This paper examines the role of inhibition in generating the receptive-field properties of local edge detector (LED) ganglion cells in the rabbit retina. We confirm that the feed-forward inhibition is largely glycinergic but, contrary to a recent report, our data demonstrate that the glycinergic inhibition contributes to temporal tuning for the OFF and ON inputs to the LEDs by delaying the onset of spiking; this delay was more pronounced for the ON inputs (∼340 ms) than the OFF inputs (∼12 ms). Blocking glycinergic transmission reduced the delay to spike onset and increased the responses to flickering stimuli at high frequencies. Analysis of the synaptic conductances indicates that glycinergic amacrine cells affect temporal tuning through both postsynaptic inhibition of the LEDs and presynaptic modulation of the bipolar cells that drive the LEDs. The results also confirm that presynaptic GABAergic transmission contributes significantly to the concentric surround antagonism in LEDs; however, unlike presumed LEDs in the mouse retina, the surround is only partly generated by spiking amacrine cells. Local edge detectors (LEDs) are small-field ganglion cells that display relatively long latency (sluggish), sustained responses at the onset and termination of a spot illuminating the centre of the receptive field (RF). LEDs have been described in a range of species Both GABA and glycine are fast-acting inhibitory transmitters that gate closely related chloride-permeable channels. It is unclear why these amino-acid transmitters are differentially distributed in the CNS, with GABA mediating most of the inhibition in the brain and glycine mediating most of the inhibition in the spinal cord Amacrine cells receive excitatory input from bipolar cells and provide inhibitory output both to the dendrites of ganglion cells and to processes of other amacrine cells, as well as back to the terminals of bipolar cells Currently the only glycinergic amacrine cell that has been well characterized is the AII amacrine cell, which mediates rod signalling under scotopic conditions, and also provides excitatory (dis-inhibitory) inputs to OFF-alpha ganglion cells in mouse and rabbit Three reports concluded that feed-forward inhibition strongly modulated the temporal response properties of LEDs, by suppressing spiking during rapid global luminance shifts, as will occur when an animal moves its head or eyes Experiments were performed on adult pigmented rabbits of either sex. Experimental procedures were in accordance with the National Institutes of Health guidelines for animal use and the protocols were approved by the Institutional Animal Care and Use Committee at OHSU.The methods for whole-cell recording of the visually evoked currents in retinal ganglion cells have been described in detail previously ClE, was about –68 mV when using QX-314-Cl, and –54 mV when using QX-314-Br, assuming that Br is 1.5 times more permeable than Cl through the chloride channels Extracellular and patch electrodes were pulled from borosilicate glass to a final resistance of 5–8 MΩ. The extracellular electrodes were filled with Ames medium while the patch electrodes contained: 135 mM Cs-methylsulfonate, 6 mM CsCl, 2 mM Na-ATP, 1 mM Na-GTP, 1 mM EGTA, 2 mM MgCl, 5 mM Na-HEPES and 5 mM QX-314-bromide or chloride. All reagents were obtained from Sigma-Aldrich  unless otherwise indicated. Cesium was used in place of potassium to block voltage-gated potassium currents and thereby improve the quality of the voltage clamp at positive potentials. The QX-314 was included to block voltage-gated sodium channels and abolished all spiking activity within 1–2 min of establishing the whole-cell configuration. A liquid junction potential of –10 mV was subtracted from all voltages. Series resistance compensation was not generally applied during recordings. The calculated chloride reversal potential, TG) and the synaptic reversal potential (rV), respectively. The excitatory component, eG, and the inhibitory component, iG, of the synaptic conductance were calculated from TG and rV, using an excitatory reversal potential, eV = 0 mV, and an inhibitory reversal potential, iV = ClE. The excitatory and inhibitory components were calculated as: e(t) = GT(t)(Vr(t) – Vi)/(Ve – Vi)G, and i (t) = GT(t)(Vr(t) – Ve)/(Vi – Ve)G.The conductance analysis methods have been described previously LBACK) was set to 150 µW/m2 at the retinal surface, which for the green phosphor of the stimulus monitor corresponds to ∼400 photons/µm2/s. Assuming a collecting area for the rabbit rods of ∼1 µm2, the background intensity was well above the scotopic range. The stimulus light intensity (LSTIM) was set to 30 µW/m2 for dark stimuli and to 270 µW/m2 for bright stimuli. Thus, the percentage stimulus contrast, defined as C = 100×(LSTIM–LBACK)/LBACK, ranged from –80% to +80%. For experiments involving strychnine, a 10× higher background light intensity was used to rule out the rod pathway contribution. The concentration of strychnine used, 0.5 µM, was low enough to obviate non-specific effects due to suppression of α-7 nicotinic acetylcholine receptors Flash stimuli were step-modulated spots, centred on the receptive field, whose intensity was increased (bright spot) or decreased (dark spot) from the background level. Flicker stimuli were sinusoidal or square-wave modulated spots centred on the receptive field. All stimuli were generated on CRT computer monitors at refresh rates of 60 or 85 Hz, using only the green gun of the CRT. The stimuli were projected through the microscope and focused onto the photoreceptor outer segments, via the 20× water-immersion objective (N.A. = 0.95). The background light intensity  Feed-forward inhibition activates as rapidly as excitation but is more transient, and hence will tend to contribute to the spike latencies in the LEDs by suppressing early spiking. 2) The duration of the spiking during a steady stimulus is determined largely by the kinetics of the excitatory inputs; this second point is evident from the finding that strychnine made the spiking responses more transient , even thFor both the ON and OFF responses, the results indicate that glycinergic pathways shift the temporal responses of the LEDs to lower temporal frequencies. The role of feed-forward inhibition in temporal tuning is also evident from responses during sinusoidal flicker stimulation. At low frequencies (<0.5 Hz), LEDs respond during positive and negative phases of the stimulus cycle as expected for ON/OFF cells  but, at The data are consistent with the notion that glycinergic mechanisms modulate spiking in LEDs both by direct inhibition and by presynaptic modulation of excitatory inputs. In order to confirm that postsynaptic inhibition was a significant factor in determining spike latencies, we performed current-clamp recordings from LEDs, using K-methanesulphonate-based intracellular solution (see Methods) and recorded spikes in response to a 1 Hz square-wave flicker of a 100µm diameter spot flashed in the centre of the receptive field. The delay to the onset of spiking for the OFF response is shown by the red vertical time-markers in A and GABAC receptors mediate surround suppression of excitation It has been shown previously that surround stimulation of LEDs strongly suppresses the centre responses by reducing both the excitatory inputs A-receptor antagonist, 10 µM SR 95531 (6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide), and the GABAC receptor antagonist, 30 µM TPMPA methylphosphinic acid), whether applied alone or together, had little effect on the centre-only response  and the ON inhibition was 115±26% (P = 0.58). However, the feed-forward inhibition at the late time-point was suppressed by 76±15% (P = 0.019) for the OFF inhibition and 79±13% (P = 0.0081) for the ON inhibition (compare black traces of lower panels in Previous studies have indicated that the suppressive surround of retinal ganglion cells is partially mediated by amacrine cells that depend on TTX-sensitive sodium channels for signal transmission The different types of retinal ganglion cells represent distinct spatio-temporal filters that transmit overlapping bands of visual information Previous studies have shown that knockout of glycine receptors in the mouse results in a loss of maintained firing and more transient spiking responses in ganglion cells The temporal tuning via glycinergic feed-forward inhibition was also demonstrated using flicker stimulation. The application of strychnine extended the spiking responses of the LEDs during flicker stimulation, an effect that also appeared to correlate with suppression of direct inhibitory inputs. However, even with glycinergic inhibition blocked, the cells responded to only a few stimulus cycles at the higher frequencies. Analysis of the synaptic conductance indicates that this is likely due to adaptation of the excitatory inputs during maintained flicker, occurring with a time-constant of several seconds . We alsoAt scotopic and mesopic light levels, when the rod-pathway is active, OFF-bipolar cells are driven by AII amacrine cells, via a glycinergic synapse We propose that the glycinergic suppression of ON and OFF excitation might reflect signalling though an intervening, presumably GABAergic, amacrine cell. A synaptic circuit that might account for this effect has tonic GABAergic inhibition onto the bipolar cell terminals that is suppressed through a glycinergic amacrine cell . Serial A or GABAC antagonists alone did not relieve the surround suppression for either the OFF or ON spiking (A and GABAC antagonists more effectively relieved the surround suppression in both OFF and ON responses (A and GABAC pathways contributed to feedback inhibition to the bipolar cells that drive the LED. During GABA block, centre stimulation with a small spot produced a peak glycinergic inhibition that was similar in magnitude to that produced by centre-plus-surround stimulation with a large spot (compare The data confirm previous observations showing that the inhibitory surround that shapes the spatial tuning of the spike output acts presynaptically to the LED  spiking . Howeveresponses , suggest compare . This in compare . Togethe compare  and othe compare , CBC1. 2 compare , CBC4. TPrevious work has shown that surround inhibition in some GCs is dependent on TTX-sensitive sodium channels, presumably because action-potentials in amacrine cells are required to rapidly convey the inhibitory signals across the retina


