bims-senagi 2021-03-28 papers

bims-senagi

Biomed News

on Senescence and aging

Issue of 2021–03–28
27 papers selected by
Maria Grazia Vizioli, Mayo Clinic

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.
The functional impact of nuclear reorganization in cellular senescence.
Fluorescence Microscopy Methods for Examining Telomeres During Cell Aging.
Role of cell competition in ageing.
Ageing of the gut microbiome: potential influences on immune senescence and inflammageing.
Chitosan hydrogel-loaded MSC-derived extracellular vesicles promote skin rejuvenation by ameliorating the senescence of dermal fibroblasts.
Vildagliptin improves vascular smooth muscle relaxation and decreases cellular senescence in the aorta of doxorubicin-treated rats.
Calorie restriction prevents age-related changes in the intestinal microbiota.
Altered Endocytosis in Cellular Senescence.
Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway.
Cellular Senescence in Liver Disease and Regeneration.
miR-23a/b suppress cGAS-mediated innate and autoimmunity.
Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis.
Chronic metformin treatment decreases cardiac injury during ischemia-reperfusion by attenuating endoplasmic reticulum stress with improved mitochondrial function.
The regulation of the DNA damage response at telomeres: focus on kinases.
DNASE1L3 arrests tumor angiogenesis by impairing the senescence-associated secretory phenotype in response to stress.
EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells.
Opportunities and Challenges in Stem Cell Aging.
IL-1β induces expression of proinflammatory cytokines and migration of human colon cancer cells through upregulation of SIRT1.
Targeting of miR-33 ameliorates phenotypes linked to age-related macular degeneration.
Gut microbiota dysbiosis promotes age-related atrial fibrillation by lipopolysaccharide and glucose-induced activation of NLRP3-inflammasome.
Senescent T Cell Induces Brown Adipose Tissue "Whitening" Via Secreting IFN-γ.
miR-30c-1 encourages human corneal endothelial cells to regenerate through ameliorating senescence.
Deficiency of telomere-associated repressor activator protein 1 precipitates cardiac aging in mice via p53/PPARα signaling.
CGAS is a micronucleophagy receptor for the clearance of micronuclei.
Lipid droplets meet aging.
cGAS knockdown promotes microglial M2 polarization to alleviate neuroinflammation by inhibiting cGAS-STING signaling pathway in cerebral ischemic stroke.

EMBO J. 2021 Mar 25. e106048

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Anthony Lagnado, Jack Leslie, Marie-Helene Ruchaud-Sparagano, Stella Victorelli, Petra Hirsova, Mikolaj Ogrodnik, Amy L Collins, Maria Grazia Vizioli, Leena Habiballa, Gabriele Saretzki, Shane A Evans, Hanna Salmonowicz, Adam Hruby, Daniel Geh, Kevin D Pavelko, David Dolan, Helen L Reeves, Sushma Grellscheid, Colin H Wilson, Sanjay Pandanaboyana, Madison Doolittle, Thomas von Zglinicki, Fiona Oakley, Suchira Gallage, Caroline L Wilson, Jodie Birch, Bernadette Carroll, James Chapman, Mathias Heikenwalder, Nicola Neretti, Sundeep Khosla, Claudio Akio Masuda, Tamar Tchkonia, James L Kirkland, Diana Jurk, Derek A Mann, João F Passos.

  Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

Keywords:  aging; neutrophils; senescence; telomeres

DOI:  https://doi.org/10.15252/embj.2020106048
Brief Funct Genomics. 2021 Mar 23. pii: elab012. [Epub ahead of print]

The functional impact of nuclear reorganization in cellular senescence.

Azucena Rocha, Audrey Dalgarno, Nicola Neretti.

  Cellular senescence is the irreversible cell cycle arrest in response to DNA damage. Because senescent cells accumulate with age and contribute to chronic inflammation, they are promising therapeutic targets for healthspan extension. The senescent phenotype can vary depending on cell type and on the specific insults that induce senescence. This variability is also reflected in the extensive remodeling of the genome organization within the nucleus of senescent cells. Here, we give an overview of the nuclear changes that occur in different forms of senescence, including changes to chromatin state and composition and to the three-dimensional organization of the genome, as well as alterations to the nuclear envelope and to the accessibility of repetitive genomic regions. Many of these changes are shared across all forms of senescence, implicating nuclear organization as a fundamental driver of the senescent state and of how senescent cells interact with the surrounding tissue.

Keywords:  aging; cellular senescence; chromatin; epigenome; nuclear organization

DOI:  https://doi.org/10.1093/bfgp/elab012
Ageing Res Rev. 2021 Mar 17. pii: S1568-1637(21)00067-2. [Epub ahead of print] 101320

Fluorescence Microscopy Methods for Examining Telomeres During Cell Aging.

Nancy Adam, Tara L Beattie, Karl Riabowol.

  Telomeres are protective structures, composed of nucleic acids and a complex protein mixture, located at the end of the chromosomes. They play an important role in preventing genomic instability and ensuring cell health. Defects in telomere integrity result in cell dysfunction and the development of diseases, including neurodegenerative disorders, cancer and premature aging syndromes, among others. Loss of telomere integrity during normal cell aging also initiates DNA damage signals that culminate in the senescence phenotype. Fluorescence microscopy has allowed researchers to study the dynamics, shape, localization, and co-distribution of telomeres with proteins of interest. The microscopy tools to investigate these structures have evolved, making it possible to understand in greater detail the molecular mechanisms affecting telomeres that contribute to cell aging and the development of age-related diseases. Using human fibroblasts as an example, we will highlight several characteristics of telomeres that can be investigated using three different microscopy systems, including wide-field microscopy, and the two super-resolution techniques called 3D Structured Illumination Microscopy (3D-SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). In this review, we will also discuss their limitations and highlight their importance in answering telomere-related scientific questions.

