Using Induced Pluripotent Stem Cell-derived POLG Astrocytes Study On Mitophagy And Drug-targeted Therapy

Background:POLG-related diseases are the most common mitochondrial diseases,accounting for approximately 25%of mitochondrial diseases.Mutations cause its pathogenesis in the POLG gene,the nuclear-encoded gene for the catalytic subunit of DNA polymerase γ(Pol-γ),the only enzyme known to be located in the mitochondria and involved in the replication and repair of mitochondrial DNA(mtDNA).The age of onset of POLG-related diseases spans a wide range,and the early and late-onset is highly correlated with the severity of the disease.POLG mutations can be found in patient brain tissue to trigger aberrant mtDNA homeostasis,resulting in neuronal loss.There is currently no cure for POLG-related diseases.Due to the lack of reliable models and the difficulty in obtaining patient tissue,the research on the disease mechanism has been slow.Induced pluripotent stem cells(iPSCs)can be applied in the fields of disease-specific cell modeling,targeted drug screening,and regenerative medicine.We previously demonstrated that neurons differentiated from patient tissue sources could mimic the pathological features found in patient brain samples and found that POLG mutations drive neuronal mitochondrial dysfunction,exacerbating elevated reactive oxygen species and cellular senescence.Astrocytes play an important role in supporting and regulating neuronal function.Studies have found that metabolic dysfunction of astrocytes is a high-risk factor for neurodegenerative diseases.We previously found mitochondrial dysfunction in POLG astrocytes,manifested by altered energy metabolism,decreased expression of mitochondrial complex I,and mtDNA depletion.However,there is no systematic study on astrocytes’ role and regulatory mechanisms in POLG-related diseases.Mitophagy,a type of autophagy,is considered to be an important regulatory mechanism for mitochondrial quality control and cellular homeostasis.It has been reported in the literature that impaired mitophagy is prevalent in neurodegenerative diseases and leads to the accumulation of defective mitochondria,aggravating the phenotype of neuronal aging and apoptosis.However,the changes,effects,and regulatory mechanisms of mitophagy in POLGrelated diseases have not been systematically studied and verified.Aim:Based on the above research foundation and our previous exploration,this study used iPSCs derived from POLG patients and iPSCs derived from healthy individuals;we induced the two groups of iPSCs to differentiate into astrocytes,compared with astrocytes in the CTRL group,to explore the effects of POLG mutations on astrocyte mitophagy and mitochondria function,the regulatory mechanisms involved,and to study drug-targeted therapies for their mechanisms of action.Methods:First part:The iPSCs of CTRL group and POLG group:1.Induce differentiation into astrocytes;2.Cell immunofluorescence staining was used to detect the expression of astrocyte molecular markers GFAP,S100β,EAAT-1,and GS;3.Applying flow cytometry to detect the positive expression of molecular markers GFAP,S100β,CD44,EAAT-1,and GS;4.Using RNA Sequencing database(RNA-Seq)to perform differential gene clustering analysis to clarify the consistency of the transcriptome of astrocytes derived from iPSCs differentiation and human normal astrocytes(HNA).The second part:The POLG group was compared with the CTRL group:1.The RNA-Seq database was used to explore the metabolic pathways,differential gene enrichment changes,and mitochondrial autophagy gene changes in POLG astrocytes;2.Applying RNA-Seq database and Western blot to analyze the mRNA and protein expressions of key molecules DNM1L,FIS1,MFF,MFN2,and OPA1 in POLG astrocytes mitochondrial fission/fusion pathway;3.Using mitochondrial morphology live-cell staining to examine mitochondrial morphology,and use flow cytometry to detect mitochondrial membrane potential to explore mitochondrial function changes in POLG astrocytes.The third