Early Changes in Exo- and Endocytosis in the EAE Mouse Model of Multiple Sclerosis Correlate with Decreased Synaptic Ribbon Size and Reduced Ribbon-Associated Vesicle Pools in Rod Photoreceptor Synapses

Abstract

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.

RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons

“…The RIBEYE B-domain antibody 2D9 also detects CtBP2 that is ubiquitously expressed as a nuclear co-repressor (Dembla et al, 2018). In the 0.5 µm thin retina sections, we did not observe a nuclear staining, similar to other studies (Dembla et al, 2018;Kesharwani et al, 2021), most likely because the focal concentration of CtBP2 in the nucleus is too low.…”

“…3D Super-Resolution Structured Illuminated Microscopy (3D SR-SIM) and Measurement and Quantification of Ribbon Contour Length 3D SR-SIM microscopy resolves immunolabeled objects beyond the diffraction limit of light (Schermelleh et al, 2010). For 3D SR-SIM microscopy, images were acquired from 1.5 µmthick immunolabeled retina sections using the ELYRA PS1 setup (Carl Zeiss Microscopy GmbH), largely as previously described (Dembla et al, 2020;Mukherjee et al, 2020;Kesharwani et al, 2021). The 1.5 µm-thick sections, which were processed for 3D SR-SIM, were labeled only with a single primary antibody (anti-RIBEYE A-domain: 6F4 or anti-RIBEYE B-domain: 2D9, as described in the respective figures) and processed for indirect immunofluorescence microscopy as described above.…”

“…The 1.5 µm-thick sections, which were processed for 3D SR-SIM, were labeled only with a single primary antibody (anti-RIBEYE A-domain: 6F4 or anti-RIBEYE B-domain: 2D9, as described in the respective figures) and processed for indirect immunofluorescence microscopy as described above. Images were acquired with a 63×/1.4 NA oil (DIC) objective using the 561 nm laser line collected by an Andor iXon EM-CCD camera (100 ms exposure time), as previously described (Wahl et al, 2013(Wahl et al, , 2016Dembla et al, 2020;Mukherjee et al, 2020;Kesharwani et al, 2021). For obtaining 3D SR-SIM images, z-stack images were taken at 125 nm intervals by using Zen 2012 software (black version).…”

“…Maximum 2D projection images were generated from the 3D images of single, cropped synaptic ribbons, as previously described (Dembla et al, 2020;Mukherjee et al, 2020). The contour length of the maximum 2D projections was measured using the open polyline tool (Dembla et al, 2020;Mukherjee et al, 2020;Kesharwani et al, 2021). Average values were calculated and plotted in Microsoft Excel.…”

“…Synaptic ribbons are large presynaptic structures with a complex threedimensional (3D) shape (Moser et al, 2020). The 3D shape and size of the ribbon are relevant for synaptic transmission/signaling because the ribbon geometry determines the pool size of ribbon-associated vesicles (Moser et al, 2020;Kesharwani et al, 2021). The photoreceptor synapse as the first synapse in the visual system transmits light stimuli to the inner retina.…”