Targeting peroxynitrite-mediated mitophagy for multiple sclerosis treatment : from mechanisms study to therapeutic intervention with radix rehmanniae

Abstract

Multiple sclerosis (MS) is a neuro-inflammatory disease in central nervous system (CNS). With poor therapeutic outcome, most MS patients eventually develop into handicap. Activated autophagy/mitophagy has been observed in neurons of the CNS lesion suffering from MS. However, the exact roles of neuronal autophagy/mitophagy and its in-depth regulatory mechanisms remain unclear yet. In this study, I have filled these gaps and elucidated the roles and potential mechanisms of mitophagy in MS, proposed a potential therapeutic herbal medicine Radix Rehmanniae (RR) and screened an active compound Acteoside (AC) to regulate excessive mitophagy activation with better therapeutic outcomes.

In mechanistic study, by using MS laboratory model of experimental autoimmune encephalomyelitis (EAE), I found that autophagy activation was coincidently increased with axonal damage, apoptosis and disease progression in EAE mice, which were reversed by autophagy inhibitor 3-Methyladenine. PINK1/Parkin-mediated mitophagy activation with Drp1 mitochondrial recruitment was predominant in neurons of CNS lesion at EAE peak time. Mitochondrial division/mitophagy inhibitor Mdivi-1 remarkably suppressed Drp1-mediated mitophagy activation and postponed EAE progression. Peroxynitrite (ONOO-), a representative RNS, could be a player in MS pathology. Increased ONOO- production was simultaneously companied with Drp1/PINK1/Parkin-mediated mitophagy activation and EAE severity, which were attenuated by FeTMPyP, a representative peroxynitrite decomposition catalyst. ONOO- induced Drp1 nitration, promoting Drp1 assembly and mitochondrial recruitment, contributing to mitophagy activation. Those results indicate that ONOO- serves as a key mediator to induce Drp1 nitration and assembly in mitophagy activation, contributing to neurological deficits in EAE pathology. Targeting ONOO- -mediated excessive mitophagy could be an important therapeutic strategy for MS.

In therapeutic study, I then investigated potential agent that targets ONOO- -mediated mitophagy with therapeutic effects in EAE. Results showed that RR ameliorated EAE severity and the underlying mechanisms were owing to its inhibitions of macrophage-derived nitrative damages through suppressing NF-kB pathway and attenuations of ONOO--induced autophagy/mitophagy activation via reducing ONOO- generation and scavenging ONOO- directly. For chemical studies, two reliable qualitative and quantitative methods for quality control of RR by using LCMS-IT-TOF or HPLC-DAD systems were established.

Finally, I screened out an active compound, AC from RR that could regulate ONOO- -induced mitophagy and exhibit neuroprotection against nitrative damages in vivo and in vitro. Both prophylactic and therapeutic AC administrations exerted beneficial improvements against EAE, attributed to its anti-inflammatory and anti-oxidant properties. Preventive AC administration attenuated the priming/activations of peripheral inflammatory cells while therapeutic AC treatment alleviated CNS infiltration of encephalitogenic cells. Moreover, AC protected neurons from ONOO- -induced nitrative stress and suppressed excessive mitophagy in EAE mice. The suppression of ONOO--induced excessive mitophagy activation could be one of critical mechanisms contributing to the efficacy of AC on EAE.

In summary, the present study suggested that ONOO- could trigger excessive mitophagy activation via inducing Drp1 nitration and assembly, leading to axonal degeneration and neuronal injury in MS pathogenesis. Targeting ONOO- -mediated excessive mitophagy could be a promising therapeutic strategy for MS. Radix Rehmanniae and its active compound Acteoside could be potential therapeutic agents to alleviate ONOO- -mediated excessive mitophagy for MS treatment.