Identification of pathological pathways centered on circRNA dysregulation in association with irreversible progression of Alzheimer's disease

Background: Circular RNAs (circRNAs) are highly stable regulators, often accumulated in mammalian brains and thought to serve as "memory molecules" that govern the long process of aging. Mounting evidence demonstrated circRNA dysregulation in the brains of Alzheimer's disease (AD) patients. However, whether and how circRNA dysregulation underlies AD progression remains unexplored.

Methods: We combined Poly(A)-tailing/RNase R digestion experimental approach with CARP, our published computational framework using pseudo-reference alignment for more sensitive and accurate circRNA detection to identify genome-wide circRNA dysregulation and their downstream pathways in the 5xFAD mouse cerebral cortex between 5 and 7 months of age, a critical window marks the transition from reversible to irreversible pathogenic progression. Dysregulated circRNAs and pathways associated with disease progression in 5xFAD cortex were systematically compared with circRNAs affected in postmortem subcortical areas of a large human AD cohort. A top-ranked circRNA conserved and commonly affected in AD patients and 5xFAD mice was depleted in cultured cells to examine AD-relevant molecular and cellular changes.

Results: We discovered genome-wide circRNA alterations specifically in 5xFAD cortex associated with AD progression, many of which are commonly dysregulated in the subcortical areas of AD patients. Among these circRNAs, circGigyf2 is highly conserved and showed the highest net reduction specifically in the 7-month 5xFAD cortex. CircGIGYF2 level in AD patients' cortices negatively correlated with dementia severity. Mechanistically, we found multiple AD-affected splicing factors that are essential for circGigyf2 biogenesis. Functionally, we identified and experimentally validated the conserved roles of circGigyf2 in sponging AD-relevant miRNAs and AD-associated RNA binding proteins (RBPs), including the cleavage and polyadenylation factor 6 (CPSF6). Moreover, circGigyf2 downregulation in AD promoted silencing activities of its sponged miRNAs and enhanced polyadenylation site processing efficiency of CPSF6 targets. Furthermore, circGigyf2 depletion in a mouse neuronal cell line dysregulated circGigyf2-miRNA and circGigyf2-CPSF6 axes and potentiated apoptotic responses upon insults, which strongly support the causative roles of circGigyf2 deficiency in AD neurodegeneration.

Conclusions: Together, our results unveiled brain circRNAs associated with irreversible disease progression in an AD mouse model that is also affected in AD patients and identified novel molecular mechanisms underlying the dysregulation of conserved circRNA pathways contributing to AD pathogenesis.