Journal of Neuroimmunology 281 (2015) 73–77
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GAD65 epitope mapping and search for novel autoantibodies in GAD-associated neurological disorders P. Fouka a,b, H. Alexopoulos a, S. Akrivou a, O. Trohatou b, P.K. Politis b, M.C. Dalakas a,⁎ a b
Neuroimmunology Unit, Department of Pathophysiology, Faculty of Medicine, National and Kapodistrian University of Athens, Greece Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, Greece
a r t i c l e
i n f o
Article history: Received 31 December 2014 Received in revised form 6 March 2015 Accepted 7 March 2015 Keywords: Anti-GAD antibodies Epilepsy Stiff person syndrome GABA Synapse Autoimmunity
a b s t r a c t Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GADspecific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Anti-glutamic acid decarboxylase (GAD) antibodies have been associated with a diverse array of neurological syndromes including stiff person syndrome, cerebellar ataxia, limbic encephalitis, epilepsy and oculomotor dysfunction (Solimena et al., 1988; Giometto et al., 1996; Peltola et al., 2000; Honnorat et al., 2001; Malter et al., 2010). Whether the antibodies have a pathophysiological role or they are only disease markers remains unclear. GAD is functional in interneurons where it catalyzes the decarboxylation of L-glutamate to γ-aminobutyric acid (GABA), the most common brain inhibitory neurotransmitter. In the central nervous system, GABA is released at inhibitory synapses and binds to two types of GABA receptors, GABAA, which are ligand-gated chloride channels and GABAB, which are G protein coupled receptors (Moss and Smart, 2001). In neurological syndromes, GAD titers are high (ranging from 2000– 5,000,000 IU/ml); in contrast, in type-1 autoimmune diabetes (T1DM), where anti-GAD antibodies are also present, the titers are low (usually below 2000). GAD has two isoforms; GAD65 (a membrane associated form) and GAD67 (a soluble form) which are 65% identical in their amino acid sequence (Butler et al., 1993). GAD65 is divided into 3 functional domains,
⁎ Corresponding author at: Neuroimmunology Unit, Department of Pathophysiology, Faculty of Medicine, National and Kapodistrian University of Athens, Greece & Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA. E-mail address: [email protected] (M.C. Dalakas).
http://dx.doi.org/10.1016/j.jneuroim.2015.03.009 0165-5728/© 2015 Elsevier B.V. All rights reserved.
an amino-terminal domain (aa 1–188), a middle domain where the catalytic site resides (aa 198–473) and a carboxy-terminal domain (aa 465–585) (Fenalti and Buckle, 2010). Epitope specificity in GAD-related neurological diseases has been examined only for stiff-person syndrome (SPS), the prototypic GADassociated disease. In SPS, the antibodies are directed against linear GAD epitopes in all 3 domains, but predominantly against the catalytic region (Fenalti and Buckle, 2010). This is in contrast to T1DM where the antibodies are directed against conformational GAD epitopes located in the middle and C-terminal domains. The epitope specificities of anti-GAD antibodies in the other GAD-positive neurological syndromes have never been investigated. The question is pertinent in understanding the pathogenic role of GAD antibodies but also for designing better assays for monitoring disease progression and response to therapy. The aim of the present study was to determine epitope specificities among all GAD-positive neurological diseases and examine whether other co-existing autoantibodies may account for the phenotypic diversity. 2. Patients and methods 2.1. Patients 21 patients with variable neurological symptoms and high-titer antiGAD antibodies were used for analysis of anti-GAD epitope specificity and for antibody testing. Six patients with classic SPS were used as controls. The test cohort included 9 patients with SPS spectrum of disorders
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(SPS plus cerebellar ataxia or downbeat nustagmus or stiff limp), 8 patients with SPS syndrome plus epilepsy (either preceding or following SPS) and 4 patients with epilepsy and encephalitis symptoms. Anti-GAD antibodies were titrated using a commercial ELISA assay (Euroimmun). One patient with NMDAR encephalitis and 1 patient with Progressive Encephalomyelitis with Rigidity and Myoclonus (PERM) positive for anti-Glycine receptor antibodies were used as positive controls for hippocampal staining assays.
2.2. Luciferase Immunoprecipitation Assay (LIPS) As previously described (Burbelo et al., 2009), the principle of the method is that the antigen is genetically tagged with the enzyme luciferase. Then the precipitated antigen–antibody complexes are quantified by light emission. A pcDNA3.1 mammalian expression vector with Renilla luciferase (RLuc) reporter gene was used for all the antigen fusion constructs. Full length and three non-overlapping fragments of
D2 rv D3 fw
D1 rv D2 fw
D1 fw 1-285 nt
286 – 1331 nt D4 fw (663 nt)
SAMPLE # GAD65
1 2 3 4 5 6 7 8 9
7.00 6.66 6.33 6.00 5.66 5.33 5.00 4.66
GAD65D1 GAD65D2 GAD65D3
D3 rv 1332 – 1858 nt
D4 rv (1331 nt)
GAD65D4
SPS (+) spectrum
10 11 12 13 14 15 16 17
SPS - Epilepsy
18 19 20 21
Autoimmune epilepsy - Limbic encephalitis
22 23 24 25 26 27
Classical SPS
4.33 4.00 3.33 3.00 2.00 1.00
Fig. 1. GAD epitope mapping. Upper panel: The four different fragments of GAD65 that were tagged with luciferase for the LIPS assay are presented. The fragment GAD65D1 represents the amino-terminal domain (1–285 nt), fragment GAD65D2 (289–1331 nt) is the central domain that contains the enzyme core and finally fragment GAD65D3 (1332–1858 nt) corresponds to the carboxyl-terminal domain. Fragment GAD65D4 (663–1331 nt) is derived from GAD65D2 and corresponds more tightly to the catalytic core. Lower panel: 21 patients with various GAD-associated neurological syndromes and high anti-GAD antibody titers and 6 with classic SPS that served as controls were assayed with LIPS to determine GAD65 epitope specificity. All anti-fragment reactivity measured by luciferase and expressed in light units (LU) was transformed to the log10 scale and the results were color-coded. Red color corresponds to the highest titers while green to the lowest. All sera reacted more strongly with the GAD65D2, indicating that this fragment is the major antigenic epitope, while 8 patients reacted with all the fragments. Further, all sera reacted strongly with the GAD65D4 fragment, where the enzyme's catalytic domain resides.
