One autoantibody associated with limbic encephalitis (LE) targets intracellular neuronal antigens (IAg) (GAD65 receptor) and three others target the neuronal surface antigens (SAg) (NMDA [N-methyl-D-aspartate) receptor, VGKC [voltage-gated potassium channel]-complex, and AMPA [α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid] receptor). 

Classically, autoimmune symptoms associated with them evolve over days to weeks and include neurocognitive and neurobehavioral manifestations as diverse as short-term memory loss, sleep disturbances, seizures, irritability, depression, hallucinations, and personality change, implicating inflammatory involvement of the limbic system, which includes the medial temporal lobes, frontobasal and cingular regions. This blog discusses anti-GAD65 autoimmune encephalitis, and forthcoming blogs will review the three others (NMDA, VGKC-complex, and AMPA receptor-associated encephalitis. 

Background: Autoimmunity targeting the 65 kilo Dalton (kDa) isoform of GAD65 encompasses diverse autoimmune disorders such as type 1 diabetes and rare neurologic disorders including LE, temporal lobe epilepsy (TLE), cerebellar ataxia, and large and small fiber peripheral and autonomic neuropathy.  

Epidemiology: A review of adult-onset SPS showed a prevalence estimate of 1 in 1,250,000 with a predominance of women, and average age of onset of 40 years.  The frequency of high titers of anti-GAD antibodies defined an RIA value >1000 IU/ml in TLE of unknown origin is 21% (57) of cases, with the highest titers related to TLE. Affected patients were typically women with type 1 diabetes, early-onset epilepsy, and concomitant hypothyroidism, psoriatic arthritis and Celiac disease, a third of whom reported onset of LE as the predominant feature, with supportive findings of amygdala and hippocampus signal intensities on brain MRI, and medial temporal hypometabolism on FDG brain PET. The levels of anti-GAD ranged from 1207 to 87 510 IU/ml, with absent OCB, and a ratio of serum/CSF GAD antibody levels >1 suggesting intrathecal synthesis.  Malter and colleagues estimated the prevalence of GAD antibodies in LE to be 17%, noting a subgroup of patients with TLE who had very high titers equivalent to those with SPS, medial temporal inflammation on MRI, and concomitant LE. In the TLE cohort, GAD antibody encephalitis proved to be equally common to VGKC-complex antibodies but differed in younger age, female sex, and presentation of first seizure, CSF oligoclonal bands, and intrathecal autoantibody synthesis.
 
A recent retrospective analysis of patients with paraneoplastic neurologic syndromes from 1995 to 2013 defined by the criteria of Graus and colleagues detected high GAD antibody levels (>2000 U/mL) by RIA in 15 patients. Six patients (40%) presented with encephalitis (5 typical LE); 6 PCA, 2 SPS, 1 POMA, 1 PEM, and 1 with a syndrome of vertigo, ataxia, axial rigidity and dysautonomia. Six patients had lung cancer, 4 neuroendocrine tumors, 2 thymic carcinoids, 2 thymoma, 2 breast cancer, and 1 patient had non-Hodgkin lymphoma. Cancer preceded the neurological diagnosis by a median delay of 3 months. In 3 tumors so studied all expressed GAD65 antigenicity. In a comparison to 106 patients with non-paraneoplastic neurological syndromes, the latter group had encephalitis in 16% (none LE); with nearly equal prevalence of PCD and SPS (67%), and 18% isolated epilepsy. Patients with high levels of GAD antibodies, and classic or other neurological syndromes not typically associated with GAD antibodies, were at higher risk for an underlying cancer.   

Clinical Aspects: Gangnon and Savard reviewed the clinical experience of 58 cases of GAD65-antibody LE beginning with the first reported case inclusively through 2016, in 7 observational studies, 3 case series, and 21 published case reports.  They provide a useful summary of the literature of anti-GAD65 associated LE. Among the 58 cases, there were 21 children and 37 adults, 59% of whom were female, with a median pediatric age of 10 years (range 1-17 years) and mean adult age of 39 years (range 19-70 years). Diabetes alone, generally T1D, was noted in 50% of cases, in association with thyroiditis, diabetes, Celiac disease, psoriasis, and common variable immune deficiency respectively in 73%, 18%, 9%, and 9%.  Cancer was noted in 6 (10%) cases, including 4 SCLC and 2 malignant thymomas, generally in men of mean age 61 years (range 38-70 years). 

