Immune-mediated epilepsy with GAD65 antibodies

Journal of Neuroimmunology 341 (2020) 577189

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Review Article

Immune-mediated epilepsy with GAD65 antibodies Xinxin Li, Qi Guo, Zhaoshi Zheng, Xiaoshuang Wang, Songyan Liu

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Department of Neurology, China–Japan Union Hospital of Jilin University, Changchun, Jilin Province, China

A B S T R A C T

Anti-GAD65 antibodies have been identified in both acute/subacute seizures (limbic encephalitis and extralimbic encephalitis) and chronic isolated epilepsy. The evidence of high serum titers and intrathecal synthesis play a fundamental role in diagnosis but poorly correlate with disease severity or response to therapies. It remains controversial whether anti-GAD65 Abs are the pathogenic entity or only serve as a surrogate marker for autoimmune disorders mediated by cytotoxic T cells. Unlike other immune-mediated epilepsy, although multiple combinations of therapeutics are used, the efficacy and prognosis of patients with GAD65-epilepsy patients are poor. Besides, GAD65-epilepsy is more prone to relapse and potentially evolve into a more widespread CNS inflammatory disorder. This article reviews the recent advances of GAD65-epilepsy, focusing on the diagnosis, epidemiology, pathophysiology, clinical features, and treatment, to better promote the recognition and provide proper therapy for this condition.

1. Introduction

2. Diagnosis

Glutamic acid decarboxylase (GAD) is a key enzyme in the dynamic regulation of neural network excitability that converts glutamate into Gamma-aminobutyric acid (GABA). There are two isoforms of this enzyme: GAD65 and GAD67, which are widely distributed within the central nervous system, pancreas, and other organs (Baizabal-Carvallo, 2019; Huang et al., 2019). In patients with neurological symptoms, antibodies can be detected against GAD65, but also less frequently against GAD67. Although the significance of anti-GAD65 antibodies (anti-GAD65 Abs) has been a focus of debate, several neurological phenotypes of anti-GAD65 associated neurological disease have been described, such as stiff-person syndrome (SPS), cerebellar ataxia(CA), ocular movement disorder and myelitis (Liu et al., 2018; Ali et al., 2011; Burbelo et al., 2008; Dubbioso et al., 2013).Lately, anti-GAD65 Abs were found in patients with seizures in the context of encephalitis, and in patients with chronic epileptic syndromes without clinical or MRI evidence of active CNS inflammation (Dubey et al., 2017; Gagnon and Savard, 2016; Spatola and Dalmau, 2017) In some cases, seizures may be the main or the only clinical presentation in encephalitis associated with anti-GAD65 Abs; thus, “subacute” encephalitis and “chronic” epilepsy seem to represent a continuum of a rare neurological syndrome (Liimatainen et al., 2010). To make consistency in terminology, we collectively refer to these diseases as “immune-mediated epilepsy with anti-GAD65 Abs “ (GAD65-epilepsy) (Malter et al., 2015; Daif et al., 2018). This article reviews the recent advances of GAD65epilepsy, focusing on the diagnosis, epidemiology, pathophysiology, clinical features, and treatment, to better promote the recognition and provide proper therapy for this condition.

The diagnosis of GAD65-epilepsy is based on clinical grounds plus the presence of high anti-GAD65 Abs titers or the detection of intrathecal synthesis (IS) in CSF. Currently, several recognized high antiGAD65 Abs levels are defined as follows: 1) usually > 20 nmol/L or > 2000 U/mL when determined by radioimmunoassay (RIA);2) > 20 nmol/L or > 1000 IU/mL by Enzyme-linked immunosorbent assay (ELISA);3) screened by immunohistochemistry (IHC) and confirm the positive cases by RIA or Western Blot (WB) (Liimatainen et al., 2010; Daif et al., 2018; Saiz et al., 2008; Peltola et al., 2000; Walikonis and Lennon, 1998; Vianello et al., 2005; Pittock et al., 2006). High anti-GAD65 Abs titers have high specificity for neurological autoimmunity, and only 0.8% of patients with T1DM detected anti-GAD65 Abs titers above 2000 U/mL (Saiz et al., 2008). 20% of patients with Abs were only present in serum, and the serum Abs titers are often higher than CSF (Liimatainen et al., 2010; Malter et al., 2010). However, in some cases, the serum Ab titers might be low, but still show clear evidence of IS in CSF. Therefore, Clinically, patients with highly suspected GAD65-epilepsy should be simultaneously screened for the titer of anti-GAD65 Abs in serum and CSF. The IS index of anti-GAD65 Abs was calculated using the values obtained by immunohistochemistry, based on the following formula: [CSF anti-GAD65 Abs titer/serum anti-GAD65 Abs titer]/ [CSF albumin/serum albumin], Values higher than the IgG index, particularly higher than 1, are strong indicators for IS of antibody-specific IgG (Saiz et al., 2008; Dalakas et al., 2001).IS suggests active and ongoing CNS inflammation that could identify patients who might be warranted an immunosuppressive therapy (Baizabal-Carvallo, 2019; Liimatainen et al., 2010).

