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Progress and stiff challenges in understanding the role of GAD-antibodies in stiff-person syndrome
Experimental Neurology 247 (2013) 303–307
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Commentary
Progress and stiff challenges in understanding the role of GAD-antibodies in stiff-person syndrome Marinos C. Dalakas ⁎ Thomas Jefferson University, Philadelphia, PA, USA Neuroimmunology Unit, University of Athens Medical School, Athens, Greece
a r t i c l e
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Article history: Received 27 December 2012 Revised 4 February 2013 Accepted 18 February 2013 Available online 26 February 2013
Introduction Stiff-person syndrome (SPS) is an autoimmune CNS disorder characterized by three sets of symptoms: 1) stiffness of truncal and proximal limb muscles due to continuous co-contracture of agonist and antagonist muscles resulting in hyperlordosis, difficulty in bending or turning, and slow, wide-base gait in an effort to improve balance; 2) episodic spasms, superimposed on the stiffness, precipitated by sudden unexpected noises, tactile and visual stimuli, or emotional upset; and 3) overt anxiety and task-specific phobias, that may lead to the erroneous diagnosis of a primary anxiety disorder (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Levy et al., 1999). If anxiety dominates the clinical picture, SPS is discovered in retrospect when the administration of anti-anxiety agents, such as diazepam or araprazolam, improves the motor symptoms. Although the phobias seem to stem from a realistic fear of falling caused by the stiffness and spasms (Ameli et al., 2005), a primary anxiety disorder due to dysfunction of the CNS inhibitory pathways, cannot be excluded. The symptoms vary in severity, from mild to severe, and can be fluctuating or fixed leading to disability. Up to 65% of SPS patients cannot independently perform daily activities because of body stiffness, phobias, anxiety-triggered spasms, and frequent falls; others use walkers or wheelchairs, and still others are bedridden due to severe stiffness (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Levy et al., 1999). At times, the spasms are prominent and continuous and, if respiratory and thoracic paraspinal muscles are affected, may cause breathing difficulty and autonomic release phenomena (“status spasticus”) requiring admission to the intensive care unit, intravenous
⁎ Neuroimmunology Unit, Dept of Pathophysiology, National University of Athens Medical School, 75 Mikras Asias Str., Athens, 11527 Greece. Fax: + 30 210 746 2664. E-mail address: [email protected]. 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.02.010
diazepam administration, hydration and supportive care (Dalakas, 2009; Dalakas et al., 2000). The diagnosis of SPS is clinical, based on the aforementioned symptomatology, the exclusion of other neurological diseases that could explain stiffness and rigidity, and the absence of extrapyramidal or pyramidal tract signs. The diagnosis is aided by normal MRI imaging of the brain and spinal cord; the electrophysiological recordings of low-frequency motor unit firing at rest, in spite of voluntary relaxation, simultaneously from agonist and antagonist muscles (normally, when the agonist muscles contract, their respective antagonists are in a state of relaxation with no electrical activity); and the presence of high-titer antibodies against glutamic acid decarboxylase (GAD)-65 (or any of the antibodies listed in Table 1 and depicted in Fig. 1) (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Folli et al., 1993; Levy et al., 1999; Murinson et al., 2004; Solimena et al., 1988). SPS is frequently associated with other autoimmune diseases; among more than 60 patients we have followed, diabetes, thyroiditis, vitiligo and pernicious anemia were the most common, with type I diabetes occurring in up to 35% of the patients (Dalakas et al., 2000). In 5% of patients, SPS is paraneoplastic associated with anti-amphiphysin or anti-gephyrin antibodies (Butler et al., 2000; Folli et al., 1993). Pathogenesis of SPS: the involvement of GABAergic inhibition The concurrent contraction of agonist and antagonist muscles and the continuous motor unit firing at rest, in spite of voluntary relaxation, suggest lack of reciprocal inhibition. The inhibition is most likely suprasegmental, as determined by paired pulse transcranial magnetic stimulation studies, implying diffuse hyperexcitability of the motor cortex and dysfunction of the inhibitory interneurons (Sandbrink et al., 2000). Because GABA is the brain's predominant inhibitory neurotransmitter, dysfunction of the GABAergic pathways could explain the muscle hyperactivity and the motor cortex hyperexcitability (Meinck et al., 1984; Sandbrink et al., 2000). Indeed, there is a reduction of GABA levels in the patients' cerebrospinal fluid (Dalakas et al., 2001) and brain (Levy et al., 2005), and strong evidence of autoimmunity directed against molecules in the GABAergic synapse (Fig. 1) that may impair GABA-ergic neurotransmission. The most common antigenic target is GAD65 (glutamic acid decarboxylase), the rate-limiting enzyme for the synthesis of GABA, because high titers of GAD antibodies are seen in the majority of the patients (Burbelo et al., 2008; Dalakas et al., 2001; Murinson et al., 2004; Solimena et al., 1988). Another autoantibody,
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Table 1 The spectrum and specificity of antibodies in stiff-person syndrome. 1. High titers anti-GAD antibodies (>10 times, compared to the low titers seen in diabetes) (Alexopoulos and Dalakas, 2010; Dalakas, 2009) - Seen in 80% of SPS (recognize linear epitopes of GAD) - Limbic encephalitis - Myoclonus and temporal lobe epilepsy - Progressive encephalomyelitis with rigidity and myoclobus (PERM) - Cerebellar ataxia - impaired eye movements with nystagmus and abnormal saccades - Neuromyotonia - Batten's disease and in the CLN3 knockout mice, the animal model of Batten's disease 2. Low-titers of anti-GAD antibodies (Kim et al., 1994) - Insulin dependent diabetes mellitus (recognize conformational GAD epitopes) - Other autoimmune disorders (of unclear significance) 3. Anti-GABARAP (Raju et al., 2006) - Seen in up to 65% of SPS (in one series) 4. Anti-amphiphysin (De Camilli et al., 1993) - Seen in up to 5% of paraneoplastic SPS 5. Anti-gephyrin (Butler et al., 2000) - Seen in a single case of paraneoplastic SPS 6. Anti-glycin receptor alpha 1 subunit (GlyRa1) antibodies (Alexopoulos et al., in press; McKeon et al., 2013) - Seen in up to 10% of SPS (especially those with prominent spasms and phobias)
