Altered calcium homeostasis in motor neurons following AMPAreceptor but not voltage-dependent calcium channels’ activationin a genetic model of amyotrophic lateral sclerosis
aFondazione S. Lucia, Centro Europeo Ricerca sul Cervello, Via del Fosso di Fiorano, 00173 Roma, ItalybDepartment of Neuroscience, University of Rome “Tor Vergata”, Via Montpellier, 1 – 00133 Roma, Italy
Received 23 March 2007; revised 28 June 2007; accepted 1 July 2007Available online 10 July 2007
and spinal cord. Approximately 10% of ALS cases are familial(FALS), and a subset of these is a result of dominantly inheritedmutations in the gene encoding the enzyme Cu,Zn superoxidedismutase (SOD1) (Rosen et al., 1993). Over 100 different muta-tions have been identified. These are associated with a clinicaldisease that is phenotypically indistinguishable from sporadic ALS(Pramatarova et al., 1994, 1995; de Belleroche et al., 1996; Shaw etal., 1998). The precise molecular pathways leading to motor neuroninjury and cell death in ALS remain not understood. There issubstantial evidence that mutant SOD1 exerts its deleterious effectsthrough a toxic gain of function rather than through a loss ofsuperoxide dismutase activity (Cleveland, 1999). However, severalpathogenic factors have been proposed, including glutamate ex-citotoxicity (Babcock et al., 1997), production of reactive oxygenspecies (ROS) (Bergmann and Keller, 2004), Ca2+-dependentformation of protein aggregates (Billups and Forsythe, 2002),axonal transport (Brini, 2003), deregulation of Ca2+ homeostasis(Budd and Nicholls, 1996), presence of highly Ca2+-permeableAMPA receptors (Billups and Forsythe, 2002; Ladewig et al., 2003;Lips and Keller, 1998), disruption of mitochondrial function (Liuand Wong-Riley, 2003; von Lewinski and Keller, 2005). Althoughthe involvement of each of these factors has been well established,their temporal and spatial interplay remains elusive.
Calcium ions are important intracellular messengers governingcellular functions, such as differentiation and growth, membraneexcitability, exocytosis and synaptic activity. Neurons control bothintracellular Ca2+ levels and the location of Ca2+ ions through acomplex interplay between Ca2+ influx, Ca2+ efflux, Ca2+ bufferingand internal Ca2+ storage. Under physiological conditions, theseprocesses enable multiple Ca2+-regulated sequences of events tooccur independently within the same cell. However, excessive Ca2+
influx or release from intracellular stores can elevate Ca2+ loads tolevels that exceed the capacity of regulatory mechanisms. This leadsto the inappropriate activation of Ca2+-dependent processes that arenormally dormant or operate at low levels, causing metabolic
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derangements and eventual cell death (Choi, 1988; Tymianski andTator, 1996; Sattler and Tymianski, 2000).
The alteration of intracellular Ca2+ homeostasis is thought tohave a key role in the disease process, independently of the cellularand molecular event initiating motor neuron degeneration in ALS.In fact, it has been reported that Ca2+-binding proteins such ascalbindin D-28k and parvalbumin were absent in motor neuronpopulations lost at an early stage in ALS (Alexianu et al., 1994; vonLewinski and Keller, 2005). These findings agree with previousobservations which identified a low cytosolic Ca2+ bufferingcapacity as an important risk factor for degeneration (Alexianuet al., 1994; Van Den Bosch et al., 2006).
It has already been reported that spinal motor neurons expressCa2+-permeable AMPARs, but no information is available on thedynamics of calcium ions within the cytoplasm of motor neurons inresponse to AMPA receptor or to voltage-dependent calcium chan-nels’ activation in a mouse model of ALS. Our previous resultsindicated that the influx of Ca2+ ions through the activated AMPAreceptors in spinal motor neurons was not different in transgenicG93A mice compared to controls (Pieri et al., 2003b). Therefore, inthe present work, we combined electrophysiological recording withmicrofluorimetric analysis to study the interaction between theintracellular Ca2+ level and the AMPA receptors’ and/or voltage-dependent calcium channels’ activation in control, SOD1 andG93A motor neurons.
