Highlights d Microglia engulf and eliminate synapses in the visual thalamus of MS patients d MS-relevant animal models show synapse engulfment and loss occur early in disease d Complement C3, but not C1q, localizes to synapses in demyelinating disease d AAV-Crry inhibits C3 and microglia-mediated synapse loss and preserves function

Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a proinflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from induced pluripotent stem cell (iPSC) lines from patients with primary progressive multiple sclerosis (PPMS) failed to promote oligodendrocyte progenitor cell (OPC) maturation, whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS (PMS) autopsy brain tissues and iPS-derived NPCs from patients with PPMS. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin, which then enhanced PPMS NPC support for oligodendrocyte (OL) differentiation. A proteomic analysis of the PPMS NPC secretome identified high-mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of OL differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in PMS, which may impact how this disease is modeled and inform development of future myelin regeneration strategies.

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....

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

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