Keywords:  fluorescence microscopy; senescence; super-resolution microscopy; telomere biology

DOI:  https://doi.org/10.1016/j.arr.2021.101320
Dev Biol. 2021 Mar 19. pii: S0012-1606(21)00072-5. [Epub ahead of print]

Role of cell competition in ageing.

Mariana Marques-Reis, Eduardo Moreno.

  Recent advances in rapid medical detection and diagnostic technology have extended both human health and life expectancy. However, ageing remains one of the critical risk factors in contributing to major incapacitating and fatal conditions, including cancer and neurodegeneration. Therefore, it is vital to study how ageing attributes to (or participates in) endangering human health via infliction of age-related diseases and what must be done to tackle this intractable process. This review encompasses the most recent literature elaborating the role of cell competition (CC) during ageing. CC is a process that occurs between two heterogeneous populations, where the cells with higher fitness levels have a competitive advantage over the neighbouring cells that have comparatively lower fitness levels. This interaction results in the selection of the fit cells, within a population, and elimination of the viable yet suboptimal cells. Therefore, it is tempting to speculate that, if this quality control mechanism works efficiently throughout life, can it ultimately lead to a healthier ageing and extended lifespan. Furthermore, the review aims to collate all the important state of the art publications that provides evidence of the relevance of CC in dietary restriction, stem cell dynamics, and cell senescence, thus, prompting us to advocate its contribution and in exploring new avenues and opportunities in fighting age-related conditions.

Keywords:  Ageing; Cell competition; Drosophila; Fitness sensing

DOI:  https://doi.org/10.1016/j.ydbio.2021.03.009
Ageing Res Rev. 2021 Mar 23. pii: S1568-1637(21)00070-2. [Epub ahead of print] 101323

Ageing of the gut microbiome: potential influences on immune senescence and inflammageing.

Jessica Conway, Niharika A Duggal.

  Advancing age is accompanied by changes in the gut microbiota characterised by a loss of beneficial commensal microbes that is driven by intrinsic and extrinsic factors such as diet, medications, sedentary behaviour and chronic health conditions. Concurrently, ageing is accompanied by an impaired ability to mount a robust immune response, termed immunesenescence, and age-associated inflammation, termed inflammaging. The microbiome has been proposed to impact the immune system and is a potential determinant of healthy aging. In this review we summarise the knowledge on the impact of ageing on microbial dysbiosis, intestinal permeability, inflammaging, and the immune system and investigate whether dysbiosis of the gut microbiota could be a potential mechanism underlying the decline in immune function, overall health and longevity with advancing age. Furthermore, we examine the potential of altering the gut microbiome composition as a novel intervention strategy to reverse the immune ageing clock and possibly support overall good health during old age.

Keywords:  Immunesenescence; ageing; inflammaging; microbiome

DOI:  https://doi.org/10.1016/j.arr.2021.101323
Stem Cell Res Ther. 2021 Mar 20. 12(1): 196

Chitosan hydrogel-loaded MSC-derived extracellular vesicles promote skin rejuvenation by ameliorating the senescence of dermal fibroblasts.

Xiangnan Zhao, Yue Liu, Pingping Jia, Hui Cheng, Chen Wang, Shang Chen, Haoyan Huang, Zhibo Han, Zhong-Chao Han, Krzysztof Marycz, Xiaoniao Chen, Zongjin Li.

   BACKGROUND: The senescence of dermal fibroblasts (DFLs) leads to an imbalance in the synthesis and degradation of extracellular matrix (ECM) proteins, presenting so-called senescence-associated secretory phenotype (SASP), which ultimately leads to skin aging. Recently, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been recognized as a promising cell-free therapy for degenerative diseases, which opens a new avenue for skin aging treatment.
METHODS: In this study, we utilized chitosan (CS) hydrogel for effective loading and sustained release of EVs. In vitro, we explored the rejuvenation effects of CS hydrogel-incorporated EVs (CS-EVs) on replicative senescence DFLs through a series of experiments such as senescence-associated β-galactosidase (SA-β-gal) staining, RT-PCR, and Western blot analysis. Besides, we employed local multi-site subcutaneous injection to treat skin aging of naturally aged mice with CS-EVs and DiI fluorescent dye was used to label EVs to achieve in vivo real-time tracking.
RESULTS: CS-EVs can significantly improve the biological functions of senescent fibroblasts, including promoting their proliferation, enhancing the synthesis of ECM proteins, and inhibiting the overexpression of matrix metalloproteinases (MMPs). Moreover, CS hydrogel could prolong the release of EVs and significantly increase the retention of EVs in vivo. After CS-EVs subcutaneous injection treatment, the aging skin tissues showed a rejuvenation state, manifested explicitly as the enhanced expression of collagen, the decreased expression of SASP-related factors, and the restoration of tissue structures.
CONCLUSIONS: CS hydrogel-encapsulated EVs could delay the skin aging processes by ameliorating the function of aging DFLs. Our results also highlight the potential of CS hydrogel-encapsulated EVs as a novel therapeutic strategy for improving aging skin to rejuvenation.

