Knockdown of NEAT1 prevents lipid droplet accumulation in microglia after ischemic stroke via autophagy
von Yongli Pan
Datum der mündl. Prüfung:2023-09-28
Betreuer:Prof. Dr. Thorsten Roland Döppner
Gutachter:Prof. Dr. Thorsten Roland Döppner
Gutachter:Prof. Dr. Michael Thumm
EnglischLipid droplets (LDs), lipid-storing organelles containing neutral lipids such as glycerolipids and cholesterol, are increasingly accepted as structural markers of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating autophagy and LDs accumulation in multiple neurological disorders. Interestingly, autophagy can modulate LDs accumulation as well. However, the knowledge about how NEAT1 modulates the formation of LDs in stroke is limited. In this study, primary microglia were isolated and identified from newborn mice. Then an in vitro oxygen-glucose deprivation and reperfusion (OGD/R) model was established, and qRT-PCR and immunofluorescence were employed to identify that NEAT1 and LDs were significantly increased in microglia after exposure to OGD/R. To further explore the mechanism, an antisense oligonucleotide (ASO NEAT1) was adopted to silence NEAT1 in microglia. It was observed that LD formation and autophagy-related protein LC3 were repressed, as exhibited in immunofluorescence and western blot analyses, respectively. Similarly, real-time PCR demonstrated the same observation on LD marker PLIN2, as well as signaling cascades related to autophagy (Atg3, Atg5, Beclin1, and STAT3). Additionally, 3-methyladenin (3-MA) and rapamycin (RAPA) were used to inhibit or activate autophagy, in turn, to illustrate the interaction between autophagy and LD accumulation. RAPA reversed the down-regulated LD accumulation and PLIN2 expression patterns induced by ASO NEAT1 under OGD/R conditions in microglia via immunofluorescence and western blot, whereas 3-MA promoted these effects. This thesis provides insight into NEAT1 knockdown as a potential novel treatment option for stroke by alleviating autophagy and further suppressing LDs.
Keywords: Stroke; NEAT1; Lipid droplet