directed against the GABA-receptor-associated protein (GABARAP) (Fig. 1), is seen in more than 50% of the patients and may be of pathogenic significance because sera with high anti-GABARAP antibodies inhibit GABAA receptor expression on the axonal processes and may impair the stability of GABAA receptors (Raju et al., 2006). Two additional autoantibodies, seen in paraneoplastic SPS, are also connected with the
GABAergic transmission; one against amphiphysin seen in 5% of SPS patients, and another against gephyrin detected in a single case (Butler et al., 2000; De Camilli et al., 1993). Not only the GABAergic pathways, but also the glycinergic inhibitory pathways may be involved because antibodies against the GlyR(alpha)1 (glycine alpha 1 receptors) (Fig. 1) are seen in 10–12% of SPS patients (Alexopoulos et al., in press; McKeon et al., 2013) and in rare cases of progressive encephalomyelitis with rigidity and myoclonus (Hutchinson et al., 2008). The GlyRa1 receptors are mostly expressed in the spinal cord, brainstem and cerebellum and may be relevant to the hypersensitivity and spasms seen in SPS patients because mutations in the GlyR cause hereditary hyperekplexia (Chung et al., 2010). In our series, 6 of the 8 anti-GlyRa1-positive SPS patients had severe hypersensitivity and spasms. All the aforementioned antibodies are currently serving as immune and diagnostic markers for the disease and, although their presence indicates that SPS is an autoimmune disease, their pathogenic role has been questioned, as discussed below. The pathogenic role of GAD antibodies: not enough evidence It has been hypothesized that GAD antibodies may be pathogenic because anti-GAD-specific IgG from SPS inhibits GAD activity in vitro and impairs the synthesis of GABA (Dinkel et al., 1998; Raju et al., 2005). The degree of inhibition, although dose dependent, is however unrelated to GAD antibody titers (Raju et al., 2005). Intracerebral and paraspinal infusions of purified IgG from GAD-positive patients to anesthetized rats, inhibit the corticomotor response and raise the motor neuron excitability, suggesting pathogenicity (Manto et al., 2007). Anti-GAD antibodies are also produced intrathecally, but there is no correlation
Fig. 1. Autoantigens associated with GABAergic synaptic transmission in patients with stiff person syndrome (SPS). The pre-synaptic antigens are glutamic acid decarboxylase (GAD) (1), the enzyme that synthesizes GABA and amphiphysin (2), a synaptic vesicle protein responsible for endocytosis of vesicle plasma membranes following GABA release. In the post-synaptic membrane, the target antigens are gephyrin (3) and GABA-A receptor-associated protein (GABARAP) (4). Gephyrin is a tubulin-binding protein needed for clustering the receptors of both inhibitory neurotransmitters, the glycine receptors and the GABA-A receptors in the brain. GABARAP is a linker protein between gephyrin and GABA-A receptors and promotes the recruitment of gephyrin and organization of the GABA receptors. The most common autoantigen in SPS is GAD, seen in 85% of patients, followed by GABARAP, seen in 65%. Amphiphysin is seen in 5% of patients, and gephyrin has been seen in only one case. Recently, antibodies against the GlyRalpha1 protein have been identified in up to 10% of patients with GAD-positive SPS. Modified from Dalakas (2009).
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between the serum and the cerebrospinal fluid GAD-specific IgG index with disease severity or symptom duration even within individual patients followed over time (Rakocevic et al., 2004); monitoring GAD antibody titers during the course of SPS is therefore not meaningful. Electrophysiological studies have demonstrated that the magnitude of intracortical facilitation is associated with high levels of GAD antibodies in the CSF (Koerner et al., 2004), as discussed later. Anti-GAD antibodies are not however unique to SPS because they are also seen in rare patients with diverse clinical symptomatology such as cerebellar ataxia, myoclonus, epilepsy, Batten's disease, limbic encephalitis or abnormal eye movements (Table 1) (Nemni et al., 2004; Rakocevic et al., 2006; Saiz et al., 2008; Vianello et al., 2008), suggesting that they either recognize different disease-specific GAD epitopes within the CNS or, more likely, they do not define a unique clinical phenotype. Low titers of non-pathogenic anti-GAD antibodies are also seen in at least 35% of insulin dependent diabetes mellitus (IDDM) because GAD is also present on the beta cells of the pancreas; in IDDM, the antibodies are against conformational GAD epitopes whereas in SPS they are against linear epitopes (Kim et al., 1994; Reetz et al., 1991; Solimena et al., 1994). Additional concerns casting doubt on the pathogenic role of GAD antibodies are based on the following observations: a) GAD65 antibodies do not transfer the disease from mothers to infants (Burns et al., 2003); b) GAD-specific T cells are inconsistently present in only a small number of SPS patients (Raju and Dalakas, unpublished data); c) GAD65, like the other antibodies mentioned earlier (against amphiphysin, gephyrin, GABARAP and GlyRa1), are cytosolic and the fundamental mechanism by which antibodies against such intracellular targets can cause disease is unclear (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Levy et al., 1999; Vincent, 2008); d) SPS has not been produced in animals after immunization with purified GAD; and e) GAD-specific SPS has not been successfully transmitted to experimental animals using patients' sera; this is in contrast to amphiphysin-specific SPS IgG that passively transferred some SPS symptomatology to experimental rats (Sommer et al., 2005). The paper by Hansen et al. (2013), along with a similar paper published by the same authors one year ago (Geis et al., 2011), attempts to fill the missing gap by providing important information on whether GAD-specific IgG from an SPS patient is pathogenic. This group, headed by Somner and Toyka, has unique expertise in this field because they have been successful in transferring SPS symptomatology associated with amphiphysin IgG to experimental rats. Although the present methodology lacks the impressive and technically demanding electrophysiological complexity of their previous work and uses intracerebral instead of systemic injections, it does provide evidence that GAD-IgG can transfer some SPS-like behavior. The topic