Keywords:  Chitosan hydrogel; Dermal fibroblasts (DFLs); Extracellular matrix (ECM); Extracellular vesicles (EVs); Skin aging

DOI:  https://doi.org/10.1186/s13287-021-02262-4
Vascul Pharmacol. 2021 Mar 17. pii: S1537-1891(21)00027-6. [Epub ahead of print] 106855

Vildagliptin improves vascular smooth muscle relaxation and decreases cellular senescence in the aorta of doxorubicin-treated rats.

Svetozár Mišúth, Marína Uhrinová, Ján Klimas, Diana Vavrincová-Yaghi, Peter Vavrinec.

   INTRODUCTION: Doxorubicin (DOX) is a chemotherapeutic agent used in cancer treatment. Its use is limited by later toxicity to the cardiovascular system (CVS). Cellular senescence has been proposed as one mechanism of DOX toxicity. It has also been suggested that senescence reduction can improve the condition in many pathologies. We hypothesised that vildagliptin treatment can reduce senescence and thus improve the relaxation of vascular smooth muscle (VSM) in the aorta of a rat DOX model.
METHODS: The rats received DOX and were treated with vildagliptin for 6 weeks. Thereafter, the rats were sacrificed, and the aorta prepared for measurements of VSM relaxation and RNA isolation to detect the level of senescence markers. To further prove the antisenescence effect of the main vildagliptin effector glucagon-like peptide 1(GLP-1), VSM cells (VSMCs) were incubated with DOX and treated with GLP-1. Subsequently, senescence was detected by senescence-associated beta-galactosidase (SA-β-gal) and by the presence of senescence markers.
RESULTS: DOX in rats caused diminished relaxation of VSM to sodium nitrate and caused an increase in the senescence mRNA markers p16Ink4a and p27Kip1 and the senescence-associated secretory phenotype (SASP) IL-6 and IL-8. Vildagliptin treatment led to improved relaxation and a reduction in senescence and SASP markers. Furthermore, in VSMCs DOX increased SA-β-gal activity, p16Ink4a, p27Kip1, IL-6 and IL-8, and GLP1 treatment led to a decrease of both senescence and SASP markers.
CONCLUSION: In summary we conclude that vildagliptin can reduce senescence and improve relaxation of vascular smooth muscle in the aorta of DOX-treated rats, and GLP-1 can reduce senescence of DOX-treated VSMCs. These data suggest that incretin-based drugs are promising candidates for patients suffering from late doxorubicin cardiovascular toxicity.

Keywords:  Cellular senescence; Doxorubicin; GLP-1; Vascular smooth muscle of aorta; Vildagliptin

DOI:  https://doi.org/10.1016/j.vph.2021.106855
Aging (Albany NY). 2021 Mar 10. 13(5): 6298-6329

Calorie restriction prevents age-related changes in the intestinal microbiota.

Kavitha Kurup, Stephanie Matyi, Cory B Giles, Jonathan D Wren, Kenneth Jones, Aaron Ericsson, Daniel Raftery, Lu Wang, Daniel Promislow, Arlan Richardson, Archana Unnikrishnan.

  The effect of calorie restriction (CR) on the microbiome, fecal metabolome, and colon transcriptome of adult and old male mice was compared. Life-long CR increased microbial diversity and the Bacteroidetes/Firmicutes ratio and prevented the age-related changes in the microbiota, shifting it to a younger microbial and fecal metabolite profile in both C57BL/6JN and B6D2F1 mice. Old mice fed CR were enriched in the Rikenellaceae, S24-7 and Bacteroides families. The changes in the microbiome that occur with age and CR were initiated in the cecum and further modified in the colon. Short-term CR in adult mice had a minor effect on the microbiome but a major effect on the transcriptome of the colon mucosa. These data suggest that CR has a major impact on the physiological status of the gastrointestinal system, maintaining it in a more youthful state, which in turn could result in a more diverse and youthful microbiome.

Keywords:  aging; calorie restriction; metabolome; microbiome; transcriptome

DOI:  https://doi.org/10.18632/aging.202753
Ageing Res Rev. 2021 Mar 19. pii: S1568-1637(21)00079-9. [Epub ahead of print] 101332

Altered Endocytosis in Cellular Senescence.

Eun-Young Shin, Nak-Kyun Soung, Martin Alexander Schwartz, Eung-Gook Kim.

  Cellular senescence occurs in response to diverse stresses (e.g., telomere shortening, DNA damage, oxidative stress, oncogene activation). A growing body of evidence indicates that alterations in multiple components of endocytic pathways contribute to cellular senescence. Clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME) represent major types of endocytosis that are implicated in senescence. More recent research has also identified a chromatin modifier and tumor suppressor that contributes to the induction of senescence via altered endocytosis. Here, molecular regulators of aberrant endocytosis-induced senescence are reviewed and discussed in context of their capacity to serve as senescence-inducing stressors or modifiers.

Keywords:  Endocytosis; ING1; amphiphysin; caveolin-1; senescence; βPAK-interacting nucleotide exchange factor (βPIX)

DOI:  https://doi.org/10.1016/j.arr.2021.101332
Nat Commun. 2021 03 23. 12(1): 1826

Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway.

Takeshi Fujino, Susumu Goyama, Yuki Sugiura, Daichi Inoue, Shuhei Asada, Satoshi Yamasaki, Akiko Matsumoto, Kiyoshi Yamaguchi, Yumiko Isobe, Akiho Tsuchiya, Shiori Shikata, Naru Sato, Hironobu Morinaga, Tomofusa Fukuyama, Yosuke Tanaka, Tsuyoshi Fukushima, Reina Takeda, Keita Yamamoto, Hiroaki Honda, Emi K Nishimura, Yoichi Furukawa, Tatsuhiro Shibata, Omar Abdel-Wahab, Makoto Suematsu, Toshio Kitamura.

