pH-Activatable Ruthenium(II) Fluorescein Salphen Schiff Base Photosensitizers for Theranostic Applications.

Ruthenium(II) polypyridyl complexes exhibit a lack of selectivity toward cancer tissues despite extensive studies as photosensitizers for photodynamic therapy (PDT). Here, we report pH-activatable RuII photosensitizers for molecularly targeted PDT by exploiting the higher acidity of tumoral tissue. The fluorescein moiety, well known for its high pH sensitivity, was connected to a RuII center to yield novel photosensitizers for pH-sensitive 1O2 photogeneration. Their ability to photosensitize molecular dioxygen was studied at various pHs and revealed a drastic enhancement from 0.07 to 0.66 of the 1O2 quantum yield under acidic conditions (pH 7.5 to pH 5.5). Their photocytotoxicity against U2OS osteosarcoma cells was also investigated at pH 5.5 and 7.5 through IC50 determination. A strong enhancement of the photocytotoxicity reaching 930 nM was observed at pH 5.5, which showed the potential of such photosensitizers for pH-activatable PDT.

Mitochondria and telomeres: hand in glove.

Born as an endosymbiont, the bacteria engulfed by the proto-eukaryotic cell more than 1.45 billion years ago progressively evolved as an important organelle with multiple interactions with the host cell. In particular, strong connections between mitochondria and the chromosome ends, the telomeres, led to propose a new theory of ageing in which dysfunctional telomeres and mitochondria are the main actors of a vicious circle reducing cell fitness and promoting cellular ageing. We review the evidences that oxidative stress and dysfunctional mitochondria damage telomeres and further discuss the interrelationship between telomere biology and mitochondria through the lens of telomerase which shuttles between the nucleus and mitochondria. Finally, we elaborate on the possible role of the mitochondrial genome on the inheritance of human telomere length through the expression of mitochondrial gene variants.

Senescence-related impairment of autophagy induces toxic intraneuronal amyloid-ß accumulation in a mouse model of amyloid pathology.

Aging is the main risk factor for Alzheimer's disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes underlying pathological aging of the nervous system are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. Senescence has also been shown to reduce the autophagic flux, a mechanism involved in clearing damaged proteins from the cell, and such impairment has been linked to AD pathogenesis. In this study, we investigated the role of cellular senescence on AD pathology by crossing a mouse model of AD-like amyloid-ß (Aß) pathology (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc-/-). We studied changes in amyloid pathology, neurodegeneration, and the autophagy process in brain tissue samples and primary cultures derived from these mice by complementary biochemical and immunostaining approaches. Postmortem human brain samples were also processed to evaluate autophagy defects in AD patients. Our results show that accelerated senescence produces an early accumulation of intraneuronal Aß in the subiculum and cortical layer V of 5xFAD mice. This correlates with a reduction in amyloid plaques and Aß levels in connecting brain regions at a later disease stage. Neuronal loss was specifically observed in brain regions presenting intraneuronal Aß and was linked to telomere attrition. Our results indicate that senescence affects intraneuronal Aß accumulation by impairing autophagy function and that early autophagy defects can be found in the brains of AD patients. Together, these findings demonstrate the instrumental role of senescence in intraneuronal Aß accumulation, which represents a key event in AD pathophysiology, and emphasize the correlation between the initial stages of amyloid pathology and defects in the autophagy flux.

Senescence and senotherapies in biliary atresia and biliary cirrhosis.

