The Effect And Mechanism Of CPT1A-mediated Fatty Acid Metabolism In Alzheimer’s Disease

Background and objectives:Alzheimer’s disease（AD）is the most common type of dementia and the fifth leading cause of death in the elderly.AD not only causes great suffering to patients,but also imposes a heavy burden on families and society.At present,the clinical diagnosis and treatment of AD is faced with low early recognition rate,low diagnosis rate,high missed diagnosis rate,high misdiagnosis rate,and there is no curative method yet.Therefore,studying the pathogenesis of AD and exploring new targets and strategies for the diagnosis and treatment of AD have become frontier issues in the field of neuroscience and medicine.The brain is one of the organs with the most abundant lipid content and diversity.With increasing research,there is growing evidence that brain lipids play an important role in the development of AD.Fatty acids are important components of brain lipids,and slight changes in fatty acid composition may lead to significant changes in neurological function,which have been an important research direction for many chronic diseases,including AD.More and more studies support that the association between fatty acids and AD,but the role of fatty acids in the clinical diagnosis and prediction of AD is inconclusive.Lipid droplets are organelles that store fatty acids,and when Alzheimer first described AD in1907,he noticed that many glial cells in the lesion contained lipid droplets,in addition to neuroinflammatory plaques and neurofibrillary tangles.The studies of Hamilton et al.found that lipid droplet accumulation preceded the formation of neuroinflammatory plaques and neural progenitor fibers tangles in a model of AD in mice.All of these studies demonstrate the importance of lipid droplets in driving AD pathology.The synthesis and catabolism of lipid droplets are closely related to fatty acid metabolism.Recent studies have shown that decreased expression levels of carnitine palmitoyltransferase 1A（CPT1A）,a key enzyme in fatty acid metabolism for mitochondrial fatty acidβ-oxidation,can lead to the accumulation of lipid droplets in astrocytes,and that knockdown of CPT1A in adult mice results in cognitive dysfunction.Current studies of CPT1A have involved a variety of central nervous system disorders,including cerebrovascular disease,multiple sclerosis,and glioblastoma,however,the role and mechanism of CPT1A in the context of AD are unclear.Based on the current research background,the following questions were raised:Can fatty acids be used as diagnostic biomarkers for AD?Is CPT1A differentially expressed in AD patients and healthy populations?Is CPT1A a key gene for AD in terms of fatty acid metabolism?What are the roles of fatty acidβ-oxidation mediated by CPT1A in the pathogenesis of AD?In order to clarify the above questions,we used publicly available databases to search for biomarkers for AD diagnosis and prediction of progression,and identified CPT1A,a key gene in AD in terms of fatty acid metabolism through bioinformatics methods,machine learning methods,etc..Constructed an animal model and applied proteomics technology to validate the expression trend of Cpt1a in AD model rats at the protein level.With CPT1A as the center of the study,through in vivo and in vitro experiments,we applied recombinant adeno-associated virus,plasmid transfection,hematoxylin-eosin（HE）staining,oil red O staining,flow cytometry,Western blot,real-time quantitative PCR（q PCR）,and enzyme-linked immunosorbent assay（ELISA）to explore the effects of CPT1A expression decline mediated fatty acidβ-oxidation impairment on the cognitive function of AD model rats and the specific mechanism involved in the pathogenesis of AD,which provide a new perspective for understanding the pathogenesis of AD,and provide a theoretical basis and new targets for the development of therapeutic and preventive drugs for AD.Methods:1.Data used in preparation of our study were obtained from the Alzheimer’s Disease Neuroimaging Initiative（ADNI）database.The basic characteristics and erythrocyte fatty acids in the cognitively normal（CN）,mild cognitive impairment（MCI）,and AD groups were analyzed,and the correlation of fatty acids（C16:0,C18:0 andω3 index）with cognitive function,cerebrospinal fluid biomarkers and imaging markers was carried out by Spearman correlation coefficient and multivariate linear regression analysis.Multivariate logistic regression analysis was used to evaluate the impact of fatty acids（C16:0,C18:0 andω3 index）on the diagnosis of AD.The receiver operating characteristic（ROC）curve was used to evaluate the performance of fatty acids.Kaplan-Meier survival analysis and Cox risk proportion regression model were