Role And Mechanism Of TRPML1/TFEB-mediated Lysosomal Biogenesis Disorders In Fluoride-induced Neurotoxicity

Part Ⅰ:Role of TRPML1/TFEB involved lysosomal biogenesis disorder in fluoride-induced developmental neurotoxicity in SD ratsObjective:Excessive fluoride exposure can cause developmental neurotoxicity,but the specific mechanism is not clear.This study aims to reveal the effect of TRPML1/TFEB involved lysosomal biogenesis on the developmental neurotoxicity of Sodium fluoride(NaF)SD rats,and provide scientific basis for further elucidation of the mechanism of TRPML1/TFEB involved lysosomal biogenesis disorder in fluoride-induced neurotoxicity.Methods:A total of 24 adult SD rats were selected,including 8 males and 16 females.After one week of adaptive feeding,the rats were randomly divided into 4 groups according to the ratio of male to female 2:1 and the pregnant rats were fed in a single cage.From the date of conception,SD rats were treated with fluorinated drinking water freely,control group(water fluorion concentration not exceeding 1 mg/L),25,50 and 100 mg/L NaF groups.F1 generation rats and mother were fed separately after weaning(21 days after birth),and the method and dose of mother’s exposure were continued until 2 months.Morris water maze experiment was conducted to test the learning and memory as well as memory retention capacities of F1 generation rats.The rats were euthanized and the hippocampus was separated for index detection.The ultrastructure of neurons was observed by transmission electron microscope.The expressions of TRPML1,nuclear TFEB,total TFEB,cytoplasmic TFEB,lysosomal associated membrane proteins LAMP2,lysosomal cathepsin CTSD and CTSB,autophagy associated proteins LC3-Ⅱ and p62,apoptosis associated protein cleaved PARP,pyroptosis associated proteins NLRP3,Caspase1,and IL-1β were detected by Western blot.Results:1.Morris water maze experiment showed that compared with the control group,the swimming speed of F1 generation rats in 100 mg/L NaF group were significantly decreased and escape latency of F1 generation rats in 100 mg/L NaF group were significantly increased on the second day(P<0.05),the swimming distance of F1 generation rats in 100 mg/L NaF group was significantly increased on the first day(P<0.05).In addition,compared with the control group,the number of crossing platforms of F1 generation rats in each NaF group were significantly reduced;the ratio of target quadrant time and the ratio of target quadrant distance of F1 generation rats in 100 mg/L NaF group were significantly reduced(P<0.05).2.The observation of transmission electron microscopy showed that the ultrastructure of hippocampal neurons of F1 generation rats in the control group had no obvious abnormal changes,and the number of lysosomes was decreased in the 100 mg/L NaF group.3.Western blot results showed that compared with the control group,the expressions of TRPML1 protein in the hippocampus of F1 generation rats in each NaF groups was significantly decreased(P<0.05),the expression levels of total TFEB,nuclear TFEB and cytoplasmic TFEB in hippocampus of F1 generation rats in 100 mg/L NaF group were significantly decreased(P<0.05),the expression levels of lysosomal associated membrane proteins LAMP2 in the hippocampus of F1 generation rats in 50 and 100 mg/L NaF groups was significantly decreased(P<0.05),the expression of lysosomal cathepsin CTSD and CTSB decreased in the hippocampus of F1 generation rats in 100 mg/L NaF group(P<0.05),autophagy associated proteins LC3-II,p62 and apoptosis associated protein cleaved PARP expression were increased in 50 and 100 mg/L NaF groups,the expression levels of pyroptosis associated proteins NLRP3 in the hippocampus of F1 generation rats in each NaF groups was significantly increased,Caspasel and IL-1βexpression were increased in 50 and 100 mg/L NaF groups(P<0.05).Conclusions:NaF exposure can lead to impaired learning and memory as well as memory retention capacities of F1 generation rats,and result in nervous system damage.In addition,NaF exposure inhibited the expression of TRPML1/TFEB in the hippocampus of F1 generation rats,resulting in lysosomal biogenesis disorder and autophagy flux blockade.At the same time,NaF exposure can lead to hippocampal apoptosis and pyroptosis.In conclusion,neurotoxicity caused by NaF exposure may be related to lysosomal biogenesis disorder.Part Ⅱ:Role and mechanism of TRPML1/TFEB mediated lysosomal biogenesis disorder in NaF-induced SH-SY5Y cytotoxicityObjective:To