Complement upregulation and activation on motor neurons and neuromuscularjunction in the SOD1 G93A mouse model of familial amyotrophic lateral sclerosis☆
Bianca Heurich a, Nawal Bahia el Idrissi c, Rossen M. Donev a, Susanne Petri d, Peter Claus e, James Neal b,B. Paul Morgan a, Valeria Ramaglia a,c,⁎a Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff, UKb Department of Histopathology, School of Medicine, Cardiff, UKc Department of Genome Analysis, Academic Medical Centre, Amsterdam, The Netherlandsd Department of Neurology, Hannover Medical School, Germanye Institute of Neuroanatomy, Hannover Medical School, Germany
a b s t r a c ta r t i c l e i n f o
Article history:Received 18 October 2010Received in revised form 22 February 2011Accepted 28 March 2011
Complement activation products are elevated in cerebrospinal fluid, spinal cord and motor cortex of patientswith amyotrophic lateral sclerosis (ALS) but are untested in models. We determined complement expressionand activation in the SOD1 G93A mouse model of familial ALS (fALS). At 126 days, C3 mRNA was upregulatedin spinal cord and C3 protein accumulated in astrocytes and motor neurons. C3 activation products C3b/iC3bwere localized exclusively on motor neurons. At the neuromuscular junction, deposits of C3b/iC3b and C1qwere detected at day 47, before the appearance of clinical symptoms, and remained detectable atsymptomatic stage (126 days). Our findings implicate complement in the denervation of the muscle endplateby day 47 and destruction of the neuromuscular junction and spinal neuron loss by day 126 in the SOD1 G93Amouse model of fALS.
Amyotrophic lateral sclerosis (ALS), the most common adult-onsetmotor neuron disease (Pasinelli and Brown, 2006), is characterized byprogressive degeneration of both upper and lower motor neurons,leading to muscle atrophy and eventually death from respiratoryparalysis (Mitchell and Borasio, 2007). With rare exceptions, thecause of disease is unknown and the mechanism of motor neuroninjury occult. Most ALS cases (90%) are sporadic (sALS) while 10% arefamilial (fALS); 15–20% of these are caused by mutations in copper/zinc superoxide dismutase 1 (SOD1) (Rosen et al., 1993). Transgenicmice expressing the commonest of these mutations, SOD1 G93A,develop a pathological and clinical phenotype resembling human ALS(Gurney, 1994a).
Complement (C), a key component of innate immunity, has thecapacity to cause damage to self and is consequently implicated inmany diseases (Walport, 2001a; Walport, 2001b). A role for C in thepathogenesis of ALS in humans is suggested by the presence of Cactivation products, including C3c, C3d, C4d and C3dg, in spinal cord
and motor cortex, and in elevated concentrations in serum and CSF(Annunziata and Volpi, 1985; Apostolski et al., 1991; Tsuboi andYamada, 1994; Goldknopf et al., 2006). In murine ALS models,upregulation of C1q and C4 in motor neurons (Lobsiger et al., 2007;Ferraiuolo et al., 2007), and C3 upregulation in the anterior horn areascontaining motor neuron degeneration (Woodruff et al., 2008), aredescribed. Surprisingly, C deposition at the neuromuscular junction(NMJ) and motor end plate (MEP), principal sites of degeneration inhuman and mouse ALS (Fischer et al., 2004), has not been reported.We examined expression, localization and activation of C3 in spinalcord and MEP, and C1q deposition at MEP, in the SOD1 G93A mousemodel of fALS at presymptomatic (47 days) and symptomatic(126 days) stages of disease progression.
2. Materials and methods
2.1. Animals
G93A transgenic familial ALS mice [high copy number; B6SJLTg(SOD1-G93A)1Gur/J] (Gurney, 1994b) and wildtype (B6SJL) litter-mates were free of microbiological infection (FELASA screened). Micewere housed in groups at 20 °C on 12:12 h light:dark cycle, with freeaccess to food and water. Experimental protocols complied withnational animal care guidelines, licensed by the responsible authority.
