Huntington’s disease: CAG RNAs induce DNA damage via NUDT16, drug candidate effective in vivo

An international team, including researchers from the Chinese University of Hong Kong (CUHK), demonstrated that neuronal DNA damage in Huntington’s disease (HD) is caused by untranslatable expanded CAG RNAs, which downregulate the expression of Nudix hydrolase 16 (NUDT16) gene. A bisamidinium compound, DB213, was subsequently shown to rescue CAG repeat RNA-induced synapse loss in a murine model of HD.

HD belongs to a group of polyglutamine (polyQ) diseases caused by the expansion of CAG triplet repeat sequences in the disease loci and is associated with neuronal DNA damage, with multiple strand breaks. “Although the transcription of expanded CAG RNAs has been reported to cause DNA damage and induce apoptosis, how mutant CAG transcripts are involved in these processes is not entirely clear,” wrote the researchers.

“In the current study, we found that the expression level of NUDT16 is downregulated in polyQ disease models, which promotes DNA damage and apoptosis. Mechanistically, NUDT16 transcripts, which bear naturally occurring CUG sequences in their messenger RNA, form heteroduplexes with neurotoxic CAG RNAs, thus causing NUDT16 silencing,” reported the researchers. [PNAS 2021;118;e2022940118]

NUDT16 is a “housecleaning” enzyme specialized in eliminating hazardous (deoxy)inosine diphosphate from the nuclear nucleotide pool. [Nucleic Acids Res 2010;38:4834-4843] To confirm its downregulation in polyQ degeneration, NUDT16 transcript and protein levels were examined in the R6/2 HD transgenic mouse model. It was found that both were reduced in these animals’ brains, corresponding with previously reported atrophy in the cortex, striatum, and hippocampus. [J Neurosci 2012;32,6456-6467] In addition, when primary cortical neurons of wild-type mice were transfected with the non-translatable expanded CAG expression constructs, NUDT16 expression was reduced and compromised synaptic activities on both pre- and postsynaptic sides.

After transfecting neuroblastoma cells with different concentrations of the synthetic CAG RNA and observing that they induced cell death in a dose-dependent manner, the researchers strove to identify a drug candidate that would disrupt the CUG-CAG heteroduplex formation and thus suppress CAG RNA–induced DNA damage and apoptosis. “We previously reported a series of bisamidinium-based RNA groove binders as potential therapeutic agents for the treatment of myotonic dystrophy type I, identifying DB213 as a weak CUG RNA binder, with preferential binding to continuous CAG RNA sequences,” they wrote. [ACS Chem Biol 2011;6:857-864]

After a series of in vitro experiments and establishing DB213’s efficacy against CAG RNA toxicity in Drosophila models, the researchers investigated its efficacy in the murine R6/2 transgenic HD model. Untreated R6/2 mice displayed mild behavioural defects in weeks 7 and 9, with a significantly more severe phenotype observed at week 11, while a 4-week daily treatment of 25 mg/kg DB213 significantly rescued the locomotor defects in the intervention group by week 11. “At the molecular level, downregulated NUDT16 protein expression was restored, and the downstream DNA damage–induced apoptotic pathway was suppressed after DB213 treatment,” added the researchers.

“Our research helps understand how DB213 neutralizes RNA toxicity and relieves HD symptoms. When the compound was administered to primary neurons and diseased mice, we found DNA damage suppression and restoration of behavioural phenotypes. Furthermore, we established that DB213 can enter the brain when applied intranasally, highlighting its therapeutic applicability,” said Professor Edwin Chan of the School of Life Sciences at CUHK. “DB213 is ready to enter the preclinical stage. Other than HD, it can be utilized in other types of rare neurological diseases, such as spinocerebellar ataxias.”