Bicentre, randomized, parallel-arm, sham-controlled trial of transcranial direct-current stimulation (tDCS) in the treatment of palliative care patients with refractory cancer pain.

BACKGROUND: Pain is a common symptom in palliative care cancer patients and is often insufficiently relieved. In recent years, transcranial direct-current stimulation (tDCS) of the motor cortex has been shown to be effective to treat chronic pain, essentially neuropathic pain. We propose to test the efficacy of tDCS in patients experiencing cancer pain in the palliative care setting. METHOD/DESIGN: This article describes the protocol of a bicentre, randomized, parallel-arm, sham-controlled clinical trial evaluating tDCS in the treatment of palliative care patients with refractory cancer pain. Seventy patients between the ages of 18 and 80 years experiencing refractory pain with a pain score of 4/10 on a numerical rating scale (NRS) ranging from 0 to 10 will be enrolled in this trial. The main exclusion criteria are patients unable to fill in the various rating scales and life expectancy less than 3 weeks. Treatment consists of 5 consecutive tDCS sessions targeting the motor cortex (one daily session for 5 days) on the contralateral side to the pain. After randomization (1:1 ratio), 35 patients will receive active stimulation and 35 patients will receive sham stimulation. The primary endpoint is the NRS score and the primary objective is a significant improvement of this score between the baseline score recorded between D-3 and D-1 and the score recorded 4 days after stopping treatment (D8). The secondary objectives are to evaluate whether this improvement is maintained 16 days after stopping treatment (D21) and whether the following scores are improved on D14 and D21: Brief Pain Inventory, Edmonton Symptom Assessment System, Hospital Anxiety and Depression scale, State-Trait Anxiety Inventory and Medication Quantification Scale. DISCUSSION: Positive results of this trial would indicate that tDCS can improve pain and quality of life of cancer patients in the palliative care setting. Reduction of analgesic consumption and improvement of activities of daily living should allow many patients to return home with a decreased workload for caregivers.

Manganese Stress Tolerance Depends on Yap1 and Stress-Activated MAP Kinases.

Understanding which intracellular signaling pathways are activated by manganese stress is crucial to decipher how metal overload compromise cellular integrity. Here, we unveil a role for oxidative and cell wall stress signaling in the response to manganese stress in yeast. We find that the oxidative stress transcription factor Yap1 protects cells against manganese toxicity. Conversely, extracellular manganese addition causes a rapid decay in Yap1 protein levels. In addition, manganese stress activates the MAPKs Hog1 and Slt2 (Mpk1) and leads to an up-regulation of the Slt2 downstream transcription factor target Rlm1. Importantly, Yap1 and Slt2 are both required to protect cells from oxidative stress in mutants impaired in manganese detoxification. Under such circumstances, Slt2 activation is enhanced upon Yap1 depletion suggesting an interplay between different stress signaling nodes to optimize cellular stress responses and manganese tolerance.

Manganese is a physiologically relevant TORC1 activator in yeast and mammals.

The essential biometal manganese (Mn) serves as a cofactor for several enzymes that are crucial for the prevention of human diseases. Whether intracellular Mn levels may be sensed and modulate intracellular signaling events has so far remained largely unexplored. The highly conserved target of rapamycin complex 1 (TORC1, mTORC1 in mammals) protein kinase requires divalent metal cofactors such as magnesium (Mg2+) to phosphorylate effectors as part of a homeostatic process that coordinates cell growth and metabolism with nutrient and/or growth factor availability. Here, our genetic approaches reveal that TORC1 activity is stimulated in vivo by elevated cytoplasmic Mn levels, which can be induced by loss of the Golgi-resident Mn2+ transporter Pmr1 and which depend on the natural resistance-associated macrophage protein (NRAMP) metal ion transporters Smf1 and Smf2. Accordingly, genetic interventions that increase cytoplasmic Mn2+ levels antagonize the effects of rapamycin in triggering autophagy, mitophagy, and Rtg1-Rtg3-dependent mitochondrion-to-nucleus retrograde signaling. Surprisingly, our in vitro protein kinase assays uncovered that Mn2+ activates TORC1 substantially better than Mg2+, which is primarily due to its ability to lower the Km for ATP, thereby allowing more efficient ATP coordination in the catalytic cleft of TORC1. These findings, therefore, provide both a mechanism to explain our genetic observations in yeast and a rationale for how fluctuations in trace amounts of Mn can become physiologically relevant. Supporting this notion, TORC1 is also wired to feedback control mechanisms that impinge on Smf1 and Smf2. Finally, we also show that Mn2+-mediated control of TORC1 is evolutionarily conserved in mammals, which may prove relevant for our understanding of the role of Mn in human diseases.

