Role Of Selenoprotein S Regulating Lysosomal Homeostasis In Cerebellar Neuronal Apoptosis Induced By Selenium Deficiency In Chicken

Selenium(Se) is an essential trace element for human s and animals.It is widely involved in many biological processes and plays a variety of important biological functions.Studies has shown that Se deficiency can cause brain damage.Selenoprotein S(SELS),as one endoplasmic reticulum localized selenoprotein,plays an important role in the protection of brain injury.Highly expressed SELS can relieve brain injury by alleviating endoplasmic reticulum stress and inf lammatory stimulus,however,inhibition of SELS expression may aggravate the brain injury.Lysosomes,as the"digestive center"of cells,play a key role in maintaining intracellular homeostasis through degrading and removing intracellular harmful substanc es.It's found that many degenerative neurological diseases are accompanied by lysosomal dysfunction.Se deficiency could decrease lysosomal stabilization and SELS expression.However,whether the imbalance of lysosomal homeostasis is involved in Se deficiency-caused cerebellar injury,and whether SELS regulates lysosomal function and homeostasis are still unclear.Based on it,we propose a scientific hypothesis that Se deficiency can mediate the lysosome imbalance and induce apoptosis through reducing SELS expression in cerebellar cells.To clarify this hypothesis,we established the Se deficient chicken cerebellar injury model,Se deficient chick embryo brain neuron model,SELS knockdown chick embryo brain neuron model and treated with the oxidative stress inhibitor N-acetyl-L-cysteine(NAC),the CTSB inhibitor E-64 and the CTSD inhibitor Pepstatin A respectively.And based on these models,ICP-MS,histopathological observation,immunofluorescence,TUNEL staining,transcriptome,RT-PCR,Western blot and flow cytometry were performed;metal ion homeostasis,pathomorphology,mRNA transcriptome,antioxidant levels(CAT,GSH-Px,SOD,T-AOC,ROS,H₂O₂,LPO and MDA),endoplasmic reticulum stress-related indexes(GRP78,IRE1,XBP1,PERK,ATF4 and ATF6),lysosomal-related indexes(p H,ATP6V1A,ATP6V1B2,ATP6V1D,CTSB and CTSD),autophagy-related indexes(LC3-2 and P62),and apoptosis-related indexes(BCL2,BAX,CAS9 and CAS3)were detected.The results showed that:(1)Histopathological observation showed that Se deficiency led to the thinning of cerebellum granular layer,increase of cerebellum white matter,decrease or disappearance of Purkinje cells,increase of eosinophilic Purkinje cells,the accumulation and tangle of nerve fibers in the Purkinje cell layer,the disorder of nerve fibers in the granular layer and the decrease of Nissl body.Immunofluorescence and TUNEL results showed that Se deficiency led to cerebellum?-syn aggregation and apoptosis.(2)The detection results of metal ions showed that the contents of K,Fe,Zn,B,Ni,Hg and Sb decreased and the contents of Ca,Cr,Mo,V and Cd increased in the cerebellar tissue of chickens under Se deficiency,which indicated that Se deficiency affected t he metal ion homeostasis in the cerebellar tissue of chickens.(3)mRNA transcriptome analysis showed that Se deficiency resulted in the differential expression of 421 genes in the cerebellum,of which 171 genes were upregulated and 250 genes were downregulated.Go and KEGG enrichment analysis showed that the differential genes were closely related to neural function,antioxidation,metal ions,lysosome and apoptosis.(4)Se deficiency resulted in the decreased expression of SELS at mRNA and protein in the chicken cerebellum and brain neurons,increased mRNA expressions of endoplasmic reticulum stress-related genes GRP78,XBP1,ATF4 and ATF6,and increased protein expressions of GRP78,IRE1,XBP1,PERK,ATF4 and ATF6.Besides,SELS Knockdown led to increased mRNA levels of SELN and DIO2,decreased mRNA levels of SELT and SEP15,and increased protein levels of GRP78,IRE1,XBP1,PERK,ATF4 and ATF6,indicating that the decreased expression of SELS could cause endoplasmic reticulum stress.(5)Se deficiency decreased the activities of CAT,GSH-PX and T-AOC,increased the contents of ROS,H₂O₂,LPO and MDA in the cerebellum and brain neurons.SELS Knockdown decreased the activities of antioxidant enzymes and increased the contents of free radicals and lipid peroxidation products in brain neurons,which indicated that the decreased expression of SELS led to oxidative stress in brain neurons.(6)Se deficiency inhibited the protein expressions of lysosomal V-ATPase,CTSB and CTSD,and increased the expressions of MCOLN1 and cytoplasm CTSB and CTSD in the cerebellum and brain neurons.SELS knockdown also caused similar changes in brain neurons.NAC treatment could effectively alleviate the lysosomal changes caused by decreased SELS expression.These results indicated that the decrease of SELS expression led to the disruption of lysosomal acidification,the decrease of enzyme activity,the release of calcium ion and the permeability of the lysosomal membrane,resulting in the imbalance of lysosomal homeostasis.(7)Se deficiency increased the expressions of autophagy markers LC3-2 and P62 in the cerebellum and brain neurons.SELS knockdown also increased the expression of LC3-2 and P62.NAC effectively restored lysosomal homeostasis and alleviated the increased expressio n of LC3-2and P62 caused by SELS knockdown,which indicated that the decreased expression of SELS inhibited autophagy substrate degradation through lysosomal dysfunction,leading to autophagosome accumulation and autophagic flux inhibition.(8)Se deficiency inhibited the expression of BCL2 in the cerebellum and brain neurons,and increased the mRNA and protein expressions of BAX,CAS9 and CAS3.SELS knockdown also caused similar changes in the above indexes.Flow cytometry showed that Se deficiency and SE LS knockdown caused the increase of apoptotic cells in brain neurons.Besides,NAC,CTSB inhibitor E-64 and CTSD inhibitor Pepstatin A could alleviate the apoptosis induced by SELS knockdown.These results indicated that the decrease of SELS expression co uld induce apoptosis via autophagic flux inhibition and leakage of CTSB and CTSD.In conclusion,Se deficiency led to the imbalance of metal ion homeostasis in the chicken cerebellum,resulted in mitochondrial pathway apoptosis,induced 171 mRNA expression upregulation and 250 mRNA expression downregulation,which were closely related to neural function,antioxidation,metal ions,lysosome and apoptosis.Se deficiency led to the decrease of SELS expression,and further investigations showed that the decreas e of SELS expression caused endoplasmic reticulum stress,promoted ROS accumulation,led to oxidative stress,induced imbalance of lysosome homeostasis,inhibited autophagic flux,and finally led to apoptosis.These results indicated that SELS could regulate lysosomal homeostasis and induce apoptosis in Se deficient chicken cerebellar neurons.These results enrich the molecular mechanism of Se deficient cerebellar injury and provide references for further research.