GAD antibody‐associated neurological illness and its relationship to gluten sensitivity

Hadjivassiliou M, Aeschlimann D, Grünewald RA, Sanders DS, Sharrack B, Woodroofe N. GAD antibody associated neurological illness and its relationship to gluten sensitivity.
Acta Neurol Scand: 2011: 123: 175–180.
© 2010 The Authors Journal compilation © 2010 Blackwell Munksgaard. Background – The high prevalence of gluten sensitivity in patients with stiff‐person syndrome (SPS) lead us to investigate the relationship between gluten sensitivity and GAD‐antibody‐associated diseases. Methods – We used ELISA assays for anti‐GAD and for serological markers of gluten sensitivity. Patients were recruited from clinics based at the Royal Hallamshire hospital, Sheffield, UK. Patients with gluten sensitivity were followed up after the introduction of a gluten‐free diet and serological testing was repeated. Results – Six of seven (86%) patients with SPS were positive for anti‐GAD, mean titre 109 U/ml; This compared with 9/90 (11%) patients with idiopathic sporadic ataxia, mean titre 32 U/ml, 16/40 (40%) patients with gluten ataxia, mean titre 25 U/ml, and 6/10 patients with type 1 diabetes only, mean titre 8 U/ml. None of 32 patients with celiac disease only, and of 40 patients with genetic ataxia were positive for anti‐GAD. The titre of anti‐GAD reduced following the introduction of a gluten‐free diet in patients with SPS who had serological evidence of gluten sensitivity. The same was observed in patients with gluten ataxia and anti‐GAD antibodies. This was also associated with clinical improvement. Conclusion – These findings suggest a link between gluten sensitivity and GAD antibody‐associated diseases.     

GAD65 IgG autoantibodies in stiff person syndrome: clonality, avidity and persistence

Background and purpose:  Persistent intrathecal production of IgG autoantibodies against glutamic acid decarboxylase 65 (GAD65 IgG) and oligoclonal IgG of undetermined specificity has been reported in stiff person syndrome (SPS).
Methods:  To chart the avidity and clonal patterns of GAD65 IgG, we performed scatchard plot of binding characteristics and isoelectric focusing-immunoblot of cerebrospinal fluid (CSF) and serum from five SPS patients.
Results:  Oligoclonal GAD65 IgG bands, predominantly restricted to the IgG1 subclass, were detected in CSF and serum in all patients. The distribution of GAD65-specific IgG bands in serum and CSF revealed intrathecal synthesis of oligoclonal GAD65 IgG in all five patients, whilst radioimmunoassay demonstrated intrathecal synthesis of GAD65 IgG in four. The binding avidity of GAD65 IgG from CSF was more than 10 times higher than in serum in two of the patients but did not differ substantially in the remaining three. These differences were not related to symptom severity. The pattern of oligoclonal GAD65 IgG bands in CSF and serum in three patients examined remained unchanged for up to 7 years after symptom debut.
Conclusion:  This study confirms the persistent systemic and intrathecal production of GAD65-specific IgG in SPS, and further shows that this immune response is oligoclonal and mediated by a stable population of affinity maturated B cell clones.     

GAD65抗体相关性小脑性共济失调一例报道及文献复习

目的探讨抗谷氨酸脱羧酶65(glutamic acid decarboxylase,GAD65)抗体相关性小脑性共济失调的临床表现、治疗及预后。方法报道一例抗GAD65抗体相关性小脑性共济失调患者的临床资料,进行诊断、鉴别诊断、治疗及预后分析。结果患者为中老年男性,急性起病,缓慢进展,主要表现为步态不稳并渐出现复视、不能独立行走;神经系统体检发现双眼上视受限、双侧跟膝胫试验及双手轮替试验欠稳准;血清及脑脊液抗GAD65抗体阳性;头颅MRI未见异常萎缩及责任病灶;大剂量甲强龙冲击后症状明显减轻。结论抗GAD65抗体相关性小脑性共济失调可有脑干及小脑受累症状,是一种可以治疗性疾病,早期诊断并积极治疗有望改善预后。     