The human nucleoporin Tpr protects cells from RNA-mediated replication stress.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

The Nup84 complex coordinates the DNA damage response to warrant genome integrity.

DNA lesions interfere with cellular processes such as transcription and replication and need to be adequately resolved to warrant genome integrity. Beyond their primary role in molecule transport, nuclear pore complexes (NPCs) function in other processes such as transcription, nuclear organization and DNA double strand break (DSB) repair. Here we found that the removal of UV-induced DNA lesions by nucleotide excision repair (NER) is compromised in the absence of the Nup84 nuclear pore component. Importantly, nup84Δ cells show an exacerbated sensitivity to UV in early S phase and delayed replication fork progression, suggesting that unrepaired spontaneous DNA lesions persist during S phase. In addition, nup84Δ cells are defective in the repair of replication-born DSBs by sister chromatid recombination (SCR) and rely on post-replicative repair functions for normal proliferation, indicating dysfunctions in the cellular pathways that enable replication on damaged DNA templates. Altogether, our data reveal a central role of the NPC in the DNA damage response to facilitate replication progression through damaged DNA templates by promoting efficient NER and SCR and preventing chromosomal rearrangements.

Efficacy of Transcranial Direct Current Stimulation Combined with Cognitive Training in the Treatment of Apathy in Patients with Alzheimer's Disease: Study Protocol for a Randomized Trial.

BACKGROUND: Apathy, commonly defined as the loss of motivation, is a symptom frequently encountered in Alzheimer's Disease (AD). The treatment of apathy remains challenging in the absence of any truly effective medications. Transcranial Magnetic Stimulation (rTMS) or Transcranial Direct Current Stimulation (tDCS) can improve cognitive disorders, but do not appear to improve apathy. Isolated cognitive training also appears to have no effect on apathy. We propose to test the efficacy of a new procedure for the treatment of apathy in AD patients consisting of a combination of tDCS and cognitive training, based on the latest guidelines for the design of therapeutic trials in this field. METHODS/DESIGN: This article primarily describes the design of a monocentre, randomized, doubleblind trial to be conducted in France to evaluate the effect of the combination of tDCS and cognitive training on apathy compared to a group treated exclusively by cognitive training (sham tDCS). Twenty- four patients under the age of 90 years with mild-to-moderate Alzheimer's disease (Mini Mental State Examination score between 15 and 26/30) (MMSE)) presenting clinically significant apathy evaluated by the Apathy Inventory (AI) and the NeuroPsychiatric Inventory (NPI) apathy subscore will be enrolled. Severe depression will be excluded by using the NPI depression subscore. Treatment will comprise 10 sessions (D0-D11) including tDCS (bilateral prefrontal, temporal and parietal targets) and Cognitive Training (Cog) (6 simple tasks involving working memory, language and visuospatial function). After randomization (ratio 2:1), 16 patients will receive the complete treatment comprising tDCS and Cog (group 1) and 8 patients will be treated exclusively by Cog (sham tDCS) (group 2). The primary endpoint will be a significant improvement of the AI score by comparing baseline measures (D-15) to those recorded one month after stopping treatment (D44). Secondary endpoints will be an improvement of this score immediately after treatment (D14), 2 weeks (D29) and 2 months (D74) after stopping treatment and improvement of the MMSE score, NPI apathy subscore, ADAS Cog (Alzheimer Disease Assessment cognitive Scale subsection), ADCS-ADL (Alzheimer Disease Cooperative Study-Activities of Daily Living), FAB (Frontal Assessment Battery) and the latency of P300 evoked potentials at the same timepoints. CONCLUSION: The purpose of our study is to check the assumption of tDCS and cognitive training efficacy in the treatment of apathy encountered in AD patients and we will discuss its effect over time.

Gene gating at nuclear pores prevents the formation of R loops.

Transcription is an important source of genetic variability. A large amount of transcription-associated genome variation arises from the unscheduled formation of R loops. We have recently found that physical proximity of chromatin to nuclear pores prevents the formation of pathological R loops during transcription. Our study opens new perspectives to understand genome stability as a function of nuclear location.

Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability.

