Motion anticipation allows the visual system to compensate for the slow speed of phototransduction so that a moving object can be accurately located. This correction is already present in the signal that ganglion cells send from the retina but the biophysical mechanisms underlying this computation are not known. Here we demonstrate that motion anticipation is computed autonomously within the dendritic tree of each ganglion cell and relies on feedforward inhibition. The passive and non-linear interaction of excitatory and inhibitory synapses enables the somatic voltage to encode the actual position of a moving object instead of its delayed representation. General rather than specific features of the retinal connectome govern this computation: an excess of inhibitory inputs over excitatory, with both being randomly distributed, allows tracking of all directions of motion, while the average distance between inputs determines the object velocities that can be compensated for.DOI:http://dx.doi.org/10.7554/eLife.06250.001 The retina is a structure at the back of the eye that converts light into nerve impulses, which are then processed in the brain to produce the images that we see. It normally takes about one-tenth of a second for the retina to send a signal to the brain after an object first moves into view. This is about the same time it takes a tennis ball to travel several meters during a tennis match, yet we are still able to see where the moving tennis ball is in real time. This is because a process called ‘motion anticipation’ is able to compensate for the delay in processing the position of a moving object. However, it was not known precisely how motion anticipation occurs.Inside the retina, cells called photoreceptors detect light and ultimately send signals (via some intermediate cell types) to nerve cells known as retinal ganglion cells. These signals can either excite a retinal ganglion cell to cause it to send an electrical signal to the brain, or inhibit it, which temporarily prevents electrical activity. Each cell receives signals from several photoreceptors, which each connect to a different site along branch-like structures called dendrites that project out of the retinal ganglion cells.Johnston and Lagnado have now investigated how motion anticipation occurs in the retina by using electrical recordings of the activity in the retinas of goldfish combined with computer simulations of this activity. This revealed inhibitory signals, sent from photoreceptors to retinal ganglion cells via a type of intermediate cell , play a key role in motion anticipation. The ability to track motion effectively in all directions requires more inhibitory signals to be sent to the dendrites of a retinal ganglion cell than excitatory signals. These two types of input must also be randomly distributed across the cell. Furthermore, it is the density of these input sites on a dendrite that determines how well the retina can compensate for the motion of a fast-moving object. The building blocks required for motion anticipation in the retina are also found in visual areas higher in the brain. Therefore, further work may reveal that higher visual areas also use this mechanism to predict the future location of moving objects.DOI:http://dx.doi.org/10.7554/eLife.06250.002 The brain estimates the location of an object in visual space by reading out which retinal ganglion cells (RGCs) respond to it. This ‘retinotopic map’ is preserved through the visual pathway and is used to guide behaviour . There iAlthough the retina is capable of processing the visual input in a variety of ways, we still do not understand how most of these transformations are achieved . One aspWe have used electrophysiology and modelling to investigate the mechanisms of motion anticipation and find that it is not usually exerted through changes in the excitatory inputs to RGCs, but rather depends on the shunting effect of inhibition that RGCs receive from amacrine cells. The non-linear interaction between excitatory and inhibitory synapses is a result of the passive properties of the dendritic tree and remains intact when active conductances are blocked. Motion anticipation operates across most ganglion cells because it depends on general rather than specific features of the retinal connectome: independent and random distributions of excitatory and inhibitory synapses on RGCs ; an exce−1 (7.5° s−1), the peak spike rate occurred 46 ± 13 ms (n = 25) before the leading edge reached the receptive field centre and then activity decayed before the bar left  and intracellular resistance (Ri) , and in 2+ channels in the dendrites of starburst amacrine cells acts to boost EPSPs moving from the soma toward distal dendrites  and 14.7° s−1 (1000 μm s−1) but not at 27.8° s−1 (2000 μm s−1) or higher  inhibitory inputs should be located on the path between the excitatory synapse and the soma , and (b) inhibitory inputs should be activated  synapse . Satisfy synapse , left, bthe soma , right. The conditions in which proximal inhibition blocks excitation arriving at the soma that were laid out by To investigate how the ratio of inhibition to excitation affected the generation of motion anticipation we carried out simulations in which we varied the number of inhibitory inputs in the model cells. When the ratio of inhibition to excitation (I/E) was increased, the resultant EPSPs became smaller  and the −1 in most RGCs in salamander, rabbit and goldfish . Extracellular recordings were made in voltage-clamp mode. For whole-cell voltage-clamp recordings, the intracellular solution contained 104 mM CsMeSO4, 8 mM CsCl, 5 mM Na2 Phosphocreatine, 4 mM HEPES, 2 mM Mg.ATP, 1 mM Na.GTP, 1 mM EGTA and 2 mM QX-314-Chloride, with this solution the calculated reversal potential for Cl− was −59 mV. Voltage-clamping at −60 mV and 0 mV isolated the EPSC and IPSC respectively. For whole-cell current-clamp measurements with high Cl− the intracellular solution contained 120 mM KCl, 10 mM Na2 Phosphocreatine, 4 mM HEPES, 2 mM Mg.ATP, 1 mM Na.GTP, 0.15 mM EGTA. For the passive dendrite recordings in 4, 8 mM KCl, 10 mM Na2 Phosphocreatine, 10 mM QX-314-Bromide, 4 mM HEPES, 2 mM MK801, 2 mM Mg.ATP, 1 mM Na.GTP, 0.15 mM EGTA. Pipettes had a resistance of 5–6 MΩ. Signals were digitized using an ITC18 A-D converter and acquired on a Mac mini using Neuromatic running in Igor Pro 6 .All procedures were carried out in accordance with the UK Animals (Scientific Procedures) act 1986. Retinae were removed from goldfish (15–20 cm long) and placed in AMES solution  diluted to 270 mOsM. Pieces of retina ∼1 cmSpikes were recorded from RGCs on a 60 channel multi-electrode array  using perforated electrode arrays. Spikes were sorted as described previously  using Wa−2 and our standard stimulus was a bar of −100% contrast measuring 160 µm by 2400 µm on the retina. To relate the retinal images to objects in the real world we measured the distance between the centre of the lens and the retina, for the goldfish used in this study (∼150 mm in length) this was ∼3.8 mm. All sizes and speeds have been converted to degrees of visual angle according to the equation:A 852 × 600 pixel monochromatic OLED micro-display  was focused onto the photoreceptor layer of the retina through an oil condenser. Pixels measured 4 × 4 µm on the retinal surface. Visual stimuli were delivered via Matlab  using psychophysics toolbox libraries. Visual stimulation and electrophysiology were synchronized by recording the times of screen refreshes and the timing precision was verified with PMTs. The mean irrandiance was 40 nW mmR/n = tanV,−1.where R is the size of the retinal image, n is the distance from the retina to the lens centre and V is visual angle. Therefore our standard stimulus of a 160 µm bar covered ∼2.4° of visual angle and moved at ∼7.5° sInitially RGCs were classified as OFF vs ON and brisk-transient vs brisk-sustained by flashing full field stimuli of −100 and 100% contrast for 0.5 s each. Brisk transient cells responded strongly to stimulus onset then adapted completely. Brisk-sustained cells responded strongly initially and then adapted slightly over the rest of the stimuli. No ON cells were encountered in our recordings. We also tested for both direction-selectivity and orientation-selectivity by moving a bar across the retina at four different angles. We encountered two orientation-selective cells but no direction-selective cells. The orientation selective cells responded strongly to a moving bar in their preferred orientation and failed to spike to a bar orthogonal to this axis.Similar to , the RF http://www.igorexchange.com/project/DendritePruner) and imported to neuroConstruct.RGCs were filled with 50 µM alexa 488 by dialysis through the patch pipette then, subsequent to electrophysiological recording, a volume containing the RGC dendrites and soma was acquired with a custom built 2-photon microscope similar to . The mor−2 and membrane resistance (Rm) = 20 MΩ cm−2, however, we found that these parameters were not critical for the appearance of motion anticipation  to each synapse, the array of bipolar and amacrine synapses were divided in to strips with a width of 30 µm. The RF of each model ganglion cell was measured by stimulating a single 30 µm strip and plotting the amplitude of the somatic EPSP vs space, similar to the performed physiological measurements. To simulate a bar moving across the retina each strip was given a set of values for t0 that reflected the time required for the leading edge of the bar to traverse the 30 µm strip, each synapse was then randomly assigned a value from this set. For example, for a bar moving at 0.5 mm s−1 the t0 values of the first strip would range from 0 ms to 60 ms and for the subsequent strip range from 60 ms to 120 ms. For the simple model used in To facilitate assignment of an activation time , Matteo Carandini (Reviewing editor), and by two reviewers, one of whom, Michael Berry, has agreed to share his identity.Thank you for sending your work entitled “General features of the retinal connectome determine the computation of motion anticipation” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.In this excellent study, Johnston