# Restricting Synaptotagmin‐3 Internalization Mitigates Cerebral Ischemia/Reperfusion Injury by Curtailed Neuronal Apoptosis and Microglial Re‐Programming

## TL;DR

A peptide that blocks Synaptotagmin-3 internalization reduces brain damage after stroke by protecting neurons and reprogramming immune cells.

## Contribution

A novel therapeutic strategy for stroke by inhibiting Synaptotagmin-3 internalization to protect neurons and reprogram microglia.

## Key findings

- Peptide 3Y reduced infarct volume, brain edema, and improved neurological deficits in stroke models.
- 3Y suppressed neuronal apoptosis and shifted microglia to an anti-inflammatory state via the cAMP pathway.
- In vitro, 3Y protected neurons and induced microglia to adopt a protective, TGF-β1-secreting phenotype.

## Abstract

Ischemic stroke is a major global cause of disability and death. Synaptotagmin‐3 (Syt3), a synaptic calcium sensor, exacerbates ischemic injury by promoting pathological glutamate release. This study investigated the potential neuroprotective effect of restriction of Syt3 internalization on neuronal apoptosis and microglial reprogramming following ischemia/reperfusion (I/R).

The effects of Tat‐GluA2‐3Y (3Y), a cell‐permeable peptide inhibitor of Syt3 internalization, were studied in a mouse model of middle cerebral artery occlusion/reperfusion (MCAO/R) and in oxygen–glucose deprivation/reperfusion (OGD/R)‐induced HT22 and BV2 cells.

The peptide 3Y treatment significantly reduced infarct volume, brain edema, and improved neurological deficits. Additionally, it reduced neuronal apoptosis and also inhibited neuroinflammation by down‐regulating the level of IL‐1b, TNF‐a, and iNOS but up‐regulating the level of TGF‐b1 and Arg‐1. Single‐nucleus RNA sequencing of the peri‐infarct region revealed a dual mechanism: 3Y suppressed pro‐apoptotic gene programs in neurons and facilitated the transformation between different phenotypes of microglia via the cAMP pathway from a pro‐inflammatory to an anti‐inflammatory and immune‐protective state. In vitro, 3Y attenuated oxygen–glucose deprivation‐induced neuronal death and instructed microglia to adopt a protective, TGF‐β1‐secreting phenotype.

Collectively, our findings establish the inhibition of Syt3 internalization as a novel therapeutic strategy that concurrently protects neurons and reprograms the microglial immune response, offering a promising dual‐mechanism approach for acute stroke therapy.

Restriction of Syt3 internalization by peptide 3Y conferred robust neuroprotection against cerebral ischemia/reperfusion injury. Mechanistically, 3Y suppressed neuronal apoptosis fundamentally, and additionally, these salvaged neurons orchestrated and remodulated the transformation of surrounding microglia toward an anti‐inflammatory and immunoregulatory‐oriented direction to mitigate neuronal damage immediately.