used to evaluate the association of fatty acids（C16:0,C18:0 andω3 index）with the prognosis of CN and MCI.2.Data used in preparation of our study were obtained from ADNI database.Bioinformatics methods and machine learning methods were used to identify the key gene related to fatty acid metabolism（CPT1A）in AD.CPT1A differential expression was verified using two datasets from the GEO database（GSE63060 and GSE63061）,RNA sequencing data from postmortem brain samples from the Mount Sinai Brain Bank,and CPT1A expression in AD was analyzed by single-cell sequencing.In terms of correlation between CPT1A and clinical indicators,Spearman correlation coefficient and multiple linear regression analysis were used to analyze the correlation between CPT1A and cognitive function,cerebrospinal fluid biomarkers and imaging markers,the logistic regression model was used to evaluate the impact of CPT1A on AD diagnosis,and the value of CPT1A to AD diagnosis was evaluated by ROC curve.3.AD rat model was prepared by injectingβ-amyloid 1-42(Aβ_1-42)into bilateral hippocampal CA1 region of Wistar rats using fully automatic brain stereotaxic apparatus,learning memory function of AD model rats was detected by Morris water maze assay,and pathological changes in hippocampal CA1 region of AD model rats were evaluated by HE staining.TMT-tagged quantitative proteomics technology and bioinformatics methods were applied to discover the down-regulation of CPT1A protein expression and the pathways involved in the hippocampal tissue of AD model rats.Western blot was used to verify the differential expression of CPT1A.4.In this study,we investigated the effects of CPT1A-mediated fatty acid metabolism on cognitive function and the specific mechanisms involved in AD through in vivo and in vitro experiments.（1）The construction of the AD model with overexpressing CPT1A was verified to be successful by using the Western blot and q PCR methods.The behavioral changes of the rats were observed by Morris water maze experiment.The pathological changes of the CA1 region of the hippocampus were observed by HE staining.Theβoxidation of fatty acids was evaluated by the level of free fatty acids.Pro-inflammatory cytokines and oxidative stress were detected by ELISA.Adenylate-activated protein kinaseα（AMPKα）were detected by Western blot and q PCR.（2）Aβ_1-42 was used to treat astrocytes to prepare AD cell models.The effect of different concentrations of Aβ_1-42 on astrocyte viability was detected by CCK-8 method to determine the concentration of Aβ_1-42 in the construction of AD cell models.The AD cell model with overexpressing CPT1A was constructed by transfection with CPT1A plasmid,and the transfection efficiency was verified by cell immunofluorescence,Western blot and q PCR.Lactate dehydrogenase assay（LDH）,flow cytometry,Western blot and q PCR methods were used to evaluate the effects of Aβ_1-42and CPT1A on astrocytes.JC-1 staining was used to observe the changes in mitochondrial membrane potential.Oil red O staining was used to evaluate fatty acidβoxidation.The pro-inflammatory cytokines and oxidative stress response were detected by ELISA.AMPKαwere detected by Western blot and q PCR.Results:1.The fatty acid C16:0,C18:0 andω3 index were significantly different between the CN and the AD group.After adjusting for potential confounders（age,sex,education,BMI,and APOEε4gene carrier status）,C16:0 was negatively correlated with the Mini-Mental State Examination,C18:0 was positively correlated with cerebrospinal fluid Ptau-181 and Ttau,ω3 index was positively correlated with the Brief Intelligent Mental State Examination Scale,the Montreal Cognitive Assessment Scale,positron emission tracer by fluorodeoxyglucose tomography,andω3index was negatively correlated with the Clinical Dementia Scale,The Alzheimer’s Disease Assessment Scale-Cognitive component.The results of multivariate logistic regression（adjusted covariate）showed that elevated C16:0 and C18:0 were risk factors for AD,and elevatedω3 index was a protective factor for AD.In order to evaluate the value of C16:0,C18:0,ω3 index and the multivariate logistic regression model for AD diagnosis,the ROC curves showed that C16:0,C18:0,ω3 index and multivariate logistic regression model had high performance in AD diagnosis.The results of Kaplan-Meier survival curve,log-rank test and multivariate Cox proportional hazards regression model analysis showed thatω3 index could predict progression from MCI to AD and was a protective factor.2.The results of bioinformatics analysis showed that the differentially expressed genes between CN and AD were involved in pathways including fatty acid metabolism,and GSEA enrichment analysis showed that fatty acid metabolism pathways were downregulated