| BackgroundAlzheimer’s disease（AD）is a common neurodegenerative disease characterized by progressive cognitive and behavioral disorders.At present,the pathogenesis of AD remains unclear.The widely accepted hypothesis is that the unbalanced generation and clearance of amyloid-β is the initial factor of neuronal degeneration and dementia.The abnormal expression of amyloid-β forms neurotoxic plaques between brain neurons,leading to neuronal degeneration and death.Mitochondrial dysfunction plays an important role in the pathogenesis and development of AD.Mitochondrial dysfunction occurs in AD patients,such as decreased mitochondrial biosynthesis,abnormal mitochondrial dynamics,and mitochondrial dysfunction.Although mitochondria have their own DNA,most of the proteins that mitochondria need are encoded by nuclear genes and are synthesized in the form of precursor proteins with mitochondrial targeting signals in the cytoplasmic ribosome,which are recognized and transported to the mitochondria by TOM complex,to perform their corresponding physiological functions.TOM70,one of the main receptor proteins of the TOM complex,is an important component of the TOM complex that functions to recognize precursor proteins.A recent study has shown that TOM70 is involved in mitochondria not only as a recognition receptor for mitochondrial precursor proteins but also as a key regulatory protein in the mitochondrial compromised protein import response（mitoCPR）.Recent studies have found that TOM70 expression changes in the brain tissue of AD patients.However,the involvement of TOM70 in the pathogenesis of AD and its related mechanism is not clear.Purpose1.To define whether the expression changes of TOM70 in AD are specific.2.To define whether the abnormal regulation of TOM70 is related to Aβ.3.To explore whether TOM70 can be used as a potential diagnostic biomarker for AD.4.To explore the influence of abnormal regulation of TOM70 in AD on mitochondrial biological function.Method1.The expression level of TOM70 in AD.1.1 Human blood samplesPeripheral blood was obtained from patients diagnosed with AD,Lewy bodies（DLB）,and post-stroke dementia（PSD）and age-sex-matched controls with normal cognitive function.The mRNA expression of TOM70 in peripheral blood was detected by RT-qPCR.Neuropsychological scales including the Minimum Mental State Examination（MMSE）,the Montreal Cognitive Assessment（MoCA）,Clinical Dementia Rating（CDR）,and media temporal lobe atrophy scale（MTA）were used to assess disease progression.The correlation between the expression of TOM70 and gender,age,and various scales were analyzed.1.2 Mouse samplesAPP/PS1 double transgenic mice（transfected with amyloid precursor protein and presenilin 1）were selected as the animal model of AD,and C57BL/6J mice of the same sex and age were selected as the control group.The hippocampus,cortex,cerebellum,skeletal muscle,liver,and peripheral blood of mice at different stages of disease（3,6,and 12 months old）were obtained.The mRNA expression of TOM70 was detected by RT-qPCR and the protein expression of TOM70 was detected by western blot.1.3 Cell samplesMouse hippocampal neuron cells（HT22 cells）added with Aβ25-35 were selected as AD cell models,TOM70 mRNA expression was detected by RT-qPCR,and TOM70 protein expression was detected by western blot.Then,detect TOM70 level after adding Aβ25-35 inhibitors.2.The influence of TOM70 level changes on mitochondrial morphology and function.2.1 In vivoAdeno-associated virus（AAV）carrying the TOM70 gene or vehicle（VEH）was injected into the hippocampus.Mouse was divided into four groups:C57 group,APP/PS1 group,APP/PS1 injected with VEH group,and APP/PS1 injected with AAV carrying TOM70 gene group.The injection of the AAV virus carrying TOM70 gene caused the overexpression of TOM70 in the hippocampus of mice.Mitochondrial morphology in mouse neurons was detected by electron microscopy.ATP and respiratory chain function in hippocampal tissue were detected by the experiment kits.Mitochondrial reactive oxygen species（ROS）levels were detected by MitoSOX.2.2 In vitroHT22 cell models were divided into 4 groups:no intervention group,Aβ25-35 intervention group,Aβ25-35 intervention,and transfected plasmids group,Aβ25-35 intervention and plasmids carrying TOM70 gene group.Plasmid carrying TOM70 gene made cells overexpress TOM70.ATP in cells was detected by the experiment kits.The level of reactive oxygen species was determined by MitoSOX.Mitochondrial membrane