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Corresponding author. E-mail address: [email protected] (S. Liu).

https://doi.org/10.1016/j.jneuroim.2020.577189 Received 11 January 2020; Received in revised form 11 February 2020; Accepted 11 February 2020 0165-5728/ © 2020 Elsevier B.V. All rights reserved.

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3. Epidemiology Up to 80% of type I diabetes(T1DM) have low-positive anti-GAD65 Abs (< 100 U/mL); furthermore, such low-positive antibodies may have been detected in 1 to 2% of healthy people and are relatively nonspecific (Meinck et al., 2001; Brooking et al., 2003). The true incidence of GAD65-epilepsy remains unknown. In the existing studies, due to the method of determination and the levels of antibodies considered relevant have been variable, discrepant results do exist, and most studies are limited by lack of CSF data. Recently, a population-based study from Olmsted County, USA, estimated the prevalence of autoimmune encephalitis to be13.7/100,000 people，of which anti-GAD65 Abs was found to be one of the most common autoantibodies, with a prevalence of 1.9/100,000 (Dubey et al., 2018).A recent hospital-based prospective study reported that 8% of new-onset or established epilepsy of unknown etiology had high titers of anti-GAD65 Abs (Dubey et al., 2017).Other studies reported that high anti-GAD65 Abs titers have been detected in 2.1 to 5.4% of adult-onset focal epilepsy (more frequently exhibiting drug-resistant seizures) and 6.5%–17% of limbic encephalitis (LE) (Liimatainen et al., 2010; Peltola et al., 2000; Malter et al., 2010; McKnight et al., 2005).Less research on children, high anti-GAD65 Abs titers have been detected in 0.5% to 0.8% in pediatric epilepsy, and 2% in pediatric patients with cryptogenic forms of epileptic encephalopathies (Borusiak et al., 2016; Veri et al., 2013; Tekturk et al., 2018).Moreover, anti-GAD65 Abs were detected 8.7% in pediatric epileptic patients with an undetermined etiology and up to 35.3% in patients with encephalitis and childhood status epilepticus, but none of them reached the high titer levels (Lin et al., 2012; Bektas et al., 2015). However, the diagnostic value of these low anti-GAD65 Abs titers to neurological disease remains unclear. Fig. 1. A) Each GAD monomeric unit，GAD65 and GAD67, comprise three domains: N-terminal, PLP-binding, and C-terminal domain. 74% identity for the middle and C-terminal domains, 25% identity for the N-terminal domain. B) Dimeric structure ofGAD65 showing the structural locations of the three domains, colored as in Fig. 1(A) Note that ctc1 includes prototypically mAb b78 and some residues from the N-terminal domain, ctc2 includes prototypically mAb 96.11 and some residues from the PLP-domain. (Fig. 1. from Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: Protean additions to the autoimmune central neuropathies. Journal of Autoimmunity. 37 (2011) 79–87. copyright 2011; Elsevier. This material is reproduced with permission from Elsevier).

4. Pathophysiology 4.1. Structural and functional of the GAD The neuroendocrine enzyme GAD catalyzes the synthesis of Gammaaminobutyric acid (GABA), which is the prime inhibitory neurotransmitter that is necessary to the control of synaptic excitation/inhibition and neural oscillation (Hwang et al., 2018) GAD involves two isoforms that catalyze GABA synthesis named according to their respective molecular weights: GAD65 and GAD67 (Kass et al., 2014). The two isoforms have the same fold and general overall sequence similarity, each having an amino (N)-terminal domain, a middle PLPbinding domain containing the active catalytic site of the enzyme, and a carboxy (C)-terminal domain, with the middle and C-terminal domains having 74% identity, 25% identity for the N-terminal domain (Ali et al., 2011) (Fig. 1). GAD67 is scattered all over the cytoplasm, is an active element and constantly active to produce basal levels of GABA, whereas GAD65 is mainly located in synaptic vesicles, is temporarily activated, undergoes auto-inactivation during enzyme activity and occurs in the cell primarily as apoenzyme, providing for a pulse in production under circumstances that demand a rapid surge of GABA synthesis and release (Kass et al., 2014; Fenalti et al., 2007).The activity and expression of the two isoforms are highly correlated with GABA levels and subsequent inhibitory neurotransmission at the synapses (Lee et al., 2019).GAD67 interneuron subtype-specific knockdown animals have shown that the reduced inhibitory neurotransmission caused by GAD67 deficiency results in behavioral changes related to anxiety and schizophrenia (Lazarus et al., 2015).In GAD65 knockout mice, the level of GABA is a greater than 50% decrease, and the mice appear to be more vulnerable to stress and seizures (Qi et al., 2018; Muller et al., 2015).However, only the GAD65 functions as an autoantigen, the GAD67 is rarely autoantigenic in isolation (Fouka et al., 2015; Arafat et al., 2009). Comparison of GAD65 and GAD67 reveals that the binding site of GAD65 for antibodies displays greater polarity and more negatively charged amino acid residues on its surface, and the C-

terminal and catalytic loop residues of GAD65 display more flexibility and mobility, which may enhance the propensities of antigenicity (Ali et al., 2011; Kass et al., 2014; Fenalti and Buckle, 2010).

