Study On The Effect And Mechanism Of High-salt Diet Aggravating AD Pathology By Acting On The Neurovascular Units

ObjectiveAging and unhealthy lifestyles（poor diet,fast-paced life,and reduced physical activity）are risk factors for the dementia,with Alzheimer’s disease（AD）being the most common type of dementia.The ineffectiveness of AD therapies targeting the existing pathogenesis suggests that alternative approaches are needed to address the pathogenesis of AD.The association of type 2 diabetes mellitus（T2DM）and other metabolic diseases with AD has been increasingly studied,and abnormalities in neuronal and glial cell energy metabolism have been found to be closely related to the pathogenesis of AD.The pathological progression of AD is often accompanied by vascular lesions,energy supply disorders,and blood brain barrier（BBB）abnormalities,and most of them appear early in the disease,suggesting that factors leading to vascular and metabolic abnormalities play a key role in the pathogenesis of AD.Although epidemiological studies on the association between high salt diet（HSD）and the incidence of AD are lacking,studies have provided evidence of the vascular and metabolic detrimental effects of HSD.the neuroenergetic hypothesis of AD pathogenesis suggests that reduced levels of neuronal energy supply are involved in AD pathogenesis,synaptic atrophy,and neuronal death.Rapid and stable neuronal energy supply depends on neurovascular coupling（NVC）mechanisms based on the neurovascular unit（NVU）.However,it is not clear whether a chronic HSD affects NVU leading to decreased cerebral blood flow（CBF）and abnormal neuronal energy metabolism,thereby accelerating brain aging,aggravating AD pathology and cognitive impairment.The aim of this study was to investigate the effects of a chronic HSD on AD and to investigate whether the development of AD pathology is related to changes in the structure and function of the NVU.Considering the close relationship between NVU and glymphatic system transport in the brain and the important role it plays in metabolism,this study further looked at the relationship between AD pathological evolution and glymphatic system transport efficiency.Methods1.Effects of chronic HSD on cognitive function and NVU in mice1.1 HSD feeding:6-month-old wild type or APP/PS1 mice fed high-salt diet（8%Na Cl,HSD）for 4 or 8 months for behavioral tests,and 8 months for biochemical and pathological tests.1.2 Behavioral tests:open-field experiment to evaluate voluntary movements;morris water maze to evaluate spatial cognition and learning memory functions;new object recognition experiment to evaluate working memory.1.3 Immunofluorescence and immunohistochemistry assays:Whole brain slices were collected,and the combined immunofluorescence and immunohistochemistry semi-quantitative method was used to detect the alterations of AD-related pathology and NVU composition in hippocampus and cortex,including the expression of Aβ₄₂,p-Tau,Iba-1,GFAP,PDGFR-β,AQP-4 and Neu N.1.4 Western Blotting（WB）assay:mouse hippocampal and cortical proteins were collected,and the expression of AD pathological features and related enzymes were detected by WB,including Aβ₄₂,p-Tau,Bace,IDE,p-GSK-3βand ALPL;alterations in NVU composition and regulatory molecules,including AQP-4,Calpain,PDGFR-βand CD31;detection of expression of energy metabolism-related molecules,including HIF-1α,Na⁺/K⁺-ATPase and Cytochrome C.1.5 RT-q PCR assay:m RNA was collected from hippocampus and cortex,and vegf,tgf-βand e NOS m RNA expression were detected by RT-q PCR.1.6 Evens Blue assay for BBB integrity:Evens Blue was injected into the tail vein,and the Evens Blue content in hippocampus was detected 2 hours later.1.7 Alizarin red staining to detect the amount of calcium deposition in the brain2.Effect of Aβ_1-42 oligomer on energy metabolism of HT22 cells2.1 Detection of cell viability（CCK-8）:Different concentrations of Aβ_1-42 were co-incubated with HT22 cells for 48 h.Cell viability was evaluated by CCK-8.2.2 Detection of cellular oxygen consumption rate:Seahorse XF-24 assayed the effect of 50μM Aβ_1-42 on the oxygen consumption rate of HT22 cells.2.3 RT-q PCR assay:The mitochondrial respiratory chain component molecules of HT22 cells was detected by RT-q PCR,including protein 1-6,protein 41,protein A61 and cyt 1-33.Analysis of glymphatic system transport efficiency in APP/PS1 mice3.1 Experimental groups:The control group was 14-month-old wild-type male mice,and the experimental group was age-matched APP/PS1 male mice.3.2 Evaluation of transport from brain parenchyma to the glymphatic system:1μL of Cascade Blue was injected into the striatum,and the brain and deep cervical lymph nodes were taken 120 minutes after the injection,and the content of Cascade Blue was measured.3.3 Evaluation of transport from CSF to brain parenchyma:4μL of Cascade Blue was injected into the lateral ventricle,and the brain and deep cervical lymph nodes were taken 120 minutes after the injection,and the amount of Cascade Blue was measured.3.4 Evaluation of transport from CSF to deep cervical lymph nodes:5μL of Cascade Blue was injected into the cisterna magna,and the brain,meninges,and deep cervical lymph nodes were taken 30 minutes after the injection,and the levels of Cascade Blue were measured.Results1.Study of the effects of chronic HSD on AD pathology and mechanisms1.1 Effects on cognitive function.After 4 months of HSD,compared with wild-type mice in normal diet（ND）,mice in HSD group had increased time to find escape platforms in the morris water maze and decreased recognition index of new objects;after 8 months of HSD,mice in the HSD group could hardly find escape platforms in the morris water maze and further decreased recognition index of new objects.After 4months of HSD,compared with APP/PS1 mice on ND,the time to find escape platform in morris water maze increased in mice on HSD group,and the recognition index of new objects decreased;after 8 months of HSD,APP/PS1 mice on both