Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons

A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered

Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus.

We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG), but not undifferentiated neuronal progenitor cells (NPCs) from ventral subventricular zone (SVZ), results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2). NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control). By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+), whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+). At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative). Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78%) expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG.This is the final version of the article. It was published by PLOS in PLOS One and can be found here: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0031547

Cell origin and culture history determine successful integration of neural precursor transplants into the dentate gyrus of the adult rat.

The success of transplants of neural tissue into the adult dentate gyrus in generating mature neurons is highly variable. Here we address the roles of the origin of the tissue and its pre-implantation preparation, and show that both are critical. We transplanted neonatal cultured or primary rat cells from either the ventral subventricular zone (vSVZ) or the dentate gyrus (DG) into the adult rat DG. Only primary DG cells robustly generated DG neurons (80% NeuN and Prox1-positive cells at 6 weeks), substantially repaired the damaged DG, and formed glutamatergic projections to the target CA3 region. Cultured DG cells expanded for 7 days showed limited neuronal differentiation after transplantation (10% NeuN and Prox1-positive cells) whereas cultured or primary vSVZ cells failed to make any Prox1-positive DG granular neurons. We found that a specific population of postmitotic young neurons (triple doublecortin/NeuN/Prox1-positive) were particularly abundant in primary DG cells, but were markedly reduced in the cultured DG cells and were absent in the cultured and primary vSVZ cells. Labelling of primary DG cells with the mitotic marker BrdU suggested that postmitotic young neurons are the source of the transplanted mature neurons in-vivo. We conclude that both the origin and pre-transplantation history of donor cells are key factors that determine the outcome of transplantation. These findings may be of therapeutic interest for cell replacement therapy in treating the damaged hippocampus.This is the published version of the article. It was published by PLOS in PLOS One and can be found here: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017072

The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice.

Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as β-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 μM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.This work was funded by grant number NC/L000741/1 from the National Council of the 3Rs (NC3Rs) and Alzheimer's Research UK (ARUK). KPR Nilsson is funded by an ERC Starting Independent Researcher Grant (Project: MUMID, number 260604).This is the final published version of the article. It was originally published in Frontiers in Neuroscience (Brelstaff J, Ossola B, Neher JJ, Klingstedt T, Nilsson KPR, Goedert M, Spillantini MG, Tolkovsky AM, Frontiers in Neuroscience, 2015, 9:184, doi:10.3389/fnins.2015.00184). The final version is available at http://dx.doi.org/10.3389/fnins.2015.0018

Suppression of the intrinsic apoptosis pathway by sinaptic activity

Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival

HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes

An inwardly rectifying K^+ current is present in atrial cardiac myocytes that is activated by acetylcholine (I_{KACh}). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch

The 'duality' of fraud in English law and practice

This thesis critically assesses the scope and method of criminalisation of the concept of fraud under the Fraud Act 2006 through the discussion of an apparent ‘duality’ between (co-existing) criminal and non-criminal resolution mechanisms. The reader will find social sciences theory and mixed-methods research techniques being used to identify and characterise a dysfunction between legislation and the social function of fraud control and its resolution. The 2006 Act appears to present a categorical and monolithic headline offence of fraud qualified by dishonesty, yet it is not clear that the Act clearly identifies the scope of effective criminalisation with respect to fraud. The dishonesty-based conduct offence provided in the Fraud Act 2006 is examined in the context of contemporary theory and practical considerations that relate to the discipline of law-enforcement. This work investigates pre-industrial modes of fraud resolution and identifies industrial-era points of divergence between the concepts of fraud and theft (a similar headline offence defined and criminalised under the Theft Act 1968). The work also offers an empirical study of survey-based data collection involving one-hundred-and-forty participants (N=140). It measured the practical extent of criminalisation of fraud in terms of participant indications of the (typically) most likely official outcome in response to sixteen hypothetical examples of fraud offences. The survey results appear to support practical, contextual, and theoretical considerations from the literature on the inhibitors to the consistent application of a conduct-based general fraud offence. The data and findings highlight the advantages of detailed actus reus-based criminalisation of types of fraud that require additional control through effective criminalisation.N/

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis.

We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKL(Ser358) and Drp1(Ser616) phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKL(Ser358), and coincidence of phospho-MLKL(ser358) and phospho-Drp1(Ser616) at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre , Kidney Research UK and NIH grant R01-HL36003.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Nature Publishing Group