Circ-sAPPα Translates the sAPPα-199aa Protein and Activates Autophagy in AD, Thereby Improving AD Markers

摘要

Objective: The A β theory is the main mechanism of Alzheimer's disease（AD） onset. We retrieved from the circular RNA professional database circBase that many circular RNAs formed by the APP gene, which is the precursor protein of A β, are highly specifically expressed in brain tissues. Among them, there is a homologous circular RNA that exists at high abundance in both human and mouse species（hum＿circ＿0115728, mmu＿circ＿0000705）. Its mature length is 597 nucleotides in both humans and mice. In this study, it is named circ-sAPPα. This study will elaborate on its functional mechanism in the process of AD. Methods: By sequencing the circular RNAs in the whole brain tissues of APP/PS1 transgenic mice and age-matched, genetically-similar C57 BL/6J male mice, we identified novel target circular RNAs related to AD, circ-SAPPα is one of the circRNAs that show significant differences. In order to study the effects and functions of circ-sAPPα on AD, we overexpressed or knocked out circ-sAPPα in cells and animals, to understand its expression and ability to translate proteins, and further investigate the role of circ-sAPPα in AD. Using AAV vectors, we packaged the pK25ssAAV-ciR plasmid containing the linear sequence of OV-circ-sAPPα and transfected BV2 cells. After transfection, we extracted RNA and proteins. In the animal experiments, we injected AAV（OV-circ-sAPPα） into 3×Tg-AD model mice and constructed KO-circ-sAPPα mice. We conducted transcriptome sequencing of 3×Tg-AD mice, OV-circ-sAPPα mice, and KO-circsAPPα mice. We used WB and RT-qPCR to verify the differential gene and protein expression of the transcriptome. We used immunoblotting technology to detect AD-related pathological indicators. We performed IP experiments using anti-sAPPα-199aa antibody to study its target site. Results: In APP/PS1 mice, a total of 5849 circRNAs were identified. Among them, 501 circRNAs showed increased expression, and 624 circRNAs showed decreased expression（Foldchange > 1.50, P < 0.05）. Among the 624 upregulated circular RNAs, the homologous circular RNA molecule（hum＿circ＿0115728） was one of them. qRT-PCR analysis revealed that the relative expression level of circ-sAPPα was lower in 3-month-old and 11-month-old APP/PS1 mice. In 11-month-old APP/PS1 mice, the ratio of circsAPPα to mAPP was significantly decreased. In the cell experiment, circ-sAPPα was successfully overexpressed in BV2 cells. The protein particles and the custom anti-sAPPα-199aa antibody were confirmed by western blotting and mass spectrometry. After treating 3×Tg-AD mice with the AAV system overexpressing circ-sAPPα(virus titer 1×10¹²), circ-sAPPα could translate the sAPPα-199aa sequence protein in the brain. The expression of memory-related genes such as Arc, c-Fos, Egr1, Npas4, Nr4a1, etc. in the brain tissue of KO-circ-sAPPα mice showed significant differences, suggesting that circ-sAPPα and/or sAPPα-199aa affect the transcription and expression of memoryrelated genes. The IP experiment showed that sAPPα-199aa could bind to HSP60 and HSP70. Transcriptome results of 3×Tg-AD mice and OV-circ-sAPPα indicated that circ-sAPPα changed the genes related to phagocytosis and autophagy in 3×Tg-AD mice, including up-regulating Hspa1b, Hspa1a, RSRC1, FOS, NPAS4, Nr4a2, Nr4a3, Nr4a1, etc., and down-regulating 394 mRNAs such as BACE2, Apbb 1ip, Pcdhga9, Lcn2, Slc11a1, etc. Immunoblotting and immunohistochemistry of mouse brain tissue revealed that the expression levels of heat shock protein family members Hsp90, Hsp70, Hsp40, LC3A/B, LAMP2, and TREM2 in the brain of OV-circ-sAPPα group mice were significantly higher than those in the 3×Tg-AD group（P < 0.05）. circ-sAPPα improved the expression of AD markers such as Aβ, Tau, p-Tau, TREM2, CD33, APOE in the brain tissue of 3×Tg-AD mice. Conclusion: The above research results indicate that circ-sAPPα is an important circRNA marker in the occurrence and development of AD. It is highly expressed in brain tissues. circ-sAPPα can translate the sAPPα-199aa sequence protein. sAPPα-199aa can bind to HSP60 and HSP70, thereby altering cell autophagy. Overexpression of circ-sAPPα can increase the level of cell autophagy in the brain tissues of AD mice, affect the expression of AD markers such as Aβ, Tau, p-Tau, TREM2, CD33, and APOE in the brain tissues of AD mice, and delay the progression of AD.

关键词

Alzheimer's disease / amyloid-beta protein / amyloid precursor protein / circRNA / autophagy