The commonest presenting clinical features were seizures in 56 (97%) cases, most commonly refractory status epilepticus; cognitive impairment in 38 (59%) mainly affecting memory, language, executive function, and attention; psychiatric symptoms in 16 (28%) cases, most commonly depression, behavior, perception, and anxiety. Less common clinical manifestations included fever, dysautonomia, cerebellar incoordination and headache respectively in 8 (12%), 7 (12%), 4 (7%), and 3 (5%) cases.  The most common seizure presentation was refractory status epilepticus.  Mild muscular rigidity and stiff person syndrome were diagnosed in 2 cases, as well as myoclonus and facial cramps in 4 others. 

Cellular and Synaptic Antibody Effects: Low titers of anti-GAD65 antibodies, generally <20 nmol/L occur in type 1 diabetes and in the general population, while cases of anti-GAD65-associated neurological disorders including LE are seen in the hundreds of nmol/L. Two GAD isoforms with distinct localizations and functions, GAD65 and GAD67, are expressed in presynaptic CNS GABAergic neurons, and in pancreatic b cells. Glutamic acid decarboxylase converts L-glutamate to GABA using pridoxal-5’-phosphate (PLP) as a cofactor. GABA is the commonest inhibitory neurotransmitter in the CNS and the ligand for the inhibitory voltage-gated chloride channel GABAA, and G-protein coupled GABAB receptors.  GAD67 commonly coexists with GAD65 antibodies in patients with neurologic autoimmunity. However GAD67 is rarely an autoantigen in isolation, found predominantly in the cytoplasm where it produces basal levels of GABA; whereas GAD65 is located predominantly in nerve terminals anchored to the cytoplasm-facing side of synaptic vesicles where it believed to synthesize GABA for neurotransmission supplementary to basal levels. The classification of high titers of anti-GAD65 autoantibodies has bene problematic in being grouped with onconeural autoantibodies directed at an IAg since they are not consistently paraneoplastic in origin.

Laboratory Investigations: A dominant clinical phenotype of seizures, neurocognitive and neuropsychiatric disturbances in most patients with anti-GAD-autoantibody associated LE has been explained by the frequent involvement of the medial temporal lobes; an inflammatory CSF with intrathecal secretion of the anti-GAD65 autoantibody, and oligoclonal bands. Bien and colleagues described a 24-year-old woman with frequent temporal lobe seizures, non-paraneoplastic LE, and a serum anti-GAD65 antibody titer of 1:32,000, in whom T2/FLAIR MRI evolved over a period of 8 months, demonstrating right hippocampal swelling and signal increase to sclerosis and atrophy on MRI commensurate with clinical progression. The same authors extended their findings to two other patients age 18 and 31 years, with a respective duration of disease of 127 and 115 months, manifesting refractory temporal lobe seizures and LE. The latter patient showed similar evolution of medial temporal atrophy in the context of T2/FLAIR hyperintensities during the disease course. The remaining patient who had unilateral encephalitic T2-hypersignal in the left amygdala was found at the time of epilepsy surgery to have a normal MRI without medial temporal brain atrophy despite a 10-year disease history. 

Among 58 literature patients, 45/58 (78%) patient MRIs were abnormal with specific involvement of the temporal lobes in 34 (59%), and multifocal abnormalities in 9 (16%); and 7 patient MRIs were normal. 

The results of electroencephalography (EEG) available in 35 cases, showed epileptiform discharges in 27 (77%), and focal temporal involvement in 19 (70%). Lumbar CSF so studied in 41 cases, showed pleocytosis in 11 (27%) with white blood cell (WBC) counts ranging from 7 to 114 cells/ul; and present oligoclonal bands in one-half of cases. Hyponatremia was identified in 3 cases. There were significantly elevated titers of anti-GAD65 antibodies in both serum and CSF in 35 patients; and in either serum (in 18) or CSF alone in 3. Concurrent antibodies were reported in 11 cases, including those to GABAA in 5 cases, and VGKC-complex or GABAB in 3 cases. Antibodies to NMDA receptor and AMPAR, and onconeural antibodies were all absent.
FDG brain PET imaging and MRI were complementary in 50% of cases (49). Combining both MRI and PET, all patients had temporal lobe abnormalities. EEG may be useful in classifying the seizure type and directing the use of anticonvulsants especially when clinical seizures lack motor convulsions.  

