Journal of Neuroimmunology 235 (2011) 104109

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Journal of Neuroimmunology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Short communication

Complement upregulation and activation on motor neurons and neuromuscular junction 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, UK Department of Histopathology, School of Medicine, Cardiff, UK c Department of Genome Analysis, Academic Medical Centre, Amsterdam, The Netherlands d Department of Neurology, Hannover Medical School, Germany e Institute of Neuroanatomy, Hannover Medical School, Germany
b

a r t i c l e

i n f o

a b s t r a c t
Complement activation products are elevated in cerebrospinal uid, spinal cord and motor cortex of patients with amyotrophic lateral sclerosis (ALS) but are untested in models. We determined complement expression and activation in the SOD1 G93A mouse model of familial ALS (fALS). At 126 days, C3 mRNA was upregulated in spinal cord and C3 protein accumulated in astrocytes and motor neurons. C3 activation products C3b/iC3b were localized exclusively on motor neurons. At the neuromuscular junction, deposits of C3b/iC3b and C1q were detected at day 47, before the appearance of clinical symptoms, and remained detectable at symptomatic stage (126 days). Our ndings implicate complement in the denervation of the muscle endplate by day 47 and destruction of the neuromuscular junction and spinal neuron loss by day 126 in the SOD1 G93A mouse model of fALS. 2011 Elsevier B.V. All rights reserved.

Article history: Received 18 October 2010 Received in revised form 22 February 2011 Accepted 28 March 2011 Keywords: Amyotrophic lateral sclerosis Complement Motor neurons Neuromuscular junction

1. Introduction Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease (Pasinelli and Brown, 2006), is characterized by progressive degeneration of both upper and lower motor neurons, leading to muscle atrophy and eventually death from respiratory paralysis (Mitchell and Borasio, 2007). With rare exceptions, the cause of disease is unknown and the mechanism of motor neuron injury occult. Most ALS cases (90%) are sporadic (sALS) while 10% are familial (fALS); 1520% of these are caused by mutations in copper/ zinc superoxide dismutase 1 (SOD1) (Rosen et al., 1993). Transgenic mice 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 the capacity to cause damage to self and is consequently implicated in many diseases (Walport, 2001a; Walport, 2001b). A role for C in the pathogenesis of ALS in humans is suggested by the presence of C activation 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 and Yamada, 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 areas containing motor neuron degeneration (Woodruff et al., 2008), are described. Surprisingly, C deposition at the neuromuscular junction (NMJ) and motor end plate (MEP), principal sites of degeneration in human and mouse ALS (Fischer et al., 2004), has not been reported. We examined expression, localization and activation of C3 in spinal cord and MEP, and C1q deposition at MEP, in the SOD1 G93A mouse model 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) littermates were free of microbiological infection (FELASA screened). Mice were housed in groups at 20 C on 12:12 h light:dark cycle, with free access to food and water. Experimental protocols complied with national animal care guidelines, licensed by the responsible authority.

This study was supported by the Wellcome Trust VIP award no. 084542 to VR and the 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: v.ramaglia@amc.uva.nl (V. Ramaglia). 0165-5728/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.03.011

B. Heurich et al. / Journal of Neuroimmunology 235 (2011) 104109 Table 1 Mouse Taqman primer sequences. Target gene C3 fH Crry DAF Accession no. NM_009778 NM_009888 NM_013499 NM_010016 Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Sequence 53 5-AAGCATCAACACACCCAACA-3 5-CTTGAGCTCCATTCGTGACA-3 5-GCACCCAGGCTACCTACAAA-3 5-AGATCCAACTGCCAGCCTAA-3 5-CCCATCACAGCTTCCTTCTG-3 5-CTTCAGCACTCGTCCAGGTT-3 5-CTTGCCTTGAGGATTTAGTATGG-3 5-CTAGCCTGTACCCTGGGTTG-3

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at 80 C until used for histology. A portion of spinal cord was fresh frozen for RNA analysis. 2.3. Molecular analyses Total RNA was extracted from spinal cords using GeneElute Mammalian Total RNA Miniprep kits (Sigma-Aldrich, Dorset, UK). cDNAs were synthesized using TaqMan Reverse Transcription reagents (Applied Biosystems, Warrington, UK). Reactions were run on the Mini Opticon Taqman (Bio-Rad, Hemel Hempstead, UK) with SYBR GREEN Supermix and primer pairs described in Table 1. C component copy number was calculated using the comparative Ct (Ct) method (Donev and Morgan, 2006) with results normalized against -actin. At least two independent experiments in triplicate were performed for each cDNA analyzed. 2.4. Immunohistochemistry Transverse sections (7 m) of lumbar spinal cord were xed (cold acetone, 10 min), endogenous peroxidases were blocked in 0.03% H2O2/PBS (RT, 20 min), and non-specic binding sites blocked in 10%

