| A robust protein quality control(PQC)system is essential for maintaining protein homeostasis and guarding the accuracy of neurodevelopment in the nervous system.Our previous study found that a conserved PQC regulator EBAX-1(Elongin BC-Binding AXon Regulator-1)restricts axon guidance errors by controlling the quality of ROBO receptor in C.elegans.ZSWIM8(Zinc-finger SWIM domain-containing protein 8),which is the mouse homolog of EBAX-1,also preferably degrade misfolded ROBO3 proteins in mammalian cells.However,the in vivo roles of ZSWIM8 were largely unknown.Therefore,this study aimed to examine whether and how ZSWIM8 functions in the nervous system.Here,we report that ZSWIM8 is critical for the development of the mammalian nervous system.ZSWIM8 acts as guardian of brain development and performs quality check for an essential signaling molecule Disabled1(Dab1).In cultured Neuro-2a cells,ZSWIM8 responded to unfolded protein stress and colocalized with the stress granule marker G3BP1.ZSWIM8 was highly expressed in the developing and neonatal mouse brain.Knocking down ZSWIM8 in primary hippocampal neurons caused a significant decrease of the numbers of excitatory synapses and AMPA receptors on the synaptic surface.Global knockout of Zswim8 in mice led to prenatal death,suggesting that Zswim8 is essential for embryonic development.Conditional knockout of Zswim8 in the neural progenitor cells(NPCs)caused perinatal death of pups.Survivors showed reduced body weights,which gradually recovered if these animals were raised separately after weaning.After examining the embryonic brain at different developmental stages,we found that the migration of NPCs and maturation of granular cells in the dentate gyrus(DG)was significantly delayed.A permanent morphological change of the DG was observed in one-month and three-month old mice.Additionally,adult CKO mice showed severely impaired locomotion learning,novel object recognition,and fear conditioning.Numerous studies have established the essential roles of the Reelin signaling pathway in NPC migration and neuronal maturation.Impaired Reelin signaling results in severe developmental defects in the cortex and hippocampal regions.Thus,we proposed that ZSWIM8 may act on a misfolding-protein member of the Reelin pathway.By analyzing the disordered contents of proteins in this pathway,we identified that the downstream effector Disabled1(Dab1)contains large intrinsically disordered regions(IDRs)in its C-terminus.ZSWIM8 interacted with the full-length isoform of Dab1 and promoted its degradation.In contrast,ZSWIM8 did not interact with a short isoform of Dab1(p45)which lacks the highly disordered C-terminus and consequentially failed to affect its degradation.These data suggest that the IDRs of Dab1 is essential for target recognition and degradation by ZSWIM8,consistent with the PQC function of ZSWIM8.Genetic deletion of ZSWIM8 in mice and overexpression of a dominant negative mutant of ZSWIM8 in cultured cells caused accumulation of Dab1 proteins.Interestingly,the phosphorylated proportion of Dab1 was significantly decreased in these experiments,suggesting that ZSWIM8 acts differently from the Cul5-dependent degradation pathway targeting phosphorylated Dab1.In cultured hippocampal neurons,overexpression of the full-length isoform or p45 isoform of Dab1 rescued the defects in synaptic formation caused by ZSWIM 8 deletion,indicating that ZSWIM8 deletion reduces the level of effective Dab1 proteins and overexpression of Dab1 can compensate this phenotype.Taken together,we propose that ZSWIM8 degrades misfolded Dab1 proteins that cannot be phosphorylated and ensures the Reeling signaling pathway can properly and timely function.By revealing this critical PQC mechanism for neurodevelopment,circuitry formation and learning and memory,our studies have provided important evidence for understanding the in vivo functions of PQC and inspired future investigations in PQC-related neurodevelopmental diseases. |
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