Hesperetin promotes longevity and delays aging via activation of Cisd2 in naturally aged mice.

BACKGROUND: The human CISD2 gene is located within a longevity region mapped on chromosome 4q. In mice, Cisd2 levels decrease during natural aging and genetic studies have shown that a high level of Cisd2 prolongs mouse lifespan and healthspan. Here, we evaluate the feasibility of using a Cisd2 activator as an effective way of delaying aging. METHODS: Hesperetin was identified as a promising Cisd2 activator by herb compound library screening. Hesperetin has no detectable toxicity based on in vitro and in vivo models. Naturally aged mice fed dietary hesperetin were used to investigate the effect of this Cisd2 activator on lifespan prolongation and the amelioration of age-related structural defects and functional decline. Tissue-specific Cisd2 knockout mice were used to study the Cisd2-dependent anti-aging effects of hesperetin. RNA sequencing was used to explore the biological effects of hesperetin on aging. RESULTS: Three discoveries are pinpointed. Firstly, hesperetin, a promising Cisd2 activator, when orally administered late in life, enhances Cisd2 expression and prolongs healthspan in old mice. Secondly, hesperetin functions mainly in a Cisd2-dependent manner to ameliorate age-related metabolic decline, body composition changes, glucose dysregulation, and organ senescence. Finally, a youthful transcriptome pattern is regained after hesperetin treatment during old age. CONCLUSIONS: Our findings indicate that a Cisd2 activator, hesperetin, represents a promising and broadly effective translational approach to slowing down aging and promoting longevity via the activation of Cisd2.

Cisd2 is essential to delaying cardiac aging and to maintaining heart functions.

CDGSH iron-sulfur domain-containing protein 2 (Cisd2) is pivotal to mitochondrial integrity and intracellular Ca2+ homeostasis. In the heart of Cisd2 knockout mice, Cisd2 deficiency causes intercalated disc defects and leads to degeneration of the mitochondria and sarcomeres, thereby impairing its electromechanical functioning. Furthermore, Cisd2 deficiency disrupts Ca2+ homeostasis via dysregulation of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) activity, resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay cardiac aging and attenuate age-related structural defects and functional decline. In addition, it results in a younger cardiac transcriptome pattern during old age. Our findings indicate that Cisd2 plays an essential role in cardiac aging and in the heart's electromechanical functioning. They highlight Cisd2 as a novel drug target when developing therapies to delay cardiac aging and ameliorate age-related cardiac dysfunction.

RNA Modifications and Epigenetics in Modulation of Lung Cancer and Pulmonary Diseases.

Lung cancer is the leading cause of cancer-related mortality worldwide, and its tumorigenesis involves the accumulation of genetic and epigenetic events in the respiratory epithelium. Epigenetic modifications, such as DNA methylation, RNA modification, and histone modifications, have been widely reported to play an important role in lung cancer development and in other pulmonary diseases. Whereas the functionality of DNA and chromatin modifications referred to as epigenetics is widely characterized, various modifications of RNA nucleotides have recently come into prominence as functionally important. N6-methyladosine (m6A) is the most prevalent internal modification in mRNAs, and its machinery of writers, erasers, and readers is well-characterized. However, several other nucleotide modifications of mRNAs and various noncoding RNAs have also been shown to play an important role in the regulation of biological processes and pathology. Such epitranscriptomic modifications play an important role in regulating various aspects of RNA metabolism, including transcription, translation, splicing, and stability. The dysregulation of epitranscriptomic machinery has been implicated in the pathological processes associated with carcinogenesis including uncontrolled cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In recent years, with the advancement of RNA sequencing technology, high-resolution maps of different modifications in various tissues, organs, or disease models are being constantly reported at a dramatic speed. This facilitates further understanding of the relationship between disease development and epitranscriptomics, shedding light on new therapeutic possibilities. In this review, we summarize the basic information on RNA modifications, including m6A, m1A, m5C, m7G, pseudouridine, and A-to-I editing. We then demonstrate their relation to different kinds of lung diseases, especially lung cancer. By comparing the different roles RNA modifications play in the development processes of different diseases, this review may provide some new insights and offer a better understanding of RNA epigenetics and its involvement in pulmonary diseases.