A significant amount of evidence suggests that the p38-mitogen-activated protein kinase (MAPK) signalling cascade plays a crucial role in synaptic plasticity and in neurodegenerative diseases. In this review we will discuss the cellular localisation and activation of p38 MAPK and the recent advances on the molecular and cellular mechanisms of its substrates: MAPKAPK 2 (MK2) and tau protein. In particular we will focus our attention on the understanding of the p38 MAPK-MK2 and p38 MAPK-tau activation axis in controlling neuroinflammation, actin remodelling and tau hyperphosphorylation, processes that are thought to be involved in normal ageing as well as in neurodegenerative diseases. We will also give some insight into how elucidating the precise role of p38 MAPK-MK2 and p38 MAPK-tau signalling cascades may help to identify novel therapeutic targets to slow down the symptoms observed in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The MAPKs are a specific class of serine/threonine kinases which respond to extracellular signals such as growth factors, mitogens, and cellular stress and mediate proliferation, differentiation, and cell survival in mammalian cells. There are 4 distinct groups of MAPKs within mammalian cells: the extracellular signal-related kinases (ERKs), the c-jun N-terminal kinases (JNKs), the atypical MAPKs , and the p38 MAPKs . The p38in vivo. In this paper we will especially focus our attention on the role of two p38 MAPK substrates in neurons: MAPK-activated protein kinase 2  and tau protein. The involvement of the p38 MAPK-MK2 and p38 MAPK-tau signalling cascades in neuroinflammation, actin remodeling, and tau hyperphosphorylation in neurodegenerative diseases will also be discussed. Highlighting the functional role of specific p38 MAPK substrates in neurodegenerative disease will be of particular importance as these could be potential signalling targets which could be exploited therapeutically to slow cognitive decline occurring in normal ageing and in neurodegenerative disease.In the present paper we will give an overview of p38 MAPK localisation, activation, and the functional role of this signalling cascade in the mammalian brain, especially the activation of the p38 MAPK cascade during synaptic plasticity in the hippocampus. Although p38 MAPK isoforms have been shown to be highly expressed in the brain, only a handful of brain-specific substrates for p38 MAPK have been characterised in vitro experiments in 293T HEK cells showed a similar distribution profile for both the endogenous and exogenous MKK3 and MKK6 proteins in that they are both nuclear and cytoplasmically localised within the cell . It. Itα/β Mammation , 62. In ytokines . This siβ plaques or NFTs formed from free aggregated neuronal tau within the brain. However the relationship between these structures and the symptoms of cognitive impairment and memory loss that is associated with the disease remains uncertain. Increasing evidence has shown that the stability of dendritic spines and actin remodelling may participate in the pathology of the disease. The loss of synapses is a common occurrence within postmortem tissue of AD patients , 83β is  neurons , contribal cells . In AD, deposits  through  in vivo . Upon thin vitro. It has been demonstrated that release of proinflammatory cytokine IL-1 from activated microglia increased the levels of tau phosphorylation in neurons. These changes are partly mediated through activation of p38 MAPK as a significant increase in the levels of phospho-p38 MAPK was observed upon application of IL-1β in cultures of neocortical neurons and microglia [in vitro that IL-1β-induced tau phosphorylation was considerably decreased in neuronal culture [Glial-neuron interactions and the effect these interactions have on tau phosphorylation have been analysed icroglia . Additio culture , again hα has been seen to localise to areas where NFTs, amyloid plaques, and glial cells are present both within human AD brain and transgenic mouse models [β. This complex, which forms through Aβ-peptide-induced dimerisation, suggests a connection between the aberrant processing of APP and the ASK-1-MKK6-p38 MAPK cascade which is involved in inflammation and abnormal tau phosphorylation [In vitro activation of MKK6-p38 MAPK has led to tau phosphorylation at specific sites, the most efficient being Ser-396, which has been suggested to have a functional role in microtubule binding. Abnormal phosphorylation at Ser-396 is observed in AD brain but not in normal functioning adult brain [β peptide, can activate the upstream MKKK ASK-1 and MKK6, consequently activating p38 MAPK, which can directly phosphorylate tau protein hence linking deposition of Aβ plaques to downstream tau phosphorylation through the activation of the p38 MAPK signalling cascade under pathophysiological conditions. Under pathophysiological conditions, activated p38e models , 89, 90.e models  and to be models , the prerylation . In vitrlt brain . Furtherlt brain . This suPick's disease is another severe neurodegenerative disorder, which involves progressive dementia and aphasia through the development of Pick bodies, which are comprised of neurofibrils formed of aggregated phosphorylated tau. It is known that oxidative stress is involved in instigating Pick's disease, and since it has been highlighted that the p38 MAPK cascade is activated upon such stimuli, it may play an important role in this disease as well. It has been observed in post-mortem brain tissue that phosphorylated p38 MAPK localises to the Pick bodies which contain highly phosphorylated tau protein, and since p38 MAPK is capable of phosphorylating tau, as described above, it emphasises the importance of p38 MAPK in this disease as well as AD and other related tauopathies .α-synuclein (SNCA), which is a protein present within the LBs of PD patients and known to play a pivotal role in the development of the disease. α-Synuclein is highly expressed in neuronal tissue and is subject to many post-translational modifications such as phosphorylation and ubiquitination, although its physiological function is poorly characterised. It has been suggested, however, that these post-translational modifications participate in neurotoxicity [α-synuclein is the predominant fibrillar component of the proteinaceous Lewy bodies seen in PD. Additionally, it has been observed that amplified α-synuclein levels are linked to an increase in neuroinflammation as it has been shown that α-synuclein activates p38 MAPK and other MAPKs in glial cells, a finding that is supported by the fact that extracellular α-synuclein released from damaged neurons interacts with microglia [α, which can induce and promote neuroinflammation as a physiological neuroprotective mechanism. If the glial cells become overactivated, however, as may occur through elevated levels of extracellular α-synuclein released from damaged Lewy body containing neurons, then chronic inflammation could be established and lead to neuronal cell death. Parkinson's disease is the second most prevalent neurodegenerative disease and around 127,000 people in the UK are currently living with the disease, which has been estimated to rise by 28% by the year 2020 . PD invotoxicity  as aggreicroglia . As descα, thus causing neuroinflammation and potentially neuronal cell death. An investigation by Culbert et al. [MAPKAP kinase-2, one of p38 MAPK more prevalent substrates has also been implicated within PD, where it has been shown that MK2-deficient mice show decreased levels of neuroinflammation and loss of dopaminergic neurons within the substantia nigra after treatment with the Parkinson's inducing neurotoxin MPTP compared to MK2 wild-type mice . MK2, aft et al.  demonstrConsiderable progress has been made in the understanding of the functional role of the p38 MAPK signalling cascade in synaptic plasticity in the hippocampus and its potential role in neurodegenerative diseases such as AD. However, less is known regarding the role of the direct targets of p38 MAPK, such as MK2 and tau, in regulating neuroinflammation and the actin cytoskeleton in dendritic spines of neuronal cells. Growing evidence suggests that remodelling of actin at dendritic spines plays a crucial role in synaptic plasticity and therefore in cognitive processes such as learning and memory. Furthermore, recent findings in animal models have linked early symptoms of AD with loss of cognitive functions, combined with a reduced number of dendritic spines in the hippocampus. Abnormal dendritic spine morphology has also been observed in brain tissue from patients suffering from AD. Therefore, with an ageing population continuing to grow and the consequent rise in AD, elucidating the precise role of p38 MAPK-MK2 and p38 MAPK-tau signalling cascades in controlling actin remodelling becomes very important as it may identify novel targets to slow down the cognitive decline observed in normal ageing and in the early stages of neurodegenerative diseases.