ND and HSD groups could not find escape platform in morris water maze,and the recognition index of new objects decreased in both groups.1.2 Effects on AD pathology.After 8 months of HSD,compared with wild-type mice on ND,p-Tau was significantly increased in the hippocampus and cortex on HSD;p-GSK-3βand ALPL,enzymes related to Tau phosphorylation,were expressed abnormally;Aβlevels did not change significantly,and only a small amount of Aβ₄₂positive expression was observed in the perivascular area;Bace and IDE,enzymes related to Aβmetabolism,were not significantly changed,and the number of hippocampal and cortical neurons was decreased.After 8 months of HSD,compared with APP/PS1 mice on ND,p-Tau and Aβexpression were significantly increased in the hippocampus and cortex of APP/PS1 mice on HSD;and abnormal accumulation of amyloid plaques was seen;Tau phosphorylation-related enzymes such as p-GSK-3βand ALPL were expressed abnormally,while Aβmetabolism-related enzymes Bace and IDE were not significantly altered in the hippocampus and cortex of APP/PS1mice on HSD.APP/PS1 mice showed loss of hippocampal and cortical neurons with increasing age.1.3 Effects on perivascular glial cells.After 8 months of HSD,compared with wild-type mice on ND,mice in HSD group showed activation of microglia in the hippocampus and cortex and insignificant changes in perivascular astrocytes,but AQP-4 expression was significantly downregulated;calpain expression,a calcium-dependent protease expressed mainly in microglia and astrocytes,is upregulated in the hippocampus and cortex;a small amount of calcium was detected in the hippocampus,cortex and thalamus.In APP/PS1 mice on a ND,microglia and astrocytes were activated in the hippocampus and cortex with increasing age.8months of HSD increased microglia activation in the hippocampus and promoted downregulation of perivascular AQP-4 expression and further upregulation of calpain expression in hippocampus and cortex.The calcium deposition in hippocampus,cortex and thalamus was further increased.1.4 Effects on intracerebral vascular structures.After 8 months of HSD,compared with wild-type mice on ND,the hippocampal vascular density on HSD was not significantly altered,but there was loss of pericytes,proliferation of endothelial cells,downregulation of m RNA levels of angiogenesis-related genes,and increased BBB permeability in the hippocampus.After 8 months of HSD,compared with APP/PS1 mice on ND,the hippocampal vascular coverage area of mice on HSD did not change significantly,but pericytes showed further loss,endothelial cells proliferated significantly,and m RNA levels of angiogenesis-related genes were further downregulated;BBB permeability increased significantly.1.5 Effects on energy metabolism.After 8 months of HSD,compared with wild-type mice on ND,mice in HSD group showed high expression of HIF-1αin the hippocampus,low expression of Na⁺/K⁺-ATPase in the hippocampus and low expression of Cytochrome C in the hippocampus and cortex.After 8 months of HSD,compared with APP/PS1 mice on ND,mice in HSD group showed showed high expression of HIF-1αin the hippocampus and cortex,low expression of Na⁺/K⁺-ATPase in the hippocampus and cortex and low expression of Cytochrome C in the hippocampus.Aβ_1-42 oligomer inhibit mitochondrial function in HT22 cells,reduce energy utilization of the oxidative phosphorylation pathway,and result in decreased expression levels of mitochondria-encoded molecules associated with oxidative phosphorylation.2.Altered transport efficiency of the glymphatic system in APP/PS1 mice2.1 After striatal injection of Cascade Blue,compared with wild type mice,the diffusion rate of Cascade Blue in the brain parenchyma of APP/PS1 mice was slowed down and the efficiency of ISF transport to the glymphatic system was decreased in APP/PS1 mice.2.2 After injection of Cascade Blue into the lateral ventricles,compared with wild mice,the diffusion rate of Cascade Blue from the ventricles to the brain parenchyma was slowed down in APP/PS1 mice,and the amount of CSF entering the deep cervical lymph nodes in the same time was reduced,and the transfer efficiency of CSF to the deep cervical lymph nodes through the glymphatic system was decreased in APP/PS1 mice.2.3 After injection of Cascade Blue into the cisterna magna,compared with wild type mice,the amount of Cascade Blue entering the deep cervical lymph nodes increased in APP/PS1 mice,and the efficiency of CSF transport through the glymphatic system to the brain parenchyma decreased in APP/PS1 mice.Conclusions1.Cognitive dysfunction in wild mice caused by HSD is related to the following factors:HSD leads to structural changes in NVU,mainly manifested by loss of pericytes and proliferation of endothelial cells,decreased blood oxygen levels and abnormal energy metabolism in hippocampus;HSD leads to hyperphosphorylation of Tau in hippocampus and cortex and loss of neuronal cells.2.APP/PS1 mice showed high expression of Aβin hippocampus and cortex and altered NVU structure in the hippocampal region,mainly manifested by proliferation of perivascular astrocytes and microglia activation.HSD aggravated AD pathology and cognitive dysfunction in APP/PS1 mice and was associated with the following factors:HSD leads to structural changes in NVU,mainly manifested by pericyte loss and endothelial cell proliferation,decreased blood oxygen levels and abnormal energy metabolism in hippocampus and cortex;HSD leads to decreased clearance and increased aggregation of Aβand increased amyloid plaques with abnormal aggregation in the hippocampus and cortex;HSD aggravated the hyperphosphorylation of Tau and activation of Calpain signaling in glial cells,which aggravated AD pathology.HSD aggravates AD pathology through the above pathways and accelerates cognitive dysfunction in APP/PS1 mice.3.The decreased efficiency of glymphatic system transport in APP/PS1 mice may further impede the clearance of Aβ,which is one of the reasons for aggravating AD pathology.