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GAD65, spanning aminoacids 2–95 (GAD65D1, amino-terminal domain), 96–444 (GAD65D2, enzymic core) and 445–585 (GAD65D3, carboxy-terminal domain), were cloned next to RLuc gene. A subfragment of the GAD65D2 domain, spanning amino acids 221–444 (GAD65D4) more tightly corresponding to the catalytic core was also cloned (Fig. 1). This method of epitope mapping has been previously validated for GAD-positive SPS (Burbelo et al., 2008). The fusion constructs were transfected to human embryonic kidney cells (HEK293T) and whole-cell extracts were used for the immunoprecipitation assay. All extracts were measured by means of light emission (light units, LU) before incubation with sera. Then, 50 μl of 1.0 × 107 LU extracts diluted in buffer A (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton) were incubated with 10 μl patient serum (1/10 or 1/20 diluted in buffer A) and 40 μl buffer A at room temperature for 1 h. The samples were then transferred to tubes containing 15 μl of 30% suspension of protein A/G agarose beads (Pierce, Thermo Scientific) and incubated for another hour. The antibody–antigen binding was measured by means of light emission. Data were transformed to log10 scale and the results were color-coded. Normal sera were used as negative controls. 2.3. Immunostaining in primary hippocampal neurons Primary hippocampal neurons were isolated from E16 mice embryos as previously described (Chang et al., 2013a, 2013b). All patient sera were diluted 1/100 in 1% normal donkey serum (NDS) in phosphatebuffered saline (PBS) and incubated with 3, 7 and 10 DIV primary hippocampal neurons for 1 h at 37 °C respectively. After extensive washes with PBS, neurons were fixed with 4% paraformaldehyde in PBS for 5 min. The cells were permeabilized with 0.3% Triton in PBS for another 5 min and then incubated with blocking solution for 1 h (10% NDS in PBS) at room temperature. The samples were then incubated with anti-human fluorochrome-conjugated secondary antibodies at 1/750 dilution (Alexa Fluor 488, Invitrogen) for another hour, mounted with a fluorescent mounting medium (Dako, Agilent Technologies) and visualized using a fluorescent microscope (Zeiss Axiophot). 2.4. Cell based assays HEK293T cells were transfected with GABAA receptor α1 and β3 units (cDNA clones were a kind gift from Prof. Dalmau, Barcelona) or both, glycine receptor-α1 (cDNA clone was a kind gift from Prof. Vincent FRS, Oxford), and GABA receptor-associated protein (GABARAP, cDNA clone obtained from Imagenes). All sera were tested on either live or pre-fixed cells. Briefly, for live assays, patient sera were diluted 1/25 in BSA 1% in PBS and applied on cells for 2 h at room temperature. Then cells were fixed with 4% PFA for 5 min and permeabilized with 0.3% Triton in PBS. After incubation with blocking solution (10% NDS) for 1 h at room temperature, cells were incubated with anti-human fluorochromeconjugated secondary antibodies at 1/750 dilutions (Alexa Fluor 488 or 568, Invitrogen) for another hour, mounted with a fluorescent mounting medium (Dako, Agilent Technologies) and visualized using a fluorescent microscope (Zeiss Axiophot). Fixed-cell assays were performed using the same protocol but with sera incubation following cell fixation. Antibody testing for extracellular neuronal antigens (NMDAR, GABAb, LGI1, CASPR2, AMPA1, AMPA2 and DPPX) was performed with commercially available assays (Euroimmun). 3. Results 3.1. Anti-GAD titers Anti-GAD titers, following serial dilutions, as determined with ELISA, ranged from 37,550 IU/ml to 4,242,000 IU/ml. No correlation was observed between titers and specific symptoms. For 6 of the control SPS
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patients and 10 of the SPS-spectrum patients, titer range was also confirmed with the LIPS method (data not shown). 3.2. Epitope specificities No differences were observed in epitope specificity between the GAD-associated syndromes using the LIPS method. All patients primarily recognized the fragment D2 (aa 96–444), which corresponds to the catalytic core of the enzyme and fragment D4 (aa 221–444), also derived from the middle portion of the enzyme (Fig. 1b). Both the amino- and carboxy-terminals were also recognized, albeit to a lesser extent. The results corresponded with the data from Burbelo et al., where the same fragments were identified as the main epitopes in SPS (Burbelo et al., 2008). The same was true for the 6 SPS patients used as controls (Fig. 1). 3.3. Search for other autoantigens of inhibitory synapses All patients (n = 21) were then tested for a series of antibodies previously reported in the SPS-spectrum disorders or other GAD-associated syndromes, which included anti-GABAA receptor (Petit-Pedrol et al., 2014), anti-GABARAP (Raju et al., 2006), anti-glycine receptor (McKeon et al., 2013) and anti-amphiphysin (De Camilli et al., 1993; Folli et al., 1993). Two previously reported SPS patients with prominent hyperexcitability and anxiety, were positive for anti-glycine receptor antibodies (Alexopoulos et al., 2013). No other patients showed any reactivity. 