L  is a serine/threonine kinase that is now firmly established as a central component in T cell activation, proliferation, differentiation, and apoptosis  have reduced incidence and severity of Th2 and Th17-mediated inflammatory disorders, including asthma, inflammatory bowel disease, multiple sclerosis, arthritis, and allograft rejection in comparison to their wild-type littermates (PKC θ+/+; Marsland and Kopf, −/− mice are still capable of mounting relatively normal Th1 and CD8+ T cell-mediated immune responses to infectious viruses (Marsland and Kopf, + T cells from PKC θ−/− mice have a survival defect following activation (Barouch-Bentov et al., −/− mice have an impaired anti-leukemic response (Garaude et al., in vivo. It is important therefore that these issues are addressed in respect of any PKC θ-targeting strategies that are developed in the future.A host of studies have now convincingly demonstrated that targeting PKC θ could be a viable therapeutic strategy to block the T cell inflammatory response in autoimmunity, allergy, and allograft rejection (Marsland and Kopf, in vitro have now been characterized (Nika et al., bona fide substrates in vivo remains to be addressed. Like many other kinases, PKC θ is also regulated by phosphorylation on a host of serine, threonine, and tyrosine residues that influence its activity and intracellular localization. Six phosphorylation sites have been mapped on PKC θ in T cells to date. Some of these sites appear to be phosphorylated by unrelated upstream kinases, while other sites are regulated via auto-phosphorylation. Three of these phosphorylation sites are highly conserved on most other PKC isoforms, which suggests that they may regulate aspects that are central to all isoforms, such as stability. In contrast, PKC θ contains three phosphorylation sites that appear to be unique to this isoform.Frontiers in T Cell Biology, Wang et al. (Although much has been learned about PKC θ in T cells, considerable gaps still exist in our knowledge as to how this kinase is regulated, including the upstream signals and interacting partners that control its intracellular localization and catalytic activation at various locations in the cell. Furthermore, although a plethora of substrates that are phosphorylated by PKC θ g et al.  summariz


Austrovenus stutchburyi is a key species on intertidal sandflats in New Zealand, affecting the appearance and functioning of these systems, but is susceptible to several environmental stressors including sedimentation. Previous studies into the effect of this species on ecosystem function have been restricted in space and time, limiting our ability to infer the effect of habitat change on functioning. We examined the effect of Austrovenus on benthic primary production and nutrient dynamics at two sites, one sandy, the other composed of muddy-sand to determine whether sedimentary environment alters this key species' role. At each site we established large (16 m2) plots of two types, Austrovenus addition and removal. In winter and summer we deployed light and dark benthic chambers to quantify oxygen and nutrient fluxes and measured sediment denitrification enzyme activity to assess denitrification potential. Rates of gross primary production (GPP) and ammonium uptake were significantly increased when Austrovenus was added, relative to removed, at the sandy site , p<0.05). Denitrification potential was also elevated in Austrovenus addition plots at the sandy site in summer . In contrast, there was no effect of Austrovenus treatment on any of these variables at the muddy-sand site, and overall rates tended to be lower at the muddy-sand site, relative to the sandy site . Our results suggest that the positive effects of Austrovenus on system productivity and denitrification potential is limited at a muddy-sand site compared to a sandy site, and reveal the importance of considering sedimentary environment when examining the effect of key species on ecosystem function.The suspension feeding bivalve  Estuaries are highly productive ecosystems that play a major role in biogeochemical cycles, but are subject to multiple stressors that will likely be exacerbated by climate change and expanding human habitation of coastal areas per se, can have a disproportionate effect on indicators of ecosystem functioning such as nutrient cycling and productivity (e.g. Austrohelice crassa) displays functional plasticity, acting as a bioturbator in sandy sediments and as a bioirrigator in muddy cohesive sediments If habitat change does lead to decreasing biodiversity, then that alone may cause shifts in ecosystem structure and function ity e.g. . Althoug3− to N2 gas, which permanently removes fixed nitrogen from an ecosystem; thus, suspension feeding bivalves can also exert a bottom-up control on phytoplankton populations (e.g. Crassostrea virginica) beds has substantially increased the incidence of phytoplankton blooms, sometimes resulting in the occurrence of deep-water hypoxia (e.g. Potamocorbula amurensis) in San Francisco Bay and the zebra mussel (Dreissena polymorpha) in many freshwater systems in the USA, has resulted in reduced phytoplankton biomass , which commonly exists in high density beds covering large areas of intertidal flats; typical bed densities average c. 1000 ind. m−2, although peak densities may be 2000–3000 ind. m−2 in some areas Austrovenus is an infaunal species that bioturbates surficial sediments through vertical and horizontal movement, but has very short siphons and so lives close to the sediment surface (<5 cm). Austrovenus beds are found across a range of sediment types, although very high levels of sedimentation adversely affect abundance Austrovenus has been shown to be a key species influencing sediment stability, solute fluxes and macrofauna community structure as well as enhancing microphytobenthos productivity In New Zealand estuaries the dominant suspension feeding bivalve is the native clam Austrovenus in situ at two estuarine sites, both with nearby high density Austrovenus beds, but with contrasting sediment properties. Our aim was to see if the role of this key species in ecosystem functioning was the same at a sandy site (a proxy for a habitat unimpacted by sedimentation) and at a muddy-sand site (a proxy for a habitat affected by a moderate level of sedimentation). In winter and summer, light and dark benthic chambers were used to quantify the effect of Austrovenus on O2 and nutrient  fluxes, and to estimate gross primary production and nutrient uptake rates. Additionally, denitrification enzyme activity (DEA) assays were used to quantify the effect of Austrovenus on maximum sediment denitrification potential. Previously, high Austrovenus densities have been shown to enhance ammonium efflux which supported higher rates of microphytobenthos (MPB) production 2) to reduce confounding edge effects  Client Number 8770024.This study complied with all existing legislation governing animal welfare and field-based experiments. Animal ethics approval/permits were not sought as benthic invertebrate fauna manipulated/sampled in this study are exempt from the Animal Welfare Act 1999. After consultation with the Bay of Plenty Regional Council permits were not required for the 2) barrier-enclosed estuary on the north-eastern coast of New Zealand. We manipulated the presence or absence of Austrovenus at two sites with differing sedimentary characteristics on lower-mid intertidal flats in the harbour (−1 (peak flow was 35 cm s−1), and at the muddy-sand site were 7.2 cm s−1 (peak flow was 18 cm s−1), as determined by deployment of a FSI current meter that included a spring and a neap tidal phase. Tides in the harbour are semi-diurnal and the mean immersion period at each site is 8 h. Water temperature in Tauranga Harbour typically fluctuates between 13°C in mid-winter (July/August) and 22°C in mid-summer (January/February) Tauranga Harbour is a large , but were within 20 m of high density Austrovenus beds. Preliminary observations indicated that densities in the natural beds were c. 600–1200 ind. m−2 at the sandy site, and c. 2000–3000 ind. m−2 at the muddy-sand site. We noted however that Austrovenus individuals were larger at the sandy site  we manually removed all Austrovenus by finger plowing the sediment, minimising the impact of the manipulation on ambient macrofauna Austrovenus. The large mesh size (1 cm) and short height (5 cm above sediment) of the fencing was used to minimise effects on water flow Austrovenus manipulation was undertaken 6 weeks prior to the winter (August 2009) and summer (February-March 2010) benthic chamber incubations (see below) to allow the sediment and resident macrofauna to recover from the effects of the manipulation In June 2009, at both sites, six 4 m×4 m plots separated by 1 m were established in a line parallel with the channel. site see . We inteAustrovenus manipulations, O2 and nutrient fluxes were measured in light and dark benthic chambers. One light and one dark chamber was deployed to each of the six plots per site on two consecutive days in both winter and summer. Chambers were placed at least 1 m inside each plot's fence to avoid edge artefacts  have been described previously Austrovenus exhibits a circatidal rhythm whereby feeding is limited to this period 2 concentration was measured immediately with a hand held dissolved O2 probe (PreSens Fibox 3 PSt3) and the water was then filtered through a Whatman GF/C filter, stored on ice in the dark and frozen that day for later analysis of nutrients  on a Thermo Scientific Aquakem 200 discrete analyzer. Water column effects on O2 and nutrient concentrations were found to be negligible based on incubation of ambient water in light and dark water bottles (1 L) for the same length of time and at the same depth as the chamber incubations. O2 and nutrient fluxes were calculated from the slope of the regression between concentration and incubation time, corrected for dilution of chamber water that occurred during each of the five 60 ml samplings. Additionally, HOBO® light meters and TidBit® temperature loggers were fitted to the outside of randomly selected chambers during the experiments.The incubation chambers (square chambers with domed lids enclosing 0.25 mAustrovenus density (sieved on a 1 mm mesh). For light chamber cores only, the surficial 5 cm of sediment was placed in airtight bags, kept cool and transported to the laboratory that evening for denitrification assays.After chamber deployment 16 surface sediment samples (1 cm depth) were taken from within each chamber footprint using a small syringe core (2.5 cm diameter). Samples were pooled and frozen for later analysis of pigments, grain size, organic matter, nitrogen and organic carbon content. One large core  was collected for macrofauna analysis, sieved on a 0.5 mm mesh and preserved in 70% isopropyl alcohol with Rose-Bengal stain. A second large core was collected for sediment denitrification and DEA assays (see below) and an additional estimate of 2H2) inhibition technique 2H2, it has proven reliable for comparison of denitrification activity among treatments, sites and seasons as well as measuring nutrient limitation of denitrification . For each sediment sample (5 cm depth core from light chambers) we combined 30 mL of homogenized sediment with 25 mL unfiltered site