Keeping the Rhythm while Changing the Lyrics: Circadian Biology in Aging.

Aging and circadian rhythms have been linked for decades, but their molecular interplay has remained obscure. Sato et al. and Solanas et al. now reveal that, while core clock components remain nearly unaltered, aging is associated with tissue-specific rewiring, which can be prevented by calorie restriction.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Cellular Metabolism and Induced Pluripotency.

The discovery of induced pluripotent stem cells (iPSCs) a decade ago, which we are celebrating in this issue of Cell, represents a landmark discovery in biomedical research. Together with somatic cell nuclear transfer, iPSC generation reveals the remarkable plasticity associated with differentiated cells and provides an unprecedented means for modeling diseases using patient samples. In addition to transcriptional and epigenetic remodeling, cellular reprogramming to pluripotency is also accompanied by a rewiring of metabolic pathways, which ultimately leads to changes in cell identities.

In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming.

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

In vivo reprogramming of wound-resident cells generates skin epithelial tissue.

Large cutaneous ulcers are, in severe cases, life threatening1,2. As the global population ages, non-healing ulcers are becoming increasingly common1,2. Treatment currently requires the transplantation of pre-existing epithelial components, such as skin grafts, or therapy using cultured cells2. Here we develop alternative supplies of epidermal coverage for the treatment of these kinds of wounds. We generated expandable epithelial tissues using in vivo reprogramming of wound-resident mesenchymal cells. Transduction of four transcription factors that specify the skin-cell lineage enabled efficient and rapid de novo epithelialization from the surface of cutaneous ulcers in mice. Our findings may provide a new therapeutic avenue for treating skin wounds and could be extended to other disease situations in which tissue homeostasis and repair are impaired.

Polyadenylation of mRNA as a novel regulatory mechanism of gene expression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common and refractory form of epilepsy in adults. Gene expression within affected structures such as the hippocampus displays extensive dysregulation and is implicated as a central pathomechanism. Post-transcriptional mechanisms are increasingly recognized as determinants of the gene expression landscape, but key mechanisms remain unexplored. Here we show, for first time, that cytoplasmic mRNA polyadenylation, one of the post-transcriptional mechanisms regulating gene expression, undergoes widespread reorganization in temporal lobe epilepsy. In the hippocampus of mice subjected to status epilepticus and epilepsy, we report >25% of the transcriptome displays changes in their poly(A) tail length, with deadenylation disproportionately affecting genes previously associated with epilepsy. Suggesting cytoplasmic polyadenylation element binding proteins (CPEBs) being one of the main contributors to mRNA polyadenylation changes, transcripts targeted by CPEBs were particularly enriched among the gene pool undergoing poly(A) tail alterations during epilepsy. Transcripts bound by CPEB4 were over-represented among transcripts with poly(A) tail alterations and epilepsy-related genes and CPEB4 expression was found to be increased in mouse models of seizures and resected hippocampi from patients with drug-refractory temporal lobe epilepsy. Finally, supporting an adaptive function for CPEB4, deletion of Cpeb4 exacerbated seizure severity and neurodegeneration during status epilepticus and the development of epilepsy in mice. Together, these findings reveal an additional layer of gene expression regulation during epilepsy and point to novel targets for seizure control and disease-modification in epilepsy.

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

High concordance between hippocampal transcriptome of the mouse intra-amygdala kainic acid model and human temporal lobe epilepsy.

OBJECTIVE: Pharmacoresistance and the lack of disease-modifying actions of current antiseizure drugs persist as major challenges in the treatment of epilepsy. Experimental models of chemoconvulsant-induced status epilepticus remain the models of choice to discover potential antiepileptogenic drugs, but doubts remain as to the extent to which they model human pathophysiology. The aim of the present study was to compare the molecular landscape of the intra-amygdala kainic acid model of status epilepticus in mice with findings in resected brain tissue from patients with drug-resistant temporal lobe epilepsy (TLE). METHODS: Status epilepticus was induced via intra-amygdala microinjection of kainic acid in C57BL/6 mice, and gene expression was analyzed via microarrays in hippocampal tissue at acute and chronic time-points. Results were compared to reference datasets in the intraperitoneal pilocarpine and intrahippocampal kainic acid model and to human resected brain tissue (hippocampus and cortex) from patients with drug-resistant TLE. RESULTS: Intra-amygdala kainic acid injection in mice triggered extensive dysregulation of gene expression that was ~3-fold greater shortly after status epilepticus (2729 genes) when compared to epilepsy (412). Comparison to samples from patients with TLE revealed a particularly high correlation of gene dysregulation during established epilepsy. Pathway analysis found suppression of calcium signaling to be highly conserved across different models of epilepsy and patients. cAMP response element-binding protein (CREB) was predicted as one of the main upstream transcription factors regulating gene expression during acute and chronic phases, and inhibition of CREB reduced seizure severity in the intra-amygdala kainic acid model. SIGNIFICANCE: Our findings suggest the intra-amygdala kainic acid model faithfully replicates key molecular features of human drug-resistant TLE and provides potential rational target approaches for disease-modification through new insights into the unique and shared gene expression landscape in experimental epilepsy.

