Small Molecule from Natural Phytochemical Mimics Dietary Restriction by Modulating FoxO3a and Metabolic Reprogramming.

Many studies utilizing animal models have revealed the genetic and pharmacogenetic modulators of the rate of organismal aging. However, finding routes for healthy aging during extended life remains one of the largest questions. With regards to an antiaging reagent, it has been shown that natural phytochemical syringaresinol (SYR) delays cellular senescence by activating sirtuin1 (SIRT1). Here, it is found that SYR treatment results in metabolic changes similar to those observed during dietary restriction (DR). The DR mimetic effects are mediated by FoxO3a-dependent SIRT1 activation and insulin/insuline growth factor-1 signaling modulation. The direct binding of SYR-FoxO3a is identified and this could partially explain the DR-like phenotype. The report gives a clue as to how the longevity gene involves the DR pathway and suggests that natural phytochemicals applied as a geroprotector mimics DR effects.

Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus.

Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.

Syringaresinol protects against hypoxia/reoxygenation-induced cardiomyocytes injury and death by destabilization of HIF-1α in a FOXO3-dependent mechanism.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of hypoxic response and has been a prime therapeutic target for ischemia/reperfusion (I/R)-derived myocardial dysfunction and tissue damage. There is also increasing evidence that HIF-1 plays a central role in regulating aging, both through interactions with key longevity factors including Sirtuins and mTOR, as well as by directly promoting longevity in Caenorhabditis elegans.We investigated a novel function and the underlying mechanism of syringaresinol, a lignan compound, in modulation of HIF-1 and protection against cellular damage and death in a cardiomyocyte model of I/R injury. Syringaresinol caused destabilization of HIF-1α following H/R and then protected against hypoxia/reoxygenation (H/R)-induced cellular damage, apoptosis, and mitochondrial dysfunction in a dose-dependent manner. Knock-down of FOXO3 by specific siRNAs completely abolished the ability of syringaresinol to inhibit HIF-1 stabilization and apoptosis caused by H/R. Syringaresinol stimulated the nuclear localization and activity of FOXO3 leading to increased expression of antioxidant genes and decreased levels of reactive oxygen species (ROS) following H/R. Our results provide a new mechanistic insight into a functional role of syringaresinol against H/R-induced cardiomyocyte injury and death. The degradation of HIF-1α through activation of FOXO3 is a potential therapeutic strategy for ischemia-related diseases.

Identification of a small molecule activator of SIRT1 gene expression.

Increased SIRT1 expression exerts beneficial effects in transgenic animal models, ameliorating the onset and progression of aging-related disease phenotypes in various organs including the heart. The potential beneficial effects of SIRT1 have made SIRT1 a prime therapeutic target for age-related diseases and considerable efforts led to the identification of small molecule activator of SIRT1 protein. Thus far, however, a small molecule activator of SIRT1 gene expression has not been reported. Here, we report that syringaresinol, isolated from Panax ginseng berry pulp, is an activator of SIRT1 gene expression. Using human umbilical endothelial cells (HUVECs), we show that syringaresinol treatment induced binding of FOXO3 to the SIRT1 promoter in a sequence-specific manner, leading to induction of SIRT1 expression. Increased SIRT1 expression in HUVECs by syringaresinol treatment delayed cellular senescence and improved various markers of endothelial functions in a FOXO3 dependent manner. Collectively, these findings bring to light a new transcription activator of SIRT1 that may have therapeutic potential.

Homeostatic imbalance between apoptosis and cell renewal in the liver of premature aging Xpd mice.

Unrepaired or misrepaired DNA damage has been implicated as a causal factor in cancer and aging. Xpd(TTD) mice, harboring defects in nucleotide excision repair and transcription due to a mutation in the Xpd gene (R722W), display severe symptoms of premature aging but have a reduced incidence of cancer. To gain further insight into the molecular basis of the mutant-specific manifestation of age-related phenotypes, we used comparative microarray analysis of young and old female livers to discover gene expression signatures distinguishing Xpd(TTD) mice from their age-matched wild type controls. We found a transcription signature of increased apoptosis in the Xpd(TTD) mice, which was confirmed by in situ immunohistochemical analysis and found to be accompanied by increased proliferation. However, apoptosis rate exceeded the rate of proliferation, resulting in homeostatic imbalance. Interestingly, a metabolic response signature was observed involving decreased energy metabolism and reduced IGF-1 signaling, a major modulator of life span. We conclude that while the increased apoptotic response to endogenous DNA damage contributes to the accelerated aging phenotypes and the reduced cancer incidence observed in the Xpd(TTD) mice, the signature of reduced energy metabolism is likely to reflect a compensatory adjustment to limit the increased genotoxic stress in these mutants. These results support a general model for premature aging in DNA repair deficient mice based on cellular responses to DNA damage that impair normal tissue homeostasis.

A haplotype-based molecular analysis of CFTR mutations associated with respiratory and pancreatic diseases.

Aberrant membrane transport caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with a wide spectrum of respiratory and digestive diseases as well as cystic fibrosis. Using a gene scanning method, we found 11 polymorphisms and mutations of the CFTR gene in the Korean population. Individual variants at these sites were analyzed by conventional DNA screening in 117 control and 75 patients having bronchiectasis or chronic pancreatitis. In a haplotype determination based on a Bayesian algorithm, 15 haplotypes were assembled in the 192 individuals tested. Several haplotypes, especially with Q1352H, IVS8 T5, and E217G, were found to have disease associations in a case-control study. Notably, a common polymorphism of M470V appears to affect the intensity of the disease association. Among the two haplotypes having IVS8 T5, the T5-V470 haplotype showed higher disease association than the T5-M470 haplotype. In addition, a Q1352H mutation found in a V470 background showed the strongest disease association. The physiological significances of the identified mutations were rigorously analyzed. Non-synonymous E217G and Q1352H mutations in the M470 background caused a 60-80% reduction in CFTR-dependent Cl(-) currents and HCO3(-) -transport activities. Surprisingly, the additional M470V polymorphic variant with the Q1352H mutation completely abolished CFTR-dependent anion transport activities. These findings provide the first evidence on the importance of CFTR mutations in the Asian population. Importantly, the results also reveal that interactions between multiple genetic variants in cis affect the final function of the gene products.