Tumor Necrosis Factor-Alpha Exacerbates Viral Entry in SARS-CoV2-Infected iPSC-Derived Cardiomyocytes.

The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.

Exercise and the Cisd2 Prolongevity Gene: Two Promising Strategies to Delay the Aging of Skeletal Muscle.

Aging is an evolutionally conserved process that limits life activity. Cellular aging is the result of accumulated genetic damage, epigenetic damage and molecular exhaustion, as well as altered inter-cellular communication; these lead to impaired organ function and increased vulnerability to death. Skeletal muscle constitutes ~40% of the human body's mass. In addition to maintaining skeletal structure and allowing locomotion, which enables essential daily activities to be completed, skeletal muscle also plays major roles in thermogenesis, metabolism and the functioning of the endocrine system. Unlike many other organs that have a defined size once adulthood is reached, skeletal muscle is able to alter its structural and functional properties in response to changes in environmental conditions. Muscle mass usually remains stable during early life; however, it begins to decline at a rate of ~1% year in men and ~0.5% in women after the age of 50 years. On the other hand, different exercise training regimens are able to restore muscle homeostasis at the molecular, cellular and organismal levels, thereby improving systemic health. Here we give an overview of the molecular factors that contribute to lifespan and healthspan, and discuss the effects of the longevity gene Cisd2 and middle-to-old age exercise on muscle metabolism and changes in the muscle transcriptome in mice during very old age.

Expression of a hepatitis B virus pre-S2 deletion mutant in the liver results in hepatomegaly and hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and has a poor prognosis and a low survival rate; its incidence is on the rise. Hepatitis B virus (HBV) infection is one of the main causes of HCC. A high prevalence of pre-S deletions of HBV surface antigen, which encompass T-cell and/or B-cell epitopes, is found in HBV carriers; antiviral therapy and viral immune escape may cause and select for these HBV mutants. In particular, the presence of pre-S2 deletion mutants is an important risk factor associated with cirrhosis and HCC. We generated Alb-preΔS2 transgenic mice that express a naturally occurring pre-S2 mutant protein containing a 33-nucleotide deletion (preΔS2); the aim was to investigate its effect on hepatocarcinogenesis. After 30 months of follow-up, the liver pathology of the mice fell into four groups: G1, chronic inflammation solely; G2, chronic inflammation and fibrosis; G3, inflammation, fibrosis, and hepatomegaly accompanied by rectal prolapse (4-12%); and G4, hepatomegaly and spontaneous HCC (12-15%). Striking degeneration of the endoplasmic reticulum (ER) was present in the mouse livers at an early stage (4 months old). At 8 months, overt ER stress and the Atf6 pathway of the unfolded protein response (UPR) were induced; at the same time, metabolic pathways associated with mevalonate and cholesterol biogenesis, involving the peroxisomes and the ER, were disturbed. At 20 months and older, the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway of the UPR was induced and the Hippo transducer Yap was activated. Together, these ultrastructural aberrations and metabolic disturbance all seem to contribute to the molecular pathogenesis and hepatocarcinogenesis present in the Alb-preΔS2 mice. These findings may contribute to the development of therapies for the liver disorders and HCC associated with pre-S2 deletion mutations among HBV carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Polygenic Risk Score Improves Cataract Prediction in East Asian Population.

Cataracts, characterized by crystalline lens opacities in human eyes, is the leading cause of blindness globally. Due to its multifactorial complexity, the molecular mechanisms remain poorly understood. Larger cohorts of genome-wide association studies (GWAS) are needed to investigate cataracts' genetic basis. In this study, a GWAS was performed on the largest Han population to date, analyzing a total of 7079 patients and 13,256 controls from the Taiwan Biobank (TWB) 2.0 cohort. Two cataract-associated SNPs with an adjustment of p < 1 × 10−7 in the older groups and nine SNPs with an adjustment of p < 1 × 10−6 in the younger group were identified. Except for the reported AGMO in animal models, most variations, including rs74774546 in GJA1 and rs237885 in OXTR, were not identified before this study. Furthermore, a polygenic risk score (PRS) was created for the young and old populations to identify high-risk cataract individuals, with areas under the receiver operating curve (AUROCs) of 0.829 and 0.785, respectively, after covariate adjustments. Younger individuals had 17.45 times the risk while older people had 10.97 times the risk when comparing individuals in the highest and lowest PRS quantiles. Validation analysis on an independent TWB1.0 cohort revealed AUROCs of 0.744 and 0.659.

