| BackgroundAlzheimer’s disease(AD)is an irreversible and progressive neurodegenerative disorder in the human brain,which is a common cause of dementia among older adults.The main clinical symptoms of AD patients are the impairment of cognition and memory,language and mental disorders,disability of judgment and discrimination of objects in time and space.In gross pathology,AD patients usually suffer obvious central nervous system atrophy.In microscopic pathology,the hallmarks of AD are amyloid palques,neurofibrillary tangles,neuronal dystrophy and loss,together with astrogliosis and vascular alterations.Amyloid plaques,also called senile plaques,are deposits formed by a protein fragment called beta-amyloid(Aβ)that aggregates in the space between nerve cells.In addition to amyloid plaques,neurofibrillary tangles are hallmarks of AD,which constitute deposits of insoluble hyperphosphorylated tau protein.Since the 1980 s,The "Amyloid cascade hypothesis" and "Tau hyperphosphorylation hypothesis" were formed from the perspective of pathological characteristics of Aβ plaques and Tau hyperphosphorylation.However,until now,drugs based on these two dominant hypotheses for the treatment of AD have not been successful.These findings suggest that the understanding of the pathogenesis of AD may be limited based on these two pathological features.Moreover,it also suggests that the mechanism of AD occurrence and development is very complex.Therefore,the study and treatment of AD have become more and more hot issues in this field,resulting in many studies on AD from different perspectives.Cell adhesion molecule L1(L1)is a transmembrane protein mainly expressed on the plasma membrane of nerve cells.In the central nervous system,it can be involved in the recognition and connection between neurons.Previous studies in our laboratory have shown that adenovirus-mediated high expression of full-length L1(originally reduced L1)in the hippocampus of AD model mice effectively promotes Aβ plaque clearance.It was also found that full-length L1 could be cleaved into a 70 k Da fragment(called L1-70)in the hippocampal tissue of wild-type mice,and it could enter the nucleus after certain molecular modification.However,the details of how L1 overexpression promotes Aβ plaque clearance in the hippocampus of AD model mice and its relationship with fragment L1-70 remains unclear.AimBased on the accumulation of studies on the biological characteristics of L1 from different perspectives in our laboratory,we realized the possibility that the mechanism of L1 promoting the clearance of Aβ is not the direct function of full-length L1 molecule,but the result of the role of its cleaved fragment L1-70.So,we point out the scientific hypothesis that "in nerve cells,full-length L1 can be cleavaged into L1-70 specially,and this fragment can regulate the expression of certain genes to a particular form,and regulation of phagocytosis of glial cells within the brain activity,thus promoting the clearance of amyloid plaques".Research StrategyIn order to test this hypothesis,we adopted the basic strategy of "tracking the behavior of L1-70 fragment of L1 in the functional activity of nerve cells" to explore the correlation between L1-70 and Aβclearance.Firstly,western blotting and immunofluorescence histochemistry were used to analyze the expressions of L1 and L1-70,as well as Aβ plaque deposition in the neurons of wild-type mice and AD model mice,in order to understand their expression characteristics and their correlation.Then,the expression of L1 and L1-70 and the degree of Aβ plaque deposition were analyzed at the protein level and tissue level of hippocampal tissues of aged AD mice at different time points(3 and 14 days after conjoined with young wild-type mice)using Parabiosis mice research system.To understand whether the expression of L1 and L1-70 in nerve cells is affected by humoral microenvironment and its correlation with Aβ plaque deposition.Furthermore,the presence of L1-70 in nerve cells was analyzed by Co IP technique,immunofluorescence staining technique and ELISA technique with antibodies to L1 intracellular segment,in order to understand whether L1-70 can combine with other specific molecules to form complexes.Then,Duolink technique was used to verify in situ the presence of the complex directly bound and its spatial distribution in neuron cells.Then,using chip-sequencing method for antibody to L1 intracellular segment,analyze the regulatory sequence of genes L1-70 can be directly combined with in nerve cells,and analyze the expression level of regulated genes.It is hoped that l1-70 can regulate the activity of cells with Aβ phagocytosis in nerve cells.Furthermore,immunofluorescence histochemistry was used to analyze in situ the regulation of the expression products of related genes on specific "cells with Aβ phagocytosis".Finally,we will use conventional molecular and cell biological methods to preliminarily explore the related factors involved in the regulation of L1-70 expression in the serum of nerve cells,and find out the related circulating factors which are transferred from young mice to old AD mice and cause reaction changes.Based on these studies,we hope to clarify the relationship between "L1-70 and Aβ plaque clearance" in the hippocampus of mice,and clarify its regulatory mechanism involved in Aβ plaque clearance.ResultsIn this study,it was found that the full-length L1 molecule was underexpressed in the hippocampal tissue of aged AD model mice,and L1-70 was also underexpressed correspondingly,accompanied by obvious Aβ plaque deposition.These results suggest that L1 and its fragment L1-70 are correlated with Aβplaque deposition.According