Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo.

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.

Poly(ADP-Ribose) Polymerase-1 Lacking Enzymatic Activity Is Not Compatible with Mouse Development.

Poly(ADP-ribose) polymerase-1 (PARP1) binds DNA lesions to catalyse poly(ADP-ribosyl)ation (PARylation) using NAD+ as a substrate. PARP1 plays multiple roles in cellular activities, including DNA repair, transcription, cell death, and chromatin remodelling. However, whether these functions are governed by the enzymatic activity or scaffolding function of PARP1 remains elusive. In this study, we inactivated in mice the enzymatic activity of PARP1 by truncating its C-terminus that is essential for ART catalysis (PARP1ΔC/ΔC, designated as PARP1-ΔC). The mutation caused embryonic lethality between embryonic day E8.5 and E13.5, in stark contrast to PARP1 complete knockout (PARP1-/-) mice, which are viable. Embryonic stem (ES) cell lines can be derived from PARP1ΔC/ΔC blastocysts, and these mutant ES cells can differentiate into all three germ layers, yet, with a high degree of cystic structures, indicating defects in epithelial cells. Intriguingly, PARP1-ΔC protein is expressed at very low levels compared to its full-length counterpart, suggesting a selective advantage for cell survival. Noticeably, PARP2 is particularly elevated and permanently present at the chromatin in PARP1-ΔC cells, indicating an engagement of PARP2 by non-enzymatic PARP1 protein at the chromatin. Surprisingly, the introduction of PARP1-ΔC mutation in adult mice did not impair their viability; yet, these mutant mice are hypersensitive to alkylating agents, similar to PARP1-/- mutant mice. Our study demonstrates that the catalytically inactive mutant of PARP1 causes the developmental block, plausibly involving PARP2 trapping.

Cell Type-Specific Role of RNA Nuclease SMG6 in Neurogenesis.

SMG6 is an endonuclease, which cleaves mRNAs during nonsense-mediated mRNA decay (NMD), thereby regulating gene expression and controling mRNA quality. SMG6 has been shown as a differentiation license factor of totipotent embryonic stem cells. To investigate whether it controls the differentiation of lineage-specific pluripotent progenitor cells, we inactivated Smg6 in murine embryonic neural stem cells. Nestin-Cre-mediated deletion of Smg6 in mouse neuroprogenitor cells (NPCs) caused perinatal lethality. Mutant mice brains showed normal structure at E14.5 but great reduction of the cortical NPCs and late-born cortical neurons during later stages of neurogenesis (i.e., E18.5). Smg6 inactivation led to dramatic cell death in ganglionic eminence (GE) and a reduction of interneurons at E14.5. Interestingly, neurosphere assays showed self-renewal defects specifically in interneuron progenitors but not in cortical NPCs. RT-qPCR analysis revealed that the interneuron differentiation regulators Dlx1 and Dlx2 were reduced after Smg6 deletion. Intriguingly, when Smg6 was deleted specifically in cortical and hippocampal progenitors, the mutant mice were viable and showed normal size and architecture of the cortex at E18.5. Thus, SMG6 regulates cell fate in a cell type-specific manner and is more important for neuroprogenitors originating from the GE than for progenitors from the cortex.

ATR regulates neuronal activity by modulating presynaptic firing.

Ataxia Telangiectasia and Rad3-related (ATR) protein, as a key DNA damage response (DDR) regulator, plays an essential function in response to replication stress and controls cell viability. Hypomorphic mutations of ATR cause the human ATR-Seckel syndrome, characterized by microcephaly and intellectual disability, which however suggests a yet unknown role for ATR in non-dividing cells. Here we show that ATR deletion in postmitotic neurons does not compromise brain development and formation; rather it enhances intrinsic neuronal activity resulting in aberrant firing and an increased epileptiform activity, which increases the susceptibility of ataxia and epilepsy in mice. ATR deleted neurons exhibit hyper-excitability, associated with changes in action potential conformation and presynaptic vesicle accumulation, independent of DDR signaling. Mechanistically, ATR interacts with synaptotagmin 2 (SYT2) and, without ATR, SYT2 is highly upregulated and aberrantly translocated to excitatory neurons in the hippocampus, thereby conferring a hyper-excitability. This study identifies a physiological function of ATR, beyond its DDR role, in regulating neuronal activity.