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.

DOI: https://doi.org/10.1038/s41467-021-22053-y
Semin Liver Dis. 2021 Jan;41(1): 50-66

Cellular Senescence in Liver Disease and Regeneration.

Sofia Ferreira-Gonzalez, Daniel Rodrigo-Torres, Victoria L Gadd, Stuart J Forbes.

Cellular senescence is an irreversible cell cycle arrest implemented by the cell as a result of stressful insults. Characterized by phenotypic alterations, including secretome changes and genomic instability, senescence is capable of exerting both detrimental and beneficial processes. Accumulating evidence has shown that cellular senescence plays a relevant role in the occurrence and development of liver disease, as a mechanism to contain damage and promote regeneration, but also characterizing the onset and correlating with the extent of damage. The evidence of senescent mechanisms acting on the cell populations of the liver will be described including the role of markers to detect cellular senescence. Overall, this review intends to summarize the role of senescence in liver homeostasis, injury, disease, and regeneration.

DOI: https://doi.org/10.1055/s-0040-1722262
Cell Mol Immunol. 2021 Mar 25.

miR-23a/b suppress cGAS-mediated innate and autoimmunity.

Qiuya Yu, Lei Chu, Yongxing Li, Quanyi Wang, Juanjuan Zhu, Chen Wang, Shufang Cui.

  Cyclic GMP-AMP synthase (cGAS), a key sensor of intracellular DNA, is essential for eliciting innate immunity against infection, whereas aberrant activation of cGAS by endogenous DNA promotes severe autoimmune diseases. However, it is largely unknown how cGAS expression is regulated during pathogen infection and autoimmunity. Here, we report that during herpes simplex virus type 1 (HSV-1) infection, two microRNAs (miR-23a and miR-23b) whose levels significantly decrease due to their interaction with the lncRNA Oasl2-209 directly regulate the expression of cGAS. Overexpression of miR-23a/b markedly dampens cytosolic DNA-induced innate immune responses, whereas inhibition of miR-23a/b enhances these responses. Mice treated with miR-23a/b agomirs exhibit increased susceptibility to HSV-1 infection. Moreover, cGAS is significantly upregulated in the Trex1-/- mouse autoimmune disease model. Administration of miR-23a/b blunts self DNA-induced autoinflammatory responses in Trex1-/- mice. Collectively, our study not only reveals a novel regulatory mechanism of cGAS expression by miRNAs but also identifies a potential therapy for cGAS-related autoimmune diseases.

Keywords:  Autoimmune disease; DNA virus; Innate immunity; cGAS; miR-23a; miR-23b

DOI:  https://doi.org/10.1038/s41423-021-00668-x
Nat Commun. 2021 03 23. 12(1): 1832

Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis.

Xiaonan Liu, Yu Chai, Guanqiao Liu, Weiping Su, Qiaoyue Guo, Xiao Lv, Peisong Gao, Bin Yu, Gerardo Ferbeyre, Xu Cao, Mei Wan.

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.

DOI: https://doi.org/10.1038/s41467-021-22131-1
Aging (Albany NY). 2021 03 22. 13

Chronic metformin treatment decreases cardiac injury during ischemia-reperfusion by attenuating endoplasmic reticulum stress with improved mitochondrial function.

Qun Chen, Jeremy Thompson, Ying Hu, Edward J Lesnefsky.

  Aging impairs mitochondrial function that leads to greater cardiac injury during ischemia and reperfusion. Cardiac endoplasm reticulum (ER) stress increases with age and contributes to mitochondrial dysfunction. Metformin is an anti-diabetic drug that protects cardiac mitochondria during acute ER stress. We hypothesized that metformin treatment would improve preexisting mitochondrial dysfunction in aged hearts by attenuating ER stress, followed by a decrease in cardiac injury during subsequent ischemia and reperfusion. Male young (3 mo.) and aged mice (24 mo.) received metformin (300 mg/kg/day) dissolved in drinking water with sucrose (0.2 g/100 ml) as sweetener for two weeks versus sucrose vehicle alone. Cytosol, subsarcolemmal (SSM), and interfibrillar mitochondria (IFM) were isolated. In separate groups, cardioprotection was evaluated using ex vivo isolated heart perfusion with 25 min. global ischemia and 60 min. reperfusion. Infarct size was measured. The contents of CHOP and cleaved ATF6 were decreased in metformin-treated 24 mo. mice compared to vehicle, supporting a decrease in ER stress. Metformin treatment improved OXPHOS in IFM in 24 mo. using a complex I substrate. Metformin treatment decreased infarct size following ischemia-reperfusion. Thus, metformin feeding decreased cardiac injury in aged mice during ischemia-reperfusion by improving pre-ischemic mitochondrial function via inhibition of ER stress.

Keywords:  electron transport chain; electron transport complex I; endoplasmic reticulum stress; mitochondrial permeability transition pore; myocardial infarction

DOI:  https://doi.org/10.18632/aging.202858
Biochem Soc Trans. 2021 Mar 26. pii: BST20200856. [Epub ahead of print]

The regulation of the DNA damage response at telomeres: focus on kinases.

Michela Galli, Chiara Frigerio, Maria Pia Longhese, Michela Clerici.

  The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.

