Human telomeres are composed of duplex TTAGGG repeats and a 3 single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.Cells with linear chromosomes must solve the following two problems: the progressive lagging strand shortening with each cycle of DNA replication and the need to protect the ends of linear chromosomes from unwanted DNA damage responses (1). As a solution to both these problems, telomeres stand at the junction between aging, genomic stability, and cancer.Telomeres are composed of the "shelterin complex" of proteins and TTAGGG repeats of duplex DNA along with an ssDNA overhang or "tail" of 50 -500 nucleotides (1). The ssDNA tail can fold into G-quadruplex DNA (G4 DNA), 4 which consists of three tetrads of four guanines that form Hoogsteen base pairs with each other (Fig. 1A). These tetrads are in a square planar conformation and are stacked atop one another with the TTA sequences forming linker loops (2, 3). The formation of G4 DNA has been shown to inhibit the telomere-lengthening enzyme complex telomerase in vitro (4), although a recent in vivo study of Saccharomyces cerevisiae telomerase found that G4 DNA can promote the activity of yeast telomerase (5).Protection of telomeres 1 (POT1) is part of the shelterin protein complex and binds to single-stranded telomeric TTAGGG repeats (6, 7). POT1 protects mammalian chromosome ends from the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage response, inhibits 5Ј end resection at telomere termini, and regulates telomerase-mediated telomere extension (8). Although POT1 was shown to trap an oligonucleotide with four telomere repeats in an unfolded state to prevent G4 formation (4), the biological significance of this result is unclear. First, POT1 could not bind the short four telomere repeat substrate when the oligonucleotide was prefolded into...

WRN protein loss causes Werner syndrome (WS), which is characterized by premature aging as well as genomic and telomeric instability. WRN prevents telomere loss, but the telomeric protein complex must regulate WRN activities to prevent aberrant telomere processing. Telomere-binding TRF2 protein inhibits telomere t-loop deletion by blocking Holliday junction (HJ) resolvase cleavage activity, but whether TRF2 also modulates HJ displacement at t-loops is unknown. In this study, we used multiplex fluorophore imaging to track the fate of individual strands of HJ substrates. We report the novel finding that TRF2 inhibits WRN helicase strand displacement of HJs with telomeric repeats in duplex arms, but unwinding of HJs with a telomeric center or lacking telomeric sequence is unaffected. These data, together with results using TRF2 fragments and TRF2 HJ binding assays, indicate that both the TRF2 B- and Myb domains are required to inhibit WRN HJ activity. We propose a novel model whereby simultaneous binding of the TRF2 B-domain to the HJ core and the Myb domain to telomeric arms promote and stabilize HJs in a stacked arm conformation that is unfavorable for unwinding. Our biochemical study provides a mechanistic basis for the cellular findings that TRF2 regulates WRN activity at telomeres.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN-DBS) has largely replaced ablative therapies for Parkinson's disease.Because of the similar efficacies of the two treatments, it has been proposed that DBS acts by creating an "informational lesion," whereby pathologic neuronal firing patterns are replaced by low-entropy, stimulus-entrained firing patterns. The informational lesion hypothesis, in its current form, states that DBS blocks the transmission of all information from the basal ganglia, including both pathologic firing patterns and normal, task-related modulations in activity. We tested this prediction in two healthy rhesus macaques by recording single-unit spiking activity from the globus pallidus (232 neurons) while the animals completed choice reaction time reaching movements with and without STN-DBS. Despite strong effects of DBS on the activity of most pallidal cells, reach-related modulations in firing rate were equally prevalent in the DBS-on and DBS-off states. This remained true even when the analysis was restricted to cells affected significantly by DBS. In addition, the overall form and timing of perimovement modulations in firing rate were preserved between DBS-on and DBS-off states in the majority of neurons (66%). Active movement and DBS had largely additive effects on the firing rate of most neurons, indicating an orthogonal relationship in which both inputs contribute independently to the overall firing rate of pallidal neurons. These findings suggest that STN-DBS does not act as an indiscriminate informational lesion but rather as a filter that permits task-related modulations in activity while, presumably, eliminating the pathological firing associated with parkinsonism.

Cardiac arrest survival rates have improved with modern resuscitation techniques, but many survivors experience impairments associated with hypoxic-ischemic brain injury (HIBI). Currently, little is understood about chronic changes in striatal dopamine (DA) systems after HIBI. Given the common empiric clinical use of DA enhancing agents in neurorehabilitation, investigation evaluating dopaminergic alterations after cardiac arrest (CA) is necessary to optimize rehabilitation approaches. We hypothesized that striatal DA neurotransmission would be altered chronically after ventricular fibrillation cardiac arrest (VF-CA). Fast-scan cyclic voltammetry was used with median forebrain bundle (MFB) maximal electrical stimulations (60Hz, 10s) in rats to characterize presynaptic components of DA neurotransmission in the dorsal striatum (D-Str) and nucleus accumbens 14 days after a 5-min VF-CA when compared to Sham or Naïve. VF-CA increased D-Str-evoked overflow [DA], total [DA] released, and initial DA release rate versus controls, despite also increasing maximal velocity of DA reuptake (V ). Methylphenidate (10 mg/kg), a DA transporter inhibitor, was administered to VF-CA and Shams after establishing a baseline, pre-drug 60 Hz, 5 s stimulation response. Methylphenidate increased initial evoked overflow [DA] more-so in VF-CA versus Sham and reduced D-Str V in VF-CA but not Shams; these findings are consistent with upregulated striatal DA transporter in VF-CA versus Sham. Our work demonstrates that 5-min VF-CA increases electrically stimulated DA release with concomitant upregulation of DA reuptake 2 weeks after brief VF-CA insult. Future work should elucidate how CA insult duration, time after insult, and insult type influence striatal DA neurotransmission and related cognitive and motor functions.

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Patients with disorders of consciousness have been at the heart of some of the most heated debates on so-called right-to-die cases such as the Terri Schiavo case. People with DOCs occupy a spectrum of disorders from coma to the minimally conscious state. Recent advances in neuroscience have led to insights on the mechanism of these disorders as well as to the revelation that some patients might have a greater degree of awareness than previously believed. These scientific developments have paralleled long-term clinical follow-ups, which have also shown more positive outcomes than expected.