Article Figures & Data
Figures
- Figure 1 Patient Antibodies Localize to the AIS and NoR
(A) Mouse brain sections were immunostained with case 1 serum (1:1,000 dilution), case 1 CSF (1:100 dilution), and case 2 CSF (1:25 dilution). In all cases, patient IgG (green) immunostained structures consistent with the AIS (arrows). Images were captured at 20× on an epifluorescent microscope. (B–D) Anatomic coimmunostaining of case 1 and 2 CSF IgG (green) with the AIS and nodal marker AnkG (magenta) in the (B) cortex, (C) optic tract, and (D) cerebellum. In D, the dotted line indicates the boundary between the cerebellar WM and GCL. Arrows indicate axon initial segments while arrowheads indicate nodes of Ranvier which are demarcated by CASPR in red. All scale bars = 10 µm. Images were captured by confocal microscopy at 60 or 100×. AIS = axon initial segment; AnkG = ankyrin G; GCL = granule cell layer; IgG = immunoglobulin G; NoR = node of Ranvier; WM = white matter.
- Figure 2 Identification and Validation of Anti–BIV-Spectrin Antibodies
(A) Heatmap of PhIP-Seq enrichments of proteins expressed in the both AIS and NoR. Enrichments are represented as length-normalized rpK (total rpK for the given protein/number of peptides that map to that protein, see supplemental methods). βIV-spectrin was detected in case 1 CSF with a length-normalized rpK = 4.8. All dark and unmarked cells had a length-normalized rpK <1. (B) Graphical representation of the approximate and relative locations of actin-binding domain (ABD, blue), spectrin repeats (SR, red, * = partial SR), specific domain (SD), and pleckstrin homology domain (PH, green) relative to PhIP-Seq peptide enrichments in the heatmap below spanning the full length of βIV-spectrin (amino acids 1–2564, overlapping peptides are laid end-to-end). Heatmap of βIV-spectrin peptide enrichments regarding AIS and nodal βIV-spectrin isoforms Σ1 and Σ6. Each peptide corresponds to a single peptide. N and C refer to the amino and carboxy termini of βIV-spectrin. (C) Left, case 1 CSF immunostaining of AdvillinCre/+ DR-AnkGfl/fl and DR-AnkG−/− shows nodal staining in the absence of AnkG, suggesting that AnkG is not the autoantigen. Right, case 2 CSF immunostaining of ChatCre/+ VR-AnkGfl/fl and VR-AnkG−/− tissue indicates that case 2 does not harbor AnkG antibodies. (D) Case 1 and 2 CSF immunostaining of AdvillinCre/+ DR-βI/βIVfl/fl and DR-βI/βIV−/− shows the disappearance of nodal staining in the absence of βI/V-spectrin, suggesting that βIV-spectrin is the autoantigen in both cases. For C and D, CSF was immunostained at 1:4 dilution. All scale bars = 5 µm. AIS = axon initial segment; AnkG = ankyrin G; IgG = immunoglobulin G; NoR = node of Ranvier; PhIP-Seq = phage display immunoprecipitation sequencing.
- Figure 3 Direct Validation of Anti-BIV Antibodies by HEK 293T Overexpression Cell-Based Assay
(A) HEK 293T cells were transfected with Myc-βIV-spectrin Σ1 and Σ6, fixed, permeabilized, and immunostained with case 1 or case 2 CSF at 1:100 dilution. In each case, CSF IgG (green) substantially overlapped with Myc immunostaining (red). (B) HEK 293T cells were transfected with rat RFP-AnkG, fixed, permeabilized and immunostained with case 1 (1:100) or serum (1:1,000). In both instances, case 1 IgG (green) failed to immunostain or colocalize with commercial AnkG immunostaining (red). AnkG = ankyrin G; IgG = immunoglobulin G.
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