Astrocytes‐derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

Abstract

Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface. K E Y WO R D Sastrocytes, cytoskeleton, extracellular vesicles, neurons, optical tweezers, prion protein, PrP knock-out  INTRODUCTIONExtracellular vesicles (EVs) are circular membrane fragments released by all cells which function as intercellular signalling vehicles. EVs influence the behaviour of target cells in multiple ways, including the transfer of bioactive cargoes and the activation of signalling events at the cell surface (Holm et al., 2018). In addition, EVs possess enzymatic activity and are capable of modifying

Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer’s disease

“…4 ), in agreement with our previous observation that large EV size is a key factor retaining EVs at the neuron surface. 54 Altogether, these data indicate that Aβ-EVs move extracellularly along axonal projections, with a prevalent anterograde direction, supporting the hypothesis that they may propagate Aβ-mediated synaptic alterations among synaptically connected neurons.…”

“…Our recent work shows that a fraction of large EVs derived from astrocytes moves at the surface of cultured neurons exploring actin protrusions and use neurites as routes to pass between connected cells. 54 Based on this evidence, we first explored whether EVs of microglial origin may similarly move at the neuron surface. Using optical tweezers, we gently placed single large EV on cell bodies and neurites of developing hippocampal neurons, cultured from 2 to 12 DIV and examined EV–neuron interaction in bright field through live microscopy.…”

“…Annexin-V, bound to phosphatidylserine residues on the EV surface, can link the EV to tether molecule(s) expressed by recipient cells, 95 thus stabilizing EV–neuron contact with axons, inside which large EVs cannot be internalized (this study and D’Arrigo et al . 54 ), and hampering extracellular EV motion; and (iii) Aβ-EVs injected in the EC impair LTP in both the EC and the DG, while more static Aβ-EVs (annexin-V coated) inhibit LTP only in the EC and cannot propagate LTP impairment to the DG.…”

“…This hypothesis has been suggested by our recent work indicating that large astrocyte-derived EVs use neurites as routes to move extracellularly among connected neurons. 54 We show that large microglial Aβ-EVs affect synaptic plasticity both in culture and in slices and, once injected in the mouse brain, propagate synaptic dysfunction in the entorhinal–hippocampal circuit through a mechanism sensitive to annexin-V, a phosphatidylserine ligand blocking EV extracellular motion.…”

“…Optical trapping and manipulation of EVs was performed following the approach previously described. 54 , 61 In order to distinguish dendrites from axons, 12 × 10 4 neurons on 24 mm glass coverslips were transfected with RFP (red fluorescent protein) using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). Before recording, neurons were washed to remove EVs constitutively released by neurons and large-EVs (10 000 g pellet) produced by ∼1 × 10 5 microglia were added to neurons and maintained in 500 μl of phenol red-free neuronal medium in a 5% CO 2 and temperature-controlled recording chamber at 37°C.…”