Publications
TREX2 Exonuclease Causes Spontaneous Mutations and Stress-Induced Replication Fork Defects in Cells Expressing RAD51K133A
Jun Ho Ko, Mi Young Son, Qing Zhou, Lucia Molnarova, Lambert Song, Jarmila Mlcouskova, Atis Jekabsons, Cristina Montagna, Lumir Krejci, Paul Hasty
Cell Reports. 2020 Dec 22;33(12):108543. doi: 10.1016/j.celrep.2020.108543.
Abstract:
DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.
Keywords: DNA damage tolerance; double-strand break repair; genomic instability; homologous recombination; replication fork maintenance.
Development of a Geropathology Grading Platform for nonhuman primates
Katie J Olstad, Denise M Imai, Rebekah I Keesler, Rachel Reader, John H Morrison, Jeffery A Roberts, John P Capitanio, Elizabeth S Didier, Marcelo J Kuroda, Heather Simmons, Shabnam Salimi, Julie A Mattison, Yuji Ikeno, Warren Ladiges
Aging Pathobiol Ther. 2020;2(1):16-19. doi: 10.31491/apt.2020.03.008.
Abstract:
A geropathology grading platform (GGP) for assessing age-related lesions has been established and validated for in inbred strain of mice. Because nonhuman primates (NHPs) share significant similarities in aging and spontaneous chronic diseases with humans, they provide excellent translational value for correlating histopathology with biological and pathological events associated with increasing age. Descriptive age-associated pathology has been described for rhesus macaques and marmosets, but a grading platform similar to the mouse GGP does not exist. The value of these NHP models is enhanced by considerable historical data from clinical, bio-behavioral, and social domains that align with health span in these animals. Successful adaptation of the mouse GGP for NHPs will include 1) expanding the range of organs examined; 2) standardizing necropsy collection, tissue trimming, and descriptive lesion terminology; 3) expanding beyond rhesus macaques and marmosets to include other commonly used NHPs in research; and 4) creating a national resource for age-related pathology to complement the extensive in-life datasets. Adaptation of the GGP to include translational models other than mice will be crucial to advance geropathology designed to enhance aging research.
Keywords: Aging; age-related diseases; geropathology; geroscience; marmosets; mice; nonhuman primates; rhesus macaques.
The CCR4-NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression
Taku Ito-Kureha, Takahisa Miyao, Saori Nishijima, Toru Suzuki, Shin-Ichi Koizumi, Alejandro Villar-Briones, Akinori Takahashi, Nobuko Akiyama, Masahiro Morita, Isao Naguro, Hiroki Ishikawa, Hidenori Ichijo, Taishin Akiyama, Tadashi Yamamoto
Nat Commun. 2020 Dec 2;11(1):6169. doi: 10.1038/s41467-020-19975-4.
Abstract:
A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.
Hepatocyte-specific PKCβ deficiency protects against high-fat diet-induced nonalcoholic hepatic steatosis
Yaoling Sh, Faizule Hassan, Vincenzo Coppola, Kedryn K Baskin, Xianlin Han, Neil K Mehta, Michael C Ostrowski, Kamal D Mehta
Mol Metab. 2021 Feb:44:101133. doi: 10.1016/j.molmet.2020.101133. Epub 2020 Nov 30.
Abstract:
Objective: Nonalcoholic hepatic steatosis, also known as fatty liver, is a uniform response of the liver to hyperlipidic-hypercaloric diet intake. However, the post-ingestive signals and mechanistic processes driving hepatic steatosis are not well understood. Emerging data demonstrate that protein kinase C beta (PKCβ), a lipid-sensitive kinase, plays a critical role in energy metabolism and adaptation to environmental and nutritional stimuli. Despite its powerful effect on glucose and lipid metabolism, knowledge of the physiological roles of hepatic PKCβ in energy homeostasis is limited.
Methods: The floxed-PKCβ and hepatocyte-specific PKCβ-deficient mouse models were generated to study the in vivo role of hepatocyte PKCβ on diet-induced hepatic steatosis, lipid metabolism, and mitochondrial function.
