Research On The Disruption Of Metabolites And Related Pathways In Alzheimer’s Disease

Alzheimer’s disease(AD),a neurodegenerative disease primarily affecting the elderly,is one of the most prevalent causes of dementia.The most typical pathological features of AD are amyloid plaques formed by abnormal deposition of amyloid-β(Aβ)and neurofibrillary tangles formed by excessive phosphorylation of tau protein in the brain.AD is clinically characterized by a progressive decline in cognitive function.Nevertheless,the specific pathogenesis of AD is not yet completely understood and there are still many trials to be conducted before clinical drugs can be developed to treat AD.This suggests that we should explore the causes of the occurrence and development of AD from more perspectives.A growing number of studies have demonstrated that AD is associated with metabolic disorders.Various metabolites produced by metabolic processes,such as amino acids and lipids,are abnormally altered in AD patients and are associated with AD progression.It is therefore crucial to comprehensively study metabolites and related pathways in AD,which may provide valuable insight into the pathology of AD as well as identify more potential diagnostic markers and drug targets.Firstly,we studied the changes in metabolite distribution in the brain during the pathogenesis of AD.We used an acid-enhanced version of the desorption electrospray ionization mass spectrometry imaging(DESI-MSI)technique to in situ detect the whole brain metabolites of wild-type(WT)mice and the APP23 mouse model of AD at different ages.Through a series of technical optimizations,this acid-enhanced DESI-MSI can detect more kinds of metabolites in the mouse brain than the traditional version,and has high specificity and repeatability for the metabolite imaging.A total of 317 compounds were detected on mouse brain slices.Of these,119 compounds could be identified as metabolite candidates,and 71 metabolites showed strong signals in the brains of all mice at different ages.A comparison of 71 metabolites in the brains of WT mice and APP23 mice showed that changes were evident in many metabolites in APP23 mice,and these changes were age-and region-specific.At 3 months of age,glutamine and pyroglutamic acid became abnormal in APP23 mouse brains.More metabolites,such as adenine,adenosine,and hypoxanthine,were changed in the brains of APP23 mice at 18 months.A simultaneous change in these metabolites was observed in many brain regions.Meanwhile,glutamate,aspartic acid,creatinine,and taurine were significantly altered in only a few brain areas of APP23 mice.In addition,we found that the spatial correlation of metabolites was distinct in certain brain regions.It suggests that metabolites are processed differently in brain regions with different functions.Furthermore,the spatial correlation of metabolites was different between WT and APP23 mice,reflecting the changes in the metabolic network of the brain in AD.Next,we analyzed metabolite-related pathways in the brain of mice using DESI-MSI,and identified metabolic pathways that were disturbed in the brain of APP23 mice at different ages.The camitine-acetylcamitine pathway,which is crucial to fatty acid metabolism,was up-regulated in brain regions such as the caudate putamen,amygdaloid nucleus,and thalamus in 3-month-old APP23 mice.Immunofluorescence imaging also showed an increase in carnitine acetyltransferase,which catalyzes this pathway.Besides,the choline-phosphocholine pathway associated with phospholipid synthesis was significantly upregulated in the piriform cortex,amygdaloid nucleus,and hippocampus of 18-month-old APP23 mice.A reduction in choline kinase,which catalyzes this pathway,was likewise observed.In addition to pathways related to lipid metabolism,APP23 mouse brains also exhibited significant changes in those related to adenosine,creatine,and polyamine.Furthermore,glutamine-glutamate/y-aminobutyric acid(GABA),an influential neurotransmitter metabolic circuit,showed a series of changes with age in APP23 mice.Based on DESI-MSI,immunofluorescence imaging,and two-photon glutamate imaging in vitro,we found that glutamate synthesis and release were decreased in glutamatergic neurons,while GABA was significantly increased in GABA-ergic neurons of aged APP23 mice.Using a multi-electrode in vivo electrophysiology study,we recorded neuronal firing in mice’s prefrontal cortex and hippocampus to determine if neuronal activity changes in response to an imbalance between glutamate and GABA.It was found that 18-month-old APP23 mice exhibited a significantly lower spontaneous firing rate and a significantly higher proportion of hypoactive neurons in the hippocampus and cortex.Therefore,we believe that although the hyperactivity of neurons exists in the early stage of AD,the disorder of metabolic pathways leads to the down-regulation of excitatory neurotransmitters and the up-regulation of inhibitory neurotransmitters with increasing age,resulting in a substantial decline in neuronal activity.Then,in order to verify the metabolite changes observed in the brain of AD mice,and to investigate the changes in peripheral blood metabolites and their bloodbrain transport,we analyzed metabolites in the blood and cerebrospinal fluid(CSF)from AD patients.We performed untargeted metabolomics analyses of serum and CSF samples collected from cognitively normal(CN),mild cognitive impairment(MCI),AD and non-AD dementia(Non-ADD)patients in the China Aging and Neurodegenerative Disorder Initiative(CANDI)cohort.The differential metabolites in serum and CSF between patients were screened out by using an intergroup variance analysis.As compared to the CN group,most metabolites with significant changes were found in AD patients.A number of these metabolites are also altered in patients with MCI.Moreover,the serum and CSF of AD patients show changes in some metabolic pathways disrupted in AD mouse brains,including glutamine,glutamate,and purine pathways.Analyzing the correlation of differential metabolites in AD patients with imaging and fluid biomarkers for AD,we investigated the relationship between metabolite changes and AD pathology.We found that many of the differential metabolites in serum and CSF of AD patients were significantly associated with CSF Aβ42/Aβ40,a core biomarker of AD that reflects Aβ pathology.Further,some differential metabolites in AD are correlated with Aβ plaque positron emission tomography imaging status and phosphorylated tau(P-tau)in CSF.This suggests that metabolite changes are closely related to Aβ and tau pathologies in AD patients.A number of common metabolites were detected in both the CSF and serum of patients,so the CSF/serum ratios of these metabolites were also analyzed for intergroup differences.This ratio of metabolites was significantly lower in AD patients compared to other groups,and we observed that most of the differential CSF metabolites of AD patients were decreased,indicating that the blood-brain transport of metabolites may be altered.Therefore,we examined the transporters responsible for metabolite uptake by the brain at the blood-brain barrier.Using postmortem brain samples of Non-ADD and AD patients,we isolated cerebral vessels and performed vascular immunofluorescence to detect transporters of several metabolites with decreased CSF/serum ratios.The transporters in the cerebral vessels of AD patients showed a downward trend,indicating a disorder in metabolite transport in the brain.Lastly,we evaluated the ability of blood metabolites to predict AD pathology.We found that serum metabolites combined with plasma P-tau,an AD biomarker,could predict CSF Aβ42/Aβ40 very closely.In addition,the models established with plasma P-tau and serum metabolites have significantly higher accuracy of predicting Aβ pathologic status based on CSF Aβ42/Aβ40 compared with plasma P-tau alone.Thus,blood metabolites can be used to improve the prediction of the pathological status of AD by biomarkers.In summary,our study revealed not only central and peripheral metabolite changes associated with AD,but also alterations in the blood-brain transportation of metabolites in AD patients.Moreover,the association between metabolite changes and AD pathology was also demonstrated,and blood metabolites were used to predict AD pathology in patients.Thus,our study contributes to a more comprehensive understanding of AD-related metabolic disorders,while providing metabolite-type biomarkers and targets for clinical diagnosis and drug development for AD.