Contribution of defective mitophagy to the neurodegeneration in DNA repair-deficient disorders

Abstract

DNA repair is a prerequisite for life as we know it, and defects in DNA repair lead to accelerated aging. Xeroderma pigmentosum group A (XPA) is a classic DNA repair-deficient disorder with patients displaying sun sensitivity and cancer susceptibility. XPA patients also exhibit neurodegeneration, leading to cerebellar atrophy, neuropathy, and hearing loss, through a mechanism that has remained elusive. Using in silico, in vitro, and in vivo studies, we discovered defective mitophagy in XPA due to PARP1 hyperactivation and NAD⁺ (and thus, SIRT1) depletion. This leads to mitochondrial membrane hyper-polarization, PINK1 cleavage and defective mitophagy. This study underscores the importance of mitophagy in promoting a healthy pool of mitochondria and in preventing neurodegeneration and premature aging.

Mitochondrial autophagy (hereafter referred to as mitophagy) is an important cellular pathway facilitating the removal of damaged or unwanted mitochondria. The process entails targeting the autophagic machinery to engulf the mitochondrion. Specificity toward damaged mitochondria may be mediated through the sensing of the mitochondrial membrane potential. Upon damage, the mitochondrial membrane depolarizes leading to the accumulation of the mitophagic kinase PINK1 at the outer membrane. PINK1 facilitates the recruitment of PARK2/Parkin and the downstream activation of mitophagy. The process of mitophagy is essential for the degradation of damaged mitochondria, removal of mitochondria in developing erythroblasts, and possibly for the elimination of paternally derived mitochondria in fertilized oocytes. While this pathway was originally shown to be defective in familial cases of Parkinson disease caused by mutations in PINK1 and PARK2, we now show defects in this pathway in human cells and animal models of neurodegenerative DNA repair disorders.

DNA is constantly challenged by reactive chemical species, exogenous toxicants, and spontaneous decay of nucleotides. To cope with all of this, organisms have evolved pathways capable of repairing their DNA. A number of rare genetic diseases are caused by mutations in genes involved in DNA repair. Whereas patients suffering from these diseases show cancer predisposition and accelerated aging, a number of these disorders are also characterized by neurodegeneration. These include xeroderma pigmentosum group A (XPA), Cockayne syndrome, and ataxia-telangiectasia. Although DNA damage does accumulate in the brains of mouse models of these diseases, the levels of DNA damage seem too low to explain the severe phenotype observed in these disorders. Notably, the neurological phenotype closely mimics what is seen in primary mitochondrial disorders with cerebellar degeneration, neuropathy, and seizures. Defective mitophagy and mitochondrial dysfunction were recently shown in Cockayne syndrome and ataxia-telangiectasia, however, the pathogenesis was unknown. We find that cells from XPA patients appear to have a similar mitochondrial phenotype as seen in ataxia-telangiectasia and Cockayne syndrome. This includes increased mitochondrial content, increased mitochondrial superoxide production, and increased mitochondrial membrane potential. Importantly, the XPA protein was not present in mitochondria indicating that the mitochondrial defects may be secondary to a nuclear DNA repair defect.

Considering that the mitochondrial membrane potential regulates mitophagy and that the membrane potential is increased in cell lines from XPA, Cockayne syndrome, and ataxia-telangiectasia patients, we speculated that this may be the underlying cause of the defective mitophagy in these DNA repair disorders. Indeed, increased import and cleavage of PINK1 and decreased colocalization of PARK2 with mitochondria is observed in cell lines from XPA patients. Uncoupling proteins (UCPs) regulate mitochondrial membrane potential, and UCP2 appears to be decreased in various cell lines and in the cerebellum of a mouse model of XPA. In support of the idea that UCP2 may be regulating the membrane potential in the DNA repair-deficient disorders, overexpression of UCP2 in XPA-deficient cells rescues the mitochondrial phenotype.

To understand what might be leading to loss of UCP2 in the DNA repair disorders we further investigated the upstream regulation of this protein. UCPs are tightly regulated by the PPARGC1/PGC1 (peroxisome proliferator-activated receptor-gamma, coactivator 1) family of transcription factors. UCP2, in particular, is regulated by PPARGC1A that in turn is regulated by the NAD⁺-dependent enzyme SIRT1. Recent findings have suggested that persistent activation of a DNA damage response leads to loss of SIRT1 activity. This is mediated through the DNA damage recognizing protein poly[ADP-ribose] polymerase 1 (PARP1). Activation of PARP1 depletes NAD⁺ and attenuates SIRT1-activity. Since XPA, Cockayne syndrome, and ataxia-telangiectasia are known DNA repair factors, we hypothesized that PARP1 may be activated leading to depletion of a NAD⁺-SIRT1-PPARGC1A-UCP2 axis. Indeed, hyper-PARylation is found in XPA-deficient human cells, worms, and mice leading to lower NAD⁺ levels. Further, inhibition of PARP1 or replenishment of NAD⁺ rescues XPA-associated phenotypes in cells, worms and mice. Thus, in XPA deficiency, SIRT1 activity is depleted due to lower availability of NAD⁺ (Fig. 1). Importantly, this mechanism appears similar in Cockayne syndrome and ataxia-telangiectasia. This novel nuclear-mitophagic crosstalk may thus be involved in the pathogenesis of these neurodegenerative diseases. It therefore appears that NAD⁺ levels have a profound impact on mitochondrial function, and syndromes characterized by chronic DNA damage and PARP1 activation are at risk for mitochondrial alterations. Further, these findings may also imply that other diseases with defects in mitophagy, such as Parkinson disease, may be treatable with NAD⁺ precursors.

Figure 1. Persistent activation of a DNA damage response by PARP1 leads to loss of NAD⁺ and attenuation of SIRT1 and PPARGC1A. This in turn leads to decreased expression of UCP2 and increased mitochondrial membrane potential. PINK1 is increasingly cleaved leading to defective mitophagy.

In summary, it is our hypothesis that persistent DNA damage triggers activation of PARP1 and subsequently NAD⁺ insufficiency which induces mitochondrial dysfunction. Importantly, our findings reveal a previously unknown nuclear-mitochondrial crosstalk that is critical for maintenance of mitochondrial health, and further that DNA repair-deficient patients may benefit from NAD⁺ supplementation.

Fang EF, Scheibye-Knudsen M, Brace LE, Kassahun H, Sengupta T, Nilsen H, Mitchell JR, Croteau DL, Bohr VA. Defective Mitophagy in XPA via PARP-1 Hyperactivation and NAD(+)/SIRT1 Reduction. Cell. 2014;157:882–96. doi: 10.1016/j.cell.2014.03.026.