water in preserve jars modified with n-butyl rubber septa in the lids . Chloramphenicol was added to the jars to suppress de novo enzyme production and the jars were purged with ultra-pure helium for 10 min to ensure anoxic conditions. Pure C2H2 was added to the jar headspace to prevent the conversion of N2O to N2 and gas samples were collected hourly beginning 10 mins after the addition of the C2H2 for 4 h. To maintain a constant pressure the headspace was replaced with a mixture of helium and C2H2 after each sample. The gas samples were analysed for N2O using a Varian CP 3800 gas chromatograph equipped with a HayeSep D column and electron capture detector. Denitrification rates were calculated from the linear increase in N2O concentration over time, normalized to the sediment surface area. To determine whether sediment denitrification was limited by nitrate or carbon we amended additional jars prepared identically to those above with additional nitrate (as potassium nitrate 10 mg N L−1), carbon (as glucose 12 mg C L−1) or both nitrate (10 mg N L−1) and carbon (12 mg C L−1). The DEA measurements were determined from the rates measured in the samples amended with nitrate and carbon (+N+C). DEA provides a measure of maximum denitrification potential by providing optimized conditions in anoxic, +N+C-amended slurries, valuable for making across-site comparisons Sediment denitrification rates were quantified within 24 h of collection using the chloramphenicol-amended acetylene (Ca (chl a) and phaeopigment content were determined by extraction in 90% acetone and measurement of fluorescence before and after acidification on a Turner Designs 10-AU fluorometer Austrovenus, other bivalves, mudflat anemones (Anthopleura aureoradiata), annelids, crustaceans and gastropods counted and weighed (blotted wet weight). The weight of the bivalves included their shells. Austrovenus density and biomass in each chamber was estimated from the mean of the two large sediment cores.Sediment chlorophyll 2 and nutrient fluxes in the light and dark chambers were analysed separately. We defined the rate of net primary production (NPP) and sediment oxygen consumption (SOC) as the O2 flux in light and dark chambers respectively and estimated gross primary production (GPP) from NPP-SOC. GPP was standardised by the sediment chl a content to account for variations in microphytobenthos biomass. We estimated nutrient uptake rates (the difference between dark chamber flux and light chamber flux) to quantify usage by microbes and microphytes living in surficial sediments.To eliminate pseudo-replication, one representative value for each chamber type per plot was obtained prior to statistical analysis by averaging the data from the two light and two dark chambers deployed per plot. Sediment OAustrovenus density and biomass between sites, seasons and treatments separately. In all tests, normality and homogeneity of variances were evaluated with Kolmogorov-Smironov tests and by plotting of residual versus predicted values. Variables were log or square root transformed where required. All statistical analyses were performed using Statistica .The response variables  were analysed using 3-factor analysis of variance (ANOVA), with treatment , site , and season  all considered as fixed factors. Any non-significant interaction terms of the highest order were removed and the analysis repeated. When the overall ANOVA was significant at α = 0.05, pairwise comparisons were performed using Tukey post-hoc tests. For sediment denitrification, 2-factor ANOVA by presence or absence of nitrate (N) or carbon (C) was used to identify the limiting nutrient. Single nutrient limitation by N or C is identified with a significant result for that treatment, and co-limitation is identified by a significant interaction term Austrovenus density in +AS plots ranged from c. 500 to 1000 ind. m−2 at the sandy site and from c. 1800 to 2500 ind. m−2 at the muddy-sand site  to affect the sediment biogeochemical environment at a scale larger than the chamber footprints (0.25m2). Although we did not achieve total removal in -AS plots, Austrovenus density and biomass were at least an order of magnitude less than in the +AS plots.and site . Small-sted e.g. . HoweverAustrovenus density and biomass were significantly greater in +AS compared to -AS plots . Densities in +AS plots were equivalent to planned densities, i.e. mean Austrovenus density in +AS plots was significantly lower at the sandy site (700 ind. m−2) compared to the muddy-sand site . Mean Austrovenus shell length (± SD) was significantly greater at the sandy site, 23.3 (±1.0) mm, compared to the muddy-sand site, 17.7 (±1.1) mm, (p<0.001). Thus, as expected, mean biomass in +AS plots (c. 2300 g ww m−2) was not significantly different between the two sites (p>0.05). There was no significant seasonal difference in Austrovenus density, size or biomass at either site (p>0.05).Regardless of site or season, Anthopleura aureoradiata  and crustaceans  at the sandy site; annelids and other bivalves dominated at the muddy-sand site  than in winter (c. 80 µmol photons m−2 s−1). Regardless of season or Austrovenus treatment, median grain size was significantly lower at the muddy-sand site (c. 220 µm), compared to the sandy site . Mud, OM, OC, N, chl a and phaeopigment content were all significantly greater at the muddy-sand site (p<0.001). We did not detect a significant effect (α = 0.05) of Austrovenus treatment on any sediment properties at the sandy site. However, at the muddy-sand site, in both winter and summer, grain size was greater (p<0.05) and mud content was lower (p<0.05) in +AS than -AS plots. Also at the muddy-sand site, OM content was lower in +AS plots than in -AS plots, although the effect was only marginally significant .There were large differences in water temperature and photosynthetically active radiation (PAR) between winter and summer, with only small differences between sites within each of the seasons. Conversely, there were large differences in sediment properties between sites, but not between winter and summer . Water t2 into the sediments, indicating sediment oxygen consumption (SOC), however, in light chambers there was always an efflux of O2 from the sediments which indicated that net primary production (NPP) was greater than zero  revealed that SOC was significantly greater (by 2.5×) in summer than in winter at the sandy site but that there was no significant difference between seasons at the muddy-sand site. Comparisons between sites within seasons demonstrated that SOC was significantly higher (by 1.7×) at the sandy than at the muddy-sand site in summer only (in winter there was no significant difference). For light chamber O2 fluxes, there was a marginally significant site*treatment interaction (p = 0.086). Closer examination suggested that NPP tended to be greater in +AS plots (compared to -AS plots) at the sandy site in summer. There was no indication of treatment effects on NPP in winter at the sandy site or at the muddy-sand site in either season. The site*season interaction was significant (p<0.05) with NPP greater (by 2.4×) in summer than in winter at the sandy site. There was no significant seasonal effect on NPP at the muddy-sand site and no significant difference between the sites in either season.In dark chambers there was always an influx of Ohan zero . There wp<0.001, . Post-ho2 m−2 h−1 at the sandy site, but the range was much smaller at the muddy-sand site (3.1 to 3.8 mmol O2 m−2 h−1). When normalised by sediment chl a content (a proxy for primary producer biomass), GPP at the sandy site was consistently greater (0.24 to 0.90 mmol O2 µg chl a g−1 m−2 h−1) than at the muddy-sand site  from the sediment, the only exception being some light chambers in the +AS plots at the sandy site in summer, when there was a small influx  showed that dark chamber NH4+ flux was greater in summer than in winter at both sites . Comparisons between sites within seasons demonstrated that dark chamber NH4+ flux was greater (by 2.3×) at the muddy-sand site than at the sandy site in winter only (in summer there was no significant difference). The effect of Austrovenus treatment on light chamber NH4+ flux was not consistent across sites and seasons  in +AS compared to -AS plots at the muddy-sand site in summer, but there was no significant difference between treatments in winter or at the sandy site in either season. Comparison between sites within seasons and treatments revealed that in +AS plots in summer light chamber NH4+ flux was significantly greater at the muddy-sand site; at the sandy site NH4+ flux was negative (−0.52 µmol m−2 h−1) indicating a small influx into the sediment, but at the muddy-sand site NH4+ flux was positive (74.6 µmol m−2 h−1) indicating a large efflux out of the sediment. In contrast, in +AS plots in winter and in -AS plots in both seasons, there was no significant difference between the sites.In dark and light chambers there was nearly always a net efflux of ammonium (NHl influx . There w4+ uptake exhibited a far greater range at the sandy (6 to 105 µmol NH4+ m−2 h−1) compared to the muddy-sand site  in +AS compared to -AS plots at the sandy site in summer, but there was no significant difference between treatments in winter. At the muddy-sand site there was no treatment effect in either season. Comparison between seasons within treatments and sites revealed that in +AS plots at the sandy site NH4+ uptake was significantly greater (by 10×) in summer compared to winter, but there was no significant difference between the seasons in -AS plots. At the muddy-sand site there was no significant difference between the seasons in +AS or -AS plots. Comparison between sites within treatments and seasons revealed that NH4+ uptake was significantly greater (by 3.4×) at the sandy site in +AS plots in summer, but there was no significant difference between the sites in +AS plots in winter. There was also no significant difference between the sites in -AS plots in either season.NH4+ is the form of dissolved inorganic nitrogen (DIN) most readily taken up by microphytobenthos (MPB) and, as is typically the case in New Zealand estuaries, comprised the majority (c. 80%) of DIN in our samples 3− and PO43− fluxes (2− (0.005 µmol L−1), and this led to uncertainty in flux estimates (mean r2<0.3). There were no obvious treatment effects and no further analyses were conducted for NO3−, NO2− or PO43− fluxes.NH− fluxes . Additio−2 h−1) were lower than the sediment denitrification potential (38 to 164 µmol N m−2 h−1), which was determined from samples amended with nitrate and carbon (DEA). Two-way ANOVA revealed a significant effect of nitrate addition at both sites in winter and in summer, indicating that denitrification was always N limited, regardless of site or season  compared to the muddy-sand site  and post-hoc analysis demonstrated that denitrification potential was significantly greater (by 2.4×) in summer compared to winter at the sandy site, but there was no significant seasonal effect at the muddy-sand site. Also, denitrification potential was significantly greater (by 3×) at the sandy site than at the muddy-sand site in summer, but there was no significant difference between the sites in winter.As with GPP and NH4+ uptake and denitrification potential) in summer, compared to