and Lagnado tackle an important and interesting phenomenon exhibited by signals in many types of retinal ganglion cells (RGCs): motion anticipation. This is the observation that peak RGC spiking activity (or that of a population of RGCs) is reached prior to a moving object arriving at the center of the receptive field. This observation is more than a decade old now, but the mechanisms that generate it are poorly understood.To elucidate these mechanisms, the authors record from RGCs in the goldfish retina, and show these cells, like RGCs in several other species, exhibit motion anticipation. They then use a combination of electrophysiological, pharmacological, and computational/modeling approaches to show that motion anticipation results from a very generic mechanism: shunting inhibition on the RGC dendrites. They further show that dendritic active conductances and excitation (with feedback inhibition to bipolar cells) alone are not sufficient to give rise to motion anticipation.The authors use models of 3 RGCs where they have measured dendritic architecture, along with synaptic densities taken from literature, to show that a 2:1 ratio of randomly placed inhibitory to excitatory synapses can account for the motion anticipation they observed in their recorded cells. Motion anticipation, therefore, can result from randomly distributed synapses as long as inhibitory synapses outnumber excitatory by about 2-to-1. Such conditions are met on many RGC dendrites  and might well be observed in other parts of the brain, making this biophysical mechanism potentially quite general. More generally, this study makes a compelling case that feedforward inhibition plays a major role in motion anticipation.Also, the paper goes on to add some intuition underlying the phenomenon: strong shunting that occurs when inhibitory synapses are activated just before excitatory synapses located distally . In particular, Overall, the manuscript makes a convincing case that feedforward inhibition is a central mechanism contributing to motion anticipation. This is a significant advance. What is less clear is whether it is the only mechanism. Below are some suggestions for improving the paper on this and other fronts. It should be possible to address these suggestions by editing the paper to highlight and discuss some of these issues, or perhaps by analyzing additional existing data, without necessarily requiring new experiments.1) The manuscript claims that a general feature of the retinal connectome is 2:1 ratio in the number of inhibitory to excitatory synapses. The literature, however, does not seem to substantiate this claim. For instance the recent paper by Abbott et al., (2012) from the laboratories of Grunert and Martin working on the marmoset retina. They show that parasol and midget RGCs across a wide range of eccentricities have a 1:1 ratio of inhibition to excitation. Several studies from old world monkeys by Calkins, Sterling, Marshak, and Kolb indicate that inhibitory synapses in the peripheral retina outnumber excitatory synapses, but that the opposite is true in the central retina. Studies of bistratified RGCs in the monkey suggest that the proportion of excitatory and inhibitory synapses depends on dendritic strata. Thus the ratio of inhibitory to excitatory synapses depends on species, ganglion cell type, and retinal location.Given that a 2:1 ratio does not appear to be a general feature, do the RGCs probed in this study exhibit a 2:1 ratio in synapse number? This seems important to determine to validate their analysis and conclusions. The authors show that the ratio of inhibition to excitation functionally is about 2:1, but doesn't synapse position/number also matter? Also, the authors show a large range of I:E ratios in different RGCs. Does the ratio of I:E correlate with the amount of motion anticipation, with low ratios exhibiting less anticipation? This seems straightforward to test on a cell by cell basis and would help to relate inhibition directly motion anticipation rather than by the process of elimination.2) In 3) It would be nice to see the degree of motion anticipation of individual RGCs before and after dialyzing the cells with MK801 and QX-314-Bromide to eliminate active conductances. The authors show active conductances are not required, but it seems like they may still augment or amplify the amount of motion anticipation. As it stands the reader is left with the impression that active conductances don't participate, but it is not clear that this is the case. This question speaks to the issue of sufficiency. Currently, 4) How do the speeds of motion used in this study relate to speeds in the real world? The authors motivate their study by the important contribution the retina makes to the behavioral phenomenon of motion anticipation. However, without knowing the size of a goldfish eye, one cannot relate any of the speeds they probed experimentally to the speed of objects in the environment. The findings in this study could be more relevant to a general readership if this relationship is made explicit and speeds are quoted in units of degrees of visual angle per second. Related, it seems important to note that this will depend on the size of the animal's eye. Thus, given an equivalent amount of motion anticipation per RGC in a small eye compared to a large eye, there will be very different amounts of motion anticipation at the level of the retina. This seems worth noting in the Discussion. 1) The manuscript claims that a general feature of the retinal connectome is 2:1 ratio in the number of inhibitory to excitatory synapses. The literature, however, does not seem to substantiate this claim. For instance the recent paper by Abbott et al., (2012) from the laboratories of Grunert and Martin working on the marmoset retina. They show that parasol and midget RGCs across a wide range of eccentricities have a 1:1 ratio of inhibition to excitation. Several studies from old world monkeys by Calkins, Sterling, Marshak, and Kolb indicate that inhibitory synapses in the peripheral retina outnumber excitatory synapses, but that the opposite is true in the central retina. Studies of bistratified RGCs in the monkey suggest that the proportion of excitatory and inhibitory synapses depends on dendritic strata. Thus the ratio of inhibitory to excitatory synapses depends on species, ganglion cell type, and retinal location.Given that a 2:1 ratio does not appear to be a general feature, do the RGCs probed in this study exhibit a 2:1 ratio in synapse number? This seems important to determine to validate their analysis and conclusions. The authors show that the ratio of inhibition to excitation functionally is about 2:1, but doesn't synapse position/number also matter? Also, the authors show a large range of I:E ratios in different RGCs. Does the ratio of I:E correlate with the amount of motion anticipation, with low ratios exhibiting less anticipation? This seems straightforward to test on a cell by cell basis and would help to relate inhibition directly motion anticipation rather than by the process of elimination.The literature concerning the ratio of inhibitory to excitatory synapses is broad and consistently indicates that for non-midget ganglion cells, inhibitory synapses outnumber their excitatory counter parts. We state that this is a general feature as it has been detailed across many species, including cat , goldfish , primates (including human) , toad , turtle  as well as the ground squirrel .The paper by Abbott et al. (2012) uses light microscopy and finds lower densities of inhibitory synapses than other studies using electron microscopy. The deficiencies in light compared to electron microscopy are well known. A point which the authors concede in their discussion: “In summary, the absolute density of the presumed amacrine input analyzed with our light microscopic methods is likely to be lower than absolute density estimated by electron microscopy.”Furthermore, the method used to identify inhibitory synapses in this paper is likely to systematically exclude some inhibitory synapses, the authors state: “Thus, amacrine synapses containing the a1 subunit of the GABAA receptor are probably not included in counts of gephyrin IR puncta and thus also contribute to underestimation of amacrine input.”The synapse positions used in our modeling reflect the known distributions of synapses; excitatory synapses are uniformly distributed across the dendrites and inhibition is distributed independently of these. This observation is also consistent across many species .We have included a new section in the Discussion on the generality of the mechanism and discuss this in light of the additional data we include showing that RF size is related to the amount of motion anticipation. We make specific reference to the lower I/E ratios observed in the paraforeal midget RGCs of primates.For your reference, in the Results section, we added the following sentences:“RGCs with larger RFs tended to display greater anticipation, with RF size accounting for 35% of the observed variance in the delays for motion  In This was a nice suggestion and we thank the editor/reviewers for it. In four of the cells we also presented a flashed bar after disrupting inhibition. There was no change in the delay to peak firing with disruption of inhibition (control = 64.4 ± 8.7 ms vs disrupted inhibition = 62.7 ± 5.2 ms). This data has been added to a panel in “Importantly, with inhibition disrupted, the location of the peak firing became delayed occurring 210 ± 26 ms after the stimuli had reached the RF centre .”3) It would be nice to see the degree of motion anticipation of individual RGCs before and after dialyzing the cells with MK801 and QX-314-Bromide to eliminate active conductances. The authors show active conductances are not required, but it seems like they may still augment or amplify the amount of motion anticipation. As it stands the reader is left with the impression that active conductances don't participate, but it is not clear that this is the case. This question speaks to the issue of sufficiency. Currently, Unfortunately, in this last set of experiments, we did not record motion-evoked spikes