Identification of ROCK1 kinase as a critical regulator of Beclin1 mediated autophagy during metabolic stress

The Ser/Thr Rho kinase 1 (ROCK1) is known to play major roles in a wide range of cellular activities, including those involved in tumor metastasis and apoptosis. Here we identify an indispensable function of ROCK1 in metabolic stress-induced autophagy. Applying a proteomics approach, we characterize Beclin1, a proximal component of the PI(3)kinase class III lipid-kinase complex that induces autophagy, as an interacting partner of ROCK1. Upon nutrient deprivation, activated ROCK1 promotes autophagy by binding and phosphorylating Beclin1 at Thr119. This results in the specific dissociation of the Beclin1-Bcl-2 complex, without affecting the Beclin1-UVRAG interaction. Conversely, inhibition of ROCK1 activity increases Beclin1-Bcl-2 association, thus reducing nutritional stress-mediated autophagy. Genetic knockout of ROCK1 function in mice also leads to impaired autophagy as evidenced by reduced autophagosome formation. These results show that ROCK1 acts as a prominent upstream regulator of Beclin1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates T oxidative DNA damage induced aging

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF- 16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation cor- responded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep- 1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats

Spontaneous DNA damage to the nuclear genome promotes senescence, T redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Spontaneous DNA damage to the nuclear genome promotes senescence,redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Frataxin deficiency promotes endothelial senescence in pulmonary hypertension

The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S–containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell–derived endothelial cells from Friedreich’s ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency–dependent senescence drove vessel inflammation, remodeling, and PH, whereas pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes

A Chemoptogenetic Tool for Spatiotemporal Induction of Oxidative DNA Lesions <i>In Vivo</i>

Oxidative nuclear DNA damage increases in all tissues with age in multiple animal models, as well as in humans. However, the increase in DNA oxidation varies from tissue to tissue, suggesting that certain cells/tissues may be more vulnerable to DNA damage than others. The lack of a tool that can control dosage and spatiotemporal induction of oxidative DNA damage, which accumulates with age, has severely limited our ability to understand how DNA damage drives aging and age-related diseases. To overcome this, here we developed a chemoptogenetic tool that produces 8-oxoguanine (8-oxoG) at DNA in a whole organism, Caenorhabditis elegans. This tool uses di-iodinated malachite green (MG-2I) photosensitizer dye that generates singlet oxygen, 1O2, upon fluorogen activating peptide (FAP) binding and excitation with far-red light. Using our chemoptogenetic tool, we are able to control generation of singlet oxygen ubiquitously or in a tissue-specific manner, including in neurons and muscle cells. To induce oxidative DNA damage, we targeted our chemoptogenetic tool to histone, his-72, that is expressed in all cell types. Our results show that a single exposure to dye and light is able to induce DNA damage, promote embryonic lethality, lead to developmental delay, and significantly reduce lifespan. Our chemoptogenetic tool will now allow us to assess the cell autonomous versus non-cell autonomous role of DNA damage in aging, at an organismal level

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates oxidative DNA damage induced aging

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself. Keywords: DNA damage, Aging, Reactive oxygen species, Oxidative stress, DAF-16/FOXO3A, Stress resistanc