winter. In contrast, for the same variables at the muddy-sand site, there was no significant difference between winter and summer measurements. Similarly, the effect of Austrovenus treatment on response variables was inconsistent between sites and seasons. Although both SOC and dark chamber NH4+ fluxes increased significantly in +AS plots regardless of site and season, GPP (and NPP to a lesser extent) were increased in +AS plots only at the sandy site. An increase in GPP indicates increased MPB productivity as water column primary production was negligible. Our results suggest that increased availability of NH4+ drives this increase in MPB productivity as NH4+ uptake was higher in +AS plots at the sandy site, especially in summer. There was also a trend for greater denitrification potential in +AS sandy site plots in summer. At the muddy-sand site there was no significant effect of Austrovenus on GPP, NH4+ uptake or denitrification potential. Furthermore, GPP and denitrification potential were both significantly lower than at the sandy site.At the sandy site, there were significant increases in many response variables . More surprising are the low rates of GPP in summer, and lack of an effect of Austrovenus on GPP, at the muddy-sand site. As dark chamber NH4+ fluxes in +AS plots were similar between the two sites it seems unlikely that the reason for low MPB productivity at the muddy-sand site was nutrient limitation.As for other suspension feeding bivalves in coastal systems worldwide, resuspended MPB are an important component of Muddy sediments, despite often having higher microalgal biomass, can be less productive (in terms of rates of photosynthesis and oxygen evolution) than sandy sediments Anthopleura aureoradiata). Previous work has described a mutualistic relationship between Austrovenus and A. aureoradiata whereby the anemones use the living bivalves as hard substrate for attachment and the bivalves gain protection from parasitic infection 4+ availability in Austrovenus beds as endosymbiotic zooxanthellae can uptake NH4+ from surrounding water 4+, but further work is needed to determine whether this species is a net source or sink of NH4+, and its effect on system productivity. Barnacles were also supported on Austrovenus shells at the sandy site. It is therefore possible that the positive effect on productivity measured in +AS plots at the sandy site is not attributable to Austrovenus alone, but to the combination of Austrovenus and the macrofaunal communities they support.At the sandy site other macrofaunal abundance and biomass in +AS plots was dominated by mudflat anemones (Nucula hartvigiana and Macomona liliana). We expected OM content to increase in +AS plots at this site, due to retention of biodeposits in the lower energy environment, but instead found the reverse to be true. Deposit-feeder abundance and biomass was higher in +AS plots and they may have utilised the increased supply of OM. Alternatively, decreased mud content and increased grain size in +AS plots suggests that Austrovenus bioturbation enhanced fine sediment and OM transport by destabilising the sediment Austrovenus biodeposits do not accumulate at either site. It is probable that OM is dispersed by currents at the sandy site, and quickly utilised by deposit feeders and/or resuspended by bivalve bioturbation at the muddy-sand site.In contrast, at the muddy-sand site other macrofaunal abundance and biomass was dominated by other bivalves  would fuel coupled nitrification-denitrification but we found that OM content was not increased in +AS plots, and denitrification remained N limited regardless of site, season or addition/removal of Austrovenus. However, sediment denitrification potential (as measured with excess nitrate and carbon) did trend towards an increase in +AS plots at the sandy site in summer. Although Austrovenus biodeposits may not accumulate at the sandy site, bivalve bioturbation and excretion may have enhanced NH4+ availability, thus providing a source of nitrogen for nitrification 4+ uptake was significantly increased in +AS plots at the sandy site in summer, which may have been partly due to increased nitrification. Without measuring sediment nitrification rates, however, it is not possible to separate uptake by nitrifiers from that by MPB . Nitrifiers are known to be poor competitors for nitrogen 4+ is not limiting 4+ at the sandy site in summer as a result of Austrovenus activity likely increases both MPB productivity and sediment denitrification, though concurrent measurements of GPP, nitrification and denitrification would be needed to confirm this.Unamended sediment denitrification rates were nil or low, likely because sediment nitrification may be a major source of NOhod e.g. . This isAustrovenus size between the two sites. Individuals were significantly larger at the sandy site (c. 23 mm shell length) than at the muddy-sand site (c. 18 mm shell length). Previous research has indicated that Austrovenus condition is enhanced in sandy compared to muddier sediments Austrovenus bioturbation alters sediment chemistry. Bioturbation by macrofauna that mix surficial sediments, such as Austrovenus, can facilitate the release of solutes from sediment porewater Austrovenus tend to be retained in unfenced high-density plots, i.e. individual bivalves display minimal horizontal movement through surface sediments when in high-density beds Austrovenus bioturbation in bivalve beds is therefore likely to be small-scale (< 2–3 cm) vertical movement as the bivalves move to the sediment-water interface to feed around high tide, and thereafter retreat to just below the sediment surface. The larger bivalves at our sandy site may have reworked sediment to a greater depth than the smaller individuals at our muddy-sand site. However, solute gradients are likely to be steeper in sediments at the muddy-sand site, potentially offsetting the size difference, and making it difficult to speculate on size-specific bioturbation effects on solute fluxes.A possible confounding factor influencing the interpretation of our results is the difference in 2 profiles, nitrate and OM availability in the sediment Austrovenus, MPB and microbial communities  inherent in our experiments, but our efforts were focused on using relatively large plots as the estuarine intertidal is dynamic and subject to substantial bedload transport and sediment reworking rates e.g. ; consequAustrovenus enhanced primary productivity and sediment denitrification potential at the sandy site, whereas there was no effect of Austrovenus on these variables at the muddy-sand site, leading us to hypothesise that increasing estuarine mud content may limit the influence of this key species on ecosystem function. However, there is a need to sample across a gradient of increasing muddiness to further explore these relationships. Similarly, there is a need for more comprehensive sampling to better resolve temporal variability. Previous research has established that high levels of sedimentation are likely to reduce Austrovenus populations


Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. Bupivacaine is an amide type local anaesthetic used in clinical pain management . Althougβ/S6K1 axis /IC50), with Graph Pad Prism Software (Version 4.0).Cytotoxicity of bupivacaine was evaluated by its effect on cell viability. Accordingly cell lines NIH-3T3, C6, and C2C12 myoblasts were allowed to grow in the absence (control) or presence of bupivacaine at various concentrations over a period of 24 hrs. While the control cells exhibited normal growth characteristics, significant dose-dependent decline in cellular density was observed in bupivacaine treated cells resulting in substantial cell death . The magGrowth inhibition and apoptosis have quite often been associated with dysregulation of signaling pathways with potential to influence S6K1 activity directly or indirectly. We therefore, sought to investigate any such possibility by analyzing activity status of S6K1 in the presence or the absence of the drug. Endogenous S6K1 was immunoprecipitated from NIH-3T3 cells grown in presence or absence of different bupivacaine concentrations for its ability to phosphorylate GST-S6. As seen in Catalytic and linker domain phosphorylations at the activation loop (AL) and hydrophobic motifs (HM) are established determinants of S6K1 enzyme activity. Accordingly their loss is a hallmark of S6K1 inhibition. We therefore sought to ascertain whether inhibition of S6K1 by bupivacaine did indeed correspond with loss of these phosphorylations. As seen in In an attempt to establish that S6K1 inactivation did indeed mediate the growth inhibitory effects associated with bupivacaine, cells transfected with HA-tagged S6K1 wild type and a constitutively active variant T412E-D3E were examined for their ability to resist bupivacaine induced toxicity. mTORC1 is considered to be a major input acting upstream of S6K1 and implicated in T412 phosphorylation of the enzyme and parallel phosphorylations of its other substrate, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Possible inactivation of mTOR pathway suggested by inhibition of S6K1 should therefore be corroborated by its inability to cause phosphorylations in 4EBP1 (Threonine 70). Phosphorylation of 4EBP1 at Threonine 70 site  remainedn = 3) indicated that the extent of Erk inhibition coincided with the inhibition of S6K1 as much as it did with the kinetics of cell proliferation, to suggest that the effect of bupivacaine may be mediated through inactivation of Erk and S6K1.In the absence of mTOR as the mediator of S6K1 inhibition by bupivacaine, it was imperative to examine other signaling pathways that may directly or indirectly influence S6K1 activity in the cell. Since extracellular signal-related kinase/s (Erk) are believed to participate in the events leading to cytotoxicity by anesthetic drugs like bupivacaine, we sought to investigate the activation status of Erks to possibly relate it with observed inhibition of cellular proliferation and S6K1 activity. The activation of Erks was evaluated by immunoblotting with a phospho specific antibody. As seen in The use of local anesthetics like bupivacaine is associated with chondrotoxicity, myotoxicity, and neurotoxicity at variable propensities , 9, 32. We chose NIH-3T3 cells for their restrained response to bupivacaine induced cytotoxicity compared to C2C12 and C6 cell lines, to help us register the dynamics of signaling events responsible for preventing cell growth and proliferation. We demonstrate that the inhibition of cell proliferation by bupivacaine strictly correlated with the inactivation of ribosomal protein S6 kinase 1 and its relief by the use of constitutively active enzyme to suggest a possible dysregulation of events that control cell division, at least in part via protein translation. With long standing implications that bupivacaine may influence protein synthesis by interfering with amino acylation of tRNAs , the relMAPK signaling has a central role in regulating cell growth and proliferation . AccordiOur data suggests that bupivacaine induced inhibition of cell proliferation is mediated at least in part through inactivation of S6 kinase 1. We also demonstrate redundant role of mTORC1 signaling in mediating this effect to suggest the possible involvement of Erk pathway.