3.4. Test for novel autoantibody specificities All sera were screened in live cultured hippocampal neurons, a standard and widely used substrate for identifying novel specificities against neuronal surface antigens (Vincent et al., 2011). Three sera from patients with autoimmune epilepsy but none of the others, showed high binding (2 patients) or low binding (1 patient) (Fig. 2). There was no correlation between anti-GAD titers and hippocampal staining, suggesting that the staining corresponds to a novel – but still unidentified – antigen, which cannot be attributed to anti-GAD antibodies. Performing the staining on live, non-fixed, neurons supports this conclusion. The 3 patients with positive hippocampal staining were subsequently tested for most known antibodies directed against extracellular antigens, including anti-NMDA receptor, anti LGI1, anti-CASPR2, anti-GABAB, anti-GABAA, anti-AMPA and anti-DPPX. All three patients were negative. One of the three patients with prominent hippocampal binding was a 20-year old female with 2 episodes of epilepsy and no notable findings on EEG and MRI. She was treated with anti-epileptics and steroids and, despite no-clinical relapse during one year of follow-up her anti-GAD antibody titers remained in the same high range (N 4,000,000 IU/ml). The second patient with high positive binding, was also female with drug-resistant multiple episodes of epilepsy and positive MRI-FLAIR signal in the temporal lobes. She was treated with plasmapheresis and steroids and significantly improved. In both cases, the positive response to immunotherapy suggests that the anti-surface antibodies may be relevant to disease pathogenesis. The third patient was a 30-year old male who presented only with epilepsy but at a later stage his disease progressed to typical SPS. 4. Discussion A range of diverse neurological syndromes, including SPS, epilepsy, cerebellar ataxia, ocular nustagmus and encephaliltis are associated with anti-GAD antibodies, suggesting a pathophysiological mechanism affecting GABAergic neurotransmission. It has been proposed that autoantibodies directed against antigens in the GABAergic synapse could impair either the GABA synthesis or the GABAergic signaling resulting in the various symptoms (Alexopoulos
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50 µm
A
B
C
D
Fig. 2. Hippocampal stainings. 21 patients with various GAD-associated neurological syndromes were tested for autoantibody binding in live cultured hippocampal neurons. Data are shown for 2 controls [A, B] with anti-NMDAR (A) and anti-Glycine receptor (B) antibodies and 2 patients (C, D) with high anti-GAD antibody titers and epilepsy. There is specific binding on neuronal cell surface, representing the presence of antibodies against a yet unknown extracellular antigen. None of the control GAD-positive SPS patients showed any such binding.
and Dalakas, 2013). This hypothesis has so far been tested mainly for SPS based on experiments of GABA inhibition by anti-GAD antibodies in vitro and by passive transfer of anti-GAD antibodies in animals, in vivo. Although, this limited evidence assigns a pathogenetic role to anti-GAD antibodies, it is unclear how the same antibody may cause syndromes of such a clinical diversity. Limited data also exist about anti-amphiphysin antibodies, which are associated with the paraneoplastic variant of SPS (Dinkel et al., 1998; Raju et al., 2005) and anti-GABARAP antibodies (Raju et al., 2006). GABARAP and amphiphysin are intracellular and it is difficult to explain how an antibody against intracellular targets can be pathogenic. It is plausible that these antigens during synaptic transmission may transiently expose epitopes in the extracellular milieu and hence be recognized by the immune system. Our first aim was to test whether possible diversity in epitope specificity could confer specific properties to anti-GAD antibodies that may account for the various clinical syndromes, as previously demonstrated for anti-GAD antibodies between SPS and T1DM (Kim et al., 1994; Solimena et al., 1994). We found no differences in epitope specificity in the antibodies from the various syndromes, which support the view that anti-GAD antibodies may not be pathogenic. This is highly possible as GAD is cytosolic and the mechanism by which antibodies can recognize intracellular targets and cause disease is not clear (Levy et al., 1999; Dalakas et al., 2000; Vincent, 2008; Dalakas, 2009). The possibility that a GAD-mediated T-cell or neuronal death mechanism may be implicated cannot be however excluded (Alexopoulos and Dalakas, 2013). Another plausible explanation is that the antibodies might be pathogenic only at disease onset or shortly thereafter and, as the disease evolves they become indolent serological markers. An alternative hypothesis is that other antibodies, targeting extracellular antigens of the inhibitory GABAergic or glycinergic synapse, may be present in GAD-positive disease subsets. Antibodies against single subunits and/or combinations of subunits of the GABAA receptor have been identified in encephalitis patients and in a small subset of SPS patients. In SPS, a study (Petit-Pedrol et al.) has identified antibodies against extracellular epitopes of the α1β3 combination of subunits.
