8-Oxoguanine (8-oxoG), a common DNA lesion caused by reactive oxygen species, is associated with carcinogenesis and neurodegeneration. Although the mechanism by which 8-oxoG causes carcinogenesis is well understood, the mechanism by which it causes neurodegeneration is unknown. Here, we report that neurodegeneration is triggered by MUTYH-mediated excision repair of 8-oxoG-paired adenine. Mutant mice lacking 8-oxo-2′-deoxyguanosine triphosphate-depleting (8-oxo-dGTP-depleting) MTH1 and/or 8-oxoG-excising OGG1 exhibited severe striatal neurodegeneration, whereas mutant mice lacking MUTYH or OGG1/MUTYH were resistant to neurodegeneration under conditions of oxidative stress. These results indicate that OGG1 and MTH1 are protective, while MUTYH promotes neurodegeneration. We observed that 8-oxoG accumulated in the mitochondrial DNA of neurons and caused calpain-dependent neuronal loss, while delayed nuclear accumulation of 8-oxoG in microglia resulted in PARP-dependent activation of apoptosis-inducing factor and exacerbated microgliosis. These results revealed that neurodegeneration is a complex process caused by 8-oxoG accumulation in the genomes of neurons and microglia. Different signaling pathways were triggered by the accumulation of single-strand breaks in each type of DNA generated during base excision repair initiated by MUTYH, suggesting that suppression of MUTYH may protect the brain under conditions of oxidative stress.
Oxidative stress and mitochondrial dysfunction are implicated in aging-related neurodegenerative disorders. 8-Oxoguanine (8-oxoG), a common oxidised base lesion, is often highly accumulated in brains from patients with neurodegenerative disorders. MTH1 hydrolyses 8-oxo-2′-deoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and pyrophosphate in nucleotide pools, while OGG1 excises 8-oxoG paired with cytosine in DNA, thereby minimising the accumulation of 8-oxoG in DNA. Mth1/Ogg1-double knockout (TO-DKO) mice are highly susceptible to neurodegeneration under oxidative conditions and show increased accumulation of 8-oxoG in mitochondrial DNA (mtDNA) in neurons, suggesting that 8-oxoG accumulation in mtDNA causes mitochondrial dysfunction. Here, we evaluated the contribution of MTH1 and OGG1 to the prevention of mitochondrial dysfunction during neuritogenesis in vitro. We isolated cortical neurons from adult wild-type and TO-DKO mice and maintained them with or without antioxidants for 2 to 5 days and then examined neuritogenesis. In the presence of antioxidants, both TO-DKO and wild-type neurons exhibited efficient neurite extension and arborisation. However, in the absence of antioxidants, the accumulation of 8-oxoG in mtDNA of TO-DKO neurons was increased resulting in mitochondrial dysfunction. Cells also exhibited poor neurite outgrowth with decreased complexity of neuritic arborisation, indicating that MTH1 and OGG1 are essential for neuritogenesis under oxidative conditions.
Accumulation of oxidized bases such as 8-oxoguanine in either nuclear or mitochondrial DNA triggers various cellular dysfunctions including mutagenesis, and programmed cell death or senescence. Recent studies have revealed that oxidized nucleoside triphosphates such as 8-oxo-dGTP in the nucleotide pool are the main source of oxidized bases accumulating in the DNA of cells under oxidative stress. To counteract such deleterious effects of nucleotide pool damage, mammalian cells possess MutT homolog-1 (MTH1) with oxidized purine nucleoside triphosphatase and related enzymes, thus minimizing the accumulation of oxidized bases in cellular DNA. Depletion or increased expression of the MTH1 protein have revealed its significant roles in avoiding programmed cell death or senescence as well as mutagenesis, and accumulating evidences indicate that MTH1 is involved in suppression of degenerative disorders such as neurodegeneration.
Neonates with congenital diaphragmatic hernia often require surgical defect closure with a patch. Alternatives to native diaphragmatic tissue are critically needed for this paediatric surgery. The clinical efficacy of mesh patches is limited by complications associated with residual foreign material and by hernia recurrence. In this study, we used a novel bio-3D printer method to generate large scaffold-free tissue patches composed of human cells. The resulting large tissue constructs had high elasticity and strength. Cellular patches were transplanted into rats with surgically created diaphragmatic defects. Rats survived for over 710 days after implantation of tissue constructs. CT confirmed complete tissue integration of the grafts during rat growth. Histology revealed regeneration of muscle structure, neovascularization, and neuronal networks within the reconstructed diaphragms. Our results demonstrate that created cellular patches are a highly safe and effective therapeutic strategy for repairing diaphragmatic defects, and thus pave the way for a clinical trial.
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