TRIP6 functions in brain ciliogenesis.

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.

HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration.

Brain homeostasis is regulated by the viability and functionality of neurons. HAT (histone acetyltransferase) and HDAC (histone deacetylase) inhibitors have been applied to treat neurological deficits in humans; yet, the epigenetic regulation in neurodegeneration remains elusive. Mutations of HAT cofactor TRRAP (transformation/transcription domain-associated protein) cause human neuropathies, including psychosis, intellectual disability, autism, and epilepsy, with unknown mechanism. Here we show that Trrap deletion in Purkinje neurons results in neurodegeneration of old mice. Integrated transcriptomics, epigenomics, and proteomics reveal that TRRAP via SP1 conducts a conserved transcriptomic program. TRRAP is required for SP1 binding at the promoter proximity of target genes, especially microtubule dynamics. The ectopic expression of Stathmin3/4 ameliorates defects of TRRAP-deficient neurons, indicating that the microtubule dynamics is particularly vulnerable to the action of SP1 activity. This study unravels a network linking three well-known, but up-to-date unconnected, signaling pathways, namely TRRAP, HAT, and SP1 with microtubule dynamics, in neuroprotection.

ATRIP protects progenitor cells against DNA damage in vivo.

The maintenance of genomic stability during the cell cycle of progenitor cells is essential for the faithful transmission of genetic information. Mutations in genes that ensure genome stability lead to human developmental syndromes. Mutations in Ataxia Telangiectasia and Rad3-related (ATR) or in ATR-interacting protein (ATRIP) lead to Seckel syndrome, which is characterized by developmental malformations and short life expectancy. While the roles of ATR in replicative stress response and chromosomal segregation are well established, it is unknown how ATRIP contributes to maintaining genomic stability in progenitor cells in vivo. Here, we generated the first mouse model to investigate ATRIP function. Conditional inactivation of Atrip in progenitor cells of the CNS and eye led to microcephaly, microphthalmia and postnatal lethality. To understand the mechanisms underlying these malformations, we used lens progenitor cells as a model and found that ATRIP loss promotes replicative stress and TP53-dependent cell death. Trp53 inactivation in Atrip-deficient progenitor cells rescued apoptosis, but increased mitotic DNA damage and mitotic defects. Our findings demonstrate an essential role of ATRIP in preventing DNA damage accumulation during unchallenged replication.

Progenitor death drives retinal dysplasia and neuronal degeneration in a mouse model of ATRIP-Seckel syndrome.

Seckel syndrome is a type of microcephalic primordial dwarfism (MPD) that is characterized by growth retardation and neurodevelopmental defects, including reports of retinopathy. Mutations in key mediators of the replication stress response, the mutually dependent partners ATR and ATRIP, are among the known causes of Seckel syndrome. However, it remains unclear how their deficiency disrupts the development and function of the central nervous system (CNS). Here, we investigated the cellular and molecular consequences of ATRIP deficiency in different cell populations of the developing murine neural retina. We discovered that conditional inactivation of Atrip in photoreceptor neurons did not affect their survival or function. In contrast, Atrip deficiency in retinal progenitor cells (RPCs) led to severe lamination defects followed by secondary photoreceptor degeneration and loss of vision. Furthermore, we showed that RPCs lacking functional ATRIP exhibited higher levels of replicative stress and accumulated endogenous DNA damage that was accompanied by stabilization of TRP53. Notably, inactivation of Trp53 prevented apoptosis of Atrip-deficient progenitor cells and was sufficient to rescue retinal dysplasia, neurodegeneration and loss of vision. Together, these results reveal an essential role of ATRIP-mediated replication stress response in CNS development and suggest that the TRP53-mediated apoptosis of progenitor cells might contribute to retinal malformations in Seckel syndrome and other MPD disorders.This article has an associated First Person interview with the first author of the paper.