Other studies have also identified anti-GABAA receptor antibodies (although only in encephalitis patients) that bind to the β3 subunit (Ohkawa et al., 2014) or the α1 and γ2 subunits (Pettingill et al., 2015). However, in all these cases, and consistent with our results, no epitope specificity has been associated with any specific symptomatology or disease subset. Although we did not detect any of the known autoantibodies against cell surface antigens, we did identify antibodies of unknown specificity using hippocampal neurons as substrate, in 3 patients with GADpositive epilepsies. In a previously published series, anti-GAD antibodies were found in 6-patients with pharmaco-resistant epilepsy (Peltola et al., 2000). The immunological profile of these patients was different from the SPS spectrum patients (e.g., negative CSF for oligoclonal bands). In a similar study, in 23 patients with temporal lobe epilepsy of unknown etiology (like in our 3 patients), 5 were GAD-positive (Falip et al., 2012), although only 2 had high GAD titers. It is unclear whether in these cases the epilepsy was a sequela of limbic encephalitis, or a primary event. In two of our large series on SPS, 5%–12% of the patients have seizures (Dalakas et al., 2001). The specificity of the antibody that binds on the surface of hippocampal neurons needs to be determined, as epitope specificity of antibodies targeting extracellular antigens is pertinent to pathogenicity. For example, in anti-AQP4 spectrum disorders, the majority of autoantibodies are directed against extracellular domains but a minority of mapped epitopes are linear and even intracellular (Kampylafka et al., 2011). This diversity and highlevel epitope spreading probably reflects the inflammatory nature of the syndrome and is spearheaded by astrocyte destruction (VakninDembinsky et al., 2014). In contrast, in NMDAR encephalitis, the antibodies only target the NR2 subunit of the NMDA receptor and exert a direct functional effect on their target (Hughes et al., 2010). These examples illustrate how epitope determination can help us understand the nature of an autoimmune response. The work demonstrates that anti-GAD antibodies do not have different specificities in a cross-sectional study of patients with diverse symptomatology encompassing all known GAD clinical associations, but some patients harbor novel antigens. Our results raise the possibility
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that the pathogenetic mechanism in GAD-associated syndromes may involve spread to antigens expressed on the surface of inhibitory neurons and their synapses. Acknowledgments We wish to thank Dr. M. Kosmidis and Dr. G. Rakocevic for providing clinical information and Prof. Angela Vincent and Dr. Josep Dalmau for providing cDNA clones. Disclosures The authors report no conflicts of interest. Funding was obtained from the Special Research Account, University of Athens and by the European Union (European Social Fund — ESF) and Greek national funds through the operational program “Education and lifelong learning” of the National Strategic Reference Framework (NSRF) – Research Funding Program: Thales, Investing in knowledge society through the European Social Fund. References Alexopoulos, H., Dalakas, M.C., 2013. Immunology of stiff person syndrome and other GAD-associated neurological disorders. Expert. Rev. Clin. Immunol. 9, 1043–1053. Alexopoulos, H., Akrivou, S., Dalakas, M.C., 2013. Glycine receptor antibodies in stiffperson syndrome and other GAD-positive CNS disorders. Neurology 81, 1962–1964. Burbelo, P.D., Groot, S., Dalakas, M.C., Iadarola, M.J., 2008. High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome. Biochem. Biophys. Res. Commun. 366, 1–7. Burbelo, P.D., Ching, K.H., Klimavicz, C.M., Iadarola, M.J., 2009. Antibody profiling by Luciferase Immunoprecipitation Systems (LIPS). J. Vis. Exp. 32, 1549. Butler, M.H., Solimena, M., Dirkx Jr., R., Hayday, A., De Camilli, P., 1993. Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome. J. Exp. Med. 178, 2097–2106. Chang, T., Alexopoulos, H., McMenamin, M., Carvajal-Gonzalez, A., Alexander, S.K., Deacon, R., Erdelyi, F., Gabor, S., Lang, B., Blaes, F., Brown, P., Vincent, A., 2013a. Neuronal surface and glutamic acid decarboxylase autoantibodies in nonparaneoplastic stiff person syndrome. JAMA Neurol. 70, 1140–1149. Chang, T., Alexopoulos, H., Pettingill, P., McMenamin, M., Deacon, R., Erdelyi, F., Szabo, G., Buckley, C.J., Vincent, A., 2013b. Immunization against GAD induces antibody binding to GAD-independent antigens and brainstem GABAergic neuronal loss. PLoS One 8, e72921. Dalakas, M.C., 2009. Stiff person syndrome: advances in pathogenesis and therapeutic interventions. Curr. Treat. Options Neurol. 11, 102–110. Dalakas, M.C., Fujii, M., Li, M., McElroy, B., 2000. The clinical spectrum of anti-GAD antibody-positive patients with stiff-person syndrome. Neurology 55, 1531–1535. Dalakas, M.C., Li, M., Fujii, M., Jacobowitz, D.M., 2001. Stiff person syndrome: quantification, specificity, and intrathecal synthesis of GAD65 antibodies. Neurology 57, 780–784. De Camilli, P., Thomas, A., Cofiell, R., Folli, F., Lichte, B., Piccolo, G., Meinck, H.M., Austoni, M., Fassetta, G., Bottazzo, G., et al., 1993. The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of stiff-man syndrome with breast cancer. J. Exp. Med. 178, 2219–2223. Dinkel, K., Meinck, H.M., Jury, K.M., Karges, W., Richter, W., 1998. Inhibition of gammaaminobutyric acid synthesis by glutamic acid decarboxylase autoantibodies in stiffman syndrome. Ann. Neurol. 44, 194–201. Falip, M., Carreno, M., Miro, J., Saiz, A., Villanueva, V., Quilez, A., Molins, A., Barcelo, I., Sierra, A., Graus, F., 2012. Prevalence and immunological spectrum of temporal lobe epilepsy with glutamic acid decarboxylase antibodies. Eur. J. Neurol. 19, 827–833. Fenalti, G., Buckle, A.M., 2010. Structural biology of the GAD autoantigen. Autoimmun. Rev. 9, 148–152.
