An Experimental Study Of The Role Of Autophagy-lysosome Pathway In Parkinson’s Disease And The Regulatory Effect And Underlying Mechanism Of Ghrelin On It

Objective:Parkinson’s disease（PD）is one of the most common neurodegenerative diseases.Progressive loss of dopaminergic neurons in the substantia nigra（SN）,aggregation ofα-synuclein andα-synuclein-containing inclusion Lewy body are the hallmarks of PD.So far,the pathogenesis of PD remains unclear and the current medical treatments can neither ameliorate neuron loss nor delay disease progression.Hence,it is critical to develop agents that can curb dopaminergic neuronal loss or promote neurogenesis.Autophagy-lysosome pathway is a major lysosomal degradative pathway in eukaryotes that is responsible for degrading long-lived or unwanted proteins that are too large to be degraded by the other proteolytic system,namely,ubiquitin-proteasome system（UPS）.The processes of autophagy include:autophagy induction,autophagosome elongation,autophagosome formation,fusion with lysosomes and content digestion.Multiple studies have suggested altered autophagy-lysosome pathway in PD.An understating of the effect of autophagy-lysosome pathway in PD will benefit future therapy development.Ghrelin is a neuropeptide that is mainly secreted upon hunger signals by X/A like cells in gastric mucosa.It can cross the blood-brain-barrier and exert multiple biological functions.It has two forms in the circulation system:acylated ghrelin（AG）and unacylated ghrelin（UAG）.In the field of PD,it remains controversial whether UAG is neuroprotective.But AG is neuroprotective in a number of disease models of central nervous system.Herein,we will focus on the neuroprotective effect of AG and its underlying mechanism.The current mechanisms of its neuroprotective effect mainly involve anti-inflammation,anti-oxidative stress and anti-apoptosis.Yet how ghrelin regulates the autophagy-lysosome pathway and the relationship with its neuroprotective effect remain largely unknown.In the current study,we will study the effect of 6-OHDA on autophagy-lysosome pathway in different autophagy stages and the impact on neuronal survival with the application of 6-OHDA-induced rat and SH-SY5Y cell models.We will also explore the regulatory effect of ghrelin on autophagy-lysosome pathway and the potential mechanism.Methods:In the current study,we firstly examined the expression of autophagy-related proteins with immunofluorescence and Western blot in6-OHDA-induced rat models.We also applied autophagy inhibitors and cysteine protease inhibitors to study the effect of autophagy activation on neuronal survival.On this basis,we further studied the state autophagic flux with the application of chloroquine in vivo and Ad-m Cherry-GFP-LC3B adenovirus infection in vitro.We also detected the expression of lysosomal-related proteins and hydrolase activities after 6-OHDA treatment.We modified autophagic flux with the application of plasmid transfection,autophagy inhibitor or small interfering RNA to explore the effect of autophagic flux state on neuron survival.After that,we confirmed the neuroprotective effect of ghrelin in 6-OHDA-induced rat and SH-SY5Y cell models and studied its effect on autophagy with immunofluorescence and Western blot.With the application of autophagy inhibitors of different stages,we further clarified the regulatory target of ghrelin on autophagy-lysosome pathway and explored its potential mechanism.Results:1,6-OHDA could induce significant upregulation of autophagy and apoptosis-related proteins within 1 day after treatment in the rat models;while autophagy inhibitor 3-MA or cysteine protease inhibitor Z-FA-fmk could inhibit6-OHDA-induced autophagosome formation and reduce apoptosis-related protein levels.In addition,3-MA and Z-FA-fmk could retain more dopaminergic neurons 1week after 6-OHDA treatment with behavior improvement,suggesting that inhibition of 6-OHDA-induced early autophagy activation could have a long-term neuroprotective effect.2,neuron loss of approximately 50%could be observed 1week after 6-OHDA treatment in the rat models with upregulated autophagy and apoptosis-related proteins within 1 weeks and p62 level significantly higher than the normal level at the 5^thweek（P<0.05）,suggesting altered autophagic flux.With the application of chloroquine in vivo,we found that chloroquine could not further increase LC3-II or p62 protein level in the SN.With the application of Ad-mCherry-GFP-LC3B in vitro,we found that SH-SY5Y cells treated with6-OHDA for 24h presented diffused yellow puncta,suggesting impaired autophagosome and lysosome fusion.By examing the level of lysosome-related proteins and functional lysosomes,we found 6-OHDA could decrease the number of lysosomes.Also we found 6-OHDA could impair cathepsin B and cathepsin D activities.Western blot and immunofluorescence further revealed that the expression and distribution of transcription factor EB（TFEB）were altered by 6-OHDA with decreased expression of both total proteins and the nuclear fraction.TFEB overexpression with plasmid transfection could improve LAMP1 mRNA level and reduce 6-OHDA-induced apoptosis.In addition,inhibiting autophagosome formation with 3-MA or small interference RNA can also reduce 6-OHDA-induced apoptosis.3,ghrelin was neuroprotective in both 6-OHDA-induced rat and SH-SY5Y models,which was closely related to its dose.Higher doses of ghrelin would weaken its neuroprotective effect or even aggravate neuron injury.In addition,ghrelin treatment could significantly upregulate autophagy-related proteins.With the application of3-MA and chloroquine to inhibit autophagy initiation or autophagosome fusion,we found that chloroquine could significantly increase apoptosis-related protein levels,while 3-MA showed little effect.SH-SY5Y cells infected with adenovirus Ad-mCherry-GFP-LC3B showed increased red fluorescence intensity and decreased number of yellow fluorescence puncta with dual application of ghrelin and 6-OHDA,suggesting improved autophagic flux state by ghrelin.The effect of ghrelin on the expression and distribution of TFEB in SH-SY5Y cells was detected with immunofluorescence,and we found that ghrelin could increase both the total fluorescence intensity of TFEB and its intensity in the nucleus compartment.Conclusion:1,6-OHDA could induce concomitant early activation of autophagy and apoptosis,and inhibition of autophagy could not only reduce 6-OHDA-induced neuronal apoptosis but also have long-term neuroprotective effect;2,6-OHDA can induce autophagic flux impairment,which is related to its negative regulation of TFEB and impairment of lysosome expression and function.Bidirectional regulation of autophagic flux with either inhibiting autophagosome formation or promoting lysosome biogenesis with TFEB overexpression could ameliorate 6-OHDA-induced apoptosis;3,ghrelin is neuroprotective in both 6-OHDA-induced rat and SH-SY5Y models,and the effect depends on its concentration.Its neuroprotective effect is closely related to autophagic flux restoration and TFEB regulation could account for this.