in the same cells prior to breaking into whole-cell mode. We had already demonstrated that the majority of RGCs display motion anticipation . Our modHowever, we have now included a comparison of the time of the peak EPSP in passive dendrites with that observed for the time of peak spike rate from the data in This paper focuses on how motion anticipation arises from a mechanism common to the majority of ganglion cells. We have not addressed whether active conductances augment motion anticipation; if this occurs it is likely to vary in a cell type specific manner. We have added our thoughts on this to the Discussion (subsection “Motion anticipation is autonomous to each ganglion cell”): “We show that motion anticipation emerges from the passive properties of dendrites, but it is possible that subtypes of RGCs could augment anticipation using active conductances. […] Additionally dendritic spikes combined with gap junction coupling allow a specific subtype of directionally selective ganglion cell to respond much earlier than expected to motion in its preferred direction .”4) How do the speeds of motion used in this study relate to speeds in the real world? The authors motivate their study by the important contribution the retina makes to the behavioral phenomenon of motion anticipation. However, without knowing the size of a goldfish eye, one cannot relate any of the speeds they probed experimentally to the speed of objects in the environment. The findings in this study could be more relevant to a general readership if this relationship is made explicit and speeds are quoted in units of degrees of visual angle per second. Related, it seems important to note that this will depend on the size of the animal's eye. Thus, given an equivalent amount of motion anticipation per RGC in a small eye compared to a large eye, there will be very different amounts of motion anticipation at the level of the retina. This seems worth noting in the Discussion.-1 corresponds to a bar covering 2.4° of visual angle moving at 7.5° s-1. We have now added this calculation to the Methods section and updated all appropriate units to degrees of visual angle.For the goldfish sizes used in this study (∼150 mm in length), the distance between the lens centre and retina was ∼3.8 mm, this was estimated by freezing and bisecting eyes from 3 fish. Our standard stimuli of a 160 µm bar moving at 500 µm s


Diacylglycerol kinases (DGKs) transform diacylglycerol (DAG) into phosphatidic acid (PA), balancing the levels of these key metabolic and signaling lipids. We previously showed that PA derived from the DGKζ isoform promotes mammalian target of rapamycin complex 1 (mTORC1) activation. This function might be crucial for the growth and survival of cancer cells, especially for those resistant to the allosteric mTOR inhibitor rapamycin. How this positive function of DGKζ coordinates with DAG metabolism and signaling is unknown. In this study, we used a rapamycin-resistant colon cancer cell line as a model to address the role of DGKζ in tumor cells. We found that DGKζ predominated over other PA sources such as DGKα or phospholipase D to activate mTORC1, and that its activity was a component of the rapamycin-induced feedback loops. We show that the DGKζ DAG-consuming function is central to cell homeostasis, as DAG negatively regulates levels of the lipogenic transcription factor SREBP-1. Our findings suggest a model in which simultaneous regulation of DAG and PA levels by DGKζ is integrated with mTOR function to maintain tumor cell homeostasis; we provide new evidence of the crosstalk between mTOR and lipid metabolism that will be advantageous in the design of drug therapies. Hyperactivation of the mTOR complex 1 (mTORC1) downstream of oncogenic mutations in cancer provides the metabolic reprogramming that allows autonomous proliferation and lipogenic tumor metabolism.11 Development and evaluation of mTOR-based anticancer therapies require better characterization of the elements that contribute to mTOR crosstalk through different pathways.Because of the central role of mTOR in cancer biology, its inhibitors are targets for the development of anticancer drugs. Many of the genetic alterations responsible for colorectal carcinogenesis act through the mTOR pathway, suggesting that mTOR inhibitors could be effective in preventing colon cancer progression. The therapeutic effectiveness of these inhibitors is nonetheless limited, due mainly to the intricate regulation of mTOR and to negative feedback loops that lead to resistance.14 that facilitates allosteric mTOR activation and displacement of the endogenous inhibitor FKBP38.15 Rapamycin, a potent mTOR inhibitor,16 associates with the immunophilin FBPK12 to form a complex that prevents FRB/PA interaction.12 Although effective against renal cancer, rapamycin derivatives have only modest effects and limited success in clinical trials for other solid tumors. In advanced cancers, rapamycin-mediated inhibition of mTORC1 releases feedback loops that activate the mTOR complex 2 (mTORC2) and sustain tumor survival via AKT18 and SREBP-1-dependent fatty acid synthesis.19 The activity of PA-generating enzymes, predicted to limit rapamycin inhibition,20 might also temper the rapamycin-triggered feedback loops responsible for resistance in advanced cancers. PA-generating enzyme expression and/or activity could thus provide a basis for cancer therapies.The regulation of mTORC1 by the lipid phosphatidic acid (PA) has been proposed to mediate resistance to pharmacological mTOR inhibition. PA activates mTOR by binding to the FKBP12-rapamycin-binding domain (FRB)21 DGKζ-derived PA promotes mTORC1 activation in several cell systems; it promotes mTORC1-mediated phosphorylation of S6K in response to serum in HEK293 cells22 and C2C12 myoblasts,23 and activates mTORC1 in response to mechanical stimulation of muscle cells.23 By controlling TOR function, the Drosophila and Caenorhabditis elegans DGKζ homologs rdgA and dgk-5 regulate lifespan and the oxidative stress response.24 DGKζ-mediated mTORC1 activation might support cancer growth and responses to mTORC1 inhibitors, which coincides with increased DGKζ expression in colon cancer compared with normal tissue,28 and with the observation that colon tumors show marked resistance to mTOR inhibition.29Diacylglycerol kinase-ζ (DGKζ) is a DGK family enzyme that, by phosphorylating diacylglycerol (DAG), produces PA.Here we used a rapamycin-resistant colon cancer cell line to examine the DGKζ contribution to mTORC1 activation and rapamycin-elicited responses. We confirm that DGKζ operates upstream of mTORC1, limiting its sensitivity to rapamycin, and we identify rapamycin-mediated DGKζ activation as a feedback regulatory circuit by which DGKζ helps to regulate SREBP-1 levels via DAG restriction. As a result, whereas DGKζ depletion sensitizes cells to rapamycin-dependent AKT activation, it severely impairs SREBP-1 stabilization, limiting cell proliferation. Our results show additional DGKζ contributions to the control of mTORC1 functions and provide evidence that DGKζ is a regulatory node in cancer metabolism.30 DGKζ expression was silenced by interfering RNA (RNAi) with a previously validated sequence.31 We determined total DGK activity in DGKζ-silenced cells and compared it with that of reducing DGKα, also expressed by this cell line. DGKζ silencing had a greater effect on total DGK activity than DGKα silencing , another PA-producing enzyme that regulates phosphorylation of the mTORC1 target p70S6K33  decreased early p70S6K phosphorylation, but was more pronounced for DGKζ  in cells with reduced DGKζ expression  are reported to be rapamycin insensitive and AKT regulated in some tumors,19 suggesting a role for mTORC2 in the control of lipid metabolism.Our data suggested that although DGKζ expression limits rapamycin-triggered AKT activation, it has additional functions that sustain long-term cancer cell growth. Activation of SREBP-1 cleavage in tumor cells helps to maintain cell-autonomous tumor growth.We first determined the effect of rapamycin and DGKζ silencing on SREBP-1 transcriptional activity in SW480 cells. DGKζ silencing diminished SREBP-1 and FASN transcription, suggesting reduced SREBP-1 function . RapamycReduced SREBP-1 transcription may be the result of impaired proteolytic cleavage of its precursor. We next analyzed whether rapamycin and DGKζ depletion modified SREBP-1 processing. Treatment of SW480 cell with low rapamycin doses increased levels of the processed SREBP-1 form, with no change in the unprocessed form . This isSREBP-1 is closely linked to the capacity of cancer cells to promote lipogenic metabolism; they synthesize the bulk of their lipids from autonomous fatty acids. Reduction of serum levels in SW480 cell culture medium decreased mTORC1 activation to levels similar to those triggered by rapamycin . SREBP-1These experiments confirmed the rapamycin insensitivity of SREBP-1 processing in SW480 cells, and suggested that DGKζ is necessary to maintain SREBP-1 precursor levels. SREBP-1 activity might require DGKζ-derived PA or be negatively modulated by a DGKζ-metabolized DAG pool. As we found that DGKζ silencing increased cellular DAG levels, we explored the DAG contribution to the maintenance of SREBP-1 levels.36 as PKD phosphorylation is very sensitive to DAG levels, we used it as an indicator of DAG-mediated function. When cells were treated with dioctanoyl (C8)-DAG, western blot analysis showed a modest reduction in SREBP-1 levels and no increase in PKD phosphorylation  is activated by direct DAG binding and by protein kinase C (PKC)-dependent phosphorylation;rylation . We alsorylation . These e2+-sensitive type I DGK, mainly DGKα, more efficiently than other isoforms.37 SW480 cells express DGKα but not type I DGKβ and γ.32 We compared the effects of DGKζ and DGKα silencing on SREBP-1 levels and PKD phosphorylation with those elicited by pharmacological inhibition of DGK. The sharp reduction in SREBP-1 levels following DGKζ depletion was isoform specific, as DGKα silencing did not alter SREBP-1 expression , whereas it sustains SREBP-1 function. To determine whether DGKζ regulation of SREBP-1 might be mediated