Chakraborty Anirban, Mandal Santi M, Mankevich Mikita, Mani Rajam S, Shahabi Shandy, Biswas Tapan, Kodavati Manohar, Sreenivasmurthy Sravan Gopalkrishnashetty, Tapryal Nisha, Krishnan Balaji, Hegde Muralidhar L, Weinfeld Michael, Ghosh Gourisankar, Hazra Tapas
Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, 77555, USA.
Department of Chemistry and Biochemistry, University of California San Diego, LA Jolla, California 92093, USA.
bioRxiv. 2025 Jun 30:2024.11.04.621834. doi: 10.1101/2024.11.04.621834.
Several reports have indicated that impaired mitochondrial function contributes to the development and progression of Huntington's disease (HD). Mitochondrial genome damage, particularly DNA strand breaks, is a potential cause for its compromised functionality. Here we show that the activity of polynucleotide kinase 3'-phosphatase (PNKP), a critical DNA end-processing enzyme, is significantly decreased in the mitochondrial extract of HD patients' brains due to a lower level of fructose-2,6 bisphosphate (F2,6BP), a biosynthetic product of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Such decrease in PNKP activity leads to persistent DNA strand breaks that are refractory to subsequent steps for repair completion. Both PFKFB3 and F2,6BP, an allosteric modulator of glycolysis, are also present in the mitochondria and PFKFB3 is part of a mitochondrial DNA repair complex containing HTT, PNKP, DNA Pol γ (POLG) and Lig IIIa. Notably, PNKP binds F2,6BP (Kd= 525±25 nM) and utilizes it as a cofactor. The levels of both F2,6BP and PFKFB3 are significantly decreased in the mitochondrial extract of HD mouse striatal neuronal cells and patients' brain. Activity of PNKP is thus severely decreased in the mitochondrial extract; however, addition of F2,6BP restored its activity. Moreover, supplementation of F2,6BP in HD cells restored PFKFB3 level, mitochondrial genome integrity and partially restored mitochondrial membrane potential, mitochondrial respiration and prevented pathogenic aggregate formation. We also observed that supplementation with F2,6BP restored mitochondrial genome integrity in an HD model. Our findings, therefore, suggest that F2,6BP-mediated restoration of PNKP activity could have a profound impact in ameliorating neurodegenerative symptoms in HD.
多项报告表明，线粒体功能受损会导致亨廷顿舞蹈病（HD）的发生和发展。线粒体基因组损伤，尤其是DNA链断裂，是其功能受损的一个潜在原因。在此我们发现，多核苷酸激酶3'-磷酸酶（PNKP）是一种关键的DNA末端加工酶，在HD患者脑线粒体提取物中的活性显著降低，这是由于6-磷酸果糖-2-激酶果糖-2,6-二磷酸酶3（PFKFB3）的生物合成产物果糖-2,6-二磷酸（F2,6BP）水平较低所致。PNKP活性的这种降低会导致DNA链持续断裂，这些断裂对后续的修复完成步骤具有抗性。PFKFB3和F2,6BP（糖酵解的变构调节剂）也存在于线粒体中，并且PFKFB3是包含HTT、PNKP、DNA聚合酶γ（POLG）和连接酶IIIa的线粒体DNA修复复合物的一部分。值得注意的是，PNKP结合F2,6BP（解离常数Kd = 525±25 nM）并将其用作辅因子。在HD小鼠纹状体神经元细胞和患者脑的线粒体提取物中，F2,6BP和PFKFB3的水平均显著降低。因此，线粒体提取物中PNKP的活性严重降低；然而，添加F2,6BP可恢复其活性。此外，在HD细胞中补充F2,6BP可恢复PFKFB3水平、线粒体基因组完整性，并部分恢复线粒体膜电位、线粒体呼吸，并防止致病性聚集体形成。我们还观察到，在HD模型中补充F2,6BP可恢复线粒体基因组完整性。因此，我们的研究结果表明，F2,6BP介导的PNKP活性恢复可能对改善HD的神经退行性症状具有深远影响。
