bims-caglex 2024-02-11 papers

Front Aging. 2023 ;4 1323194

Initiation phase cellular reprogramming ameliorates DNA damage in the ERCC1 mouse model of premature aging.

Patrick Treat Paine, Cheyenne Rechsteiner, Francesco Morandini, Gabriela Desdín-Micó, Calida Mrabti, Alberto Parras, Amin Haghani, Robert Brooke, Steve Horvath, Andrei Seluanov, Vera Gorbunova, Alejandro Ocampo.

Unlike aged somatic cells, which exhibit a decline in molecular fidelity and eventually reach a state of replicative senescence, pluripotent stem cells can indefinitely replenish themselves while retaining full homeostatic capacity. The conferment of beneficial-pluripotency related traits via in vivo partial cellular reprogramming in vivo partial reprogramming significantly extends lifespan and restores aging phenotypes in mouse models. Although the phases of cellular reprogramming are well characterized, details of the rejuvenation processes are poorly defined. To understand whether cellular reprogramming can ameliorate DNA damage, we created a reprogrammable accelerated aging mouse model with an ERCC1 mutation. Importantly, using enhanced partial reprogramming by combining small molecules with the Yamanaka factors, we observed potent reversion of DNA damage, significant upregulation of multiple DNA damage repair processes, and restoration of the epigenetic clock. In addition, we present evidence that pharmacological inhibition of ALK5 and ALK2 receptors in the TGFb pathway are able to phenocopy some benefits including epigenetic clock restoration suggesting a role in the mechanism of rejuvenation by partial reprogramming.

Keywords: DNA damage; TGFb; aging; cellular reprogramming; ercc1

DOI: https://doi.org/10.3389/fragi.2023.1323194

Aging (Albany NY). 2024 Feb 02. 16

MicroRNA-146a gene transfer ameliorates senescence and senescence-associated secretory phenotypes in tendinopathic tenocytes.

Che-Chia Hsu, Shih-Yao Chen, Po-Yen Ko, Fa-Chuan Kwan, Wei-Ren Su, I-Ming Jou, Po-Ting Wu.

OBJECTIVE: Tendinopathy is influenced by multiple factors, including chronic inflammation and aging. Senescent cells exhibit characteristics such as the secretion of matrix-degrading enzymes and pro-inflammatory cytokines, collectively known as senescence-associated secretory phenotypes (SASPs). Many of these SASP cytokines and enzymes are implicated in the pathogenesis of tendinopathy. MicroRNA-146a (miR-146a) blocks senescence by targeting interleukin-1β (IL-1β) receptor-associated kinase 4 (IRAK-4) and TNF receptor-associated factor 6 (TRAF6), thus inhibiting NF-κB activity. The aims of this study were to (1) investigate miR-146a expression in tendinopathic tendons and (2) evaluate the role of miR-146a in countering senescence and SASPs in tendinopathic tenocytes.
METHODS: MiR-146a expression was assessed in human long head biceps (LHB) and rat tendinopathic tendons by in situ hybridization. MiR-146a over-expression in rat primary tendinopathic tenocytes was achieved by lentiviral vector-mediated precursor miR-146a transfer (LVmiR-146a). Expression of various senescence-related markers was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunoblotting and immunofluorescence. MiR-146a expression showed a negative correlation with the severity of tendinopathy in human and rat tendinopathic tendons (p<0.001).
RESULTS: Tendinopathic tenocyte transfectants overexpressing miR-146a exhibited downregulation of various senescence and SASP markers, as well as the target molecules IRAK-4 and TRAF6, and the inflammatory mediator phospho-NF-κB. Additionally, these cells showed enhanced nuclear staining of high mobility group box 1 (HMGB1) compared to LVmiR-scramble-transduced controls in response to IL-1β stimulation.
CONCLUSIONS: We demonstrate that miR-146a expression is negatively correlated with the progression of tendinopathy. Moreover, its overexpression protects tendinopathic tenocytes from SASPs and senescence through the IRAK-4/TRAF6/NF-kB pathway.

Keywords: lentiviral vector; microRNA-146a; senescence; senescence-associated secretory phenotypes; tendinopathy

DOI: https://doi.org/10.18632/aging.205505

Biogerontology. 2024 Feb 04.

A CRISPR base editing approach for the functional assessment of telomere biology disorder-related genes in human health and aging.

Gustavo Borges, Yahya Benslimane, Lea Harrington.

