Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

Abstract

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on axon loss in MS, far less is known about synaptic changes. Here, in striking similarity to other neurodegenerative diseases, we identify in postmortem human MS tissue and in nonhuman primate and mouse MS models profound synapse loss and microglial synaptic engulfment. These events can occur independently of local demyelination, neuronal degeneration, and peripheral immune cell infiltration, but coincide with gliosis and increased localization of complement component C3, but not C1q, at synapses. Finally, we use AAV9 to overexpress the complement inhibitor Crry at activated C3-bound synapses in mice and demonstrate robust protection of synapses and visual function. These results mechanistically dissect synapse loss as an early pathology in MS. We further provide a novel gene therapy approach to prevent synapse loss by microglia, which may be broadly applicable to other neurodegenerative diseases.1 1 induced in these mice ( Fig. 6C). Mice were then analyzed at the onset of moderate clinical scores (average: AAV-Crry: 1.4±0.3; AAV-EGFP 1.5±0.3), which were typically observed around day 11 post-immunization (AAV-Crry: 11.2±0.6; AAV-EGFP: 11.4±0.4) ( Fig. 7A). Using confocal imaging, we first determined the degree of EGFP colocalization with VGluT2 + -retinogeniculate terminals in the LGN and found ~45% of VGluT2 + -retinogeniculate terminals in the LGN colocalized with EGFP after injection of both AAVs (Fig. 6D). We then immunostained for Crry protein and identified that Crry was highly enriched in presynaptic bouton structures in EGFPlabeled retinogeniculate arbors vs. other fine processes (i.e. axons) within the LGN of AAV-Crry injected mice (Fig. 6E). Previous work has demonstrated that these boutons represent VGluT2 +presynaptic terminals along retinogeniculate arbors (Hong et al., 2014), precisely where we originally identified enrichment of C3 (Fig. 5). These data demonstrate successful CR2-mediated Crry targeting to retinogeniculate synapses tagged by activated C3. In contrast, AAV-EGFP injected mice showed only diffuse Crry immunoreactivity with no concentration on or around retinogeniculate synapses in the LGN following EAE. Regardless of AAV treatment, mice showed comparable development of EAE clinical scores, infiltration of peripheral immune cells, and micro-and astrogliosis (Fig. 7A-D). These data suggest that retinogeniculate overexpression of Crry did not have global, systemic effects, which is important when considering the beneficial and detrimental effects of inflammation in the development of new therapeutic strategies. Crry-mediated inhibition of activated C3 at retinogeniculate synapses blocks microglial synapse engulfment, rescues synapse loss, and restores visual functionFollowing validation that Crry is successfully expressed using our AAV9 strategy and localizes to retinogeniculate presynaptic terminals, we asked if Crry overexpression reduced deposition of C3 following EAE....

Meningeal inflammation in multiple sclerosis induces phenotypic changes in cortical microglia that differentially associate with neurodegeneration

“…5j). Similar to what happens in the thalamus of MS patients 31 , we found a significant increase in synaptic phago-cytosis specifically in MS1 cortical microglia ( Fig. 5j-k), which is in line with an overall increased phagocytic capacity of MS1 microglia as indicated by increased CD68 expression ( Fig.…”

“…Surprisingly, we did not detect an overall decrease in pre-synaptic density in SPMS cortex, despite evidence for increased synapse phagocytosis by microglia. Although loss of synapses has been extensively reported in hippocampus 39,40 , thalamus 31 and spinal cord of MS patients 41 , evidence for a loss of synapses in the cortex is more ambiguous. Reduced spine density on apical dendrites, indicating post-synaptic loss, has been shown in both myelinated and demyelinated cortex of MS patients 9 , whereas other studies, using similar methods to ours, only found a significant loss of synaptophysin in leukocortical demyelinated lesions but not in normal appearing MS cortex 42 and no correlation with cortical atrophy 43 .…”

CUB and Sushi Multiple Domains 1 (CSMD1) opposes the complement cascade in neural tissues

“…This raises the question of whether Csmd1 could affect which ages, regions, neurons, and synapses are more or less vulnerable to complement-dependent processes. As a recent paper found that inducing retinal ganglion cells to exogenously express Crry, a known complement inhibitor, protects not only the retinogeniculate synapses from complement deposition and microglial engulfment, but also neighboring synapses (though to a lesser degree) (Werneburg et al, 2020), non-cell autonomous effects could add another layer of complexity. Much of our mechanistic study focused on glutamatergic cells and synapses, and we found Csmd1 to be preferentially co-localized with glutamatergic synaptic markers in early postnatal mouse cortex.…”

An Antibody to RGMa Promotes Regeneration of Cochlear Synapses after Noise Exposure

“…Upregulation of RGMa in spinal cord injury results in a block to regrowth of motor neurons, but treatment with anti-RGMa antibody could overcome this inhibition (Hata et al, 2006). Synaptic loss is thought to play a role in declining cognitive function in Alzheimer's disease (Hong et al, 2016;Werneburg et al, 2020), and anti-RGMa had an effect on neural growth and connectivity in multiple sclerosis and spinal cord injury (Demicheva et al, 2015;Hata et al, 2006). Intracellular signaling elicited by neurotrophins is mediated in part by protein prenylation (Li et al, 2013), suggesting a link between neurotrophins and RGMa which has been shown to act through a prenylation pathway (Siebold et al, 2017).…”