The Mechanism Of ANKHD1 Binding LINC00115 Regulating 18S RRNA Pseudouridylation To Mediate Blood Brain Barrier Permeability In Alzheimer’s Disease

Objective: Alzheimer’s disease is the most common cause of dementia and seriously affects human health and quality of life.With the further aging of the population,the number of people with Alzheimer’s disease is expanding and tending to spread to younger ages,and early onset cases have been reported.Alzheimer’s disease causes great suffering to patients and caregivers and imposes a huge economic burden on families and society.Currently,symptomatic treatment of Alzheimer’s disease provides modest,clinically measurable effects in cognition along with it,but etiologic treatment is urgently needed.The pathological mechanisms of Alzheimer’s disease are complex and involve a variety of factors including abnormalβ-amyloid metabolism,tau protein hyperphosphorylation,oxidative stress,and blood-brain barrier disruption.The blood-brain barrier in normal physiological state can restrict peripheral neurotoxic substances,inflammatory factors and other harmful substances from entering the central nervous system and maintain the normal microenvironment.The blood-brain barrier is composed of brain microvascular endothelial cells,pericytes,astrocyte peduncles and extracellular matrix,of which the most basic is the tight junction structure formed between microvascular endothelial cells,which is an important part to maintain the structural and functional integrity of the blood-brain barrier.Occludin and claudins are the membrane proteins that make up the tight junctions,and ZO proteins anchor the aforementioned membrane proteins to the cytoskeleton and forming the tight junction structure.Therefore,the expression level of tight junction-related proteins is closely related to the integrity and permeability of the blood-brain barrier.Long non-coding RNAs(lncRNAs)have become biomarkers and therapeutic targets for a variety of diseases,and their role in the physiopathology of the central nervous system has also becoming the direction of exploration by researchers.LINC00115 has been identified as a potential biological marker for several tumors,but whether it plays a role in neurodegenerative diseases remains to be investigated.The physiological functions and roles of small nucleolarRNAs(sno RNAs)have received increasing attention from researchers.sno RNAs are mainly found in the nucleus and regulate post-transcriptional modifications of ribosomal RNAs(rRNAs).Among them,H/ACA box sno RNAs bind to four highly conserved proteins GAR1,NHP2,NOP10 and NAP57(dyskerin)to form an RNA-protein complex to regulate pseudouridylation modifications of rRNAs.NAF1 is an assembly cofactor of the H/ACA sno RNP complex,which binds to NAP57 during its forming of the sno RNP complex in assembly.SNORA4 has not been reported in neurodegenerative diseases.The predicted modification site of SNORA4 is located in 18 S rRNA,a major subunit component of the 40 S ribosome.The eukaryotic initiation factor(EIF)complex is responsible for regulating the protein translation initiation step,and EIF5 A is associated with maintaining the active state of the 40 S ribosome.ANKHD1 is an RNA-binding protein.Studies have confirmed that RNA-binding proteins can bind non-coding RNAs and further influence the biological functions of non-coding RNAs.High expression of ANKHD1 in glioma has been reported in the literature,but its relevance in Alzheimer’s disease has not been seen.In this study,we firstly clarified the expression of LINC00115,SNORA4,NAF1,EIF5 A,and ANKHD1 in Abeta-incubated human brain microvascular endothelial cells,and further investigated the possible regulatory relationships between the above factors and their effects on the integrity and permeability of the blood-brain barrier in Alzheimer’s disease,aiming to provide new ideas for the pathology and treatment of Alzheimer’s disease from the direction of the blood-brain barrier.Methods: 1.The establishment of BBB model in vitro.2.q RT-PCR was used to detect the expression of LINC00115,SNORA4 and NAF1.Western blot was used to detect the expression levels of NAF1,EIF5 A,ANKHD1,ZO-1,occludin and claudin-5.3.Abeta-incubated endothelial cells were stably transfected with overexpression and knockdown plasmids of LINC00115,SNORA4 and NAF1.and EIF5 A,respectively.4.RNA-RNA pull down assay was applied to detect the binding of LINC00115 to SNORA4.RNA binding protein immunoprecipitation assay was applied to detect specific binding of LINC00115 to NAF1,SNORA4 to NAF1,and LINC00115 to ANKHD1.5.