Laser microbeams and optical tweezers in ageing research.

We show how a technique developed within the framework of physics and physical chemistry-in a true interdisciplinary approach-can answer questions in life sciences that are not solvable by using other techniques. Herein, we focus on blood-pressure regulation and DNA repair in ageing studies. Laser microbeams and optical tweezers are now established tools in many fields of science, particularly in the life sciences. A short glimpse is given on the wide field of non-age-research applications in life sciences. Then, optical tweezers are used to show that exerting a vertical pressure on cells representing the inner lining of blood vessels results in bursts of NO liberation concomitant with large changes in cell morphology. Repeated treatment of such human umbilical vein endothelial cells (HUVEC) results in stiffening, a hallmark of manifest high blood pressure, a disease primarily of the elderly. As a second application in ageing research, a laser microbeam is used to induce, with high spatial and temporal resolution, DNA damages in the nuclei of U2OS human osteosarcoma cells. A pairwise study of the recruitment kinetics of different DNA repair proteins reveals that DNA repair starts with non-homologous end joining (NHEJ), a repair pathway, and may only after several minutes switch to the error-free homologous recombination repair (HRR) pathway. Since DNA damages-when incorrectly repaired-accumulate with time, laser microbeams are becoming well-used tools in ageing research.

A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.

In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.

RINT1 functions as a multitasking protein at the crossroads between genomic stability, ER homeostasis, and autophagy.

RINT1 was first identified as an RAD50-interacting protein and its function was therefore linked to the maintenance of genomic stability. It was also shown that RINT1 was a key player in ER-Golgi trafficking as a member of an ER tethering complex interacting with STX18. However, due to early embryonic lethality of rint1-null mice, the in vivo functions of RINT1 remained for the most part elusive. We recently described the consequences of Rint1 inactivation in various neuronal cells of the central nervous system. We observed that lack of RINT1 in vivo triggers genomic instability and ER stress leading to depletion of the neural progenitor pool and neurodegeneration. Surprisingly, we also observed inhibition of autophagy in RINT1-deficient neurons, indicating an involvement of RINT1 in the regulation of neuronal autophagy. Here, we summarize our main RINT1 findings and discuss its putative roles in autophagy.

Epidermal Nbn deletion causes premature hair loss and a phenotype resembling psoriasiform dermatitis.

Nijmegen Breakage Syndrome is a disease caused by NBN mutations. Here, we report a novel function of Nbn in skin homeostasis. We found that Nbn deficiency in hair follicle (HF) progenitors promoted increased DNA damage signaling, stimulating p16Ink4a up-regulation, Trp53 stabilization and cytokines secretion leading to HF-growth arrest and hair loss. At later stages, the basal keratinocytes layer exhibited also enhanced DNA damage response but in contrast to the one in HF progenitor was not associated with pro-inflammatory cytokines expression, but rather increased proliferation, lack of differentiation and immune response resembling psoriasiform dermatitis. Simultaneous Nbn and Trp53 inactivation significantly exacerbated this phenotype, due to the lack of inhibition of pro-inflammatory cytokines secretion by Trp53. Altogether, we demonstrated novel functions of Nbn in HF maintenance and prevention of skin inflammation and we provide a mechanistic explanation that links cell intrinsic DNA maintenance with large scale morphological tissue alterations.

The tumor suppressor PML specifically accumulates at RPA/Rad51-containing DNA damage repair foci but is nonessential for DNA damage-induced fibroblast senescence.

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.

Nbn and atm cooperate in a tissue and developmental stage-specific manner to prevent double strand breaks and apoptosis in developing brain and eye.

Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in NBN or ATM result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show in vivo consequences of conditional inactivation of both genes in neural stem/progenitor cells using Nestin-Cre mice. Genetic inactivation of Atm in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with Nbn deletion was prevented by simultaneous inactivation of Atm in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation.