Keywords:   Saccharomyces cerevisiae ; Mec1/ATR; Tel1/ATM; checkpoint; replicative senescence; telomeres

DOI:  https://doi.org/10.1042/BST20200856
Aging (Albany NY). 2021 Mar 19. 13

DNASE1L3 arrests tumor angiogenesis by impairing the senescence-associated secretory phenotype in response to stress.

Deliang Guo, Dong Ma, Pengpeng Liu, Jianwei Lan, Zhisu Liu, Quanyan Liu.

  Hepatocellular carcinoma (HCC) is one of the most challenging and aggressive cancers with limited treatment options because of tumor heterogeneity. Tumor angiogenesis is a hallmark of HCC and is necessary for tumor growth and progression. DNA damage stress and its associated deoxyribonuclease1-like 3 (DNASE1L3) are involved in HCC progression. Here, we explored the influence mechanism of DNASE1L3 on tumor angiogenesis under DNA damage stress in vitro and in vivo. DNASE1L3 was found downregulated and negatively correlated with poor prognosis of resectable and unresectable HCC patients. The tissue microarray of HCC revealed the negative association between DNASE1L3 and cancer vasculature invasion. Mechanistically, DNASE1L3 was found to relieve cytoplasmic DNA accumulation under DNA damage stress in HCC cell lines, in turn cell senescence and senescence-associated secretory phenotype were arrested via the p53 and NF-κB signal pathway, and hence, tumor angiogenesis was impaired. Furthermore, we found that DNASE1L3 excised these functions by translocating to the nucleus and interacting with H2BE under DNA damage stress using co-immunoprecipitation and fluorescence resonance energy transfer assay. In conclusion, DNASE1L3 inhibits tumor angiogenesis via impairing the senescence-associated secretory phenotype in response to DNA damage stress.

Keywords:  DNASE1L3; angiogenesis; hepatocellular carcinoma; senescence

DOI:  https://doi.org/10.18632/aging.202740
BMC Cancer. 2021 Mar 24. 21(1): 310

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells.

Akihiko Miyanaga, Masaru Matsumoto, Jessica A Beck, Izumi Horikawa, Takahiro Oike, Hirokazu Okayama, Hiromi Tanaka, Sandra S Burkett, Ana I Robles, Mohammed Khan, Delphine Lissa, Masahiro Seike, Akihiko Gemma, Hiroyuki Mano, Curtis C Harris.

   BACKGROUND: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells.
METHODS: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes.
RESULTS: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice.
CONCLUSIONS: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

Keywords:  Anchorage-independent growth; DNA damage; EML4-ALK; Lung cancer; Senescence; hTERT

DOI:  https://doi.org/10.1186/s12885-021-07905-6
Adv Exp Med Biol. 2021 Mar 22.

Opportunities and Challenges in Stem Cell Aging.

Bagher Larijani, Najmeh Foroughi-Heravani, Setareh Alaei, Mostafa Rezaei-Tavirani, Sepideh Alavi-Moghadam, Moloud Payab, Parisa Goodarzi, Akram Tayanloo-Beik, Hamid Reza Aghayan, Babak Arjmand.

  Studying aging, as a physiological process that can cause various pathological phenotypes, has attracted lots of attention due to its increasing burden and prevalence. Therefore, understanding its mechanism to find novel therapeutic alternatives for age-related disorders such as neurodegenerative and cardiovascular diseases is essential. Stem cell senescence plays an important role in aging. In the context of the underlying pathways, mitochondrial dysfunction, epigenetic and genetic alterations, and other mechanisms have been studied and as a consequence, several rejuvenation strategies targeting these mechanisms like pharmaceutical interventions, genetic modification, and cellular reprogramming have been proposed. On the other hand, since stem cells have great potential for disease modeling, they have been useful for representing aging and its associated disorders. Accordingly, the main mechanisms of senescence in stem cells and promising ways of rejuvenation, along with some examples of stem cell models for aging are introduced and discussed. This review aims to prepare a comprehensive summary of the findings by focusing on the most recent ones to shine a light on this area of research.

Keywords:  Aging; Model; Partial reprogramming; Rejuvenation; Stem cell

DOI:  https://doi.org/10.1007/5584_2021_624
Arch Biochem Biophys. 2021 Mar 22. pii: S0003-9861(21)00097-7. [Epub ahead of print] 108847

IL-1β induces expression of proinflammatory cytokines and migration of human colon cancer cells through upregulation of SIRT1.

Jaekyung Jung, Yeon-Hwa Lee, Xizhu Fang, Su-Jung Kim, Sung-Hoon Kim, Do-Hee Kim, Na-Young Song, Hye-Kyung Na, Jeong-Heum Baek, Young-Joon Surh.

  SIRT1 is a mammalian NAD+-dependent deacetylase, which is known to be involved in varous physiological events, such as adaptive response to environmental stresses including caloric restriction as well as in aging and cellular senescence. However, recent studies have revealed overexpression of SIRT1 in many different types of human malignancies, particularly colon cancer. Interleukin-1β (IL-1β) is a proinflammatory cytokine that plays a major role in invasiveness, stemness and progression of colon cancer. However, the interaction between IL-1β and SIRT1 in the tumor development and progression remains elusive. In this study, we found that IL-1β induces SIRT1 protein expression in human colon cancer HCT-116 cells. IL-1β-induced SIRT1 upregulation led to enhanced expression of mRNA transcripts of pro-inflammatory cytokines, IL-6 and IL-8. Knockdown of SIRT1 prevented IL-1β-induced nuclear accumulation as well as phosphorylation of c-Jun. Furthermore, pharmacologic inhibition of SIRT1 abrogated clonogenicity and migrative capability of human colon cancer cells stimulated with IL-1β. In summary, IL-1β-induced SIRT1 upregulation stimulates production of proinflammatory cytokines via a nuclear accumulation of c-Jun, leadng to colon cancer growth and progression.