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GAD65 epitope mapping and search for novel autoantibodies in GAD-associated neurological disorders P. Fouka a,b, H. Alexopoulos a, S. Akrivou a, O. Trohatou b, P.K. Politis b, M.C. Dalakas a,⁎ a b
Neuroimmunology Unit, Department of Pathophysiology, Faculty of Medicine, National and Kapodistrian University of Athens, Greece Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, Greece
a r t i c l e
i n f o
Article history: Received 31 December 2014 Received in revised form 6 March 2015 Accepted 7 March 2015 Keywords: Anti-GAD antibodies Epilepsy Stiff person syndrome GABA Synapse Autoimmunity
a b s t r a c t Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GADspecific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Anti-glutamic acid decarboxylase (GAD) antibodies have been associated with a diverse array of neurological syndromes including stiff person syndrome, cerebellar ataxia, limbic encephalitis, epilepsy and oculomotor dysfunction (Solimena et al., 1988; Giometto et al., 1996; Peltola et al., 2000; Honnorat et al., 2001; Malter et al., 2010). Whether the antibodies have a pathophysiological role or they are only disease markers remains unclear. GAD is functional in interneurons where it catalyzes the decarboxylation of L-glutamate to γ-aminobutyric acid (GABA), the most common brain inhibitory neurotransmitter. In the central nervous system, GABA is released at inhibitory synapses and binds to two types of GABA receptors, GABAA, which are ligand-gated chloride channels and GABAB, which are G protein coupled receptors (Moss and Smart, 2001). In neurological syndromes, GAD titers are high (ranging from 2000– 5,000,000 IU/ml); in contrast, in type-1 autoimmune diabetes (T1DM), where anti-GAD antibodies are also present, the titers are low (usually below 2000). GAD has two isoforms; GAD65 (a membrane associated form) and GAD67 (a soluble form) which are 65% identical in their amino acid sequence (Butler et al., 1993). GAD65 is divided into 3 functional domains,
⁎ Corresponding author at: Neuroimmunology Unit, Department of Pathophysiology, Faculty of Medicine, National and Kapodistrian University of Athens, Greece & Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA. E-mail address: [email protected] (M.C. Dalakas).
http://dx.doi.org/10.1016/j.jneuroim.2015.03.009 0165-5728/© 2015 Elsevier B.V. All rights reserved.
an amino-terminal domain (aa 1–188), a middle domain where the catalytic site resides (aa 198–473) and a carboxy-terminal domain (aa 465–585) (Fenalti and Buckle, 2010). Epitope specificity in GAD-related neurological diseases has been examined only for stiff-person syndrome (SPS), the prototypic GADassociated disease. In SPS, the antibodies are directed against linear GAD epitopes in all 3 domains, but predominantly against the catalytic region (Fenalti and Buckle, 2010). This is in contrast to T1DM where the antibodies are directed against conformational GAD epitopes located in the middle and C-terminal domains. The epitope specificities of anti-GAD antibodies in the other GAD-positive neurological syndromes have never been investigated. The question is pertinent in understanding the pathogenic role of GAD antibodies but also for designing better assays for monitoring disease progression and response to therapy. The aim of the present study was to determine epitope specificities among all GAD-positive neurological diseases and examine whether other co-existing autoantibodies may account for the phenotypic diversity. 2. Patients and methods 2.1. Patients 21 patients with variable neurological symptoms and high-titer antiGAD antibodies were used for analysis of anti-GAD epitope specificity and for antibody testing. Six patients with classic SPS were used as controls. The test cohort included 9 patients with SPS spectrum of disorders
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(SPS plus cerebellar ataxia or downbeat nustagmus or stiff limp), 8 patients with SPS syndrome plus epilepsy (either preceding or following SPS) and 4 patients with epilepsy and encephalitis symptoms. Anti-GAD antibodies were titrated using a commercial ELISA assay (Euroimmun). One patient with NMDAR encephalitis and 1 patient with Progressive Encephalomyelitis with Rigidity and Myoclonus (PERM) positive for anti-Glycine receptor antibodies were used as positive controls for hippocampal staining assays.
2.2. Luciferase Immunoprecipitation Assay (LIPS) As previously described (Burbelo et al., 2009), the principle of the method is that the antigen is genetically tagged with the enzyme luciferase. Then the precipitated antigen–antibody complexes are quantified by light emission. A pcDNA3.1 mammalian expression vector with Renilla luciferase (RLuc) reporter gene was used for all the antigen fusion constructs. Full length and three non-overlapping fragments of
D2 rv D3 fw
D1 rv D2 fw
D1 fw 1-285 nt
286 – 1331 nt D4 fw (663 nt)
SAMPLE # GAD65
1 2 3 4 5 6 7 8 9
7.00 6.66 6.33 6.00 5.66 5.33 5.00 4.66
GAD65D1 GAD65D2 GAD65D3
D3 rv 1332 – 1858 nt
D4 rv (1331 nt)
GAD65D4
SPS (+) spectrum
10 11 12 13 14 15 16 17
SPS - Epilepsy
18 19 20 21
Autoimmune epilepsy - Limbic encephalitis
22 23 24 25 26 27
Classical SPS
4.33 4.00 3.33 3.00 2.00 1.00
Fig. 1. GAD epitope mapping. Upper panel: The four different fragments of GAD65 that were tagged with luciferase for the LIPS assay are presented. The fragment GAD65D1 represents the amino-terminal domain (1–285 nt), fragment GAD65D2 (289–1331 nt) is the central domain that contains the enzyme core and finally fragment GAD65D3 (1332–1858 nt) corresponds to the carboxyl-terminal domain. Fragment GAD65D4 (663–1331 nt) is derived from GAD65D2 and corresponds more tightly to the catalytic core. Lower panel: 21 patients with various GAD-associated neurological syndromes and high anti-GAD antibody titers and 6 with classic SPS that served as controls were assayed with LIPS to determine GAD65 epitope specificity. All anti-fragment reactivity measured by luciferase and expressed in light units (LU) was transformed to the log10 scale and the results were color-coded. Red color corresponds to the highest titers while green to the lowest. All sera reacted more strongly with the GAD65D2, indicating that this fragment is the major antigenic epitope, while 8 patients reacted with all the fragments. Further, all sera reacted strongly with the GAD65D4 fragment, where the enzyme's catalytic domain resides.