Histopathologic Correlation: The clinicopathologic features of anti-GAD antibody LE were described in a woman who presented with frequent complex temporal lobe seizures at age 23. Over 8 months there was an evolution of right hippocampal swelling and signal increase on T2/FLAIR MRI to sclerosis and atrophy. At the time of epilepsy brain surgery CSF showed 10 WBC  and oligoclonal bands. Right-sided selective amygdalohippocampectomy of the sclerotic hippocampus showed strong encephalitic features. High-dose corticosteroid therapy administered over several months resulted in seizure-free status until she was weaned off them 2.4 years later when she developed Cushing syndrome. She lapsed into a series of temporal lobe seizures and memory impairment accompanied by temporal left hippocampal swelling for which long-term corticosteroid and azathioprine therapy was started with slow clinical improvement. At follow-up 3.7 years later, she was still experiencing frequent partial temporal lobe seizures with memory deficits.  

Bien and colleagues summarized the histopathologic features of selective amygdalohippocampectomy of the sclerotic hippocampus in this patient that included neuronal loss and astrogliosis and a strong accumulation of inflammatory cells in the resected hippocampus. There was marked invasion of the hippocampus by lymphocytes that were mainly CD8+T-cells with the cytotoxic effector molecule GrB, in addition to CD20+ B-cells and CD138+ plasma cells. The pattern of pyramidal cell loss was severe in sectors CA4 and CA3, with selective sparing of CA1 and 2.  Surviving neurons were positive for MHC class I, fulfilling the prerequisite for attack by CD8+ T-cells. 

Bien and colleagues quantitated the number of parenchymal T-, B-, and plasma cells, macrophages and glial cells in 3 cases of anti-GAD65 autoantibody LE. Their analysis which included one previously reported case, differentiated anti-GAD65 cases from others with IAg-onconeural autoantibodies (Ma2 in 3 cases; Hu in 4 cases); SAg autoantibodies targeting VGKC-complex (4 cases) and NMDA receptor (3 cases), compared to cases of Rasmussen encephalitis (22 cases) and neurodegeneration controls (25 cases).  The percentage of CD8 T-cells in the IAg-GAD cases was intermediate (54%) between the IAg-onconeural and SAg cases. The CD8+/CD3+ ratio of the SAg cases was significantly different from the Rasmussen encephalitis controls. That ratio was lower than in the parenchyma confirming that CD8+ T-cells migrated into the parenchyma more readily than CD4+ T cells. Apposition of multiple GrB+ lymphocytes to single neurons, consistent with a specific cytotoxic T-cell attack in case GAD/3.  Bien and colleagues noted diffuse cytoplasmic IgG detected by anti-human IgG in both neurons and astrocytes in all cases similar to that of controls, which they attributed to leakiness of damaged neuronal membranes. Staining of C9neo indicative of complement activation was negative in the IAg GAD cases. CD68+ cells comprised 0.2% in the IAg-GAD cases. 

The finding of a reduced CD8+/CD3+ ratio of the IAg-GAD cases, all without concomitant tumor, was lower than the IAg-onconeural group. It was also within the highest ratios of the SAg group of NMDAR cases compatible with the hypothesis that T-cells were a necessary aspect for neuronal loss and hippocampal atrophy in the 2 GAD-associated LE (in GAD cases 2 and 3). Notwithstanding, the absence of an underlying malignancy could contribute to the long duration of disease and overall less intense inflammation.  
As noted by Bien and coworkers, the pathogenic role of GAD antibodies has not been certain because of the different associated clinical syndromes (SPS, cerebellar ataxia, TLE and LE). Multiple antibodies against IAg and SAg may explain the relatively low CD8+/CD3+ and GrB+/CD3+ ratios in the IAg-GAD cases. 

Diagnosis and Treatment:  The diagnosis of anti-GAD LE is justified in patients with a clinical syndrome of temporal lobe seizures, and cognitive and psychiatric disturbances, brain MRI abnormalities on T2FLAIR MRI implicating the medial temporal lobes; CSF pleocytosis and OCB; and EEG revealing temporal lobe epileptic or slow-wave activity, in association with high levels of anti-GAD65 autoantibodies by RIA. Most cases will fail to disclose an underlying malignancy. 
In the series of 58 literature cases summarized by Gangnon and Savard, follow-up was available in 53 cases. Full recovery was noted in 4 (8%) cases, 3 of whom received corticosteroids alone, with intravenous (IV) immunoglobulins, or in combination with PE; a fourth case received no immunosuppressant therapy and recovered.  Death occurred in 4 (8%) cases, 3 of whom had an associated cancer. Sustained improvement was noted in 23 (43%) cases with follow-up of 96 months.  A favorable outcome was noted in 45% of cases with positive CSF GAD65 antibodies compared to 56% of those with antibodies only in serum.