Alzheimer’s disease (AD) is an age-related detrimental dementia. Amyloid beta peptides (Aβ) play a crucial role in the pathology of AD. In familial AD, Aβ are generated from the full-length amyloid beta precursor protein (APP) via dysregulated proteolytic processing; however, in the case of sporadic AD, the mechanism of Aβ biogenesis remains elusive. circRNAs are a class of transcripts preferentially expressed in brain. We identified a circRNA harboring the Aβ-coding region of the APP gene termed circAβ-a. This circular RNA was detected in the brains of AD patients and non-dementia controls. With the aid of our recently established approach for analysis of circRNA functions, we demonstrated that circAβ-a is efficiently translated into a novel Aβ-containing Aβ175 polypeptide (19.2 KDa) in both cultured cells and human brain. Furthermore, Aβ175 was shown to be processed into Aβ peptides—a hallmark of AD. In summary, our analysis revealed an alternative pathway of Aβ biogenesis. Consequently, circAβ-a and its corresponding translation product could potentially represent novel therapeutic targets for AD treatment. Importantly, our data point to yet another evolutionary route for potentially increasing proteome complexity by generating additional polypeptide variants using back-splicing of primary transcripts that yield circular RNA templates.

Circular RNAs (circRNAs) are an emerging class of RNA molecules that have been linked to human diseases and important regulatory pathways. Their functional roles are still under investigation, often hampered by inefficient circRNA formation in and ex vivo. We generated an intron-mediated enhancement (IME) system that—in comparison to previously published methods—increases circRNA formation up to 5-fold. This strategy also revealed previously undetected translation of circRNA, e.g., circRtn4. Substantiated by Western blots and mass spectrometry we showed that in mammalian cells, translation of circRtn4 containing a potential “infinite” circular reading frame resulted in “monomers” and extended proteins, presumably “multimer” tandem repeats. In order to achieve high levels of circRNA formation and translation of other natural or recombinant circRNAs, we constructed a versatile circRNA expression vector—pCircRNA-DMo. We demonstrated the general applicability of this method by efficiently generating two additional circRNAs exhibiting high expression levels. The circRNA expression vector will be an important tool to investigate different aspects of circRNA biogenesis and to gain insights into mechanisms of circular RNA translation.

Prader-Willi syndrome (PWS) is a neurogenetic multifactorial disorder caused by the deletion or inactivation of paternally imprinted genes on human chromosome 15q11-q13. The affected homologous locus is on mouse chromosome 7C. The positional conservation and organization of genes including the imprinting pattern between mice and men implies similar physiological functions of this locus. Therefore, considerable efforts to recreate the pathogenesis of PWS have been accomplished in mouse models. We provide a summary of different mouse models that were generated for the analysis of PWS and discuss their impact on our current understanding of corresponding genes, their putative functions and the pathogenesis of PWS. Murine models of PWS unveiled the contribution of each affected gene to this multi-facetted disease, and also enabled the establishment of the minimal critical genomic region (PWScr) responsible for core symptoms, highlighting the importance of non-protein coding genes in the PWS locus. Although the underlying disease-causing mechanisms of PWS remain widely unresolved and existing mouse models do not fully capture the entire spectrum of the human PWS disorder, continuous improvements of genetically engineered mouse models have proven to be very powerful and valuable tools in PWS research.

Alzheimer’s disease (AD) is an age-related detrimental dementia. Amyloid beta peptides (Aβ) play a crucial role in the pathology of AD. In familial AD, Aβ is generated from the full-length amyloid precursor protein (APP) via dysregulated proteolytic processing. However, Aβ biogenesis in case of sporadic AD remains so far elusive. circRNAs are a class of transcripts, which are preferentially expressed in brain. Here, we identified a circRNA (circAβ-a) derived from the Aβ-coding region of the APP gene. circAβ-a is expressed in brains of AD and nondementia controls. With the aid of our recently established approach for analysis of circRNA functions, we demonstrated that circAβ-a is efficiently translated into a novel Aβ-related Aβ175 protein in living cells. Importantly, Aβ175 was further processed to Aβ peptides, the hallmark of AD. In summary, our analysis revealed alternative route of Aβ biogenesis. circAβ-a and its corresponding protein might represent novel therapeutic targets for AD treatment.

CircRNAs are expressed in many important biological processes. Studying their function requires an effective expression method. When we used intron-mediated enhancement (IME) to improve circRNA expression of mouse Rtn4 (Nogo, a key protein in Nogo-Rho pathways) circRNA as a test case, we achieved a 4-6-fold improvement compared to an existing method. We further developed this approach into a general circRNA expression vector pCircRNA-DMo. An unexpected feature of our approach is its ability to promote translation of circRNA into detectable amounts of proteins. Intriguingly, both monomer and multimer repeating peptides can be observed as a result of rolling circle translation of RTN4 circRNA. We also confirmed the presence of both peptide forms in human and mouse brains, highlighting the significance of circRNA translation in vivo . In summary, we demonstrate the significant advantage of IME in enhancing circRNA biogenesis and hence our vector offers a robust platform for exploring potential circRNA peptide-encoding functions.

Mechanisms that trigger Aβ production in sporadic Alzheimer’s disease are still obscure. We recently reported the expression of a human circular RNA (circAβ-a) encoded Aβ peptide precursor variant (Aβ175). Presently, we demonstrated that AAV9 virus-expressed circAβ-a gave rise to extensive extracellular Aβ plaque depositions and microglial activation in mouse brain; this recapitulates critical pathogenic hallmarks within a sporadic AD mouse model. Specifically developed antibodies detected robust endogenous Aβ175 expression in HEK293 cells and hNSC-derived human neurons, underscoring the potential of Aβ175 as a salient Aβ precursor. Furthermore, we detected high levels of Aβ175 oligomers in young-adult human brains. In intermediate and old-age human brain samples, accumulation of soluble Aβ175 pentamers was reduced and Aβ175 oligomers were components of most insoluble Aβ plaques in older human brain. We propose a causal relationship between human circAβ-a RNA expression, dysregulation of Aβ175 oligomer processing/aggregation and Aβ plaque accumulation in sporadic AD.