Rogelj, B. | Search bioRxiv

bioRxiv2025

Fast MAS NMR Spectroscopy Can Identify G-Quartets and Double-Stranded Structures in Aggregates Formed by GGGGCC RNA Repeats

The expansion of GGGGCC repeats within the C9orf72 gene has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In neurons from patients with expanded repeats, C9orf72 GGGGCC repeat RNA predominantly forms nuclear foci, and in vitro, repeat-containing RNA can self-aggregate. Two structural motifs have been proposed to provide the interstrand interactions that drive aggregation: G-quartet (G4) structures and double-strand interactions with GG mismatches. Using in vitro transcribed RNA with pathologically relevant number of repeats, we were able to form gel-like aggregates suitable for investingation using fast MAS NMR spectroscopy. This approach enabled us to characterize the dominant interstrand interactions within the RNA gels. Both Watson-Crick and Hoogsteen base pairs were identified in RNA gels formed by RNA with 48 GGGGCC repeats. Their relative abundance shifted upon reconstitution in the presence of different divalent cations or nuclear extracts, underscoring the dynamic equilibrium between G-quadruplex and duplex interactions in GGGGCC RNA aggregation.

biophysics↗

bioRxiv2023

Neuroimmune characterization of optineurin insufficiency mouse model during ageing

Optineurin is a multifunctional polyubiquitin-binding protein implicated in inflammatory signalling. Optineurin mutations are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerative diseases characterised by neuronal loss, neuroinflammation, and peripheral immune disbalance. However, the pathogenic role of optineurin mutations is unclear. We previously observed no phenotype in the unmanipulated young optineurin insufficiency mice (Optn470T), designed to mimic ALS/FTD-linked truncations deficient in polyubiquitin binding. The purpose of this study was to investigate whether ageing would trigger neurodegeneration. We performed a neuroimmune characterization of ageing wild-type (WT) and Optn470T mice. No motor or cognitive differences were detected between the genotypes. Neuropathological analyses demonstrated signs of ageing including lipofuscin accumulation and microglial activation. However, this was not worsened in Optn470T mice, and they did not exhibit TAR DNA-binding protein 43 (TDP-43) aggregation or neuronal loss. Spleen immunophenotyping uncovered T cell immunosenescence at two years but without notable differences between the WT and Optn470T mice. Conventional dendritic cells (cDC) and macrophages exhibited increased expression of activation markers in two-year-old Optn470T males but not females, although the numbers of innate immune cells were similar between genotypes. Altogether, a combination of optineurin insufficiency and ageing did not induce ALS/FTD-like neuropathology in mice.

immunology↗