to our laboratory’s previous discovery that "L1-70 exists in mouse brain tissue and can enter the nucleus",this study focused on the phenomenon of "decreased expression level of L1-70" in AD model mouse brain tissue.Then,used the couplet of symbiotic system of research in mice,the old AD model(18 months)and young C57 BL / 6 mice of couplet of wild type mice(2 months),and let them live together after period of time,found that the old AD mice in the hippocampus of couplet of "the expression levels of endogenous L1-70" there is an obvious increase of change,"Reduced Aβ plaque deposition" was observed in the hippocampus of these mice.On this basis,we further investigated the biological behavior of L1-70 in hippocampus of aged AD mice.First of all,according to the previous mass spectrometry analysis information of "related proteins that full-length L1 molecule may bind" in our laboratory,the total protein of brain tissue of wild-type mice from 5 to 7 days after birth was taken as material by using Co IP and ELISA methods.The Topoisomerase1(Top1)with high binding probability was analyzed,and it was proved that l1-70 had the characteristic of interbinding with Top1.Subsequently,Duolink method was used to prove that L1-70 could indeed combine with Top1 to form a complex(i.e.,"L1-70/Top1")at the molecular level and coexist in the nucleus of nerve cells.Furthermore,chip-sequencing method was used to identify some candidate genes that may be regulated by L1-70/Top1 complex.Based on the existing knowledge that glial cells are directly involved in the clearance of Aβ plaque deposition in brain tissues,as well as the associated inflammatory factors,we identified these candidate genes The expression of macrophage migration inhibitory factor(MIF)and the function of microglia were analyzed.In this study,the expression of L1-70 in hippocampus of 18-month-old AD mice was significantly increased after 3 days of conjoined with young wild-type mice,and A large number of microglias clustered around Aβ plaques.Moreover,it was found that the expression level of MIF in the brain tissue of AD model mice was significantly higher than that in the brain tissue of non-united AD model mice.After 14 days of conglutination,it was found that Aβ deposition in the hippocampus of AD model mice decreased compared with that at 3 days of conglutination.Based on the existing knowledge that MIF activates microglia and promotes Aβ clearance,these results not only further confirm that high expression of L1 fragment L1-70 promotes Aβ plaque clearance in vivo,but also logically suggest that L1-70 may be mediated by "regulation of MIF high expression,Activation of microglia"mechanism to achieve Aβ clearance in brain tissue.On this basis,the related factors that may regulate L1 expression in serum were also discussed.According to the research hints of Geismann’s laboratory on "the expression of L1 in tumor tissues is regulated by TGFβ-1",we tentatively analyzed the combination of aged AD model mice and young wild-type mice.Is the phenomenon of "increased expression level of L1-70" in the brain tissue of the aged AD model mice caused by "high level of TGFβ-1 in the blood of the young wild-type mice as a circulating factor transferred to the aged AD model mice through capillaries,so that the level of TGFβ-1 in the blood of the aged AD model mice was significantly increased? Therefore,it is possible to regulate the expression of L1-70 in nerve cells through a specific mechanism.First,we used ELISA to demonstrate that TGFβ-1 levels in serum of elderly AD model mice in conjoined mice were significantly increased compared with that in non-conjoined elderly AD model mice.Furthermore,we used si RNA technology to Knock down the expression of TGFβ-1 in N2 a cells cultured in vitro,and found that the expression levels of L1-70 and MIF were decreased accordingly.These results suggest that TGFβ-1 in serum may be involved in the regulation of L1-70 expression in nerve cells.ConclusionBased on these results and the existing understanding of L1 biological characteristics,this study proposed a new model of L1-70 role in the hippocampus of AD model mice for Aβ plaque clearance "L1,located on the membrane of neurons,is cleaved by the properties of serine-dependent proteases to generate L1-70,which transfer into the cell via endocytosis--L1-70 binds Top1 to form a complex L1-70/Top1,which enters the nucleus through the nuclear pore,regulating the expression of the MIF gene,which promotes Aβ plaque clearance by activating microglia.At the same time,based on the results that "the expression of L1-70 in brain tissue and the level of TGFβ-1 in blood of aged AD model mice is low,and the expression of L1-70 in brain tissue can be improved by upregating the level of TGFβ-1 in blood".This project proposed the scientific hypothesis that "TGFβ-1 may be a starting point to explore the pathogenesis of AD from the perspective of aging".Research ValuesThe scientific innovations of this study are as follows: 1)The phenomenon of low expression of L1-70 in hippocampus of AD model mice and its concomitative relationship with Aβ plaque deposition were revealed for the first time.2)The regulation mechanism of L1-70 promoting Aβ plaque clearance in hippocampus of AD model mice was elucidated for the first time;3)It was found for the first time that TGFβ-1 in the humoral microenvironment can regulate the expression of L1-70 in nerve cells,thereby promoting the clearance of Aβ plaques.These advances in this study provide a new perspective for the discussion of the pathogenesis of AD and a new idea for the clinical prevention and treatment of AD. |
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