Telomere Biology Disorders (TBDs) are a group of rare diseases characterized by the presence of short and/or dysfunctional telomeres. They comprise a group of bone marrow failure syndromes, idiopathic pulmonary fibrosis, and liver disease, among other diseases. Genetic alterations (variants) in the genes responsible for telomere homeostasis have been linked to TBDs. Despite the number of variants already identified as pathogenic, an even more significant number must be better understood. The study of TBDs is challenging since identifying these variants is difficult due to their rareness, it is hard to predict their impact on the disease onset, and there are not enough samples to study. Most of our knowledge about pathogenic variants comes from assessing telomerase activity from patients and their relatives affected by a TBD. However, we still lack a cell-based model to identify new variants and to study the long-term impact of such variants on the genes involved in TBDs. Herein, we present a cell-based model using CRISPR base editing to mutagenize the endogenous alleles of 21 genes involved in telomere biology. We identified key residues in the genes encoding 17 different proteins impacting cell growth. We provide functional evidence for variants of uncertain significance in patients with TBDs. We also identified variants resistant to telomerase inhibition that, similar to cells expressing wild-type telomerase, exhibited increased tumorigenic potential using an in vitro tumour growth assay. We believe that such cell-based approaches will significantly advance our understanding of the biology of TBDs and may contribute to the development of new therapies for this group of diseases.

Keywords: CRISPR Base editing; Telomerase inhibitor resistance; Telomerase reverse transcriptase; Telomere biology disorders

DOI: https://doi.org/10.1007/s10522-024-10094-x

Free Radic Biol Med. 2024 Feb 06. pii: S0891-5849(24)00065-0. [Epub ahead of print]

Sestrin2 reduces ferroptosis via the Keap1/Nrf2 signaling pathway after intestinal ischemia-reperfusion.

Le-le Zhang, Ke Ding, Shi-Shi Liao, Yi-Guo Zhang, Hui-Yang Liao, Rong Chen, Qing-Tao Meng.

Sestrins are metabolic regulators that respond to stress by reducing the levels of reactive oxygen species (ROS) and inhibiting the activity of target of rapamycin complex 1 (mTORC1). Previous research has demonstrated that Sestrin2 mitigates ischemia-reperfusion (IR) injury in the heart, liver, and kidneys. However, its specific role in IIR injury remains unclear. To elucidate the role of Sestrin2 in intestinal ischemia-reperfusion injury, we conducted an experimental study using a C57BL/6J mouse model of IIR. We noticed an increase in the levels of Sestrin2 expression and indicators associated with ferroptosis. Our study revealed that manipulating Sestrin2 expression in Caco-2 cells through overexpression or knockdown resulted in a corresponding decrease or increase, respectively, in ferroptosis levels. Furthermore, our investigation revealed that Sestrin2 alleviated ferroptosis caused by IIR injury through the activation of the Keap1/Nrf2 signaling pathway. This finding highlights the potential of Sestrin2 as a therapeutic target for alleviating IIR injury. These findings indicated that the modulation of Sestrin2 could be a promising strategy for managing prolonged IIR injury.

Keywords: Ferroptosis; Intestinal ischemia-reperfusion; Keap1; Nrf2; Sestrin2

DOI: https://doi.org/10.1016/j.freeradbiomed.2024.02.003

Cells. 2024 Jan 29. pii: 247. [Epub ahead of print]13(3):

Simplifying Genotyping of Mutants from Genome Editing with a Parallel qPCR-Based iGenotype Index.

Liezhen Fu, Shouhong Wang, Lusha Liu, Yuki Shibata, Morihiro Okada, Nga Luu, Yun-Bo Shi.

Targeted genome editing is a powerful tool in reverse genetic studies of gene function in many aspects of biological and pathological processes. The CRISPR/Cas system or engineered endonucleases such as ZFNs and TALENs are the most widely used genome editing tools that are introduced into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, triggering cellular DNA repair through either homologous recombination or non-homologous end joining (NHEJ). DNA repair through the NHEJ mechanism is usually error-prone, leading to point mutations or indels (insertions and deletions) within the targeted region. Some of the mutations in embryos are germline transmissible, thus providing an effective way to generate model organisms with targeted gene mutations. However, point mutations and short indels are difficult to be effectively genotyped, often requiring time-consuming and costly DNA sequencing to obtain reliable results. Here, we developed a parallel qPCR assay in combination with an iGenotype index to allow simple and reliable genotyping. The genotype-associated iGenotype indexes converged to three simple genotype-specific constant values (1, 0, -1) regardless of allele-specific primers used in the parallel qPCR assays or gene mutations at wide ranges of PCR template concentrations, thus resulting in clear genotype-specific cutoffs, established through statistical analysis, for genotype identification. While we established such a genotyping assay in the Xenopus tropicalis model, the approach should be applicable to genotyping of any organism or cells and can be potentially used for large-scale, automated genotyping.

Keywords: Xenopus tropicalis; genome editing; genotyping; iGenotype index; parallel qPCR

DOI: https://doi.org/10.3390/cells13030247