Journal of Neuroimmunology 235 (2011) 104–109
☆ This study was supported by the Wellcome Trust VIP award no. 084542 to VR andthe Wellcome Trust Programme Grant no. 068590 to BPM.⁎ Corresponding author at: Valeria Ramaglia, Department of Infection, Immunity and
Biochemistry, School of Medicine, Cardiff, UK.E-mail address: [email protected] (V. Ramaglia).
j ourna l homepage: www.e lsev ie r.com/ locate / jneuro im
2.2. Tissue processing
SOD1 G93A and wildtype mice were killed at post-natal day 47(presymptomatic stage SOD1 G93A n=5; wildtype n=5) and post-natal day 126 (symptomatic stage SOD1 G93A n=7; wildtype n=5)by CO2 inhalation. Spinal cord and gastrocnemius muscle weredissected, post-fixed overnight in 4% paraformaldehyde/PBS at 4 °C,cryoprotected in 30% sucrose/PBS for 72 h at 4 °C, then embedded inOCT (Sakura, Zoeterwoude, NL), frozen in liquid nitrogen and stored
at −80 °C until used for histology. A portion of spinal cord was freshfrozen for RNA analysis.
2.3. Molecular analyses
Total RNA was extracted from spinal cords using GeneEluteMammalian Total RNA Miniprep kits (Sigma-Aldrich, Dorset, UK).cDNAs were synthesized using TaqMan Reverse Transcription re-agents (Applied Biosystems, Warrington, UK). Reactions were run onthe Mini Opticon Taqman (Bio-Rad, Hemel Hempstead, UK) withSYBR GREEN Supermix and primer pairs described in Table 1. Ccomponent copy number was calculated using the comparative Ct(ΔΔCt) method (Donev and Morgan, 2006) with results normalizedagainst β-actin. At least two independent experiments in triplicatewere performed for each cDNA analyzed.
2.4. Immunohistochemistry
Transverse sections (7 μm) of lumbar spinal cord were fixed (coldacetone, 10 min), endogenous peroxidases were blocked in 0.03%H2O2/PBS (RT, 20 min), and non-specific binding sites blocked in 10%
Table 2Antibodies for immunohistochemistry.
Antibody Clone Source Concentration/dilution
Primary antibodiesMonoclonal rat anti-mouse C3 11H9 HyCult biotechnology (NL) 2 μg/mlMonoclonal rat anti-mouse iC3b/C3b/C3c 3/26 HyCult biotechnology 200 μg/mlMonoclonal mouse anti-mouse C1q JL-1 HyCult biotechnology 2 μg/mlMonoclonal mouse anti-mouse NeuN A60 Millipore (UK) 2 μg/mlPolyclonal rabbit anti-rat/mouse C9 Made in house 2 μg/mlRabbit polyclonal antiserum cocktail to neurofilaments Biotrend (UK) 1:80
Fig. 1. Complement C3, factor H, DAF and Crry mRNA relative levels in spinal cords of wildtype (n=5) and SOD1 G93A (n=7) mice at 126 days, showing up-regulation of C3 mRNAin SOD1 G93A spinal cord compared to wildtypes but no changes in the mRNA levels of fH, DAF and Crry regulators of the C3 convertase.
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normal goat serum (NGS)/PBS (RT, 20 min). Slides were incubatedwith appropriate primary antibodies (Table 2) diluted in 1% bovineserum albumin (BSA) (90 min, RT), followed by biotinylated second-ary antibody (Table 2) in 1% BSA (30 min, RT), and peroxidase–polystreptavidin (Sigma-Aldrich; 20 min RT) diluted 1:400 in 1% BSA.Controls included irrelevant antibody of identical isotype andomission of primary antibody. To visualize peroxidase activity, slideswere incubated in 3,3-diaminobenzidine tetrahydrochloride (DAB;Peroxidase Substrate Kit, VectorLabs, Peterborough, UK; 3 min) thencounterstained with hematoxylin. Slides were dehydrated in ethanoland mounted in Pertex (Histolab, Gothenburg, Sweden).
For fluorescent immunostaining, sectionswere pre-incubatedwithImage-iT FX Signal Enhancer (Invitrogen, Renfrew, UK; 0.1 ml, 30 min,RT), then with mouse anti-NeuN in PBS/BSA , followed by rat anti-C3antibody (each diluted in 1% BSA, incubated 60 min, RT). Boundantibody was detected using the mouse-on-mouse ImmunodetectionKit (Vector) according to manufacturer's instructions, followedsequentially by FITC-labeled polystreptavidin (Sigma-Aldrich; 1:400in PBS/BSA) and rhodamine (TRITC)-conjugated goat anti-rat immu-
noglobulin (Table 2). Slides were counterstained with Dapi (Sigma;1:1000 in PBS/BSA) and mounted in anti-fade medium (Fluor Save™,Calbiochem, Nottingham, UK).