NAD+ salvage pathway proteins suppress proteotoxicity in yeast models of neurodegeneration by promoting the clearance of misfolded/oligomerized proteins.

Increased levels of nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT) act as a powerful suppressor of Wallerian degeneration and ataxin- and tau-induced neurodegeneration in flies and mice. However, the nature of the suppression mechanism/s remains controversial. Here, we show that in yeast models of proteinopathies, overexpression of the NMNAT yeast homologs, NMA1 and NMA2, suppresses polyglutamine (PolyQ) and α-synuclein-induced cytotoxicities. Unexpectedly, overexpression of other genes in the salvage pathway for NAD(+) biosynthesis, including QNS1, NPT1 and PNC1 also protected against proteotoxicity. Our data revealed that in all cases, this mechanism involves extensive clearance of the non-native protein. Importantly, we demonstrate that suppression by NMA1 does not require the presence of a functional salvage pathway for NAD(+) biosynthesis, SIR2 or an active mitochondrial oxidative phosphorylation (OXPHOS) system. Our results imply the existence of histone deacetylase- and OXPHOS-independent crosstalk between the proteins in the salvage pathway for NAD(+) biosynthesis and the proteasome that can be manipulated to achieve cellular protection against proteotoxic stress.

Simple high-throughput analytical method using ultra-performance liquid chromatography coupled with tandem mass spectrometry to quantify total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine.

BACKGROUND: Since the urinary concentration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a reliable biomarker of exposure to tobacco smoke, we developed a relatively simple high-throughput chromatographic method to quantify total urinary NNAL concentrations in the general population. METHODS: The high-throughput analytical method was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) to identify and quantify total urinary NNAL concentrations in 10 non-smokers and 15 otherwise healthy smokers. RESULTS: Loss of nitric oxide at m/z 30 was found to be the predominant mass transitioned, and therefore was used as the SIM transition to quantify both NNAL and NNAL-methyl-d3 in urine. The analytical method did not require sample derivatization. Standard curves for total NNAL concentrations were linear between 20 and 1500 pg/mL, with coefficients of determination >0.95. Precision and accuracy ranged from 2.2% to 8.6% (CV) and from -5.6% to 10.9% (percent error), respectively. The lowest limit of quantification was 6.7 pg/mL, and 2.0 pg/mL the lowest limit of detection (LLOD). Total urinary NNAL concentrations in non-smoker subjects were

Dose-response and time-response analysis of total fatty acid ethyl esters in meconium as a biomarker of prenatal alcohol exposure.

OBJECTIVES: Little is known on how the dose and timing of exposure co-influence the cumulative concentration of fatty acid ethyl esters (FAEEs) in meconium. The objective of the study was to assess the cumulative concentration of FAEEs in meconium as a biomarker of light, moderate, or heavy prenatal alcohol exposure occurring at either first, second, or third trimesters of pregnancy. METHODS: History of prenatal alcohol exposure was obtained in the 34th week of gestation from 294 pregnant women. Meconium was collected from their babies within the first 6 to 12 h after birth and examined for the presence of nine FAEEs. RESULTS: No significant differences were identified between the cumulative levels of FAEEs in the meconium from the babies born to abstainers and those born to mothers with history of light-to-moderate prenatal alcohol exposure during their pregnancy. CONCLUSIONS: Light-to-moderate prenatal alcohol exposure cannot be reliably predicted by the cumulative FAEE concentrations in meconium of exposed babies. A cumulative FAEE level of >10 nmol/g would be required to consider that prenatal alcohol exposure during the second to third trimesters occurred at risky levels in the absence of reliable maternal history of ethanol exposure.

Addressing the needs of Latinos with type 2 diabetes through online patient education.

Patients with Type 2 diabetes are increasingly turning to the Web for information about diabetes and self-management. These sites, however, fail to address the cultural and linguistic needs of the growing community of Latinos with diabetes. The Juntos Controlamos la Diabetes Web site was designed as a low-cost patient education tool to be used by patients, caregivers, and healthcare providers to provide ongoing information about diabetes self-management tailored to the needs of the regional Latino community.

Partial cellular reprogramming: A deep dive into an emerging rejuvenation technology.

Aging and age-associated disease are a major medical and societal burden in need of effective treatments. Cellular reprogramming is a biological process capable of modulating cell fate and cellular age. Harnessing the rejuvenating benefits without altering cell identity via partial cellular reprogramming has emerged as a novel translational strategy with therapeutic potential and strong commercial interests. Here, we explore the aging-related benefits of partial cellular reprogramming while examining limitations and future directions for the field.

ATAC-clock: An aging clock based on chromatin accessibility.

