Identification of DAGLA as an autoantibody target in cerebellar ataxia

Abstract

Background: We aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis.

Methods: Serum and cerebrospinal fliud (CSF) samples from four index patients were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IIFA). Immunoprecipitation, mass spectrometry and recombinant protein assays were used to identify the autoantigen. Sera from 101 patients with various neurological symptoms and a similar tissue staining pattern as the index patient samples, and 102 healthy donors were analysed in recombinant cell-based IIFA (RC-IIFA) with the identified protein. Epitope characterisation of all positive samples was performed via ELISA, immunoblot, immunoprecipitation and RC-IIFA using different DAGLA fragments.

Results: All index patients were relatively young (age: 18-34) and suffered from pronounced gait ataxia, dysarthria and visual impairments. Paraclinical hallmarks in early-stage disease were inflammatory CSF changes and cerebellar cortex hyperintensity in MRI. Severe cerebellar atrophy developed in three of four patients within 6 months. All patient samples showed the same unclassified IgG reactivity with the cerebellar molecular layer. DAGLA was identified as the target antigen and confirmed by competitive inhibition experiments and DAGLA-specific RC-IIFA. In RC-IIFA, serum reactivity against DAGLA was also found in 17/101 disease controls, including patients with different clinical phenotypes than the one of the index patients, and in 1/102 healthy donors. Epitope characterisation revealed that 17/18 anti-DAGLA-positive control sera reacted with a C-terminal intracellular DAGLA 583-1042 fragment, while the CSF samples of the index patients targeted a conformational epitope between amino acid 1 and 157.

Conclusions: We propose that anti-DAGLA autoantibodies detected in CSF, with a characteristic tissue IIFA pattern, represent novel biomarkers for rapidly progressive cerebellitis.

Keywords: CSF; cerebellar ataxia; movement disorders; neuroimmunology.

Figure 1. Transverse T2 sections of the infratentorial brain (T1 for patient 3 and fluid-attenuated inversion recovery (FLAIR) for patient 4 at 18–28 weeks). At onset, T2-hyperintense signal changes in the cerebellar hemispheres were present in all patients (arrows). At follow-up, patients 1–3 showed a marked cerebellar atrophy (open arrows), whereas patient 4 showed only a mild atrophy.

Figure 2. Indirect immunofluorescence assays with brain tissues. Rat cerebellum (A), primate cerebellum (B), rat hippocampus (C), sagittal sections of murine whole brain (D), murine cerebellum (E) or murine hippocampus (F) permeabilised cryosections incubated with CSF of patient 1 followed by an incubation with Alexa488-labelled anti-human IgG. Nuclei were counterstained by incubation with TO-PRO-3 iodide or 4',6-diamidino-2-phenylindole (DAPI). A dense fine-speckled staining of the cerebellar molecular (A, B) and a weaker staining of the hippocampal molecular layer (C) were observed. On murine whole brain sections the patient CSF reacted mainly with the molecular layer of the cerebellum and also showed weaker reactivity against the hippocampus. No obvious reactivity against other parts of the brain was observed (scale bar A–C: 100 µm, E, F: 500 µm).

Figure 3. Identification of DAGLA as the target autoantigen. (A) Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis of IP eluates of serum/CSF of patient 2 (P2) or serum of a healthy control (HC1) with homogenised rat cerebellum. In each lane a band (1-3) of ~100 kDa was selected for liquid chromatography/mass spectrometry (LC/MS). (B) Mass spectrometry analysis identified DAGLA in the IP eluate of the patient serum (band 1, seven DAGLA peptides identified) and CSF (band 2, 22 DAGLA peptides identified) but not in the eluate of the healthy control serum (band 3, no DAGLA peptides identified). (C) The IP eluates of the patient serum or CSF or the healthy control serum were analysed in immunoblots using a commercial rabbit antibody binding to DAGLA. A band at ~100 kDa was detected in the eluate of the patient serum and CSF (arrow) but not in the eluate of the healthy control serum. (D) Indirect immunofluorescence with rat and primate cerebellum tissue using patient serum (P1) and a specific polyclonal anti-DAGLA antibody revealed an exact overlap of the molecular layer staining. (E) In competitive inhibition experiments immunofluorescence on rat and primate cerebellum the tissue reaction of the patient’s autoantibodies could be abolished by preincubation with HEK293 lysate containing DAGLA, whereas antibody binding was unaffected when a comparable fraction from empty vector-transfected HEK293 cells was used (scale bar 100 µm). DAGLA, diacylglycerol lipase alpha; IP, immunoprecipitation; LC/MS, liquid chromatography/mass spectrometry.

Figure 4. Indirect immunofluorescence using transfected HEK293 cells expressing DAGLA. Acetone-fixed recombinant HEK293 cells expressing DAGLA or an empty vector-transfected control were incubated with the patient’s CSF/serum or control CSF/serum (1:10) in the first step, followed by an incubation with Alexa488-labelled anti-human IgG. Patient’s CSF/serum reacted positively, while control CSF/serum was negative. Nuclei were counterstained with TO-PRO-3 iodide (scale bar: 100 µm). DAGLA, diacylglycerol lipase alpha.

Figure 5. Anti-DAGLA epitope characterisation with patient samples and anti-DAGLA RC-IIFA positive control sera. (A) In an ELISA with the purified C-terminal DAGLA 583–1042 fragment patient sera/CSFs (P1–P4), DC or HC sera (serum 1:100, CSF 1:10) or anti-His were incubated in the first step, followed by an incubation with anti-human or anti-mouse IgG-POD. The patient sera/CSFs, DC9 and all healthy control sera except HC29 were negative, while 16/17 DC sera and HC29 showed a positive reaction. (B) In an immunoblot with full length DAGLA patient sera/CSFs, DC or HC sera, tissue IIFA negative CSF controls (1:200) or anti-DAGLA rabbit antibody were incubated in the first step, followed by anti-human or anti-rabbit IgG-AP. All patient samples and DC9 (*) were negative or showed only a weak positive reaction (serum P4) while 16/17 DC sera and HC29 were positive. (C) Eluates of immunoprecipitates of HEK293-DAGLA 1–582 lysates and anti-His, patient or control sera were analysed in immunoblots with anti-His mouse antibody and anti-mouse IgG-AP. All patient sera and DC9 (*) were able to precipitate the C-terminal truncated DAGLA 1–582 variant, whereas the other control sera were not. (D) HEK293 cells expressing DAGLA, the C-terminal truncated DAGLA 1–582 or DAGLA 1–157 variants or an empty vector-transfected control were incubated with patient CSF, control CSF (1:1) or HC29 (1:10) in the first step, followed by an incubation with Alexa488-labelled anti-human IgG. The patient CSF also reacted with the C-terminal truncated DAGLA variants, while CSF of DC5 (arrow shows week positive reaction with HEK293 DAGLA cells), control CSF and HC29 did not. Nuclei were counterstained with TO-PRO-3 iodide (scale bar: 50 µm). DAGLA, diacylglycerol lipase alpha; DC, disease cohort; HC, healthy control; IIFA, indirect immunofluorescence assay; RC-IIFA, recombinant cell-based IIFA.