Alzheimer's disease pathogenetic progression is associated with changes in regulated retained introns and editing of circular RNAs

Abstract

Introduction: The molecular changes leading to Alzheimer's disease (AD) progression are poorly understood. A decisive factor in the disease occurs when neurofibrillary tangles (NFT) composed of microtubule associated protein tau (MAPT) form in the entorhinal cortex and then spread throughout the brain.

Methods: We therefore determined mRNA and circular RNA changes during AD progression, comparing Braak NFT stages I-VI. Total RNA was isolated from human brain (entorhinal and frontotemporal cortex). Poly(A)+ RNA was subjected to Nanopore sequencing, and total RNA was analyzed by standard Illumina sequencing. Circular RNAs were sequenced from RNase R treated and rRNA depleted total RNA. The sequences were analyzed using different bioinformatic tools, and expression constructs for circRNAs were analyzed in transfection experiments.

Results: We detected 11,873 circRNAs of which 276 correlated with Braak NFT stages. Adenosine to inosine RNA editing increased about threefold in circRNAs during AD progression. Importantly, this correlation cannot be detected with mRNAs. CircMAN2A1 expression correlated with AD progression and transfection experiments indicated that RNA editing promoted its translation using start codons out of frame with linear mRNAs, which generates novel proteins.

Discussion: Thus, we identified novel regulated retained introns that correlate with NFT Braak stages and provide evidence for a role of translated circRNAs in AD development.

Keywords: Alzheimer; Braak stage; alternative splicing; circular RNAs; gene expression; retained intron.

Figure 1

Changes in gene expression in entorhinal cortex, temporal cortex white and gray matter.

Figure 2

Cluster analysis from PSI and Braak stages. The Euclidian distance in splice site usage, measured as log₂(changes in per cent spliced in) was calculated for each Braak stage. (A) Entorhinal cortex, (B) gray temporal cortex, and (C) white temporal cortex.

Figure 3

Properties of human regulated retained introns. (A) Algorithm to identify regulated retained introns from Illumina RNAseq data. (B) Position of the regulated retained introns in pre-mRNA. All mRNAs were normalized to a length of 100 and the position of each regulated retained intron tabulated. (C) Per cent of introns containing at least one Alu-element in regulated retained introns compared to control. The difference is significant, Chi2 p−value = 7.79e⁻²⁰⁷.

Figure 4

Regulatory elements in human regulated retained introns. (A) Comparison of regulated retained intron length that were validated by Nanopore sequencing (validated) with regulated retained introns determined by RNAseq (reference) and constitutively spliced introns (constitutive). (B) Occurrence of enhancer splicing regulatory elements (SRE) in introns. The box plots show the proportion of intron bases that overlap with enhancers. (C) Occurrence of silencer splicing regulatory elements (SRE) in introns. The box plots show the proportion of intron bases that overlap with silencers. (D) 3′ splice site quality of regulated retained introns and (E) 5′ splice site quality of regulated retained introns, (F) Combined splice site quality of regulated retained introns.

Figure 5

Clustering of circRNAs of different Braak stages in entorhinal cortex. The normalized Eucledian expression distance was compared with Braak NFT stages.

Figure 6

Change of expression of selected circRNAs in entorhinal cortex according to Braak stages. The relative expression levels of the genes indicated are plotted against the Braak stages. Structures of various circRNA isoforms are shown on the right. (A) circMAN2A1, (B) circDOCK1; (C) circHOMER1; (D) circST18; (E) circRTN4.

Figure 7

RNA editing of mRNAa and circular RNAs in the entorhinal cortex. (A) A > G changes in circular and linear RNAs from entorhinal cortex were calculated and normalized to the total sequence count at the individual sites. r² for the circular RNAs is 0.89. (B) Per cent of genes expressing at least one regulated retained intron and one circular RNA (blue) or no circular RNAs (gray).

Figure 8

After adenosine to inosine RNA editing, circMAN2A1 is expressed as protein. (A) Structure of the genomic region of MAN2A1 leading to backsplicing and (B) structure of the expression construct that is flanked by ZKSCAN1 introns (Welden et al., 2022), a curved arrow indicates the backsplicing. (C) 400 nt circMAN2A1 formed by the expression construct. The three start codons M1-M3 are indicated. The inner thinner circle depicts the predicted protein. Blue: novel circMAN2A1-specific peptides, yellow: Flag tag, red: proteins shared with the linear MAN2A1 that encompass the N-terminal catalytic domain of MAN2A1 and a predicted Rho binding domain. The small arrow indicates the clockwise direction of translation. The nucleotide and amino acid sequences are in Supplementary Figure 3. (D) The circMAN2A1 expression construct was cotransfected with expression clones for GFP, ADAR1, ADAR2 and ADAR3 in HEK293 cells. Left: The immunoprecipitation and detection was with anti-Flag antibody. Right: Western blot of the cell lysates. (E) Detection of ADAR overexpression in the cell lysates. Lysates were analyzed in Western Blot using anti-GFP. All constructs were GFP-tagged, ADAR3 exposure is 4× the rest of the blot, the expected size is indicated with a hexagon.

Figure 9

Proposed role of circular RNAs in Alzheimer’s disease. (A,B) AD progression leads to an increase of several circRNAs and generally to an increase of Adenosine to inosine RNA editing of these circRNAs (indicated as “I”). (C) The RNA editing leads to translation of the circRNAs. circRNAs can use reading frames different from linear RNAs, leading to the translation of novel peptides that could be Alzheimer-specific and could contribute to inflammation during AD progression.