Protective Effect And Mechanism Of IGF-1 On Mitochondria In ALS Mice And Cell Model

Amyotrophic lateral sclerosis(ALS)a common neurodegenerative disorder,motor neurons in the brainstem,cortex,and spinal cord progressively degenerated which leads to muscle paralysis.Most of patients die from respiratory failure within three to five years from disease onset.The majority of ALS cases are sporadic(s ALS),while 10% are familial(fALS).20% of fALS are caused by mutant superoxide dismutase 1(SOD1)gene.Other important ALS-causing mutations including transactive response DNA binding protein 43(TDP-43)and Chromosome 9 Open Reading Frame 72(C9ORF72)have been described.There are no effective treatment methods therefore understanding the mechanism of this disease is helpful to explore new therapies.Increasing evidence suggest mitochondrial dysfunction play a central role in the etiopathogenesis of neurodegenerative diseases,including Huntington's disease,Parkinson's disease,Alzheimer's disease and Amyotrophic lateral sclerosis.Mitochondria play a pivotal role in the oxidative stress,mitophagy,glutamate excitotoxicity,and intrinsic pathway of apoptosis.Little is known about the exactly causes and pathophysiology of this disease.In transgenic mouse models of ALS,the appearance of swollen and vacuolar mitochondria was present before the motor dysfunction.Mitochondria abnormalities develop during the disease progression.Mitochondria are the main intracellular organelles which contribute to cell death and survival.Intrinsic apoptosis pathway is initiated in this organelle,the old and dysfunctional mitochondria are removed by autophagy.Lately,gene therapy appears to be an effective treatment,human insulin-like growth factor-1(hIGF-1)delivering through self-complementary(sc)adeno-associated virus(AAV)vectors has shown positive effect on ALS mouse model.In this study,we used the adenovirus-associated virus serotype 9(AAV9)to carry the double-stranded human IGF-1 gene(scAAV9-hIGF-1)on the TDP-25 stable cell line and through the intramuscular route to interfere with G93A-SOD1 transgenic mice,we observed intrinsic apoptosis process was modified through up-regulating the expression of anti-apoptotic agents(Bcl-xl,Bcl2)and down-regulating the expression of pro-apoptotic agents(Bax,Bak,Cytochrome C),mitophagy was also activated after scAAV9-hIGF-1 treatment in vivo and vitro experiments.We demonstrated that the mitochondrial electrical transmembrane potential was increased and abnormal mitochondria was transformed.Furthermore,to confirm these findings,CRISPR-Cas9 system was used to delete IGF-1 in SOD1G93 A mouse model.We found the protective effect of IGF-1 on mitochondria was declined after genetic deletion.These novel findings suggest that IGF-1 strongly protect mitochondria in ALS mouse and cell models and therapies targeted at protecting mitochondrial function maybe promising in treating ALS.We did not find similar research or reports at home or abroad.Part one The effect of scAAV9-hIGF-1 on mitochondria in ALS cell model and its mechanismObjective: To investigate the effects of scAAV9-hIGF-1 and AAV9-GFP on ALS cell model TDP-25 stable transfected cell lines,we studied the changes of mitochondrial membrane potential,mitochondrial morphology,protein levels of mitochondrial-related apoptotic pathway and autophagic pathway in ALS cell model TDP-25 cell lines,and explored the protective effect of gene transduction of hIGF-1 on mitochondria in ALS cell model and its mechanism.Methods: We selected TDP-25 cell line as a cell model,and treated with different concentrations of scAAV9-hIGF-1 in cell culture medium.Cell viability was determined by cell viability assay kit CCK8.Concentrations of hIGF-1 in cells and culture media were measured by enzyme-linked immunosorbent assay ELISA.The expressions of GFP and VDAC were determined by Western blotting in scAAV9-hIGF-1 and AAV9-GFP treated cells at different time points,and the optimum concentration and time point were selected.We use that concentration and time for the following test.The AAV9-hIGF-1 was added in cell culture medium of experimental group,AAV9-GFP was administered to the cell culture medium of the control group,and the mitochondrial morphological changes were observed by transmission electron microscopy.The mitochondrial membrane potential was analyzed by flow cytometry.The mitochondrial and cytosolic protein were axtracted freshly and the protein contents of Bcl2,Bcl-xl,Bax,Bak,Cytochrome C,LC3 and P62 were analyzed by western blotting.Results:1 Different concentrations of scAAV9-hIGF-1 were intervented in the TDP-25 cell line culture medium,CCK8 assay was used to determine the best cell viability was at the concentration of 1 * 107 vg / ul,and it was higher than the control group,the difference was statistically significant(P <0.05).The levels of hIGF-1 in the cells and culture medium were significantly higher than those in the control group by enzyme-linked immunosorbent assay(ELISA).The difference was statistically significant(P <0.05).The expression of GFP was time-dependent and increased with time,while the expression