Analysis Of Neuronal Autophagy Degradation Landscape And The Mechanism In Regulating Neuronal Activity

Background : Autophagy is a lysosomal degradation pathway which mediates the degradation of large molecules and organalles and plays an important role in maintaining neuronal homeostasis.Previous studies have shown that autophagy is often disrupted in neurodegeneration diseases and neurodevelopmental disorders.However,the precise process of autophagy and autophagy cargos in neurons remain poorly defined.Studies aims in elucidating autophagy cargos in neurons in an un-biased way,with profund depth are still limited.Due to the difficulty obtaining neurons from human beings and ethnic issues,most of the studies focused on only cancer cell lines or mouse models.Research on autophagy substrates and their regulatory mechanisms on neuronal function is very limited.In recent years,the rapid development of directed differentiation methods from human stem cells into mature neurons and gene editing technology provide effective methods for exploring the autophagy substrates in neurons,especially in human neurons.The development of high-throughput quantitative proteomics also provides a basis for efficient and accurate detection of protein expression landscape in neurons.By applying integrated proteomics from multiple autophagy-deficient models,LC3 interactome and experimental validation of candidate autophagy caogos,our study performed systematic investigation in autophagy cargos and their functions in regulating neuronal activity.Our study provided a basis for understanding the autophagy degradation in neurons at basal level and the mechanisms underlying autophagy deficiency-related neurological diseases.Objective: To study the autophagy substrates landscape in neurons and investigate the mechanism of autophagy in regulating neuronal activity.Methods:1.We used multiple autophagy-deficiency models to enrich and profile autophagy cargos in human and mouse neurons,including ATG7knockdown(KD)and ATG14 KD human stem cell derived glutermatergic neurons,neuron-specific Atg7 or Atg14 conditional knockout(c KO)mice,and GFP-LC3 transgenic mice.We performed intergrated quantitative proteomics,GFP-LC3 interactome and transcriptomics in various autophagy-deficient models to investigate the landscape of autophagic substrates in neurons.2.We validated potential autophagic substrates in neurons through immunoblotting,immunofluescence staining,immunoprecipitation,and protein structure analysis.3.We identified a new selective autophagy receptor through LC3 interactome and explored its function in regulating autophagy.4.We applied small chemical treatment through a timecourse to activate PKA pathway,and deteched the downstream phosphorylated PKA substrates and transcription factor expression by immunoblotting and immunofluescence staining to investigate the role of autophagy in regulating neuronal activity.Results:1.Quantitative proteomics detected 1113 and 637 proteins with significantly increased expression levels in ATG7 KD and ATG14 KD human embryonic stem cell-induced glutamatergic neurons,respectively,of which 339 proteins were shared by both ATG7 KD and ATG14 KD neurons;the main autophagy substrates in human stem cell-derived neurons are endoplasmic reticulum(ER),Golgi apparatus,synaptic vesicles(synaptic vesicles,SV),mitochondria and endosomes and proteins in c AMP-PKA pathway.Among the organalles degraded by autophagy pathway,the proportions of ER-related proteins in ATG7 KD and ATG14 KD neurons were 29.7% and 32.9% respectively;the proportions of SV-related proteins were 9.2% and 15.3%.2.ER proteins,such as RTN3,ATL1,SEC62,TEX264 etc.,were validated by immunoblotting to be autophagy substrates in neurons;in ATG7 KD neurons,abnormally accumulated ER proteins aggregated in neuronal axons and neurites and formed bead-like enlarged protein aggregates.3.Synaptic proteins are degraded by autophagy,among which are mainly pre-synaptic proteins,including SV and pre-synaptic membrane proteins,such as SV2 A,SV2C,SYNGR3,SYNGR1,and SYP1.4.459 and 343 significantly accumulated proteins were identified by quantitative proteomics in the Atg7 c KO and Atg14 c KO mice brain,respectively.102 proteins were enriche and shared by both Atg7 c KO and Atg14 c KO mouse brain.The main autophagy substrates in mouse neurons are ER,Golgi apparatus,SV,mitochondria,etc.Among them,ER is the most abundant autophagy substrate in mouse neurons.In Atg7 c KO and Atg14 c KO mice brain,among all the enriched proteins,ERrelated proteins accounted for 37% and 31% of the total enriched proteins,respectively;SV-related proteins accounted for 7% and 10%,respectively.5.GFP-LC3 interactome from the Atg7 c KO;GFP-LC3 mouse background detected 1967 significantly enriched proteins in the brain.Some SV and PKA pathway-related proteins,such as SV2 A,SYNGR3and AKAP11 were validated by GFP-LC3 co-immunoprecipitation.6.From the significantly enriched proteins in autophagy-deficient human neurons,Calumenin(CALU)was identified and validated as a new selective autophagy receptor located on the endoplasmic reticulum through screening and functional verification experiments.CALU is recruited and degraded by the autophagy receptor LC3 through LC3 interacting motif(LIR)-mediated binding.Its possible function is mediating endoplasmic reticulum autophagy(ER-phagy)and maturation of autophagosomes.7.Autophagy regulates the kinase activity of PKA through the degradation of PKA subunits RⅠα and Cα mediated by the autophagy receptor AKAP11 in human and mouse neurons.Autophagy regulates neuronal activity by regulating the phosphorylation level of PKA substrates and downstream transcription factor expression,such as c-FOS.Conclusion:Our work revealed the landscape of autophagy degradation and regulation in ATG7 KD,ATG14 KD human neurons,Atg7 c KO and Atg14 c KO mouse brains.Organalles such as ER,glogi apparatus,SV,mitochondria and proteins in PKA pathway are autophagy substrates.Our study identified an ER-resident protein Calumenin as a new selective autophagy receptor,which may mediate ER-phagy and autophagosome formation.Our study suggests a particularly important role of autophagy in regulating neuronal activity by the degradation of the autophagy receptor AKAP11,PKA-RⅠα and Cα subunits in the PKA pathway.