through DAG-activated kinases, we analyzed SREBP-1 levels in cells treated with Bim (a pan-PKC inhibitor) or with Gö6976 . Both drugs increased SREBP-1 levels, suggesting negative control of SREBP-1-mediated transcription by classical PKC/PKD kinases . In agreDGKζ helps to maintain SREBP-1 levels in rapamycin-treated SW480 cells, suggesting that DGK activation is a consequence of rapamycin-triggered mTORC1 inactivation. Rapamycin treatment decreased DAG levels in SW480 cells, and this effect was reversed by pharmacological DGK inhibition . Measure38 mTORC2 phosphorylates AKT at Ser473 and PKCα at its hydrophobic motif, Ser657.39 We reasoned that rapamycin triggering of mTORC2 could activate the PKCα/DGKζ axis. Rapamycin treatment of SW480 cells triggered early activation of mTORC2, determined as phosphorylation of AKT and PKCα at their hydrophobic motifs, observed as early as 15 min post rapamycin addition  activation,18 two signals that mediate DGKα activation. The recent characterization of the DGKα contribution to Src activation in SW480 cells suggests that DGKα activation helps to sustain the feedback circuits released by mTORC1 inhibition.32Rapamycin competes with PA for mTORC1 binding that correlates with the enhanced sensitivity of DGKζ-silenced SW480 cells to rapamycin, including activation of mTORC1-controlled negative feedback. These data confirm DGKζ-derived PA regulation of mTOR,43 that is both an effector and a component of mTORC2 signaling.45 Rapamycin short-term kinetics confirmed concomitant phosphorylation of AKT and PKCα at their mTORC2-regulated sites. PKCα phosphorylation of DGKζ triggers its activation, thus terminating DAG-regulated signals.38 The sustained AKT phosphorylation contrasted with the transient PKCα phosphorylation and that of its substrates. These results suggest that mTORC2 activation by rapamycin triggers DGKζ that limits DAG-mediated signals. Analysis of torin-treated cells confirmed DGKζ activation downstream of mTORC2, indicating activation of DAG metabolism as component of rapamycin-triggered feedback. This mutual DGKζ and mTOR regulation resembles that of mTOR and PI3K; whereas in normal cells, such axes help to maintain homeostasis, in cancer cells they mediate the cell rewiring that contributes to drug resistance.DGKζ is a substrate of PKCα46 SREBP-1 cleavage by AKT-dependent and -independent mechanisms suggest that mTORC2 is also a key controller of lipid metabolism in tumors such as glioblastoma.19 We demonstrate that DGKζ silencing regulates SREBP-1 levels while promoting AKT phosphorylation, showing additional controls on SREBP-1. Concomitant addition of DAG and DGK inhibitor also reduced SREBP-1 levels, indicating that DGK-mediated DAG consumption is an AKT-independent mechanism for SREBP-1 regulation downstream of mTOR. R59949 has been reported to block more efficiently the type I DGK isoforms, and from these, α is the only DGK expressed in SW480 cells. The discrepancy between DGKα silencing and the DGK inhibitor effects might be because of the distinct molecular mechanisms that operate in each case, as a reduction in enzyme levels differs from maintaining the enzyme in an inactive conformation. It also probably reflects that R59949 has additional targets, including some unidentified R59949-sensitive DGK isoforms, including DGKζ,The nature of the pathways under the control of mTORC1 feedback loops is understood only incompletely, but these pathways modify cell metabolism and increase survival to sustain cancer cell growth. The mTORC2 is a critical regulator of glycolytic metabolism in cancer, mainly via AKT activation.47 Specific loss of lipin phosphatase activity in adipocytes decreases DAG, with increased PA and mTORC1 activation.48 Our findings support these two studies and suggest a similar DGKζ contribution to the regulation of the mTOR/SREBP axis via modulation of the PA/DAG equilibrium. The mechanism by which increased DAG levels impair SREBP-1 is not clear. Pharmacological classical PKC and/or PKD-1 inhibitors promoted SREBP-1-mediated transcription, strongly suggesting the involvement of these DAG-regulated kinases in control of SREBP-1. DAG might also have a direct effect by altering membrane lipid composition and/or transport.The negative correlation between DAG and SREBP-1 coincides with a report that SREBP-1 activity in liver is negatively regulated by lipin, a PA phosphatase whose nuclear localization is governed by mTORC1-induced phosphorylation.49 but might also target PI3K and trigger feedback mechanisms involving tyrosine signaling.32 Determination of tumor DGKζ levels might be a useful marker for therapy assignment and could improve interpretation of therapeutic effectiveness. Our results indicating that cancer cells require DGKζ to maintain lipogenic functions suggest new strategies to better cope with drug resistance in cancer.The activation of DGK lipid kinases in response to mTORC1 inhibition indicates additional feedback mechanisms that restore cell homeostasis. DGKζ activation downstream of mTORC2 in rapamycin-resistant tumors helps to maintain the DAG/PA balance necessary for cell survival. DGKζ reduction potentiates the growth inhibitory effect of rapamycin. In tumors, DGKζ levels could thus control the magnitude of mTORC1-mediated feedback triggered by rapamycin analogs as well as by second-generation mTOR inhibitors. These inhibitors target the mTOR catalytic site and block the mTORC2 complex, preventing AKT activation. They appear to have a biphasic effect on AKT, particularly at Thr308,M L-Gln  and maintained at 37 °C and 5% CO2. Cell line identity was confirmed throughout culture by STR genotyping .The SW480 colon cancer cell line was purchased from the American Type Culture Collection , cultured in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum  and 2 mEscherichia coli (D3065), PMSF, leupeptin and aprotinin were from Sigma-Aldrich. Adenosine 5′triphosphate [γ32P] was from Hartman Analytic . All lipids were purchased from Avanti Polar Lipids . Organic reagents were from Merck .Antibodies to p-S6K Thr389 (9205), S6K (9202), p-AKT Ser473 (9271), AKT (2920), p-S6 Ser240/4 (2215), S6 (2317), p-PKD Ser744/8 (2054) and p-PKC substrate (2261) were from Cell Signaling , anti-PKCα Ser657 from US Biologicals  (P9103-16A), anti-PKCα and PKD from Santa Cruz Biotechnology  , anti-SREBP-1 from Millipore  (04-469), anti-DGKζ from Abcam  (ab105195), anti-DGKα from Abnova  (BO1P), anti-tubulin DM1A (T9026) from Sigma-Aldrich  and anti-phosphotyrosine (4G10) from Upstate . Horseradish peroxidase-coupled polyclonal goat anti-mouse and -rabbit immunoglobulins were from DakoCytomation  . DGK inhibitor II , bisindolylmaleimide and Gö6976 were from Calbiochem , . Rapamycin was from LC Laboratory  (R-5000). Torin was from Tocris  (4247). Recombinant DGK from 50 and knockdown efficiency was assessed.51 For DGKζ targeting, we used the previously validated sequences 5′-CUAUGUGACUGAAGAUCGCATT-3′ and 5′-GGUGAAGA GCUGAUUGAGGTT-3′.52 DGKα was silenced with validated sequences;32 either a scrambled  or the equivalent mouse sequence was used as control. For transient targeting, distinct siRNAs were transfected in cells using Oligofectamine . For stable targeting, sequences that target murine (control) or human DGKζ were cloned in the pSuperRetro vector , and SW480 cells stably expressing shRNAi were obtained by infection with pSuperRetro-cloned sequence-containing retroviruses using standard protocols.For PLD1 targeting, previously described oligos were used,32For protein extraction from cultured cells and western blot analyses of extracted proteins, we used the ECL system  as previously described.32 Primers for the genes analyzed were SREBP-1, forward 5′-TCAGCGAGGCGGCTTTGGAGCAG-3′, reverse 5′-GAGTCTGCCTTGATGAAGTG-3′ FASN, forward 5′-GAAACTGCAGGAGCTGTC-3′, reverse 5′-CACGGAGT TGAGCCGCAT-3′ ACACA, forward 5′-GCCTCTTCCTGACAAACGAG-3′, reverse 5′-GACTGCCGAAACATCTCTG-3′ GAPDH, forward 5′-ACAGCCTCAAGATCATCA GCAA-3′, reverse 5′-ATGGCATGGACTGTGGTCATG-3′ actin, forward 5′-GGCACCACACCTTCTACAATG-3′, reverse 5′-GTGGTGGTGAAGCTGTAGCC-3′.Whole RNA extraction, complementary DNA generation, PCR and quantitative PCR were performed as previously described.32 using 1,2-dioleoyl (C18:1)-DAG or 1,2 dioctanoyl (C8)-DAG as substrate. For DAG measurement, cell lipids were extracted with a modification of the method of Bligh and Dyer53 that uses CHCl3/MeOH/HCl 12N (50:100:1 v/v/v) in the first extraction step, and the lipid-containing organic phase was dried in a nitrogen stream. DAG and phospholipid-associated phosphate were measured as previously described.54DGK activity was measured as previously reported,M Tris-HCl pH 8.0, 150 mM NaCl; 5 mM final concentration). Lipids were sonicated to form micelles  and added to cell cultures . DAG or buffer were added every 90 min throughout the treatment period.C8-DAG was nitrogen dried and resuspended in vesicle buffer (1 2/well) were seeded in 12-well plates; medium was replaced with medium containing fetal bovine serum (10 or 1%) after 48 h. Drugs were added at indicated concentrations every 48 h for 7 days. Colony formation was determined as previously described.32Cells (5 × 10a2 × b)/2, where a is the tumor width and b is tumor length in mm. Pharmacological treatment was begun when tumors reached at least 150 mm3 (~15 days). The rapamycin dose, selected based on previously determined criteria,55 was administered intraperitoneally (10 mg/kg every 48 h for 10 days) and mouse weight was recorded to rule out toxic effects of the treatment. Mice were killed, tumors removed and weighed.For all animal work, we used a protocol approved by the CNB/CSIC Ethical Committee for Animal Experimentation , in conformity with national and EU guidelines. Female BALB/c SCID mice aged 6 to 8 weeks received subcutaneous injections of SW480 cells. Tumor volume was estimated as volume = .Data are represented as mean±s.e.m.; statistical analysis was done with GraphPad Prism 5 Software . Student's


Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage.p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants  Prooxidant function has also been suggested for signaling through JNK1/237–41 and phosphorylation of S36 of p66Shc by JNK has been reported following UV-irradiation4021Although no inhibitors of p66Shc’s oxidoreductase activity are available, understanding the complex mode of p66Shc activation will provide suitable targets for therapeutic interference. PKCß phosphorylation of serine 36 has been implicated in the mitochondrial import of p66Shc, ROS production and cell death induction32333435In the work presented here we thus systematically addressed a possible role of JNK in controlling the activation of p66Shc, ROS production and cell death in a setting close to ischemia and early reperfusion. Our experiments demonstrate that JNK1/2 regulate p66Shc S36 phosphorylation and mitochondrial ROS production under the conditions studied here and blocking this molecular route may provide a therapeutic mean to prevent cellular damage and death under oxidative stress.2O2 and HR. HR reproduces the early events of ischemia/reperfusion, where mainly the changes in oxygen and nutrient provision are the key triggers for cellular responses. Using phosphorylation-specific and total protein antibodies for p66Shc and JNK1/2 as well as for the p38 substrate MAPKAP kinase 2 (MK2) and the JNK1/2 target ATF2 changes in signaling were analyzed. As shown in in vitro and in vivo settings21Mouse embryonic fibroblasts (MEFs) were exposed to two different treatments commonly used to mimic ischemia/reperfusion (IR), prooxidant treatment with H2O2 and the effect on mitochondrial ROS production was monitored using MitoTracker Red CM-H2XRos. As shown in 2O2 application resulted in increased ROS production, which was suppressed by JNK as well as by the p38 inhibitor. Similarly, HL-1 cells also showed reduced ROS production under sIR in presence of JNK inhibitor  are characterized by the phosphorylation of the histone γH2AX, which can be monitored by appropriate antibodiesupon sIR .2O2  will rescue ROS production following prooxidant treatment or HR. MEF JNK1/2pression . Expresspression , confirmith FACS 2O2 treatment in the presence of Gö6976 and only a marginal effect on JNK phosphorylation upon HR. Under both conditions p66ShcS36 phosphorylation was completely abolished by the JNK inhibitor  or 8 h of hypoxia (b) resulted in increased association of both proteins. These data demonstrate that association occurs, which depends on the presence of a stress stimulus. Finally recombinant PKCß and JNK were used to phosphorylate bacterially expressed p66Shc under defined conditions. Phosphorylation was monitored by p66ShcS36 antibody. As shown in To obtain further support for a crosstalk between JNK and p66Shc in the regulation of p66Shc activation we checked for direct interaction by performing co-immunoprecipitation assays. Both, an HA-tagged version of p66Shc and FLAG-tagged constructs for either JNK1 or JNK2 were expressed in HEK293 cells and a possible association was tested by immunoprecipitating FLAG-tagged JNK with anti-FLAG M2 affinity gel. As shown in +/+ MEFs this treatment resulted in the detachment and rounding up of cells, no such effect was observed in JNK1/2-deficient cells (Following the demonstration of a link between JNK activation and p66ShcS36 phosphorylation we went to address its role in cell damage and death. For this we used the hypoxia/reoxygenation protocol (HR) described in Material and Methods. While in JNK1/2nt cells . This prnt cells , Annexinnt cells , caspasent cells . In bothnt cells  as well nt cells  and γH2Ant cells .c leading to the production of H2O245While the contribution of excessive ROS production to the pathogenesis of many diseases is realized, approaches for the clinical management of redox stress are unsatisfactory. In particular the use of antioxidants has been faced with only moderate clinical benefits4In the work presented here we show that in a cellular model of ischemia/reperfusion the stress kinases  get activated along with enhanced p66ShcS36 phosphorylation. We go on to demonstrate that S36 phosphorylation by JNK1/2 is critical for unleashing the pro-oxidant and pro-apoptotic function of p66Shc. p66Shc possesses oxidoreductase activity and causes oxidative damage in many disease settings29et al. manuscript in preparation). The evidence that JNK1/2 phosphorylation of this site is critical is based on inhibitor experiments, but also on the use of knockout cells, and most convincingly shown by the use of rescue mutant, where the phosphorylation site was exchanged to a glutamic acid (S36E).Published work stressed the importance of S36 phosphorylation in the activation of p66Shc. Work by the group of Rizzuto showed for the first time that PKCß may be the kinase phosphorylating this site2632333435373821223738393738et al. manuscript in preparation), which may be required for p66Shc activation under the conditions described here. The situation may be even more complex, as we demonstrate interaction of JNK1/2 with p66Shc  mouse embryonic fibroblasts (MEFs) or MEFs deficient in p66ShcFor the generation of mutant p66Shc constructs, the following primers were used: p66Shc S36A , p66Shc S36E (Forward: CCGGAGGAGCTGCCTGAGCCATCAGCTTCATCC and Reverse: GGATGAAGCTGATGGCTCAGGCAGCTCCTCCG) . Site-directed mutagenesis was performed using the QuikChange II Site-directed mutagenesis kit from Stratagene . Amino acid exchanges were confirmed by DNA sequencing .+/+, JNK1/2−/− (3.5 × 105 cells/well) and HEK293 cells (6.5 × 105) were transfected using Lipofectamine . Transiently transfected cells were used 48 hours (h) after transfection while stable cells lines were produced by appropriate antibiotic selection.The following plasmids were used in this study: pBABEpuro p66Shc (kindly provided by Marco Giorgio), pcDNA3 FLAG JNK1a1 (ID 13798), pcDNA3 FLAG JNK2a2-(ID 13755) , and pRec_p66Shc-HA-His , pHyPer-dMito/pLenti6/V5-DEST Gateway lentiviral vector (kindly provided by Pidder Jansen- Dürr)4P2O7, 25 mM glycerol-2-phosphate, 10% glycerol, 0.75% NP-40, 25 mM NaF) supplemented with Protease Inhibitor Cocktail Set . 40 μl of the lysate were mixed with 8 μl 6 x Laemmli buffer and incubated for 5 minutes at 95 °C to be used for expression control. Pre-clearing was performed with 40 μl Protein G Agarose beads  or mouse IgG agarose  and incubated for 1 hour at 4 °C, shaking at 300 rpm. Immunoprecipitation was done for 3 h with 40 μl anti FLAG M2 affinity gel . Proteins were isolated by boiling with 2 x Laemmli buffer for 5 minutes and were detected with immunoblotting.HEK293 cells were transiently transfected with equal amounts of pcDNA3 FLAG JNK1a1 or pcDNA3 FLAG JNK2a2 and pRec_p66Shc-HA-His. 48–72 h after transfection, cells were lysed in 1 ml NP-40 lysis buffer  or HL-1 (6.5 × 105) cells/well were subjected to hypoxia/reoxygenation and HL-1 with sIR as previously described2PO4, 1.25 mM MgSO4, 1.2 mM CaCl2, 6.25 mM NaHCO3, 20 mM 2-deoxyglucose, 5 mM sodium lactate, 20 mM HEPES, pH 6.6), respectively, in a hypoxia chamber (Billups-Rothenberg). Oxygen concentration was reduced to 0.5% by flushing the oxygen with nitrogen (N2) and was maintained for 1–6 h controlled by an oxygen sensor . For reoxygenation starvation medium was replaced with full serum medium or reperfusion buffer  and cells were incubated in normoxic culture conditions. Inhibitors for PKC , p38 , MEK  and JNK  were applied 1 h prior to stress application and maintained during stress conditions. H2O2 were obtained from Sigma-Aldrich  and directly added to the culture medium at the final concentrations indicated.MEFs  in serum-free DMEM as described previously4–6 × 104) cells per well were seeded in 8-well chambered Lab-Tek cover glasses . Cells were stained for 30 minutes at 37 °C after stress application according to experimental settings. Images were taken using an Olympus IX-70 inverted microscope. Grey values were measured using Scion Image software  for Windows. For the detection of mitochondrial H2O2 levels we also used a protein-based detection system. Cells were stably or transiently transfected with pHyPer-dMito/pLenti6/V5-DEST Gateway lentiviral vector. Fluorescence was detected by confocal microscopyMitochondrial reactive oxygen species (ROS) were imaged by fluorescence microscopy after staining the cells with 100 nM MitoTracker Red CM-HProteins were isolated and detected as described previously23245 JNK1/2+/+ or JNK1/2−/− MEFs were seeded in 6 well plates  for 24 h before stressing them with hypoxia (3 h) and reperfusion . Apoptosis was determined using FACS after staining with Annexin V-FITC  and propidium iodide . Cells were collected, centrifuged and incubated for 10 minutes on ice in 30 μl Annexin staining solution, containing 1.5 μg propidium iodide and 1 μg Annexin V-FITC. 400 μl Annexin binding buffer  was added and centrifuged. For FACS measurement cells were resuspended in 300 μl full-serum DMEM. The percentage of vital cells, Annexin V- and PI-negative, was determined using CellQuest software for FACSCalibur . Cell viability was also determined by staining dead cells with trypan blue and counting unstained cells with a microscope. Briefly, after stress, cells were collected and centrifuged for 5 minutes at 200× g and resuspended in 200 ul 10% DMEM. 1:1 dilution was made with filter sterile trypan blue . Stained and unstained cells were counted by using Neubauer counting chamber. DNA damage and cell death was also monitored by using antibodies detecting phosphorylation of γH2AX, cleaved caspase 3 and PARP fragments respectively by western blotting.7 × 10Purified recombinant p66Shc-GST fusion protein and 100 ng recombinant GST-JNK1  or PKCß were incubated in 50 μl of kinase assay buffer  for 15, 30 and 90 minutes at 30 °C. The reaction was stopped by adding 5 × SDS-PAGE sample buffer and proteins were resolved on SDS-PAGE gels and probed by Western blotting with p66ShcS36 and total p66Shc antibody.t-test or ANOVA. Statistical analysis was done using GraphPad Prism 5 . Significance values were designated as follows: *p < 0.05, **p < 0.005, ***p < 0.0005.All data are presented as mean ± SD using How to cite this article: Khalid, S. et al. cJun N-terminal kinase (JNK) phosphorylation of serine 36 is critical for p66Shc activation. Sci. Rep.6, 20930; doi: 10.1038/srep20930 (2016).


Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins.Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs.In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding. •Bisphosphonate side chain nitrogen interaction with side chain of Thr201 is essential for maximal inhibition by ZOL•Tyr204 is essential for maximal inhibition by RIS but not aminoalkyl bisphosphonates•Active site residues Thr201 and Tyr204 aid formation of the second substrate IPP binding site Subsequently, a second head-to-tail condensation of GPP and IPP within this enzyme yields the C15 farnesyl pyrophosphate (FPP). The reaction is thought to proceed by a highly ordered three step ionization-condensation-elimination mechanism, via the formation of a carbocation intermediate, which is stabilized by the presence of the OH group of Thr201, the main-chain carbonyl oxygen of Lys200 and the side chain oxygen of glutamine 240 2 + cluster where all three Mg2 + ions form salt-bridges with the un-esterified oxygens of the pyrophosphate, enhancing the juxtaposition of the hydrophobic C5 isoprenoid tail of the IPP into the correct conformation for subsequent catalysis 3–C5 double bond of IPP. Finally, elimination of the isoprenoid reaction product, GPP, is achieved by de-protonation of the condensed intermediate by the free pyrophosphate oxygen FPPS is a homodimeric enzyme, made up of two 42 kDa monomers, that initially catalyse a head-to-tail condensation of the 5-carbon allylic substrate dimethylallyl pyrophosphate (DMAPP) with isopentenyl pyrophosphate (IPP) to yield the C2 + ions with the same aspartate-rich motifs of FPPS that bind the pyrophosphate moieties of the allylic substrate isom = (Ki − Ki*) / Ki*.The structures of human FPPS co-crystallised with several clinically utilised N-BPs demonstrated that N-BPs occupy the “allylic substrate binding pocket” (DMAPP/GPP) of FPPS, in agreement with the proposed kinetic model The biological effects of the bisphosphonates are still being extensively studied and many new applications are emerging, including improvements to current therapies Having initially identified the importance of the conserved Thr201 and Tyr204 as two pivotal active site residues contributing to the inhibitory action of the more potent N-BPs we have sought to define their roles in greater detail. This has been enabled by the generation of the threonine-to-serine (T201S) and tyrosine-to-phenylalanine (Y204F) mutants, which we hypothesised would not disrupt the proposed Thr201:N-BP hydrogen bond and hydrophobic interactions respectively whereas a Thr201 to alanine (T201A) and Tyr204 to alanine (Y204A) mutant should abolish such interactions.214C) triammonium salt was purchased from American Radiochem Corp UK Ltd . All BPs were obtained from Procter and Gamble Pharmaceuticals , except PAM and ALN which were from Sigma.Oligonucleotides were purchased from Eurofins MWG. Modified standard Expression LIC vectors were all based on pET-28a  and were provided by Structural Genomics Consortium, Oxford, UK. Unlabelled geranyl pyrophosphate (GPP) and isopentenyl pyrophosphate (IPP) were purchased from Isoprenoids LC . Isopentenyl Pyrophosphate  and by ESI-TOF mass spectrometry (Agilent).2.2FPPS was expressed as described previously 2.3FPPS activity was measured as described previously 2.4Data were fitted to the appropriate kinetic models by nonlinear regression analysis using Graphpad Prism®. Determination of tight-binding ligand characteristics were analysed based on the equation developed by Morrison Ki), overall inhibition constant (Ki*) and isomerisation constant (Kisom) values were calculated as previously described Initial inhibition constant 2SO4, 0.1 M Acetate, pH 4.6 for all other structures the precipitant consisted of 0.2 M NH4Cl, 20% (w/v) PEG 6000, 10% ethylene glycol, pH 7.5. A single crystal was transferred to cryoprotectant solution composed of 20% (v/v) ethylene glycol and 80% well solution and flash cooled in liquid nitrogen.Tobacco etch virus protease-cleaved FPPS was concentrated up to 15 mg/ml. N-BPs were prepared as a 20 mM stock solution in 100 mM Tris–HCl pH 7.7 and were added to a final concentration of 2 mM. MgCl2.6Indexing and integration of collected data was performed using MOSFLM 3i, Ki* and Kisom were analysed for significance using one way Anova with a Tukey's post hoc test and also by Students T-Test. Inhibition kinetic curves were further differentiated by comparing best fit values for IC50 to selected data sets using an F-test. All statistical analysis was performed with Graphpad Prism®K44.1m for the allylic GPP substrate comparable to that of the wtFPPS , with the exception of T201S FPPS where the KmGPP was slightly lower (p < 0.01). The KmIPP was significantly increased in all mutants with Y204A showing the greatest (24-fold) increase. Consequently, the catalytic efficiency (kcat/Km) for the Thr201 mutants was unaffected with respect to GPP but decreased for the other mutants, whereas all the other mutants showed reduced catalytic efficiency with regard to IPP.Determination of kinetic parameters for all mutants  revealed4.2The side chain of Tyr204 has a hydroxyl moiety which could conceivably act as a proton donor during catalysis. This ability will vary with the pH of the surrounding solvent. To investigate this possible role the catalytic activity for wtFPPS and Tyr204 mutated FPPS constructs was evaluated at nine different pH conditions . The pH 4.3mIPP observed for the mutant enzymes.To investigate the reduction in IPP binding affinity for the FPPS mutants, we compared structures of wtFPPS and mutant FPPS. In all the crystallographic models of FPPS:BP:IPP ternary complexes, IPP is held in place by hydrogen-bonding and salt-bridge interactions with Gln96 and Arg60. In addition, Gln96 is held in place by a hydrogen bond with Tyr204 and Arg60 is positioned by a hydrogen bond with Ser205 A. A comp4.42 + ions and the aspartate motifs  remained unaffected by the mutation. The RIS position did not shift significantly in the binding pocket and it is unclear which interactions contribute to the observed increased inhibition. Interestingly, the T201A mutation, which was predicted to have reduced inhibition by RIS, had no effect on Ki but showed a small increase in overall inhibition . Disruption of the Thr201:RIS hydrogen bond in the T201A FPPS did not affect the orientation of the heterocyclic ring of RIS or the relative positions of key amino acids in the allylic binding pocket.The conservative T201S mutation, which was expected to have little effect on N-BP inhibition, significantly increased both the initial competitive inhibition and also the final isomerized inhibition with a reduction in Ki and Ki*(p < 0.01) of RIS . The cry4.5isom.The role of Thr201:N-BP hydrogen bond in enzyme inhibition was further investigated via kinetic studies with RIS analogues NE58018, NE58022, 8, 9 . The laci* and approximately 30-fold reduction in the Kisom relative to wtFPPS. Compound NE58025 1S6R was a much weaker inhibitor and overall inhibition was virtually unaffected by the T201A replacement  . However4.7isom (p < 0.001) by ZOL but does not have any impact on the initial Ki value  and it was predicted that the replacement of this residue with an Ala would disrupt this bonding and reduce the overall inhibition of the enzyme. The replacement of Thr201 by Ala resulted in a significant increase in Ki* and reduction in KKi value . The repKi value . The lacKi value B.50) corresponded to a value lower than half of the enzyme concentration, making the determination of Ki* and Kisom impossible. The IC50 was the only available way of determining the strength of ZOL inhibition  does not offer any insights as to why this would be so since in that structure the PAM nitrogen is within hydrogen-bonding distance of the Tyr204 hydroxyl (3.0 Å) and does not interact directly with Thr201.In enzyme inhibition experiments, all of the mutants examined with the exception of Y204F showed that the final inhibition of FPPS by PAM, ALN and IBN was narrowly affected by changes to Thr201 or Tyr204. The competitive inhibition of IBN and ALN was increased in both the T201S and T201A enzymes . The impInterestingly, the Ki values obtained for both Tyr204 mutants with PAM were similar to the wtFPPS, indicating that, even after disruption of any putative hydrogen bond between PAM and the Tyr residue, the BP is still capable of competing with GPP. Finally, the overall inhibition of Y204F FPPS was significantly increased by ALN and less so by PAM and IBN (p < 0.01). One possible explanation is that the IPP forms a tighter complex, with the amino-alkyl BPs in Y204F. In the wtFPPS ternary structure, the IPP tail is sterically constrained between Asp243 (3.4 Å) and the Tyr204 hydroxyl (3.6 Å). Without the hydroxyl present, the IPP C4 can shift 0.9 Å toward Phe204 and into a more hydrophobic environment, as seen in Y204F-RIS-IPP C. For PA5The availability of crystal structures of FPPS in complex with bisphosphonates identified several active site residues that could play a role in N-BP binding and also in catalysis. Here we use mutants of human FPPS complexed with BPs to elucidate the involvement of Tyr204 and the aliphatic portion of the Thr201 side chain in FPPS substrate binding, in the catalytic mechanism, and in interactions with N-BPs that can influence the strength of drug binding.mGPP was observed in all the FPPS mutants examined here even though in the Escherichia coli FPPS calculations of the stabilization effect of Thr201 on the carbocation species  suggest a more substantial role of the Thr201 residue in catalysis than the one reported here Given the high degree of conservation of Thr201 and Tyr204 among different species, along with computer modelling and structure activity relationship studies of BP inhibitors mIPP for all the mutants suggesting a role of the methyl group of Thr201 in the formation of the IPP binding site; however it was the Tyr204 mutants that showed a greater reduction in affinity for IPP. This reduced binding of IPP in both Tyr204 mutants suggests involvement of this residue in the isomerisation event responsible for the formation of the IPP binding site, the orientation of the IPP substrate, or in the stabilization of the carbocation. Our crystal structures of Tyr204 FPPS mutants provide evidence that the Tyr204 hydroxyl forms a hydrogen bond network that correctly orients the IPP-binding residues Gln96 and Arg60 making Tyr204 an essential residue in the second substrate binding event/catalytic process.Unexpectedly, there was an increased KIn our early experiments we detected the abolition of the acidic pKa value in Tyr204 mutants, a result which indicates the disruption of a possible ionization mechanism 50 for the inhibition indicates that one molecule of ZOL was inhibiting more than one molecule of the mutant FPPS. It is possible that by binding to one member of the dimer pair, one ZOL molecule prevents both from turning over substrate, maybe by locking the FPPS into a rigid conformation