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 postnatal day 126 (symptomatic stage SOD1 G93A n = 7; wildtype n = 5) by CO2 inhalation. Spinal cord and gastrocnemius muscle were dissected, post-xed overnight in 4% paraformaldehyde/PBS at 4 C, cryoprotected in 30% sucrose/PBS for 72 h at 4 C, then embedded in OCT (Sakura, Zoeterwoude, NL), frozen in liquid nitrogen and stored
Table 2 Antibodies for immunohistochemistry. Antibody Primary antibodies Monoclonal rat anti-mouse C3 Monoclonal rat anti-mouse iC3b/C3b/C3c Monoclonal mouse anti-mouse C1q Monoclonal mouse anti-mouse NeuN Polyclonal rabbit anti-rat/mouse C9 Rabbit polyclonal antiserum cocktail to neurolaments Secondary antibodies Biotinylated-polyclonal goat anti-rat Alexa-Fluor 488-conjugated goat anti-rat Alexa-Fluor 488-conjugated goat anti-mouse Rhodamine (TRITC)-conjugated donkey anti-rabbit Clone 11H9 3/26 JL-1 A60

Source HyCult biotechnology (NL) HyCult biotechnology HyCult biotechnology Millipore (UK) Made in house Biotrend (UK)

Concentration/dilution 2 g/ml 200 g/ml 2 g/ml 2 g/ml 2 g/ml 1:80

Vector labs (UK) Invitrogen (UK) Invitrogen Jackson Immuno Research (UK)

7.5 g/ml 20 g/ml 20 g/ml 1 g/ml

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 mRNA in 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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Fig. 2. (AC) Representative C3 immunostaining of ventral horn of the spinal cord in wildtype (A) (n = 5) and SOD1 G93A (BC) (n = 7) at 126 days, showing trace C3 immunoreactivity in wildtypes contrasting with abundant C3 immunoreactivity in the SOD1 G93A cord localizing with astrocytes (C, arrows) and neurons (C, arrowheads). (DF) Double immunouorescent staining of NeuN and C3, showing co-localization in the SOD1 G93A cord at 126 days (DF, arrows and F, in yellow). Note C3-positive/NeuN-negative astrocytes. (G,H) Representative C3b/iC3b immunostaining of spinal cord ventral horn showing blood vessel immunoreactivity in the wildtype (G, arrow head), contrasting with strong neuronal immunoreactivity in SOD1 G93A at 126 days (H, arrow head). (I) Quantication of C3 immunoreactive area, showing higher level in SOD1 G93A spinal cords compared to wildtypes at 126 days. Data represent mean SD. Statistical signicance is for p 0.05.

normal goat serum (NGS)/PBS (RT, 20 min). Slides were incubated with appropriate primary antibodies (Table 2) diluted in 1% bovine serum albumin (BSA) (90 min, RT), followed by biotinylated secondary 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 and omission of primary antibody. To visualize peroxidase activity, slides were incubated in 3,3-diaminobenzidine tetrahydrochloride (DAB; Peroxidase Substrate Kit, VectorLabs, Peterborough, UK; 3 min) then counterstained with hematoxylin. Slides were dehydrated in ethanol and mounted in Pertex (Histolab, Gothenburg, Sweden). For uorescent immunostaining, sections were pre-incubated with Image-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-C3 antibody (each diluted in 1% BSA, incubated 60 min, RT). Bound antibody was detected using the mouse-on-mouse Immunodetection Kit (Vector) according to manufacturer's instructions, followed sequentially by FITC-labeled polystreptavidin (Sigma-Aldrich; 1:400 in 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 m muscle sections were permeabilized with 1% TritonX-100 in PBS and blocked with 5% BSA for 1 h at RT, then incubated with primary and secondary antibodies (Table 2; each overnight at RT). End-plates were labeled with Alexa Fluor 647-conjugated anti--bungarotoxin (Invitrogen; 5 g/ml in PBS/ BSA, 1 h, RT). Sections were mounted as above and images captured with a digital camera attached to a light/uorescent microscope (DM LB2, Leica Microsystems, Bucks, UK) or from a confocal imaging system (TCS SP2, Leica).

2.5. Quantitative analysis of immunohistochemistry Quantitative analysis of C3 immunostaining was performed using Image Pro Plus 6.00 (Media Cybernatics, Wokingham, UK). Four nonconsecutive sections of spinal cord were scored for each animal in

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each group. Percentage immunoreactive area per section was scored and expressed as mean SD. 2.6. Statistical analysis Two tailed t test was performed for the analysis of qPCR and C3 immunoreactivity data. Statistical signicance was accepted when p 0.05.

3. Results C component expression is not up-regulated in the spinal cord of the SOD1 G93A mouse at presymptomatic stage, but is up-regulated in the late symptomatic stage (Ferraiuolo et al., 2007; Woodruff et al., 2008). Here we determined whether C3 up-regulation at symptomatic stage, day 126, was also paralleled by C3 activation in the spinal cord.