Keywords:  Human colon cancer HCT-116 cells; IL-1β; Proinflammatory cytokines; SIRT1; c-Jun

DOI:  https://doi.org/10.1016/j.abb.2021.108847
Mol Ther. 2021 Mar 17. pii: S1525-0016(21)00143-X. [Epub ahead of print]

Targeting of miR-33 ameliorates phenotypes linked to age-related macular degeneration.

Gopalan Gnanaguru, Alexandre Wagschal, Justin Oh, Kahira L Saez-Torres, Tong Li, Ryan E Temel, Mark E Kleinman, Anders M Näär, Patricia A D'Amore.

  Abnormal cholesterol/lipid homeostasis is linked to neurodegenerative conditions such as age-related macular degeneration (AMD), which is a leading cause of blindness in the elderly. The most prevalent form, termed "dry" AMD, is characterized by pathological cholesterol accumulation beneath the retinal pigment epithelial (RPE) cell layer and inflammation-linked degeneration in the retina. We show here that the cholesterol-regulating microRNA miR-33 was elevated in the RPE of aging mice. Expression of the miR-33 target ATP-binding cassette transporter (ABCA1), a cholesterol efflux pump genetically linked to AMD, declined reciprocally in the RPE with age. In accord, miR-33 modulated ABCA1 expression and cholesterol efflux in human RPE cells. Subcutaneous delivery of miR-33 antisense oligonucleotides (ASO) to aging mice and non-human primates fed a Western-type high fat/cholesterol diet resulted in increased ABCA1 expression, decreased cholesterol accumulation, and reduced immune cell infiltration in the RPE cell layer, accompanied by decreased pathological changes to RPE morphology. These findings suggest that miR-33 targeting may decrease cholesterol deposition and ameliorate AMD initiation and progression.

Keywords:  ABCA1; age-related macular degeneration; cholesterol; geographic atrophy; inflammation; microRNA; retinal pigment epithelial cells

DOI:  https://doi.org/10.1016/j.ymthe.2021.03.014
Cardiovasc Res. 2021 Mar 23. pii: cvab114. [Epub ahead of print]

Gut microbiota dysbiosis promotes age-related atrial fibrillation by lipopolysaccharide and glucose-induced activation of NLRP3-inflammasome.

Yun Zhang, Song Zhang, Bolin Li, Yingchun Luo, Yongtai Gong, Xuexin Jin, Jiawei Zhang, Yun Zhou, Xiaozhen Zhuo, Zixi Wang, Xinbo Zhao, Xuejie Han, Yunlong Gao, Hui Yu, Desen Liang, Shiqi Zhao, Danghui Sun, Dingyu Wang, Wei Xu, Guangjin Qu, Wanlan Bo, Dan Li, Yue Wu, Yue Li.

   AIMS: Aging is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). The gut microbiota dysbiosis is involved in age-related diseases. However, whether the aged-associated dysbiosis contributes to age-related AF is still unknown. Direct demonstration that the aged gut microbiota is sufficient to transmit the enhanced AF susceptibility in a young host via microbiota-intestinal barrier-atria axis has not yet been reported. This study aimed to determine whether gut microbiota dysbiosis affects age-related AF.
METHODS AND RESULTS: Herein, by using a fecal microbiota transplantation (FMT) rat model, we demonstrated that the high AF susceptibility of aged rats could be transmitted to a young host. Specially, we found the dramatically increased levels of circulating lipopolysaccharide (LPS) and glucose led to the up-regulated expression of NLR family pyrin domain containing 3 (NLRP3)-inflammasome, promoting the development of AF which depended on the enhanced atrial fibrosis in recipient host. Inhibition of inflammasome by a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, resulted in a lower atrial fibrosis and AF susceptibility. Then we conducted cross-sectional clinical studies to explore the effect of aging on the altering trends with glucose levels and circulating LPS among clinical individuals in two China hospitals. We found that both of serum LPS and glucose levels were progressively increased in elderly patients as compared with those young. Furthermore, the aging phenotype of circulating LPS and glucose levels, intestinal structure and atrial NLRP3-inflammasome of rats were also confirmed in clinical AF patients. Finally, aged rats colonized with youthful microbiota restored intestinal structure and atrial NLRP3-inflammasome activity, which suppressed the development of aged-related AF.
CONCLUSIONS: Collectively, these studies described a novel causal role of aberrant gut microbiota in the pathogenesis of age-related AF, which indicates that the microbiota-intestinal barrier-atrial NLRP3 inflammasome axis may be a rational molecular target for the treatment of aged-related arrhythmia disease.
TRANSLATIONAL PERSPECTIVE: The current study demonstrates that aged-associated microbiota dysbiosis promotes AF in part through a microbiota-gut-atria axis. Increased AF susceptibility due to enhanced atrial NLRP3-inflammasome activity by LPS and high glucose was found in an aged FMT rat model, and also confirmed within elderly clinical individuals. In a long-term FMT rat study, the AF susceptibility was ameliorated by treatment with youthful microbiota. The present findings can further increase our understanding of aged-related AF and address a promising therapeutic strategy that involves modulation of gut microbiota composition.

