The progeroid phenotype of Ku80 deficiency Is dominant over DNA-PK CS deficiency

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80-/- mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs -/- mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80-/-, dna-pkcs -/- and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver a

Common Variants in the Type 2 Diabetes KCNQ1 Gene Are Associated with Impairments in Insulin Secretion During Hyperglycaemic Glucose Clamp

Background: Genome-wide association studies in Japanese populations recently identified common variants in the KCNQ1 gene to be associated with type 2 diabetes. We examined the association of these variants within KCNQ1 with type 2 diabetes in a Dutch population, investigated their effects on insulin secretion and metabolic traits and on the risk of developing complications in type 2 diabetes patients. Methodology: The KCNQ1 variants rs151290, rs2237892, and rs2237895 were genotyped in a total of 4620 type 2 diabetes patients and 5285 healthy controls from the Netherlands. Data on macrovascular complications, nephropathy and retinopathy were available in a subset of diabetic patients. Association between genotype and insulin secretion/action was assessed in the additional sample of 335 individuals who underwent a hyperglycaemic clamp. Principal Findings: We found that all the genotyped KCNQ1 variants were significantly associated with type 2 diabetes in our Dutch population, and the association of rs151290 was the strongest (OR 1.20, 95% CI 1.07-1.35, p = 0.002). The risk C-allele of rs151290 was nominally associated with reduced first-phase glucose-stimulated insulin secretion, while the non-risk T-allele of rs2237892 was significantly correlated with increased second-phase glucose-stimulated insulin secretion (p = 0.025 and 0.0016, respectively). In addition, the risk C-allele of rs2237892 was associated with higher LDL and total cholesterol levels (p = 0.015 and 0.003, respectively). We found no evidence for an association of KCNQ1 with diabetic complications. Conclusions: Common variants in the KCNQ1 gene are associated with type 2 diabetes in a Dutch population, which can be explained at least in part by an effect on insulin secretion. Furthermore, our data suggest that KCNQ1 is also associated with lipid metabolism

Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70-/- cells and ku80 -/- cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80-/- mice had a shorter life span than dna-pkcs-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER. © 2014 Choi et al

Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair

Abstract Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PK CS ) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70 -/-cells and ku80 -/-cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase b (Pol b). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80 -/-mice had a shorter life span than dna-pkcs -/-mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol b. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER

Mean body weights.

<p>(A) Males. (B) Females.</p

Breeding strategy to generate the knockout cohorts.

<p><i>Ku80^+/−</i> and <i>dna-pk_cs</i>^+/− animals are backcrossed for multiple generations to C57Bl6/J-pUR288 (left of dashed line) and FVB (right of dashed line) background. Double heterozygous knock out C57BL6/J male mice are crossed with double heterozygous knock out FVB female mice. Using this strategy all four desired cohorts are generated as F1 hybrids (grey boxes).</p

Histopathology neoplastic lesions.

<p>Mentioned n-numbers are tumor bearing animals available for microscopical examination.</p><p><i>k</i>-/-;<i>d</i>-/-: <i>ku80</i>-/-; <i>dna-pk_cs^−/−</i>.</p><p>mes. lm. nd.: mesenterial lymph node.</p><p>ax. lm. nd.: axillary lymph node.</p><p>*∼5 thymus tumor preparations were microscopically examined per group and found to be all lymphomas. All other macroscopic neoplastic lesions observed in thymus are assumed to be lymphomas as well.</p><p>** analyzed in femur.</p><p>Tumor incidence based on all animals from longevity cohort, including those not microscopically examined.</p

Survival curves.

<p>Median survivals are shown in the figure legends in parentheses. <i>dna-pk_cs^−/−</i> mice live significantly longer compared to <i>ku80^−/−</i> mice in both male (<i>p</i> = 0.01) and females (<i>p</i> = 8.7*10⁻⁶) mice. Survival of double knock out mice is identical to <i>ku80^−/−</i>. (A) Males. (B) Females.</p

The use of progeroid DNA repair-deficient mice for assessing anti-aging compounds, illustrating the benefits of nicotinamide riboside.

Despite efficient repair, DNA damage inevitably accumulates with time affecting proper cell function and viability, thereby driving systemic aging. Interventions that either prevent DNA damage or enhance DNA repair are thus likely to extend health- and lifespan across species. However, effective genome-protecting compounds are largely lacking. Here, we use Ercc1 Δ/- and Xpg -/- DNA repair-deficient mutants as two bona fide accelerated aging mouse models to test propitious anti-aging pharmaceutical interventions. Ercc1 Δ/- and Xpg -/- mice show shortened lifespan with accelerated aging across numerous organs and tissues. Previously, we demonstrated that a well-established anti-aging intervention, dietary restriction, reduced DNA damage, and dramatically improved healthspan, strongly extended lifespan, and delayed all aging pathology investigated. Here, we further utilize the short lifespan and early onset of signs of neurological degeneration in Ercc1 Δ/- and Xpg -/- mice to test compounds that influence nutrient sensing (metformin, acarbose, resveratrol), inflammation (aspirin, ibuprofen), mitochondrial processes (idebenone, sodium nitrate, dichloroacetate), glucose homeostasis (trehalose, GlcNAc) and nicotinamide adenine dinucleotide (NAD+) metabolism. While some of the compounds have shown anti-aging features in WT animals, most of them failed to significantly alter lifespan or features of neurodegeneration of our mice. The two NAD+ precursors; nicotinamide riboside (NR) and nicotinic acid (NA), did however induce benefits, consistent with the role of NAD+ in facilitating DNA damage repair. Together, our results illustrate the applicability of short-lived repair mutants for systematic screening of anti-aging interventions capable of reducing DNA damage accumulation