GAD在颞叶癫痫大鼠海马内源性促痫机制中的作用

目的:探讨GAD65、GAD67在颞叶癫痫发生后海马内源性促痫机制中的作用。方法:112只雄性SD大鼠随机分为实验组(n=70)与对照组(n=42),实验组大鼠选用海人酸腹腔注射法建立颞叶癫痫模型,对照组大鼠腹腔注射无菌生理盐水。选取腹腔注射后3小时、6小时、12小时、24小时、48小时、7天、30天为研究的时间点,颞叶海马的CA1区、CA3区、齿状回为研究部位。腹腔给药后每天观察大鼠的行为学变化,大鼠处死前进行EEG描记。用原位杂交方法检测不同时间点海马不同区域GAD65、GAD67mRNA的表达,免疫组织化学法检测GAD65、GAD67蛋白的表达。结果:实验组大鼠海马GAD65 mRNA及其蛋白的表达随时间呈逐渐增高趋势,致痫后48小时～30天,GAD65 mRNA及其蛋白表达较对照组增高(48小时P<0.05;7～30天P<0.01);海人酸致痫后6小时、24小时实验组大鼠海马的GAD67mRNA及其蛋白表达较对照组增高(分别为P<0.01、P<0.05)。结论:颞叶癫痫急性期海马GAD67表达的增高及慢性期海马GAD65表达的增高是癫痫发生后机体的内源性抗痫机制。     

GAD67/GAD65在颞叶癫痫大鼠海马内源性促痫机制中的作用

目的探讨GAD67/GAD65在颞叶癫痫发生后大鼠海马内源性促痫机制中的作用.方法112只雄性SD大鼠随机分为实验组(n=70)与对照组(n=42),实验组大鼠选用海人酸腹腔注射法建立颞叶癫痫模型,对照组大鼠腹腔注射无菌生理盐水.选取腹腔注射后3 h、6 h、12 h、24 h、48 h、7 d、30 d为研究的时间点,颞叶海马的CA1区、CA3区、齿状回为研究部位.腹腔给药后每天观察大鼠的行为学变化,大鼠处死前进行EEG描记.免疫组织化学法检测GAD65、GAD67蛋白的表达.结果海人酸致痫后6 h,实验组大鼠海马CA1区、CA3区GAD67/GAD65的比率较对照组升高(P＜0.01);海人酸致痫后30 d,实验组大鼠海马齿状回GAD67/GAD65的比率较对照组降低(P＜0.05).结论颞叶癫痫急性期CA1区、CA3区GAD67/GAD65比率的增高及慢性期齿状回GAD67/GAD65比率的降低与颞叶癫痫发生及癫痫发生后机体的内源性抗痫机制密切相关.     

GAD antibody positive paraneoplastic stiff person syndrome in a patient with renal cell carcinoma

Stiff person syndrome (SPS) is an unusual cause of muscle rigidity and spasms. It is believed to have an autoimmune pathogenesis and is associated with autoantibodies to glutamic acid decarboxylase (GAD). Paraneoplastic SPS (PSPS) has been described mainly in relation to breast cancer and is associated with antibodies to amphiphysin. Few reports of PSPS document the finding of GAD autoantibodies. We present the first reported case of anti‐GAD positive PSPS in a 53‐year‐old male with occult renal carcinoma. Clinical benefit was marked following nephrectomy and intravenous immunoglobulin treatment. Renal carcinoma should be considered in patients with SPS. © 2007 Movement Disorder Society     

GAD65 antibody‐associated autoimmune epilepsy with unique independent bitemporal‐onset ictal asystole

Antibodies against the 65‐kDa isoform of the intracellular enzyme, glutamate decarboxylase (GAD65), have been found in patients with limbic encephalitis and drug‐resistant autoimmune epilepsy. We report a 22‐year‐old female who presented with new‐onset seizures and neuropsychiatric symptoms. Video‐EEG captured unique, independent bitemporal‐onset focal seizures with impaired awareness and ictal asystole. An autoimmune epilepsy panel revealed elevated GAD65 antibodies in the serum (225 nmol/l) and CSF (2.78 nmol/l), while [¹⁸F]‐fluoro‐deoxy‐glucose positron emission tomography showed bitemporal hypometabolism (left > right). The patient was diagnosed with GAD65 antibody‐associated autoimmune epilepsy. Our observation adds to the spectrum of neurocardiac syndromes associated with autoimmune epilepsy.     

GAD67 and GAD65 mRNA and protein expression in cerebrocortical regions of elderly patients with schizophrenia

Gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter of CNS, has been consistently implicated in the pathophysiology of schizophrenia. GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD). Two isoforms of GAD have been identified and have been named GAD65 and GAD67 based on their apparent molecular weights. In this study, GAD65 and GAD67 mRNA and protein levels were measured by using real-time RT-PCR and immunoblotting, respectively, in post-mortem brain tissue from the dorsolateral prefrontal cortex (DLPFC) and the occipital cortex of the elderly persons with schizophrenia and matched normal controls. In addition, the mRNA expression of GAT-1, one of the principal transporters of GABA, was also studied in the same subjects. Expression of GAD65 and GAD67 mRNA in the DLPFC and in the occipital cortex was significantly elevated in patients with schizophrenia, whereas the expression of the corresponding proteins and GAT-1 mRNA was unchanged. Although the levels of GAD65 and GAD67 messages were increased in schizophrenia subjects, the proportion of the two GAD isoforms remained constant in controls and schizophrenics. In the human DLPFC, GAD65 mRNA was found to be expressed significantly less than the message for GAD67, approximately 16% of that observed for GAD67. On the contrary, the abundance of GAD65 protein in the DLPFC was about 350% of that observed for GAD67. The results suggest a substantial dysregulation of GAD mRNA expression in schizophrenia and, taken together with the results of protein expression studies, raise the possibility that both cortical and subcortical GABA function may be compromised in the disease.     

GAD65‐Ab encephalitis and subtle focal status epilepticus

Aims. To delineate common epilepsy features associated with the presence of glutamic acid decarboxylase autoantibodies (GAD65‐Ab). Methods. Three consecutive cases of GAD65‐Ab encephalitis patients, followed in our neurological department, were investigated with regards to clinical semiology and EEG. Results. These patients presented new‐onset subtle ictal clinical features. Patients 1 and 2 described prolonged and transitory feelings of “déjà vu ‐ déjà vécu” and a “dreamy state”. Patient 3 was admitted for subsequent transient aphasia events followed by paroxysmal behavioural disturbances. Epileptic origin of the symptoms was confirmed using either a standard EEG (observation of temporal status epilepticus in one case) or a prolonged EEG (focal epileptiform activity during an asymptomatic period for two patients). All patients suffered from clinical focal status epilepticus. Patients 1 and 2 presented with temporo‐mesial seizures in agreement with the definition for limbic encephalitis, whereas Patient 3 presented with neocortical (lateral temporal and frontal lobe) seizures arguing for a non‐limbic encephalitis. A high level of GAD65‐Ab was found in cerebral spinal fluid, confirming a diagnosis of epilepsy associated with GAD65‐Ab encephalitis. Conclusion. Encephalitis seems to be a frequent neurological syndrome associated with GAD65‐Ab disorders. Epilepsy may be more frequent and severe than currently suggested, as ictal semiology may be subtle for these outpatients in whom standard EEG is commonly falsely reassuring. Subtle focal status epilepticus is a particular semiology of the GAD65‐Ab encephalitis spectrum.     

大鼠GAD65基因慢病毒载体的构建及其在MSCs中的表达

目的构建谷氨酸脱羧酶65(glutamic acid decarboxylase 65,GAD65)重组慢病毒表达载体,感染间充质干细胞(mesenchymal stem cells,MSCs)并进行鉴定。方法 PCR法扩增GAD65基因,构建LV5-GFP-GAD65慢病毒载体;与包装质粒共转染293T细胞包装病毒;将慢病毒感染大鼠MSCs,荧光显微镜鉴定转染率,Western blot检测GAD65的表达。结果双酶切及测序结果表明LV5-GFP-GAD65慢病毒载体构建成功;包装病毒产生的病毒液滴度为5×107TU/ml;慢病毒感染大鼠MSCs的转染率高于90%,Western blot结果显示GAD65蛋白表达比对照组明显升高。结论 GAD65重组慢病毒载体构建成功,包装得到高浓度病毒液,感染大鼠MSCs能稳定过表达GAD65蛋白,为进一步探索侧脑室注射基因化的MSCs治疗癫痫奠定实验基础。     

Familial autoimmunity in neurological patients with GAD65 antibodies: an interview-based study

Journal of Neurology - The common co-occurrence of autoimmune systemic diseases in patients with neurological disorders and antibodies against glutamic acid decarboxylase 65 (GAD65) suggests a...     

全脑缺血-再灌注对海马CA1区GAD65表达的影响及意义

目的探讨全脑缺血-再灌注对成年大鼠海马CA1区GAD65表达的影响及意义。方法成年雄性SD大鼠24只,随机分为3组:假手术组(SH)、缺血-再灌注3d组(IR-3)及缺血-再灌注7d组(IR-7),每组8只。采用四动脉阻断法制作全脑缺血-再灌注模型,应用免疫组织化学方法检测海马CA1区谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)同工酶GAD65的表达变化。结果与假手术组相比,IR-3组GAD65的表达明显增多,IR-7组恢复正常。结论GABA能中间神经元对缺血相对耐受;全脑缺血-再灌注3dGAD65的表达增多可能是一种代偿性的机制,以减轻脑缺血后的高兴奋性。     