in AD.By machine learning methods,a key gene related to fatty acid metabolism in AD was obtained.The expression trend of CPT1A was verified using independent datasets and single-cell sequencing data,and CPT1A expression levels were found to be elevated in astrocytes compared to neurons by single-cell sequencing analysis.In order to clarify the association between CPT1A and clinical indicators,multiple linear regression analysis（adjusted for potential confounders）showed that CPT1A was positively correlated with cognitive function and total hippocampal volume,and negatively correlated with AβPET.The results of multivariate logistic regression（adjusted for potential confounders）showed that CPT1A was a protective factor for AD disease,and the ROC curve was plotted based on the multivariate logistic regression model,and the results showed that the combined CPT1A,C16:0 andω3 index had good diagnostic performance for AD.3.The behavioral evaluations and the pathological results suggested that the AD model we prepared was reliable.The proteomics results further confirmed that CPT1A is a key gene related to fatty acid metabolism in AD,and KEGG analysis showed that AMPK signaling pathway,fatty acid metabolism and other signaling pathways were associated with CPT1A.The expression trend of CPT1A in the hippocampus of rats was verified using Western blot.4.In this study,we investigated the effects of CPT1A-mediated fatty acid metabolism on cognitive function and the specific mechanisms involved in AD through in vivo and in vitro experiments.（1）Morris water maze experiment showed that AD model rats have reduced learning and memory functions.HE staining showed neuronal degeneration in the CA1 region of the hippocampus in AD rats.Western blot and q PCR for GAFP showed that Aβ_1-42 induced abnormal activation of astrocytes,and LDH and Flow cytometry showed that Aβ_1-42 induced astrocyte injury.The results of JC-1 mitochondrial membrane potential detection showed that Aβ1-42 induced a decrease in mitochondrial membrane potential in astrocytes.The results of free fatty acid content assay and oil red O staining results suggested that fatty acidβdisorder in AD rats’hippocampal tissue and AD astrocytes.The results of pro-inflammatory cytokine test suggested that neuroinflammation in AD rats’hippocampal tissues and AD astrocytes.The results of oxidative stress indicator indicated that oxidative stress response occurred in AD rats’hippocampal tissue and AD astrocytes.The decrease of protein p-AMPKα/AMPKαand the m RNA expression level of AMPKα,indicating that the AMPKαwas inhibited in hippocampal tissue and astrocytes of AD.（2）The results of Western blot and q PCR showed that the AD model with overexpressing CPT1A was successfully prepared,and the AD cell model with overexpressing CPT1A was successfully constructed based on the results of cellular immunofluorescence,Western blot and q PCR.Morris water maze experiment showed that overexpression of CPT1A improved the learning and memory functions of AD model rats.The results of HE staining showed that overexpression of CPT1A improved the morphology of neurons in the hippocampal CA1 region of AD rats.Western blot and q PCR results showed that overexpression of CPT1A alleviated the abnormal activation of AD astrocytes,and the results of LDH and Flow cytometry showed that overexpression of CPT1A reduced the degree of damage of AD astrocytes.JC-1 results showed that overexpression of CPT1A could increase the mitochondrial membrane potential of AD astrocytes.The results of free fatty acid content detection and oil red O staining showed that overexpression of CPT1A alleviated theβoxidation disorder of fatty acids in the AD model.The results of pro-inflammatory cytokine indicated that overexpression of CPT1A attenuated neuroinflammatory response.The results of oxidative stress indicator indicated that the overexpression of CPT1A alleviated oxidative stress.In AD models,the increase of m RNA expression level of AMPKα,suggesting that expression level of CPT1A could affect the activation of AMPKα.Conclusions:1.C16:0,C18:0 andω3 index are expected to be developed as biomarkers for AD diagnosis,andω3 index can be used to predict progression from MCI to AD.2.CPT1A is a key gene for AD in fatty acid metabolism.The combination of CPT1A,C16:0andω3 index is helpful in the diagnosis of AD.3.The expression of CPT1A was significantly inhibited in animal and cell models of AD,and the decrease in CPT1A expression mediated fatty acidβoxidation disorder can lead to nerve cell damage,promote oxidative stress and neuroinflammation,aggravate cognitive impairment,and affect the onset of AD.Therefore,CPT1A may become a new target for the treatment and prevention of AD.