is of great immunological and neurobiological interest because if GAD, or another antibody within the IgG, successfully transmits the patients' symptoms to live animals, not only proves that the implicated antibodies are pathogenic but it also provides the opportunity to explore at the molecular level how a circulating antibody can reach intracellular targets. Does serum IgG with high anti-GAD-antibodies transfer SPS symptomatology? Hansen et al. (2013) injected the purified IgG fraction from one SPS patient with high anti-GAD titers and severe motor impairment, but without anxious comorbidity, into the lateral ventricle (i.c.v.) or intrathecally (i.th.) in experimental rats. Serum IgG from one CIDP patient served as control. Animals injected i.c.v. with SPS-IgG, but not control IgG, showed stiffness-like behavior with impaired walking ability and reduced grip strength; like the patient, the rats did not exhibit anxiety behavior. The injected SPS-IgG was localized predominantly in GABAergic interneurons in globus pallidus, internal capsule, striatum and anterior thalamus. In contrast, rats injected i.th. did not exhibit any obvious motor symptoms or abnormal synaptic transmission at
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the spinal level, suggesting that the induced GABAergic dysfunction was in the supraspinal motor pathways. The study is clearly a step forward in suggesting that IgG- anti GAD antibodies may be pathogenic in situ. The data, a compilation of clinical, electrophysiological and neuropathological observations, clearly advance our understanding on the role of these antibodies in SPS but generate a number of questions critical for the design of future experiments, as discussed below. Symptomatology As mentioned earlier, SPS is a syndrome comprising a constellation of symptoms including stiffness, muscle spasms and phobic anxiety that fluctuate from day to day and are influenced by various external factors difficult to control for. Observing and quantifying these fluctuating phenomena in the tiny rats may not be easy. The authors are arguably experienced and careful by recruiting two independent observers, but it is the nature of the symptoms that poses difficulties not only in quantifying fluctuating stiffness and postural tone but also in controlling for the external factors that trigger these phenomena. The run bridge stepping apparatus is a useful tool, often used for demonstrating weakness in experimental allergic encephalomyelitis (EAE), experimental allergic neuritis (EAN) or experimental allergic myasthenia gravis (EAMG) models, but whether it can also capture the complexities of SPS symptomatology is unclear. In a previous experiment of disease transmission to rats done by the same authors using the same tools, only the anxiety, but not the motor, phenotype was “transmitted” to rats after repetitive intrathecal application of IgG from an SPS patient who had prominent anxiety (Geis et al., 2011). This is puzzling as high anti-GAD IgG from two different SPS patients seems to transfer a different disease phenotype; anxiety and emotional behavior in one and motor phenomena in the other, even though in both experiments the infused IgG bound to the same areas of the rat amygdala, hippocampus and frontal cortex. Does SPS IgG possess unique disease-causing epitopes and is behavior or anxiety antibody-mediated and transferable? Given the complexity of the experimental conditions, the heterogeneous nature of SPS patients, and the inherent unexplained variability, a larger patient sample will be needed in future experiments to assess the factors that define which phenotype is consistently transferable. Because high anti-GAD antibody titers are also seen in patients with cerebellar ataxia, myoclonus or epilepsy, it is essential to examine whether anti-GAD-IgG from such patients can also transfer their respective symptomatology. These experiments – although demanding – are necessary because ataxia and myoclonus are quantifiable and represent more common disorders where autoimmunity remains still uncertain. Clinicopathologic correlations There is no doubt that the injected animals in the Hansen et al. experiments developed clinical symptoms and neuropathological signs, but were these signs typical of SPS? The significant reduction in forelimb strength, for example, is not seen in SPS and the noted impaired walking ability and posture were not associated with stiffness or muscle spasms, the two sine qua non features of human disease. The staining of infused IgG is of interest in understanding which brain region is clinically relevant because in SPS patients brain imaging is normal and the clinical phenomena are thought to be due to a functional blockade of GABergic pathways rather than to an identifiable structural lesion (Dalakas, 2009). In both experimental studies however (Geis et al., 2011; Hansen et al., 2013), the infused IgG immunolocalized in areas that did not correlate with the observed symptomatology because the same staining pattern in the amygdala, dentate gyrus and hippocampus, areas associated with abnormal behavior, was seen not only in rats exhibiting motor symptoms and no anxiety (Hansen et al., 2013), but also in the rats exhibiting only anxiety and no motor phenomena (Geis et al., 2011). It is possible
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that the more specific staining for GABAergic interneurons, noted with i.c.v injections in the rat globus pallidus, capsule and anterior thalamus, might best define the pathogenetically relevant GABAergic pathways corresponding to motor phenomena, but this possibility requires more studies. In human SPS, other rich-in-GABA brain regions, such as cerebellum, temporal or motor cortex, were the most relevant because they exhibited reduced levels of GABA in vivo as measured with magnetic resonance spectroscopy (Levy et al., 2005). Since the specific area responsible for SPS symptomatology remains still elusive, additional experiments with IgG from more patients are needed for clinicopathologic and functional correlations. Electrophysiology An interesting observation in the Hansen study was the transfer of disease only with i.c.v. but not with i.th. injections. This, along with the lack of inhibition of GABAergic presynaptic transmission based on H-reflex recordings that measure excitability of spinal neurons, supports suprasegmental inhibition, a conclusion in agreement with the paired pulse transcranial magnetic stimulation studies performed in SPS patients (Sandbrink et al., 2000). In