Introduction: Polyadenylation is the process in which the pre-mRNA is cleaved at the poly(A) site and a poly(A) tail is added – a process necessary for normal mRNA formation. Genes with multiple poly(A) sites can undergo alternative polyadenylation (APA), producing distinct mRNA isoforms with different 3′ untranslated regions (3′ UTRs) and in some cases different coding regions. Two thirds of all human genes undergo APA. The efficiency of the polyadenylation process regulates gene expression and APA plays an important part in post-transcriptional regulation, as the 3′ UTR contains various cis-elements associated with post-transcriptional regulation, such as target sites for micro-RNAs and RNA-binding proteins.Implications of alterations in polyadenylation for endocrine disease: Alterations in polyadenylation have been found to be causative of neonatal diabetes and IPEX  and to be associated with type I and II diabetes, pre-eclampsia, fragile X-associated premature ovarian insufficiency, ectopic Cushing syndrome, and many cancer diseases, including several types of endocrine tumor diseases.Perspectives: Recent developments in high-throughput sequencing have made it possible to characterize polyadenylation genome-wide. Antisense elements inhibiting or enhancing specific poly(A) site usage can induce desired alterations in polyadenylation, and thus hold the promise of new therapeutic approaches.Summary: This review gives a detailed description of alterations in polyadenylation in endocrine disease, an overview of the current literature on polyadenylation and summarizes the clinical implications of the current state of research in this field. Almost all eukaryotic pre-messenger RNAs (pre-mRNAs) and several non-coding transcripts have poly(A) sites and are polyadenylated  tail] is beyond the scope of this review.Pre-mRNAs are transcribed from protein-coding genes by the RNA-polymerase II (Pol-II). The nascent pre-mRNA molecule emerging from the Pol-II goes through the co-transcriptional processes of 5′ capping, splicing, and polyadenylation before transcription is terminated. Polyadenylation consists of two steps: (1) Cleavage of the pre-mRNA at a poly(A) site and (2) Addition of an untemplated 3′ poly(A) tail to the upstream cleavage product. The specific cleavage position and the efficiency of the process depend on the interaction between trans-acting polyadenylation factors and cis-elements present in the pre-mRNAs. All eukaryotic pre-mRNAs, except some replication-dependent histone pre-mRNAs, as well as several non-coding transcripts, including micro-RNAs (miRNAs), have poly(A) sites and are polyadenylated  and miRNAs. RBPs and miRNAs mainly bind target sequences in the 3′ UTR of mRNAs, thereby controlling mRNA turnover and translation rate Keene, . An examAs RBPs and miRNAs mainly target the 3′ UTR, alterations to the 3′ UTR affect the post-transcriptional regulation. More than two thirds of all mammalian genes encode alternative mRNA isoforms with different 3′ UTRs through APA  site can only be constitutively polyadenylated Figure , top, asFor genes with multiple poly(A) sites, APA can take place  sites located in the 3′ UTR of the last exon is called UTR-APA. It results in mRNAs with the same CR, but with different 3′ UTR length Figure , middle,CALCA gene encoding calcitonin/CGRP  sites located in introns or in the CR of exons is called CR-APA. It results in mRNAs with both different C-terminal CRs and 3′ UTRs Figure , bottom,CALCA gene encoding calcitonin/CGRP  site Figure  reviewe. The polThe human 3′-end processing complex consists of more than 80 proteins  The strength of the cis-elements. (2) The concentration and activity of polyadenylation factors. (3) The concentration and activity regulatory factors  sites, encountered earlier during transcription Figure . This isSequence changes in genes encoding polyadenylation factors, page 15).During colorectal carcinoma progression and in breast cancer cell lines, upregulated levels of CPSF-subunits CPSF-73, CPSF-160, CPSF-30, and symplekin correlate with proximal poly(A) site usage  gene in male germ cells  site usage, during colorectal carcinoma progression and in breast cancer cell lines  site usage by enhancing the recruitment of CPSF  site usage  and Star-PAP associates with both PIPKIα, which can generate new PI4,5P2, and with the PI4,5P2 sensitive protein kinase CKIα, which can phosphorylate Star-PAP  site usage -binding protein (PABPN1) is upregulated during the induction of stem cells from somatic cells, correlating with a general shift toward proximal poly(A) site usage  site dominantly fold into a hairpin structure, thereby inhibiting polyadenylation  sites, most significantly around the actively used ones , as a result of DNA-methylation. A CpG island separates polyA sites utilized in H13. Alleles without methylation of the CpG island utilize a proximal poly(A) site, generating a truncated H13, whereas alleles with methylation of the CpG island, utilize downstream poly(A) sites  site usage is seen in an imprinted mouse gene  mRNA, encodes a protein important for the leptin signaling pathway. SOCS3 mRNA is expressed in two isoforms due to APA. The two isoforms are expressed in an oscillating pattern and in opposite phases  sites in human were done by aligning complementary DNAs (cDNAs) and expressed sequence tags (ESTs) to the genome, thereby identifying the 3′ end of genes. These analyses revealed that more than half of human genes have multiple poly(A) sites and undergo APA  site usage genome-wide  sites by APA, altering the 3′ UTRs, and in some cases the coding potential of the mRNAs. A shift toward more proximal poly(A) site usage associates with increased proliferation  site usage is seen during increased proliferation, leading to 3′ UTR shortening. Enhanced usage of proximal intronic poly(A) sites is also seen, leading to changes in mRNA CRs  site usage differs according to tissue type, developmental stage, genotype, and cancer subtype  site is required for polyadenylation and thus transcription termination  site (AAUAAA to AAUAAG), reducing insulin mRNA expression to less than 3 × 10-4 percent . The mutation changes the PAS (AAUAAA to AAUGAA), leading to diminished FOXP3 mRNA expression  site in the SF3a2 gene into the AMH gene, due to failed polyadenylation of the SF3a2 gene, is normally seen for a small proportion of transcripts  site leading to reduced polyadenylation with more read-through transcription, could cause Mayer–Rokitanski–Küster–Hauser (MRKH) syndrome. MRKH syndrome is characterized by the Müllerian ducts failing to develop into the uterus, cervix, and upper vagina. Polymorphisms around the SF3a2 gene poly(A) site were however not found in 30 MRKH patients examined in one study   gene a PAS U/G polymorphism exists in the distal, more canonical poly(A) site. The G-allele changes the PAS (AUUAAC to AGUAAC) leading to a lesser usage of the distal poly(A) site. This reduces total SERT expression to about half in the brain , leading to impaired polyadenylation and transcription termination. This results in decreased p53 mRNA expression and read-through trancription. The change of the PAS sequence (AAUAAA to AAUACA) reduces polyadenylation efficiency about 89%  site in the F2 gene encoding thrombin  sites, can lead to enhanced gene expression caused by improved polyadenylation efficiency and to changes in APA. Such gain of function changes are associated with various diseases. They were first described in the CA dinucleotide immediately 5′ of the cleavage site in the GIMAP5 gene has two poly(A) sites in its 3′ UTR. In the PAS of the proximal poly(A) site a G/A polymorphism exists. The minor A-allele changes the PAS to a canonical one (AAUAGA to AAUAAA) leading to enhanced proximal poly(a) site usage  gene has been found. Usage of this poly(A) site leads to production of a C-terminally truncated protein isoform that repress T-cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes  site.A proximal intronic poly(A) site in the transcription factor 7-like 2 (IGF-1) gene 3′ UTR has been described. It changes the PAS (AAUAUA to AAAAUA), leading to expression of a shorter than normal IGF-1 mRNA. The U/A polymorphism has been reported in a child born small for gestation age with IGF-1 deficiency  site PAS of the insulin-like growth factor 1  site PAS of intron 19 exists  tumors. These mutations create new proximal poly(A) sites, giving rise to expression of truncated mRNAs lacking most of the 3′ UTR. These truncated mRNAs are associated with strongly proliferative MCL tumors and inferior survival  gene premutation alleles compared to normal alleles  site by altering the transcription start site  site usage due to UTR-APA is seen in the fragile X mental retardation 1 . Expression of this isoform is seen in low levels in normal subjects. In medullary thyroid carcinomas expression is more abundant, leading to higher plasma levels of CCP II   arises from CR-APA utilizing an intronic poly(A) site in the Ectopic Cushing syndrome is cause by adrenocorticotropic hormone (ACTH) production in non-pituitary tumors, such as small cell lung cancers, which is normally non-suppressible  protein express two mRNA isoforms (1.6 and 3.5 kb) differing only in the length of their 3′ UTR due to UTR-APA  sites can be induced by targeting the upstream 5′ splice site with an antisense oligonucleotide, thereby inhibiting U1 binding  site usage. They include antisense oligonucleotides  site, changes the PAS (AUUAAC to AGUAAC). This leads to lesser usage of the poly(A) site, reducing total SERT expression to about half  site in these genes, lead to an almost diminished gene expression  sites have been shown to cause disease  sites exists  Poly(A) site usage differs according to tissue type, developmental stage, genotype, and cancer subtype , critical revision of the manuscript , and obtained funding .