4.2. Epitope features of anti-GAD65 abs Anti-GAD65 Abs autoimmunity is associated with a variety of syndromes, from T1DM to neurological diseases such as SPS, CA, as well as limbic encephalitis (LE) and epilepsy. Immunodominant epitope-specific recognition is considered to be one of the causes of different clinical symptoms. Studies disclosed that antibodies epitopes were tightly clustered in the region surrounding the C-terminal flexible region, and segregated into two distinct C-terminal clusters (b78 and 96.11) located to opposite faces of the C-terminal domain (Ali et al., 2011; Fenalti and Rowley, 2008) (Fig. 1). Anti-GAD65 Abs from patients with neurological disorders have been shown to preferentially recognize the b78 epitope specificity, which tended to be enzyme inhibitory, while that in T1DM patients recognize more commonly the b96.11 epitope specificity, which tended to be non-enzyme inhibitory (Ganelin-Cohen et al., 2016; Manto et al., 2015; Cheramy et al., 2013). Antibodies against the b78 epitope have demonstrated to interferes with the association of GAD65 with the cytosolic face of GABA-containing synaptic vesicles, inhibit the enzymatic activity of GAD65. Besides, cerebellar slice experiments suggested a gradual but sustained 2

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and ipsilateral interpositus nucleus (Manto et al., 2015; Manto et al., 2011; Chang et al., 2013a; Chang et al., 2013b). However, it was not confirmed in a study involving patients with LE and epilepsy (GresaArribas et al., 2015).Furthermore, GAD65 can become membrane-associated or anchored to synaptic vesicles through protein complex formation with the heat shock protein 70 families (Hsu et al., 2000). Thus, GAD65 could be transiently exposed on the cell surface during exocytosis from GABAergic neurons. Alternatively, the antibodies may be endocytosed and transported internally to their cytoplasmic antigen, allowing a pathogenic antibody-antigen interaction to occur (Errichiello et al., 2009; Alexopoulos and Dalakas, 2019; Dalakas, 2013). Moreover, anti-GAD65 Abs may coexist with additional cellsurface antibody, such as anti-NMDA-R, anti-VGKC, anti-GABAA-R, or GABAB-R (Gagnon et al., 2016; Kammeyer and Piquet, 2019; Hoftberger et al., 2013).These antibodies might alter neuronal function by receptor competition and internalization and are directly pathogenic to cells. At this time, anti-GAD65 Abs might rather be a bystander effect generated by blood-brain barrier permeability and widespread neuronal destruction (Kammeyer and Piquet, 2019).

inhibitory effect of the b78 epitope on the GABAergic synaptic transmission between basket cells and Purkinje cells. These effects were not observed with Abs against the b96.11 epitope (Baizabal-Carvallo, 2019; Manto et al., 2015; Manto et al., 2019; Hampe et al., 2013) In neurological disorders linked to anti-GAD65 Abs, researches on specific epitopes recognition was contradictory. Vianello et al., 2006 used immunofluorescence on cultured hippocampal neurons, and staining patterns differed among patients with SPS, CA，and epilepsy-related to anti-GAD65 Abs; the tentative suggestion was that differences in GAD65 epitope recognition differentiated clinical phenotypes. But in two other studies of GAD65 epitope mapping, no differences in specific epitopes recognized was found among various symptoms, and only reported that the serum of patients with LE was more likely to react with the N-terminal domain, whereas epileptic patients showed more reactivity against the C-terminal domain, but this difference of epitope recognition was insignificant and not observed in the CSF (Fouka et al., 2015; Gresa-Arribas et al., 2015). A recent study showed that SPS patients could recognize a linear epitope at the N-terminal region of GAD65 ，preferably, and inhibited GAD65 enzyme activity better compared to epilepsy patients (Liimatainen et al., 2018), but these are not sufficient, and further specific identification studies of the epitope of GAD65 are necessary.

5. Clinical features GAD65-epilepsy may be found in two different settings: acute/ subacute onset of seizures or chronic epilepsy, and the clinical manifestations are heterogeneous with a wide clinical spectrum ranging from mild non-pharmacoresistant epilepsy to refractory TLE, LE, and also extralimbic encephalitis (ELE). Most cases were young adult patients, and women were more frequently affected than men; however, in the pediatric population, males have a slight predominance (Lin et al., 2012; Lilleker et al., 2014).

