Early auto‐immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis

Abstract Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto‐immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well‐validated mouse models of optic neuritis that ribbon synapses in the myelin‐free retina are targeted by an auto‐reactive immune system even before alterations in the optic nerve have developed. The auto‐immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.


Figure legend: Appendix Figure S2
Mouse retinal lysates were probed with the indicated antibodies for Western blot. (A) Two different independent antibodies against CASPR1 detect a single protein band at the expected running position of CASPR1 at 180kDa (lanes 1,2). Similarly, two independent antibodies against contactin-1 (CNTN1) detect a single protein band at the expected running position of CNTN1 at 120kDa (lanes 3,4). (B) shows a control incubation in which the polyclonal CASPR1 antibody was pre-absorbed either with the CASPR1 peptide against which it was generated (B1, lane 2) or pre-absorbed with a control peptide (B2, lane 4). If the polyclonal CASPR1 antibody was preabsorbed with the CASPR1 peptide, the 180kDa band was completely abolished (Appendix Fig. S2, B1, lane 2), while the 180kDa band was completely unaffected if the antibody was pre-absorbed with a control peptide (Appendix Fig. 2, B2, lane 4).
In lane 1 (in B1) and lane 3 (in B2), the unblocked CASPR1 antibody was applied for comparison. Immunodetection of synaptophysin served as loading control. (C) Retina tissue from control mice (lane 1) and RIBEYE knockout mice (lane 2) were probed by the monoclonal antibody 2D9 against RIBEYE in Western blot. The ≈120kDa RIBEYE band was completely abolished in the RIBEYE knockout. As expected, CtBP2 is still present in the RIBEYE knockout tissue (Maxeiner et al., 2016). Probing the same nitrocellulose membrane with antibodies against dynamin1xb (Appendix Fig. 2Cb) served as loading control. WB, Western blot.

Figure legend: Appendix Figure S3
Cryostat section (A; 10µm in thickness) and semi-thin section (B,C; 0.5µm in thickness) immunolabelled with the indicated antibodies. The cryostat section in A) was immunolabelled with the rabbit polyclonal antibody against CASPR1. In (B,C). the retina was double-immunolabelled with rabbit polyclonal CASPR1 antibody and mouse monoclonal antibody 2D9 against RIBEYE. CASPR1 is strongly expressed in the synaptic layers of the retina, the OPL and IPL, respectively. High magnification analyses revealed that the CASPR1 immunosignal was particularly enriched in close vicinity to the synaptic ribbon (C). Appendix Fig. 3 was obtained by confocal microscopy. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20µm (A,B); 1µm (C).

Figure legend: Appendix Figure S4
Semi-thin (0.5µm-thin) sections of the mouse retina immunolabelled with the rabbit polyclonal CASPR1 antibody that was pre-absorbed either with a control peptide (peptide from the Tulp1 protein; Wahl et al., 2016) (B) or with the CASPR1 peptide against which the polyclonal CASPR1 antibody was raised (A). Sections were double-immunolabelled with anti-RIBEYE antibodies to visualize synaptic ribbons. The strong CASPR1 immunosignal in the OPL is completely absent if the rabbit polyclonal CASPR1 antibody was pre-absorbed by the CASPR1 peptide (A) whereas the synaptic CASPR1 immunolabel is completely unaffected if a control peptide was used for pre-absorption (B). The RIBEYE immunolabelling was unaffected by both of these treatments. Appendix Fig. S4 was obtained by confocal microscopy. ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 5µm. Figure S5 (A,B) High-resolution analysis of semi-thin sections of the mouse retina doubleimmunolabelled with the rabbit polyclonal antibody against CASPR1 and the mouse monoclonal antibody against RIBEYE (2D9) obtained by super-resolution structuredillumination-microscopy (SR-SIM). The CASPR1 immunosignal was particularly enriched around the synaptic ribbon. OPL, outer plexiform layer. (C) Contactin-1 (CNTN1) co-localizes with CASPR1 at photoreceptor synapses in the OPL in close vicinity to the synaptic ribbon. High-resolution confocal analyses of rod photoreceptor synapses of the mouse retina (0.5µm sections) triple-immunolabelled with rabbit polyclonal antibody against CASPR1, mouse monoclonal antibody against CNTN1 and rabbit polyclonal antibodies against RIBEYE (U2656). CNTN1 largely has a similar distribution as CASPR1 in photoreceptor synapses of the OPL and is highly enriched at the synaptic ribbon (see also Appendix Fig. S9). In (C4-C6), two immunosignals indicated in the respective figures were overlayered; In C7, all three immunosignals were overlayered to each other. Scale bars: 1µm (A-C).

