LAMP3 exacerbates autophagy-mediated neuronal damage through NF-kB in microglia

Abstract

Background and purpose

Cerebral ischemia/reperfusion (IR) after ischemic stroke causes deleterious microglial activation. Lysosomal associated membrane protein 3 (LAMP-3) has been indicated play a role in autophagy, yet the specific role of LAMP3 in microglia autophagy during cerebral ischemia and reperfusion (I/R) injury (CIRI) is unknown.

Methods

The oxygen-glucose deprivation/reperfusion (OGD/R) model and middle cerebral artery occlusion/reperfusion (MCAO/R) model were established. Changes in autophagy levels were detected through Western blot, immunohistochemistry, transmission electron microscopy, and laser scanning confocal microscopy. Oxidative stress damage in neurons was assessed using ROS and LDH assays. Cytokine levels (IL-6, IL-10, TNF-α, and IL-13) were measured using RT-qPCR and ELISA assays. HMC3, SH-SY5Y cell viability was evaluated using CCK8, EdU staining, Calcein/PI staining, and Transwell assays. Apoptosis was detected via TUNEL staining and flow cytometry. The role of LAMP3 in neuronal function post-cerebral ischemia-reperfusion was further investigated by administering rapamycin and BAY 11–7082.

Results

LAMP3 expression is decreased in IS, and negatively correlated with LC3B expression. In the HMC3 OGD/R model, LAMP3 inhibits microglial autophagy, and induces oxidative stress damage and inflammatory response in HMC3 cells through the NF-κB pathway. In co-culture system of HMC3 and SH-SY5Y cells, LAMP3 inhibits neuronal autophagy and activity through the NF-κB pathway under OGD/R conditions. In vivo, overexpression of LAMP3 inhibits autophagy and exacerbates brain tissue damage after MCAO/R.

Conclusions

During cerebral ischemia-reperfusion, LAMP3 inhibits autophagy in microglia and neurons by activating the NF-κB pathway, thereby inducing oxidative stress and inflammatory factor release, promoting neuronal death. Treatment targeting microglial LAMP3 might be a potential therapeutic strategy for ischemic stroke treatment.