Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination

Impact of telomerase ablation on organismal viability, aging, and tumorigenesis in mice lacking the DNA repair proteins PARP-1, Ku86, or DNA-PKcs

The DNA repair proteins poly(ADP-ribose) polymerase-1 (PARP-1), Ku86, and catalytic subunit of DNA-PK (DNA-PKcs) have been involved in telomere metabolism. To genetically dissect the impact of these activities on telomere function, as well as organismal cancer and aging, we have generated mice doubly deficient for both telomerase and any of the mentioned DNA repair proteins, PARP-1, Ku86, or DNA-PKcs. First, we show that abrogation of PARP-1 in the absence of telomerase does not affect the rate of telomere shortening, telomere capping, or organismal viability compared with single telomerase-deficient controls. Thus, PARP-1 does not have a major role in telomere metabolism, not even in the context of telomerase deficiency. In contrast, mice doubly deficient for telomerase and either Ku86 or DNA-PKcs manifest accelerated loss of organismal viability compared with single telomerase-deficient mice. Interestingly, this loss of organismal viability correlates with proliferative defects and age-related pathologies, but not with increased incidence of cancer. These results support the notion that absence of telomerase and short telomeres in combination with DNA repair deficiencies accelerate the aging process without impacting on tumorigenesis

Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA

To examine the role of telomerase in normal and neoplastic growth, the telomerase RNA component (mTR) was deleted from the mouse germline. mTR-/- mice lacked detectable telomerase activity yet were viable for the six generations analyzed. Telomerase-deficient cells could be immortalized in culture, transformed by viral oncogenes, and generated tumors in nude mice following transformation. Telomeres were shown to shorten at a rate of 4.8+/-2.4 kb per mTR-/- generation. Cells from the fourth mTR-/- generation onward possessed chromosome ends lacking detectable telomere repeats, aneuploidy, and chromosomal abnormalities, including end-to-end fusions. These results indicate that telomerase is essential for telomere length maintenance but is not required for establishment of cell lines, oncogenic transformation, or tumor formation in mice

Prenatal Treatment of Thyroid Hormone Cell Membrane Transport Defect Caused by MCT8 Gene Mutation

[Background]: Mutations of the thyroid hormone (TH)-specific cell membrane transporter, monocarboxylate transporter 8 (MCT8), produce an X-chromosome-linked syndrome of TH deficiency in the brain and excess in peripheral tissues. The clinical consequences include brain hypothyroidism causing severe psychoneuromotor abnormalities (no speech, truncal hypotonia, and spastic quadriplegia) and hypermetabolism (poor weight gain, tachycardia, and increased metabolism, associated with high serum levels of the active TH, T3). Treatment in infancy and childhood with TH analogues that reduce serum triiodothyronine (T3) corrects hypermetabolism, but has no effect on the psychoneuromotor deficits. Studies of brain from a 30-week-old MCT8-deficient embryo indicated that brain abnormalities were already present during fetal life. [Methods]: A carrier woman with an affected male child (MCT8 A252fs268*), pregnant with a second affected male embryo, elected to carry the pregnancy to term. We treated the fetus with weekly 500 μg intra-amniotic instillation of levothyroxine (LT4) from 18 weeks of gestation until birth at 35 weeks. Thyroxine (T4), T3, and thyrotropin (TSH) were measured in the amniotic fluid and maternal serum. Treatment after birth was continued with LT4 and propylthiouracil. Follow-up included brain magnetic resonance imaging (MRI) and neurodevelopmental evaluation, both compared with the untreated brother. [Results]: During intrauterine life, T4 and T3 in the amniotic fluid were maintained above threefold to twofold the baseline and TSH was suppressed by 80%, while maternal serum levels remained unchanged. At birth, the infant serum T4 was 14.5 μg/dL and TSH 8 mU/L, respectively. MRI at six months of age showed near-normal brain myelination compared with much reduced in the untreated brother. Neurodevelopmental assessment showed developmental quotients in receptive language and problem-solving, and gross motor and fine motor function ranged from 12 to 25 at 31 months in the treated boy and from 1 to 7 at 58 months in the untreated brother. [Conclusions]: This is the first demonstration that prenatal treatment improved the neuromotor and neurocognitive function in MCT8 deficiency. Earlier treatment with TH analogues that concentrate in the fetus when given to the mother may further rescue the phenotype.This work was supported by grants from the National Institutes of Health, USA, DK15070 to Samuel Refetoff and DK110322 to Alexandra M. Dumitrescu, and by funds from the Esformes Thyroid Research Fund to Roy E. Weiss

Disease-Related Changes in the Cerebrospinal Fluid Metabolome in Amyotrophic Lateral Sclerosis Detected by GC/TOFMS

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease

Severity-adjusted evaluation of liver transplantation on health outcomes in urea cycle disorders

Purpose: Liver transplantation (LTx) is performed in individuals with urea cycle disorders when medical management (MM) insufficiently prevents the occurrence of hyperammonemic events. However, there is a paucity of systematic analyses on the effects of LTx on health-related outcome parameters compared to individuals with comparable severity who are medically managed. Methods: We investigated the effects of LTx and MM on validated health-related outcome parameters, including the metabolic disease course, linear growth, and neurocognitive outcomes. Individuals were stratified into “severe” and “attenuated” categories based on the genotype-specific and validated in vitro enzyme activity. Results: LTx enabled metabolic stability by prevention of further hyperammonemic events after transplantation and was associated with a more favorable growth outcome compared with individuals remaining under MM. However, neurocognitive outcome in individuals with LTx did not differ from the medically managed counterparts as reflected by the frequency of motor abnormality and cognitive standard deviation score at last observation. Conclusion: Whereas LTx enabled metabolic stability without further need of protein restriction or nitrogen-scavenging therapy and was associated with a more favorable growth outcome, LTx—as currently performed—was not associated with improved neurocognitive outcomes compared with long-term MM in the investigated urea cycle disorders.</p

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words