BACKGROUND: Premature senescence occurs in adult hepatobiliary diseases and worsens the prognosis through deleterious liver remodeling and hepatic dysfunction. Senescence might also arises in biliary atresia (BA), the first cause of pediatric liver transplantation. Since alternatives to transplantation are needed, our aim was to investigate premature senescence in BA and to assess senotherapies in a preclinical model of biliary cirrhosis. METHODS: BA liver tissues were prospectively obtained at hepatoportoenterostomy (n=5) and liver transplantation (n=30) and compared to controls (n=10). Senescence was investigated through spatial whole transcriptome analysis, SA-ß-gal activity, p16 and p21 expression, γ-H2AX and senescence-associated secretory phenotype (SASP). Human allogenic liver-derived progenitor cells (HALPC) or dasatinib and quercetin (D+Q) were administrated to two-month-old Wistar rats after bile duct ligation (BDL). RESULTS: Advanced premature senescence was evidenced in BA livers from early stage and continued to progress until liver transplantation. Senescence and SASP were predominant in cholangiocytes, but also present in surrounding hepatocytes. HALPC but not D+Q reduced the early marker of senescence p21 in BDL rats and improved biliary injury (serum γGT and Sox9 expression) and hepatocytes mass loss (Hnf4a). CONCLUSIONS: BA livers displayed advanced cellular senescence at diagnosis that continued to progress until liver transplantation. HALPC reduced early senescence and improved liver disease in a preclinical model of BA, providing encouraging preliminary results regarding the use of senotherapies in pediatric biliary cirrhosis.

Photo-induced telomeric DNA damage in human cancer cells.

Herein we report on the study of novel dinuclear ruthenium(ii) complexes designed to target and to photo-react with G-quadruplex telomeric DNA. Upon irradiation, complexes efficiently generate guanine radical cation sites as photo-oxidation products. The compounds also display efficient cell penetration with localization to the nucleus and show strong photocytotoxicity toward osteosarcoma cells. Thanks to a microscopic-based telomere dysfunction assay, which allows the direct visualization of DNA damage in cells, we brought the first evidence of forming photo-oxidative damage at telomeres in cellulo. This emphasizes interesting prospects for the development of future cancer phototherapies.

TERRA and RAD51AP1 at the R&D-loop department of ALT telomeres.

In this issue of Molecular Cell, Kaminski et al. and Yadav et al. demonstrate that RAD51AP1 and TERRA non-coding RNA positively regulate the alternative mechanism of telomere maintenance by promoting D-loop formation and chromatin-directed mechanisms to suppress transcription-collision events during ALT telomere synthesis.

Lung Microenvironments and Disease Progression in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis (fHP) is an interstitial lung disease caused by sensitization to an inhaled allergen. Objectives: To identify the molecular determinants associated with progression of fibrosis. Methods: Nine fHP explant lungs and six unused donor lungs (as controls) were systematically sampled (4 samples/lung). According to microcomputed tomography measures, fHP cores were clustered into mild, moderate, and severe fibrosis groups. Gene expression profiles were assessed using weighted gene co-expression network analysis, xCell, gene ontology, and structure enrichment analysis. Gene expression of the prevailing molecular traits was also compared with idiopathic pulmonary fibrosis (IPF). The explant lung findings were evaluated in separate clinical fHP cohorts using tissue, BAL samples, and computed tomography scans. Measurements and Main Results: We found six molecular traits that associated with differential lung involvement. In fHP, extracellular matrix and antigen presentation/sensitization transcriptomic signatures characterized lung zones with only mild structural and histological changes, whereas signatures involved in honeycombing and B cells dominated the transcriptome in the most severely affected lung zones. With increasing disease severity, endothelial function was progressively lost, and progressive disruption in normal cellular homeostatic processes emerged. All six were also found in IPF, with largely similar associations with disease microenvironments. The molecular traits correlated with in vivo disease behavior in a separate clinical fHP cohort. Conclusions: We identified six molecular traits that characterize the morphological progression of fHP and associate with in vivo clinical behavior. Comparing IPF with fHP, the transcriptome landscape was determined considerably by local disease extent rather than by diagnosis alone.

Endurance training alleviates MCP-1 and TERRA accumulation at old age in human skeletal muscle.