4.3. Do anti-GAD65 abs play a pathogenic role in GAD65-epilepsy? It has been postulated that the humoral immune response to antiGAD65 Abs could result in reduced amounts of presynaptic available GABA content through reducing GABA synthesis in nerve terminals and interfering with exocytosis of GABA in synaptic vesicles (Manto et al., 2015; Vega-Flores et al., 2014; Manto et al., 2007; Manto et al., 2011; Ishida et al., 2008). In an MRI-spectroscopy study, low cortical GABA levels were reported in epileptic patients with high levels of serum antiGAD65 Abs (Stagg et al., 2010). Nevertheless, it remains controversial that anti-GAD65 Abs are the pathogenic entity of GAD65-epilepsy. Firstly, the results of studies on the direct pathogenic influence of antiGAD65 Abs affecting inhibitory synaptic transmission in central GABAergic pathways are inconsistent. Increased spontaneous activity of a network of hippocampal neurons in culture caused by registering on cultured hippocampal neurons following application of serum from epileptic patients with anti-GAD65 Abs while no effect was noted using sera from negative controls (Vianello et al., 2008).However, stereotactic injection of human CSF containing anti-GAD65 Abs into the hippocampus of rats in vivo did not alter evoked and spontaneous GABAergic synaptic transmission intact in the recent two studies (Hackert et al., 2016; Stemmler et al., 2015). Secondly, GAD65 is cytosolic and, therefore, not readily accessible for circulating antibodies. It generally acts as a biomarker for cytotoxic T-cell mediated neuronal damage (Dalmau and Graus, 2018). Nowadays, the effector functions of T cells in GAD65-epilepsy have attracted attention. In pathological studies of LE and unilateral TLE who underwent amygdalohippocampectomy, patchy inflammation by T cells was observed in the hippocampi. Instead, immunoglobulin and complement deposition was completely absent (Widman et al., 2015; Bien et al., 2012; Kumar et al., 2013). Experimental studies have shown that cytotoxic t-lymphocyte attack on neurons may lead to perforin-dependent electrical silencing, thereby causing the cluster of differentiation CD8+ T cells to play a major role in neuronal damage in CNS inflammation, but this damage does not necessarily cause the death of neurons (Meuth et al., 2009). Thus, these nonlethal cytotoxic attacks against GAD65-expressing GABAergic interneurons and ensuing cellular silence/dysfunction could lead to chronic epilepsy (Daif et al., 2018; Widman et al., 2015).Thirdly, if anti-GAD65 Abs are pathogenic, they need to be internalized and reach the intracellular targets. Some experiments have confirmed the internalization of human monoclonal anti-GAD65Ab b78 in cultured cells or rat cerebral cortex, and have observed b78 in CA1 interneurons and Purkinje neurons in the medial septum/diagonal band

5.1. Chronic epilepsy Historically, Giometto was the first to describe the presence of antiGAD65 Abs in patients with chronic epilepsy in 1998 (Giometto et al., 1998).Most patients were focal seizures with clinical localization of the seizure focus to the temporal lobe, and onset in adult life with no history of antecedent cerebral insults and no epileptogenic lesions identified on MRI scanning using appropriate imaging protocols (Lilleker et al., 2014).The most common focal seizures were psychic, followed by somatosensorial, motor, and visual symptoms (Falip et al., 2018a).Moreover, recent studies reported that 9% of to15%of patients with GAD65 epilepsy presented music-induced reflex seizures (MRS), it seems to be a distinctive seizure type of GAD65 epilepsy (Falip et al., 2018a; Falip et al., 2018b). Also, some patients experienced different seizure types during disease evolution. 75% of patients found no epileptogenic lesions identified on the first MRI scanning (Falip et al., 2018a). During the follow-up, the most common imaging abnormality was disproportionate cerebral or cerebellar volume loss for age. Although a minority of patients had some degree of hippocampal involvement, hippocampal sclerosis (HS) seemed to be not a dominant feature (Malter et al., 2015; Fredriksen et al., 2018).In the FDG-PET study, most patients presented hypometabolism in MTLE and insular structures, and insular involvement could be an important diagnostic clue for GAD65-epilepsy, especially in patients with MTLE epilepsy of unknown origin (Falip et al., 2018a). 5.2. Limbic encephalitis (LE) and extralimbic encephalitis (ELE) LE is subacute onset with progression in less than 3 months，defined by pathological temporal structural abnormalities on neuroimaging, temporal lobe seizures, and in part, rapidly progressive memory and affective disturbances (Graus et al., 2016; Bien and Elger, 2007). Sub-forms of LE is differentiated based on the underlying antigen. One form of LE based on neuronal cell-surface antibodies such as voltagegated potassium channel (VGKC) complex Abs (currently known to 3

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concerning for co-existing anti-GABAA-R antibodies (Gagnon et al., 2016). Also, GAD65-epilepsy may co-exist with visual disturbances or movement disorder when anti-GlyR antibodies are present, or warning a paraneoplastic and refractory disease in the presence of anti-GABAB-R antibodies (Hoftberger et al., 2013; Boronat et al., 2011; Piquet et al., 2019). Nevertheless, in some cases, the titer of anti-GAD65 Abs was present at a much lower than that in neurologic syndromes and was not related to disease activity (Kammeyer and Piquet, 2019). Moreover, some patients with GAD65-epilepsy may evolve into a more widespread CNS inflammatory disorder at some point in their illness, such as SPS, CA, optic neuritis and transverse myelitis, these syndromes may represent a continuum of anti-GAD65 Abs -associated CNS disease (Lilleker et al., 2014; Schaefer and Moeller, 2015; Flores-Cantu et al., 2015; Dubey et al., 2014).