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.The green biflagellate alga  Eukaryotic cilia or flagella are microtubule-based organelles that are highly conserved in protein composition and structural organization from protozoa to mammals. They are structurally characterized by nine microtubular doublets surrounding two central microtubular singlets . SubstruChlamydomonas reinhardtii, whose genome has been sequenced, is used as a model to study flagella structure, assembly, formation, and motility [C. reinhardtii uses flagella for motility in aqueous environments, for attaching to surfaces and for cell-cell recognition during mating. A proteomic analysis of Chlamydomonas flagella revealed more than 600 proteins [Since many years, the green biflagellate alga motility . C. reinproteins  that incproteins . But in C. reinhardtii, several proteomes and phosphoproteomes of subcellular compartments . But thiewed in , 8) wereiewed in , the eyeiewed in , and the domains . Chlamydomonas flagella anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility by reversible protein phosphorylation [C. reinhardtii named Rhythm of Chloroplast (ROC) and a homologue of Constans (CrCO) have in parallel severe effects on hatching, flagella formation, and/or movement, underlining that these processes are interconnected in C. reinhardtii [The axoneme of rylation . One of rylation –16. In prylation , 15. Morrylation . Interesnhardtii –19. C. reinhardtii, we were interested in the identification of CK1 targets in flagella beside IC138. In a comparative phosphoproteomic approach using wild-type cells with and without CKI-7 treatment, we determined the targets of CK1 in the flagellum. In the CKI-7-treated cells, several phosphoproteins were missing or were identified with a reduced number of phosphorylation sites, compared to untreated wild-type cells. Also novel phosphopeptides or additional phosphorylation sites of known phosphopeptides were identified in the CKI-7-treated cells, suggesting that CK1 is part of a signaling network in the flagellum.Regarding the multiple functions of CK1 in flagella formation and motility along with its regulatory role in the circadian system in Standard molecular biology methods were done according to .C. reinhardtii strain 137c (nit1 nit2) was used with whom the flagellar proteome and phosphoproteome were analyzed [μE m−2 sec−1 (1 E = 1 mol of photons) at 24°C. The beginning of the light period is defined as time zero (LD0) and the beginning of the dark period is LD12. In some cases, cells were released after growth in LD into constant conditions (LL) of dim light (15 μE m−2 sec−1). analyzed , 11. Celanalyzed  under a E. coli according to the Qiagen protocol. Antibodies were raised by the “Pineda-Antikörper-Service,” Berlin, Germany. Immunoblots were done as described [Protein extracts were prepared as described previously . The conescribed  using thQuantifications were done with the Image Master 2D Elite (version 4.01) software from GE Healthcare (formerly Amersham Pharmacia Biotech). Measurement of swimming velocity was done by using a hemocytometer and a differential interference contrast microscope with a total magnification of 400 including a personal computer with a digital video recording system to measure displacement versus time. The swimming velocity was determined manually by measuring the linear displacement of cells on the scale of the micrometer. 10 samples were measured to obtain the average velocity of a given sample.6 cells mL−1 by centrifugation .Cells were resuspended in one-half volume of minimal medium [μM following the shift to LL conditions. Isolation of the matrix membrane axoneme fraction (MMA) of flagella, tryptic digestion of MMA proteins, and enrichment of flagellum phosphopeptides by IMAC were done as previously described [Cells were grown in a LD cycle and harvested at the end of the night LD24) at a cell density of 2-3 × 10l medium  and then at a celescribed . −1 coupled online with a linear ion trap ESI-MS . The instrument was run by the data-dependent neutral loss method, cycling between one full MS and MS/MS scans of the four most abundant ions. After each cycle, these peptide masses were excluded from the analysis for 10 sec. The detection of a neutral loss fragment  in the MS2 scans triggered an MS3 scan of the neutral loss ion representing the dephosphorylated peptide. nLC-ESI-MS and data analysis were carried out as described before . Briefly2 and MS3 spectra. For the database searches with MS3 data, modifications of −18.00 Da on Ser and Thr residues representing the neutral loss were additionally used. Further, detection of a modification of 16 Da on Met representing its oxidized form was enabled and carboxyamidomethylation of Cys residues was enabled as a static modification. Peptide mass tolerance was set to 1.5 Da in MS mode. In MS2 and MS3 modes, fragment ion tolerance was set up to 1 Da. The parameters for all database searches were set to achieve a false discovery rate (FDR) of not more than 1% for each individual analysis. Data were searched against the flagellar proteome database [http://labs.umassmed.edu/chlamyfp/index.php). Additionally, NCBI and the Joint Genome Institute C. reinhardtii databases (Version 2 and Version 3) were used for data evaluation.Data analysis was done using the Proteome Discoverer software (Version 1.0) from Thermo Electron Corp. including the SEQUEST algorithm . The sofdatabase   compared to untreated cells   . These dThe targets of CK1 in the flagellum are of high interest with regard to flagella formation as well as for clock control events. They are largely unknown. An exception is IC138 that is suggested as a direct target of CK1 based on experimental data (summarized in ). In a next step, the direct and indirect targets of CK1 were analyzed by a functional proteome approach. For that purpose, we compared the already existing phosphoproteome  with onein vitro, as done in another study [in vivo under physiological conditions See Supplemental Figures 1(a), 1(b) in Supplementary Material available online at doi: 1155/2012/581460. We avoided to add high amounts of ATP to isolated flagella and to postincubate them at elevated temperatures to induce kinase activities er study . We foun The further analysis of the phosphoproteome in CKI-7-treated cells was carried out with the same procedure and criteria as applied before for the flagellum phosphoproteome . Three iIn the CKI-7-treated cells, phosphopeptides from 14 phosphoproteins were missing . Four adDrosophila [Also novel phosphopeptides or additional phosphorylation sites of known phosphopeptides were identified in the proteome of CKI-7-treated cells, suggesting that CK1 is part of a signaling network in the flagellum. They belong to either 15 new phosphoproteins or six already known phosphoproteins . Two othosophila . MoreoveThe identification of targets of CK1 in the flagellum will help understanding flagella formation as well as clock control events related to flagella –19. The One of the direct targets of CK1 was suggested to be IC138, the Inner Dynein Arm I1 Intermediate Chain 138. It was shown that phosphorylation of IC138 correlates with the inhibition of dynein activity and that PKA beside CK1 as well as the Protein Phosphatases PP2A and PP1 are involved there (summarized in ). IC138 Another structural phosphoprotein previously identified with two phosphopeptides and variable phosphorylation sites is ODA-DC1. The outer dynein arm docking complex (ODA-DC), which is composed of three proteins, designated DC1, DC2, and DC3, is associated with microtubules and targets the outer dynein arms to its binding site on the flagellum axoneme . In bothC. reinhardtii is composed of at least 23 proteins, and not all of them have been characterized at the molecular level [in vivo phosphorylation site at Thr-35, which is located directly in the RIIa domain , for example, the mponents . In mammmponents . Especiaydomonas . Interesydomonas , 26. ThiSupplemental Figure 1: Influences on the phosphorylation pattern of flagellum proteins by ATP treatment of isolated flagella.Supplemental Table 1: Newly identified phosphopeptides or phosphorylation sites of already known phosphopeptides in CKI-7-treated cells.Click here for additional data file.


In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent  cAMP-specific pathway that is partially transduced by EPAC.  The ubiquitous second messenger cyclic adenosine monophosphate (cAMP) is a key regulator of metabolic activity, survival, proliferation and differentiation in a wide variety of cell types. In particular, isolated cultured Schwann cells (SCs), the myelinating glia in the peripheral nervous system, are strongly dependent on the intracellular levels of cAMP. On one hand, cAMP is an instructive signal for cell cycle exit and differentiation into a phenotype that resembles that of the myelinating SC -3. On th In SCs, the transition from a proliferative (immature) to a differentiated (myelinating) stage is a developmentally regulated highly coordinated process that culminates with the production of a myelin sheath, a multispiraled extension of the plasma membrane that surrounds axons and allows the rapid conduction of electrical impulses. An early event in the process of differentiation is the upregulation of the transcription factor Krox-20/Egr-2 , a maste Accumulated evidence has indicated that cAMP controls complex cellular processes via changes in target gene transcription primarily through the activation of two downstream effectors, the cAMP-dependent protein kinase (PKA) and the newly discovered exchange protein activated by cAMP (EPAC) . Upon bi It has also became apparent that PKA and EPAC are able to simultaneously control multiple processes within the same cell and that the outcome of cAMP signaling may depend on the particular set of downstream effectors activated. Thus, we sought to investigate the differential contribution of PKA and EPAC to the cAMP-dependent regulation of SC proliferation and differentiation. To discriminate between the actions of PKA and EPAC, we used a variety of cAMP-stimulating agents, including pathway-selective cAMP analogs, along with pharmacological inhibitors to selectively interfere with PKA and EPAC signaling. Experiments were carried out using purified primary SCs growing in isolation (SC monocultures) and together with purified dorsal root ganglion (DRG) neurons (SC-neuron co-cultures). In turn, co-culture experiments were performed under conditions supportive of active cell division and myelin sheath formation, respectively. The side-by-side comparison of the mitogenic and differentiating responses of isolated and axon-related SCs allowed us to conclude that cAMP controls proliferation and myelination via different and independent downstream signaling mediators. Whereas the cAMP-dependent synergism of growth factor-induced proliferation relies on the activation of the canonical PKA pathway, differentiation and myelin formation are controlled non-canonically by cAMP through an EPAC-dependent mechanism. 3H]-thymidine into nuclear DNA, a reliable measure of S-phase entry, in neuregulin-stimulated SCs. Differentiation assays evaluated the expression of markers typical of myelinating SCs, including the myelin proteins MAG and P0 and the myelin lipid galactocerebroside (cell surface O1) by means of immunofluorescence microscopy and western blot. We also evaluated the levels of expression and nuclear localization of the transcription factor Krox-20 along with the expression of immature/non-myelinating SC markers such as GFAP and c-Jun, which are both strongly reduced by treatment with cAMP -thymidine in the absence (control) or presence of PKA inhibitors. The incorporation of [3H]-thymidine into dividing cells was evaluated in cultures consisting of neurons and SCs alone, as controls for basal levels of tritium incorporation in each cell population, along with SCs together with neurons  which was used as a specificity and negative control for EPAC activation. PKA-cAMP strongly induced PKA activity in SCs, as shown by the enhancement in phospho-PKA substrate immunoreactivity  redin vitro ; and (3)in vitro . Consistin vitro  and SC-nin vitro  without ing GFAP .  