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GAD65, spanning aminoacids 2–95 (GAD65D1, amino-terminal domain), 96–444 (GAD65D2, enzymic core) and 445–585 (GAD65D3, carboxy-terminal domain), were cloned next to RLuc gene. A subfragment of the GAD65D2 domain, spanning amino acids 221–444 (GAD65D4) more tightly corresponding to the catalytic core was also cloned (Fig. 1). This method of epitope mapping has been previously validated for GAD-positive SPS (Burbelo et al., 2008). The fusion constructs were transfected to human embryonic kidney cells (HEK293T) and whole-cell extracts were used for the immunoprecipitation assay. All extracts were measured by means of light emission (light units, LU) before incubation with sera. Then, 50 μl of 1.0 × 107 LU extracts diluted in buffer A (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton) were incubated with 10 μl patient serum (1/10 or 1/20 diluted in buffer A) and 40 μl buffer A at room temperature for 1 h. The samples were then transferred to tubes containing 15 μl of 30% suspension of protein A/G agarose beads (Pierce, Thermo Scientific) and incubated for another hour. The antibody–antigen binding was measured by means of light emission. Data were transformed to log10 scale and the results were color-coded. Normal sera were used as negative controls. 2.3. Immunostaining in primary hippocampal neurons Primary hippocampal neurons were isolated from E16 mice embryos as previously described (Chang et al., 2013a, 2013b). All patient sera were diluted 1/100 in 1% normal donkey serum (NDS) in phosphatebuffered saline (PBS) and incubated with 3, 7 and 10 DIV primary hippocampal neurons for 1 h at 37 °C respectively. After extensive washes with PBS, neurons were fixed with 4% paraformaldehyde in PBS for 5 min. The cells were permeabilized with 0.3% Triton in PBS for another 5 min and then incubated with blocking solution for 1 h (10% NDS in PBS) at room temperature. The samples were then incubated with anti-human fluorochrome-conjugated secondary antibodies at 1/750 dilution (Alexa Fluor 488, Invitrogen) for another hour, mounted with a fluorescent mounting medium (Dako, Agilent Technologies) and visualized using a fluorescent microscope (Zeiss Axiophot). 2.4. Cell based assays HEK293T cells were transfected with GABAA receptor α1 and β3 units (cDNA clones were a kind gift from Prof. Dalmau, Barcelona) or both, glycine receptor-α1 (cDNA clone was a kind gift from Prof. Vincent FRS, Oxford), and GABA receptor-associated protein (GABARAP, cDNA clone obtained from Imagenes). All sera were tested on either live or pre-fixed cells. Briefly, for live assays, patient sera were diluted 1/25 in BSA 1% in PBS and applied on cells for 2 h at room temperature. Then cells were fixed with 4% PFA for 5 min and permeabilized with 0.3% Triton in PBS. After incubation with blocking solution (10% NDS) for 1 h at room temperature, cells were incubated with anti-human fluorochromeconjugated secondary antibodies at 1/750 dilutions (Alexa Fluor 488 or 568, Invitrogen) for another hour, mounted with a fluorescent mounting medium (Dako, Agilent Technologies) and visualized using a fluorescent microscope (Zeiss Axiophot). Fixed-cell assays were performed using the same protocol but with sera incubation following cell fixation. Antibody testing for extracellular neuronal antigens (NMDAR, GABAb, LGI1, CASPR2, AMPA1, AMPA2 and DPPX) was performed with commercially available assays (Euroimmun). 3. Results 3.1. Anti-GAD titers Anti-GAD titers, following serial dilutions, as determined with ELISA, ranged from 37,550 IU/ml to 4,242,000 IU/ml. No correlation was observed between titers and specific symptoms. For 6 of the control SPS
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patients and 10 of the SPS-spectrum patients, titer range was also confirmed with the LIPS method (data not shown). 3.2. Epitope specificities No differences were observed in epitope specificity between the GAD-associated syndromes using the LIPS method. All patients primarily recognized the fragment D2 (aa 96–444), which corresponds to the catalytic core of the enzyme and fragment D4 (aa 221–444), also derived from the middle portion of the enzyme (Fig. 1b). Both the amino- and carboxy-terminals were also recognized, albeit to a lesser extent. The results corresponded with the data from Burbelo et al., where the same fragments were identified as the main epitopes in SPS (Burbelo et al., 2008). The same was true for the 6 SPS patients used as controls (Fig. 1). 3.3. Search for other autoantigens of inhibitory synapses All patients (n = 21) were then tested for a series of antibodies previously reported in the SPS-spectrum disorders or other GAD-associated syndromes, which included anti-GABAA receptor (Petit-Pedrol et al., 2014), anti-GABARAP (Raju et al., 2006), anti-glycine receptor (McKeon et al., 2013) and anti-amphiphysin (De Camilli et al., 1993; Folli et al., 1993). Two previously reported SPS patients with prominent hyperexcitability and anxiety, were positive for anti-glycine receptor antibodies (Alexopoulos et al., 2013). No other patients showed any reactivity. 