Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

Multiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ∼20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5′ OH on the 3′ product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei

High Speed Two-Photon Imaging of Calcium Dynamics in Dendritic Spines: Consequences for Spine Calcium Kinetics and Buffer Capacity

Rapid calcium concentration changes in postsynaptic structures are crucial for synaptic plasticity. Thus far, the determinants of postsynaptic calcium dynamics have been studied predominantly based on the decay kinetics of calcium transients. Calcium rise times in spines in response to single action potentials (AP) are almost never measured due to technical limitations, but they could be crucial for synaptic plasticity. With high-speed, precisely-targeted, two-photon point imaging we measured both calcium rise and decay kinetics in spines and secondary dendrites in neocortical pyramidal neurons. We found that both rise and decay kinetics of changes in calcium-indicator fluorescence are about twice as fast in spines. During AP trains, spine calcium changes follow each AP, but not in dendrites. Apart from the higher surface-to-volume ratio (SVR), we observed that neocortical dendritic spines have a markedly smaller endogenous buffer capacity with respect to their parental dendrites. Calcium influx time course and calcium extrusion rate were both in the same range for spines and dendrites when fitted with a dynamic multi-compartment model that included calcium binding kinetics and diffusion. In a subsequent analysis we used this model to investigate which parameters are critical determinants in spine calcium dynamics. The model confirmed the experimental findings: a higher SVR is not sufficient by itself to explain the faster rise time kinetics in spines, but only when paired with a lower buffer capacity in spines. Simulations at zero calcium-dye conditions show that calmodulin is more efficiently activated in spines, which indicates that spine morphology and buffering conditions in neocortical spines favor synaptic plasticity

Developmental profile of localized spontaneous Ca2+ release events in the dendrites of rat hippocampal pyramidal neurons

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Cell Calcium 52 (2012): 422-432, doi:10.1016/j.ceca.2012.08.001.Recent experiments demonstrate that localized spontaneous Ca2+ release events can be detected in the dendrites of pyramidal cells in the hippocampus and other neurons (J. Neurosci. 29:7833-7845, 2009). These events have some properties that resemble ryanodine receptor mediated “sparks” in myocytes, and some that resemble IP3 receptor mediated “puffs” in oocytes. They can be detected in the dendrites of rats of all tested ages between P3 and P80 (with sparser sampling in older rats), suggesting that they serve a general signaling function and are not just important in development. However, in younger rats the amplitudes of the events are larger than the amplitudes in older animals and almost as large as the amplitudes of Ca2+ signals from backpropagating action potentials (bAPs). The rise time of the event signal is fast at all ages and is comparable to the rise time of the bAP fluorescence signal at the same dendritic location. The decay time is slower in younger animals, primarily because of weaker Ca2+ extrusion mechanisms at that age. Diffusion away from a brief localized source is the major determinant of decay at all ages. A simple computational model closely simulates these events with extrusion rate the only age dependent variable.Supported in part by NIH grant NS-016295

Dendritic signals from rat CA1 neurons during coincident pre- and post-synaptic activity : a combined voltage- and calcium-imaging study

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Physiological Society for personal use, not for redistribution. The definitive version was published in Journal of Physiology 580 (2007): 463-484, doi:10.1113/jphysiol.2006.125005.The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca2+-imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca2+]i changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca2+]i-signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP-AP pairing evoked supra-linear summation of [Ca2+]i-transients. We suggest that modulations in the bAP baseline-to- peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca2+-influx through NMDA receptors (0 to -15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.This work was supported by NIH grant RO1NS42739 and by Kavli Institute for Neuroscience, Yale University School of Medicine

Testing the relative sensitivity of 102 ecological variables as indicators of woodland condition in the New Forest, UK.

Forests globally are facing an increasing number of threats from modified disturbance regimes, novel stressors and changing environmental conditions. This has ultimately resulted in declines in the ecological condition of many forest and woodland ecosystems, leading to widespread tree mortality and stand dieback. Effective indicators of overall woodland ecological condition are therefore needed for environmental monitoring and to support management responses. To test the effectiveness of different variables that could potentially be used as indicators of woodland condition, 102 variables that describe woodland structure, composition, functioning, edaphic conditions and disturbance regimes were assessed along 12 replicate gradients of beech stand dieback. Results indicated that 35 variables differed significantly between at least two stages of the dieback gradient, indicating their sensitivity to stand dieback. Seven of these indicators related to woodland species composition, two to functional processes, 20 to structural features, four to edaphic conditions, and two to disturbance regimes. These results demonstrate that effective indicators can potentially be identified for each of the ecological categories. Effective composition indicators included species richness of ectomycorrhizal fungi, ground flora and epiphytic lichens; functional indicators were soil respiration rate and net nitrification rate; edaphic conditions included soil Na:Ca ratio, exchangeable sodium, total carbon, Ca:Al ratio; structural indicators included canopy openness, litter cover, sward height, and volume of deadwood, and for disturbance the indicator was Equus dung density. Other measures, such as shrub cover and species richness of carabid beetles and spiders, were not found to vary significantly along the dieback gradients, and were therefore not identified as effective indicators. These results demonstrate the value of gradient analysis for evaluating indicators of woodland condition, but also highlight the need for multi-site studies to identify indicators with widescale applicability