For confocal microscopy, 40 μm muscle sections were permeabi-lized with 1% TritonX-100 in PBS and blocked with 5% BSA for 1 h atRT, then incubated with primary and secondary antibodies (Table 2;each overnight at RT). End-plates were labeled with Alexa Fluor®647-conjugated anti-α-bungarotoxin (Invitrogen; 5 μg/ml in PBS/BSA, 1 h, RT). Sections were mounted as above and images capturedwith a digital camera attached to a light/fluorescent microscope (DMLB2, Leica Microsystems, Bucks, UK) or from a confocal imagingsystem (TCS SP2, Leica).
2.5. Quantitative analysis of immunohistochemistry
Quantitative analysis of C3 immunostaining was performed usingImage Pro Plus 6.00 (Media Cybernatics, Wokingham, UK). Four non-consecutive sections of spinal cord were scored for each animal in
Fig. 2. (A–C) Representative C3 immunostaining of ventral horn of the spinal cord in wildtype (A) (n=5) and SOD1 G93A (B–C) (n=7) at 126 days, showing traceC3 immunoreactivity in wildtypes contrasting with abundant C3 immunoreactivity in the SOD1 G93A cord localizing with astrocytes (C, arrows) and neurons (C, arrowheads). (D–F)Double immunofluorescent staining of NeuN and C3, showing co-localization in the SOD1 G93A cord at 126 days (D–F, arrows and F, in yellow). Note C3-positive/NeuN-negativeastrocytes. (G,H) Representative C3b/iC3b immunostaining of spinal cord ventral horn showing blood vessel immunoreactivity in the wildtype (G, arrow head), contrasting withstrong neuronal immunoreactivity in SOD1 G93A at 126 days (H, arrow head). (I) Quantification of C3 immunoreactive area, showing higher level in SOD1 G93A spinal cordscompared to wildtypes at 126 days. Data represent mean±SD. Statistical significance is for p≤0.05.
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each group. Percentage immunoreactive area per section was scoredand expressed as mean±SD.
2.6. Statistical analysis
Two tailed t test was performed for the analysis of qPCR and C3immunoreactivity data. Statistical significance was accepted whenp≤0.05.
3. Results
C component expression is not up-regulated in the spinal cord ofthe SOD1 G93A mouse at presymptomatic stage, but is up-regulatedin the late symptomatic stage (Ferraiuolo et al., 2007; Woodruff et al.,2008). Here we determined whether C3 up-regulation at symptom-atic stage, day 126, was also paralleled by C3 activation in the spinalcord.
Fig. 3. Representative confocal microscopy images of the NMJ from wildtype (n=10) and SOD1 G93A mice (n=12) at 47 (A–P) and 126 days (Q–AF), immunostained for C3b/iC3b(A–H; Q–X) and C1q (I–P; Y–AF), showing deposition of C3b/iC3b (G andW) and C1q (O and AE arrows) on themuscle end-plate and nerve terminals (O, arrows) in SOD1G93Amice.The nerve terminal is labeledwith polyclonal antiserum to neurofilaments (A, E, I, M, Q, U, Y, and AC in red). Themuscle end-plate is labeledwithα-bungarotoxin (B, F, J, N, R, V, Z, andAD in magenta).
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Expression of mRNA encoding C3 and C3 convertase regulators wasmeasured in spinal cords from 126 days old SOD1 G93A and wildtypemice. C3 mRNA was elevated 12-fold in SOD1 G93A mice compared towildtype (p≤0.05), whereas expression of the C3 convertase regulatorsCfH, DAF and Crry were not different (Fig. 1). Abundant C3 proteinimmunoreactivity was detected in SOD1 G93A mice in neurons andastrocytes in the ventral horn of the spinal cord, while wildtype cordshowed only faint neuronal staining (Fig. 2A-C and I, pb0.05). Neuronallocalization was confirmed by co-staining with NeuN (Fig. 2D-F). C3activation product immunoreactivity, detected with the C3b/iC3b/C3c-specific antibody (clone 3/26), was observed on ventral horn neuronsbut not astrocytes in SOD1G93A spinal cord;wildtype cord stained only
in blood vessels (Fig. 2G and H). Staining for membrane attack complex(MAC) using a proven anti-mouse C9 antibody, was consistentlynegative in all spinal cord sections (not shown).