Taken together, these results suggest that EPAC signaling is sufficient to reduce proliferation but not drive SC differentiation in connection to myelin formation. Despite the availability of a number of PKA inhibitors, up until recently there were no reported EPAC antagonists to pharmacologically interfere with EPAC signaling in living cells. In the last year, novel cell permeant EPAC-specific inhibitors have become commercially available. Thus, we tested the effect of ESI-09, an inhibitor of the guanine nucleotide exchange activity of EPAC, on the induction of the expression of myelin-related markers in SCs. SCs growing in isolation and in co-culture with DRG neurons were induced to differentiate by the addition of CPT-cAMP  and asco In summary, these results are consistent with the idea that the induction of SC differentiation and myelin sheath formation is a cAMP-dependent event that relies at least in part on EPAC activation.  Our studies have shown that elevated intracellular cAMP in SCs controls proliferation and differentiation via different signaling effectors. Whereas proliferation requires activation of PKA rather than EPAC, differentiation into myelin-forming cells requires activation of EPAC rather than PKA. It is therefore possible that an increase in the balance between EPAC and PKA switches the action of cAMP from a signal that enhances growth factor-induced proliferation into one that drives a program of differentiation into a myelinating phenotype. Yet, EPAC activation alone is neither sufficient to drive a full differentiating response, nor does it account for the induction of the expression of the master regulator Krox-20, altogether indicating that a still unidentified cAMP effector is likely to be required at the onset of differentiation. These novel findings have been summarized in the schematic diagram presented in in vitro. On one hand, results using PKA-selective analogs of cAMP showed no correlation between the magnitude of the PKA activity signal and the induction of differentiation. On the other hand, it was interesting to observe an increased rather than decreased expression of Krox-20, O1, MAG, P0 and even MBP under conditions in which PKA activity was pharmacologically suppressed. This observation leads to the interesting but yet to be explored possibility that PKA exerts a negative regulation on the myelination process. The PKA-independent control of SC differentiation was confirmed by using a battery of PKA agonists and antagonists acting through different molecular mechanisms. Results were also validated by using isolated adult and postnatal SCs in combination with organotypic SC-neuron cultures that faithfully recapitulate essential features of SC development including axon contact-stimulated proliferation and the formation of myelin sheaths -thymidine (6.7 Ci/mmol) and SolvableTM were from Perkin-Elmer . O1 hybridoma cells -thymidine into nuclear DNA was assayed as a measure of S-phase entry, as reported previously [3H]-thymidine (0.25 µCi/well). In some experiments, a mitogenic concentration of forskolin (2 µM) was used to enhance growth factor-induced SC proliferation, and thereby achieve a maximal response in [3H]-thymidine incorporation [ The incorporation of [eviously . Brieflyporation . 3H]-thymidine. For co-culture initiation, subconfluent cultures of mitogen- and serum-starved SCs were trypsin-dissociated to obtain a single cell suspension  prior to being placed on top of cultures of purified DRG neurons [3H]-thymidine. The incorporation of tritium into the proliferating SCs was determined by liquid scintillation counting 2-3 days after mitogenic stimulation or co-culture initiation. Stocks of cAMP analogs were prepared in DMEM and inhibitors in DMSO, according to the manufacturer’s instructions. The final concentration of DMSO in the culture medium was calculated to be less than 0.2%. Cells in the control condition were stimulated with the appropriate volume of vehicle (i.e. DMEM or DMSO).  Axon contact-induced SC proliferation was measured by plating purified SCs on purified dissociated DRG neurons in the presence of -x-[S*/T*]) in target proteins . It has been shown previously that this antibody detects both cytoplasmic and nuclear phosphorylated PKA substrates in SCs and that the extent of phospho-PKA substrate immunoreactivity is directly proportional to the levels of PKA activation and intracellular cAMP . The act To measure the nucleotide exchange activity of EPAC, we assayed the GTP-binding activity of its downstream effector, Rap1. For this, we used a standard method that involves the precipitation of GTP-bound Rap1 molecules by affinity binding to purified recombinant Ral-GDS-agarose beads, according to the manufacturer’s recommendations (Roche). Purified samples containing GTP-bound Rap1 were denatured in SDS loading buffer, resolved by 15% denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by western blotting using anti-Rap1 antibodies (Santa Cruz). Samples from total cell lysates were also analyzed as controls for total Rap1 expression.  Total cell lysates were prepared as reported previously . Equal p The statistical significance of the different treatments was analyzed relative to each control group by a one-way analysis of variance (ANOVA) followed by a post-hoc Tukey test using IBM SPSS statistics software. A p-value <0.05 was interpreted as statistically significant. Data from a representative experiment out of at least three independent experiments performed are shown.


Leucine-rich repeat kinase 2 (LRRK2), a large protein kinase containing multi-functional domains, has been identified as the causal molecule for autosomal-dominant Parkinson's disease (PD). In the present study, we demonstrated for the first time that (i) LRRK2 interacts with tau in a tubulin-dependent manner; (ii) LRRK2 directly phosphorylates tubulin-associated tau, but not free tau; (iii) LRRK2 phosphorylates tau at Thr181 as one of the target sites; and (iv) The PD-associated LRRK2 mutations, G2019S and I2020T, elevated the degree of tau-phosphorylation. These results provide direct proof that tau is a physiological substrate for LRRK2. Furthermore, we revealed that LRRK2-mediated phosphorylation of tau reduces its tubulin-binding ability. Our results suggest that LRRK2 plays an important role as a physiological regulator for phosphorylation-mediated dissociation of tau from microtubules, which is an integral aspect of microtubule dynamics essential for neurite outgrowth and axonal transport. Tau is a microtubule-associated protein found predominantly in the central nervous system and expressed mainly in neuronal axons Leucine-rich repeat kinase 2 (LRRK2) is the causative molecule of familial Parkinson's disease (PD) It has been reported that neurite length is reduced in LRRK2-deficient cultured mouse neurons 32P]ATP (3000 Ci/mmol) was obtained from Perkin Elmer Inc. ; dithiothreitol (DTT) was from Wako Pure Chemical ; recombinant N-terminal glutathione S-transferase (GST)-tagged LRRK2  were from Invitrogen ; recombinant tau protein 441  was from Signal Chem Pharmaceuticals ; purified porcine tubulin was from Cytoskeleton ; and bovine myelin basic protein (MBP) was from Sigma-Aldrich .. In another experiment, recombinant tau was mixed with GST-LRRK2 or GST alone and incubated with glutathione agarose beads in the presence or absence of tubulin in 100 µL of TNE buffer overnight at 4°C. After centrifugation, the supernatant was removed, and the beads were washed three times with TNE buffer. The bound proteins were eluted from the beads by boiling in 50 µL of SDS-PAGE sample buffer. The GST-LRRK2 , tau and tubulin in the eluted samples were detected by Western blotting.2+, 3 µCi of [γ-32P]ATP] in the presence or absence of tubulin (1 µg). After incubation for 30 min at 30°C, the reaction was stopped by adding SDS-PAGE sample buffer and boiling. The 32P-labeled proteins in the reaction mixture were detected by autoradiography followed by SDS-PAGE, as described previously Recombinant tau was used as a substrate for GST-LRRK2. Briefly, 2 µg of tau was incubated with 50 ng of GST-LRRK2  in 25 µL of standard reaction mixture ATP in the presence or absence of tubulin. We found that LRRK2 markedly phosphorylated tau in the presence of tubulin, whereas it phosphorylated tau only very slightly in the absence of tubulin   . These fts (a–d) . These rin vivo, suggesting that tau is likely the physiological substrate for LRRK2.To confirm the tau-phosphorylation activity of LRRK2 in neural cells, we next analyzed a SH-SY5Y clone, WT4-D33, overexpressing V5-wild-type LRRK2 It has been reported that increase of tau phosphorylation at Thr181 correlate with neurite retraction in SH-SY5Y cells 32P-phosphorylated tau was not detected in the precipitated fraction (upper panel), although non-phosphorylated tau was detectable in this fraction (lower panel). In contrast, 32P-phosphorylated tau was detected only in the LRRK2-unbound supernatant. Furthermore, we investigated whether tau phosphorylated at Thr181 interacts with LRRK2 using a co-immunoprecipitation assay with a WT4-D33 cell lysate. We found that phosphorylated tau (Thr181) was not detected in the fraction containing LRRK2, whereas immunoreactivity for phospho-independent tau and tubulin was detected in the same fraction (As the association of tau with tubulin is well known to be modulated in a phosphorylation-dependent manner, we investigated the effect of LRRK2-mediated phosphorylation on the tubulin-binding capacity of tau. After full phosphorylation of recombinant tau by GST-LRRK2 in the presence of tubulin, we performed a GST pull-down assay. As shown in fraction . These rIn the present study, we found for the first time that LRRK2 directly phosphorylates tubulin-associated tau and reduces its tubulin-binding ability, whereas LRRK2 does not phosphorylate the free tau molecule. Thus, LRRK2 would serve as a regulator of association/dissociation between tau and tubulin . In neurTau has not been reportedly detected as the LRRK2 binding protein in other studies involving proteomic analysis In the present study, we identified a Thr181 as one of the direct target sites of tau for LRRK2. It has been shown in mouse models that the level of tau phosphorylation at Ser202/Thr205 in the brain was elevated in R1441G and G2019S LRRK2 transgenic mice and diminished in LRRK2-knockout mice The present results indicating that LRRK2-mediated phosphorylation of tau enhances its dissociation from tubulin suggest that this process is one of the important regulatory mechanisms for microtubule disassembly, which may lead to reduced neurite outgrowth. In mouse neurons, however, there has been some controversy as to whether kinase activity of LRRK2 reduces or promotes neurite outgrowth. Neurite length and branching are reportedly increased by LRRK2-knockdown or LRRK2-kinase inactivation Several candidate LRRK2-substrate molecules and various mechanisms of neurodegeneration caused by LRRK2 mutations have been reported