Glutamate decarboxylase (GAD67 and GAD65) gene expression is increased in a subpopulation of neurons in the putamen of parkinsonian monkeys

The cellular distribution of the mRNAs encoding for the two isoforms of glutamate decarboxylase, GAD67 and GAD65, was analyzed by in situ hybridization histochemistry in the caudate nucleus and putamen of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian squirrel monkeys. On brain sections processed with a radioactive and a digoxigenin-labeled cRNA probe, the GAD67 and GAD65 mRNAs were colocalized in virtually all labeled neurons of the caudate nucleus and putamen, in both control and MPTP-treated monkeys. Furthermore, neurons labeled with the GAD cRNAs constituted at least 90% of all striatal neurons, as estimated on adjacent Nissl-stained sections. In the two groups of monkeys, double-labeling experiments using a combination of radioactive GAD67 or GAD65 and digoxigenin-labeled preproenkephalin (PPE) cRNA probes showed that roughly half of all neurons labeled with the GAD cRNAs were also labeled with the PPE cRNA probe. When compared to controls, GAD67 and GAD65 mRNA levels were higher in the putamen, and to a lesser extent in the caudate nucleus, of MPTP-treated monkeys. Further analysis of labeling at the cellular level in a dorsolateral sector of the putamen revealed that GAD67 and GAD65 mRNA levels in MPTP-treated monkeys were increased in PPE-labeled (presumed striato-pallidal) neurons but not in PPE-unlabeled (presumed striato-nigral) neurons. Our results demonstrate that most neurons in the caudate nucleus and putamen of squirrel monkeys contain the mRNAs encoding for the two GAD isoforms. In addition, the selective increase in GAD mRNA levels in PPE-labeled neurons provides further evidence that striato-pallidal GABAergic neurons are hyperactive in MPTP-treated parkinsonian monkeys. Synapse 27:122–132, 1997. © 1997 Wiley-Liss, Inc.     

Autoimmune stiff person syndrome and related myelopathies: understanding of electrophysiological and immunological processes

Stiff person syndrome (SPS) is a disabling autoimmune central nervous system disorder characterized by progressive muscle rigidity and gait impairment with superimposed painful spasms that involve axial and limb musculature, triggered by heightened sensitivity to external stimuli. Impaired synaptic GABAergic inhibition resulting from intrathecal B-cell-mediated clonal synthesis of autoantibodies against various presynaptic and synaptic proteins in the inhibitory neurons of the brain and spinal cord is believed to be an underlying pathogenic mechanism. SPS is most often idiopathic, but it can occur as a paraneoplastic condition. Despite evidence that anti-GAD and related autoantibodies impair GABA synthesis, the exact pathogenic mechanism of SPS is not fully elucidated. The strong association with several MHC-II alleles and improvement of symptoms with immune-modulating therapies support an autoimmune etiology of SPS. In this review, we discuss the clinical spectrum, neurophysiological mechanisms, and therapeutic options, including a rationale for agents that modulate B-cell function in SPS.     

Different distributions of GAD65 and GAD67 mRNAS suggest that the two glutamate decarboxylases play distinctive functional roles

Two genes encode two forms of glutamate decarboxylase, GAD₆₅ and GAD₆₇. Because the two GADs differ in subcellular distribution and interactions with the cofactor pyridoxal phosphate, the two enzymes may play different roles in gamma-aminobutyric acid (GABA) production. In this study we have used in situ hybridization to compare the regional and cellular distributions of the two GAD mRNAs in rat brain. Both GAD mRNAs are abundant in olfactory bulb, olfactory tubercle, zona incerta, reticular nucleus of the thalamus, oculomotor nuclei, and pontine tegmental area. GAD₆₅ mRNA is more abundant in several structures of the visual system, including the lateral geniculate nuclei, superior colliculi, and olivary pretectal nucleus, as well as in several hypothalamic and pontine nuclei. In contrast, GAD₆₇ mRNA is more abundant in neocortex, the granular layer of olfactory bulb, lateral and medial septum, globus pallidus, inferior colliculi, and cerebellar cortex. Both GAD mRNAs are present in interneurons as well as in projection neurons, and both are present in neurons with different types of synapses, including dendrodendiritic, axosomatic, and axodendritic synapses. GAD₆₅ mRNA predominates in the visual and the neuroendocrine systems, which are more subject to phasic changes, while GAD₆₇ is present at relatively higher concentrations in many tonically active neurons. GAD₆₅ and GAD₆₇ together may provide more flexibility in the regulation of GABA synthesis than either could alone. © 1993 Wiley-Liss, Inc.