C deposition and activation have never been tested at the NMJ ofSOD1 G93Amice. Therefore we examined the NMJ/MEP of SOD1 G93Amice for C deposition and activation at the presymptomatic (47 days)and symptomatic (126 days) stages. NMJ/MEP was detected bydouble immunofluorescent staining with anti-neurofilament (detectsnerve terminals) and α-bungarotoxin (detects MEP). Confocalmicroscopy showed abundant C3b/iC3b immunoreactivity in SOD1G93Amuscle, co-localized with degenerated MEP at post-natal day 47(Fig. 3A–H) and 126 (Fig. 3Q–X); C1q immunoreactivity was present
Fig. 3 (continued).
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at the MEP (Fig. 3O) and nerve terminals (Fig. 3O, arrows) at 47 daysand still detectable at the MEP edge at 126 days (Fig. 3AE–AF),implicating classical pathway activation, although IgG deposition wasabsent from this site. No MAC staining was detected in SOD1 G93Amuscle. In contrast, wildtypes displayed no C3b/iC3b or C1q or MACimmunoreactivity in muscle.
4. Discussion
The data show upregulation of C3mRNA and protein in spinal cordventral horn neurons and astrocytes, and C3 activation productdeposition restricted to ventral horn neurons in the 126 days SOD1G93A fALS model. C3 activation products and C1q were also depositedon the denervated and degenerated SOD1 G93A MEP at 47 and126 days.
Motor neuron pathology in the SOD1 G93A mouse begins distallywith denervation of NMJ by day 47, followed by motor axon lossbetween days 47–80, and loss of lumbar cord neuronal cell bodiesafter day 80 (Fischer et al., 2004). Axons express C components butlack C regulators (de Jonge et al., 2004), rendering them vulnerable toC attack (Ramaglia et al., 2007). Presynaptic neurons and perisynapticSchwann cells are also sensitive to C attack and undergo C-mediateddamage in peripheral neuropathy models (O'Hanlon et al., 2001;Halstead et al., 2004). C3 fragment and C1q deposition at thedenervated MEP in SOD1 G93Amice implicate the classical C pathwayin degeneration of distal axons. A novel role for C1q/C3 in selectiveelimination of synaptic connections during development was recentlydescribed (Stevens et al., 2007). Developmental elimination ofsynapses involves tagging by C3 fragments and phagocytosis byresident macrophages; our data suggest that this process is mimickedat the NMJ in the SOD1 G93Amouse, leading to synapse degeneration.
Although C3 biosynthesis was up-regulated in bothmotor neuronsand astrocytes in SOD1 G93A mice, C3 activation products depositonly on motor neurons, driving their loss. Neighboring astrocytesresist C3 activation because they abundantly express C3 convertaseregulators (Griffiths et al., 2009), but contribute to neuronal loss byincreasing local synthesis of C components. Both neurons andastrocytes must express mutant SOD1 for development of ALSpathology (Wang et al., 2005; Gong et al., 2000; Pramatarova et al.,2001). If expression of the mutant SOD1 directly or indirectly drivesup-regulation of C component synthesis, then it may be that local Cbiosynthesis only breaches the threshold necessary for neuronaldamage when both neurons and astrocytes are involved.
Treatment with C inhibitors protects from early axonal degener-ation and facilitates regeneration and recovery in peripheral nerveinjury (Ramaglia et al., 2009). Because C is deposited and activated atthe NMJ before denervation, and axonal loss precedes motor neuronloss in ALS (Fischer et al., 2004), early intervention with C inhibitorsmay protect the NMJ and stop further neuronal degeneration, offeringthe prospect of therapy for this currently untreatable disorder.
Acknowledgments
We thank Dr. J. Verhaagen for kindly providing the mouse tissuefor this study.
References
Annunziata, P., Volpi, N., 1985. High levels of C3c in the cerebrospinal fluid fromamyotrophic lateral sclerosis patients. Acta Neurol. Scand. 72, 61–64.
de Jonge, R.R., van Schaik, I.N., Vreijling, J.P., Troost, D., Baas, F., 2004. Expression ofcomplement components in the peripheral nervous system. Hum. Mol. Genet. 13,295–302.
Donev, R.M., Morgan, B.P., 2006. A quantitative method for comparison of expression ofalternatively spliced genes using different primer pairs. J. Biochem. Biophys.Methods 66, 23–31.