of VDAC reached a peak at 60 hours and then decreased.Cells were observed under a microscope and the results were the same as those of the Western blotting,and the concentration of 1 * 107 vg / ul and 60 hours were selected.2 The mitochondrial rupture,fragmentation,vacuolization,cytoplasmic edema and uuclear chromatin loose were observed in the GFP group.The mitochondrial morphology of the IGF-1 group was long oval,and no ridge disappeared or vacuolized.3 The mitochondrial membrane potential of GFP group was lower than that of IGF-1 group,and the difference was statistically significant(P <0.05).4 The protein contents of Bcl2,Bcl-xl and LC3-? in the mitochondrial fraction of IGF-1 group were increased analyzed by Western blotting.The protein contents of Bax,Bak and P62 were decreased,Cytochrome C was increased in the mitochondrial component and decreased in the cytosolic fraction,suggesting that Cytochrome C in the IGF-1 group was mainly present in the mitochondria,whereas in the GFP group,Cytochrome C decreased in the mitochondrial component,and increased in the cytosolic fraction,suggesting that Cytochrome C release into the cytoplasm,the difference was statistically significant(P <0.05).Conclusions: In this study,IGF-1 can protect the mitochondria in ALS cell model by comparing the results of previous studies.IGF-1 can protect the mitochondria by improving the mitochondrial membrane potential,improving the mitochondrial morphology,inhibiting the pro-apoptosis protein and increasing the expression of antiapoptotic proteins in the endogenous apoptosis pathway and promoting mitochondrial autophagy,thereby protecting the mitochondria.Part two Effects of intramuscular injection of scAAV9-hIGF-1 on mitochondria in ALS mouse model and its mechanismObjective: We used neurotropic adeno-associated virus serotype 9(AAV9)carried duplex encoding human insulin-like growth factor 1-IGF-1 gene(scAAV9-hIGF-1)by intramuscular injection in G93A-SOD1 transgenic female mice of 60 days of age.We observed the mitochondrial morphological changes,mitochondrial membrane potential changes and mitochondrial-related apoptotic pathway and autophagy pathway protein content after intervention,and explored the protective effect of gene transduction of hIGF-1 on mitochondria in ALS mice and its mechanism.Methods: We used G93A-SOD1 and wild-type WT-SOD1 as the animal model of ALS,which were purchased in Jackson laboratory of the United States,and the DNA genotype was identified by PCR.The SOD1 positive of G93A-SOD1 female mice and same age wild-type WT mice were randomly selected as subjects.13 pairs of 60-day age of G93A-SOD1 and WT-SOD1 mice were used,the G93A-SOD1 transgenic female mice were randomly assigned to the treatment group injected with scAAV9-hIGF-1,the control group was injected with AAV9-GFP.Negative control was the same age of WT-SOD1 mice.About 40-50 days after intramuscular injection,3 pairs of them were perfused with 4% paraformaldehyde solution by heart perfusion.The expression of IGF-1 in lumbar medulla neurons was observed by immunohistochemical staining.Immunofluorescence staining was used to observe the expression of lumbar GFP expression.1 pairs were fixed in 2.5% glutaraldehyde solution and mitochondrial morphological changes was observed by transmission electron microscopy in the spinal cord anterior horn motor neuron,3 pairs were extracted freshly to extract the cells in spinal cord,the changes of mitochondrial membrane potential were analyzed by flow cytometry.The mitochondrial and cytosolic protein were axtracted freshly of 6 pairs and the changes of protein contents of Bcl2,Bcl-xl,Bax,Bak,Cytochrome C,LC3 and P62 were analyzed by protein Western blotting.Results:1 Mice in all experimental groups were identified by DNA identification through PCR,the SOD1 gene-positive G93A-SOD1 and wild-type WT-SOD1 were verified.2 ScAAV9-hIGF-1 and AAV9-GFP were injected intramuscularly in 60-day old G93A-SOD1 transgenic mice.After about 40 days,4% paraformaldehyde solution was perfused and fixed by heart.The expression of GFP in lumbar medulla was mainly observed in neurons by immunofluorescence staining.The expression of IGF-1 in lumbar medulla neurons was also observed by immunohistochemical staining.3 G93A-SOD1 mice injected with scAAV9-hIGF-1 and AAV9-GFP and control wild-type WT-SOD1 mice were fixed with 2.5% glutaraldehyde solution at 100-110 days,and the mitochondrial morphological changes were observed by transmission electron microscopy.The mitochondria of GFP group were rupture,fragmentation,vacuolization,cytoplasmic edema and nuclear chromatin were loose.The mitochondrial morphology of IGF-1 group was similar to that of WT group,which were long oval,no ridge disappeared,vacuolization or so on.4 In the G93A-SOD1 mice injected with scAAV9-hIGF-1 and AAV9-GFP together with control wild-type WT-SOD1 mice,the spinal cord cells were extracted at 100-110-day of age.Flow cytometry analysis of mitochondrial membrane potential were evaluated.The mitochondrial membrane potential of GFP group was lower than that of IGF-1 group and WT group,the differences were statistically