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Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.

Membrane nanotubes are cytosolic protrusions with diameters in vivo relevance, and most studies have been confined to cell culture systems. Here, we introduce lattice-light sheet imaging of MDA-MB-231 human breast cancer cells genetically engineered to brightly express membrane–targeted GFP as a promising approach to visualize membrane nanotubes in vitro and in situ. We demonstrate that cultured cells form multiple nanotubes that mediate intercellular communication of Ca2+ signals and actively traffic GFP-tagged membrane vesicles along their length. Furthermore, we directly visualize nanotubes in situ, interconnecting breast cancer cells in live acute brain slices from an experimental mouse model of breast cancer brain metastasis. This amenable experimental system should facilitate the transition of the study of intercellular communication by membrane nanotubes from cell culture to the whole animal.

This paper outlines the current state of play in the attempts by the British government to regulate psychotherapy and to define different modalities of psychotherapeutic practice, including psychoanalysis. The response of the United Kingdom Council for Psychotherapy (UKCP) to government attempts to regulate practice through the Health Professions Council is described, and the internal debates among psychoanalysts and psychoanalytic psychotherapists in the UKCP are located in the context of relationships with other professional organizations.

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo‐active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 ‘set points’ among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention. Chemical agents provide powerful tools for dissecting complex physiological functions mediated through diverse receptor subtypes. In particular, selective agonist and antagonist pairs that are active in vivo have the distinct advantage of enabling acute, reversible modulation of molecular function while circumventing the developmental compensations that can arise in gene deletion studies. We have targeted this approach to the signaling pathway mediated by S1P and have thereby showed that in vivo–active reversible chemical tools can be used to address a series of mechanistic and therapeutic questions. S1P is a pleiotropic autocrine and paracrine signaling lipid 1 that mediates graded rheostat control of numerous physiological functions through a family of G protein–coupled receptors. Small variations in ligand concentration are amplified by selective high-affinity receptors to acutely regulate vital functions such as heart rate 2,3 ,v ascular and stromal barrier integrity 4 and lymphocyte egress 5 .T he functioning of S1P receptors in the maintenance and modulation of biological barrier activity is of profound biological importance and has therapeutic implications 4 , including prevention of transplant rejection and treatment of multiple sclerosis and perhaps adult respiratory distress syndrome as well 6 .

Sphingosine 1-phosphate type 1 (S1P(1)) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S1P(1) receptors on lymphocytes; a second hypothesis proposes instead that S1P(1) agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S1P(1) agonists or antagonists has provided insight into the mechanism by which S1P(1) agonists function. The selective S1P(1) agonist SEW2871 caused reversible slowing and 'log-jamming' of T cells between filled medullary cords and empty sinuses, whereas motility was unaltered in diffuse cortex. Removal or antagonist competition of SEW2871 permitted recovery of T cell motility in the parenchyma of the medulla and resumption of migration across the stromal endothelial barrier, leading to refilling of sinuses. Our results provide visualization of transendothelial migration of T cells into lymphatic sinuses and suggest that S1P(1) agonists act mainly on endothelial cell S1P(1) receptors to inhibit lymphocyte migration.

The writings of Slavoj Žižek have a disorienting effect on readers, and attempts to understand and utilize his ideas in cultural theory have often come to grief. One way into an interpretation of what Žižek is up to is to trace the cultural–political interventions of some fellow travellers still active in the production and disturbance of our ideological enjoyment. This commentary follows some of the contours of the landscape of Slovenian theory.

Autism spectrum disorder (ASD) affects 2% of children, and is characterized by impaired social and communication skills together with repetitive, stereotypic behavior. The pathophysiology of ASD is complex due to genetic and environmental heterogeneity, complicating the development of therapies and making diagnosis challenging. Growing genetic evidence supports a role of disrupted Ca2+ signaling in ASD. Here, we report that patient-derived fibroblasts from three monogenic models of ASD—fragile X and tuberous sclerosis TSC1 and TSC2 syndromes—display depressed Ca2+ release through inositol trisphosphate receptors (IP3Rs). This was apparent in Ca2+ signals evoked by G protein-coupled receptors and by photoreleased IP3 at the levels of both global and local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given the ubiquitous involvement of IP3R-mediated Ca2+ signaling in neuronal excitability, synaptic plasticity, gene expression and neurodevelopment, we propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect. These findings highlight potential pharmaceutical targets, and identify Ca2+ screening in skin fibroblasts as a promising technique for early detection of individuals susceptible to ASD.

Sodium channels and receptors to serotonin and kainate were 'transplanted' from human brain into frog oocytes, by isolating messenger RNA from a fetal brain, and injecting it into Xenopus laevis oocytes. The mRNA was translated by the oocyte and induced the appearance of functional receptors and channels in its membrane. This approach renders drug- and voltage-operated channels of the human brain more amenable to detailed study.

MANY muscles in the frog contain slow as well as twitch muscle fibres, which differ in their innervation, electrical and contractile properties and fine structure1. Slow fibres give graded slow contractions with nerve stimulation, and can maintain a prolonged contracture when depolarised. There is evidence that contractile activation in slow fibres is mediated by a rise in myoplasmic calcium concentration2, but it is not clear whether this calcium originates from the sarcoplasmic reticulum, as in twitch fibres, or enters the fibre from the external solution1,3–5. We have used the calcium indicator dye arsenazo III (refs 6–9) to follow changes in intracellular free calcium concentration occurring during depolarisation of slow fibres, and find that the membrane potential dependence of these calcium transients in slow fibres is very similar to that observed in twitch fibres9. The time courses of the calcium transients in slow fibres are, however, very much slower than in twitch fibres9,10, and may be a major factor in determining the time courses of tension development and relaxation.

TWITCHES in skeletal muscle are preceded and accompanied by changes in optical properties of the muscle fibres1–11. Measurements of birefringence give large signals which can be separated into two main components6–8: the first begins on the falling phase of the action potential, and reaches a peak at the onset of tension development; this is followed by a late signal which is thought to be associated with development of tension by the contractile proteins2,3,6. Since the early signal precedes tension development, it could provide an important tool for studying intervening steps in excitation–contraction coupling. This signal has recently been attributed to potential changes in the sarcoplasmic reticulum (SR) membrane, associated with Ca2+ release into the sarcoplasm6,10. We have simultaneously recorded birefringence signals and changes in intracellular Ca2+ concentration, using arsenazo III, in frog muscle fibres. The results show that the onset of the early birefringence signal coincides with the rise in sarcoplasmic Ca2+ concentration, and that both can be abolished by injecting EGTA to chelate the sarcoplasmic Ca2+. This suggests that the early birefringence signal arises from some process dependent on the rise in sarcoplasmic Ca2+, and is not caused by events in the SR associated with the calcium release mechanism.

THE mechanism by which depolarisation of the muscle fibre membrane leads to release of stored Ca2+ ions from the sarcoplasmic reticulum, although crucial, is perhaps the least understood stage in excitation–contraction coupling1,2. We report here our investigation of the temperature dependence of this mechanism, using intracellular injection of arsenazo III (refs 3,4), a Ca2+ indicator dye with a fast response time5. We have found that the latency between depolarisation of the fibre and the onset of the rise in intracellular free Ca2+ is proportional to the reciprocal temperature, but that on an Arrhenius plot this relationship shows a change in slope at a temperature which depends upon the Ca2+ concentration in the bathing solution.