During transcription, the mRNA may hybridize with DNA, forming an R loop, which can be physiological or pathological, constituting in this case a source of genomic instability. To understand the mechanism by which eukaryotic cells prevent harmful R loops, we used human activation-induced cytidine deaminase (AID) to identify genes preventing R loops. A screening of 400 Saccharomyces cerevisiae selected strains deleted in nuclear genes revealed that cells lacking the Mlp1/2 nuclear basket proteins show AID-dependent genomic instability and replication defects that were suppressed by RNase H1 overexpression. Importantly, DNA-RNA hybrids accumulated at transcribed genes in mlp1/2 mutants, indicating that Mlp1/2 prevents R loops. Consistent with the Mlp1/2 role in gene gating to nuclear pores, artificial tethering to the nuclear periphery of a transcribed locus suppressed R loops in mlp1∆ cells. The same occurred in THO-deficient hpr1∆ cells. We conclude that proximity of transcribed chromatin to the nuclear pore helps restrain pathological R loops.

Transcription as a Threat to Genome Integrity.

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant.

Replication stress and cancer.

Genome instability is a hallmark of cancer, and DNA replication is the most vulnerable cellular process that can lead to it. Any condition leading to high levels of DNA damage will result in replication stress, which is a source of genome instability and a feature of pre-cancerous and cancerous cells. Therefore, understanding the molecular basis of replication stress is crucial to the understanding of tumorigenesis. Although a negative aspect of replication stress is its prominent role in tumorigenesis, a positive aspect is that it provides a potential target for cancer therapy. In this Review, we discuss the link between persistent replication stress and tumorigenesis, with the goal of shedding light on the mechanisms underlying the initiation of an oncogenic process, which should open up new possibilities for cancer diagnostics and treatment.

Methods to study transcription-coupled repair in chromatin.

The effect of endogenous and exogenous DNA damage on the cellular metabolism can be studied at the genetic and molecular level. A paradigmatic case is the repair of UV-induced pyrimidine dimers (PDs) by nucleotide excision repair (NER) in Saccharomyces cerevisiae. To follow the formation and repair of PDs at specific chromosome loci, cells are irradiated with UV-light and incubated in the dark to allow repair by NER. Upon DNA isolation, cyclobutane pyrimidine dimers, which account for about 90 % of PDs, can be cleaved in vitro by the DNA nicking activity of the T4 endonuclease V repair enzyme. Subsequently, strand-specific repair in a suitable restriction fragment is determined by denaturing gel electrophoresis followed by Southern blot and indirect end-labeling using a single-stranded DNA probe. Noteworthy, this protocol could potentially be adapted to other kind of DNA lesions, as long as a DNA nick is formed or a lesion-specific endonuclease is available.Transcription-coupled repair (TC-NER) is a sub-pathway of NER that catalyzes the repair of the transcribed strand of active genes. RNA polymerase II is essential for TC-NER, and its occupancy on a damaged template can be analyzed by chromatin immunoprecipitation (ChIP). In this chapter, we provide an up-dated protocol for both the DNA repair analysis and ChIP approaches to study TC-NER in yeast chromatin.

Transcription and recombination: when RNA meets DNA.

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics.

Cleavage factor I links transcription termination to DNA damage response and genome integrity maintenance in Saccharomyces cerevisiae.

During transcription, the nascent pre-mRNA undergoes a series of processing steps before being exported to the cytoplasm. The 3'-end processing machinery involves different proteins, this function being crucial to cell growth and viability in eukaryotes. Here, we found that the rna14-1, rna15-1, and hrp1-5 alleles of the cleavage factor I (CFI) cause sensitivity to UV-light in the absence of global genome repair in Saccharomyces cerevisiae. Unexpectedly, CFI mutants were proficient in UV-lesion repair in a transcribed gene. DNA damage checkpoint activation and RNA polymerase II (RNAPII) degradation in response to UV were delayed in CFI-deficient cells, indicating that CFI participates in the DNA damage response (DDR). This is further sustained by the synthetic growth defects observed between rna14-1 and mutants of different repair pathways. Additionally, we found that rna14-1 suffers severe replication progression defects and that a functional G1/S checkpoint becomes essential in avoiding genetic instability in those cells. Thus, CFI function is required to maintain genome integrity and to prevent replication hindrance. These findings reveal a new function for CFI in the DDR and underscore the importance of coordinating transcription termination with replication in the maintenance of genomic stability.