Materials and methods
Animal model
B6SJL-TgN (SOD1-G93A)1Gur mice expressing the G93Amutant SOD1 (G93A) and B6SJL-TgN (SOD1)2Gur mice expres-sing wild-type human SOD1 (SOD1) constructed by Gurney et al.(1994) were originally obtained from Jackson Laboratories (BarHarbor, USA) and then housed in our animal facilities. Screeningfor the presence of the human transgene was performed on tail tipsfrom adult mice and on the head from each embryo after removal ofthe spinal cord (Pieri et al., 2003b). Procedures involving animalsand their care were conducted in strict accordance with the Policyon Ethics approved by the Society for Neuroscience and with theEuropean Communities Council Directive for Experimental Pro-cedures. Every effort was made to minimize the number of animalsused and their suffering.
Cell cultures
Mixed spinal cord cultures were prepared as previously des-cribed (Carriedo et al., 1996; Pieri et al., 2003b; Zona et al., 2006).Briefly, spinal cord cultures were prepared from 15-day-oldembryos of a control female mated with a G93A male. Each neuraltube was dissected and individually incubated for 10 min in 0.025%trypsin and then dissociated by gentle trituration. The resultingmixed cultures were plated on poly-L-lysine-coated glass coverslips (four cover slips for each spinal cord) and maintained inDMEM supplemented with 5% FBS and 5% HS. About 2 to 3 hafter plating, the medium was replaced with neurobasal supple-mented with B-27 (1:50), 0.5 mM glutamine, 25 μM glutamic acidand 100 μg/mL gentamycin.
All experiments were performed on neurons maintained in vitroand used between the 8th and the 12th day. Cortical cultures wereprepared as previously described and used between the 8th and the
11th day (Carunchio et al., 2007). For both cultures, the embryoswere analyzed for the presence of the human SOD1. At least threecultures were used for the measurement of each parameter given inthe Results.
Electrophysiology
Spinal cord motor neurons were visually identified by theirmorphological appearance: a large cell body, long axon, and exten-sive dendritic arborization (Carriedo et al., 1996; Pieri et al., 2003b;Vandenberghe et al., 2000; Zona et al., 2006) and by a thresholddiameter criteria (N28 μm; Carriedo et al., 1996; Takahashi, 1990;Vandenberghe et al., 2000; Zona et al., 2006). Whole-cell configur-ation of the patch-clamp technique (Hamill et al., 1981) was used torecord currents in voltage-clamp mode. Cells plated on the glasscoverslip were placed into a recording chamber of an uprightmicroscope (Axioscope FS, Carl Zeiss) equipped for infra red videomicroscopy (Hamamatsu, Japan) and video microfluorometry(ImproVision, Coventry, UK). To record the kainate-induced cur-rents, the standard external solution contained (in mM): NaCl 130,KCl 3, MgCl2 2, CaCl2 1.5, HEPES 10, D-glucose 6, tetraethyl-ammonium (TEA) Cl 10, pH 7.4 with NaOH. D-Amino-phos-phonovaleriate (APV, 50 μM) and nifedipine (50 μM) were added toprevent calcium entry through N-methyl-D-aspartate (NMDA)receptors and voltage-gated calcium channels (VGCCs), respec-tively, whereas tetrodotoxin (TTX, 1 μM) was added to avoid actionpotential-dependent glutamate release by surrounding neurons.Neurons were approached by means of borosilicate glass pipettes(resistance 3–4 MΩ) made with a PP 83 Narishige puller and filledwith a solution containing (mM): CsCl 140, EGTA 0.75, HEPES10, D-glucose 6, fura-2 0.25, pH 7.4 with CsOH. Experiments wereperformed at room temperature (22–24 °C). Data were acquiredwith an Axopatch 1D amplifier (Axon Instruments, USA) andpClamp 8 and Axoscope software (Axon Instruments, USA). Afterthe establishment of a gigaseal, the pipette resistance and capa-citance were compensated electronically. After seal rupture, thewhole cell capacitance was assessed on-line from the integral ofthe current transient after a 10-mV voltage step (membrane testfunction, pClamp 8; Axon Instruments). Cells with a capacitanceof b25 pF were rejected, in accordance with the dimensioncriteria previously described, assuming 1 μF/cm2 as specificcapacitance.
Drug application
Kainate (KA, 200 μM) was dissolved in the extracellular solu-tion and applied by means of a patch pipette positioned in the closevicinity of the cell body, connected to a pressure application system(Picospritzer, b10 psi, 5 s). Nifedipine, APV, TEACl and TTX wereadded directly into the perfusing solution. Cyclopiazonic acid(30 μM) was applied through a syringe connected to the recordingchamber via a three-way tap.
