ALS is an intractable disease that affects the motor cortex. It begins in middle age and leads to death. It is caused by the accumulation of a protein (TDP-43) in the cells. However, its amount is usually strictly controlled. Our group has shown that TDP-43 regulates the amount of TDP-43 by altering the splicing of its mRNA. Furthermore, we have demonstrated that ALS-like pathology can be induced by artificially damaging this splicing in mice. However, it was not clear why TDP-43 accumulates in the motor cortex with age, despite this regulatory mechanism.

DNA methylation varies with age and tissue. Methylation affects splicing. Therefore, we hypothesized that aging alters gene expression levels in a site-specific manner by changing DNA methylation.

To prove this hypothesis, we examined TDP-43, one of the causative proteins of ALS and FTD, and focused on the 3'UTR of TARDBP, which auto-regulates the TDP-43 levels via the alternative splicing ^1-4.

First, we used the dCas9-TET1 fusion system to selectively demethylate the CpG region of the alternative splicing-related site in the TARDBP 3'UTR. We found that the demethylation of this region inhibits the alternative splicing and increases the expression of TARDBP mRNA. Next, we analyzed the characteristic of the DNA methylation status of this autoregulatory region in each human brain region. We showed the demethylation of TARDBP 3'UTR and the amount of TRADBP mRNA increase with age only in the human motor cortex. Furthermore, we demonstrated that the DNA methylation age calculated from the methylation status of the TARDBP 3'UTR is associated with the age of onset in ALS patients, suggesting a link between DNA methylation of the TARDBP 3'UTR and the pathogenesis of ALS.

In the central nervous system, diverse mRNA isoforms produced via alternative splicing contribute to neuronal functions and pathological mechanisms ⁵. Identifying the DNA methylation sites that can regulate these alternative splicing may contribute to understanding the pathogenesis of ALS and other age-dependent neurodegenerative diseases. Our findings may explain system selectivity and the effects of aging, which are big mysteries in neurodegenerative diseases.

References

1. Ayala, Y. M. et al. TDP-43 regulates its mRNA levels through a negative feedback loop. EMBO J. 30, 277–288 (2011).
2. Polymenidou, M. et al. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat. Neurosci. 14, 459–468 (2011).
3. Koyama, A. et al. Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43. Nucleic Acids Res. 44, 5820–5836 (2016).
4. Sugai, A. et al. Non-genetically modified models exhibit TARDBP mRNA increase due to perturbed TDP-43 autoregulation. Neurobiol. Dis. 130, 104534 (2019).
5. Thalhammer, A., Jaudon, F. & Cingolani, L. A. Emerging Roles of Activity-Dependent Alternative Splicing in Homeostatic Plasticity. Front. Cell. Neurosci. 14, 1–9 (2020).