react with LGI-1 and CASPR-2 antigens), and the other form of LE with antibodies targeting intracellular antigens like GAD65 (Hansen et al., 2016).The main clinical manifestation with LE associated with antiGAD65 Abs are epilepsy and memory impairment, and psychiatric disorders, orientation, executive functions, and language disorders were also possible (Gagnon and Savard, 2016).But a “craniofacial dystonia or orofacial dyskinesia” pattern, as described in VGKC or NMDAR antibodies receptor encephalitis, is lacking in anti-GAD65 Absrelated LE (Gagnon and Savard, 2016). Compared to patients with LE associated with anti-VGKC complex Abs, those with anti-GAD65 Abs were younger, present first with seizures rather than cognitive symptoms, had oligoclonal bands in the CSF, along with intrathecal synthesis, and showed an unremitting chronic disease course with antibodies persistence (Malter et al., 2010; Feyissa et al., 2017; Wagner et al., 2015a).Rare cases may present with prominent dysautonomia, severe neuropsychological impairments，dementia,ictal asystole, and may have drug-resistant TLE for several years prior to diagnosis (Ben Achour et al., 2018; D'Souza and Feyissa, 2018; Witt et al., 2015; Finelli, 2011; Takagi et al., 2011). Cases involving mixed LE and ELE subtypes have also been described, but the seizure features were mesial temporal despite ELE involvement (Najjar et al., 2011).Isolated ELE subtype is uncommon, and only a few have been reported. ELE subtype comprises, clinically and radiographically, a more heterogeneous syndrome, varying according to the location and extent of ELE involvement, some patients developed non-convulsive status epilepticus and convulsive status epilepticus (Najjar et al., 2011; Incecik et al., 2018; Triplett et al., 2018; Cikrikcili et al., 2013). In the setting of LE, brain MRI usually shows MRI T2-weighted hyperintensity and “swelling” in mesial temporal structure (bilaterally in most cases), and automated mesiotemporal volumetry only displays amygdala enlargement (Wagner et al., 2015a; Wagner et al., 2015b). Follow up MRI in patients with prolonged and frequent seizures may demonstrate hippocampal sclerosis and atrophy (Kumar et al., 2013). The brain MRI of ELE present with hyperintense cortical/subcortical lesions in T2W/FLAIR MRI sequences, progressive changes predominantly affecting the frontal lobes, the occipito-parietal region with some extension into the temporal lobes and other cortical regions, and usually without contrast enhancement (Incecik et al., 2018; Triplett et al., 2018). FDG-PET may show hypermetabolism corresponding to the MRI lesions, and DTI may show widespread white matter damage already at relatively early disease stages (Kojima et al., 2014; Wagner et al., 2016) (Fig. 2).

5.4. Paraneoplastic associations Previous studies have suggested that GAD65-epilepsy often associated with non-paraneoplastic forms (Daif et al., 2018), little is known of anti-GAD65 Abs in the paraneoplastic context. However, anti-GAD65 Abs have been detected in patients with lung carcinomas (small-cell and non-small cell), thymoma, breast cancer, and testicular seminoma (Zekeridou et al., 2019; Arino et al., 2015; McKeon and Tracy, 2017). Although this antibody has less sensitivity and specificity for the presence of malignancy, and are not included as a “classic” paraneoplastic antibody, anti-GAD65 Abs are already included in commercially available paraneoplastic line-blots (Olberg et al., 2019).Paraneoplastic etiology in anti-GAD65 Abs associated isolate epilepsy is extremely rare (Serafini et al., 2016). The probability of malignancy with anti-GAD65 Abs increases if the presentation matches a classic paraneoplastic syndrome or typical of LE with rapid disease progression (Arino et al., 2015). Furthermore, it has been shown that the probability of an underlying malignancy was a 7-fold increased risk in patients with antiGAD65 Abs and co-existing neuronal cell-surface antibodies, particularly in male patients older than 50 years of age with concomitant antiGABAB receptor Abs (Boronat et al., 2011; Arino et al., 2015; McKeon and Tracy, 2017).In these cases, appropriate and targeted malignancy screening should be performed. Paraneoplastic antibodies may be earlier than any evidence of primary malignancy, so continued, regular malignancy screening is necessary, it is recommended to be at least once every six months for four years (Kannoth, 2012; Pittock et al., 2004).

5.3. Co-existing autoimmune antibodies and diseases 6. Treatment Patients with GAD65-epilepsy frequently coexist one or more systemic autoimmune disorders, including diabetes，thyroiditis，psoriasis, celiac disease, vitiligo, common variable immune deficiency, and others (Gagnon and Savard, 2016). In addition, some patients are more likely to have serological evidence of multiple autoantibodies, but without any clinical signs of autoimmune diseases, such as anti-TPO, anti-gliadin, antinuclear antibody, antiphospholipid antibodies (Liimatainen et al., 2010; Quek et al., 2012). In Gagnon's report, almost half of cases have co-existing systemic autoimmune conditions, mainly T1DM, followed by autoimmune thyroiditis (Gagnon and Savard, 2016). These systemic autoimmune conditions and high anti-GAD65 Abs levels can precede the clinical manifestations of GAD65-epilepsy for several years (Fauser et al., 2015).Furthermore, it is necessary to screen and address endocrinologic abnormalities, particularly in patients with GAD65-epilepsy that are less responsive to immunosuppressive therapy. One case reported that exogenous testosterone replacement had played an important role in improving the seizures in a patient with GAD65-epilepsy and low testosterone (Heiry et al., 2015). Rare cases have reported the co-existence of anti-GAD65 Abs and cell-surface antibodies, and this co-existence may result in the variability seen in the clinical and radiographic features. For example, multifocal asynchronous sub-cortical/cortical hyperintensities are

