MicroRNA 223 Targeting ATG16L1 Affects Microglial Autophagy in the Kainic Acid Model of Temporal Lobe Epilepsy

Abstract

This study aimed to explore whether microRNA (miR) 223 affects microglial autophagy by targeting autophagy-related 16-like 1 (ATG16L1) in the kainic acid (KA) model of temporal lobe epilepsy (TLE). The miRNA and mRNA expression levels were quantified using quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expression was investigated using western blotting. A dual-luciferase reporter assay was used to test the direct interaction between miR 223 and ATG16L1. In situ hybridization was performed to measure the hippocampal expression of miR 223. We used immunofluorescence staining to assess the expression of ATG16L1 and microtubule-associated protein light chain 3 (LC3) in the murine hippocampal microglia. Inhibitor of miR 223 was utilized to investigate the role of miR 223 in TLE, and the epileptic activity was assessed using electroencephalography (EEG). The autophagosomes were observed by transmission electron microscopy. In patients with TLE, the murine KA model of TLE, and the KA-stimulated BV2 cells, miR 223, and sequestosome 1 (SQSTM1/P62) expressions were remarkably increased, whereas ATG16L1 and LC3 levels were significantly decreased. Using a dual-luciferase reporter assay, ATG16L1 was determined as a direct target of miR 223. Treatment with antagomir 223 alleviated epilepsy, prevented abnormalities in EEG recordings and increased the ATG16L1 and LC3 levels in KA-treated mice. Inhibition of miR 223 induced increased autophagy in BV2 cells upon Rapamycin stimulation. These findings show that miR 223 affects microglial autophagy via ATG16L1 in the KA model of TLE. The miR 223/ATG16L1 pathway may offer a new treatment option for TLE.

IncRNA UCA1 induces autophagic gene expression via epigenetic regulation mediated by ATG16L1 and miR‐132‐3p in SH‐SY5Y cells treated with retinoic acid

“…In this study, miR-132-3p levels were elevated and ATG16L1 decreased in the serum of epileptic patients and RA-treated SH-SY5Y cells, consistent with previous studies. ATG16L1 is an autophagic protein that is poorly expressed in epilepsy patients and animal/cell models that may promote autophagy and affect the progression of epilepsy [24]. In a kainic acid model of temporal lobe epilepsy, miR-223 affects microglia autophagy through ATG16L1 [24].…”

“…ATG16L1 is an autophagic protein that is poorly expressed in epilepsy patients and animal/cell models that may promote autophagy and affect the progression of epilepsy [24]. In a kainic acid model of temporal lobe epilepsy, miR‐223 affects microglia autophagy through ATG16L1 [24]. miR‐223 also inhibits autophagy and facilitates central nervous system inflammation by targeting ATG16L1 [38].…”

“…Autophagy-associated 16-like protein 1 (ATG16L1) is a recombinant protein with multiple functions in autophagy and related processes [23]. ATG16L1 is underexpressed in epilepsy and ATG16L1 can activate downstream signalling pathways, the activation of which can promote autophagy, thus affecting the progression of epilepsy [24]. In addition, miR-132 is predicted to bind ATG16L1, based on Starbase.…”

Molecular Mechanism and Regulation of Autophagy and Its Potential Role in Epilepsy

“…Some genetic sequencing studies for epilepsy patients and animal models suggest that the mutations in TBCK (encoding the TBC1-domain-containing kinase and inhibiting mTORC1 signaling), VSP15 (perturbing endosomal-lysosomal trafficking and autophagy flux), EPG5 (encoding a tethering factor that regulates the specific fusion of APs with late lysosomes/endosomes), SNX14 (encoding the sorting nexin family protein that mediating lysosome-AP fusion), ATP6V1A (encoding for the “A” subunit of the v1 sub-complex of V-ATPase, affecting lysosomal homeostasis and autophagy), ATP6AP2 (encoding a key regulator of v-ATPase, and its loss leads to lysosomal and autophagic defects), DMXL2 (encoding a member of the WD40 protein family that regulates v-ATPase trafficking and activity), or FAM134B (encoding the ER autophagy receptor) may contribute to the occurrence of epilepsy in space- and time-dependent manners [ 268 , 269 ] ( Table 1 ). Notably, recent studies have demonstrated that microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) involve the post-transcriptional regulation of autophagy-related proteins and the development of epilepsy pathophysiologies, such as miR-101, miR-181b, miR-134, miR-142, miR-421, miR-223, and Zinc finger antisense 1 (ZFAS1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), respectively [ 270 , 271 , 272 , 273 , 274 , 275 ]. Moreover, glia including astrocytes and microglia also plays a multi-faceted role in autophagy-mediated mechanisms that determine seizures and epileptogenesis [ 276 , 277 ].…”

Specific microRNAs for Modulation of Autophagy in Spinal Cord Injury

“…As previously mentioned for its role in neural autophagy, miR-223 has also been implicated in reducing neuroinflammation in microglia by targeting ATG16L [ 140 , 141 ]. Interestingly, it has been found that FOXO3a is yet another autophagy-related target of miR-223, with a negative correlation between the miR-223 levels and FOXO3a expression reported in several cell types [ 142 , 143 , 144 ].…”