Fructose-2,6-bisphosphate restores TDP-43 pathology-driven genome repair deficiency in motor neuron diseases

Abstract TAR DNA-binding protein 43 (TDP-43) proteinopathy plays a critical role in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We recently reported that TDP-43 plays an essential role in DNA double-strand break (DSB) repair via the non-homologous end-joining (NHEJ) pathway. Here, we provide evidence that the brain of patients with ALS exhibit persistent DNA damage in the transcribed regions of the genome. While investigating the mechanistic basis, we found that the activity of polynucleotide kinase 3’-phosphatase (PNKP) was severely impaired in the nuclear extracts of patient brains and TDP-43-depleted cells. PNKP is a key player in DSB repair within the transcribed genome, where its 3’-phosphate termini processing activity is crucial for preventing persistent DNA strand breaks and neuronal death. The inactivation of PNKP was due to the reduced level of its interacting partner, phosphofructo-2-kinase fructose 2,6 bisphosphatase (PFKFB3), and its biosynthetic product, fructose-2,6-bisphosphate (F2,6BP), an allosteric modulator of glycolysis. Recently, we have demonstrated that F2,6BP acts as a positive modulator of PNKP activity in vivo. Furthermore, F2,6BP supplementation in cultured ALS patient-derived neural progenitor stem cells (NPSCs) reduced the toxic aggregation of polyubiquitinated TDP-43 and cytosolic pTDP-43 (S409/410). Notably, F2,6BP supplementation restored the PNKP activity in the nuclear extracts from autopsied ALS/FTD brain tissues and patient iPSC-derived NPSCs harboring TDP-43 mutations. Importantly, F2,6BP administration significantly restored the genome integrity and motor phenotypes in a Drosophila model of ALS-TDP-43. Collectively, these findings underscore the therapeutic potential of F2,6BP in TDP-43 pathology-associated motor neuron diseases. Competing Interest Statement The authors have declared no competing interest. Footnotes The manuscript has been updated to include additional experiments that further validate the key findings and strengthen the overall conclusions. We have also increased the number of biological samples analyzed to enhance statistical robustness and reproducibility of the results.