INTRODUCTION: Insulin-like growth factors 1 and 2 (IGF1 & 2) together with their receptor (IGF1R) have emerged as a critical autocrine system regulating dendritic spine structural plasticity. However, outside of the cell, IGF exists mostly in a complex with its binding protein 2 (IGFBP2). IGFBP2 which seems to be released mostly by astrocytes prevents IGF from degradation but also from binding to the IGF1R.

AIM(S): Here we aim to describe an extracellular system which involves Matrix Metalloproteinase- 9 (MMP-9) release from neurons, digestion of astrocytic IGFBP2 and release of IGF1 leading to IGF1R activation and synaptic plasticity.

METHOD(S): We have combined two-photon imaging and glutamate uncaging in to show structural long-term potentiation (sLTP)of dendritic spines in organotypic hippocampal cultures. We have used transgenic models of MMP-9 knockout as well as CRISPR/Cas9 knockdown of astrocytic IGFBP2 to elucidate role of those proteins in described system. Additionally, we have used immunofluorescent staining to localize IGFBP2 and validate gRNAs leading to IGFBP2 knockdown.

RESULTS: Using immunostaining we show that IGFBP2 is astrocytic protein and that it can be found in vicinity of synapses. We identify gRNAs leading to knockdown of IGFBP2. We show that blocking of IGFBP2 either with broad chemical inhibitor or with specific antibody reduces sLTP and that this effect can be rescued by application of IGF1. We show that MMP-9 cleaves IGFBP2 and is able to release IGF1. Moreover we show that MMP-9 knockout has decreases sLTP, which can be rescued by application of IGF1.

CONCLUSIONS: 1. IGFBP2 is a astrocytic protein which plays crucial role in sLTP. 2. IGFBP2 is important as a reservoir of IGF1 outside the cell 3. MMP-9 cleaves IGFBP2, leads to IGF1 release from the complex and activation of IGF1R.

FINANCIAL SUPPORT: National Science Centre, Poland grant: 2023/51/B/NZ4/02135