potential was detected by TMRM.Mitochondrial respiration condition was measured using Seahorse Cell Mito Stess Test.3.The influence of TOM70 level changes on the cognition and behavior of mice.Mice was divided into four groups:C57 group,APP/PS1 group,APP/PS1 injected with VEH group,and APP/PS1 injected with AAV carrying TOM70 gene group.Morris water maze experiment was used to detect the spatial learning and memory ability of mice.Results1.The expression of TOM70 in the peripheral blood of AD patients decreased.TOM70 mRNA levels were significantly reduced in AD patients compared with controls.However,we did not find a significant difference between the DLB group,the PSD group,and their age-sex-matched control group.Receivers operating characteristic curve（ROC）analysis was performed to analyze the specificity and sensitivity of TOM70 in distinguishing dementia patients from controls.In AD,the area under the curve（AUC）of peripheral blood TOM70 levels was significantly increased,well over 0.5.However,the AUC level of TOM70 in the peripheral blood of DLB and PSD groups was not significant.This indicates that peripheral blood TOM70 has good specificity and sensitivity in AD patients.2.The decreased TOM70 level correlates with the progression of AD.We further analyzed the correlation between the expression of TOM70 levels and clinical features in AD patients.We examined the association of TOM70 with sex,age,neuropsychological test scores,and MTA scores.There was no correlation between the level of TOM70 and sex.The level of TOM70 was negatively correlated with age.The mRNA level of TOM70 was negatively correlated with the MTA score and CDR score but positively correlated with the MMSE score and MoCA score.MMSE and MoCA scores were negatively correlated with AD progression,while CDR and MTA scores were positively correlated with AD progression.Our data suggest that decreased TOM70 expression in peripheral blood is associated with the progression of AD.3.The TOM70 level in AD decreased in specific tissue and overlapped with AD-affected sites.Expression levels of TOM70 mRNA and protein were detected in peripheral blood,brain（including hippocampus,cortex,and cerebellum）,liver and skeletal muscle of APP/PS1 mice at 3,6,and 12 months old.The levels of TOM70 in the hippocampus and peripheral blood of APP/PS1 mice were significantly decreased at 6 and 12 months and were associated with age.However,TOM70 levels in the cortex,cerebellum,skeletal muscle and liver of APP/PS1 mice were not decreased.4.Overexpression of TOM70 in AD models can save mitochondrial function.In vivo,data showed that in APP/PS1 mice,the mitochondrial structure was damaged,mitochondrial ridge loss,rupture,mitochondrial swelling,and vacuole-like.Mitochondrial morphology improved after TOM70 overexpression.Compared with APP/PS1 mice,over-expression of TOM70 increased the content of ATP in hippocampal tissue,improved the function of the mitochondrial respiratory chain,and decreased the level of mitochondrial reactive oxygen species.However,overexpression of TOM70 in could not reduce the levels of Aβ1-40 and Aβ1-42 in the hippocampus of APP/PS1 mice,and could not reduce the deposition of senile plaques.In vitro,we found that HT22 cells transfected TOM70 increased ATP content,decreased reactive oxygen species,increased mitochondrial membrane potential,and improved mitochondrial respiration functions compared with HT22 cells treated with Aβ.5.Overexpression of TOM70 alleviated the symptoms of impaired spatial learning and memory in AD mice.In the water maze experiment,the incubation period of the mice each day was shortened as the experiment progressed,due to their improved memory and learning ability.Compared with C57 mice,the incubation period of APP/PS1 mice was significantly prolonged,and overexpression of TOM70 significantly shortened the incubation period of APP/PS1 mice.Compared with C57 mice,APP/PS1 mice significantly reduced the number of platform crossing and the stay time in the target quadrant,while overexpression of TOM70 increased the number of platform crossing and the stay time in the target quadrant of APP/PS1 mice.Conclusion1.The expression of TOM70 was decreased in AD and overlapped with the AD-affected organs.2.Decreased TOM70 level was closely related to Aβ toxicity.3.TOM70 can be used as a potential biomarker for the diagnosis of AD and the determination of disease progression.4.Overexpression of TOM70 in AD improve mitochondrial function. |