explore the role and mechanism of TRPML1/TFEB mediated lysosomal biogenesis disorder in NaF-induced SH-SY5Y cytotoxicity,and to provide scientific basis for exploring the mechanism of fluoride neurotoxicity.Methods:SH-SY5Y cell models were constructed with different doses of NaF(0,20,40 and 60 mg/L NaF).TRPML1,total TFEB,nuclear TFEB,cytoplasmic TFEB,lysosomal associated membrane proteins LAMP2,lysosomal cathepsin CTSD and CTSB,autophagy associated proteins LC3-Ⅱ and p62,apoptosis associated protein cleaved PARP,pyroptosis associated proteins NLRP3,Caspase1,and IL-1β were detected by western blot.Nuclear translocation of TFEB was detected by immunofluorescence method.Lysosomal content was observed by fluorescence microscope after Lyso-Tracker Red staining.Intracellular Ca2+ was observed by fluorescence microscope after Fluo-3AM staining.Autophagy flux was detected after transfection of the autophagy double-labeled adenovirus mRFP-GFP-LC3.TRPML1 agonists ML-SA1 and TFEB overexpressed adenovirus treated cells to construct intervention models(ML-SA1 intervention:control group,60 mg/L NaF group,ML-SA1 group,ML-SA1+60 mg/L NaF group;TFEB overexpressed adenovirus intervention:control group,60 mg/L NaF group,Ad-null group,Ad-null+60 mg/L NaF group,Ad-TFEB group,Ad-TFEB+60 mg/L NaF group),the expression of nuclear TFEB,lysosomal associated membrane proteins LAMP2,lysosomal cathepsin CTSB,autophagy associated proteins LC3-II and p62,apoptosis associated protein cleaved PARP,and pyroptosis associated protein Caspase1 were detected by western blot.Results:1.Immunofluorescence results showed that the fluorescence intensity of TFEB in SH-SY5Y cells of each NaF group decreased gradually compared with the control group,and the colocalization of TFEB with nucleus decreased with the increase of NaF dose(P<0.05).2.Lyso-Tracker Red staining showed that,compared with the control group,the number of red fluorescent spots in SH-SY5Y cells of each NaF group gradually decreased,and the lysosomal content decreased.3.Fluo-3AM staining showed that the fluorescence intensity of Ca2+ in NaF groups was decreased in SH-SY5Y cells compared with the control group.4.Autophagy flux detection results showed that after transfection of SH-SY5Y cells with mRFP-GFP-LC3 adenovirus,compared with the control group,the number of yellow and green spots were increased in each NaF infected group and the number of red spots were decreased(P<0.05).5.Western blotting results showed that,after different doses of NaF infected SH-SY5Y cells,compared with the control group,the protein expression levels of TRPML1,nuclear TFEB and cytoplasmic TFEB in 40 and 60 mg/L NaF groups were significantly decreased(P<0.05);the expression level of total TFEB protein in 60 mg/L NaF group was observably decreased(P<0.05);the expression level of lysosomal associated membrane proteins LAMP2 was remarkably decreased in 40 and 60 mg/L NaF groups(P<0.05);the protein expression levels of lysosomal cathepsin CTSD in each NaF groups and CTSB in 60 mg/L NaF groups were significantly decreased(P<0.05);the expression levels of LC3-II in 40 and 60 mg/L NaF groups and p62 in 60 mg/L NaF groups were remarkably increased(P<0.05);the expression levels of apoptosis associated protein cleaved-PARP in each NaF groups were observably increased(P<0.05);the expression levels of pyroptosis associated proteins Caspase1 in each NaF groups and NLRP3,IL-1β in 40 and 60 mg/L NaF groups were remarkably increased(P<0.05).After intervention with ML-SA1,the protein levels of nuclear TFEB,LAMP2,CTSB and LC3-Ⅱ in ML-SA1+60 mg/L NaF group were significantly increased compared with those in the 60 mg/L NaF group in SH-SY5Y cells,the protein levels of p62,cleaved PARP,and Caspase1 were remarkably decreased(P<0.05).After intervention with Ad-TFEB,the protein levels of nuclear TFEB,LAMP2,and CTSB in Ad-TFEB+60 mg/L NaF group were observably increased compared with those in the Ad-null+60 mg/L NaF group in SH-SY5Y cells,the protein levels of LC3-Ⅱ,p62,cleaved PARP,and Caspase1 were significantly decreased(P<0.05).Conclusions:NaF inhibited the expression of TRPML1/TFEB signaling pathway in SH-SY5Y cells,resulting in lysosomal biogenesis disorder and autophagy flux blockade,leading to cell apoptosis and pyroptosis.TRPML1 activation and TFEB overexpression promote lysosomal biogenesis by increasing TFEB nuclear translocation,improving autophagy flux,alleviating apoptosis and pyroptosis,and thereby alleviating NaF-induced neurotoxicity.