Both oxidative stress and telomere transcription are up-regulated by acute endurance exercise in human skeletal muscle. Whether and how life-long exercise training influences the antioxidant system response at transcriptional level and TERRA expression is unknown, especially during aging. Response to acute endurance exercise was investigated in muscle biopsies of 3 male subjects after 45 min of cycling. MCP-1 and SOD1 mRNA levels increased up to, 15-fold and 63%, respectively, after the cycling session while the mRNA levels of SOD2 were downregulated by 25%. The effects of chronic endurance exercise and aging were tested in the blood and muscle of 34 male subjects divided into four groups: young (YU) or old (OU) untrained, young (YT) or old (OT) trained cyclists. Long-term endurance training limited the age-dependent elevation in SOD1 (OT vs OU, -26%, P = 0.03) and the decline in SOD2 mRNA levels (OU vs YU, -41%, P = 0.04). A high endurance training status alleviated the age-related increase in the aging biological marker MCP-1 in plasma (OU vs YU, +48%, P = 0.005). Similar results were observed for telomeric transcription as the age-associated increase in 16p TERRA levels (OU vs YU, +39%, P = 0.001) was counteracted by a high endurance training status (OT vs OU, -63%, P = 0.0005). In conclusion, as MCP-1, we propose that the age-related TERRA accumulation might represent a novel biological marker of aging. Those aging-related increase expression might be alleviated by a high endurance training status. Whether those biological markers of aging are linked to an elevation of oxidative stress is still an open question. Therefore, whether the positive adaptations provided by endurance training indeed reduce oxidative stress, including at telomeres, and whether TERRA plays any role in this, need to be further investigated.

The multifaceted hTR telomerase RNA from a structural perspective: Distinct domains of hTR differentially interact with protein partners to orchestrate its telomerase-independent functions.

Human telomerase progressively emerged as a multifaceted ribonucleoprotein complex with additional functions beyond telomeric repeat synthesis. Both the hTERT catalytic subunit and the hTR long non-coding RNA (lncRNA) subunit are engaged in highly regulated cellular pathways that, together, contribute to cell fitness and protection against apoptosis. We recently described a new role for hTR in regulating the abundance of replication protein A at telomeres, adding to the growing repertoire of hTR's functions. Here, we focus on the non-canonical roles of hTR and discuss them in the context of the structural elements of the lncRNA. We propose that some functions of hTR may compete amongst each other through distinct interactions with its partners, proteins or mRNAs. We postulate that hTR's non-canonical functions may be highly relevant in the context of normal somatic cells that naturally silence hTERT gene, while keeping hTR expression.

NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres.

About 10% of cancer cells employ the "alternative lengthening of telomeres" (ALT) pathway instead of re-activating the hTERT subunit of human telomerase. The hTR RNA subunit is also abnormally silenced in some ALT+ cells not expressing hTERT, suggesting a possible negative non-canonical impact of hTR on ALT. Indeed, we show that ectopically expressed hTR reduces phosphorylation of ssDNA-binding protein RPA (p-RPAS33 ) at ALT telomeres by promoting the hnRNPA1- and DNA-PK-dependent depletion of RPA. The resulting defective ATR checkpoint signaling at telomeres impairs recruitment of the homologous recombination protein, RAD51. This induces ALT telomere fragility, increases POLD3-dependent C-circle production, and promotes the recruitment of the DNA damage marker 53BP1. In ALT+ cells that naturally retain hTR expression, NHP2 H/ACA ribonucleoprotein levels are downregulated, likely in order to restrain DNA damage response (DDR) activation at telomeres through reduced 53BP1 recruitment. This unexpected role of NHP2 is independent from hTR's non-canonical function in modulating telomeric p-RPAS33 . Collectively, our study shines new light on the interference between telomerase- and ALT-dependent pathways and unravels a crucial role for hTR and NHP2 in DDR regulation at ALT telomeres.

Detection of alternative lengthening of telomeres mechanism on tumor sections.

