Duloxetine ameliorates cerebral ischemic injury by inhibiting autophagy

Ischemic stroke is a severe medical condition characterized by diminished blood flow to the brain, resulting in a shortage of oxygen and nutrients. During ischemia, neurons surrounding the cerebral infarct initiate macroautophagy. However, the implications of this activation for neuronal cell survival are still debated. The identification of new autophagy modulators could aid in understanding autophagy’s role in brain ischemia and lay the groundwork for innovative therapeutic strategies aimed at minimizing brain damage in this life-threatening neurological emergency. In this study, we developed a robust and sensitive screening platform to identify autophagy modulators from a library of bioactive compounds. Selected compounds underwent further in vitro validation, leading to the identification of duloxetine, a Food and Drug Administration (FDA)-approved drug, as an effective autophagy inhibitor at low-micromolar concentrations. Following its original characterization, the molecule, a serotonin-norepinephrine re-uptake inhibitor (SNRI) family member, was subsequently tested in young and aged mice subjected to photothrombotic stroke. Our results demonstrated that duloxetine significantly reduced infarct size and improved locomotor performance in mice that had undergone a stroke. Similar protective effects were observed in transgenic mice lacking the autophagy gene Atg5 (autophagy related 5) in SLC17A6/Vglut2⁺ (solute carrier family 17 member 6) excitatory cortical neurons. Finally, we elucidated the underlying mechanism of action that involves duloxetine-mediated inhibition of TRPM2 (transient receptor potential cation channel subfamily M member 2) ion channels. Altogether, our findings suggest that early autophagy inhibition is neuroprotective in stroke, and duloxetine serves as an effective means of achieving this inhibition. Abbreviation: AMPK - AMP-activated protein kinase; ATG5 - autophagy related 5; AVs - autophagic vacuoles; Baf -bafilomycin A₁; BBB - blood-brain barrier; BECN1 -beclin 1; CAMK2 -calcium/calmodulin dependent protein kinase II; cCASP3 -cleaved CASP3; cKO -conditional knockout; CNS -central nervous system; DMPK - drug metabolism and pharmacokinetics; DMSO -dimethyl sulfoxide; DIV - days in vitro; DMEM - Dulbecco’s modified Eagle’s medium; FDA - Food and Drug Administration; FBS -fetal bovine serum; GFP -green fluorescent protein; GFAP -glial fibrillary acidic protein; HIF1A/HIF-1α - hypoxia inducible factor 1 subunit alpha; HMGCR - 3-hydroxy-3-methylglutaryl-CoA reductase; IHC - immunohistochemistry; I/R – ischemia-reperfusion; LAMP1 - lysosomal associated membrane protein 1; MAP1LC3B/LC3B -microtubule associated protein 1 light chain 3 beta; MCAO - middle cerebral artery occlusion; MFI -mean fluorescence intensity; MTT - 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; ND -nutrient deprivation; NPCs -neural precursor cells; NVU -neuro-vascular unit; OGD - oxygen-glucose deprivation; PI - propidium iodide; PIKK - phosphatidylinositol 3-kinase-like; PRKDC/DNA-PKcs - protein kinase, DNA-activated, catalytic subunit; SQSTM1/p62 -sequestosome 1; PIK3C3/VPS34 - phosphatidylinositol 3-kinase catalytic subunit type 3; PK-hLC3 -pHluorin-mKate2-human LC3; PLP1 - proteolipid protein 1; PT - photothrombotic; ROS -reactive oxygen species; SLC17A6/Vglut2 - solute carrier family 17 member 6; S:N –signal-to-noise; SSMD - strictly standardized mean difference; SNRIs - serotonin and norepinephrine reuptake Inhibitors; TTC - 2,3,5-triphenyltetrazolium chloride; TEER - transendothelial electrical resistance; TEM - transmission electron microscopy; TRPM2 -transient receptor potential cation channel subfamily M member 2; TBI -traumatic brain injury; ULK1 - unc-51 like autophagy activating ainase 1; V-ATPase - vacuolar-type H⁺-translocating ATPase.