Rap1 prevents fusions between long telomeres in fission yeast.

The conserved Rap1 protein is part of the shelterin complex that plays critical roles in chromosome end protection and telomere length regulation. Previous studies have addressed how fission yeast Rap1 contributes to telomere length maintenance, but the mechanism by which the protein inhibits end fusions has remained elusive. Here, we use a mutagenesis screen in combination with high-throughput sequencing to identify several amino acid positions in Rap1 that have key roles in end protection. Interestingly, mutations at these sites render cells susceptible to genome instability in a conditional manner, whereby longer telomeres are prone to undergoing end fusions, while telomeres within the normal length range are sufficiently protected. The protection of long telomeres is in part dependent on their nuclear envelope attachment mediated by the Rap1-Bqt4 interaction. Our data demonstrate that long telomeres represent a challenge for the maintenance of genome integrity, thereby providing an explanation for species-specific upper limits on telomere length.

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe.

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive, correlates with Pol II transcription and mRNA expression levels. Changes in Pol II occupancy were detected in a Upf1 deficient (upf1Δ) strain, prevalently at genes showing a high Upf1 relative to Pol II association in wild-type. Additionally, an increased Ser2 Pol II signal was detected at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1Δ cells are hypersensitive to the transcription elongation inhibitor 6-azauracil. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1Δ. These data envisage that by operating on the nascent transcript, Upf1 might influence Pol II phosphorylation and transcription.

Minishelterins separate telomere length regulation and end protection in fission yeast.

The conserved shelterin complex is critical for chromosome capping and maintaining telomere length homeostasis. In fission yeast, shelterin is comprised of five proteins. Taz1, Rap1, and Poz1 function as negative regulators of telomere elongation, whereas Pot1 and Tpz1 are critical for end capping and telomerase recruitment. How the five proteins work together to safeguard chromosome ends and promote telomere length homeostasis is a matter of great interest. Using a combination of deletions, fusions, and tethers, we define key elements of shelterin important for telomere length regulation. Surprisingly, deletion of the entire Rap1 and Poz1 proteins does not impair telomere length regulation as long as a static bridge is provided between Taz1 and Tpz1. Cells harboring minishelterin display wild-type telomere length and intact subtelomeric silencing. However, protection against end fusions in G1 is compromised in the absence of Rap1. Our data reveal a remarkable plasticity in shelterin architecture and separate functions in length regulation and end protection.

The Aging Enteric Nervous System.

The gut and the brain communicate via the nervous system, hormones, microbiota-mediated substances, and the immune system. These intricate interactions have led to the term "gut-brain axis". Unlike the brain-which is somewhat protected-the gut is exposed to a variety of factors throughout life and, consequently, might be either more vulnerable or better adapted to respond to these challenges. Alterations in gut function are common in the elder population and associated with many human pathologies, including neurodegenerative diseases. Different studies suggest that changes in the nervous system of the gut, the enteric nervous system (ENS), during aging may result in gastrointestinal dysfunction and initiate human pathologies of the brain via its interconnection with the gut. This review aims at summarizing the contribution of normal cellular aging to the age-associated physiological changes of the ENS. Morphological alterations and degeneration of the aging ENS are observed in different animal models and humans, albeit with considerable variability. The aging phenotypes and pathophysiological mechanisms of the aging ENS have highlighted the involvement of enteric neurons in age-related diseases of the central nervous system such as Alzheimer's or Parkinson's disease. To further elucidate such mechanisms, the ENS constitutes a promising source of material for diagnosis and therapeutic predictions, as it is more accessible than the brain.

A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition.

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRß subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

TERRA -a calling card for telomerase.

Transcription of telomeric DNA has long been thought to inhibit telomerase. Using live cell imaging, in this issue, Cusanelli et al. (2013) now reveal a different scenario: telomeric RNA is preferentially generated at short telomeres and delivers telomerase to the chromosome end from which the transcript originated.

Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast.