Spine Calcium Transients Induced by Synaptically-Evoked Action Potentials Can Predict Synapse Location and Establish Synaptic Democracy

CA1 pyramidal neurons receive hundreds of synaptic inputs at different distances from the soma. Distance-dependent synaptic scaling enables distal and proximal synapses to influence the somatic membrane equally, a phenomenon called “synaptic democracy”. How this is established is unclear. The backpropagating action potential (BAP) is hypothesised to provide distance-dependent information to synapses, allowing synaptic strengths to scale accordingly. Experimental measurements show that a BAP evoked by current injection at the soma causes calcium currents in the apical shaft whose amplitudes decay with distance from the soma. However, in vivo action potentials are not induced by somatic current injection but by synaptic inputs along the dendrites, which creates a different excitable state of the dendrites. Due to technical limitations, it is not possible to study experimentally whether distance information can also be provided by synaptically-evoked BAPs. Therefore we adapted a realistic morphological and electrophysiological model to measure BAP-induced voltage and calcium signals in spines after Schaffer collateral synapse stimulation. We show that peak calcium concentration is highly correlated with soma-synapse distance under a number of physiologically-realistic suprathreshold stimulation regimes and for a range of dendritic morphologies. Peak calcium levels also predicted the attenuation of the EPSP across the dendritic tree. Furthermore, we show that peak calcium can be used to set up a synaptic democracy in a homeostatic manner, whereby synapses regulate their synaptic strength on the basis of the difference between peak calcium and a uniform target value. We conclude that information derived from synaptically-generated BAPs can indicate synapse location and can subsequently be utilised to implement a synaptic democracy

High fat diet enhances stemness and tumorigenicity of intestinal progenitors

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we find that high fat diet (HFD)-induced obesity augments the numbers and function of Lgr5+ intestinal stem-cells (ISCs) of the mammalian intestine. Mechanistically, HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-d) signature in intestinal stem and (non-ISC) progenitor cells, and pharmacologic activation of PPAR-d recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-d dependent manner. Interestingly, HFD- and agonist-activated PPAR-d signaling endow organoid-initiating capacity to progenitors, and enforced PPAR-d signaling permits these progenitors to form in vivo tumors upon loss of the tumor suppressor Apc. These findings highlight how diet-modulated PPAR-d activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumors

Calcium control of triphasic hippocampal STDP

Bush D, Jin Y. Calcium control of triphasic hippocampal STDP. Journal of Computational Neuroscience. 2012;33(3):495-514.Synaptic plasticity is believed to represent the neural correlate of mammalian learning and memory function. It has been demonstrated that changes in synaptic conductance can be induced by approximately synchronous pairings of pre- and post- synaptic action potentials delivered at low frequencies. It has also been established that NMDAr-dependent calcium influx into dendritic spines represents a critical signal for plasticity induction, and can account for this spike-timing dependent plasticity (STDP) as well as experimental data obtained using other stimulation protocols. However, subsequent empirical studies have delineated a more complex relationship between spike-timing, firing rate, stimulus duration and post-synaptic bursting in dictating changes in the conductance of hippocampal excitatory synapses. Here, we present a detailed biophysical model of single dendritic spines on a CA1 pyramidal neuron, describe the NMDAr-dependent calcium influx generated by different stimulation protocols, and construct a parsimonious model of calcium driven kinase and phosphatase dynamics that dictate the probability of stochastic transitions between binary synaptic weight states in a Markov model. We subsequently demonstrate that this approach can account for a range of empirical observations regarding the dynamics of synaptic plasticity induced by different stimulation protocols, under regimes of pharmacological blockade and metaplasticity. Finally, we highlight the strengths and weaknesses of this parsimonious, unified computational synaptic plasticity model, discuss differences between the properties of cortical and hippocampal plasticity highlighted by the experimental literature, and the manner in which further empirical and theoretical research might elucidate the cellular basis of mammalian learning and memory function