GLX-351322

Sleep deprivation (SD) can negatively affect the central nervous system owing to its detrimental effects on learning and memory. l-theanine, known for its antioxidative properties, can confer neuroprotection by enhancing learning and memory. Here, we explored whether l-theanine could improve learning and memory by inhibiting the NOX4-mediated ferroptosis in hippocampal neurons caused by SD. The Morris water maze (MWM) was employed to assess learning and memory ability. MDA, SOD, GSH, LDH, and Fe²⁺ levels were measured using their specific assay kits. The degree of damage in hippocampal neurons was examined by Nissl staining. The process of ferroptosis in the hippocampus of SD mice and HT22 cells was analyzed using transmission electron microscopy (TEM), immunohistochemistry, flow cytometry, and Western blotting assays. l-Theanine treatment significantly increased the frequency of mice crossing the platform. Moreover, it conferred protection on hippocampal neurons and mitochondria in SD mice. Furthermore, l-theanine mitigated Erastin-induced oxidative stress and ferroptosis in HT22 cells. In addition, it reversed the abnormal expression of proteins including PSD-95, SYN, NOX4, TFRC, ACSL4, Nrf2, Keap1, SLC7A11, P53, HO-1, NQO1, FTH1, and GPX4 in the hippocampus of SD mice and in the HT22 cells induced by Erastin. Moreover, the neuroprotection of l-theanine was reversed by the GLX351322 agent. l-Theanine treatment inhibited hippocampal ferroptosis by attenuating NOX4, suggesting that it can be used to mitigate the adverse effects of SD.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder with uncleared mechanisms. Short-chain enoyl-CoA hydratase 1 (ECHS1) is a mitochondrial enzyme critical for the β-oxidation of fatty acids and ATP production. This study aims to explore the roles of ECHS1 in PD by using rotenone-induced experimental PD models.

To evaluate the role of ECHS1 in rotenone-induced dopaminergic neurodegeneration, adeno-associated virus (AAV)-ECHS1 was stereotactically injected into the substantia nigra region of mice to overexpress ECHS1. Motor function of mice among groups was detected by rotarod test and gait analysis. Neurodegeneration, mitochondrial dysfunction and apoptosis were determined by immunohistochemistry, immunofluorescence staining, Western blot or kits, respectively.

The expression and activity of ECHS1 were decreased in PD mice and positive correlations between ECHS1 reduction and dopaminergic neurodegeneration were observed. Overexpression of ECHS1 by AAV delivery attenuated loss of dopaminergic neuron and motor deficits in PD mice. Mechanistically, ECHS1 attenuated rotenone-induced mitochondrial swelling and loss of cristae as well as decrease of ATP production, mitochondrial membrane potential, complex I/IV activities and oxygen consumption rate (OCR). Mitochondrial ROS (mtROS)-targeted antioxidant mito-TEMPO prevented ECHS1 silence-mediated mitochondrial dysfunction. Furthermore, we found that ECHS1 interacted with NADPH oxidase 4 (NOX4), resulting in decrease of NOX4 activation and subsequent reduction of mtROS production and mitochondrial dysfunction. Finally, inhibition of NOX4 by GLX351322 or mtROS production by mito-TEMPO greatly reduced ECHS1 silence-mediated apoptosis in rotenone-treated SH-SY5Y cells.

ECHS1 counteracted dopaminergic neurodegeneration through inhibition of mtROS and restoration of mitochondrial function via interaction with NOX4. Given the central role of mitochondrial dysfunction in PD pathogenesis, elucidating the role of ECHS1 holds great promise for uncovering novel therapeutic targets.