The fission yeast telomerase RNA (TER1) precursor harbors an intron immediately downstream from its mature 3' end. Unlike most introns, which are removed from precursor RNAs by the spliceosome in two sequential but tightly coupled transesterification reactions, TER1 only undergoes the first cleavage reaction during telomerase RNA maturation. The mechanism underlying spliceosome-mediated 3' end processing has remained unclear. We now demonstrate that a strong branch site (BS), a long distance to the 3' splice site (3' SS), and a weak polypyrimidine (Py) tract act synergistically to attenuate the transition from the first to the second step of splicing. The observation that a strong BS antagonizes the second step of splicing in the context of TER1 suggests that the BS-U2 snRNA interaction is disrupted after the first step and thus much earlier than previously thought. The slow transition from first to second step triggers the Prp22 DExD/H-box helicase-dependent rejection of the cleaved products and Prp43-dependent "discard" of the splicing intermediates. Our findings explain how the spliceosome can function in 3' end processing and provide new insights into the mechanism of splicing.

Widespread failure to complete meiosis does not impair fecundity in parthenogenetic whiptail lizards.

Parthenogenetic species of whiptail lizards in the genus Aspidoscelis constitute a striking example of speciation by hybridization, in which first-generation hybrids instantly attain reproductive isolation and procreate as clonal all-female lineages. Production of eggs containing a full complement of chromosomes in the absence of fertilization involves genome duplication prior to the meiotic divisions. In these pseudo-tetraploid oocytes, pairing and recombination occur exclusively between identical chromosomes instead of homologs; a deviation from the normal meiotic program that maintains heterozygosity. Whether pseudo-tetraploid cells arise early in germ cell development or just prior to meiosis has remained unclear. We now show that in the obligate parthenogenetic species A. neomexicana the vast majority of oocytes enter meiosis as diploid cells. Telomere bouquet formation is normal, but synapsis fails and oocytes accumulate in large numbers at the pairing stage. Pseudo-tetraploid cells are exceedingly rare in early meiotic prophase, but they are the only cells that progress into diplotene. Despite the widespread failure to increase ploidy prior to entering meiosis, the fecundity of parthenogenetic A. neomexicana is similar to that of A. inornata, one of its bisexual ancestors.

Fluorescent blood-brain barrier tracing shows intact leptin transport in obese mice.

BACKGROUND/OBJECTIVES: Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance. METHODS: We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting. RESULTS: Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in microdissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment. CONCLUSIONS: Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.

Calcineurin A beta deficiency ameliorates HFD-induced hypothalamic astrocytosis in mice.

ᅟ: Astrocytosis is a reactive process involving cellular, molecular, and functional changes to facilitate neuronal survival, myelin preservation, blood brain barrier function and protective glial scar formation upon brain insult. The overall pro- or anti-inflammatory impact of reactive astrocytes appears to be driven in a context- and disease-driven manner by modulation of astrocytic Ca2+ homeostasis and activation of Ca2+/calmodulin-activated serine/threonine phosphatase calcineurin. Here, we aimed to assess whether calcineurin is dispensable for astrocytosis in the hypothalamus driven by prolonged high fat diet (HFD) feeding. Global deletion of calcineurin A beta (gene name: Ppp3cb) led to a decrease of glial fibrillary acidic protein (GFAP)-positive cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) of mice exposed chronically to HFD. The concomitant decrease in Iba1-positive microglia in the VMH further suggests a modest impact of Ppp3cb deletion on microgliosis. Pharmacological inhibition of calcineurin activity by Fk506 had no impact on IBA1-positive microglia in hypothalami of mice acutely exposed to HFD for 1 week. However, Fk506-treated mice displayed a decrease in GFAP levels in the ARC. In vivo effects could not be replicated in cell culture, where calcineurin inhibition by Fk506 had no effect on astrocytic morphology, astrocytic cell death, GFAP, and vimentin protein levels or microglia numbers in primary hypothalamic astrocytes and microglia co-cultures. Further, adenoviral overexpression of calcineurin subunit Ppp3r1 in primary glia culture did not lead to an increase in GFAP fluorescence intensity. Overall, our results point to a prominent role of calcineurin in mediating hypothalamic astrocytosis as response to acute and chronic HFD exposure. Moreover, discrepant findings in vivo and in cell culture indicate the necessity of studying astrocytes in their "natural" environment, i.e., preserving an intact hypothalamic microenvironment with neurons and non-neuronal cells in close proximity.

