Nodding syndrome may be an autoimmune reaction to the parasitic worm Onchocerca volvulus

Abstract

Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is an epidemiological association with Onchocerca volvulus, the parasitic worm that causes onchocerciasis (river blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoantibodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cortical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and suggests that patients may benefit from immunomodulatory therapies.

Fig. 1. Leiomodin-1 autoantibodies in patients with nodding syndrome

(A) Log₁₀-fold distribution plot depicting autoantibody reactivity differences between patients with nodding syndrome (NS) and unaffected village controls (UVC). Annotated on the graph are four proteins observed to have a >100-fold difference between nodding syndrome and unaffected village controls. (B) Immunoblot analyses of leiomodin-1 and DJ-1 immunoreactivity in sera from unaffected village controls or nodding syndrome patients. (C) Immunoblot analysis of recombinant DJ-1, recombinant leiomodin-1, and human brain homogenate probed with CSF pooled from 16 patients with nodding syndrome. In lane 1, there is no immunoreactivity to recombinant DJ-1. In lane 2, there is immunoreactivity to histidine-tagged leiomodin-1 (arrow; ~80 kDa). Lane 3 shows immunoreactivity to a single protein in brain homogenate at ~60 kDa, the molecular mass of leiomodin-1 (arrow). (D) Scatterplot depicting optical density (OD) of individual patient serum’s immunoreactivity to leiomodin-1 as determined by ELISA. The cutoff for determining a positive sample was set at 3 SD above the mean for U.S. healthy control sera (HC; n = 20). Data were analyzed by analysis of variance (ANOVA) that showed an overall significant difference (P = 0.0001), and a Sidak-Holms correction was applied to correct for multiple comparisons: nodding syndrome (n = 55) versus unaffected village controls (n = 55), P = 0.04. Data were also log-transformed and reanalyzed with consistent findings. (E) Coimmunostaining with patient sera (green) and rabbit anti–leiomodin-1 antibody (red). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 20 μm.

Fig. 2. Leiomodin-1 is expressed in human brain

(A) Cultured human neurons immunostained with rabbit anti–leiomodin-1 antibody (green channel). Vybrant Dil labels the cell membrane (red), and DAPI (blue) indicates the nucleus. Vybrant Dil and secondary antibody (2nd) only (rightmost). Scale bars, 20 μm. (B) Immunoblot analysis of human brain homogenates with rabbit anti–leiomodin-1 antibody and recombinant leiomodin-1 plus histidine-tag as a positive control. (C) Immunohistochemistry demonstrates leiomodin-1 staining in specific areas of the murine brain. Scale bar, 50 μm. (D) Murine skeletal muscle (top; scale bar, 100 μm) and murine smooth muscle in the wall of a blood vessel (bottom; scale bar, 50 μm) as positive controls. (E) Quantitative polymerase chain reaction (qPCR) measuring leiomodin-1 transcripts in HeLa cells (as a low-expressing positive control), human neuronal stem cells (NSCs), human neurons, brain homogenates from two individuals (brain 1 and brain 2), and H9C2 myoblast cells (as a high-expressing positive control). Data are expressed as the mean leiomodin-1 transcript levels [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] ± SD from three independent replicates. (Tabulated data are available in the Supplementary Materials.)

Fig. 3. Leiomodin-1 antibodies are neurotoxic

(A) Viability of primary human neurons treated with rabbit polyclonal anti–leiomodin-1 antibody compared to normal rabbit sera. Saponin was used as a positive neurotoxic control. Data shown are percent viability relative to treatment with vehicle only (P = 3.4 × 10⁻⁹; analyzed by repeated-measures ANOVA with Scheffe’s procedure), with the horizontal bars indicating the mean viability ± SD for n = 10 replicates. (B) Viability of neurons treated with sera from patients with nodding syndrome with detectable anti–leiomodin-1 antibodies compared to antibody-depleted sera from the same patient (P = 0.0003, two-way Student’s paired t test). Horizontal bars are the mean percent viability ± SD relative to cells in culture medium only of n = 5 replicates. (C) Neurotoxicity induced by each patient’s sera (PS) was compared to sera specifically depleted of leiomodin-1 antibodies (PS-depleted). Data shown are percent toxicity relative to saponin (n = 4; P = 0.0048, two-way Student’s paired t test). [Tabulated data are available for (A) to (C) in the Supplementary Materials.]

Fig. 4. Leiomodin-1 autoantibodies from the CSF of patients with nodding syndrome cross-react with O. volvulus proteins

(A) Western blot analyses of O. volvulus lysates probed with sera from unaffected village controls and patients with nodding syndrome. Red boxes indicate the region of the corresponding gel that was excised and analyzed by mass spectrometry for protein identification. (B) Sequence homology between human leiomodin-1 (accession number NP_036266) and O. volvulus tropomyosin (accession number Q25632) by pairwise alignment (EMBOSS, EMBL EBI with Smith-Waterman algorithm). Conserved tropomyosin regions 1 and 2 and their corresponding leiomodin-1 sequences are indicated by red boxes. (C) Depiction of structural homology between human leiomodin-1 (Hu_Leiomodin1) and O. volvulus tropomodulin (Ov_Tropomodulin) (accession number A0A044S1K6) (left) and between human DJ1 (Hu_DJ) (accession number Q99497) and the O. volvulus ortholog (Ov_DJ) (accession number A0A044RAJ4) (right). (D) Leiomodin-1 antibodies purified from patients reacted to a single band in HEK293 cells overexpressing tagged human leiomodin-1 (left). The same purified anti–leiomodin-1 antibodies demonstrated strong immunoreactivity to multiple proteins from O. volvulus whole-organism lysate (right). (E) Inhibition of patient sera immunoreactivity to leiomodin-1 by O. volvulus whole-organism lysate (oncho) compared to bovine serum albumin (BSA) (n = 4; P = 0.042, one-way Student’s unpaired t test). (Tabulated data are available in the Supplementary Materials.)