SNX8 Enhances Lysosome Reformation To Rescue Lysosomal Storage Disease Phenotypes

Lysosomal storage diseases(LSDs)are inherited metabolic disorders caused by mutations in genes encoding lysosomal enzymes,lysosomal membrane proteins,transporters,and lysosomal ion channels,which result in lysosome dysfunction and the accumulation of substrates inside the lysosomes.It has been found that the functional defects of more than 70 genes can result in LSDs.Although each type of lysosomal storage disease is rare,the total incidence of lysosomal storage diseases is about 1/5000-1/5500.LSDs share common phenotypes including enlarged lysosomes,defective lysosome reformation,substrate storage.Currently,several therapeutic methods,such as hematopoietic stem cell transplantation,pharmacological chaperone therapy,enzyme replacement therapy,and gene therapy,are applied to LSDs.However,all of these options are disease type-specific and most of them have important limitations,such as limited foreign DNA packaging capability,donor limitation,and protein structure-dependent.At present,there is still a lack of a broad-spectrum therapy for LSD treatment.To maintain intracellular lysosome homeostasis,lysosomes undergo tubularization(also known as lysosome tubulation)to generate new lysosomes,a process known as lysosomal regeneration.However,lysosome reformation is significantly reduced in many LSDs.Therefore,lysosome reformation may serve as a broad-spectrum target for the treatment of LSDs.But,the detailed mechanism of lysosome tubulation is unknown yet.Therefore,we aim to search for a specific factor that regulates lysosome tubulation and verify whether the regulation of lysosomal tubulation can alleviate LSDs.The Sorting nexins(SNXs)protein family is a class of proteins involved in protein sorting and transport functions.Members of the SNX-BAR subfamily SNX1 and SNX2,which contain a Bin-Amphiphysin-Rvs(BAR,membrane-curving domain)domain,have been reported to bend early endosomal membranes and promote early endosome tubulation.Therefore,we wanted to explore whether SNX-BAR family members are involved in the regulation of lysosomal tube formation.Firstly,we screened which members of the SNX-BAR subfamily can obviously localize on lysosomes.We found that SNX2 and SNX8 had significant lysosomal localization.Further starvation-induced lysosome tubulation found that both SNX2 and SNX8 could localize to lysosomal tubular structures.He La cell lines harboring single or double knockout of the SNX2 and/or SNX8 genes significantly inhibited lysosome tubulation,and this phenomenon was more pronounced in SNX2/8DKO cells.Knockdown of SNX2 alone did not affect lysosome tubulation.Further analysis found that SNX8,as the main SNX-BAR protein,regulates the initiation of lysosome tubulation.SNX8 consists of a BAR domain and a PHOX homology(PX)domain.Mutation of lysine 135 on the SNX8-PX domain to alanine(SNX8 K135A)to lose its lysosomal localization ability did not affect lysosome tubulation.Overexpression of the SNX8-BAR truncation(removing the PX domain of SNX8)also induced lysosome tubulation.This indicates that BAR domain of SNX8 can directly promote lysosome tubulation.In subsequent functional experiments,we found that knockout of SNX8 disrupted lysosomal function and resulted in lysosomal storage disease(LSD)phenotypes.Lysosomal storage diseases(LSDs)lead to inhibited lysosome tubulation,which in turn leads to LSDs.This prompted us to investigate whether up-regulating lysosome tubulation could alleviate LSDs phenotypes.We constructed three LSD cell models(NPC1KO,HEXAKO and GLAKO cells)and found that overexpressing SNX8 upregulated lysosome tubulation and alleviated LSDs phenotypes in LSD cell models.However,overexpression of HEXA or GLA in SNX8 KO cells did not alleviate LSD phenotypes.Subsequently,we overexpression of SNX8 in primary fibroblast of three LSD model mice also alleviated LSDs phenotypes.In the end,we overexpressed SNX8 in Sandhoff disease mice ameliorated their LSD phenotypes.Here,we confirmed that SNX8 acts as the major SNX-BAR protein to regulate lysosome tubulation.SNX8 deficiency impairs lysosome tubulation and leads to lysosomal dysfunction and LSD phenotypes.Overexpression of SNX8 alleviated LSD phenotypes in multiple LSD model cell lines and ameliorated Sandhoff diseases at animal level by enhancing lysosome tubulation.Thus,our study suggests that upregulating lysosome tubulation through overexpressing SNX8 as a potential broad-spectrum and specific therapeutic solution for many types of LSD.