Results: We report that hepatocyte-specific PKCβ deficiency protects mice from development of hepatic steatosis induced by high-fat diet, without affecting body weight gain. This protection is associated with attenuation of SREBP-1c transactivation and improved hepatic mitochondrial respiratory chain. Lipidomic analysis identified significant increases in the critical mitochondrial inner membrane lipid, cardiolipin, in PKCβ-deficient livers compared to control. Moreover, hepatocyte PKCβ deficiency had no significant effect on either hepatic or whole-body insulin sensitivity supporting dissociation between hepatic steatosis and insulin resistance.
Conclusions: The above data indicate that hepatocyte PKCβ is a key focus of dietary lipid perception and is essential for efficient storage of dietary lipids in liver largely through coordinating energy utilization and lipogenesis during post-prandial period. These results highlight the importance of hepatic PKCβ as a drug target for obesity-associated nonalcoholic hepatic steatosis.
Keywords: Dietary fats; Hepatic steatosis; Mitochondria respiratory chain; Protein kinase cβ.
Canagliflozin extends life span in genetically heterogeneous male but not female mice
Richard A Miller, David E Harrison, David B Allison, Molly Bogue, Lucas Debarba, Vivian Diaz, Elizabeth Fernandez, Andrzej Galecki, W Timothy Garvey, Hashan Jayarathne, Navasuja Kumar, Martin A Javors, Warren C Ladiges, Francesca Macchiarini, James Nelson, Peter Reifsnyder, Nadia A Rosenthal, Marianna Sadagurski, Adam B Salmon, Daniel L Smith Jr, Jessica M Snyder, David B Lombard, Randy Strong
JCI Insight. 2020 Nov 5;5(21):e140019. doi: 10.1172/jci.insight.140019.
Abstract:
Canagliflozin (Cana) is an FDA-approved diabetes drug that protects against cardiovascular and kidney diseases. It also inhibits the sodium glucose transporter 2 by blocking renal reuptake and intestinal absorption of glucose. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing Cana at 180 ppm at 7 months of age until their death. Cana extended median survival of male mice by 14%. Cana also increased by 9% the age for 90th percentile survival, with parallel effects seen at each of 3 test sites. Neither the distribution of inferred cause of death nor incidental pathology findings at end-of-life necropsies were altered by Cana. Moreover, although no life span benefits were seen in female mice, Cana led to lower fasting glucose and improved glucose tolerance in both sexes, diminishing fat mass in females only. Therefore, the life span benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in male mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e., slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.
Keywords: Aging; Drug therapy; Glucose metabolism; Mouse models.
Early disruption of nerve mitochondrial and myelin lipid homeostasis in obesity-induced diabetes.
Juan P Palavicini, Juan Chen, Chunyan Wang, Jianing Wang, Chao Qin, Eric Baeuerle, Xinming Wang, Jung A Woo, David E Kang, Nicolas Musi, Jeffrey L Dupree, Xianlin Han.
JCI Insight. 2020 Nov 5;5(21):137286. doi: 10.1172/jci.insight.137286.
Abstract:
Diabetic neuropathy is a major complication of diabetes. Current treatment options alleviate pain but do not stop the progression of the disease. At present, there are no approved disease-modifying therapies. Thus, developing more effective therapies remains a major unmet medical need. Seeking to better understand the molecular mechanisms driving peripheral neuropathy, as well as other neurological complications associated with diabetes, we performed spatiotemporal lipidomics, biochemical, ultrastructural, and physiological studies on PNS and CNS tissue from multiple diabetic preclinical models. We unraveled potentially novel molecular fingerprints underlying nerve damage in obesity-induced diabetes, including an early loss of nerve mitochondrial (cardiolipin) and myelin signature (galactosylceramide, sulfatide, and plasmalogen phosphatidylethanolamine) lipids that preceded mitochondrial, myelin, and axonal structural/functional defects; started in the PNS; and progressed to the CNS at advanced diabetic stages. Mechanistically, we provided substantial evidence indicating that these nerve mitochondrial/myelin lipid abnormalities are (surprisingly) not driven by hyperglycemia, dysinsulinemia, or insulin resistance, but rather associate with obesity/hyperlipidemia. Importantly, our findings have major clinical implications as they open the door to novel lipid-based biomarkers to diagnose and distinguish different subtypes of diabetic neuropathy (obese vs. nonobese diabetics), as well as to lipid-lowering therapeutic strategies for treatment of obesity/diabetes-associated neurological complications and for glycemic control.