The treatment of patients with GAD65-epilepsy is comprised of symptomatic therapy (including antiepileptic drugs), immunotherapy, and surgical therapies. Most patients received multiple therapeutic interventions in varying combinations, but the response and prognosis were poor. Moreover, current treatment recommendations lack randomized controlled trials and are mainly based on case series and clinical experience. Recently, a special study emphasis on treatment timing and the relationship between immunotherapy and anti-epileptic drugs (Makela et al., 2018).They divided the clinical course of GAD65-epilepsy into three main stages: In the first stage, acute reversible immune-activation causes the first seizure, there is no permanent brain damage, and no visible brain MRI changes (Liimatainen et al., 2013). The main focus of treatment should be on immunotherapy, and early initiation of immunotherapy can prevent from developing refractory epilepsy even provides complete seizure freedom. In the second stage, there is already subtle irreversible brain damage, which causes MRI abnormalities and refractory epilepsy (Bien et al., 2012). Immunotherapy is still effective and can resolve edema and abnormal signal changes of MRI. However, after that, focus management should shift to refractory epilepsy. In the third stage, there was post-inflammatory astrogliosis causing secondary 4

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Fig. 2. Brain MRI of GAD65-epilepsy. Patient 1. A 61-year-old man experienced left lower limb paroxysmal convulsion and numbness, associated with brief heart palpitations, tongue stiffness, and sweats several times per day, for two months. Brain MRI: FLAIR sequence demonstrating bilateral medial temporal hyperintensities on axial (A) and sagittal (B) sections. Patient 2. A 23-year-old woman presented with left face and tongue twitching, behavioral changes for 20 days, and progressed to confusion and bilateral tonic-clonic seizures 3 days. FLAIR sequence(C) and DWI image (D) demonstrating right temporoparietal lobe slightly hyperintensities on axial.

refractory status epilepticus, aggressive progression or preventing relapses, although variable combination immunotherapy of IVMP, PE and IVIG are utilized, there is no or only moderate and short-lived effect for the clinical. Previous studies have demonstrated that multimodality immunotherapy of combination of both first- and second-line therapies are effective (Dubey et al., 2014; Kanter et al., 2008; Saidha et al., 2010; Korff et al., 2011).But it remains not unequivocal which immunosuppressant may have provided the greatest impact on the outcome since all of the treatments were administered over a short time. Second-line therapy commonly consists of Mycophenolate mofetil, Azathioprine, Rituximab, and Cyclophosphamide (Daif et al., 2018).Azathioprine and mycophenolate mofetil, as mild immunosuppressants, are mainly used to maintain remission and reduce dependence on steroids, with varying results. Saidha described two patients with GAD65-epilepsy who had a partial response to IVIG and prednisolone and successfully improved symptoms with mycophenolate mofetil (Saidha et al., 2010). In one case report, the patient's condition remained unchanged after 5 years of azathioprine use (Millet et al., 2015).In another case report, azathioprine was effective but had a lower clinical response, and was suspended after about 40 days due to severe anemia (Mazzi et al., 2008). Rituximab and cyclophosphamide can be used as first aid therapy in the rapid progression of symptoms, and in some cases, remarkable and lasting improvement or remission were observed (Ben Achour et al., 2018; Kanter et al., 2008; Korff et al., 2011; Grilo et al., 2016). But compared to rituximab, it is not welcome given the adverse effects of cyclophosphamide. Besides, Triplett reported a case of GAD65-epilepsy presenting with epilepsia partialis continua progressing to a fulminant encephalopathy and responding to rituximab combined with cyclophosphamide (Triplett et al., 2018). Other immunosuppressive agents such as Natalizumab and Basiliximab

hippocampal sclerosis or to more diffuse brain damage, and it was often considered the underlying “structural” cause of the seizures. Immunotherapy seems to be ineffective, and treatment should focus on seizure control (Wagner et al., 2016; Gillinder et al., 2018). However, for patients with pharmacoresistant epilepsy without an acute onset, the mentioned above three stages do not seem to explain the clinical course, and the routine use of immunotherapy has not been efficacious (Lilleker et al., 2014; Falip et al., 2012). 6.1. Immunotherapy regimens The immunotherapy protocol may be subdivided into first-line (immunomodulatory treatment) and second-line therapies (immunosuppressive treatment) (Table 1).First-line therapies include highdose intravenous methylprednisolone (IVMP), intravenous immune globulin (IVIG), or plasma exchange (PLEX) (Husari and Dubey, 2019). High-dose IVMP (1 g per day for 5 days, followed by monthly pulsatile treatment or oral prednisone) were the most frequent applied first-line therapies, with 45% of patients achieving seizure response (50% reduction). However, relevant side effects were observed in 50% of the patients. IVIG (0.4 g/kg per day for 5 consecutive days) and immunoadsorption were less effective in seizure response but with better tolerability than corticosteroids (Malter et al., 2015; Dogan Onugoren et al., 2016). In some cases, PLEX (5–7 cycles; 1 exchange every other day spread over 10–14 days)can effectively diminish anti-GAD65 antibody titers and improve seizures (Farooqi et al., 2015; Mazzi et al., 2008). For patients with improvement, gradual reduction in dosing may be required, including a slow taper of oral prednisone, or a gradual lengthening of the intervals between intravenous treatments (McKeon and Tracy, 2017; Husari and Dubey, 2019). In some scenarios, such as 5

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Table 1 Review of recommending dose, mechanism of action, adverse effects of various immune therapies: First-line and Second-line.

