Necl2/3-mediated mechanism for tripartite synapse formation

Abstract

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner and synaptically released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl-2/CADM1 trans-interacted with axonal Necl-3/CADM2, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl-2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl-2, synaptically released glutamate, and axonal Necl-3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl-3 knockout and Necl-2/3 double knockout mice confirmed these novel mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain

“… 21 , 41 More than 95% of the cells expressed astrocyte markers, such as AQP4 and EAAT1 ( Figure S13 A), consistent with previous reports. 21 , 41 , 42 As primary astrocytes formed the intricate structure of terminal processes, and endogenous expression levels of Kirrel2 were low, both membrane-anchored GAP-43 fused to GFP (GFP-mem) and Kirrel2 proteins were overexpressed in primary astrocytes ( Figure S13 B). The intense Kirrel2 signal was observed at the boundary between neighboring astrocytes ( Figure 12 A).…”

“…Mouse astrocyte cultures were prepared by differentiation of neurospheres derived from the mouse ganglionic eminence of male or female brain at embryonic day 18.5 as described previously. 21 , 41 For differentiation of astrocytes, neurospheres were treated with 1% FBS.…”