Melatonin attenuates kainic acid‐induced neurotoxicity in mouse hippocampus via inhibition of autophagy and α‐synuclein aggregation

Abstract

In this study, the protective effect of melatonin on kainic acid (KA)-induced neurotoxicity involving autophagy and α-synuclein aggregation was investigated in the hippocampus of C57/BL6 mice. Our data showed that intraperitoneal injection of KA (20 mg/kg) increased LC3-II levels (a hallmark protein of autophagy) and reduced mitochondrial DNA content and cytochrome c oxidase levels (a protein marker of mitochondria). Atg7 siRNA transfection prevented KA-induced LC3-II elevations and mitochondria loss. Furthermore, Atg7 siRNA attenuated KA-induced activation of caspases 3/12 (biomarkers of apoptosis) and hippocampal neuronal loss, suggesting a pro-apoptotic role of autophagy in the KA-induced neurotoxicity. Nevertheless, KA-induced α-synuclein aggregation was not affected in the Atg7 siRNA-transfected hippocampus. The neuroprotective effect of melatonin (50 mg/kg) orally administered 1 hr prior to KA injection was studied. Melatonin was found to inhibit KA-induced autophagy-lysosomal activation by reducing KA-induced increases in LC3-II, lysosomal-associated membrane protein 2 (a biomarker of lysosomes) and cathepsin B (a lysosomal cysteine protease). Subsequently, KA-induced mitochondria loss was prevented in the melatonin-treated mice. At the same time, melatonin reduced KA-increased HO-1 levels and α-synuclein aggregation. Our immunoprecipitation study showed that melatonin enhanced ubiquitination of α-synuclein monomers and aggregates. The anti-apoptotic effect of melatonin was demonstrated by attenuating KA-induced DNA fragmentation, activation of caspases 3/12, and neuronal loss. Taken together, our study suggests that KA-induced neurotoxicity may be mediated by autophagy and α-synuclein aggregation. Moreover, melatonin may exert its neuroprotection via inhibiting KA-induced autophagy and a subsequent mitochondrial loss as well as reducing α-synuclein aggregation by enhancing α-synuclein ubiquitination in the CNS.

Melatonin attenuates MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/α-synuclein aggregation

“…Consistent with previous observations that melatonin can restore morphine-induced autophagy 45 and kainic acid-induced autophagy, 61 we found that melatonin inhibited MPTP-induced autophagy and cell death in vitro. This salvaging effect of melatonin was CDK5-dependent, and knockdown or overexpression of CDK5 abolished the protective effect of melatonin on MPTP-induced autophagy and cell death.…”

Involvement of autophagy in hypoxic-excitotoxic neuronal death

“…Increased numbers of autophagosomes have been described in different in vitro situations of excitotoxicity such as long-term treatment and/or high doses of NMDA 30,31 or Ka, 19 or exposure to a specific glutamate uptake inhibitor. 32 The injection of toxic doses of Ka into the adult mice hippocampus 20,33 or striatum 34,35 also induces autophagy. Numerous in vivo studies of cerebral ischemia or HI over the past 20 y have likewise shown enhanced neuronal autophagy (autophagosome formation and lysosomal activity).…”

The role of autophagy in neurotoxicity caused by extracellular ASYN

“…Kao što je ranije pomenuto, ATG7 je jedan od proteina bitnih za formiranje i sazrevanje autofagozoma, tako da je, ukoliko nema ATG7, inhibiran proces autofagije (22)(23)(24)(25)(26). Da bismo utvrdili da li je autofagija u našem modelu protektivna ili pomaže umiranju ćelija, ekspresija ATG7 proteina je inhibirana transfekcijom sa malom interferirajućom RNK (siRNA) u SH-SY5Y ćelijama.…”

“…Tačan molekularni mehanizam, međutim, i dalje ostaje nepoznanica. Kao što je ranije rečeno, autofagija je regulisana proizvodima ATG gena, a jedan od njih, ATG7, ima bitnu ulogu u formiranju i sazrevanju autofagozoma (22)(23)(24)(25)(26). Postoji nekoliko istraživanja o povezanosti ATG7 gena i procesa neurodegeneracije.…”

“…Postoji nekoliko istraživanja o povezanosti ATG7 gena i procesa neurodegeneracije. Pokazano je da nakon delecije ATG7 gena u neuronima miševa dolazi do degeneracije i smrti neurona usled povećane akumulacije ASYN (22)(23)(24)(25). Pored ovoga, u neuronima nije registrovan nijedan autofagozom, što nesumnjivo govori o značaju autofagije za funkcionisanje neurona i uklanjanje viška ASYN (26).…”