part:Comparison of POLG group and CTRL group:1.Using the mitophagy box to explore the mitophagy function,lysosome function,and mitochondrial membrane potential changes of POLG astrocytes;2.Western blot was used to analyze the protein expressions of LAMP2A,LC3B,p62,PINK1,and Parkin,and to explore the changes of mitophagy initiation pathway in POLG astrocytes;3.Western blot was used to examine the protein expressions of p-mTOR,mTOR,p-ULK1,ULK1,p-AKT,AKT,p-AMPK,and AMPK,and to explore the effect of POLG mutation on the astrocyte mitophagy regulation pathway.The fourth part:1.Western blot analysis of p-SIRT1,SIRT1,p-mTOR,mTOR,p-AMPK,AMPK,LC3B,and p62 protein expression,to explore the effects of nicotinamide ribose and metformin on mitophagy of POLG astrocytes when treated cells are used alone(in combination with treated cells);2.Apply the mitophagy kits to explore the changes of mitophagy and lysosome when nicotinamide ribose and metformin are combined at the optimal concentration;3.The protein expressions of PGC-1α and NDUFB10 were detected by Western blot,and mitochondrial membrane potential was detected by flow cytometry,to investigate the effects of nicotinamide ribose and metformin on mitochondrial function in POLG astrocytes when treated cells are used alone(in combination with treated cells).Results:First part:iPSCs-derived astrocytes have:1.Characterization of typical morphological and functional proteins;2.The positive rate of cell identification is high,and the results of RNASeq analysis are highly similar to HNA.The second part:POLG astrocytes vs.CTRL astrocytes:1.Significant changes in differentially expressed genes involving mitophagy/autophagy pathway and PI3K/AKT pathway;2.Both mitophagy/autophagy-related genes and OXPHOS,especially the NADH dehydrogenase pathway,were down-regulated,and the two were closely related;3.The key molecules of mitochondrial fission/fusion did not change significantly at the mRNA and protein levels;4.Mitochondrial membrane potential decreased,mitochondrial functional protein expression decreased.The third part:POLG astrocytes vs.CTRL astrocytes:1.Impaired initiation of mitophagy,down-regulated lysosomal function,and decreased PINK1/Parkin pathway protein expression;2.Reduced expression of AKT/mTOR/AMPK/ULK1 pathway proteins.The fourth part:1.SIRT1/AMPK pathway protein increased in POLG astrocytes under nicotinamide riboside treatment;2.Under metformin treatment,the AMPK/mTOR pathway was activated in POLG astrocytes,and the autophagy-related proteins LC3B-Ⅱ/LC3B-Ⅰ and p62 were elevated at the same time.3.Under the combined treatment of nicotinamide ribose and metformin,the SIRTl/AMPK/mTOR pathway of POLG astrocytes was significantly improved,and the autophagy-related proteins LC3B-Ⅱ/LC3B-Ⅰ and p62 were increased;4.The optimal concentration of nicotinamide ribose and metformin combined treatment of POLG astrocytes,the function of mitophagy and lysosome was significantly improved.5.The optimal concentration of nicotinamide ribose and metformin combined treatment of POLG astrocytes significantly improved mitochondrial function.Conclusion:To sum up the above,we used astrocytes differentiated from iPSCs carrying POLG mutations in this study and found down-regulation of mitophagy/autophagy-related genes based on the RNA-Seq database.This study demonstrated defects in mitophagosome formation,downregulation of the mitophagy receptor p62,decreased lipidation of LC3B-Ⅱ and decreased expression of the lysosomal functional protein LAMP2A under exogenous stress conditions.These changes are regulated by the PINK1/Parkin pathway and the AKT/mTOR/AMPK/ULK1 pathway.Most importantly,our results demonstrate for the first time that combination therapy with nicotinamide ribose and metformin improves mitophagy and function in POLG mutant astrocytes.This provides a new possibility for using mitophagy-enhancing drugs to treat POLGrelated diseases,suggesting that mitophagy-enhancing drugs are potential solutions for the treatment of mitochondrial diseases.