Immunocytochemistry and western blotting
SMI-32 staining (Sternberg Monoclonals; Carriedo et al., 1996)was performed. Cultures were fixed with 4% paraformaldehyde in0.1 M phosphate buffer (PB; pH 7.4) for 30 min at room tem-perature, and then left for 20 min in the blocking solution (2%serum in PB with 0.3% Triton X-100). Cultures were incubated inprimary antibody (1:6000 dilution) for 48 h at 4 °C, and visualized
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using a fluorescein cyanine-conjugated secondary antibody (Jack-son ImmunoResearch, West Grove, PA). Fluorescence images wereacquired through a confocal laser scanning microscope (Zeiss, LSM510, Germany).
Western blotting experiments were performed to verify an equalexpression of hSOD1 protein in the two transgenic strains. The tailswere homogenized on ice in a buffer containing 50 mM Tris,pH 7.5, 0.5 mM EDTA, pH 8 containing a 1:1000 dilution ofprotease inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Anequal volume of a solution containing 50 mM Tris, pH 7.5, 150 mMNaCl, 1% NP-40, 1% sodium deoxycholate, and 2% SDS wasadded, and the homogenates were sonicated for 20 s, boiled for10 min, and clarified by centrifugation at 16,000×g for 5 min.Protein content was determined using Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were subjected toSDS–PAGE on 10% gels. After electroblotting to nitrocellulosemembranes (Amersham Pharmacia, Piscataway, NJ), proteins werevisualized using appropriate primary antibodies. ImmunoreactiveSOD1 was detected with a rabbit polyclonal anti-SOD1 antibody(1:1000) (Stressgen, San Diego, CA). β-Actin, used as loadingcontrol, was detected with a mouse monoclonal antibody(1:10,000) (Sigma-Aldrich, St. Louis, MO). All primary antibodieswere diluted in 0.5% (w/v) nonfat dry milk and incubated with thenitrocellulose blot 2 h at room temperature. The filter wasincubated with the appropriate peroxidase-conjugated secondaryantibodies (Bio-Rad) and developed using the HRP chemilumi-nescence detection system (Roche, Indianapolis, IN). Immuno-blots were digitalized and quantified with Image Quant TL soft-ware v2003.02 (Amersham Pharmacia Biosciences, Piscataway,NJ).
Microfluorometry
Motor neurons were filled with calcium-sensitive dye throughthe patch pipette, as previously described for other neurons(Guatteo et al., 1998, 2005), within a few minutes after establishingthe whole-cell configuration. The fluorescent calcium indicator(fura-2 pentapotassium salt, Molecular Probes) was excited via a40× water immersion objective (Axioscope FS, C. Zeiss) by illu-mination with light provided by a 75-W Xenon lamp. Excitationlight was band-pass-filtered alternatively at 340 or 380 nm whereasemission light passed a barrier filter (500 nm) and was detected by aCCD camera (Photonic Science, Millham, UK). A set of imagesobtained at the two excitation wavelengths was acquired at 3-, 6- or12-s intervals. Time courses of fluorescence values, obtained byexcitation at 340 and 380 nm, were calculated over the cell soma(region of interest, ROI) and corrected for background fluorescence(BK), measured from a region N100 μm away from the soma.Changes in fluorescence corresponding to calcium concentrations[Ca2+]i are reported as ratio values and were calculated from thefollowing equation: R= (F340ROI−F340BK) / (F380ROI−F380BK),where F340 and F380 are the specific fluorescence values emittedby ROI and background at 340 and 380 nm excitation wavelengths(Tozzi et al., 2003; Guatteo et al., 2005). The [Ca2+]i wascalculated according to the following equation (Grynkiewicz et al.,1985): [Ca2+]i=KD×β×(R−Rmin) / (Rmax−R), where KD is theeffective dissociation constant, β is the ratio of 380 nm excitationflorescence at zero and saturating calcium levels, Rmin is the ratio340/380 of fura-2 in the calcium-free solution, Rmax is the ratio offura-2 in the presence of saturating calcium concentration (1.5 mMCaCl2).
Data analysis
Data were analyzed with Origin 7 (OriginLab Corporation,Northampton, USA) and Clampfit 8 (Axon Instruments, USA)software. All results are expressed as means±SE. The homogeneityof the variances was tested by Leven’s test. The lack of differenceswas tested (two-way analysis) before uniting data from differentcultures. ANOVA followed by Bonferroni’s test was used to deter-mine the significance of the differences between Control, SOD1,and G93A. When necessary, a paired t-test was used. A p value ofb0.05 was considered significant.