Ψ-CMC addition combined with Sanger sequencing assay to detect pseudouridylation levels of 18 S rRNA in Abeta-incubated ECs and Abeta-incubated ECs with knockdown of SNORA4 and overexpression of LINC00115,respectively.6.The BBB integrity was determined by Trans-endothelial electric resistance(TEER)assay.7.The BBB permeability was determined by horseradish peroxidase(HRP)flux assay.8.The expression and distribution of tight junction-associated proteins ZO-1,occludin and claudin-5 were detected by immunofluorescence assay.9.Statistical analysis was performed by one-way ANOVA as well as t-test.P < 0.05 was used as the criterion for statistical significance of differences.Results: 1.LINC00115 was highly expressed in AD-ECs,and knockdown of LINC00115 expression significantly increased TEER values and decreased HRP flux.It also promoted the expression of tight junction-related proteins ZO-1,occludin and claudin-5.and further changed their distribution on the cell membrane from a discontinuous state to a relatively continuous state.Overexpression of LINC00115 significantly reduced TEER values and increased HRP flux,It also reduced the expression of ZO-1,occludin and claudin-5,and further changed their distribution on the cell membrane from relatively continuous to discontinuous.2.SNORA4,NAF1 and EIF5A were lowly expressed.Overexpression of SNORA4,NAF1,and EIF5 A significantly increased TEER values and decreased HRP flux.It also promoted the expression of tight junction-related proteins ZO-1,occludin,and claudin-5,and further changed their distribution on the cell membrane from a discontinuous state to a relatively continuous state.Knockdown of SNORA4,NAF1,and EIF5 A significantly decreased TEER values and increased HRP flux.The expression of ZO-1,occludin and claudin-5 was reduced and their distribution on the cell membrane was changed from relatively continuous to discontinuous.3.RNA-RNA pull down assay showed that LINC00115 and SNORA4 had binding effect.RIP assay showed binding effect of NAF1 and LINC00115,NAF1 and SNORA4,LINC00115 and ANKHD1.4.Overexpression of SNORA4 and overexpression of LINC00115 simultaneously could significantly reversed the effects of overexpression of LINC00115 and SNORA4 alone on BBB integrity and permeability.Overexpression of LINC00115 and overexpression of NAF1 simultaneously could significantly reversed the effects of overexpression of LINC00115 and overexpression of NAF1 alone on BBB integrity and permeability.Compared to knockdown of LINC00115,overexpression of SNORA4 or overexpression of NAF1 alone,knockdown of LINC00115 and overexpression of SNORA4 and overexpression of NAF1 triple treatment increased TEER values,reduced HRP flux and improved BBB blood-brain barrier integrity and reduced permeability.5.Knockdown of SNORA4 significantly shortened the half-life of 18 S rRNA in AD-ECs compared with the blank control;Ψ-CMC adduct combined with Sanger sequencing showed the pseudouridylation of18 S rRNA in AD-ECs.6.Overexpression of LINC00115 significantly reduced the binding of SNORA4 and NAF1 in AD-ECs and the level of pseudouridylation at the U1347 site of 18 S rRNA in AD-ECs.7.Knockdown of SNORA4 expression significantly decreased EIF5 A protein expression in ECs.Overexpression of SNORA4 significantly increased EIF5 A protein expression in ECs.Co-overexpression of SNORA4 and LINC00115 significantly reversed the effects of overexpression of LINC00115 alone and SNORA4 alone on EIF5 A expression in AD-ECs.8.ANKHD1 was highly expressed in AD-ECs,and knockdown of ANKHD1 significantly increased TEER values,decreased HRP flux,and decreased the expression of EIF5 A in AD-ECs.simultaneously decreased the expression of LINC00115 in AD-ECs.Overexpression of ANKHD1 significantly decreased TEER values,increased HRP flux,and increased the expression of LINC00115 in AD-ECs.Conclusion: 1.LINC00115 is highly expressed in AD-ECs and SNORA4 and NAF1 are lowly expressed in AD-ECs,affecting BBB permeability in the AD microenvironment by regulating the translation of tight junction-related proteins ZO-1,occludin,and claudin-5.2.LINC00115 act as a scaffold molecule to bind NAF1 and attenuates NAF1 recruitment of SNORA4 involved in sno RNP complex composition,thereby reducing 18 S rRNA pseudouridylation and downregulating EIF5 A expression.3.Knockdown of EIF5 A decreases the expression levels of tight junction-related proteins ZO-1,occludin,and claudin-5 and increases BBB permeability.4.ANKHD1 targeted binding to LINC00115 and promoted the stability of LINC00115,which in turn affected the expression of tight junction-related proteins in AD-ECs.