3.4. Test for novel autoantibody specificities All sera were screened in live cultured hippocampal neurons, a standard and widely used substrate for identifying novel specificities against neuronal surface antigens (Vincent et al., 2011). Three sera from patients with autoimmune epilepsy but none of the others, showed high binding (2 patients) or low binding (1 patient) (Fig. 2). There was no correlation between anti-GAD titers and hippocampal staining, suggesting that the staining corresponds to a novel – but still unidentified – antigen, which cannot be attributed to anti-GAD antibodies. Performing the staining on live, non-fixed, neurons supports this conclusion. The 3 patients with positive hippocampal staining were subsequently tested for most known antibodies directed against extracellular antigens, including anti-NMDA receptor, anti LGI1, anti-CASPR2, anti-GABAB, anti-GABAA, anti-AMPA and anti-DPPX. All three patients were negative. One of the three patients with prominent hippocampal binding was a 20-year old female with 2 episodes of epilepsy and no notable findings on EEG and MRI. She was treated with anti-epileptics and steroids and, despite no-clinical relapse during one year of follow-up her anti-GAD antibody titers remained in the same high range (N 4,000,000 IU/ml). The second patient with high positive binding, was also female with drug-resistant multiple episodes of epilepsy and positive MRI-FLAIR signal in the temporal lobes. She was treated with plasmapheresis and steroids and significantly improved. In both cases, the positive response to immunotherapy suggests that the anti-surface antibodies may be relevant to disease pathogenesis. The third patient was a 30-year old male who presented only with epilepsy but at a later stage his disease progressed to typical SPS. 4. Discussion A range of diverse neurological syndromes, including SPS, epilepsy, cerebellar ataxia, ocular nustagmus and encephaliltis are associated with anti-GAD antibodies, suggesting a pathophysiological mechanism affecting GABAergic neurotransmission. It has been proposed that autoantibodies directed against antigens in the GABAergic synapse could impair either the GABA synthesis or the GABAergic signaling resulting in the various symptoms (Alexopoulos
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50 µm
A
B
C
D
Fig. 2. Hippocampal stainings. 21 patients with various GAD-associated neurological syndromes were tested for autoantibody binding in live cultured hippocampal neurons. Data are shown for 2 controls [A, B] with anti-NMDAR (A) and anti-Glycine receptor (B) antibodies and 2 patients (C, D) with high anti-GAD antibody titers and epilepsy. There is specific binding on neuronal cell surface, representing the presence of antibodies against a yet unknown extracellular antigen. None of the control GAD-positive SPS patients showed any such binding.
and Dalakas, 2013). This hypothesis has so far been tested mainly for SPS based on experiments of GABA inhibition by anti-GAD antibodies in vitro and by passive transfer of anti-GAD antibodies in animals, in vivo. Although, this limited evidence assigns a pathogenetic role to anti-GAD antibodies, it is unclear how the same antibody may cause syndromes of such a clinical diversity. Limited data also exist about anti-amphiphysin antibodies, which are associated with the paraneoplastic variant of SPS (Dinkel et al., 1998; Raju et al., 2005) and anti-GABARAP antibodies (Raju et al., 2006). GABARAP and amphiphysin are intracellular and it is difficult to explain how an antibody against intracellular targets can be pathogenic. It is plausible that these antigens during synaptic transmission may transiently expose epitopes in the extracellular milieu and hence be recognized by the immune system. Our first aim was to test whether possible diversity in epitope specificity could confer specific properties to anti-GAD antibodies that may account for the various clinical syndromes, as previously demonstrated for anti-GAD antibodies between SPS and T1DM (Kim et al., 1994; Solimena et al., 1994). We found no differences in epitope specificity in the antibodies from the various syndromes, which support the view that anti-GAD antibodies may not be pathogenic. This is highly possible as GAD is cytosolic and the mechanism by which antibodies can recognize intracellular targets and cause disease is not clear (Levy et al., 1999; Dalakas et al., 2000; Vincent, 2008; Dalakas, 2009). The possibility that a GAD-mediated T-cell or neuronal death mechanism may be implicated cannot be however excluded (Alexopoulos and Dalakas, 2013). Another plausible explanation is that the antibodies might be pathogenic only at disease onset or shortly thereafter and, as the disease evolves they become indolent serological markers. An alternative hypothesis is that other antibodies, targeting extracellular antigens of the inhibitory GABAergic or glycinergic synapse, may be present in GAD-positive disease subsets. Antibodies against single subunits and/or combinations of subunits of the GABAA receptor have been identified in encephalitis patients and in a small subset of SPS patients. In SPS, a study (Petit-Pedrol et al.) has identified antibodies against extracellular epitopes of the α1β3 combination of subunits.