Results
To verify whether the amount of human SOD1 protein wassimilar in SOD1 or G93A cultures, western blotting analysis wasperformed. Cultured motor neurons expressed similar levels ofeither human SOD1 or mutant human SOD1 (G93A) enzyme, asshown in Fig. 1A. Spinal cord motor neurons in control, SOD1 andG93A culture were identified by means of criteria previouslyreported, including morphological appearance of their large soma(≥28 μm), membrane capacitance (≥25 pF), and intense stainingwith the motor neuron marker SMI-32 (Pieri et al., 2003a; Zona etal., 2006; Figs. 1B and C).
In the cells that matched these criteria, we performed electro-physiological recordings at 8–12 DIV from control, SOD1 andG93A cultures. In accordance with previous reports (Pieri et al.,2003a; Kuo et al., 2004), the resting membrane potential isunaffected by over expression of the human transgene, either wildtype or the mutated form G93A (control: −49±2.95, n=11; SOD1:−46.5±3.6, n=6; G93A: −49±2, n=14). In addition, in all motorneurons tested, the application of 200 μM kainate (KA) at theholding potential of −60 mV, evoked a non desensitizing inwardcurrent (Figs. 1C and 2B). The currents were completely blocked byCNQX (10 μM, not shown) or GYKI53655 (100 μM, Fig. 1C), acompetitive antagonist of AMPA/KA receptors and a potent non-competitive AMPAR antagonist, respectively, indicating that theKA-evoked responses were mediated primarily through theactivation of AMPARs.
Calcium dynamics in response to KA application
To study the calcium dynamics in response to AMPA receptors’activation in the three groups of neurons, we performed voltage-clamp recordings associated with microfluorometry, utilizing acesium-based ATP-free pipette solution with the calcium-sensitivedye fura-2 added (see Materials and methods). In all tested neurons(control: n=40, SOD1: n=35 and G93A: n=39), the localapplication of KA (200 μM, 5–8 s) at the holding potential of−60 mVevoked an inward current that was associated to a transientincrease of [Ca2+]i (Fig. 2). These experiments were performed inthe presence of APV (50 μM), nifedipine (50 μM) and TTX (1 μM)in the external solution to prevent calcium entry via NMDAreceptors, voltage-gated calcium channels and glutamate releasefrom surrounding neurons that was action potential-dependent,respectively. Under these experimental conditions, calcium ions canenter into the cell specifically through calcium-permeable AMPAreceptors (Ca-AMPARs). Calcium signals (Fig. 2A) reached a peakwithin 18.5±1.5 s in controls (n=16), 16.8±1.3 s in SOD1 (n=9)and 19.8±1.3 s in G93A (n=16) motor neurons from the start of KAperfusion. These values were not significantly different (pN0.05).
Fig. 1. Immunoblot analysis and cultured motor neurons on day 9 in vitro. (A) Levels of human wild type and mutant (G93A) SOD1 enzyme in transgenic mice.Total tissue extracts from mouse tail were fractionated on 10% SDS–polyacrylamide gels and electroblotted to nitrocellulose. SOD1 was detected with a rabbitpolyclonal anti-SOD1 antibody (1:1000). Amounts of loaded proteins were controlled for homogeneity by probing the membrane with an anti β-actinmonoclonal antibody. Results show a representative experiment that was repeated twice. No difference in the amount of human SOD1 is detectable in SOD1 andmutant SOD1 (G93A) transgenic mice. (B) Phase-contrast micrograph of a dissociated spinal cord culture. (C) Cell immunolabeling by SMI-32, identifying amotor neuron. Scale bar=50 μm. (D) Examples of whole-cell currents evoked by 200 μM kainate administration in murine naïve (control), wild type humanSOD1-overexpressing (SOD1), and mutant human SOD1-overexpressing (G93A) motor neurons under voltage-clamp conditions (Vholding=−60 mV). Theinward currents were reversibly blocked by the selective antagonist GYKI53655 (100 μM).
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The inward currents induced by KA perfusion, in the threepopulations of neurons, promptly initiated and terminated uponwash-in and wash-out of agonist, respectively (Fig. 2B).