Telomerase RNA biogenesis involves sequential binding by Sm and Lsm complexes.

In most eukaryotes, the progressive loss of chromosome-terminal DNA sequences is counteracted by the enzyme telomerase, a reverse transcriptase that uses part of an RNA subunit as template to synthesize telomeric repeats. Many cancer cells express high telomerase activity, and mutations in telomerase subunits are associated with degenerative syndromes including dyskeratosis congenita and aplastic anaemia. The therapeutic value of altering telomerase activity thus provides ample impetus to study the biogenesis and regulation of this enzyme in human cells and model systems. We have previously identified a precursor of the fission yeast telomerase RNA subunit (TER1) and demonstrated that the mature 3'-end is generated by the spliceosome in a single cleavage reaction akin to the first step of splicing. Directly upstream and partly overlapping with the spliceosomal cleavage site is a putative binding site for Sm proteins. Sm and like-Sm (LSm) proteins belong to an ancient family of RNA-binding proteins represented in all three domains of life. Members of this family form ring complexes on specific sets of target RNAs and have critical roles in their biogenesis, function and turnover. Here we demonstrate that the canonical Sm ring and the Lsm2-8 complex sequentially associate with fission yeast TER1. The Sm ring binds to the TER1 precursor, stimulates spliceosomal cleavage and promotes the hypermethylation of the 5'-cap by Tgs1. Sm proteins are then replaced by the Lsm2-8 complex, which promotes the association with the catalytic subunit and protects the mature 3'-end of TER1 from exonucleolytic degradation. Our findings define the sequence of events that occur during telomerase biogenesis and characterize roles for Sm and Lsm complexes as well as for the methylase Tgs1.

Apollo-taking the lead in telomere protection.

The single-stranded overhangs at the ends of telomeres are thought to be critical for telomere maintenance, but how they are generated has been largely unclear. Two studies (one in this issue of Molecular Cell, Wu et al., 2010) have now implicated the Apollo nuclease in maintaining the overhang specifically at those telomeres generated by leading-strand DNA synthesis.

Sister chromosome pairing maintains heterozygosity in parthenogenetic lizards.

Although bisexual reproduction has proven to be highly successful, parthenogenetic all-female populations occur frequently in certain taxa, including the whiptail lizards of the genus Aspidoscelis. Allozyme analysis revealed a high degree of fixed heterozygosity in these parthenogenetic species, supporting the view that they originated from hybridization events between related sexual species. It has remained unclear how the meiotic program is altered to produce diploid eggs while maintaining heterozygosity. Here we show that meiosis commences with twice the number of chromosomes in parthenogenetic versus sexual species, a mechanism that provides the basis for generating gametes with unreduced chromosome content without fundamental deviation from the classic meiotic program. Our observation of synaptonemal complexes and chiasmata demonstrate that a typical meiotic program occurs and that heterozygosity is not maintained by bypassing recombination. Instead, fluorescent in situ hybridization probes that distinguish between homologues reveal that bivalents form between sister chromosomes, the genetically identical products of the first of two premeiotic replication cycles. Sister chromosome pairing provides a mechanism for the maintenance of heterozygosity, which is critical for offsetting the reduced fitness associated with the lack of genetic diversity in parthenogenetic species.

Chromosome fusions following telomere loss are mediated by single-strand annealing.

Progressive telomere shortening eventually results in chromosome fusions and genome instability as the cell's ability to distinguish chromosome ends from DNA double-strand breaks is compromised. In fission yeast, such events frequently produce stable survivors with all circular chromosomes. To shed light on the repair pathways that mediate chromosome end fusions and generate circular chromosomes, we have examined a diverse array of DNA repair factors. We show that telomere attrition-induced chromosome fusions are dependent on the fission yeast homologs of Rad52, the ERCC1/XPF endonuclease, the single-stranded DNA-binding protein RPA, and the Srs2 and Werner/Bloom helicases, but not Ku and ligase 4. Consistent with a recombinational mechanism of single-strand annealing, cloned junctions map to four of five homology regions in subtelomeric DNA. A comparison with telomere uncapping caused by the absence of the double-stranded telomere-binding protein Taz1 demonstrates that the circumstances and cause of telomere dysfunction profoundly affect which DNA repair pathway is engaged.