Other studies have also identified anti-GABAA receptor antibodies (although only in encephalitis patients) that bind to the β3 subunit (Ohkawa et al., 2014) or the α1 and γ2 subunits (Pettingill et al., 2015). However, in all these cases, and consistent with our results, no epitope specificity has been associated with any specific symptomatology or disease subset. Although we did not detect any of the known autoantibodies against cell surface antigens, we did identify antibodies of unknown specificity using hippocampal neurons as substrate, in 3 patients with GADpositive epilepsies. In a previously published series, anti-GAD antibodies were found in 6-patients with pharmaco-resistant epilepsy (Peltola et al., 2000). The immunological profile of these patients was different from the SPS spectrum patients (e.g., negative CSF for oligoclonal bands). In a similar study, in 23 patients with temporal lobe epilepsy of unknown etiology (like in our 3 patients), 5 were GAD-positive (Falip et al., 2012), although only 2 had high GAD titers. It is unclear whether in these cases the epilepsy was a sequela of limbic encephalitis, or a primary event. In two of our large series on SPS, 5%–12% of the patients have seizures (Dalakas et al., 2001). The specificity of the antibody that binds on the surface of hippocampal neurons needs to be determined, as epitope specificity of antibodies targeting extracellular antigens is pertinent to pathogenicity. For example, in anti-AQP4 spectrum disorders, the majority of autoantibodies are directed against extracellular domains but a minority of mapped epitopes are linear and even intracellular (Kampylafka et al., 2011). This diversity and highlevel epitope spreading probably reflects the inflammatory nature of the syndrome and is spearheaded by astrocyte destruction (VakninDembinsky et al., 2014). In contrast, in NMDAR encephalitis, the antibodies only target the NR2 subunit of the NMDA receptor and exert a direct functional effect on their target (Hughes et al., 2010). These examples illustrate how epitope determination can help us understand the nature of an autoimmune response. The work demonstrates that anti-GAD antibodies do not have different specificities in a cross-sectional study of patients with diverse symptomatology encompassing all known GAD clinical associations, but some patients harbor novel antigens. Our results raise the possibility
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that the pathogenetic mechanism in GAD-associated syndromes may involve spread to antigens expressed on the surface of inhibitory neurons and their synapses. Acknowledgments We wish to thank Dr. M. Kosmidis and Dr. G. Rakocevic for providing clinical information and Prof. Angela Vincent and Dr. Josep Dalmau for providing cDNA clones. Disclosures The authors report no conflicts of interest. Funding was obtained from the Special Research Account, University of Athens and by the European Union (European Social Fund — ESF) and Greek national funds through the operational program “Education and lifelong learning” of the National Strategic Reference Framework (NSRF) – Research Funding Program: Thales, Investing in knowledge society through the European Social Fund. References Alexopoulos, H., Dalakas, M.C., 2013. Immunology of stiff person syndrome and other GAD-associated neurological disorders. Expert. Rev. Clin. Immunol. 9, 1043–1053. Alexopoulos, H., Akrivou, S., Dalakas, M.C., 2013. Glycine receptor antibodies in stiffperson syndrome and other GAD-positive CNS disorders. Neurology 81, 1962–1964. Burbelo, P.D., Groot, S., Dalakas, M.C., Iadarola, M.J., 2008. High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome. Biochem. Biophys. Res. Commun. 366, 1–7. Burbelo, P.D., Ching, K.H., Klimavicz, C.M., Iadarola, M.J., 2009. Antibody profiling by Luciferase Immunoprecipitation Systems (LIPS). J. Vis. Exp. 32, 1549. Butler, M.H., Solimena, M., Dirkx Jr., R., Hayday, A., De Camilli, P., 1993. Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome. J. Exp. Med. 178, 2097–2106. Chang, T., Alexopoulos, H., McMenamin, M., Carvajal-Gonzalez, A., Alexander, S.K., Deacon, R., Erdelyi, F., Gabor, S., Lang, B., Blaes, F., Brown, P., Vincent, A., 2013a. Neuronal surface and glutamic acid decarboxylase autoantibodies in nonparaneoplastic stiff person syndrome. JAMA Neurol. 70, 1140–1149. Chang, T., Alexopoulos, H., Pettingill, P., McMenamin, M., Deacon, R., Erdelyi, F., Szabo, G., Buckley, C.J., Vincent, A., 2013b. Immunization against GAD induces antibody binding to GAD-independent antigens and brainstem GABAergic neuronal loss. PLoS One 8, e72921. Dalakas, M.C., 2009. Stiff person syndrome: advances in pathogenesis and therapeutic interventions. Curr. Treat. Options Neurol. 11, 102–110. Dalakas, M.C., Fujii, M., Li, M., McElroy, B., 2000. The clinical spectrum of anti-GAD antibody-positive patients with stiff-person syndrome. Neurology 55, 1531–1535. Dalakas, M.C., Li, M., Fujii, M., Jacobowitz, D.M., 2001. Stiff person syndrome: quantification, specificity, and intrathecal synthesis of GAD65 antibodies. Neurology 57, 780–784. De Camilli, P., Thomas, A., Cofiell, R., Folli, F., Lichte, B., Piccolo, G., Meinck, H.M., Austoni, M., Fassetta, G., Bottazzo, G., et al., 1993. The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of stiff-man syndrome with breast cancer. J. Exp. Med. 178, 2219–2223. Dinkel, K., Meinck, H.M., Jury, K.M., Karges, W., Richter, W., 1998. Inhibition of gammaaminobutyric acid synthesis by glutamic acid decarboxylase autoantibodies in stiffman syndrome. Ann. Neurol. 44, 194–201. Falip, M., Carreno, M., Miro, J., Saiz, A., Villanueva, V., Quilez, A., Molins, A., Barcelo, I., Sierra, A., Graus, F., 2012. Prevalence and immunological spectrum of temporal lobe epilepsy with glutamic acid decarboxylase antibodies. Eur. J. Neurol. 19, 827–833. Fenalti, G., Buckle, A.M., 2010. Structural biology of the GAD autoantigen. Autoimmun. Rev. 9, 148–152.
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