Fig. 3A shows the mean of the [Ca2+]i in response to KAperfusion in control, SOD1 and G93A motor neurons (n=16, 9 and16, respectively). Basal [Ca2+]i before KA application was com-parable in control and G93A groups, whereas SOD1 motor neuronsshowed significantly higher resting [Ca2+]i than control (one-wayANOVA followed by Bonferroni’s test, F=6.99, df=38, pb0.01)and G93A (pb0.001, Fig. 3B). The amplitude of KA-evoked cal-cium transients, calculated as the difference between the peak andthe basal calcium level (Δ[Ca2+]i), was also similar in all groups(Fig. 3C). Accordingly, the ratio Δ[Ca2+]i / (current area) was alsonot statistically significant (pN0.05, Fig. 3D). Using the β, Rmin
and Rmax values determined in our experimental condition, thebasal calcium concentration for SOD1 motor neurons (164.23±23 nM) was significantly higher compared to control (100.36±13.07 nM, pb0.03) and G93A (90.13±12.32 nM, pb0.01) motorneurons (F=5.63, df=38). Analogously, the Δ[Ca2+]i was notsignificantly different between control (136.24±33.31 nM), SOD1(258.48±70.58 nM) and G93A (126.29±21.4 nM) motor neurons.
These results support our previous finding obtained with histo-chemical labeling for KA-stimulated cobalt uptake and withelectrophysiological experiments showing that AMPA receptorsare permeable to calcium in the motor neurons and that calciumpermeability through Ca-AMPARs is not augmented in G93Aneurons compared to SOD1 and control motor neurons (Pieri et al.,2003a).
Slower recovery to basal level of calcium accumulation in G93Amotor neurons following AMPA receptors’ activation but notvoltage-dependent calcium channels’ activation
The analysis of the time course of KA-induced calcium tran-sients revealed a significant increase in the duration of the calciumsignal in G93A motor neurons compared to the other groups.Indeed, [Ca2+]i fully recovered to baseline (dotted lines) in controland SOD1 motor neurons after KA application, whereas in G93A itremained higher than baseline level at the time point when it fullyreturned to basal level in control and SOD1 neurons (approximately60 s, Fig. 3A). Thus, the mean value of [Ca2+]i, calculated at thesame time point of 3 min after KA perfusion in G93A motor
Fig. 2. Increase of [Ca2+]i in control, SOD1 and G93A motor neurons in response to 200 μM kainate application. (A) Time course of [Ca2+]i in representativecontrol, SOD1 and G93A motor neurons (9, 10, and 9 days old in culture, respectively) in response to AMPA receptors' activation. (B) Corresponding inwardcurrents elicited by perfusion of 200 μM kainate in control, SOD1 and G93A motor neurons shown in upper panel. APV (50 μM), nifedipine (50 μM) and TTX(1 μM) were present in the bath.
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neurons, was significantly higher than [Ca2+]i before KA applica-tion (paired t-test, pb0.02, Fig. 4A). This finding is supported alsoby measuring the time necessary to reduce the calcium signal to 1/3of its maximum value. In fact, this time constant was significantlylonger for G93A motor neurons (30.9±7.3 s, n=11) compared tocontrol (9.1±1.7 s, n=11) and SOD1 (9.1±2.7 s, n=6; one-wayANOVA followed by Bonferroni’s test, F=6.56, df=25, Fig. 4B).All these data suggest that G93A motor neurons have a lowercapacity to handle KA-induced calcium increases than the othergroups.
To assess if the observed differences in the time-course of thecalcium transients in the G93A motor neurons compared to controland SOD1motor neurons were due to a different functionality of thesarcoplasmatic reticulum/endoplasmatic reticulum calcium ATPase(SERCA) pump, we performed experiments (n=3 for each group)in the presence of cyclopiazonic acid (CPA, 30 μM), a selectiveinhibitor of the SERCA (Seidler et al., 1989). In this experimentalcondition, no differences in the [Ca2+]i amplitude were stillobserved in the three groups of neurons, and the difference in therecovery kinetic of the calcium transient in G93A motor neuronscompared to the control and SOD1 neurons was still present (datanot shown). These data indicates that KA-induced Ca2+ transientsare independent from Ca2+ release from endoplasmatic reticulum
(ER) and that the ER ATPase pumps are not involved in theobserved different time courses of the calcium concentrationfollowing the Ca2+ entry through AMPARs.