that prevents its twin from moving.The position of the nitrogen relative to the phosphonate groups in the bisphosphonate is critical for inhibitor potency as we have shown previously PAM and IBN were not predicted to form any interaction with Thr201 and this is reflected in the lack of change in their inhibition seen in the mutants of this residue. However ALN was predicted to form an H-bond with Thr201 but not the carbonyl of Lys200 In conclusion, we have identified novel functions of the human FPPS active site residues Thr201 and Tyr204 in catalysis as well as substrate and inhibitor binding. This study provides new insights into the molecular mechanisms of actions of N-BPs by illustrating the involvement of multiple interactions between the side chain of N-BPs with Thr201 and Tyr204 residues, which contribute to their strength of binding. Further understanding of bisphosphonate interaction with FPPS aids in explaining the clinical potency of the bisphosphonates. We hope that this information would also enable the future development of bisphosphonates or other specific FPPS inhibitors for other applications.Maria Tsoumpra, Aaron Kwassi, James Dunford and Artem Evdokimov designed and undertook experimental work. Maria Tsoumpra, Joao Muniz, Bobby Barnett, Richard Walters, Frank von Delft, Ewa Pilka, and Kathryn Kavanagh all participated in the crystal mounting, shooting, model building, proof reading and deposition of the X-ray structures. Maria Tsoumpra, Kathryn Kavanagh and James Dunford analysed the data. James Dunford, Frank H. Ebetino, Udo Oppermann and R. Graham Russell were involved in the conceptualisation of the project. The paper was written by Maria Tsoumpra, Kathryn Kavanagh, Frank H. Ebetino, Udo Oppermann, R. Graham Russell and James Dunford.Oxford NIHR Biomedical Research Unit, Arthritis Research UK (program grant 20522), the Rosetrees Trust, Medical Research Council and BBSRC. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from Abbvie, Bayer Healthcare, Boehringer Ingelheim, the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Eli Lilly and Company, Genome Canada, GlaxoSmithKline, the Ontario Ministry of Economic Development and Innovation, Janssen, the Novartis Research Foundation, Pfizer, Takeda, and the Wellcome Trust [092809/Z/10/Z]. We thank the Alliance for better Bone Health  for financial support of this work through an unrestricted educational grant.Research was supported through funding from the


GSK3 inhibition is associated with reduced mTORC1 signalling that impacts negatively on cell growth, protein synthesis and promotes cellular autophagy.Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of raptor on Ser 859. We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1  and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation.The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser Activat+-ATPase . The Rag+-ATPase . Once ac+-ATPase ,10.863, is postulated to be phosphorylated by mTOR itself and is considered necessary for promoting the hierarchical phosphorylation of neighbouring residues (Ser859 and Ser855) by other, as yet, unidentified protein kinases ATP and Protein G–Sepharose were from GE Healthcare. Cycloheximide and puromycin were from Abcam. Ku-0063794 was a gift from Professor Doreen Cantrell (University of Dundee). Oligonucleotides were purchased from the University of Dundee Oligo Synthesis Service. All other reagents and chemicals were purchased from either Sigma–Aldrich or VWR, unless stated.Antibodies directed against GSK3α/β were purchased from Santa Cruz Biotechnology. Anti-GSKβ antibody was purchased from BD Biosciences. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and anti-FLAG antibodies were purchased from Sigma–Aldrich. Anti-LAMP2 antibody was from Abcam. Antibodies against GSK3α and raptor were obtained from the Division of Signal Transduction Therapy (DSTT), University of Dundee. Anti-light chain 3 (LC3) antibody was purchased from MBL. Anti-puromycin antibody was from Kerafast. Antibodies against raptor-SerBamHI/KpnI sites (GSK3β hairpins only). Generation of recombinant lentiviruses and transduction of L6 cell cultures were carried out as described previously ATP} and incubated shaking for 30 min at 30°C. Thirty microlitres of the reaction mix was spotted on to P81 filter paper. Papers were washed three times in 1% phosphoric acid then once in acetone before being dried and [32P]ATP incorporation was measured using a Beckman LS 6000IC scintillation counter.L6 cells were treated as described in the text and cells extracted from 10 cm dishes using lysis buffer . GSK3 wa15 primers and Moloney murine leukaemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. Real-time quantitative PCR was performed using a StepOne Plus real-time thermocycler (Applied Biosystems), SYBR Green JumpStart kit (Sigma–Aldrich) and primers targeting GSK3α, GSK3β, L-type (leucine) amino acid transporter 1 (LAT1), SNAT2 and GAPDH as a control. Sequences of primers used are shown in Supplementary Table S1. PCR conditions were as follows: initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 15 s, 55°C for 15 s and extension at 68°C for 30 s. The ratio of GSK3, LAT1 and SNAT2 expression to GAPDH mRNA expression was calculated using a method described previously ATP} and incubated on a shaker for 30 min at 30°C. Reactions were boiled with 10 μl of 6× Laemmli buffer for 15 min and 30 μl of separated by SDS/PAGE and transferred on to PVDF membrane, as described above. Incorporation of [32P]ATP was detected using exposure to radiographic film. Following this, membranes were blocked and probed with anti-GST antibody to confirm equal protein loading.Mutation of pEBG6/raptor  was carried out at the indicated sites using site-directed mutagenesis to generate triple alanine mutants of SerP-value of <0.05.Statistical analysis was performed using GraphPad Prism. Data are presented as means ± S.E.M. with quantitative graphics and statistical assessments being derived from a minimum of three biological replicates per group. Values used to normalize results in each assay were excluded from statistical analysis, which involved ANOVA and Tukey's post-test for multiple comparisons. Significance was set at a Thr389 and 4E-BP1Thr37/46, whereas subsequently refeeding cells with a physiological 1× AA mix re-instates phosphorylation of both proteins to a level comparable to that seen in AA-replete cells. The phosphorylation status of ribosomal S6 is respectively down- and up-regulated when cells were either AA-deprived or-refed following AA depletion. The ability of AAs to promote p70S6K1Thr389 and S6Ser240/244 phosphorylation in all three cell lines was severely blunted by the mTOR inhibitor rapamycin  and S1(B), can be rapidly down- or up-regulated in cells irrespective of their lineage when they are subjected to withdrawal or resupply of extracellular AAs respectively. In an attempt to study whether GSK3 participates in the regulation of mTORC1 signalling, we initially investigated the effect of GSK3 inhibition upon AA-dependent phosphorylation of downstream mTORC1 substrates. Consistent with the findings shown in Supplementary Figure S1, the data presented in apamycin A–1C. Alt3T cells B and 1C.e assays  and CDK1e assays , we alsoe assays , which de assays . Figure Thr389 by mTORC1 was augmented in HEK293T cells exposed to serum, but that this enhanced stimulation remains sensitive to SB415286. In contrast, it is noteworthy that SB415286 did not suppress serum-induced phosphorylation of the Ser473 site on Akt, which serves as a target for mTORC2  and the SNAT2 (SLC38A2) transporter reveals that for the latter there was no change in transport activity as a result of silencing GSK3, whereas uptake and accumulation of leucine  by the modus operandi, we found that when HeLa cells were subject to AA depletion, there was a modest increase in nuclear localization of GFP-labelled TFEB, whereas refeeding cells with AAs after the depletion period led to significantly greater retention of the transcription factor to the cytosol  when incubated in AA-containing buffer. A marked loss in ULK1Ser757 phosphorylation was observed when cells were AA-depleted, but this was rapidly reversed upon cellular AA repletion. This AA-induced rephosphorylation of ULK1Ser757 was blunted if either rapamycin or SB415286 (but not roscovitine) were present in the AA-repletion buffer  that map to its central region and which are conserved in human, mouse and rat raptor homologues  constructs in HEK293T cells . Irrespective of whether insulin was present or not, phosphorylation of Ser859 (but not Ser863) was reduced substantially by the presence of SB415286 and, under these circumstances, we also observed an attendant reduction in p70S6KThr389 and 4E-BP1 phosphorylation (compare lanes 5 and 6 as well as lanes 8 and 9).Since insulin also positively regulates mTORC1 signalling via the Akt–TSC2–Rheb axis, we assessed whether phosphorylation of Ser859 may influence mTORC1 signalling by modulating the interaction between raptor and mTOR. To test this possibility further, we expressed FLAG-tagged constructs of wt raptor and S859A in HEK293T cells and assessed mTOR activity in FLAG immunoprecipitates. The data presented in 371 within its turn motif, which subsequently enhances Thr389 phosphorylation by mTOR [Hitherto, the idea that GSK3 may influence mTORC1 signalling has primarily been based on evidence indicating an indirect regulatory input that the kinase has upon this key nutrient signalling axis. The co-ordinated phosphorylation and activation of TSC2 by AMPK and GSK3, for example, has been reported to negatively regulate mTORC1 signalling . However by mTOR . It is u by mTOR . Althougconserved) domain that is followed by a central region containing three HEAT domains and then seven WD40 repeats near the C-terminus [722 and Ser792) located within its central region. Phosphorylation of these sites leads to raptor association with 14-3-3 protein and consequential loss of mTORC1 signalling that, in turn, induces cell cycle arrest [2/M transition when raptor is phosphorylated by cdc2 [859 by GSK3 and that phosphorylation of this site supports AA-dependent phosphorylation of multiple mTORC1 targets. In line with previous work [859 is critically dependent upon phosphorylation of Ser863, which we believe primes raptor for phosphorylation on Ser859 by GSK3. Since GSK3 is normally active in cells [859 most probably tracks that of Ser863 by an AA-regulated kinase activity. Although evidence exists in the literature showing that mTOR can mediate phosphorylation of Ser863 [863 in our hands implies that this site is targeted by another, as yet unknown, kinase(s) whose activity may be regulated in an AA-dependent manner. The finding that enhanced raptor phosphorylation of Ser863 in response to osmotic stress in HEK293 cells [863 kinase. Alternatively, the enhanced phosphorylation status of Ser863 in response to nutrient provision/osmotic stress may be critically dependent upon the inactivation