Keywords: Demyelinating disorders; Diabetes; Metabolism; Neurological disorders; Neuroscience.
Rapamycin-mediated mouse lifespan extension: Late-life dosage regimes with sex-specific effects
Randy Strong, Richard A Miller, Molly Bogue, Elizabeth Fernandez, Martin A Javors, Sergiy Libert, Paul Anthony Marinez, Michael P Murphy, Nicolas Musi, James F Nelson, Michael Petrascheck, Peter Reifsnyder, Arlan Richardson, Adam B Salmon, Francesca Macchiarini, David E Harrison
Aging Cell. 2020 Nov;19(11):e13269. doi: 10.1111/acel.13269. Epub 2020 Nov 4.
Abstract:
To see if variations in timing of rapamycin (Rapa), administered to middle aged mice starting at 20 months, would lead to different survival outcomes, we compared three dosing regimens. Initiation of Rapa at 42 ppm increased survival significantly in both male and female mice. Exposure to Rapa for a 3-month period led to significant longevity benefit in males only. Protocols in which each month of Rapa treatment was followed by a month without Rapa exposure were also effective in both sexes, though this approach was less effective than continuous exposure in female mice. Interpretation of these results is made more complicated by unanticipated variation in patterns of weight gain, prior to the initiation of the Rapa treatment, presumably due to the use of drug-free food from two different suppliers. The experimental design included tests of four other drugs, minocycline, β-guanidinopropionic acid, MitoQ, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), but none of these led to a change in survival in either sex.
Keywords: 17-DMAG; MitoQ; minocycline; rapamycin; survival; β-GPA.
APOE2 is associated with longevity independent of Alzheimer’s disease
Mitsuru Shinohara, Takahisa Kanekiyo, Masaya Tachibana, Aishe Kurti, Motoko Shinohara, Yuan Fu, Jing Zhao, Xianlin Han, Patrick M Sullivan, G William Rebeck, John D Fryer, Michael G Heckman, Guojun Bu
Elife. 2020 Oct 19:9:e62199. doi: 10.7554/eLife.62199.
Abstract:
Although the ε2 allele of apolipoprotein E (APOE2) benefits longevity, its mechanism is not understood. The protective effects of the APOE2 on Alzheimer’s disease (AD) risk, particularly through their effects on amyloid or tau accumulation, may confound APOE2 effects on longevity. Herein, we showed that the association between APOE2 and longer lifespan persisted irrespective of AD status, including its neuropathology, by analyzing clinical datasets as well as animal models. Notably, APOE2 was associated with preserved activity during aging, which also associated with lifespan. In animal models, distinct apoE isoform levels, where APOE2 has the highest, were correlated with activity levels, while some forms of cholesterol and triglycerides were associated with apoE and activity levels. These results indicate that APOE2 can contribute to longevity independent of AD. Preserved activity would be an early-observable feature of APOE2-mediated longevity, where higher levels of apoE2 and its-associated lipid metabolism might be involved.
Keywords: APOE; Alzheimer’s disease; human; longevity; medicine; mouse; neuroscience.
Primary neuron and astrocyte cultures from postnatal Callithrix jacchus: a non-human primate in vitro model for research in neuroscience, nervous system aging, and neurological diseases of aging
Angela O Dorigatti, Stacy A Hussong, Stephen F Hernandez, Aubrey M Sills, Adam B Salmon, Veronica Galvan
Geroscience. 2021 Feb;43(1):115-124. doi: 10.1007/s11357-020-00284-z. Epub 2020 Oct 15.