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

T cell activation is driven by an ADP-dependent glucokinase linking enhanced glycolysis with mitochondrial reactive oxygen species generation.

Mitochondria-originating reactive oxygen species (ROS) control T cell receptor (TCR)-induced gene expression. Here, we show that TCR-triggered activation of ADP-dependent glucokinase (ADPGK), an alternative, glycolytic enzyme typical for Archaea, mediates generation of the oxidative signal. We also show that ADPGK is localized in the endoplasmic reticulum and suggest that its active site protrudes toward the cytosol. The ADPGK-driven increase in glycolytic metabolism coincides with TCR-induced glucose uptake, downregulation of mitochondrial respiration, and deviation of glycolysis toward mitochondrial glycerol-3-phosphate dehydrogenase(GPD) shuttle; i.e., a metabolic shift to aerobic glycolysis similar to the Warburg effect. The activation of respiratory-chain-associated GPD2 results in hyperreduction of ubiquinone and ROS release from mitochondria. In parallel, mitochondrial bioenergetics and ultrastructure are altered. Downregulation of ADPGK or GPD2 abundance inhibits oxidative signal generation and induction of NF-κB-dependent gene expression, whereas overexpression of ADPGK potentiates them.

The excited-state chemistry of protochlorophyllide a: a time-resolved fluorescence study.

The excited-state processes of protochlorophyllide a, the precursor of chlorophyll a in chlorophyll biosynthesis, are studied using picosecond time-resolved fluorescence spectroscopy. Following excitation into the Soret band, two distinct fluorescence components, with emission maxima at 640 and 647 nm, are observed. The 640 nm emitting component appears within the time resolution of the experiment and then decays with a time constant of 27 ps. In contrast, the 647 nm emitting component is built up with a 3.5 ps rise time and undergoes a subsequent decay with a time constant of 3.5 ns. The 3.5 ps rise kinetics are attributed to relaxations in the electronically excited state preceding the nanosecond fluorescence, which is ascribed to emission out of the thermally equilibrated S(1) state. The 27 ps fluorescence, which appears within the experimental response of the streak camera, is suggested to originate from a second minimum on the excited-state potential-energy surface. The population of the secondary excited state is suggested to reflect a very fast motion out of the Franck-Condon region along a reaction coordinate different from the one connecting the Franck-Condon region with the S(1) potential-energy minimum. The 27 ps-component is an emissive intermediate on the reactive excited-state pathway, as its decay yields the intermediate photoproduct, which has been identified previously (J. Phys. Chem. B 2006, 110, 4399-4406). No emission of the photoproduct is observed. The results of the time-resolved fluorescence study allow a detailed spectral characterization of the emission of the excited states in protochlorophyllide a, and the refinement of the kinetic model deduced from ultrafast absorption measurements.

Readout of protein microarrays using intrinsic time resolved UV fluorescence for label-free detection.

Detecting protein-protein interactions other than those of antibody-antigen pairs still represents a demanding and tedious task. In the present work, a novel method as an alternative to current molecular biology-based detection procedures is established. It solely relies on the change of fluorescence decay times of the protein's intrinsic fluorophores tryptophan and tyrosine due to protein-protein interaction. Unlike previously utilized related methods, no labelling of the binding partners is required. This opens the possibility to detect proteins and their natural interactions without perturbation due to chemical alteration. The technique uses immobilization of one of the protein partners onto solid supports, which allows performance of protein binding studies in the microarray format. Fluorescence lifetime experiments of proteins in their different binding states have been applied to protease/protease-substrate pairs, as well as to the tubulin/kinesin system. Different binding behavior of proteins in solution towards protein partners immobilized on protein microarrays was detected with regard to binding specificity and protein amount. This label-free method for analyzing protein microarrays offers broad applicability ranging from principal investigations of protein interactions to applications in molecular biology and medicine.