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Original Investigation |

Neuronal Surface and Glutamic Acid Decarboxylase Autoantibodies in Nonparaneoplastic Stiff Person Syndrome

Thashi Chang, MD, DPhil1,6; Haris Alexopoulos, PhD1; Mary McMenamin, DPhil2; Alexander Carvajal-González, MD1; Sian K. Alexander, MD, DPhil1; Robert Deacon, PhD3; Ferenc Erdelyi, PhD4; Gabor Szabó, PhD4; Bethan Lang, PhD1; Franz Blaes, MD5; Peter Brown, FRCP1; Angela Vincent, FRCPath1
[+] Author Affiliations
1Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, England
2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, England
3Department of Experimental Psychology, University of Oxford, Oxford, England
4Department of Gene Technology and Developmental Neurobiology, Institute of Experimental Medicine, Budapest, Hungary
5Neurologische Klinik, Kreiskrankenhaus Gummersbach, Gummersbach, Germany
6currently with Department of Clinical Medicine, University of Colombo, Colombo, Sri Lanka
JAMA Neurol. 2013;70(9):1140-1147. doi:10.1001/jamaneurol.2013.3499.
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Importance  High titers of autoantibodies to glutamic acid decarboxylase (GAD) are well documented in association with stiff person syndrome (SPS). Glutamic acid decarboxylase is the rate-limiting enzyme in the synthesis of γ-aminobutyric acid (GABA), and impaired function of GABAergic neurons has been implicated in the pathogenesis of SPS. Autoantibodies to GAD might be the causative agent or a disease marker.

Objective  To investigate the characteristics and potential pathogenicity of GAD autoantibodies in patients with SPS and related disorders.

Design  Retrospective cohort study and laboratory investigation.

Setting  Weatherall Institute of Molecular Medicine, University of Oxford.

Participants  Twenty-five patients with SPS and related conditions identified from the Neuroimmunology Service.

Exposures  Neurological examination, serological characterization and experimental studies.

Main Outcomes and Measures  Characterization of serum GAD antibodies from patients with SPS and evidence for potential pathogenicity.

Results  We detected GAD autoantibodies at a very high titer (median, 7500 U/mL) in 19 patients (76%), including all 12 patients with classic SPS. The GAD autoantibodies were high affinity (antibody dissociation constant, 0.06-0.78 nmol) and predominantly IgG1 subclass. The patients’ autoantibodies co-localized with GAD on immunohistochemistry and in permeabilized cultured cerebellar GABAergic neurons, as expected, but they also bound to the cell surface of unpermeabilized GABAergic neurons. Adsorption of the highest titer (700 000 U/mL) serum with recombinant GAD indicated that these neuronal surface antibodies were not directed against GAD itself. Although intraperitoneal injection of IgG purified from the 2 available GAD autoantibody–positive purified IgG preparations did not produce clinical or pathological evidence of disease, SPS and control IgG were detected in specific regions of the mouse central nervous system, particularly around the lateral and fourth ventricles.

Conclusions and Relevance  Autoantibodies to GAD are associated with antibodies that bind to the surface of GABAergic neurons and that could be pathogenic. Moreover, in mice, human IgG from the periphery gained access to relevant areas in the hippocampus and brainstem. Identification of the target of the non-GAD antibodies and peripheral and intrathecal transfer protocols, combined with adsorption studies, should be used to demonstrate the role of the non-GAD IgG in SPS.

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Figure 1.
Glutamic Acid Decarboxylase (GAD) Autoantibody Characteristics and Immunohistochemical Staining of Rat Brain Sections With Stiff Person Syndrome (SPS) Serum

A. Typical serum GAD autoantibody titration curves for 2 representative patients with SPS (75 000 U/mL [patient 3] and 836 U/mL [patient 12]) and a healthy control using iodine 125–labeled GAD65 precipitation. B, All GAD autoantibody titers (log scale) in patients with SPS (n = 25), patients with type 1 diabetes mellitus (T1DM) (n = 5), and controls (n = 9). C, Immunoreactive dots outlining pyramidal cells in the cortex of rat brain sections (arrowheads). D, Intense immunoreactivity of lateral globus pallidus (LGP) relative to the striatum of rat brain sections. E, Staining around pyramidal cells in CA2 of the hippocampus of rat brain sections. SO indicates stratum oriens; SR, stratum radiatum. F, Dense immunoreactivity at the axon hillock (arrows) and puncta outlining the perikaryon and dendritic tree of Purkinje cells (PC) with punctate staining in the molecular (ML) and granular (GL) layers of rat brain sections. G, Representative binding curve and Scatchard plot (inset) for a serum sample from a patient with SPS. H, Subclasses of GAD autoantibody IgG in serum samples of patients seropositive for SPS (n = 19), compared with acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) antibodies.

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Figure 2.
Confocal Images of Serum IgG Binding (Red) to Permeabilized Cerebellar Neurons in Primary Cultures Derived From Glutamic Acid Decarboxylase 65–Enhanced Green Fluorescent Protein (GAD65-EGFP)–Expressing (Green) Transgenic Mice

A, Stiff person syndrome (SPS) serum antibodies bind intracellularly to EGFP-expressing neurons. B, After adsorption against GAD65, the same SPS serum (Ad SPS) still binds to the surface of the neurons. C, Normal control (NC) serum samples (red) do not bind to the neurons. D, Some serum samples showed weak nonspecific binding to nonneuronal cells. E, The SPS serum antibodies bind to the surface of EGFP- and non–EGFP-expressing neurons. F, The SPS serum binds to γ-aminobutyric acid (GABA)–expressing (blue) neurons. G, After adsorption against GAD65, the same SPS serum continues to demonstrate surface-binding antibodies. Scale bar indicates 10 µm.

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Figure 3.
Results of Passive Transfer of Stiff Person Syndrome (SPS) IgG Positive for Glutamic Acid Decarboxylase (GAD) Autoantibody and Control IgG Into Mice

Nine mice were injected with purified SPS IgG (from GAD antibody–positive patient 12 and patient 25) and compared with 9 mice injected with purified control IgG. Measurements were obtained at baseline (day 0) and days 6 (1 day after lipopolysaccharide [LPS] injection) and 9 (4 days after LPS injection). Mice were randomized on day 0 based on the burrowing test results. Lipopolysaccharide was given at days 3 and 5. A, Levels of GAD autoantibodies in the injected mice at days 10 and 18 after injection on days 1 through 5. Numbers in parentheses indicate numbers of mice undergoing sampling and testing for GAD antibodies. B, Results (mean [SEM]) of observed burrowing and accelerating rotarod test measuring mouse-specific behavior and motor power and coordination, respectively. C, Results of light-dark box test measuring anxiety and response to stressful environments. D, Results of white open-field test measuring anxiety and response to stressful environments.

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Figure 4.
Diffusion of IgG and Quantification of Enhanced Green Fluorescent Protein (EGFP)–Labeled Neurons After Stiff Person Syndrome (SPS) IgG Injection Into Mice

A, Diffusion of human IgG into the mouse brain after IgG injections. Coronal sections are seen through hippocampus (Hp), striatum (CPu), and the fourth ventricle (4V). The human IgG was found mainly in the Hp and septum, adjacent to the ventricles, and in the brainstem lateral to the 4V. B, Diffusion appeared weaker in control IgG–injected mouse brains. C, Five representative areas in which neurons were counted. D, Examples of neuronal nuclei antibody (NeuN)–positive neurons in the brainstem of mice. E, Examples of EGFP-expressing neurons in the brainstem of mice. F and G, Mean (SEM) total counts for NeuN-positive and EGFP-expressing neurons. LV indicates lateral ventricle; 3V, third ventricle. Scale bar indicates 100 µm.

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