human SPS, there is normal spinal neuronal excitability but markedly increased intracortical facilitation, due to excitability in the intracortical circuits (Sandbrink et al., 2000), that has been attributed to GAD antibodies (Koerner et al., 2004). There is also an interesting difference in the electrophysiology between the GAD-IgG and amphiphysin-IgG transfer models as performed by the same group. In contrast to GAD-IgG that induced GABAergic dysfunction in the supraspinal motor pathways, the antiamphiphysin IgG diminished GABAergic inhibition at the spinal cord level (Geis et al., 2009, 2010), implying different mechanisms for stiffness between amphiphysin-SPS and GAD-SPS. Although this is curious because both, amphiphysin and GAD, are more abundant in the brain than the spinal cord, inhibition limited to the spinal circuits may explain the less severe symptomatology we see clinically in SPS patients with anti-amphiphysin antibodies compared to SPS with anti-GAD antibodies. Routes of transfer In spite of the enlightening observations of Hansen et al. (2013) one would have liked to see a real transmission via the systemic delivery, which best mimics the human disease as autosensitization takes place in the periphery and then affects the brain. The pathogenicity of antibodies in most antibody-mediated conditions has been proven by the systemic passive transfer models (Vincent, 2008). Breaking the blood brain barrier (osmotically or via encephalitogenic T cells) will not be difficult and the present authors have been successful in these types of experiments. It is of concern however why this classic route of disease transfer that proves beyond any doubt the pathogenicity of circulation antibodies, has not been so far successful with GAD IgG. The intrathecal GAD antibody synthesis, presumably by GAD-producing plasma cells sheltered within the CNS, may explain disease chronicity but it does not prove a causal effect of GAD-IgG, as autosensitization takes place in the periphery. Impact and relevance of high anti-GAD antibody titers Whether in the Hansen et al. experiment, the impact of disease transfer was related to the high-GAD IgG titers is unclear because in SPS patients there is no correlation between GAD-antibody titers, even those produced intrathecally, with clinical symptomatology. Injecting enough of GAD-specific IgG therefore, may not be sufficient to prove anti-GAD-pathogenicity. Perhaps more pertinent information could have been obtained if the transfer experiments had been repeated comparing the i.cv effect of pre-treatment with the postrecovery IgG. Because anti-GAD titers do not substantially change
after successful therapy, such an experiment could have explained what is the main culprit, the GAD-specific IgG or the IgG against another GABAergic antigen. The future The seminal observations made not only by Hansen et al. (2013) but by the whole Wurzburg group headed by Somner and Toyka in a series of studies performed in the last 3 years, opens the way for further work. Transferring only a subset of symptoms suggests that more GAD antibody-positive patients should be tested not only with SPS but also with the other GAD-antibody-positive disorders such as myoclonus, seizures, cerebellar ataxia or limbic encephalitis that present with symptomatology easier to document and quantify. Exploring the effect of internalized GAD-IgG at the cell firing in vitro may help us understand the molecular events of neuronal excitability. In SPS we do not seem to have structural changes in the brain, implying a functional blockade by the putative pathogenic antibodies necessitating more clinicopathologic correlations. Does anti-GAD or other SPS-IgG inhibit certain cellular machineries within the GABAergic neurons leading to dysfunction in GABAergic synapse but not to actual loss of GABA neurons? Most importantly, the question as to whether anti-GAD IgG is the responsible autoantigen remains still uncertain. Some of the molecules, alone or with their anchoring proteins depicted in the figure, need to be explored; it is likely that the extracellular domain of these or other related proteins might be the autoantigenic targets inducing disease. In spite of the progress made and the fascinating work done by this group, the hunt for the pathogenic antibody in SPS still continues. References Alexopoulos, H., Dalakas, M.C., 2010. A critical update on the immunopathogenesis of stiff person syndrome. Eur. J. Clin. Invest. 40 (11), 1018–1025. Alexopoulos, H., Akrivou, S., Dalakas, M.C., in press. Search for anti-Glycine receptor antibodies in Stiff-Person Syndrome (SPS). Neurology. Ameli, R., Snow, J., Rakocevic, G., Dalakas, M.C., 2005. A neuropsychological assessment of phobias in patients with stiff person syndrome. Neurology 64, 1961–1963. 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. Burns, T.M., Jones, H.R., Phillips II, L.H., Bugawan, T.L., Erlich, H.A., Lennon, V.A., 2003. Clinically disparate stiff-person syndrome with GAD65 autoantibody in a father and daughter. Neurology 61, 1291–1293. Butler, M.H., Hayashi, A., Ohkoshi, N., Villmann, C., Becker, C.M., Feng, G., De Camilli, P., Solimena, M., 2000. Autoimmunity to gephyrin in stiff-man syndrome. Neuron 26, 307–312. Chung, S.K., Vanbellinghen, J.F., Mullins, J.G., Robinson, A., Hantke, J., Hammond, C.L., Gilbert, D.F., Freilinger, M., Ryan, M., Kruer, M.C., Masri, A., Gurses, C., Ferrie, C., Harvey, K., Shiang, R., Christodoulou, J., Andermann, F., Andermann, E., Thomas, R.H., Harvey, R.J., Lynch, J.W., Rees, M.I., 2010. Pathophysiological mechanisms of dominant and recessive GLRA1 mutations in hyperekplexia. J. Neurosci. 30 (28), 9612–9620. 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., Bates, D., Cartlidge, N., Solimena, M., Kilimann, M.W., 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 (6), 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 stiff-man syndrome. Ann. Neurol. 44, 194–201. Folli, F., Solimena, M., Cofiell, R., Austoni, M., Tallini, G., Fassetta, G., Bates, D., Cartlidge, N., Bottazzo, G.F., Piccolo, G., De Camilli, P., et al., 1993. Autoantibodies to a 128-kd synaptic protein in three women with the stiff-man syndrome and breast cancer. N. Engl. J. Med. 328, 546–551. Geis, C., Beck, M., Jablonka, S., Weishaupt, A., Toyka, K.V., Sendtner, M., Sommer, C., 2009. Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca(2+)]i rise in embryonic motoneurons. Neurobiol. Dis. 36, 191–199. Geis, C., Weishaupt, A., Hallermann, S., Grünewald, B., Wessig, C., Wultsch, T., Reif, A., Byts, N., Beck, M., Jablonka, S., Boettger, M.K., Üçeyler, N., Fouquet, W., Gerlach,
