NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies.

Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.

Chemical screening identifies ATM as a target for alleviating senescence.

Senescence, defined as irreversible cell-cycle arrest, is the main driving force of aging and age-related diseases. Here, we performed high-throughput screening to identify compounds that alleviate senescence and identified the ataxia telangiectasia mutated (ATM) inhibitor KU-60019 as an effective agent. To elucidate the mechanism underlying ATM's role in senescence, we performed a yeast two-hybrid screen and found that ATM interacted with the vacuolar ATPase V1 subunits ATP6V1E1 and ATP6V1G1. Specifically, ATM decreased E-G dimerization through direct phosphorylation of ATP6V1G1. Attenuation of ATM activity restored the dimerization, thus consequently facilitating assembly of the V1 and V0 domains with concomitant reacidification of the lysosome. In turn, this reacidification induced the functional recovery of the lysosome/autophagy system and was coupled with mitochondrial functional recovery and metabolic reprogramming. Together, our data reveal a new mechanism through which senescence is controlled by the lysosomal-mitochondrial axis, whose function is modulated by the fine-tuning of ATM activity.

Disruption of nucleocytoplasmic trafficking as a cellular senescence driver.

Senescent cells exhibit a reduced response to intrinsic and extrinsic stimuli. This diminished reaction may be explained by the disrupted transmission of nuclear signals. However, this hypothesis requires more evidence before it can be accepted as a mechanism of cellular senescence. A proteomic analysis of the cytoplasmic and nuclear fractions obtained from young and senescent cells revealed disruption of nucleocytoplasmic trafficking (NCT) as an essential feature of replicative senescence (RS) at the global level. Blocking NCT either chemically or genetically induced the acquisition of an RS-like senescence phenotype, named nuclear barrier-induced senescence (NBIS). A transcriptome analysis revealed that, among various types of cellular senescence, NBIS exhibited a gene expression pattern most similar to that of RS. Core proteomic and transcriptomic patterns common to both RS and NBIS included upregulation of the endocytosis-lysosome network and downregulation of NCT in senescent cells, patterns also observed in an aging yeast model. These results imply coordinated aging-dependent reduction in the transmission of extrinsic signals to the nucleus and in the nucleus-to-cytoplasm supply of proteins/RNAs. We further showed that the aging-associated decrease in Sp1 transcription factor expression was critical for the downregulation of NCT. Our results suggest that NBIS is a modality of cellular senescence that may represent the nature of physiological aging in eukaryotes.

Chemical screening identifies ROCK as a target for recovering mitochondrial function in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high-throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho-associated protein kinase (ROCK) inhibitor (Y-27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two-hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y-27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y-27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double-strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation.