Abstract
Purpose :
To determine the properties of primary optic nerve head astrocyte (ONHA)-derived extracellular vesicles (exosomes), their uptake into primary neurons, and to identify the functional effects of exosomes on neuronal survival and neurite outgrowth.
Methods :
Exosomes were prepared by ultracentrifugation from primary rat ONHAs. To determine the functional effects of lysyl oxidase like-1 (Loxl1) knockdown, we transduced ONHA with lentivirus harboring either control or Loxl1-targeting small hairpin RNA. A stable line with ~50% Loxl1 knockdown was generated. Exosomes were characterized by nanoparticle tracking analysis and immunoblotting. Uptake of exosomes into primary cortical neurons was quantified by quantitative microscopy using RNA-based labeling. Functional effects of exosomes were determined by testing their antioxidative properties in primary cortical neuronal culture. To quantify effects on neuronal differentiation, exosomes were added to neuronal cultures on days 3 and 5 in vitro and neurite outgrowth was quantified on day 7.
Results :
Exosomes derived from wildtype, control- and Loxl1 shRNA-transduced ONHAs were of similar size (119.8±4.3, 121.9±2.8 and 121.1±2.5 nm, respectively, n=3, P=0.89). ONHA exosomes expressed the prototypical markers Alix and CD63, but were deficient of CD81. Exosomes were taken up by primary neurons, and this uptake was time-dependent and peaked at 4 h after addition (n=3, P<0.05). Viability of rat primary cortical neuronal cultures in response to oxidative stress insult did not change in the presence of exosomes derived from wild-type, control- or Loxl1 shRNA-transduced ONHA cultures, suggesting lack of antioxidative properties. Wild-type ONHA exosomes increased neurite outgrowth of primary rat cortical neurons by 20.1±3.3% (from 57.6±4.4 to 77.6±6.5 µm, n=5, P<0.05); similarly, exosomes from control shRNA-transduced ONHAs increased neurite outgrowth by 18.7±2.0% (to 76.3±4.6 µm, n=5, P<0.05). In contrast, exosomes from Loxl1 shRNA-transduced ONHAs prevented any increase in neurite outgrowth (2.2±4.0% change; 59.8±3.3 µm, n=5, P=0.98).
Conclusions :
Our data provide evidence that ONHA-derived exosomes can exert functional effects on neurons and identify Loxl1 as a putative mediator of these effects. Altered exosome-mediated neuron-glia signaling in exfoliation glaucoma, where LOXL1 expression is chronically reduced, may be of pathological importance.
This is a 2021 ARVO Annual Meeting abstract.