Neuron-astroglial communication : exosomal heparanase isoforms released from astrocyte modulate synaptic strength

Abstract

The recent finding of heparanase 2 (HPSE2) expression in an astrocyte cluster primed this work to pursue if HPSE2 as sourced from peri-synaptic astrocytes serves to modulate synaptic transmission in the tripartite synapse. This was heralded by finding that peri-synaptic delivery of pro-heparanase (an HPSE2 analogue) triggered dose-dependent declines in both basal synaptic strength and long-term potentiation (LTP) in the Schaffer collateral (SC, CA3-CA1) pathway of the rat hippocampus (Cham 2014). Indeed, declines in both LTP and basal synaptic strength in the SC pathway of HPSE2 -treated hippocampal slices were observed. Cell surface protein internalization assay further found that HPSE2 treatment facilitated internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-syndecan-3 complexes from post-synaptic terminals among highly purified and networking hippocampal neurons (21 DIV).

With purified astrocytes in culture, low-dose NMDA (glutamate analogue) treatment of astrocytes in culture yielded significantly higher levels of exosomes, proHPSE1 and HPSE2 as recoverable from the culture medium, and revealed with Western blot and Nanoparticle tracking analysis. Transmission electron microscopy coupled with immunogold labelling that targeted HPSE1, HPSE2 and HS further provided evidence that the HPSE isoforms were associated with the astrocyte-derived exosomes (ADExo, 30-150 nm in diameter). With use of CD81-antibody coupled magnetic beads to capture the CD81-bearing HPSE-ADExo and subsequent exposure to treatment with heparinase III, decreased levels of HPSE isoforms provided evidence that the HPSE isoforms were associated with HS moieties on ADExo.

HPSE-ADExo were fluorophore-tagged and then incubated with hippocampal neurons in culture to study exosomal communication vis-à-vis astroglia-neurons. Confocal imaging revealed internalization of HPSE-ADExo by the neurons in culture but not by astrocytes in culture. Electrophysiological studies further showed declines in both basal synaptic strength and LTP at SC/CA1 synapses in the hippocampal slices treated with HPSE-ADExo. Enhanced internalization of AMPA receptor and syndecan-3 was observed following treatment of neurons in culture with HPSE-ADExo.

This study provides evidence that astrocytes respond to the spill-over of glutamate during neuronal transmission via release of HPSE2 and HPSE-ADExo which act to enhance bulk internalization of AMPA receptors and syndecan-3 at the post-synapse., A new means by which astrocytes in the tripartite synapse modulate synaptic strength is thus delineated. (352 words)