significant(P <0.05).5 In the intramuscular injection of scAAV9-hIGF-1 and AAV9-GFP in G93A-SOD1 mice and control wild-type WT-SOD1 mice,the mitochondrial and cytoplasmic proteins were extracted at 100-110 days of age.The protein contents of Bcl2,Bcl-xl and LC3-? were increased in the mitochondrial components of IGF-1 group and WT group,and the protein contents of Bax,Bak and P62 decreased.Cytochrome C increased in mitochondrial fraction,decreased in cellular components,suggesting that Cytochrome C was predominantly present in mitochondria in IGF-1 and WT groups,whereas in GFP group,Cytochrome C decreased in mitochondrial components and increased in cellular components,suggesting that Cytochrome C was released to cytoplasm,the difference was statistically significant(P <0.05).Conclusions: In this study,IGF-1 can protect the mitochondria in ALS animal model by comparing the results of previous studies.IGF-1 can protect the mitochondria by improving the mitochondrial membrane potential,improving the mitochondrial morphology,inhibiting the pro-apoptosis protein and increasing the expression of antiapoptotic proteins in the endogenous apoptosis pathway and promoting mitochondrial autophagy,thereby protecting the mitochondria.Part three Effect of intrathecal injection of scAAV9-sgRNA-IGF-1-Cas9 on mitochondria in ALS mice and its mechanismObjective: We used the CRISPR-Cas9 system to delete IGF-1 mRNA through intrathecal injection of scAAV9-sgRNA-IGF-1-Cas9 into G93A-SOD1 transgenic female mice.The effect of IGF-1 mRNA deletion on G93A-SOD1 transgenic female mice were studied by measuring the mitochondrial morphological changes,mitochondrial membrane potential changes,protein contents of mitochondrial-related apoptotic pathway and autophagy pathway,and the protective effect of gene transduction of hIGF-1 on mitochondria and its mechanism were explored in ALS mice.Methods: We used G93A-SOD1 as an animal model of ALS purchased from Jackson laboratory in USA.DNA gene was identified by PCR technique.The SOD1 positive G93A-SOD1 female mice were chose as research target.In the G93A-SOD1 mice at 30 days old,10 pairs co-infected female mice were randomly assigned to the experimental group which were intrathecal injected scAAV9-sgRNA-IGF-1-C9,the control group was injected with scAAV9-sg RNA-LacZ-Cas9.About 50 to 60 days after intrathecal injection,one pair were fixed by 2.5% glutaraldehyde solution and the morphological changes of mitochondria in the anterior horn motor neurons were observed by transmission electron microscopy.The mitochondrial membrane potential was analyzed by flow cytometry in 3 pairs.The expression of IGF-1,Bcl2,Bcl-xl,Bax,Bak,Cytochrome C,LC3,P62 and optineurin were analyzed by Western blotting in 3 pairs.Results:1 We knocked down IGF-1 in the G93A-SOD1 transgenic mice,the mitochondria in the experimental group ruptured,vacuolization,cytoplasmic edema,nuclear chromatin loose,while the control group mitochondrial morphology were long oval,no ridge disappeared,vacancy or so on.2 The mitochondrial membrane potential of the spinal cord cells were analyzed by flow cytometry.The mitochondrial membrane potential of the experimental group was significantly lower than that of the control group(P <0.05).3 In the experimental group and the control group,the mitochondrial and cytoplasmic proteins of the lumbar spinal cord were extracted freshly at 80-90 days of age.After the Western blotting analysis,the mitochondrial components of IGF-1,Bcl2,Bcl-xl,LC3-?,optineurin decreased,protein content of Bax increased,protein content of P62 did not change significantly.Cytochrome C decreased in mitochondrial components and increased in cytosolic fraction,suggesting that Cytochrome C was released into the cytoplasm.Whereas in the control group,Cytochrome C was increased in mitochondrial components and decreased in the cell fraction,suggesting that Cytochrome C was mainly present in mitochondria,the difference was statistically significant(P <0.05).Conclusions: In contrast,knocking down of IGF-1 in the spinal cord of ALS mice further aggravated mitochondrial dysfunction,decreased mitochondrial membrane potential,vacuolization of mitochondria,increased expression of pro-apoptotic protein,decreased expression of anti-apoptotic protein,inhibited mitophagy,worsened mitochondrial damage,therefore the disease progression was further aggravated.According to theresults above,we conclude that mitochondrial dysfunction is a certain factor in the pathogenesis of ALS.Mitochondria are the energy factory in cells.Once mitochondrial dysfunction occured,cells can not be metabolized normally,initiation of apoptosis cascade can not be achieved,abnormal mitochondria could not be removed by mitophagy,the disease will deteriorate exacerbated.And IGF-1 can protect this process,but its role in mitophagy need to be further studied.Expression of hIGF-1 through gene vectors to protect abnormal mitochondria in ALS mice and cell models provided us an experimental basis for the treatment of clinical ALS disease,and the therapy targeted to mitochondrial function may be promising for the treatment of ALS.