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Commentary
Progress and stiff challenges in understanding the role of GAD-antibodies in stiff-person syndrome Marinos C. Dalakas ⁎ Thomas Jefferson University, Philadelphia, PA, USA Neuroimmunology Unit, University of Athens Medical School, Athens, Greece
a r t i c l e
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Article history: Received 27 December 2012 Revised 4 February 2013 Accepted 18 February 2013 Available online 26 February 2013
Introduction Stiff-person syndrome (SPS) is an autoimmune CNS disorder characterized by three sets of symptoms: 1) stiffness of truncal and proximal limb muscles due to continuous co-contracture of agonist and antagonist muscles resulting in hyperlordosis, difficulty in bending or turning, and slow, wide-base gait in an effort to improve balance; 2) episodic spasms, superimposed on the stiffness, precipitated by sudden unexpected noises, tactile and visual stimuli, or emotional upset; and 3) overt anxiety and task-specific phobias, that may lead to the erroneous diagnosis of a primary anxiety disorder (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Levy et al., 1999). If anxiety dominates the clinical picture, SPS is discovered in retrospect when the administration of anti-anxiety agents, such as diazepam or araprazolam, improves the motor symptoms. Although the phobias seem to stem from a realistic fear of falling caused by the stiffness and spasms (Ameli et al., 2005), a primary anxiety disorder due to dysfunction of the CNS inhibitory pathways, cannot be excluded. The symptoms vary in severity, from mild to severe, and can be fluctuating or fixed leading to disability. Up to 65% of SPS patients cannot independently perform daily activities because of body stiffness, phobias, anxiety-triggered spasms, and frequent falls; others use walkers or wheelchairs, and still others are bedridden due to severe stiffness (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Levy et al., 1999). At times, the spasms are prominent and continuous and, if respiratory and thoracic paraspinal muscles are affected, may cause breathing difficulty and autonomic release phenomena (“status spasticus”) requiring admission to the intensive care unit, intravenous
⁎ Neuroimmunology Unit, Dept of Pathophysiology, National University of Athens Medical School, 75 Mikras Asias Str., Athens, 11527 Greece. Fax: + 30 210 746 2664. E-mail address: [email protected]. 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.02.010
diazepam administration, hydration and supportive care (Dalakas, 2009; Dalakas et al., 2000). The diagnosis of SPS is clinical, based on the aforementioned symptomatology, the exclusion of other neurological diseases that could explain stiffness and rigidity, and the absence of extrapyramidal or pyramidal tract signs. The diagnosis is aided by normal MRI imaging of the brain and spinal cord; the electrophysiological recordings of low-frequency motor unit firing at rest, in spite of voluntary relaxation, simultaneously from agonist and antagonist muscles (normally, when the agonist muscles contract, their respective antagonists are in a state of relaxation with no electrical activity); and the presence of high-titer antibodies against glutamic acid decarboxylase (GAD)-65 (or any of the antibodies listed in Table 1 and depicted in Fig. 1) (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Folli et al., 1993; Levy et al., 1999; Murinson et al., 2004; Solimena et al., 1988). SPS is frequently associated with other autoimmune diseases; among more than 60 patients we have followed, diabetes, thyroiditis, vitiligo and pernicious anemia were the most common, with type I diabetes occurring in up to 35% of the patients (Dalakas et al., 2000). In 5% of patients, SPS is paraneoplastic associated with anti-amphiphysin or anti-gephyrin antibodies (Butler et al., 2000; Folli et al., 1993). Pathogenesis of SPS: the involvement of GABAergic inhibition The concurrent contraction of agonist and antagonist muscles and the continuous motor unit firing at rest, in spite of voluntary relaxation, suggest lack of reciprocal inhibition. The inhibition is most likely suprasegmental, as determined by paired pulse transcranial magnetic stimulation studies, implying diffuse hyperexcitability of the motor cortex and dysfunction of the inhibitory interneurons (Sandbrink et al., 2000). Because GABA is the brain's predominant inhibitory neurotransmitter, dysfunction of the GABAergic pathways could explain the muscle hyperactivity and the motor cortex hyperexcitability (Meinck et al., 1984; Sandbrink et al., 2000). Indeed, there is a reduction of GABA levels in the patients' cerebrospinal fluid (Dalakas et al., 2001) and brain (Levy et al., 2005), and strong evidence of autoimmunity directed against molecules in the GABAergic synapse (Fig. 1) that may impair GABA-ergic neurotransmission. The most common antigenic target is GAD65 (glutamic acid decarboxylase), the rate-limiting enzyme for the synthesis of GABA, because high titers of GAD antibodies are seen in the majority of the patients (Burbelo et al., 2008; Dalakas et al., 2001; Murinson et al., 2004; Solimena et al., 1988). Another autoantibody,
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Table 1 The spectrum and specificity of antibodies in stiff-person syndrome. 1. High titers anti-GAD antibodies (>10 times, compared to the low titers seen in diabetes) (Alexopoulos and Dalakas, 2010; Dalakas, 2009) - Seen in 80% of SPS (recognize linear epitopes of GAD) - Limbic encephalitis - Myoclonus and temporal lobe epilepsy - Progressive encephalomyelitis with rigidity and myoclobus (PERM) - Cerebellar ataxia - impaired eye movements with nystagmus and abnormal saccades - Neuromyotonia - Batten's disease and in the CLN3 knockout mice, the animal model of Batten's disease 2. Low-titers of anti-GAD antibodies (Kim et al., 1994) - Insulin dependent diabetes mellitus (recognize conformational GAD epitopes) - Other autoimmune disorders (of unclear significance) 3. Anti-GABARAP (Raju et al., 2006) - Seen in up to 65% of SPS (in one series) 4. Anti-amphiphysin (De Camilli et al., 1993) - Seen in up to 5% of paraneoplastic SPS 5. Anti-gephyrin (Butler et al., 2000) - Seen in a single case of paraneoplastic SPS 6. Anti-glycin receptor alpha 1 subunit (GlyRa1) antibodies (Alexopoulos et al., in press; McKeon et al., 2013) - Seen in up to 10% of SPS (especially those with prominent spasms and phobias)