Transcription coupled repair at the interface between transcription elongation and mRNP biogenesis.

During transcription, the nascent pre-mRNA associates with mRNA-binding proteins and undergoes a series of processing steps, resulting in export competent mRNA ribonucleoprotein complexes (mRNPs) that are transported into the cytoplasm. Throughout transcription elongation, RNA polymerases frequently deal with a number of obstacles that need to be removed for transcription resumption. One important type of hindrance consists of helix-distorting DNA lesions. Transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair, ensures a fast repair of such transcription-blocking lesions. While the nucleotide excision repair reaction is fairly well understood, its regulation and the way it deals with DNA transcription remains largely unknown. In this review, we update our current understanding of the factors involved in TC-NER and discuss their functional interplay with the processes of transcription elongation and mRNP biogenesis. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Genome-wide function of THO/TREX in active genes prevents R-loop-dependent replication obstacles.

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome-wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5' → 3' gradient. This is accompanied by a genome-wide impact of THO-Sub2 deletions on expression of highly expressed, long and high G+C-content genes. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.

Methods to study transcription-coupled repair in chromatin.

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair that allows for the enhanced repair of the transcribed strand of active genes. A classical method to study DNA repair in vivo consists in the molecular analysis of UV-induced DNA damages at specific loci. Cells are irradiated with a defined dose of UV light leading to the formation of DNA lesions and incubated in the dark to allow repair. About 90% of the photoproducts consist of cyclobutane pyrimidine dimers, which can be cleaved by the DNA nicking activity of the T4 endonuclease V (T4endoV) repair enzyme. Strand-specific repair in a suitable restriction fragment is determined by alkaline gel electrophoresis followed by Southern blot transfer and indirect end-labeling using a single-stranded probe. Recent approaches have assessed the role of transcription factors in TCR by analyzing RNA polymerase II occupancy on a damaged template by chromatin immunoprecipitation (ChIP). Cells are treated with formaldehyde in vivo to cross-link proteins to DNA and enrichment of a protein of interest is done by subsequent immunoprecipitation. Upon reversal of the protein-DNA cross-links, the amount of coprecipitated DNA fragments can be detected by quantitative PCR. To perform ChIP on UV-damaged templates, we included an in vitro photoreactivation step prior to PCR analysis to ensure that all precipitated DNA fragments serve as substrates for the PCR reaction. Here, we provide a detailed protocol for both the DNA repair analysis and the ChIP approaches to study TCR in chromatin.

Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair.

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.

A novel class of mRNA-containing cytoplasmic granules are produced in response to UV-irradiation.

Nucleic acids are substrates for different types of damage, but little is known about the fate of damaged RNAs. We addressed the existence of an RNA-damage response in yeast. The decay kinetics of GAL1p-driven mRNAs revealed a dose-dependent mRNA stabilization upon UV-irradiation that was not observed after heat or saline shocks, or during nitrogen starvation. UV-induced mRNA stabilization did not depend on DNA repair, damage checkpoint or mRNA degradation machineries. Notably, fluorescent in situ hybridization revealed that after UV-irradiation, polyadenylated mRNA accumulated in cytoplasmic foci that increased in size with time. In situ colocalization showed that these foci are not processing-bodies, eIF4E-, eIF4G-, and Pab1-containing bodies, stress granules, autophagy vesicles, or part of the secretory or endocytic pathways. These results point to the existence of a specific eukaryotic RNA-damage response, which leads to new polyadenylated mRNA-containing granules (UV-induced mRNA granules; UVGs). We propose that potentially damaged mRNAs, which may be deleterious to the cell, are temporarily stored in UVG granules to safeguard cell viability.

Biogenesis of mRNPs: integrating different processes in the eukaryotic nucleus.

Transcription is a central function occurring in the nucleus of eukaryotic cells in coordination with other nuclear processes. During transcription, the nascent pre-mRNA associates with mRNA-binding proteins and undergoes a series of processing steps, resulting in export-competent mRNA ribonucleoprotein complexes (mRNPs) that are transported into the cytoplasm. Experimental evidence increasingly indicates that the different processing steps (5'-end capping, splicing, 3'-end cleavage) and mRNP export are connected to each other as well as to transcription, both functionally and physically. Here, we review the overall process of mRNP biogenesis with particular emphasis on the functional coupling of transcription with mRNP biogenesis and export and its relationship to nuclear organization.