The establishment of aging clocks highlighted the strong link between changes in DNA methylation and aging. Yet, it is not known if other epigenetic features could be used to predict age accurately. Furthermore, previous studies have observed a lack of effect of age-related changes in DNA methylation on gene expression, putting the interpretability of DNA methylation-based aging clocks into question. In this study, we explore the use of chromatin accessibility to construct aging clocks. We collected blood from 159 human donors and generated chromatin accessibility, transcriptomic, and cell composition data. We investigated how chromatin accessibility changes during aging and constructed a novel aging clock with a median absolute error of 5.27 years. The changes in chromatin accessibility used by the clock were strongly related to transcriptomic alterations, aiding clock interpretation. We additionally show that our chromatin accessibility clock performs significantly better than a transcriptomic clock trained on matched samples. In conclusion, we demonstrate that the clock relies on cell-intrinsic chromatin accessibility alterations rather than changes in cell composition. Further, we present a new approach to construct epigenetic aging clocks based on chromatin accessibility, which bear a direct link to age-related transcriptional alterations, but which allow for more accurate age predictions than transcriptomic clocks.

DNA repair-deficient premature aging models display accelerated epigenetic age.

Several premature aging mouse models have been developed to study aging and identify interventions that can delay age-related diseases. Yet, it is still unclear whether these models truly recapitulate natural aging. Here, we analyzed DNA methylation in multiple tissues of four previously reported mouse models of premature aging (Ercc1, LAKI, Polg, and Xpg). We estimated DNA methylation (DNAm) age of these samples using the Horvath clock. The most pronounced increase in DNAm age could be observed in Ercc1 mice, a strain which exhibits a deficit in DNA nucleotide excision repair. Similarly, we detected an increase in epigenetic age in fibroblasts isolated from patients with progeroid syndromes associated with mutations in DNA excision repair genes. These findings highlight that mouse models with deficiencies in DNA repair, unlike other premature aging models, display accelerated epigenetic age, suggesting a strong connection between DNA damage and epigenetic dysregulation during aging.

Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21.

Dietary restriction promotes resistance to surgical stress in multiple organisms. Counterintuitively, current medical protocols recommend short-term carbohydrate-rich drinks (carbohydrate loading) prior to surgery, part of a multimodal perioperative care pathway designed to enhance surgical recovery. Despite widespread clinical use, preclinical and mechanistic studies on carbohydrate loading in surgical contexts are lacking. Here we demonstrate in ad libitum-fed mice that liquid carbohydrate loading for one week drives reductions in solid food intake, while nearly doubling total caloric intake. Similarly, in humans, simple carbohydrate intake is inversely correlated with dietary protein intake. Carbohydrate loading-induced protein dilution increases expression of hepatic fibroblast growth factor 21 (FGF21) independent of caloric intake, resulting in protection in two models of surgical stress: renal and hepatic ischemia-reperfusion injury. The protection is consistent across male, female, and aged mice. In vivo, amino acid add-back or genetic FGF21 deletion blocks carbohydrate loading-mediated protection from ischemia-reperfusion injury. Finally, carbohydrate loading induction of FGF21 is associated with the induction of the canonical integrated stress response (ATF3/4, NF-kB), and oxidative metabolism (PPARγ). Together, these data support carbohydrate loading drinks prior to surgery and reveal an essential role of protein dilution via FGF21.

Fetal and neonatal outcomes in women reporting ingestion of licorice (Glycyrrhiza uralensis) during pregnancy.

Maternal intake of licorice from dietary sources has been associated with adverse maternal and fetal outcomes. We prospectively studied the outcome of 185 singleton pregnancies who took over-the-counter or naturopathic formulations containing licorice during their pregnancy, and 370 age-matched singleton pregnant controls that were not exposed to any potential teratogen. The indication in 56.8% of the women taking licorice was for cough and cold control, with the maximum dose of 2104 mg/day and exposure occurring between the 4th day and 25th week of gestation. The rate of stillbirths was marginally higher among women who took licorice than those who did not (OR = 7.9; 95% CI 0.9-71.5; p = 0.048), and significantly higher when compared to the general population in the Republic of Korea (OR = 13.3; 95% CI 4.9-35.8; p < 0.001). Other fetal outcomes assessed in the study were similar between the two study groups, e.g., the OR of major malformations was 3.9 (95% CI 0.4-43.5; p = 0.27). In conclusion, the present study suggests that licorice is not a major teratogen. However, whether licorice may increase the risk of stillbirths requires careful consideration in further studies with a larger sample size.

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

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

In vivo reprogramming leads to premature death linked to hepatic and intestinal failure.

The induction of cellular reprogramming via expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) can drive dedifferentiation of somatic cells and ameliorate age-associated phenotypes in multiple tissues and organs. However, the benefits of long-term in vivo reprogramming are limited by detrimental side-effects. Here, using complementary genetic approaches, we demonstrated that continuous induction of the reprogramming factors in vivo leads to hepatic and intestinal dysfunction resulting in decreased body weight and contributing to premature death (within 1 week). By generating a transgenic reprogrammable mouse strain, avoiding OSKM expression in both liver and intestine, we reduced the early lethality and adverse effects associated with in vivo reprogramming and induced a decrease in organismal biological age. This reprogramming mouse strain, which allows longer-term continuous induction of OSKM with attenuated toxicity, can help better understand rejuvenation, regeneration and toxicity during in vivo reprogramming.