directed against the GABA-receptor-associated protein (GABARAP) (Fig. 1), is seen in more than 50% of the patients and may be of pathogenic significance because sera with high anti-GABARAP antibodies inhibit GABAA receptor expression on the axonal processes and may impair the stability of GABAA receptors (Raju et al., 2006). Two additional autoantibodies, seen in paraneoplastic SPS, are also connected with the
GABAergic transmission; one against amphiphysin seen in 5% of SPS patients, and another against gephyrin detected in a single case (Butler et al., 2000; De Camilli et al., 1993). Not only the GABAergic pathways, but also the glycinergic inhibitory pathways may be involved because antibodies against the GlyR(alpha)1 (glycine alpha 1 receptors) (Fig. 1) are seen in 10–12% of SPS patients (Alexopoulos et al., in press; McKeon et al., 2013) and in rare cases of progressive encephalomyelitis with rigidity and myoclonus (Hutchinson et al., 2008). The GlyRa1 receptors are mostly expressed in the spinal cord, brainstem and cerebellum and may be relevant to the hypersensitivity and spasms seen in SPS patients because mutations in the GlyR cause hereditary hyperekplexia (Chung et al., 2010). In our series, 6 of the 8 anti-GlyRa1-positive SPS patients had severe hypersensitivity and spasms. All the aforementioned antibodies are currently serving as immune and diagnostic markers for the disease and, although their presence indicates that SPS is an autoimmune disease, their pathogenic role has been questioned, as discussed below. The pathogenic role of GAD antibodies: not enough evidence It has been hypothesized that GAD antibodies may be pathogenic because anti-GAD-specific IgG from SPS inhibits GAD activity in vitro and impairs the synthesis of GABA (Dinkel et al., 1998; Raju et al., 2005). The degree of inhibition, although dose dependent, is however unrelated to GAD antibody titers (Raju et al., 2005). Intracerebral and paraspinal infusions of purified IgG from GAD-positive patients to anesthetized rats, inhibit the corticomotor response and raise the motor neuron excitability, suggesting pathogenicity (Manto et al., 2007). Anti-GAD antibodies are also produced intrathecally, but there is no correlation
Fig. 1. Autoantigens associated with GABAergic synaptic transmission in patients with stiff person syndrome (SPS). The pre-synaptic antigens are glutamic acid decarboxylase (GAD) (1), the enzyme that synthesizes GABA and amphiphysin (2), a synaptic vesicle protein responsible for endocytosis of vesicle plasma membranes following GABA release. In the post-synaptic membrane, the target antigens are gephyrin (3) and GABA-A receptor-associated protein (GABARAP) (4). Gephyrin is a tubulin-binding protein needed for clustering the receptors of both inhibitory neurotransmitters, the glycine receptors and the GABA-A receptors in the brain. GABARAP is a linker protein between gephyrin and GABA-A receptors and promotes the recruitment of gephyrin and organization of the GABA receptors. The most common autoantigen in SPS is GAD, seen in 85% of patients, followed by GABARAP, seen in 65%. Amphiphysin is seen in 5% of patients, and gephyrin has been seen in only one case. Recently, antibodies against the GlyRalpha1 protein have been identified in up to 10% of patients with GAD-positive SPS. Modified from Dalakas (2009).
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between the serum and the cerebrospinal fluid GAD-specific IgG index with disease severity or symptom duration even within individual patients followed over time (Rakocevic et al., 2004); monitoring GAD antibody titers during the course of SPS is therefore not meaningful. Electrophysiological studies have demonstrated that the magnitude of intracortical facilitation is associated with high levels of GAD antibodies in the CSF (Koerner et al., 2004), as discussed later. Anti-GAD antibodies are not however unique to SPS because they are also seen in rare patients with diverse clinical symptomatology such as cerebellar ataxia, myoclonus, epilepsy, Batten's disease, limbic encephalitis or abnormal eye movements (Table 1) (Nemni et al., 2004; Rakocevic et al., 2006; Saiz et al., 2008; Vianello et al., 2008), suggesting that they either recognize different disease-specific GAD epitopes within the CNS or, more likely, they do not define a unique clinical phenotype. Low titers of non-pathogenic anti-GAD antibodies are also seen in at least 35% of insulin dependent diabetes mellitus (IDDM) because GAD is also present on the beta cells of the pancreas; in IDDM, the antibodies are against conformational GAD epitopes whereas in SPS they are against linear epitopes (Kim et al., 1994; Reetz et al., 1991; Solimena et al., 1994). Additional concerns casting doubt on the pathogenic role of GAD antibodies are based on the following observations: a) GAD65 antibodies do not transfer the disease from mothers to infants (Burns et al., 2003); b) GAD-specific T cells are inconsistently present in only a small number of SPS patients (Raju and Dalakas, unpublished data); c) GAD65, like the other antibodies mentioned earlier (against amphiphysin, gephyrin, GABARAP and GlyRa1), are cytosolic and the fundamental mechanism by which antibodies against such intracellular targets can cause disease is unclear (Alexopoulos and Dalakas, 2010; Dalakas, 2009; Dalakas et al., 2000; Levy et al., 1999; Vincent, 2008); d) SPS has not been produced in animals after immunization with purified GAD; and e) GAD-specific SPS has not been successfully transmitted to experimental animals using patients' sera; this is in contrast to amphiphysin-specific SPS IgG that passively transferred some SPS symptomatology to experimental rats (Sommer et al., 2005). The paper by Hansen et al. (2013), along with a similar paper published by the same authors one year ago (Geis et al., 2011), attempts to fill the missing gap by providing important information on whether GAD-specific IgG from an SPS patient is pathogenic. This group, headed by Somner and Toyka, has unique expertise in this field because they have been successful in transferring SPS symptomatology associated with amphiphysin IgG to experimental rats. Although the present methodology lacks the impressive and technically demanding electrophysiological complexity of their previous work and uses intracerebral instead of systemic injections, it does provide evidence that GAD-IgG can transfer some SPS-like behavior. The topic is of great immunological and neurobiological interest because if GAD, or another antibody within the IgG, successfully