Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases

Abstract

Objective: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases.

Methods: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples.

Results: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments.

Conclusions: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder.

Classification of evidence: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

Figure 1. Identification of AGO-Abs

(A) Immunostaining of the adult rat brain with CSF (1:10) of patient XI resulted in a strong reactivity with the stratum pyramidale of the hippocampus, granule cells of the cerebellum, and cerebral cortex. The scale bar is indicated for each magnification. (B) Immunoprecipitation of the antigens with patient XI's CSF. Note the specifically enriched band around 100 kDa (*) and the less intense band around 280 kDa (**) obtained after immunoprecipitation with patient XI's CSF (E+) compared with control CSF (E−). Protein bands were visualized via silver staining. (C) Quantified reactivities of 12 patients with SNN, 22 patients with ONPs, and 9 HCs with 7 AGO variants on protein microarrays. Red dots represent significantly positive samples (z-score > 4). AGO-Abs = antibodies against the Argonaute protein family; HC = healthy control; ONP = other peripheral neuropathy; SNN = sensory neuronopathy.

Figure 2. Confirmation of AGO-Abs Using Cell-Based Assay

HEK293 cells were transfected with VP5-AGO1-4 or VP5-TNRC6B plasmid, for a transient overexpression. Fixed and permeabilized cells were then immunostained with anti-HA antibody (in green) and patient XI's CSF (1:10; in red). Only AGO-transfected cells (in green) reacted with patient XI's CSF. No reactivity was observed on TNRC6B-transfected cells or nontransfected cells. Scale bar = 50 μm. AGO-Abs = antibodies against the Argonaute protein family.

Figure 3. Neuroimaging Findings in 2 Patients With AGO2-Abs

(A) Axial (top) and coronal (bottom) fluid-attenuated inversion recovery (FLAIR) brain MRI of patient XII with limbic encephalitis, showing bilateral swelling and hyperintensity of medial temporal lobes, extending also to a lesser degree to the lateral temporal cortex. (B) Axial FLAIR (top) brain MRI of patient XVII presenting with cerebellar ataxia, showing vermis atrophy and left cerebellar hemisphere hyperintensity. Pancerebellar atrophy is better demonstrated on the sagittal T2 brain MRI (bottom) of the same patient.