The Effect Of Exogenous Mitochondria On Cognitive Improvement Of Alzheimer’s Disease Model Mice And Its Mechanism

Alzheimer’s disease has become an important problem affecting brain health due to its high prevalence,mortality and lack of effective drugs.It was found that mitochondrial dysfunction is one of the most significant features of AD,and is closely related to the energy metabolism impairment,oxidative stress injury,autophagy disorder and Aβdeposition,which promote the progression of AD.Mitochondrial transplantation,as a therapy to repair mitochondrial function,has been extensively studied in neurodegenerative diseases and proved to improve cognitive and motor function in elderly mice,but its role and mechanism in AD remain unclear.And studies have also reported that the expression of silent information regulator 1 is negatively correlated with the neuropathological changes of AD.SIRT1 is a histone deacetylase whose activation depends on NAD⁺/NADH levels.NAD⁺and NADH are important redox regulators in mitochondria,and the ratio of NAD⁺/NADH is closely related to mitochondrial function and activity.Therefore,it is not clear whether SIRT1 is involved in the benefits of mitochondrial transplantation therapy and the regulation of AD progression.This study intends to study the cognitive improvement effect and mechanism of exogenous mitochondria on AD,and explore the regulatory role of SIRT1 in mitochondrial therapy,so as to provide a new method for the treatment of Alzheimer’s disease.In vitro,mitochondrial membrane potential,electron transport chain complex and electron microscopy were detected.The results showed that mitochondria had complete morphological structure,high membrane potential and good activity.Then,β-amyloid peptide 1-42 was used to damage SH-SY5Y cells to construct AD cell models,and the effect of exogenous mitochondria on the activity of Aβ-damaged cells was detected by using the Alamar Blue.The results showed that the cell activity of Aβ-damaged cells was enhanced with the increase of mitochondrial concentration in the range of 50-300μg/m L in a time-dependent and dose-dependent manner.After being treated with 200μg/m L mitochondria for 4 hours,the activity of Aβ-damaged cells was significantly increased.In order to study the mechanism of exogenous mitochondria on Aβ-damaged cells,we examined tricarboxylic acid cycle,ETC and redox related indexes.The results showed that the activities of PDH,SDH,α-KGDH,ICDHm,mitochondrial complex I-IV and ATP/ADP ratio were significantly increased in the mitochondrial group.In addition,NAD⁺/NADH ratio,GSH/GSSG ratio,T-AOC,SOD and GSH-Px activities were also significantly increased in the mitochondrial group,while ROS and MDA levels were significantly decreased.The results showed that exogenous mitochondrial transplantation could promote mitochondrial function and improve energy metabolism.Moreover,mitochondria can also increase the activities of GSH-Px and SOD,increase the ratios of NAD⁺/NADH and GSH/GSSG to enhance the antioxidant capacity of cells,inhibit oxidative stress and reduce ROS levels,so as to improve the redox imbalance.Then,we used transcriptomics to studied the mechanism of exogenous mitochondria on Aβ-damaged cells.The m RNA of mitochondria treatment group was compared with that of Aβ-damaged cells.GO and KEGG enrichment and specific differential expression gene analysis were found that the genes related to neuron growth,repair,connection and autophagy were significantly different after mitochondrial therapy,and the key gene of differentially expressed SIRT1 was found.Since SIRT1 is a key protein in the reverse regulation of mitochondrial NAD⁺signaling,we speculate that exogenous mitochondria may regulate downstream neurotrophic factors and autophagic proteins by activating the activity of NAD⁺/NADH-dependent SIRT1,thus playing a neuroprotective role and eliminate damaged mitochondria and misfolded proteins.In order to study the mechanism and explore whether SIRT1 plays an important role in the treatment of AD by mitochondrial transplantation,we measured the activity of SIRT1 and examined the changes of mitochondria in Aβ-damaged cells after exogenous mitochondria treatment by using electron microscopy.And also used immunofluorescence method to detect the expressions of SIRT1,BDNF,and Aβ.In addition,WB was also used to determine the expression levels of neuron growth,repair,connection and autophagy related proteins.The results showed that the activity of SIRT1and the expression level of BDNF were significantly increased after mitochondrial therapy,while the expression level of Aβwas significantly decreased.After treatment,the morphology of mitochondria in Aβ-damaged cells returned to normal,the number of mitochondria increased,and autophagy appeared.WB results showed that mitochondria could up-regulate the expression of SIRT1 downstream proteins of FOXO3,BNIP3,LC3Ⅱ/LC3Ⅰ,BDNF,and p-ERK/ERK.The results suggest that exogenous mitochondria can promote autophagy through SIRT1/FOXO3/BNIP3/LC3 pathway,and effectively eliminate damaged organelle and Aβ.Moreover,mitochondria can also regulate the growth,repair and connection of neuronal cells through SIRT1/BDNF/ERK pathway to enhance the plasticity of protrusion.In vivo,Aβ_1-42 was injected into the hippocampus of mice to construct Alzheimer’s disease mouse model.The mice were treated with mitochondria through the tail vein for4 days.Then,we used Morris water maze to test the cognitive,learning,and memory abilities of mice,and used Thioflavin T to stain the hippocampus of mice.The results showed that after mitochondrial therapy,the escape latency of mice was significantly shortened,the movement trajectory was more concentrated in the target quadrant,and the residence time in the target quadrant was significantly prolonged.In addition,the hippocampal Aβdeposition was significantly lower in the mitochondrial treatment group.These results fully indicate that exogenous mitochondria can improve the learning and cognitive memory ability of AD mice,reduce Aβdeposition,and alleviate the symptoms of AD mice.To study the cognitive improvement mechanism of mitochondria in animals.First,we examined the TCA cycle,ETC,and redox related indicators.The results showed that exogenous mitochondria could increase the activities ofα-KGDH,SDH,mitochondrial complexⅠandⅣ,increase the ratios of ATP/ADP,NAD⁺/NADH,GSH/GSSG,and reduce the ROS content.These results are consistent with the results of cell experiments,fully demonstrating that exogenous mitochondria can promote mitochondrial function,improve energy metabolism,increase antioxidant capacity,reduce ROS levels,and alleviate oxidative stress damage.Secondly,the activity of SIRT1 in mouse hippocampus was measured.And WB was used to determine the expression of SIRT1 downstream proteins of FOXO3,BNIP3,LC3Ⅱ/LC3Ⅰ,BDNF,and p-ERK/ERK.The results showed that SIRT1 activity increased significantly after mitochondrial therapy.SIRT1 can up-regulate the expression of FOXO3 and its downstream autophagy proteins of BNIP3,LC3Ⅱ/LC3Ⅰ.Moreover,the increase of SIRT1 activity can also up-regulate the expression of BDNF and p-ERK/ERK.In summary,the results showed that mitochondrial transplantation could improve cognitive function and reduce Aβdeposition in Alzheimer’s disease model mice.Meanwhile,mitochondrial transplantation can also repair mitochondrial function,increase intracellular ATP/ADP,improve energy metabolism,increase antioxidant capacity,reduce ROS level,inhibit oxidative stress,and improve redox imbalance.Moreover,the results also revealed that mitochondria can promote autophagy through the SIRT1/FOXO3/BNIP3/LC3 pathway,and effectively eliminate damaged organelle and misfolded protein.In addition,the study also clarified that mitochondria can regulate the growth,repair and interneuronal connection of neurons through SIRT1/BDNF/ERK pathway,increase synaptic plasticity,enhance learning and memory ability,and improve AD symptoms.