To assess if G93A motor neurons can recover the [Ca2+]iincrease mediated by voltage-gated calcium channels’ activation,we measured the amplitude and kinetic of the calcium transientsobtained by a depolarizing voltage command, from −60 to−10 mV for 25 s in the three neuronal populations. These expe-riments, in voltage-clamp condition, were performed omittingnifedipine in the extracellular solution. As shown in Fig. 5, thecalcium transient was not significantly different for the threeneuronal populations but it was significantly bigger than thoseinduced by AMPA receptors’ activation (Figs. 5A, C). The basallevel was still higher in SOD1 compared to control and G93Amotor neurons (Fig. 5B), as has been observed in the calciumsignal induced by AMPA receptors’ activation (Fig. 3B). More-over, the decay time to the basal level of the calcium signal was notdifferent in the three populations (control: 9.51±1.54, n=10;SOD1: 10.94±1.44, n=7; G93A: 11.13±1.3, n=12) because allmotor neurons recovered to the basal level (Fig. 5D) with a similarkinetic (Fig. 5E). This observation suggests that expression of themutated human gene affects the ability to buffer calcium accu-mulations, occurring through AMPA receptors and not by voltage-
Fig. 3. Mean kinetic of [Ca2+]i in motor neurons from the three neuronal populations. (A) Time courses of [Ca2+]i in response to kainate perfusion (200 μM) incontrol (n=16), SOD1 (n=9) and G93A (n=16) motor neurons. All neurons were clamped at −60 mVand were 8–12 days old in culture. (B) Mean values of thebasal [Ca2+]i before KA application in control (0.83±0.06), SOD1 (1.16±0.08) and G93A (0.79±0.06) motor neurons. The value for SOD1 neurons wassignificantly different compared to control (pb0.01) and G93A (pb0.001) motor neurons. (C) The values of the difference between peak and basal calcium level(Δ[Ca2+]i) were not statistically different in control (0.52±0.09), SOD1 (0.73±0.15) and G93A (0.53±0.07) motor neurons. (D) Δ[Ca2+]i divided by currentarea induced by kainate in control (0.028±0.003), SOD1 (0.027±0.005) and G93A (0.03±0.005) motor neurons were still not statistically different.
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activated calcium channels, whereas expression of the wild geneincreases the [Ca2+]i basal level.
To verify whether the observed altered recovery to basal level ofcalcium is observable also in other AMPA receptor-expressingneurons from G93A mice, we performed experiments on culturedcortical neurons. In these neurons, our preliminary data indicate thatthe values of [Ca2+]i before (basal, 72.46±6.16 nM, n=4) and afterthe AMPA receptor activation (post puff, 3 min, 83.42±8.68 nM),were not significantly different in G93A cortical neurons (pN0.05,paired t test). Also, the [Ca2+]i values before (basal: 63.06±7.34 nM, n=5) and after (post depolarization, 3 min, 69.1±9.76 nM) voltage-dependent calcium channel activation (pN0.05,paired t test) were not significantly different. These data seem toindicate that the impairment of calcium buffering following AMPAreceptor activation is only linked to spinal motor neurons.
Discussion
The etiology of the selective motor neuron loss in ALS has yet tobe elucidated. One of the most widely accepted pathogenetic hypo-theses is a disturbance in the glutamatergic neurotransmitter system(Rothstein et al., 1990, 1992, 1993, Rothstein, 1996; Caramia et al.,2000; Plaitakis and Caroscio, 1987; Hugon et al., 1989a; Leigh and
Meldrum, 1996; Shaw and Ince, 1997). In particular, it has beenproposed that excitotoxic cell damage arises as a consequence ofexcessive intracellular calcium ions entering cells via AMPARs(Carriedo et al., 1998,2000) because kainate exposures preferen-tially injure motor neurons both in vivo (Hugon et al., 1989b) and invitro (Carriedo et al., 1995, 1996; Rothstein and Kuncl, 1995). Inaddition, AMPA/kainate receptor antagonists protect against motorneuron degeneration caused by a chronic blockade of glutamateuptake in both spinal cord slice cultures (Rothstein et al., 1993) anddissociated cultures (Carriedo et al., 1996).
AMPA receptors, major mediators for fast excitatory neurotrans-mission in the mammalian central nervous system, are composed ofa heteromeric complex of four subunits GluR1–GluR4, and theabsence of GluR2 renders the receptors Ca2+-permeable (Hollmannand Heinemann, 1994). However, motor neurons in ALS patientsshow no differences in the expression levels of GluR2 mRNA incomparison to a control group (Kawahara et al., 2003) and, inaddition, previous papers from our laboratory have shown thatneither the AMPARs calcium permeability is increased (Pieri et al.,2003b), nor the GluR2 mRNA is modified in a mouse model offamilial ALS compared to control mice (Spalloni et al., 2004). It isconsequently unclear and still remains to be elucidated whether andin what manner the AMPA receptors’ activation in the ALSpathology can lead to motor neurons degeneration and death.
