Mechanistic study of C9orf72 G4C2 G-quadruplex in the pathogenesis of ALS/FTD

Abstract

Guanine rich DNA or RNA under physiological conditions, can fold to form secondary DNA/RNA G-quadruplex (G4) structures that are stabilized by stacking of two or more G-tetrads coordinated with a cation. Specifically, the hexanucleotide repeat expansion (HRE) of G4C2 in the C9orf72 can form G4 structures which induce toxic effects that accumulate into ALS/FTD.

The structure of the G4C2 2 repeats (G4C2 2R) has been previously solved by our lab and the structure of G4C2 4 repeats (G4C2 4R) was solved in this study. Initially, the structure elucidation of crystals of the G4C2 4R was not possible due to the phase problem of the crystal. A new crystal with heavy atom replacement with barium in place of potassium as the cation was used to solve the issue allowing for structural elucidation. The d(G4C2)₂ structure was found to form an eight-layer parallel tetrameric G-quadraplex, in which two d(G4C2)₂ form a parallel dimeric G-quadraplex and interact via 5’ to 5’ conformation with another dimeric G-quadraplex to form a tetramer. This research reveals key structural conformations that G4C2 repeats adopt that can help in optimization of small molecules designed to treat ALS/FTD.

Considering that a potential disease mechanism of Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) includes sequestering of RNA binding proteins, we explored the binding mechanism of RNA recognition motif (RRM) of the Heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins, which has been implicated ALS/FTD. In this study, we demonstrate hnRNPH 1 is able to specifically interact with C9orf72 HRE G4 through RRM domain and characterized the interaction. The interaction of HNRNPs with G4 indicates a possible alternative functions of HNRNPs outside their conventional RNA processing roles and provide an insight into C9orf72 HRE G4 mediated pathogenesis of ALS/FTD.

Notably, we discovered three small molecules extracted from marine products, that specially targeting C9orf72 HRE G4, can ameliorate some phenotypes associated with C9 ALS/FTD. The interaction of the small molecules with G4C2 repeats were characterized and through NMR titration the potential for the molecules to interfere/compete with G4 binding of hnRNP H1 was investigated. These small compounds provide promising therapeutic agents for treating C9 ALS/FTD.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Guang ZHU (Supervisor)