The vast majority of adult cancer cells achieve cellular immortality by activating a telomere maintenance mechanism (TMM). While this is mostly achieved by the de-silencing of hTERT telomerase gene expression, an alternative homologous recombination-based and telomerase-independent mechanism, known as ALT (Alternative Lengthening of Telomeres), is frequently activated in a subset of tumors, including paediatric cancers. Being absent from normal cells, the ALT mechanism offers interesting perspectives for new targeted cancer therapies. To date, however, the development of better translationally applicable tools for ALT detection in tumor sections is still needed. Here, using a newly derived ALT-positive cancer cell mouse xenograft model, we extensively examined how the previously known ALT markers could be used as reliable tools for ALT diagnosis in tumor sections. We found that, together with the detection of ultra-bright telomeric signals (UBS), an ALT hallmark, native telomeric FISH, that detects single-stranded C-rich telomeric DNA, provides a very sensitive and robust tool for ALT diagnosis in tissues. We applied these assays to paediatric tumor samples and readily identified three ALT-positive tumors for which the TMM was confirmed by the gold-standard C-circle amplification assay. Although the latter offers a robust assay for ALT detection in the context of research laboratories, it is more difficult to set up in histopathological laboratories and could therefore be conveniently replaced by the combination of UBS detection and native telomeric FISH.

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Short telomeres increase the risk of severe COVID-19.

Telomeres are non-coding DNA sequences that protect chromosome ends and shorten with age. Short telomere length (TL) is associated with chronic diseases and immunosenescence. The main risk factor for mortality of coronavirus disease 2019 (COVID-19) is older age, but outcome is very heterogeneous among individuals of the same age group. Therefore, we hypothesized that TL influences COVID-19-related outcomes. In a prospective study, we measured TL by Flow-FISH in 70 hospitalized COVID-19 patients and compared TL distribution with our reference cohort of 491 healthy volunteers. We also correlated TL with baseline clinical and biological parameters. We stained autopsy lung tissue from six non-survivor COVID-19 patients to detect senescence-associated ß-galactosidase activity, a marker of cellular aging. We found a significantly higher proportion of patients with short telomeres (<10th percentile) in the COVID-19 patients as compared to the reference cohort (P<0.001). Short telomeres were associated with a higher risk of critical disease, defined as admission to intensive care unit (ICU) or death without ICU. TL was negatively correlated with C-reactive protein and neutrophil-to-lymphocyte ratio. Finally, lung tissue from patients with very short telomeres exhibit signs of senescence in structural and immune cells. Our results suggest that TL influences the severity of the disease.

No effect of the endurance training status on senescence despite reduced inflammation in skeletal muscle of older individuals.

The aim of the present study was to determine if the training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle in older compared with younger subjects, with a specific focus on satellite cells. Analyses were conducted on skeletal muscle and cultured satellite cells from vastus lateralis biopsies (n = 34) of male volunteers divided into four groups: young sedentary (YS), young trained cyclists (YT), old sedentary (OS), and old trained cyclists (OT). The senescence state and inflammatory profile were evaluated by telomere dysfunction-induced foci (TIF) quantification, senescence-associated ß-galactosidase (SA-ß-Gal) staining, and quantitative (q)RT-PCR. Independently of the endurance training status, TIF levels (+35%, P < 0.001) and the percentage of SA-ß-Gal-positive cells (+30%, P < 0.05) were higher in cultured satellite cells of older compared with younger subjects. p16 (4- to 5-fold) and p21 (2-fold) mRNA levels in skeletal muscle were higher with age but unchanged by the training status. Aging induced higher CD68 mRNA levels in human skeletal muscle (+102%, P = 0.009). Independently of age, both trained groups had lower IL-8 mRNA levels (-70%, P = 0.011) and tended to have lower TNF-α mRNA levels (-40%, P = 0.10) compared with the sedentary subjects. All together, we found that the endurance training status did not slow down senescence in skeletal muscle and satellite cells in older compared with younger subjects despite reduced inflammation in skeletal muscle. These findings highlight that the link between senescence and inflammation can be disrupted in skeletal muscle.

Flexible RuII Schiff Base Complexes: G-Quadruplex DNA Binding and Photo-Induced Cancer Cell Death.