Fig. 4. Analysis of the [Ca2+]i kinetic in response to AMPA receptor activation. (A) Bar graph: The values of [Ca2+]i before (basal) and after (post puff, 3 min) theAMPA receptor activation were significantly different in G93A motor neurons (pb0.02) and not in control and SOD1 neurons. Values were taken at the timepoints indicated in the inset. (B) Bar graph: Decay time values of the [Ca2+]i signals in G93A neurons (30.89±7.3) were statistically higher compared to control(9.1±1.77; pb0.02) and SOD1 (9.1±2.7; pb0.05) motor neurons. Decay time values were calculated as indicated in the inset.
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Therefore, the objective of this study has been to investigate thecalcium homeostasis in control, SOD1 and G93A motor neuronsunder physiological stimulation of AMPA receptors and voltage-dependent Ca2+ channels, using calcium imaging techniques andpatch-clamp recordings, simultaneously. The reported data showthat an altered homeostasis of calcium occurs in G93A motorneurons following AMPA receptors’ activation but not voltage-dependent calcium channels’ activation. In addition, in SOD1motor neurons, the basal level of [Ca2+]i is higher compared tocontrol and G93A.
The evidence that the basal intracellular concentration of cal-cium ions in neurons overexpressing the human SOD1 enzyme isabout 50% higher compared to control and G93A neurons remainsto be explained. However, the link between the cellular overex-pression of hSOD1 and the alterations of different cellularproperties reported by us and other authors is still not entirelyclear (Levkovitz et al., 1999; Celsi et al., 2004; Pieri et al., 2003b;Spalloni et al., 2004).
It is known that the route of Ca2+ entry and the intracellularlocalization of Ca2+ microdomains give rise to the activation ofdistinct biochemical signaling pathways that mediate independentphysiological responses (Ghosh and Greenberg, 1995; Bading etal., 1993). Previous papers have shown that Ca-mediated neuro-toxicity requires distinct signaling pathways to be triggered incells, and such pathways are more efficiently triggered when Ca2+
ions enter neurons at specific entry points, particularly at Ca-permeable glutamate receptors (Tymianski et al., 1993; Sattler etal., 1998). In fact, it has been reported that Ca2+ loading through
L-type voltage-sensitive Ca2+ channels was not harmful, whereassimilar Ca2+ loads produced via NMDA receptors were highlyneurotoxic (Tymianski et al., 1993; Sattler et al., 1998). Theseresults suggested that rate-limiting enzymes or substrates respon-sible for excitotoxicity must be co-localized with NMDAreceptors. Analogous mechanisms may therefore also participatein toxicity produced by other glutamate receptor subtypes, espe-cially AMPA receptors. Our reported results, in fact, are inagreement with this previous evidence. In fact, we showed that therecovery to basal level of the calcium increase, following thevoltage-activated Ca2+ channels, is similar in control, SOD1 andG93A motor neurons, whereas the recovery to basal level of thecalcium concentration when AMPA receptors were activated, issignificantly slower in G93A motor neurons. The abnormalrecovery to basal level of calcium concentration following AMPAreceptors’ activation in G93A motor neurons, does not seem to bedependent on the modified levels of calcium binding proteinsshown by previous papers. In fact, specifically in our mouse modelof ALS, a higher expression of calbindin in the resistant motorneurons (Knirsch et al., 2001), a severe loss of parvalbuminimmunoreactivity and an absence of calbindin D-28k immunor-eactivity has been reported (Sasaki et al., 2006). Since our dataindicate a not modified recovery to basal level of calcium con-centration following voltage-dependent calcium channels’ activa-tion, it is plausible that the altered expression of these proteins isnot directly involved in our reported evidences. Moreover, theobserved impairment of calcium homeostasis seems to be specificto the motor neurons. This is because in cultured cortical neurons,
Fig. 5. [Ca2+]i increase following depolarization in motor neurons. (A) Mean values of [Ca2+]i variations following membrane depolarization in voltage-clampedmotor neurons (control, SOD1 and G93A) from an holding potential of −60 mV to −10 mV for 25 s (top trace). (B) Basal values of [Ca2+]i in control (0.72±0.05,n=10), SOD1 (1.02±0.08, n=7) and G93A (0.72±0.045, n=12) motor neurons. The value for SOD1 motor neuron is still significantly different compared tocontrol (pb0.02) and G93A (pb0.02, one-way ANOVA followed by Bonferroni's test, F=7.7, df=26) motor neuron. (C) Amplitudes of [Ca2+]i calculated as thedifference between peak and basal calcium level (Δ[Ca2+]i) were not statistically different in control (1.73±0.28), SOD1 (1.5±0.24) andG93A (1.72±0.19)motorneurons. (D) The values of [Ca2+]i before and after (3 min) the calcium channels' activation were not significantly different in control, SOD1 and G93A motorneurons. (E) Decay time values of the [Ca2+]i signals were not significantly different between control (9.51±1.54), SOD1 (10.94±1.44) and G93A (11.13±1.3)motor neurons.