transmits the patients' symptoms to live animals, not only proves that the implicated antibodies are pathogenic but it also provides the opportunity to explore at the molecular level how a circulating antibody can reach intracellular targets. Does serum IgG with high anti-GAD-antibodies transfer SPS symptomatology? Hansen et al. (2013) injected the purified IgG fraction from one SPS patient with high anti-GAD titers and severe motor impairment, but without anxious comorbidity, into the lateral ventricle (i.c.v.) or intrathecally (i.th.) in experimental rats. Serum IgG from one CIDP patient served as control. Animals injected i.c.v. with SPS-IgG, but not control IgG, showed stiffness-like behavior with impaired walking ability and reduced grip strength; like the patient, the rats did not exhibit anxiety behavior. The injected SPS-IgG was localized predominantly in GABAergic interneurons in globus pallidus, internal capsule, striatum and anterior thalamus. In contrast, rats injected i.th. did not exhibit any obvious motor symptoms or abnormal synaptic transmission at
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the spinal level, suggesting that the induced GABAergic dysfunction was in the supraspinal motor pathways. The study is clearly a step forward in suggesting that IgG- anti GAD antibodies may be pathogenic in situ. The data, a compilation of clinical, electrophysiological and neuropathological observations, clearly advance our understanding on the role of these antibodies in SPS but generate a number of questions critical for the design of future experiments, as discussed below. Symptomatology As mentioned earlier, SPS is a syndrome comprising a constellation of symptoms including stiffness, muscle spasms and phobic anxiety that fluctuate from day to day and are influenced by various external factors difficult to control for. Observing and quantifying these fluctuating phenomena in the tiny rats may not be easy. The authors are arguably experienced and careful by recruiting two independent observers, but it is the nature of the symptoms that poses difficulties not only in quantifying fluctuating stiffness and postural tone but also in controlling for the external factors that trigger these phenomena. The run bridge stepping apparatus is a useful tool, often used for demonstrating weakness in experimental allergic encephalomyelitis (EAE), experimental allergic neuritis (EAN) or experimental allergic myasthenia gravis (EAMG) models, but whether it can also capture the complexities of SPS symptomatology is unclear. In a previous experiment of disease transmission to rats done by the same authors using the same tools, only the anxiety, but not the motor, phenotype was “transmitted” to rats after repetitive intrathecal application of IgG from an SPS patient who had prominent anxiety (Geis et al., 2011). This is puzzling as high anti-GAD IgG from two different SPS patients seems to transfer a different disease phenotype; anxiety and emotional behavior in one and motor phenomena in the other, even though in both experiments the infused IgG bound to the same areas of the rat amygdala, hippocampus and frontal cortex. Does SPS IgG possess unique disease-causing epitopes and is behavior or anxiety antibody-mediated and transferable? Given the complexity of the experimental conditions, the heterogeneous nature of SPS patients, and the inherent unexplained variability, a larger patient sample will be needed in future experiments to assess the factors that define which phenotype is consistently transferable. Because high anti-GAD antibody titers are also seen in patients with cerebellar ataxia, myoclonus or epilepsy, it is essential to examine whether anti-GAD-IgG from such patients can also transfer their respective symptomatology. These experiments – although demanding – are necessary because ataxia and myoclonus are quantifiable and represent more common disorders where autoimmunity remains still uncertain. Clinicopathologic correlations There is no doubt that the injected animals in the Hansen et al. experiments developed clinical symptoms and neuropathological signs, but were these signs typical of SPS? The significant reduction in forelimb strength, for example, is not seen in SPS and the noted impaired walking ability and posture were not associated with stiffness or muscle spasms, the two sine qua non features of human disease. The staining of infused IgG is of interest in understanding which brain region is clinically relevant because in SPS patients brain imaging is normal and the clinical phenomena are thought to be due to a functional blockade of GABergic pathways rather than to an identifiable structural lesion (Dalakas, 2009). In both experimental studies however (Geis et al., 2011; Hansen et al., 2013), the infused IgG immunolocalized in areas that did not correlate with the observed symptomatology because the same staining pattern in the amygdala, dentate gyrus and hippocampus, areas associated with abnormal behavior, was seen not only in rats exhibiting motor symptoms and no anxiety (Hansen et al., 2013), but also in the rats exhibiting only anxiety and no motor phenomena (Geis et al., 2011). It is possible
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that the more specific staining for GABAergic interneurons, noted with i.c.v injections in the rat globus pallidus, capsule and anterior thalamus, might best define the pathogenetically relevant GABAergic pathways corresponding to motor phenomena, but this possibility requires more studies. In human SPS, other rich-in-GABA brain regions, such as cerebellum, temporal or motor cortex, were the most relevant because they exhibited reduced levels of GABA in vivo as measured with magnetic resonance spectroscopy (Levy et al., 2005). Since the specific area responsible for SPS symptomatology remains still elusive, additional experiments with IgG from more patients are needed for clinicopathologic and functional correlations. Electrophysiology An interesting observation in the Hansen study was the transfer of disease only with i.c.v. but not with i.th. injections. This, along with the lack of inhibition of GABAergic presynaptic transmission based on H-reflex recordings that measure excitability of spinal neurons, supports suprasegmental inhibition, a conclusion in agreement with the paired pulse transcranial magnetic stimulation studies performed in SPS patients (Sandbrink et al., 2000). In human SPS, there is normal spinal neuronal excitability but markedly increased intracortical facilitation, due to