Figure legend: Appendix Figure S6
Semi-thin sections of the mouse retina double-immunolabelled with the mouse monoclonal antibody against CASPR1 and rabbit polyclonal antibody against RIBEYE (U2656; Schmitz et al., 2000). The CASPR1 antibody predominantly labelled the synaptic layers of the retina, the outer plexiform layer (OPL) and the inner plexiform layer (IPL). The synaptic layers were visualized by doubleimmunolabelling with the antibody against RIBEYE. In the synaptic layers, the CASPR1 immunosignal displayed discrete spot-like distribution enriched in close vicinity to the synaptic ribbon (A). Appendix Fig. S6A was obtained by confocal microscopy. High-resolution SR-SIM microscopy (B,C) of the CASPR1 immunosignals in the OPL where photoreceptor ribbon synapses are located revealed that the CASPR1 immunosignals were highly enriched in close vicinity to the synaptic ribbon. In the incubations in A), B) and C) different secondary antibodies conjugated to different fluorophores were used on purpose to document that the immunolabelling results are independent from the selected secondary antibody/ fluorophore. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bars: 20µm (A); 1µm (B,C).

Figure legend: Appendix Figure S7
Confocal images of semi-thin sections of the mouse retina triple-immunolabelled with mouse monoclonal antibodies against CASPR1, rabbit polyclonal antibodies against PSD-95 (L667) and rabbit polyclonal antibodies against RIBEYE (U2656), as described in the Materials and Methods section. In A4,A4 and B4,B5 the indicated two immunosignals are overlayered on each other; in A6, B6, all three immunosignals were overlayered on each other. ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 2µm.

Figure legend: Appendix Figure S8
CASPR1 is located pre-synaptically in close vicinity to the synaptic ribbon. A) Highresolution confocal analysis of rod photoreceptor synapses in the OPL of the mouse retina (0.5µm-thin sections) that were triple-immunolabelled with the rabbit polyclonal antibody against CASPR1, mouse monoclonal antibody 2D9 against RIBEYE and the indicated third primary antibodies (A-F). In (A), the outline of a single presynaptic terminal was visualized by immunolabelling with antibodies against PSD95 (A2). The CASPR1 immunosignal is located within the presynaptic terminal in close vicinity to the synaptic ribbon. Furthermore, presynaptic CASPR1 was found in close vicinity to the active zone markers RIM2 (B) and CASK (C,D,E). In contrast, the CASPR1 signal did not overlap with the postsynaptic signal for mGluR6 that is localized at the tip of invaginating ON-bipolar cells that are located also in close vicinity to the synaptic ribbon (Appendix Fig. S8F). Appendix Figs. S8B,C,D,F were obtained by confocal microscopy; Appendix Figs. S8A,E by super-resolution structuredillumination-microscopy (SR-SIM). OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bars: 1µm (A-F).

Figure legend: Appendix Figure S9
Contactin-1 (CNTN1) co-localizes with CASPR1 at photoreceptor synapses in the OPL in close vicinity to the synaptic ribbon. (A) High-resolution confocal analyses of rod photoreceptor synapses of the mouse retina (0.5µm-thin sections) tripleimmunolabelled with rabbit polyclonal antibody against CASPR1, mouse monoclonal antibody against CNTN1 and rabbit polyclonal antibodies against RIBEYE (U2656). CNTN1 largely has a similar distribution as CASPR1 in photoreceptor synapses of the OPL and is highly enriched at the synaptic ribbon. (B,C) High-resolution confocal analysis of semi-thin (0.5µm-thin) sections of the mouse retina doubleimmunolabelled with the rabbit polyclonal antibody against CNTN1 and mouse monoclonal antibody against dynamin1xb (clone 1E10; Eich et al., 2017). CNTN1 has a similar distribution as dynamin1xb which is enriched in the presynaptic, periactive zone in rod photoreceptor synapses (Wahl et al., 2016;Eich et al., 2017). ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 1µm. Figure S10 (A) Western blot analyses of HEK cell extracts that were transfected with mCherry cDNA or CASPR1 mCherry cDNA with the monoclonal anti-CASPR1 antibody (5F9). 5F9 detects CASPR1 only in the CASPR1m-Cherry-transfected COS cells but not in the mCherry-transfected COS cells. A single band at the expected running position was observed. In (B), the same nitrocellulose membranes were re-probed with antiactin antibodies (C4, 1:2,000 dilution) as loading control. In C), mouse retina was probed with the 5F9 anti-CASPR1 monoclonal antibody in Western blot. The typical protein at ≈190kDa was detected as a single band. (D) 5F9 monoclonal antibody against CASPR1 produced a strong synaptic staining in the OPL. (E) High-resolution immunolabelling of a single synaptic ribbon with monoclonal anti-CASPR1 (5F9, E1) and polyclonal anti-RIBEYE antibody (E2). Overlay image is shown in (E3). This synaptic staining was blocked by pre-absorption with the CASPR1 peptide (F1) but not by pre-absorption with a control peptide (G1). F2,G2 represents an independent reference immunolabelling (anti-RIBEYE); F3,G3 are the corresponding overlay images. ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 5µm.