Urolithin A-activated autophagy but not mitophagy protects against ischemic neuronal injury by inhibiting ER stress in vitro and in vivo

CNS Neurosci Ther. 2019 Sep;25(9):976-986. doi: 10.1111/cns.13136. Epub 2019 Apr 11.


Aim: Mitochondrial autophagy (mitophagy) clears damaged mitochondria and attenuates ischemic neuronal injury. Urolithin A (Uro-A) activates mitophagy in mammal cells and Caenorhabditis elegans. We explored neuroprotection of Uro-A against ischemic neuronal injury.

Methods: Mice were subjected to middle cerebral artery occlusion. The brain infarct and neurological deficit scores were measured. The N2a cells and primary cultured mice cortical neurons were subjected to oxygen-glucose deprivation and reperfusion (OGD/R). Uro-A was incubated during OGD/R, and cell injury was determined by MTT and LDH. Autophagosomes were visualized by transfecting mCherry-microtubule-associated protein 1 light chain 3 (LC3). The protein levels of LC3-II, p62, Translocase Of Inner Mitochondrial Membrane 23 (TIMM23), and cytochrome c oxidase subunit 4 isoform 1 (COX4I1) were detected by Western blot. The ER stress markers, activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), were determined by reverse transcription-polymerase chain reaction (RT-PCR).

Results: Urolithin A alleviated OGD/R-induced injury in N2a cells and neurons and reduced ischemic brain injury in mice. Uro-A reinforced ischemia-induced autophagy. Furthermore, Uro-A-conferred protection was abolished by 3-methyladenine, suggesting the requirement of autophagy for neuroprotection. However, mitophagy was not further activated by Uro-A. Instead, Uro-A attenuated OGD/R-induced ER stress, which was abolished by 3-methyladenosine. Additionally, neuroprotection was reversed by ER stress inducer.

Conclusion: Urolithin A protected against ischemic neuronal injury by reinforcing autophagy rather than mitophagy. Autophagy activation by Uro-A attenuated ischemic neuronal death by suppressing ER stress.

Keywords: autophagy/mitophagy; cerebral ischemia; endoplasmic reticulum stress; neuroprotection; urolithin A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy / drug effects*
  • Autophagy / physiology
  • Brain Ischemia / metabolism
  • Brain Ischemia / pathology
  • Brain Ischemia / prevention & control*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Coumarins / pharmacology
  • Coumarins / therapeutic use*
  • Endoplasmic Reticulum Stress / drug effects*
  • Endoplasmic Reticulum Stress / physiology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mitophagy / drug effects*
  • Mitophagy / physiology
  • Neuroprotective Agents / pharmacology
  • Neuroprotective Agents / therapeutic use*


  • Coumarins
  • Neuroprotective Agents
  • 3,8-dihydroxy-6H-dibenzo(b,d)pyran-6-one