Use of Stroke-Related Income Supplements and Predictors of Use in a Working-Aged Finnish Ischemic Stroke Cohort.

BACKGROUND: Compared with the direct costs, the indirect costs of stroke may be larger contributors to the socioeconomic burden of stroke, and the need to better understand the indirect costs of stroke is well established. We investigated the indirect costs of stroke according to a novel outcome, the use of stroke-related income supplements, in a Finnish cohort of working-aged patients. METHODS: Consecutive patients (n = 230) who experienced a first-ever ischemic stroke were recruited. Demographic, clinical, and cognitive function data (which were measured using clinical neuropsychological assessments) were collected at baseline and at 6-month and 2-year follow-ups. Data on the use of income supplements within the first 3 years of the stroke were retrieved from national insurance registry files and used to construct survival models. RESULTS: Stroke patients used a mean of 11 months of stroke-related income supplements; this use was associated with atrial fibrillation, cognitive impairment, prestroke income supplement use, higher National Institutes of Health Stroke Scale scores, lower Barthel Index scores, and increased lesion sizes. In multivariate survival models, atrial fibrillation and cognitive impairment were the factors most strongly associated with the use of stroke-related income supplements. CONCLUSIONS: Using stroke-related income supplement data to quantify poststroke productivity losses allowed a working-aged cohort to be investigated without inclusion restrictions based on occupational status or other factors; the use of these data as an outcome emphasized the well-known detrimental effects of atrial fibrillation and cognitive impairment on stroke outcome. The results support stroke-related income supplement use as a complementary outcome for understanding stroke-related productivity losses.

Unisexual reproduction among vertebrates.

The past decade has seen a remarkable revision of perspectives on unisexual reproduction in vertebrates. One can no longer view it as a rare curiosity far outside the mainstream of evolution. More than 80 taxa of fish, amphibians, and reptiles are now known to reproduce by parthenogenesis (Greek for 'virgin birth') or its variants, and they persist in nature as all-female lineages. Other lower vertebrates that ordinarily rely on sexual reproduction can resort to facultative parthenogenesis under extenuating circumstances that isolate females from males. Molecular tools have now been applied to the study of unisexual organisms, and fascinating insights have emerged regarding the molecular mechanisms that preserve heterozygosity and increase genetic diversity in all-female populations. A deeper understanding of the underlying genetics increasingly calls into question the assumption that unisexuality in vertebrates is an evolutionary dead-end.

The cognitive burden of stroke emerges even with an intact NIH Stroke Scale Score: a cohort study.

BACKGROUND: We aim to facilitate recognition of the cognitive burden of stroke by describing the parallels between cognitive deficits and the National Institutes of Health Stroke Scale (NIHSS), a widely used measure of stroke severity. METHODS: A consecutive cohort of 223 working-age patients with an acute first-ever ischaemic stroke was assessed neuropsychologically within the first weeks after stroke and at a 6-months follow-up visit and compared with 50 healthy demographic controls. The NIHSS was administered at the time of hospital admittance and upon discharge from the acute care unit. The associations between total NIHSS scores and domain-specific cognitive deficits were analysed correlatively and with a binary logistic regression. RESULTS: Of the NIHSS measurements (admittance median=3, range 0-24; discharge median=1, range 0-13), the total score at the time of discharge had systematically stronger correlations with cognitive impairment. Adjusted for demographics, the NIHSS discharge score stably predicted every cognitive deficit with ORs ranging from 1.4 (95% CI 1.2 to 1.6) for episodic memory to 1.9 (95% CI 1.5 to 2.3) for motor skills. The specificities of the models ranged from 89.5-97.7%, but the sensitivities were as low as 11.6-47.9%. Cognitive deficits were found in 41% of patients with intact NIHSS scores and in all patients with NIHSS scores ≥4, a finding that could not be accounted for by confounding factors. CONCLUSIONS: Cognitive deficits were common even in patients with the lowest NIHSS scores. Thus, low NIHSS scores are not effective indicators of good cognitive outcomes after stroke.

