Pseudoginsenoside-F11 Improves Endosomal-autophagy-lysosomal System Dysfunction In Microglia Treated By OAβ

Alzheimer’s disease(AD)is characterized by the extracellular deposition of β-amyloid(Aβ)and the intracellular neurofibrillary tangles.Accumulation of intracellular Aβ is an early event in the pathology of AD and appears prior to nerofibrillary tangles and neuronal loss.Meanwhile,intracellular Aβ implicates in a set of cellular physiology including axonal transport,apoptosis and autophagy,further documenting the pivotal role of intracellular Aβ in AD.Aβ monomers,generated from the enzymatic cleavage of amyloid precursor protein(APP),assemble into three conformations including fibrillar,oligomeric and aggregated forms.It has become increasingly clear that soluble oligomeric Aβ(oAβ)contributes to synaptic and memory deficits at young ages prior to the formation of senile plaques in the brain.Therefore,the clearance of soluble oligomeric Aβ in the early stages is more effective than the fibrillar Aβ.The endosomal-autophagy-lysosomal system is made up of a variety of intracellular membranous compartments interconvert dynamically,which is comprised of early endosomes,autophagosomes,recycling endosomes,late endosomes,and the lysosome.Maturation of endosomes or autophagosomes fuses with the lysosome for proteolysis.In AD,the endosomal-autophagy-lysosomal system,which is a dynamic,interconnected vesicular network,may be impaired at both autophagosome formation and lysosome degradation according to the different stages of the disease,and this system is essential for clearance of Aβ.However,how the soluble oligomeric Aβ affects endosomal-autophagy-lysosomal system is largely unknown.Pseudoginsenoside-F11(PF11),an ocotillol-type saponin,is isolated from Panax pseudoginseng subsp.Himalaicus Hara(Himalayan Panax).The previous studies have shown that PF11 significantly decreased the expressions of Aβ1-40 in AβP/PS1 mice brain and the central nervous system microglia cells were clustered around the amyloid plaque.By contrast,the mechanisms of PF11 on accumulation of Aβ are still unknown.Accordingly,in this thesis,two parts were studied:(ⅰ)The effects of oAβ on the endosomalautophagy-lysosomal system in microglia;(ⅱ)The effects of PF11 on oAβ uptake and degradation and the protective effects of PF11 on oAβ stimulated microglia.The main contents are listed as below:1.oAβ influences the endosomal-autophagy-lysosomal system.The transcription factor EB(TFEB),a positive regulator for lysosomal biogenesis,mediates lysosomal biogenesis by controlling expression of autophagy and lysosomal genes.In this study,TFEB nuclear translocation was increased under oAβ(5 μM)for 9 h,but this phenomenon disappeared over time.Simultaneously,mTOR activity was increased under oAβ(5 μM)for 24 h.Low-concentration oAβ increased the levels of autophagy related protein Vps34,Beclin 1,Atg5 and LC3.Intriguingly,the levels of these proteins were back to the same level as the control at oAβ concentrations up to 5-10 μM.The transition between early and late endosomes could be mediated by Rab conversion,a process in which Rab5 is rapidly replaced with Rab7.To confirm that the Rab conversion is involved in oAβ mediated dysfunction of endocytosis,microglia cells cotransfected with Rab5-cherry-pERFP and RFP-Rab7 were imaged during continuous endocytosis of HiLyte FluorTM 488-labeled Aβ1-42 from the medium.The results showed that oAβ blocked the Rab conversion at 5 μM.Meanwhile,the protein levels of Rab5 and Rab7 were back to the control level.Moreover,the influence of oAβ on the expressions of lysosomal membrane proteins and the activity of cathepsinB(CatB)were similar to that of autophagy-related proteins.Considering the above,high dose oAβcaused the insufficiency of endosomal-autophagy-lysosomal system.2.PF11 causes an increase in oAβ uptake and degradation.In this study,we adopt both Confocal microscopy and Western Blot to detect the level of oAβ in PF11 treated microglia.The results showed that PF11 could obviously increase the oAβ uptake and degradation with positive correlation to concentration.It is well known that the clearance of the Aβ dependends on the endosomal-autophagy-lysosomal system,suggesting PF11 may be involved in regulation of this system.3.Effects of PF11 on endosomal-autophagy-lysosomal system.Our results showed that PF11 increased TFEB nuclear translocation through suppression of mTOR activity,suggesting PF11 promoted lysosomal biogenesis.To ensure the effects of PF11 on autophagosome formation,we tested the levels of autophagy-related proteins in microglia.The results showed that the levels of these proteins were unchanged by increasing PF11 dose.Notably,PF11 had the ability to promote the maturation of autophagosomes and endosomes.First,PF11 promoted the fusion of autophagosomes(marked by LC3)and endosomes/lysosomes(marked by LAMP1).Second,PF11 impacted the Rab5 to Rab7 conversion and upregulated the expressions of Rab5 and Rab7.Next,we studied the effects of PF11 on lysosomal function.The expressions of V-ATPase and LAMP2 were significantly increased by 30 μM PF11 in oAβ treated microglia.Moreover,PF11 lowered the accumulation of p62 in chloroquine treated microglia.The above results indicated that PF11 ameliorated the functions of endosomal-autophagy-lysosomal system,which damaged by high dose oAβ,via promoting autophagic/endosomal maturation and improving lysosomal function.For the first time,we found that TFEB nuclear translocation was increased under low dose oAβ,linked by upregulation of some key proteins in endosomal-autophagy-lysosomal system.Interestingly,high dose oAβ made the endosomal-autophagy-lysosomal system inadequacy.On this basis,we create a model for endosomal-autophagy-lysosomal system dysfunction in vitro and provided an example that PF11 was capable of improving the function of endosomal-autophagy-lysosomal system in primary microglia under high dose oAβ.Therefore,this thesis will declare that therapeutics targeting intracellular Aβ,especially oligomeric Aβ,could be effective treatment for AD.Besides,our results inspired the development of oligomeric Aβ clearance-targeting drugs,such as PF11,which effects by controlling endosomal-autophagy-lysosomal system.