Fig. 3. Representative confocal microscopy images of the NMJ from wildtype (n = 10) and SOD1 G93A mice (n = 12) at 47 (AP) and 126 days (QAF), immunostained for C3b/iC3b (AH; QX) and C1q (IP; YAF), showing deposition of C3b/iC3b (G and W) and C1q (O and AE arrows) on the muscle end-plate and nerve terminals (O, arrows) in SOD1 G93A mice. The nerve terminal is labeled with polyclonal antiserum to neurolaments (A, E, I, M, Q, U, Y, and AC in red). The muscle end-plate is labeled with -bungarotoxin (B, F, J, N, R, V, Z, and AD in magenta).

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Fig. 3 (continued).

Expression of mRNA encoding C3 and C3 convertase regulators was measured in spinal cords from 126 days old SOD1 G93A and wildtype mice. C3 mRNA was elevated 12-fold in SOD1 G93A mice compared to wildtype (p 0.05), whereas expression of the C3 convertase regulators CfH, DAF and Crry were not different (Fig. 1). Abundant C3 protein immunoreactivity was detected in SOD1 G93A mice in neurons and astrocytes in the ventral horn of the spinal cord, while wildtype cord showed only faint neuronal staining (Fig. 2A-C and I, p b 0.05). Neuronal localization was conrmed by co-staining with NeuN (Fig. 2D-F). C3 activation product immunoreactivity, detected with the C3b/iC3b/C3cspecic antibody (clone 3/26), was observed on ventral horn neurons but not astrocytes in SOD1 G93A 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 consistently negative in all spinal cord sections (not shown). C deposition and activation have never been tested at the NMJ of SOD1 G93A mice. Therefore we examined the NMJ/MEP of SOD1 G93A mice for C deposition and activation at the presymptomatic (47 days) and symptomatic (126 days) stages. NMJ/MEP was detected by double immunouorescent staining with anti-neurolament (detects nerve terminals) and -bungarotoxin (detects MEP). Confocal microscopy showed abundant C3b/iC3b immunoreactivity in SOD1 G93A muscle, co-localized with degenerated MEP at post-natal day 47 (Fig. 3AH) and 126 (Fig. 3QX); C1q immunoreactivity was present

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at the MEP (Fig. 3O) and nerve terminals (Fig. 3O, arrows) at 47 days and still detectable at the MEP edge at 126 days (Fig. 3AEAF), implicating classical pathway activation, although IgG deposition was absent from this site. No MAC staining was detected in SOD1 G93A muscle. In contrast, wildtypes displayed no C3b/iC3b or C1q or MAC immunoreactivity in muscle.

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4. Discussion The data show upregulation of C3 mRNA and protein in spinal cord ventral horn neurons and astrocytes, and C3 activation product deposition restricted to ventral horn neurons in the 126 days SOD1 G93A fALS model. C3 activation products and C1q were also deposited on the denervated and degenerated SOD1 G93A MEP at 47 and 126 days. Motor neuron pathology in the SOD1 G93A mouse begins distally with denervation of NMJ by day 47, followed by motor axon loss between days 4780, and loss of lumbar cord neuronal cell bodies after day 80 (Fischer et al., 2004). Axons express C components but lack C regulators (de Jonge et al., 2004), rendering them vulnerable to C attack (Ramaglia et al., 2007). Presynaptic neurons and perisynaptic Schwann cells are also sensitive to C attack and undergo C-mediated damage in peripheral neuropathy models (O'Hanlon et al., 2001; Halstead et al., 2004). C3 fragment and C1q deposition at the denervated MEP in SOD1 G93A mice implicate the classical C pathway in degeneration of distal axons. A novel role for C1q/C3 in selective elimination of synaptic connections during development was recently described (Stevens et al., 2007). Developmental elimination of synapses involves tagging by C3 fragments and phagocytosis by resident macrophages; our data suggest that this process is mimicked at the NMJ in the SOD1 G93A mouse, leading to synapse degeneration. Although C3 biosynthesis was up-regulated in both motor neurons and astrocytes in SOD1 G93A mice, C3 activation products deposit only on motor neurons, driving their loss. Neighboring astrocytes resist C3 activation because they abundantly express C3 convertase regulators (Grifths et al., 2009), but contribute to neuronal loss by increasing local synthesis of C components. Both neurons and astrocytes must express mutant SOD1 for development of ALS pathology (Wang et al., 2005; Gong et al., 2000; Pramatarova et al., 2001). If expression of the mutant SOD1 directly or indirectly drives up-regulation of C component synthesis, then it may be that local C biosynthesis only breaches the threshold necessary for neuronal damage when both neurons and astrocytes are involved. Treatment with C inhibitors protects from early axonal degeneration and facilitates regeneration and recovery in peripheral nerve injury (Ramaglia et al., 2009). Because C is deposited and activated at the NMJ before denervation, and axonal loss precedes motor neuron loss in ALS (Fischer et al., 2004), early intervention with C inhibitors may protect the NMJ and stop further neuronal degeneration, offering the prospect of therapy for this currently untreatable disorder.

Acknowledgments We thank Dr. J. Verhaagen for kindly providing the mouse tissue for this study.