Figure 2 Identification of synapsin Ia, Ib, and IIa as antigenic targets of IgA antibodies in CSF (A) Homogenates of mouse cerebellum and hippocampus as.

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Figure 2 Identification of synapsin Ia, Ib, and IIa as antigenic targets of IgA antibodies in CSF (A) Homogenates of mouse cerebellum and hippocampus as well as subcellular fractions from whole brain progressively enriched for synaptic vesicles (homogenate, synaptosomes, synaptic membranes, synaptic vesicles) were blotted and incubated with patient CSF. Bound immunoglobulin A (IgA) was detected using a peroxidase-labeled antihuman IgA secondary antibody and enhanced chemoluminescence (ECL), demonstrating proteins between 70 and 100 kDa with enrichment in synaptic vesicles. Identification of synapsin Ia, Ib, and IIa as antigenic targets of IgA antibodies in CSF (A) Homogenates of mouse cerebellum and hippocampus as well as subcellular fractions from whole brain progressively enriched for synaptic vesicles (homogenate, synaptosomes, synaptic membranes, synaptic vesicles) were blotted and incubated with patient CSF. Bound immunoglobulin A (IgA) was detected using a peroxidase-labeled antihuman IgA secondary antibody and enhanced chemoluminescence (ECL), demonstrating proteins between 70 and 100 kDa with enrichment in synaptic vesicles. Incubation with control CSF yielded no signals. glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin served as loading controls. (B) Mouse hippocampus homogenate was immunoprecipitated with antihuman IgA (heavy chain) agarose beads preincubated with patient CSF. Pelleted beads were blotted and membranes incubated with patient CSF and antihuman IgA to detect the precipitated antigens. ECL detection revealed proteins of 70–100 kDa. Beads preincubated with control CSF or buffer did not precipitate proteins. (C) Coomassie-stained gel of the immunoprecipitated proteins. The boxed area, corresponding to the upper 3 bands seen in lane 2 in (B), was excised from the gel and subjected to mass spectrometry. (D) Mass spectrometry (MS) identified synapsin (Syn) I and Syn II as the 2 proteins with the highest Mascot scores. Amino acid sequences of Syn I and Syn II are shown with the identified peptides highlighted in green. (E) Brain extracts from wild-type, Syn I, Syn II, or Syn I/II/III knockout (KO) mice were blotted and incubated with patient CSF. Bound IgA was detected using a FITC-labeled antihuman IgA secondary antibody. Corresponding membranes were developed with commercial Syn I and Syn II antibodies. Syn Ia, Ib, and IIa bands were abolished in the respective KO mice. Syn IIb was not detected by the patient's antibodies. An approximately 80 kDa band visible in Syn I/II/III KO mice could represent an additional autoantigen of unknown identity. (F) Brain sections of wild-type and Syn I/II/III KO mice were incubated with a pan-Syn antibody, patient CSF, control CSF, or no primary antibody (negative control) and developed with a FITC-labeled antihuman IgA secondary antibody. Immunoreactivity of patient CSF in wild-type was abrogated in Syn I/II/III KO mice. IP = immunoprecipitation; pI = isoelectric point. Johannes Piepgras et al. Neurol Neuroimmunol Neuroinflamm 2015;2:e169 © 2015 American Academy of Neurology