A series of new RuII Schiff base complexes built on the salphen moiety has been prepared. This includes four flexible monometallic RuII compounds and six rigid bimetallic analogues that contain NiII , PdII or PtII cations into the salphen complexation site. Steady state luminescence titrations illustrated the capacity of the compounds to photoprobe G-quadruplex (G4) DNA. Moreover, the vast array of the Schiff base structural changes allowed to extensively assess the influence of the ligand surface, flexibility and charge on the interaction of the compounds with G4 DNA. This was achieved thanks to circular dichroism melting assays and bio-layer interferometry studies that pointed up high affinities along with good selectivities of RuII Schiff base complexes for G4 DNA. In cellulo studies were carried out with the most promising compounds. Cellular uptake with location of the compounds in the nucleus as well as in the nucleolus was observed. Cell viability experiments were performed with U2OS osteosarcoma cells in the dark and under light irradiation which allowed the measurements of IC50 values and photoindexes. They showed the substantial role played by light irradiation in the activity of the drugs in addition to the low cytotoxicity of the molecules in the dark. Altogether, the reported results emphasize the promising properties of RuII Schiff base complexes as a new class of candidates for developing potential G4 DNA targeting diagnostic or therapeutic compounds.

An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue.

Senescence-associated beta-galactosidase (SA-ß-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (ß-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for ß-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-ß-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in ß-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p<0.01) and after overnight staining at pH 5.8 (24.09 ± 6.88 vs 0.12 ± 0.08; p<0.01). Although we noticed that ß-gal activity staining decreased with cryopreservation time (from 4 to 12 months of storage at -80C; p<0.05), the enhanced staining observed in BDR compared with controls remained detectable up to 12 months after cryopreservation (p<0.01). In conclusion, we provide an optimized protocol for SA-ß-gal activity histochemical detection at physiological pH 4.0 on long-term cryopreserved liver tissue.

Telomere maintenance mechanisms in cancer: telomerase, ALT or lack thereof.

Cancer cells acquire replicative immortality by activating a telomere maintenance mechanism (TMM), either the telomerase or the Alternative Lengthening of Telomeres (ALT) mechanism. ALT is frequently activated in tumors derived from mesenchymal cells, which are more frequent in childhood cancers. Recent studies showed that, occasionally, cancer cells can arise without any TMM activation. Here, we discuss the challenge in assessing which TMM is activated in tumors. We also evaluate the prevalence of ALT mechanism in pediatric cancers and review the associated survival prognosis in different tumor types. Finally, we discuss about possible anti-TMM therapies for new emerging cancer treatments.

Specific death of ALT cells through TSPYL5 depletion.

Some tumors acquire replicative immortality by activating an ALTernative telomerase-independent telomere maintenance mechanism. ALT offers interesting therapeutic perspectives but lacks any known specific targets. We discovered a crucial role for TSPYL5 (Testis-Specific Y-encoded-Like Protein 5) in keeping ALT cell viability by protecting POT1 (Protection Of Telomeres 1) from proteasomal degradation.

Exploring the Phototoxicity of Hypoxic Active Iridium(III)-Based Sensitizers in 3D Tumor Spheroids.

Among all molecules developed for anticancer therapies, photodynamic therapeutic agents have a unique profile. Their maximal activity is specifically triggered in tumors by light, and toxicity of even systemically delivered drug is prevented in nonilluminated parts of the body. Photosensitizers exert their therapeutic effect by producing reactive oxygen species via a light-activated reaction with molecular oxygen. Consequently, the lowering of pO2 deep in solid tumors limits their treatment and makes essential the design of oxygen-independent sensitizers. In this perspective, we have recently developed Ir(III)-based molecules able to oxidize biomolecules by type I processes under oxygen-free conditions. We examine here their phototoxicity in relevant biological models. We show that drugs, which are mitochondria-accumulated, induce upon light irradiation a dramatic decrease of the cell viability, even under low oxygen conditions. Finally, assays on 3D tumor spheroids highlight the importance of the light-activation step and the oxygen consumption rate on the drug activity.

Impaired telomere integrity and rRNA biogenesis in PARN-deficient patients and knock-out models.

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.