First-line therapies Corticosteroids

IVIG

Plasmapheresis

Second-line therapies Cyclophosphamide

Recommended dose

Mechanism of action

Adverse effects

Initial dose: IV. methylprednisolone (MP) pulses. 1000 mg/day on 3–5 consecutive days ↓ 1000 mg/day on 3 consecutive days followed by once weekly for 5 weeks or 1000 mg/day on 3 consecutive days ，followed by once weekly for 5 weeks, then every 2 weeks for 6 weeks. ↓ Oral. 60–80 mg prednisone daily, duration of tapering dose Initial dose: 0.4/kg body-weight/day on 3–5 consecutive days ↓ 0.4/kg body-weight/day on 3 consecutive days followed by once weekly for 5 weeks or 0.4/kg body-weight/day on 3 consecutive days followed by once weekly for 5 weeks, then every 2 weeks for 6 weeks. 1 exchange every other day spread over 5–7 cycles,10–14 days

Modulates chemokine and cytokine production, adhesion molecule expression and reduces migration of leukocytes to the target tissue

Insomnia, psychiatric symptoms, hyperglycemia, hypertension obesity, electrolyte imbalances, hypertension, peptic ulcer, Cushing syndrome, cataracts, infection and osteoporosis, avascular necrosis

Modulates complement activation, suppress idiotypic antibodies, saturate Fc receptors on macrophages, and suppress cytokines, chemokines, and metalloproteinases

Anaphylactic reactions (IgA-deficient patients) acute renal failure, thromboembolic events, hemolytic anemia, aseptic meningitis，headache

An extracorporeal blood purification technique，removes large molecular-weight molecules, such as immunoglobulins, immune complex, complements and filters out plasma membrane proteins

Hypotension and electrolyte imbalance, infection, hemorrhage, thrombosis, and pneumothorax

An alkylating agent which interferes with DNA synthesis, causing cell death by DNA–RNA cross-linking and inhibition of protein synthesis A humanized anti–α4 integrin monoclonal antibody that prevents entry of T and B cells into the CNS.

Gastrointestinal symptoms, alopecia, mucositis, ovarian failure, neutropenia, infections, hemorrhagic cystitis

IV: 500–800 mg/m2/ month for 3–6 months Oral: 1–2 mg/kg/day

Natalizumab

IV: monthly single day infusions of 300 mg

Rituximab

IV: 1000 mg followed by once weekly for 2 weeks, or 375 mg/m 2 weekly for 4 weeks.

Mycophenolate mofetil

Oral: initially 500 mg twice daily, target 1000 mg twice daily.

Azathioprine

Oral: Initially 1.5 mg/kg/day, target 2.5–3 mg/ kg/ day

Basiliximab

IV: 20 mg/month

A humanized monoclonal IgG against CD20positive B cells which leads to B cell depletion and reduces autoantibody generation Inhibition of inosine monophosphate dehydrogenase mediated de novo synthesis of guanosine nucleotides, leading to selective inhibition of lymphocyte proliferation. Converted to cytotoxic 6-thioguanine nucleotides which intercalates into replicating DNA and can block the de novo pathway of purine synthesis inducing lymphocyte apoptosis hair loss, cytopenia, hepatotoxicity, lymphoma, and infections An interleukin-2 receptor Ab that inhibits T cell-mediated immune reactions.

Progressive multifocal leukoencephalopathy (PML), gastrointestinal symptoms, allergic reaction, liver problems, headache, tired feeling, cold symptoms, joint pain, vaginal itching or discharge Allergic reaction, infections, reactivation of tuberculosis infection, or hepatitis B infection, late-onset neutropenia. Gastrointestinal symptoms, hypertension, hypercholesterolemia, hepatotoxicity, peripheral edema, infections, myelosuppression, lymphoma, and other malignancies Gastrointestinal symptoms, hypersensitivity reactions, alopecia, fatigue, lymphopenia, hepatotoxicity, opportunistic infection.