Abstract:
The ability to generate in vitro cultures of neuronal cells has been instrumental in advancing our understanding of the nervous system. Rodent models have been the principal source of brain cells used in primary cultures for over a century, providing insights that are widely applicable to human diseases. However, therapeutic agents that showed benefit in rodent models, particularly those pertaining to aging and age-associated dementias, have frequently failed in clinical trials. This discrepancy established a potential “translational gap” between human and rodent studies that may at least partially be explained by the phylogenetic distance between rodent and primate species. Several non-human primate (NHP) species, including the common marmoset (Callithrix jacchus), have been used extensively in neuroscience research, but in contrast to rodent models, practical approaches to the generation of primary cell culture systems amenable to molecular studies that can inform in vivo studies are lacking. Marmosets are a powerful model in biomedical research and particularly in studies of aging and age-associated diseases because they exhibit an aging phenotype similar to humans. Here, we report a practical method to culture primary marmoset neurons and astrocytes from brains of medically euthanized postnatal day 0 (P0) marmoset newborns that yield highly pure primary neuron and astrocyte cultures. Primary marmoset neuron and astrocyte cultures can be generated reliably to provide a powerful NHP in vitro model in neuroscience research that may enable mechanistic studies of nervous system aging and of age-related neurodegenerative disorders. Because neuron and astrocyte cultures can be used in combination with in vivo approaches in marmosets, primary marmoset neuron and astrocyte cultures may help bridge the current translational gap between basic and clinical studies in nervous system aging and age-associated neurological diseases.
Keywords: Callithrix jacchus; Nervous system aging; Neurological diseases; Neuroscience.
Diagnostic value of circulating cell-free mtDNA in patients with suspected thyroid cancer: ND4/ND1 ratio as a new potential plasma marker
Zhiying Jiang, Tyler Bahr, Chen Zhou, Tao Jin, Hao Chen, Shujie Song, Yuji Ikeno, Hengli Tian, Yidong Bai
Mitochondrion. 2020 Nov:55:145-153. doi: 10.1016/j.mito.2020.09.007. Epub 2020 Oct 7.
Abstract:
Thyroid cancer is the most common endocrine malignancy, and its incidence continues to rise. For clinicians with cancer patients, choosing and interpreting diagnostic laboratory studies has become increasingly important. Previously, changes in plasma free mitochondrial DNA levels have been found in colorectal, breast, lung, and urinary cancers, and have demonstrated diagnostic value. In this study, we investigated whether the occurrence and development of thyroid cancer might be predicted using mtDNA copy number (ND1), mtDNA integrity (ND4/ND1) and levels of cell-free nDNA (GAPDH). We analyzed ND1, ND4, and GAPDH levels in plasma and blood cells from 75 patients with thyroid cancer, 40 patients with nodular goiter, and 107 normal controls using real-time PCR. Although both the thyroid nodule and thyroid cancer patients had significantly increased ND1 levels, the ND4/ND1 ratio in the thyroid cancer group was higher than the thyroid nodule group (P < 0.05), and significantly higher than the normal control group (P < 0.01). Plasma levels of nuclear DNA (GAPDH) in the thyroid cancer group were also higher compared to normal (P < 0.05). These results indicate that increased intactness of plasma free mtDNA is associated with increased levels of plasma cell-free nDNA, and that the ND4/ND1 ratio has the potential to be a new detection indicator in thyroid cancer. Furthermore, we classified thyroid cancer patients according to clinical data including age, tumor size, and metastasis. We found significantly higher levels of GAPDH in malignant tissues. Because ND4/ND1 correlated with plasma GAPDH in the plasma studies, this also suggests a potential relationship between ND4 intactness and thyroid tumor tissue size. Taken together, our findings suggest a tumor-specific process involving increased release of intact mtDNA, detectable in the plasma, which differentiates normal patients from patients with thyroid cancer.
Keywords: Cell free mtDNA; ND4/ND1; Thyroid cancer; mtDNA copy number; mtDNA integrity.