Keywords:  Aging; Atrial fibrillation; Gut microbiota; Lipopolysaccharide; NLRP3-inflammasome

DOI:  https://doi.org/10.1093/cvr/cvab114
Front Cell Dev Biol. 2021 ;9 637424

Senescent T Cell Induces Brown Adipose Tissue "Whitening" Via Secreting IFN-γ.

Xiao-Xi Pan, Kang-Li Yao, Yong-Feng Yang, Qian Ge, Run Zhang, Ping-Jin Gao, Cheng-Chao Ruan, Fang Wu.

  Aging-associated chronic inflammation is a key contributing factor to a cluster of chronic metabolic disorders, such as cardiovascular disease, obesity, and type 2 diabetes. Immune cells particularly T cells accumulate in adipose tissue with advancing age, and there exists a cross talk between T cell and preadipocyte, contributing to age-related adipose tissue remodeling. Here, we compared the difference in morphology and function of adipose tissue between young (3-month-old) and old (18-month-old) mice and showed the phenomenon of brown adipose tissue (BAT) "whitening" in old mice. Flow cytometry analysis suggested an increased proportion of T cells in BAT of old mice comparing with the young and exhibited senescent characteristics. We take advantage of coculture system to demonstrate directly that senescent T cells inhibited brown adipocyte differentiation of preadipocytes in adipose tissue. Mechanistically, both in vitro and in vivo studies suggested that senescent T cells produced and released a higher level of IFN-γ, which plays a critical role in inhibition of preadipocyte-to-brown adipocyte differentiation. Taken together, the data indicate that senescent T cell-derived IFN-γ is a key regulator in brown adipocyte differentiation.

Keywords:  IFN-γ; T cell; adipose tissue; brown adipocyte differentiation; preadipocyte; senescence

DOI:  https://doi.org/10.3389/fcell.2021.637424
Aging (Albany NY). 2021 Mar 19. 13

miR-30c-1 encourages human corneal endothelial cells to regenerate through ameliorating senescence.

Younghwan Bae, Jin Sun Hwang, Young Joo Shin.

  In the present study, we studied the role of microRNA-30c-1 (miR-30c-1) on transforming growth factor beta1 (TGF-β1)-induced senescence of hCECs. hCECs were transfected by miR-30c-1 and treated with TGF-β1 to assess the inhibitory effect of miR-30c-1 on TGF-β1-induced senescence. Cell viability and proliferation rate in miR-30c-1-transfected cells was elevated compared with control. Cell cycle analysis revealed that cell abundance in S phase was elevated in miR-30c-1-treated cells compared with control. TGF-β1 increased the senescence of hCECs; however, this was ameliorated by miR-30c-1. TGF-β1 increased the size of hCECs, the ratio of senescence-associated beta-galactosidase-stained cells, secretion of senescence-associated secretory phenotype factors, the oxidative stress, and arrested the cell cycle, all of which were ameliorated by miR-30c-1 treatment. miR-30c-1 also suppressed a TGF-β1-induced depolarization of mitochondrial membrane potential and a TGF-β1 stimulated increase in levels of cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase 3, and microtubule-associated proteins 1A/1B light chain 3B II. In conclusion, miR-30c-1 promoted the proliferation of hCECs through ameliorating the TGF- β1-induced senescence of hCECs and reducing cell death of hCECs. Thus, miR-30c-1 may be a therapeutic target for hCECs regeneration.

Keywords:  TGF-β; human corneal endothelial cells; miR-30c-1; proliferation; senescence

DOI:  https://doi.org/10.18632/aging.202719
Theranostics. 2021 ;11(10): 4710-4727

Deficiency of telomere-associated repressor activator protein 1 precipitates cardiac aging in mice via p53/PPARα signaling.

Yin Cai, Hao Liu, Erfei Song, Lin Wang, Jindong Xu, Yi He, Dengwen Zhang, Liyan Zhang, Kenneth King-Yip Cheng, Leigang Jin, Min Wu, Shiming Liu, Dake Qi, Liangqing Zhang, Gary D Lopaschuk, Sheng Wang, Aimin Xu, Zhengyuan Xia.

  Background: Telomere shortening and dysfunction may cause metabolic disorders, tissue damage and age-dependent pathologies. However, little is known about the association of telomere-associated protein Rap1 with mitochondrial energy metabolism and cardiac aging. Methods: Echocardiography was performed to detect cardiac structure and function in Rap1+/+ and Rap1-/- mice at different ages (3 months, 12 months and 20 months). Telomere length, DNA damage, cardiac senescence and cardiomyocyte size were analyzed using the real-time PCR, Western blotting, senescence associated β-galactosidase assay and wheat germ agglutinin staining, respectively. Western blotting was also used to determine the level of cardiac fatty acid metabolism related key enzymes in mouse and human myocardium. Chromatin immunoprecipitation assay was used to verify the direct link between p53 and PPARα. The p53 inhibitor, Pifithrin-α and PPARα activator WY14643 were utilized to identify the effects of Rap1/p53/PPARα signaling pathway. Results: Telomere was shortened concomitant with extensive DNA damage in aged Rap1-/- mouse hearts, evidenced by reduced T/S ratios and increased nuclear γH2AX. Meanwhile, the aging-associated phenotypes were pronounced as reflected by altered mitochondrial ultrastructure, enhanced senescence, cardiac hypertrophy and dysfunction. Mechanistically, acetylated p53 and nuclear p53 was enhanced in the Rap1-/- mouse hearts, concomitant with reduced PPARα. Importantly, p53 directly binds to the promoter of PPARα in mouse hearts and suppresses the transcription of PPARα. In addition, aged Rap1-/- mice exhibited reduced cardiac fatty acid metabolism. Pifithrin-α alleviated cardiac aging and enhanced fatty acid metabolism in the aged Rap1-/- mice. Activating PPARα with WY14643 in primarily cultured Rap1-/- cardiomyocytes restored maximal oxygen consumption rates. Reduced Rap1 expression and impaired p53/PPARα signaling also presented in aged human myocardium. Conclusion: In summary, Rap1 may link telomere biology to fatty acid metabolism and aging-related cardiac pathologies via modulating the p53/PPARα signaling pathway, which could represent a therapeutic target in preventing/attenuating cardiac aging.

