiments. The beta3 signalwas normalized to GAPDH and values of SOD1G93A samples werecompared to WT and expressed as n-fold increase. Quantification wasdone on n=3 mice (P75 group) and n=4 mice (P120 group).

Immunofluorescence procedures and densitometric analysis

Lumbar spinal cord sections, derived from the same tissuesprocessed for LCM, were used for double and triple immunolabellingprocedures. Three sections were randomly selected from every 20serially cut ones from the L3–L6 tract, rinsed in PB and mounted ongelatin-coated slides. Sections from both wild-type and SOD1G93Amice were sorted on the same slide, in order to subject them to iden-tical incubation conditions. Theywere processed at room temperature

Reverse primer Product lenght

5′-ATGTAGGCCATGAGGTCCAC-3′ 143 bp5′-ACTGTGCTGTGCTCGTCGT-3′ 148 bp5′-CCAGGTATTCTGAGGCGTTC-3′ 146 bp5′-CTGGCTGGCTATCCATCATT-3′ 158 bp5′-GCCTGTAGAACCACTCCACTACA-3′ 150 bp

ta3 subunit of voltage-gated Na+ channels in the spinal cord of the.03.005

10 M. Nutini et al. / Molecular and Cellular Neuroscience xxx (2011) xxx–xxx

as follows: 5% donkey serum and 0.3% Triton for 40 min in PB,followed by 5% BSA and 0.3% Triton X-100 for 40 min in PB. After that,sections were incubated over-night at +4 °C in a mix solution of twoor three primary antibodies (1% donkey serum, 0.1% X-100 Triton inPB) selected from the following: anti-choline acetyltransferase (goatanti-ChAT, Chemicon, working dilution 1:100), anti-beta3 (rabbitanti-beta3, working dilution 1:50; characterized in Maier et al., 2004)and anti-GFAP (mouse anti-GFAP, working dilution 1:200, Chemicon)antibodies. After three 10 min rinses in PB, sections were incubated3 h at RT with a cocktail of the appropriate secondary antibodies(Jackson ImmunoResearch Laboratories), including Cy2-conjugateddonkey anti-rabbit IgG, Cy3-conjugated donkey anti-goat IgG andCy5-conjugated donkey anti-mouse IgG all at 1:50 working dilution.The last step was three 10 min rinses in PB and a 40 min incubationwith the Hoechst solution (1 ng/ml, Molecular Probes, Invitrogen) fornuclei visualization, followed by a further rinse in water. After that,sections were coverslipped using gel mount (Biomeda Corp., FosterCity, CA, USA). Controls included sections treated with secondaryantibody alone; no specific staining was observed (Supp. Fig. 2A). Thespecificity of the beta3 antibody was also tested in crude spinal cordpreparations by Western blot (Supp. Fig. 2B).

Sections were examined under a confocal laser scanning micro-scope (Leica SP5, Leica Microsystems, Wetzlar, Germany) equippedwith four laser lines: violet diode emitting at 405 nm, argon emittingat 488 nm, helium/neon emitting at 543 nm and helium/neonemitting at 633 nm. Images were acquired in sequential scan modeby using the same acquisitions parameters for all the images (laserintensities, gain photomultipliers, pinhole aperture, objective 63×,zoom 1). For visualization purposes channel colors were paletteassigned and may not reflect the true fluorochrome color. Forproduction of figures, brightness and contrast of images wereadjusted by taking care to leave a light tissue fluorescence backgroundfor visual appreciation of the lowest fluorescence intensity featuresand to help comparison among the different experimental groups.Final figures were assembled by using Adobe Photoshop CS3 andAdobe Illustrator 10. Laminar boundaries of spinal cord structureswere delineated according to Hoechst staining (and for LCM accordingto Nissl staining).

Quantification of the beta3 signal was performed on ChAT-positivecells, selected in lamina IX, to provide a reliable identification of MNs.This strategy allowed more direct comparison between LCM andimmunofluorescence material. Changes in the beta3 immunoreactiv-ity (IR) were measured through densitometric analyses, by experi-menters blinded as to the treatment groups, by using the ImageJsoftware (version 1.41, National Institutes of Health, USA). Afterbackground subtraction in the beta3 channel, the beta3 IR wasquantified by manually outlining individual ChAT+ cells andmeasuring cell associated fluorescence intensity. The ratio, F/A,defines mean fluorescence of individual cells (F) normalized to totalcellular surface for image (A). Quantification was done on 120 cellsper group (n=4mice per group for each experimental groupWT P75,WT P120, SOD1G93A P75, SOD1G93A P120).

Data analysis

Data are expressed as arithmetic mean±SEM. Statistically sig-nificant differenceswere determined using the Student's t-test; valuesof *pb0.05 and **pb0.01 were considered statistically significant.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.mcn.2011.03.005.

Acknowledgments

This work was supported by a grant from the “Compagnia SanPaolo” (protocol number 4932, file 2008.2395) to PL.

Please cite this article as: Nutini, M., et al., Increased expression of the beSOD1G93A mouse, Mol. Cell. Neurosci. (2011), doi:10.1016/j.mcn.2011

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