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a similar recovery kinetic to basal level of calcium was observed,both following AMPA receptor activation and voltage-dependentcalcium channel activation.
These data, besides indicating a distinct calcium buffer pathwayin the cell following the activation of voltage-dependent calciumchannels and AMPA receptors, show that the route downstream toAMPA receptors is altered in G93A motor neurons and conse-quently this may explain the controversial involvement of AMPAreceptors in the ALS pathology.
Each AMPA receptor subunit has four hydrophobic membranespanning domains with an extracellular N-terminal domain and acytoplasmically disposed C-terminal tail (Hollmann and Heine-mann, 1994). These subunits also show divergence from each otherin the sequence and length of their C-terminal tail, a region that iscurrently understood to mediate interactions with a variety ofintracellular proteins. These interactions are considered to beparamount in regulating AMPA receptor functioning, targeting andtrafficking (Kim and Huganir, 1999; Garner et al., 2000; Scannevinand Huganir, 2000).
For example, the common C-terminal sequence (t-SVKI)shared by both GluR2 and GluR3 interacts with glutamatereceptor-interacting proteins 1 an 2 (GRIP1 and GRIP2; Kimand Sheng, 2004). In particular, it has been recently reported thatthe protein alsin interacts with GRIP1 both in vitro and in vivo,and colocalizes with GRIP1 in neurons (Lai et al., 2006).Interestingly, a form of juvenile onset autosomal recessive ALS(ALS2) has been linked to the loss of function of the ALS2 genewhich encodes for the alsin (Hadano et al., 2001, Yang et al.,2001), thus indicating a link between this form of ALS and theglutamate receptor interacting proteins. Still more interestingly, anincreased susceptibility to glutamate receptor-mediated excitotoxi-city in spinal motor neurons from ALS2 knock-out mice has beenreported (Lai et al., 2006). Since interactions between AMPAreceptors and submembrane proteins are salient to AMPA recep-tor-mediated physiological signaling, the hypothesis that AMPA-interacting proteins could have an important role in mediatingexcitotoxicity is highly plausible and our data validate thishypothesis.
It is then reasonable to hypothesize that some type of proteininteracting with AMPA receptors, or some series of sequences oforganelles activated following the AMPA activation, and involvedin regulating calcium homeostasis, is altered in G93A motorneurons. In fact, although our data do not exclude the role for Ca2+
ions in AMPA receptor-mediated excitotoxicity (Iihara et al.,2001), they strongly suggests that Ca2+ permeability is not the soledeterminant of neurotoxic vulnerability. One possibility is thatAMPA receptor-mediated ion fluxes could be coupled to down-stream neurotoxic second messengers via interactions with sub-membrane proteins.
In conclusion, we have shown that the expression of themutated human gene G93A affects the ability to buffer calciumaccumulations, occurring through AMPA receptors’ activation andnot through voltage-activated calcium channels in a mouse modelof ALS and that this impairment seems to be specific to motorneurons. These data indicate an involvement of the AMPA-relatedsubmembrane proteins and, consequently, the molecular character-ization of the signal–transduction pathways linked to the AMPAreceptors’ activation may open new prospects to the ALS studies.These results may also provide targets for future pharmacologicaltherapies for ALS disease as well as for other neurodegenerativediseases.
Acknowledgments
This research was supported by grants from the MiUR (Firb:RBAU01A7T) and the Telethon Foundation (No. 1185) to CZ.
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