Circular RNAs Modulate Cancer Hallmark and Molecular Pathways to Support Cancer Progression and Metastasis.

Circular RNAs (circRNAs) are noncoding products of backsplicing of pre-mRNAs which have been established to possess potent biological functions. Dysregulated circRNA expression has been linked to diseases including different types of cancer. Cancer progression is known to result from the dysregulation of several molecular mechanisms responsible for the maintenance of cellular and tissue homeostasis. The dysregulation of these processes is defined as cancer hallmarks, and the molecular pathways implicated in them are regarded as the targets of therapeutic interference. In this review, we summarize the literature on the investigation of circRNAs implicated in cancer hallmark molecular signaling. First, we present general information on the properties of circRNAs, such as their biogenesis and degradation mechanisms, as well as their basic molecular functions. Subsequently, we summarize the roles of circRNAs in the framework of each cancer hallmark and finally discuss the potential as therapeutic targets.

CISD2 maintains cellular homeostasis.

CDGSH Iron Sulfur Domain 2 (CISD2) is the causative gene for the disease Wolfram syndrome 2 (WFS2; MIM 604928), which is an autosomal recessive disorder showing metabolic and neurodegenerative manifestations. CISD2 protein can be localized on the endoplasmic reticulum (ER), outer mitochondrial membrane (OMM) and mitochondria-associated membrane (MAM). CISD2 plays a crucial role in the regulation of cytosolic Ca2+ homeostasis, ER integrity and mitochondrial function. Here we summarize the most updated publications and discuss the central role of CISD2 in maintaining cellular homeostasis. This review mainly focuses on the following topics. Firstly, that CISD2 has been recognized as a prolongevity gene and the level of CISD2 is a key determinant of lifespan and healthspan. In mice, Cisd2 deficiency shortens lifespan and accelerates aging. Conversely, a persistently high level of Cisd2 promotes longevity. Intriguingly, exercise stimulates Cisd2 gene expression and thus, the beneficial effects offered by exercise may be partly related to Cisd2 activation. Secondly, that Cisd2 is down-regulated in a variety of tissues and organs during natural aging. Three potential mechanisms that may mediate the age-dependent decrease of Cisd2, via regulating at different levels of gene expression, are discussed. Thirdly, the relationship between CISD2 and cell survival, as well as the potential mechanisms underlying the cell death control, are discussed. Finally we discuss that, in cancers, CISD2 may functions as a double-edged sword, either suppressing or promoting cancer development. This review highlights the importance of the CISD2 in aging and age-related diseases and identifies the urgent need for the translation of available genetic evidence into pharmaceutic interventions in order to alleviate age-related disorders and extend a healthy lifespan in humans.

Cisd2 Protects the Liver from Oxidative Stress and Ameliorates Western Diet-Induced Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) and its more severe form, nonalcoholic steatohepatitis (NASH), are the most common chronic liver diseases worldwide. However, drugs to treat NAFLD and NASH are an unmet clinical need. This study sought to provide evidence that Cisd2 is a molecular target for the development of treatments targeting NAFLD and NASH. Several discoveries are pinpointed. The first is that Cisd2 dosage modulates the severity of Western diet-induced (WD-induced) NAFLD. Specifically, Cisd2 haploinsufficiency accelerates NAFLD development and exacerbates progression toward NASH. Conversely, an enhanced Cisd2 copy number attenuates liver pathogenesis. Secondly, when a WD is fed to mice, transcriptomic analysis reveals that the major alterations affecting biological processes are related to inflammation, lipid metabolism, and DNA replication/repair. Thirdly, among these differentially expressed genes, the most significant changes involve Nrf2-mediated oxidative stress, cholesterol biosynthesis, and fatty acid metabolism. Finally, increased Cisd2 expression protects the liver from oxidative stress and reduces the occurrence of mitochondrial DNA deletions. Taken together, our mouse model reveals that Cisd2 plays a crucial role in protecting the liver from WD-induced damages. The development of therapeutic agents that effectively enhance Cisd2 expression is one potential approach to the treatment of WD-induced fatty liver diseases.