Ferraiuolo, L., Heath, P.R., Holden, H., Kasher, P., Kirby, J., Shaw, P.J., 2007. Microarrayanalysis of the cellular pathways involved in the adaptation to and progression ofmotor neuron injury in the SOD1 G93Amouse model of familial ALS. J. Neurosci. 27,9201–9219.
Fischer, L.R., Culver, D.G., Tennant, P., Davis, A.A., Wang, M., Castellano-Sanchez, A.,Khan, J., Polak, M.A., Glass, J.D., 2004. Amyotrophic lateral sclerosis is a distalaxonopathy: evidence in mice and man. Exp. Neurol. 185, 232–240.
Goldknopf, I.L., Sheta, E.A., Bryson, J., Folsom, B., Wilson, C., Duty, J., Yen, A.A., Appel, S.H.,2006. Complement C3c and related protein biomarkers in amyotrophic lateralsclerosis and Parkinson's disease. Biochem. Biophys. Res. Commun. 342, 1034–1039.
Gong, Y.H., Parsadanian, A.S., Andreeva, A., Snider, W.D., Elliott, J.L., 2000. Restrictedexpression of G86R Cu/Zn superoxide dismutase in astrocytes results inastrocytosis but does not causemotoneuron degeneration. J. Neurosci. 20, 660–665.
Griffiths, M.R., Neal, J.W., Fontaine, M., Das, T., Gasque, P., 2009. Complement factor H, amarker of self protects against experimental autoimmune encephalomyelitis. J.Immunol. 182, 4368–4377.
Gurney, M.E., 1994a. Transgenic-mouse model of amyotrophic lateral sclerosis. N. Engl.J. Med. 331, 1721–1722.
Gurney, M.E., 1994b. Transgenic-mouse model of amyotrophic lateral sclerosis. N. Engl.J. Med. 331, 1721–1722.
Halstead, S.K., O'Hanlon, G.M., Humphreys, P.D., Morrison, D.B., Morgan, B.P., Todd, A.J.,Plomp, J.J., Willison, H.J., 2004. Anti-disialoside antibodies kill perisynapticSchwann cells and damage motor nerve terminals via membrane attack complexin a murine model of neuropathy. Brain 127, 2109–2123.
Lobsiger, C.S., Boillee, S., Cleveland, D.W., 2007. Toxicity from different SOD1 mutantsdysregulates the complement system and the neuronal regenerative response inALS motor neurons. Proc. Natl. Acad. Sci. U. S. A 104, 7319–7326.
Pasinelli, P., Brown, R.H., 2006. Molecular biology of amyotrophic lateral sclerosis:insights from genetics. Nat. Rev. Neurosci. 7, 710–723.
Pramatarova, A., Laganiere, J., Roussel, J., Brisebois, K., Rouleau, G.A., 2001. Neuron-specific expression of mutant superoxide dismutase 1 in transgenic mice does notlead to motor impairment. J. Neurosci. 21, 3369–3374.
Ramaglia, V., King, R.H., Nourallah, M., Wolterman, R., de Jonge, R., Ramkema, M., Vigar,M.A., Van der, W.S., Morgan, B.P., Troost, D., Baas, F., 2007. The membrane attackcomplex of the complement system is essential for rapid Wallerian degeneration. J.Neurosci. 27, 7663–7672.
Ramaglia, V., Tannemaat, M.R., de, K.M., Wolterman, R., Vigar, M.A., King, R.H., Morgan,B.P., Baas, F., 2009. Complement inhibition accelerates regeneration in a model ofperipheral nerve injury. Mol. Immunol. 47, 302–309.
Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D.,Goto, J., O'Regan, J.P., Deng, H.X., 1993. Mutations in Cu/Zn superoxide dismutasegene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62.
Tsuboi, Y., Yamada, T., 1994. Increased concentration of C4d complement protein in CSFin amyotrophic lateral sclerosis. J. Neurol. Neurosurg. Psychiatry 57, 859–861.
Walport, M.J., 2001a. Complement. First of two parts. N. Engl. J. Med. 344, 1058–1066.Walport, M.J., 2001b. Complement. Second of two parts. N. Engl. J. Med. 344,
Woodruff, T.M., Costantini, K.J., Crane, J.W., Atkin, J.D., Monk, P.N., Taylor, S.M., Noakes,P.G., 2008. The complement factor C5a contributes to pathology in a rat model ofamyotrophic lateral sclerosis. J. Immunol. 181, 8727–8734.
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