Establishment of induced pluripotent stem cells from centenarians for neurodegenerative disease research.

Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimer's disease and familial Parkinson's disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.

Essential roles of Xenopus TRF2 in telomere end protection and replication.

TRF1 and TRF2 are double-stranded (ds) telomere DNA-binding proteins and the core members of shelterin, a complex that provides the structural and functional basis of telomere functions. We have reported that unlike mammalian TRF1 that constitutively binds to chromatin, Xenopus TRF1 (xTRF1) associates with mitotic chromatin but dissociates from interphase chromatin reconstituted in Xenopus egg extracts. This finding raised the possibility that xTRF1 and Xenopus TRF2 (xTRF2) contribute to telomere functions in a manner different from mammalian TRF1 and TRF2. Here, we focused on the role of xTRF2. We prepared chromatin reconstituted in egg extracts immunodepleted for xTRF2. Compared to mock-depleted nuclei, DNA damage response at telomeres was activated, and bulk DNAs were poorly replicated in xTRF2-depleted nuclei. The replication defect was rescued by inactivating ATR through the addition of anti-ATR neutralizing antibody, suggesting that ATR plays a role in the defect. Interestingly, the bulk DNA replication defect, but not the DNA damage response at telomeres, was rescued by supplementing the xTRF2-depleted extracts with recombinant xTRF2 (rTRF2). We propose that xTRF2 is required for both efficient replication of bulk DNA and protection from the activation of the DNA damage checkpoints pathway, and that those two functions are mechanistically separable.

Alternative lengthening of telomeres pathway: recombination-mediated telomere maintenance mechanism in human cells.

Unlimitedly proliferating cells need to acquire the telomere DNA maintenance mechanism, to counteract possible shortening through multiple rounds of replication and segregation of linear chromosomes. Most human cancer cells express telomerase whereas the other cells utilize the alternative lengthening of telomeres (ALT) pathway to elongate telomere DNA. It is suggested that ALT depends on the recombination between telomere repetitive DNAs. However, the molecular details remain unknown. Recent studies have provided evidence of special structures of telomere DNA and genes essential for the phenotypes of ALT cells. The molecular models of the ALT pathway should be validated to elucidate recombination-mediated telomere maintenance and promote the applications to anti-cancer therapy.

Multiple-animal MR imaging using a 3T clinical scanner and multi-channel coil for volumetric analysis in a mouse tumor model.

PURPOSE: Multiple small-animal magnetic resonance (MR) imaging to measure tumor volume may increase the throughput of preclinical cancer research assessing tumor response to novel therapies. We used a clinical scanner and multi-channel coil to evaluate the usefulness of this imaging to assess experimental tumor volume in mice. METHODS: We performed a phantom study to assess 2-dimensional (2D) geometric distortion using 9-cm spherical and 32-cell (8×4 one-cm(2) grids) phantoms using a 3-tesla clinical MR scanner and dedicated multi-channel coil composed of 16 5-cm circular coils. Employing the multi-channel coil, we simultaneously scanned 6 or 8 mice bearing sarcoma 180 tumors. We estimated tumor volume from the sum of the product of tumor area and slice thickness on 2D spin-echo images (repetition time/echo time, 3500/16 ms; in-plane resolution, 0.195×0.195×1 mm(3)). After MR acquisition, we excised and weighed tumors, calculated reference tumor volumes from actual tumor weight assuming a density of 1.05 g/cm(3), and assessed the correlation between the estimated and reference volumes using Pearson's test. RESULTS: Two-dimensional geometric distortion was acceptable below 5% in the 9-cm spherical phantom and in every cell in the 32-cell phantom. We scanned up to 8 mice simultaneously using the multi-channel coil and found 11 tumors larger than 0.1 g in 12 mice. Tumor volumes were 1.04±0.73 estimated by MR imaging and 1.04±0.80 cm(3) by reference volume (average±standard deviation) and highly correlated (correlation coefficient, 0.995; P<0.01, Pearson's test). CONCLUSION: Use of multiple small-animal MR imaging employing a clinical scanner and multi-channel coil enabled accurate assessment of experimental tumor volume in a large number of mice and may facilitate high throughput monitoring of tumor response to therapy in preclinical research.

RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway.

Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.

A crucial role for adipose tissue p53 in the regulation of insulin resistance.

Various stimuli, such as telomere dysfunction and oxidative stress, can induce irreversible cell growth arrest, which is termed 'cellular senescence'. This response is controlled by tumor suppressor proteins such as p53 and pRb. There is also evidence that senescent cells promote changes related to aging or age-related diseases. Here we show that p53 expression in adipose tissue is crucially involved in the development of insulin resistance, which underlies age-related cardiovascular and metabolic disorders. We found that excessive calorie intake led to the accumulation of oxidative stress in the adipose tissue of mice with type 2 diabetes-like disease and promoted senescence-like changes, such as increased activity of senescence-associated beta-galactosidase, increased expression of p53 and increased production of proinflammatory cytokines. Inhibition of p53 activity in adipose tissue markedly ameliorated these senescence-like changes, decreased the expression of proinflammatory cytokines and improved insulin resistance in mice with type 2 diabetes-like disease. Conversely, upregulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance. Adipose tissue from individuals with diabetes also showed senescence-like features. Our results show a previously unappreciated role of adipose tissue p53 expression in the regulation of insulin resistance and suggest that cellular aging signals in adipose tissue could be a new target for the treatment of diabetes (pages 996-967).

Unusual telomeric DNAs in human telomerase-negative immortalized cells.

A significant fraction of human cancer cells and immortalized cells maintain telomeres in a telomerase-independent manner called alternative lengthening of telomeres (ALT). It has been suggested that ALT involves homologous recombination that is expected to generate unique intermediate DNAs. However, the precise molecular mechanism of ALT is not known. To gain insight into how telomeric DNAs (T-DNAs) are maintained in ALT, we examined the physical structures of T-DNAs in ALT cells. We found abundant single-stranded regions in both G and C strands of T-DNAs. Moreover, two-dimensional gel electrophoreses and native in-gel hybridization analyses revealed novel ALT-specific single-stranded T-DNAs, in addition to previously reported t-circles. These newly identified ALT-specific T-DNAs include (i) the t-complex, which consists of highly branched T-DNAs with large numbers of internal single-stranded portions; (ii) ss-G, which consists of mostly linear single-G-strand T-DNAs; and (iii) ss-C, which consists of most likely circular single-C-strand T-DNAs. Cellular-DNA fractionation by the Hirt protocol revealed that t-circles and ss-G exist in ALT cells as extrachromosomal and chromatin-associated DNAs. We propose that such ALT-specific T-DNAs are produced by telomere metabolism specific to ALT, namely, homologous recombination and the rolling-circle replication mechanism.

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha.

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.

Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body.

Telomere maintenance is essential for continued cell proliferation. Although most cells accomplish this by activating telomerase, a subset of immortalized tumors and cell lines do so in a telomerase-independent manner, a process called alternative lengthening of telomeres (ALT). DNA recombination has been shown to be involved in ALT, but the precise mechanisms remain unknown. A fraction of cells in a given ALT population contain a unique nuclear structure called APB (ALT-associated promyelocytic leukemia (PML) body), which is characterized by the presence of telomeric DNA in the PML body. Here we describe that hRad9, hHus1, and hRad1, which form a DNA clamp complex that is associated with DNA damage, as well as its clamp loader, hRad17, are constitutive components of APB. Phosphorylated histone H2AX (gamma-H2AX), a molecular marker of double-strand breaks (DSBs), also colocalizes with some APBs. The results suggest that telomeric DNAs at APBs are recognized as DSBs. PML staining and fluorescence in situ hybridization analyses of mitotic ALT cells revealed that telomeric DNAs present at APBs are of both extrachromosomal and native telomere origins. Furthermore, we demonstrated that DNA synthesis occurs at APBs and is significantly inhibited by caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases. Taken together, we suggest that telomeric DNAs at APBs are recognized and processed as DSBs, leading to telomeric DNA synthesis and thereby contributing to telomere maintenance in ALT cells.

The mouse Murr1 gene is imprinted in the adult brain, presumably due to transcriptional interference by the antisense-oriented U2af1-rs1 gene.

The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.