Evaluating the biocompatibility and biological performance of FePt-decorated nano-epoxy nanoparticles in mesenchymal stem cells.

BACKGROUND: Nanoparticles have wide potential applications in biolabeling, bioimaging, and cell tracking. Development of dual functional nanoparticles increases the versatility. METHODS: We combined the fluorescent property of nano-epoxy (N-Epo) and the magnetic characteristic of FePt to fabricate the FePt-decorated N-Epo (N-Epo-FePt). The size in diameter of N-Epo-FePt (177.38 ± 39.25 nm) was bigger than N-Epo (2.28 ± 1.01 nm), both could be absorbed into mesenchymal stem cells (MSCs) via clathrin-mediated endocytosis and have multiple fluorescent properties (blue, green, and red). RESULTS: N-Epo-FePt prevented N-Epo-induced platelet activation, CD68+-macrophage differentiation in blood, and intracellular ROS generation in MSCs. The induction of apoptosis and the inhibitory effects of N-Epo-FePt on cell migration, MMP-9 activity, and secretion of SDF-1α were less than that of N-Epo in MSCs. CONCLUSION: N-Epo-FePt was more biocompatible without altering biological performance than N-Epo in MSCs. These results suggest that N-Epo-FePt nanoparticle can be used for fluorescence labeling of MSCs and is potential to apply to bioimaging and cell tracking of MSCs in vivo by magnetic resonance imaging or computed tomography.

Analyses of the pericyte transcriptome in ischemic skeletal muscles.

BACKGROUND: Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients' life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions. METHODS: In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing. RESULTS: Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells. CONCLUSIONS: Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.

Progerin in muscle leads to thermogenic and metabolic defects via impaired calcium homeostasis.

Mutations in lamin A (LMNA) are responsible for a variety of human dystrophic and metabolic diseases. Here, we created a mouse model in which progerin, the lamin A mutant protein that causes Hutchinson-Gilford progeria syndrome (HGPS), can be inducibly overexpressed. Muscle-specific overexpression of progerin was sufficient to induce muscular dystrophy and alter whole-body energy expenditure, leading to premature death. Intriguingly, sarcolipin (Sln), an endoplasmic reticulum (ER)-associated protein involved in heat production, is upregulated in progerin-expressing and Lmna knockout (Lmna-/- ) skeletal muscle. The depletion of Sln accelerated the early death of Lmna-/- mice. An examination at the molecular level revealed that progerin recruits Sln and Calnexin to the nuclear periphery. Furthermore, progerin-expressing myoblasts presented enhanced store-operated Ca2+ entry, as well as increased co-localization of STIM1 and ORAI1. These findings suggest that progerin dysregulates calcium homeostasis through an interaction with a subset of ER-associated proteins, resulting in thermogenic and metabolic abnormalities.

Muscle atrophy-related myotube-derived exosomal microRNA in neuronal dysfunction: Targeting both coding and long noncoding RNAs.