Allergic reaction, lymphopenia, opportunistic infection

decisions (Di Giacomo et al., 2019). Furthermore, Gagnon et al. (Gagnon and Savard, 2016) reviewed a total of 58 published cases and did not find a significant difference in outcome between cases whether or not positive anti-GAD65 Abs were reported in CSF. We advocate that the value of anti-GAD65 Abs titer needs to be interpreted in the overall clinical context of the patient under evaluation, patients with positive anti-GAD65 Abs in CSF or high serum titers have a higher risk of clinical adverse effect or recurrence. So long-term follow-up examinations are necessary and increase the effectiveness of treatment by closely monitoring the correlation between changes in clinical symptoms and antibody titers.

have also been tried without long-term improvement (Malter et al., 2015; Widman et al., 2015). 6.2. Titer of anti-GAD65 value in guiding immunomodulatory treatment anti-GAD65 Abs levels/titers showed poor correlation with the clinical presentation of GAD65-epilepsy, and the value of the change of anti-GAD65 Abs titer in guiding long-term treatment is still unclear. In some cases, serum antibodies remained high titer during and after adequate treatment; however, patients became rapidly asymptomatic without the need for long-term immunomodulatory therapy (Di Giacomo et al., 2019).In some cases, sustained high levels of antiGAD65 Abs titers are often associated with clinical adverse effect, and reduced antibody titers are often observed in patients who clinically respond to treatment, but antibodies titers rarely disappear, and often elevated again with clinical symptom recurrence during follow-up. (Errichiello et al., 2009; Kwan et al., 2000). Thus, Di Giacomo et al. propose that clinical responses and “relative” trends in antibodies titers overtime should be used instead of “absolute” values to guide treatment

6.3. Antiepileptic drugs Even though seizures in GAD65-epilepsy are resistant to antiepileptic drugs (AEDs), some patients may respond adequately to treatment with only AEDs from the beginning of the inflammatory disease or isolated seizures, and some patients can benefit from AEDs after Immunotherapy failure (Malter et al., 2015; Falip et al., 2012). For 6

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Funding

this reason, AEDs should be used throughout the GAD65-epilepsy. In the Cabezudo-Garcia, et al. Systematic review, 8% of patients with GAD65-epilepsy responded to AEDs and usually required a combination of various AEDs (Cabezudo-Garcia et al., 2018). There is no randomized clinical trial data to support one AED over another; theoretically, they should be more responsive to AEDs that enhance GABA function.

This work was supported by the Jilin Province Science and technology development plan project in China (Grant No.20180311075yy). Declaration of Competing Interest None of the authors has any conflict of interest to disclose.

6.4. Epilepsy surgery

References

The surgical outcomes of GAD65-epilepsy seem to be worse than with other etiologies of refractory epilepsy. In a retrospective study,8 patients with GAD65-associated TLE underwent anterior temporal lobectomy (ATL); 5 patients improve in seizure frequency but not seizure freedom (Carreno et al., 2017).In another study, selective amygdalohippocampectomy (SAH) resection for this disease reduced seizures in 2 of 3 patients (Malter et al., 2015), but it should be noted that SAH combined with immunotherapy may lead to worse performance of graphics and language memory compared with immunotherapy alone (Hansen et al., 2018). But, as mentioned above, In the third stage of GAD65-epilepsy, refractory epilepsy appears to be related to underlying “structural” etiology rather than ongoing inflammatory processes. In these conditions, especially when FDG-PET scanning revealed no hypermetabolic areas or serological and CSF markers have returned to normal, epilepsy surgery can still be considered as a useful treatment option to reduce seizure frequency and improved quality of life. Additionally, cases successfully treated with vagal stimulation (VNS) or Deep brain stimulation (DBS) have been reported. (Makela et al., 2018; Gillinder et al., 2018).

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7. Conclusions High anti-GAD65 Abs levels have been identified in many patients with acute/subacute-onset, chronic, or cryptogenic epilepsy. AntiGAD65 Abs are directed against the rate-limiting enzyme for the synthesis of GABA. Although anti-GAD65 Abs have been demonstrated to inhibit GAD65 enzyme activity and reduce amounts of available GABA content in some experiments, the pathogenic role of anti-GAD65 Abs remains unclear due to the intracellular location of the antigen and the controversial evidence of Abs internalization into neurons. Patients with GAD65-epilepsy frequently coexist multiple autoimmune disorders or auto-antibodies and may evolve into a more widespread CNS inflammatory disorder at some point in their illness. This condition often associated with not paraneoplastic, but if co-existing neuronal cellsurface antibodies are present, the risk of an underlying malignancy will greatly increase. Although patients with GAD65-epilepsy received multiple therapeutic interventions in varying combinations, the efficacy and prognosis were poor. Early identification of anti-GAD65 Abs and aggressive immunosuppressive treatment has the potential to prevent the development of permanent brain tissue damage, but it is usually only a temporary therapeutic success, so more aggressive and long-term immunosuppressive therapies are needed to prevent disease progression and relapse. Improvement of clinical symptoms may parallel a “relative” reduction in antibody titers, and patients with positive antiGAD65 Abs in CSF or extremely high serum titers have a higher risk of clinical adverse effect or recurrence, so monitoring antibodies titers may be used to guide immunotherapy. In the future, prospective studies recruiting patients with GAD65-epilepsy are needed to elucidate better the spectrum of epilepsy-related to anti-GAD65 Abs, and further studies on underlying pathophysiology may influence therapeutic management strategies. Contributors All authors planned the manuscript, did the literature search, contributed to the figures, and wrote, edited, and approved the manuscript. 7

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