Spliceosomal cleavage generates the 3' end of telomerase RNA.

Telomeres cap the ends of chromosomes and provide a means to complete replication. The DNA portion of telomeres is synthesized by the enzyme telomerase using part of an RNA subunit as a template for reverse transcription. How the mature 3' end of telomerase RNA is generated has so far remained elusive. Here we show that in Schizosaccharomyces pombe telomerase RNA transcripts must be processed to generate functional telomerase. Characterization of the maturation pathway uncovered an unexpected role for the spliceosome, which normally catalyses splicing of pre-messenger RNA. The first spliceosomal cleavage reaction generates the mature 3' end of telomerase RNA (TER1, the functional RNA encoded by the ter1(+) gene), releasing the active form of the RNA without exon ligation. Blocking the first step or permitting completion of splicing generates inactive forms of TER1 and causes progressive telomere shortening. We establish that 3' end processing of TER1 is critical for telomerase function and describe a previously unknown mechanism for RNA maturation that uses the ability of the spliceosome to mediate site-specific cleavage.

Laboratory synthesis of an independently reproducing vertebrate species.

Speciation in animals commonly involves an extrinsic barrier to genetic exchange followed by the accumulation of sufficient genetic variation to impede subsequent productive interbreeding. All-female species of whiptail lizards, which originated by interspecific hybridization between sexual progenitors, are an exception to this rule. Here, the arising species instantaneously acquires a novel genotype combining distinctive alleles from two different species, and reproduction by parthenogenesis constitutes an effective intrinsic barrier to genetic exchange. Fertilization of diploid parthenogenetic females by males of sexual species has produced several triploid species, but these instantaneous speciation events have neither been observed in nature nor have they been reconstituted in the laboratory. Here we report the generation of four self-sustaining clonal lineages of a tetraploid species resulting from fertilization of triploid oocytes from a parthenogenetic Aspidoscelis exsanguis with haploid sperm from Aspidoscelis inornata. Molecular and cytological analysis confirmed the genetic identity of the hybrids and revealed that the females retain the capability of parthenogenetic reproduction characteristic of their triploid mothers. The tetraploid females have established self-perpetuating clonal lineages which are now in the third generation. Our results confirm the hypothesis that secondary hybridization events can lead to asexual lineages of increased ploidy when favorable combinations of parental genomes are assembled. We anticipate that these animals will be a critical tool in understanding the mechanisms underlying the origin and subsequent evolution of asexual amniotes.

Post-meiotic mechanism of facultative parthenogenesis in gonochoristic whiptail lizard species.

Facultative parthenogenesis (FP) has historically been regarded as rare in vertebrates, but in recent years incidences have been reported in a growing list of fish, reptile, and bird species. Despite the increasing interest in the phenomenon, the underlying mechanism and evolutionary implications have remained unclear. A common finding across many incidences of FP is either a high degree of homozygosity at microsatellite loci or low levels of heterozygosity detected in next-generation sequencing data. This has led to the proposal that second polar body fusion following the meiotic divisions restores diploidy and thereby mimics fertilization. Here, we show that FP occurring in the gonochoristic Aspidoscelis species A. marmoratus and A. arizonae results in genome-wide homozygosity, an observation inconsistent with polar body fusion as the underlying mechanism of restoration. Instead, a high-quality reference genome for A. marmoratus and analysis of whole-genome sequencing from multiple FP and control animals reveals that a post-meiotic mechanism gives rise to homozygous animals from haploid, unfertilized oocytes. Contrary to the widely held belief that females need to be isolated from males to undergo FP, females housed with conspecific and heterospecific males produced unfertilized eggs that underwent spontaneous development. In addition, offspring arising from both fertilized eggs and parthenogenetic development were observed to arise from a single clutch. Strikingly, our data support a mechanism for facultative parthenogenesis that removes all heterozygosity in a single generation. Complete homozygosity exposes the genetic load and explains the high rate of congenital malformations and embryonic mortality associated with FP in many species. Conversely, for animals that develop normally, FP could potentially exert strong purifying selection as all lethal recessive alleles are purged in a single generation.