In mammals, microRNAs can be actively secreted from cells to blood. miR-29b-3p has been shown to play a pivotal role in muscle atrophy, but its role in intercellular communication is largely unknown. Here, we showed that miR-29b-3p was upregulated in normal and premature aging mouse muscle and plasma. miR-29b-3p was also upregulated in the blood of aging individuals, and circulating levels of miR-29b-3p were negatively correlated with relative appendicular skeletal muscle. Consistently, miR-29b-3p was observed in exosomes isolated from long-term differentiated atrophic C2C12 cells. When C2C12-derived miR-29b-3p-containing exosomes were uptaken by neuronal SH-SY5Y cells, increased miR-29b-3p levels in recipient cells were observed. Moreover, miR-29b-3p overexpression led to downregulation of neuronal-related genes and inhibition of neuronal differentiation. Interestingly, we identified HIF1α-AS2 as a novel c-FOS targeting lncRNA that is induced by miR-29b-3p through down-modulation of c-FOS and is required for miR-29b-3p-mediated neuronal differentiation inhibition. Our results suggest that atrophy-associated circulating miR-29b-3p may mediate distal communication between muscle cells and neurons.

Comparative proteomic profiling reveals a role for Cisd2 in skeletal muscle aging.

Skeletal muscle has emerged as one of the most important tissues involved in regulating systemic metabolism. The gastrocnemius is a powerful skeletal muscle composed of predominantly glycolytic fast-twitch fibers that are preferentially lost among old age. This decrease in gastrocnemius muscle mass is remarkable during aging; however, the underlying molecular mechanism is not fully understood. Strikingly, there is a ~70% decrease in Cisd2 protein, a key regulator of lifespan in mice and the disease gene for Wolfram syndrome 2 in humans, within the gastrocnemius after middle age among mice. A proteomics approach was used to investigate the gastrocnemius of naturally aged mice, and this was compared to the autonomous effect of Cisd2 on gastrocnemius aging using muscle-specific Cisd2 knockout (mKO) mice as a premature aging model. Intriguingly, dysregulation of calcium signaling and activation of UPR/ER stress stand out as the top two pathways. Additionally, the activity of Serca1 was significantly impaired and this impairment is mainly attributable to irreversibly oxidative modifications of Serca. Our results reveal that the overall characteristics of the gastrocnemius are very similar when naturally aged mice and the Cisd2 mKO mice are compared in terms of pathological alterations, ultrastructural abnormalities, and proteomics profiling. This suggests that Cisd2 mKO mouse is a unique model for understanding the aging mechanism of skeletal muscle. Furthermore, this work substantiates the hypothesis that Cisd2 is crucial to the gastrocnemius muscle and suggests that Cisd2 is a potential therapeutic target for muscle aging.

Hepatocellular carcinoma mouse models: Hepatitis B virus-associated hepatocarcinogenesis and haploinsufficient tumor suppressor genes.

The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine.

Sorafenib Action in Hepatitis B Virus X-Activated Oncogenic Androgen Pathway in Liver through SHP-1.

BACKGROUND: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) shows a higher incidence in men, mainly because of hepatitis B X (HBx)-mediated enhancement of androgen receptor (AR) activity. We aimed to examine this pathway in hepatocarcinogenesis and to identify drug(s) specifically blocking this carcinogenic event in the liver. METHODS: HBx transgenic mice that spontaneously develop HCC (n = 28-34 per group) were used, either by knockout of hepatic AR or by castration. Efficacy of several HCC-targeted drugs in suppressing HBx-induced AR activity was evaluated, and cellular factors mediating suppression were investigated in cultured cells. Tissue specificity of the candidate drug was validated using mouse tissues. Data were analyzed with Chi-square and Student's t tests. All statistical tests were two-sided. RESULTS: The androgen pathway was shown to be important in early stage hepatocarcinogenesis of HBx transgenic mice. The tumor incidence was decreased from 80% to 32% by AR knockout (P < .001) and from 90% to 25% by early castration (P < .001). Sorafenib markedly inhibited the HBx-enhanced AR activity through activating the SHP-1 phosphatase, which antagonized the activation of Akt/GSK3ß and c-Src by HBx. Moreover, SHP-1 protein level was much higher in the liver than in testis, which enabled sorafenib to inhibit aberrant AR activity in the HBx-expressing liver, while not affecting the physiological AR function in normal liver or testis. CONCLUSIONS: The androgen pathway may be a druggable target for the chemoprevention of HBV-related HCC, and sorafenib might be used as a tissue- and disease-specific regimen for the chemoprevention of HBV-related HCC.