excitability in the intracortical circuits (Sandbrink et al., 2000), that has been attributed to GAD antibodies (Koerner et al., 2004). There is also an interesting difference in the electrophysiology between the GAD-IgG and amphiphysin-IgG transfer models as performed by the same group. In contrast to GAD-IgG that induced GABAergic dysfunction in the supraspinal motor pathways, the antiamphiphysin IgG diminished GABAergic inhibition at the spinal cord level (Geis et al., 2009, 2010), implying different mechanisms for stiffness between amphiphysin-SPS and GAD-SPS. Although this is curious because both, amphiphysin and GAD, are more abundant in the brain than the spinal cord, inhibition limited to the spinal circuits may explain the less severe symptomatology we see clinically in SPS patients with anti-amphiphysin antibodies compared to SPS with anti-GAD antibodies. Routes of transfer In spite of the enlightening observations of Hansen et al. (2013) one would have liked to see a real transmission via the systemic delivery, which best mimics the human disease as autosensitization takes place in the periphery and then affects the brain. The pathogenicity of antibodies in most antibody-mediated conditions has been proven by the systemic passive transfer models (Vincent, 2008). Breaking the blood brain barrier (osmotically or via encephalitogenic T cells) will not be difficult and the present authors have been successful in these types of experiments. It is of concern however why this classic route of disease transfer that proves beyond any doubt the pathogenicity of circulation antibodies, has not been so far successful with GAD IgG. The intrathecal GAD antibody synthesis, presumably by GAD-producing plasma cells sheltered within the CNS, may explain disease chronicity but it does not prove a causal effect of GAD-IgG, as autosensitization takes place in the periphery. Impact and relevance of high anti-GAD antibody titers Whether in the Hansen et al. experiment, the impact of disease transfer was related to the high-GAD IgG titers is unclear because in SPS patients there is no correlation between GAD-antibody titers, even those produced intrathecally, with clinical symptomatology. Injecting enough of GAD-specific IgG therefore, may not be sufficient to prove anti-GAD-pathogenicity. Perhaps more pertinent information could have been obtained if the transfer experiments had been repeated comparing the i.cv effect of pre-treatment with the postrecovery IgG. Because anti-GAD titers do not substantially change
after successful therapy, such an experiment could have explained what is the main culprit, the GAD-specific IgG or the IgG against another GABAergic antigen. The future The seminal observations made not only by Hansen et al. (2013) but by the whole Wurzburg group headed by Somner and Toyka in a series of studies performed in the last 3 years, opens the way for further work. Transferring only a subset of symptoms suggests that more GAD antibody-positive patients should be tested not only with SPS but also with the other GAD-antibody-positive disorders such as myoclonus, seizures, cerebellar ataxia or limbic encephalitis that present with symptomatology easier to document and quantify. Exploring the effect of internalized GAD-IgG at the cell firing in vitro may help us understand the molecular events of neuronal excitability. In SPS we do not seem to have structural changes in the brain, implying a functional blockade by the putative pathogenic antibodies necessitating more clinicopathologic correlations. Does anti-GAD or other SPS-IgG inhibit certain cellular machineries within the GABAergic neurons leading to dysfunction in GABAergic synapse but not to actual loss of GABA neurons? Most importantly, the question as to whether anti-GAD IgG is the responsible autoantigen remains still uncertain. Some of the molecules, alone or with their anchoring proteins depicted in the figure, need to be explored; it is likely that the extracellular domain of these or other related proteins might be the autoantigenic targets inducing disease. In spite of the progress made and the fascinating work done by this group, the hunt for the pathogenic antibody in SPS still continues. References Alexopoulos, H., Dalakas, M.C., 2010. A critical update on the immunopathogenesis of stiff person syndrome. Eur. J. Clin. Invest. 40 (11), 1018–1025. Alexopoulos, H., Akrivou, S., Dalakas, M.C., in press. Search for anti-Glycine receptor antibodies in Stiff-Person Syndrome (SPS). Neurology. Ameli, R., Snow, J., Rakocevic, G., Dalakas, M.C., 2005. A neuropsychological assessment of phobias in patients with stiff person syndrome. Neurology 64, 1961–1963. 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. Burns, T.M., Jones, H.R., Phillips II, L.H., Bugawan, T.L., Erlich, H.A., Lennon, V.A., 2003. Clinically disparate stiff-person syndrome with GAD65 autoantibody in a father and daughter. Neurology 61, 1291–1293. Butler, M.H., Hayashi, A., Ohkoshi, N., Villmann, C., Becker, C.M., Feng, G., De Camilli, P., Solimena, M., 2000. Autoimmunity to gephyrin in stiff-man syndrome. Neuron 26, 307–312. Chung, S.K., Vanbellinghen, J.F., Mullins, J.G., Robinson, A., Hantke, J., Hammond, C.L., Gilbert, D.F., Freilinger, M., Ryan, M., Kruer, M.C., Masri, A., Gurses, C., Ferrie, C., Harvey, K., Shiang, R., Christodoulou, J., Andermann, F., Andermann, E., Thomas, R.H., Harvey, R.J., Lynch, J.W., Rees, M.I., 2010. Pathophysiological mechanisms of dominant and recessive GLRA1 mutations in hyperekplexia. J. Neurosci. 30 (28), 9612–9620. 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., Bates, D., Cartlidge, N., Solimena, M., Kilimann, M.W., 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 (6), 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 stiff-man syndrome. Ann. Neurol. 44, 194–201. Folli, F., Solimena, M., Cofiell, R., Austoni, M., Tallini, G., Fassetta, G., Bates, D., Cartlidge, N., Bottazzo, G.F., Piccolo, G., De Camilli, P., et al., 1993. Autoantibodies to a 128-kd synaptic protein in three women with the stiff-man syndrome and breast cancer. N. Engl. J. Med. 328, 546–551. Geis, C., Beck, M., Jablonka, S., Weishaupt, A., Toyka, K.V., Sendtner, M., Sommer, C., 2009. Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca(2+)]i rise in embryonic motoneurons. Neurobiol. Dis. 36, 191–199. Geis, C., Weishaupt, A., Hallermann, S., Grünewald, B., Wessig, C., Wultsch, T., Reif, A., Byts, N., Beck, M., Jablonka, S., Boettger, M.K., Üçeyler, N., Fouquet, W., Gerlach,
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