Keywords:  PPARα; Rap1; cardiac aging; fatty acid metabolism; p53

DOI:  https://doi.org/10.7150/thno.51739
Autophagy. 2021 Mar 22. 1-17

CGAS is a micronucleophagy receptor for the clearance of micronuclei.

Mengmeng Zhao, Fei Wang, Juehui Wu, Yuanna Cheng, Yajuan Cao, Xiangyang Wu, Mingtong Ma, Fen Tang, Zhi Liu, Haipeng Liu, Baoxue Ge.

  Micronuclei are constantly considered as a marker of genome instability and very recently found to be a trigger of innate immune responses. An increased frequency of micronuclei is associated with many diseases, but the mechanism underlying the regulation of micronuclei homeostasis remains largely unknown. Here, we report that CGAS (cyclic GMP-AMP synthase), a known regulator of DNA sensing and DNA repair, reduces the abundance of micronuclei under genotoxic stress in an autophagy-dependent manner. CGAS accumulates in the autophagic machinery and directly interacts with MAP1LC3B/LC3B in a manner dependent upon its MAP1LC3-interacting region (LIR). Importantly, the interaction is essential for MAP1LC3 recruitment to micronuclei and subsequent clearance of micronuclei via autophagy (micronucleophagy) in response to genotoxic stress. Moreover, in contrast to its DNA sensing function to activate micronuclei-driven inflammation, CGAS-mediated micronucleophagy blunts the production of cyclic GMP-AMP (cGAMP) induced by genotoxic stress. We therefore conclude that CGAS is a receptor for the selective autophagic clearance of micronuclei and uncovered an unprecedented role of CGAS in micronuclei homeostasis to dampen innate immune surveillance.

Keywords:  CGAS; MAP1LC3B/LC3B; autophagy receptor; micronuclei; micronucleophagy

DOI:  https://doi.org/10.1080/15548627.2021.1899440
Aging (Albany NY). 2021 Mar 23.

Lipid droplets meet aging.

Thomas Felder, Florian Geltinger, Mark Rinnerthaler.



Keywords:  aggregates; cancer; lipid droplets; mitochondria; protein homeostasis

DOI:  https://doi.org/10.18632/aging.202883
Brain Res Bull. 2021 Mar 18. pii: S0361-9230(21)00086-1. [Epub ahead of print]

cGAS knockdown promotes microglial M2 polarization to alleviate neuroinflammation by inhibiting cGAS-STING signaling pathway in cerebral ischemic stroke.

Guo-Liang Jiang, Xing-Long Yang, Hou-Jun Zhou, Jiang Long, Bin Liu, Lin-Ming Zhang, Di Lu.

  Inflammation plays a pivotal role in promoting the pathophysiology of ischemic stroke (IS). Microglia is the major immunocompetent cells involved in different neuropathologies. The activation of cyclic GMP-AMP synthase (cGAS) and its downstream signaling protein-stimulator of interferon genes (STING) is increasingly recognized as a crucial determinant of neuropathophysiology. However, the mechanisms underlying cGAS-STING signaling regulating inflammatory response during IS remains to be elucidated. In this study, HT22 cells was used to establish an oxygen-glucose deprivation (OGD) cell model in vitro, and then this cell culture supernatant containing OGD-induced DAMPs (OIDs) was employed to stimulate BV2 microglia. Furthermore, a middle cerebral artery occlusion (MCAO) mouse model was established. Cells and MCAO mice were treated with si-cGAS or si-NC lentivirus. The expression levels of STING, cGAS and p-IRF3 in BV2 cells or MCAO mouse brain; the microglial M1/M2 polarization of BV2 microglia or isolated microglial cells from MCAO mouse brain; the contents of iNOS, TNF-α, TGF-β and IL-10 in the culture medium of BV2 cells or in murine brain homogenates, were all detected. In addition, the severity of cerebral infarction with or without the knockdown of cGAS in a MCAO mouse model was also determined by TTC staining. Results showed that OGD-induced DAMPs strongly activated cGAS-STING pathway and triggered microglia polarization in BV2 cells, reflecting as the accumulation of a plethora of pro-inflammatory factors in activated microglia. However, these effects could be inhibited by cGAS knockdown. In the MCAO mouse model, the inhibition of cGAS-STING pathway resulted from cGAS knockdown could effectively diminish cell apoptosis in mouse brain stimulated by MIDs (MCAO-induced DAMPs), reduced the area ratio of cerebral infarction and ultimately improved the injured nerve function during IS. Taken together, our elucidation of underlying mechanisms involved in the microglial inflammatory response, triggered by cGAS-STING signaling, highlights this pathway as a potential therapeutic target in IS.

Keywords:  Cerebral ischemic stroke; Microglial M1/M2 polarization; Neuroinflammation; cGAS-STING pathway

DOI:  https://doi.org/10.1016/j.brainresbull.2021.03.010