of a phosphatase that normally targets this residue. Understanding how Ser863 phosphorylation may be modulated (i.e. whether it involves an AA-regulated priming kinase or phosphatase) represents an important investigative goal of future work if we are to fully understand how AAs enhance mTORC1 signalling.Although considerable progress has recently been made in identifying the many proteins involved in signalling AA sufficiency to mTORC1, there is growing appreciation that modulation of raptor phosphorylation on specific sites may serve an important ‘gate-keeping’ function that either promotes or inhibits mTORC1 signalling. Raptor possesses a unique conserved N-terminal half known as the RNC  insulin targets a discrete subcellular pool of GSK3 that is not directly linked to regulation of the mTORC1 complex, and/or (ii) the extent to which the hormone inhibits GSK3 is insufficient to impact upon mTOR activity. Insulin only causes a partial inactivation (∼30%) of GSK3 , which is substantially less than the 80–90% inhibition seen in cells treated with SB415286 ,54. Alth859 phosphorylation) is still able to interact with mTOR [859 may induce conformational changes in mTORC1 that permit more effective engagement and phosphorylation of raptor-bound mTOR substrates.Although previous work has demonstrated that a raptor S863A mutant (in which there would also be an attendant loss of Serith mTOR ,43, our ith mTOR  and so i859 and suggest that this is crucially dependent upon phosphorylation of Ser863 by an, as yet unknown, priming kinase that is likely to be regulated in a nutrient-dependent manner. Phosphorylation of Ser859 may stabilize the interaction between mTOR and raptor as reduced phosphorylation of this site promotes their dissociation and a fall in mTORC1-directed signalling (In summary, the present study demonstrates that GSK3 activity is required for supporting mTORC1 signalling in response to nutrient availability in a variety of cell types. We propose a model in which GSK3 mediates phosphorylation of raptor on Sergnalling . The fin


In the literature, ‘below and lateral to the superior S1 facet’ is defined as the basic technique for screw introduction. Until a recently published modification, no analysis for alternative starting point has been proposed nor evaluated, although some surgeons claim to use some modifications. In this study, we analyse the data from anatomical and radiological studies for optimal starting point in transpedicular S1 screw placement.A Medline search for key word combination: sacrum, anatomy, pedicle, screws and bone density resulted in 26 publications relevant to the topic. After a review of literature, two articles were chosen, as those including the appropriate set of data. The data retrieved from the articles is used for the analysis. The spatial relation of S1 facet, pedicles and vertebral body with cortical thickness and bone density in normal, osteopenic and osteoporotic sacrum is analysed.Presented data advocates for more medial placement of the screws due to higher bone density and lower bone loss in osteoporosis. Medial shift of the starting point does not increase the risk of spinal canal perforation. Osteoarthritic changes within the facet can augment the posterior supporting point for screw. The facet angular orientation is similar to convergent screw trajectory.Modified technique for S1 screw placement takes advantage of latest anatomical and clinical data. In our opinion, technique modification improves the reproducibility and may increase stability and the screws within the posterior cortex of the S1 vertebra. Further biomechanical and clinical study should be performed to prove its superiority to classical technique. The lumbosacral junction is the anatomical region where degeneration changes of the spine are most often observed. One of the theories which justifies the above is the biomechanical analysis, making junction of mobile lumbar spine and immobile sacrum responsible for acceleration of degenerative process. Another factor aggravating the local biomechanics is spatial configuration, causing shear forces, whose effects are best seen in spondylolisthesis .Stabilization procedures extending fusion over the lumbosacral junction is usually performed in treatment of degenerative disc disease, degenerative scoliosis, spondylolisthesis, neoplastic lesions and trauma. Operative stabilization of the lumbar-sacral junction is associated with risk of complications such as non-union and pseudarthrosis. These complications are most often secondary to cortical fracture of the sacrum around the screws, or pullout of screws . It is lDespite the above-mentioned modification, the major supporting point is the screws in the S1 vertebrae. There are two major types of techniques for introducing screws in S1 vertebra: converging midsacral or medial (most often used) and laterally directed that may be used as a salvage option .The first one was initially described by Roy-Camill et al. in 1986 . The autThe main drawback of the technique is poor definition of the starting point. Definition such as ‘lateral aspect of the facet joint’ as well as ‘2 mm lateral to and below the lower end of the S1 facet joint’ is not precise. Facet joint, per continuum, turns into the posterior cortex of the sacrum with interindividual existence of defined border of the facet represented by the fold of the cortex.Recently published technical note describes modified medial entry point located at the rim of the S1 facet . In persIn the reviewed modification, a starting point for transpedicular screw introduction is, in comparison to classical method, more cranial and medial. The screw enters S1 through the superior facet at the rim of the articular surface after its osteotomy  of the English language literature from 1990 through 2010 was performed using the two sets of key words . Submitting the query resulted in 54 and 56 items for both sets, respectively. The preliminary 44 articles for set 1 and 40 for set 2 were rejected as not relevant to the topic based on the abstract revision. The remaining 10 for set 1 and 16 for set 2 articles were retrieved in full text version. None of the full text reviewed described the operative technique modification for the S1 screw placement resembling the one above . After rThe spatial relation of S1 facet, pedicles and vertebral body with cortical thickness and bone density in normal, osteopenic and osteoporotic sacrum was analysed. We have analysed the imposition and inclination of the facets with respect to pedicles with risk estimation for violation of the medial cortex and spinal canal penetration. Further analysis of facet orientation is correlated with optimal screw trajectory. Finally, bone mineral density and cortical thickness are referred to the modification of transpedicular screw entry point on the posterior cortex of the S1 vertebrae.For the posterior pedicle height, measured between the first posterior sacral foramen and superior border of the sacrum, the mean value is 20.98 ± 2.34 . There is no data regarding the width of the S1 pedicle. The pedicle in the sacral vertebra is not a clearly defined space, contrary to lumbar, thoracic or cervical ones, because it extends into sacral ala laterally. We may presume its width, knowing the diameter of adjacent posterior sacral foramina which for the first one is 8.14 ± 1.97 and decreases with each segment. The mean height of the S1 facet joint is 14.62 ± 1.83. Its mean width is 16.37 ± 2.14. As we see, the height of the S1 superior facet joint is smaller than the height of the pedicle. We cannot compare the width of the facet and pedicles due to undefined width of the S1 pedicle. The pedicle of S1 can be mostly defined by its medial margin which is the lateral border of the spinal canal, and this relation will be discussed later. For the median trajectory of the screw placement, the important information is the S1 vertebral body height and transverse diameter which are 30.22 ± 2.35 and 49.40 ± 5.89, respectively.In operative anatomy of S1, we need to be aware of two values:– transverse diameter of the spinal canal at the superior aperture of the sacrum (31.31 ± 3.16) and– distance between the S1 facet joints (25.68 ± 3.80).Particular landmark relation is presented in Figure As for projections of the facet joints over the pedicles, we assume that– from the anatomical observation, the centres of the facet joints are positioned slightly cranial in relation to centre of the pediclesp vs. v on Figure – from the anatomical analysis, the centres of the facet joints are more medial than centre of pedicles  will ever overlap the spinal canal in the frontal plane, with lateral rim of the joint surface, for the entry point, placed even more lateral.When projecting the pedicles and joints over the vertebral body,r − v) / 2 = 9.045 mm,– the pedicles on both sides overlap the vertebral body at an average of (r − p) / 2 = 11.86 mm,– the facet joints overlap the vertebral body at average (p + 2o] − v} / 2) of each lateral part of the facet joint overlaps the pedicle,– approximately 13.63 mm ({[p + 2o] − r}2) laterally over the lateral margin of the vertebral body.– lateral margin of the S1 facet joint falls approximately 4.58 mm . Pedicle screws in the anteromedial trajectory, measured by the authors, are 35.20° ± 9.62° and 33.80° ± 4.34° for the right- and left-side, respectively.When we look at the trajectory separately for both sides, a difference is more visible on the left side, compared to the right side . Theoretically, the worst anatomical discrepancy between the facet and screw trajectory can be expected on the left side. The maximal difference between the facet and ideal screw angles may reach 16.36° on the left side and 14.44° on the right side.Cortical bone thickness in the anterior medial part of the S1 body is 2.01 mm; moving more lateral, it falls down to 1.58 mm in the para-medial region and in proximity to the sacro-iliac joint is 1.32 mm in average. The posterior cortex closest to facet joint is 1.23 mm thick. Close to the sacro-iliac joint, it is only 1.13 mm. With bone mass loss, the thickness of the cortex closest to facet joint decreases in osteopenia and osteoporosis, respectively, at 1.15 and 0.86 mm.In contrast, the anterio-medial cortex, at the place where the tip of the transpedicular screws anchors, is observed to be thicker in patients with osteoporosis (2.32 mm). It is probably caused by the degeneration process of the region of anterior longitudinal ligament and spurs formation. There was no evaluation of the cortex within the facet joint itself, but based on the above-mentioned clinical observation, we can assume that also in degenerative process, cortical thickness within the facet joint will tend to increase despite the global bone mineral density (BMD) loss.3. Moving more laterally, the BMD drops significantly; measured in three areas, starting from the posterior cortex, it has a value of 61.46, 31.33 in the middle and 81.76 in the area adjacent to the anterior cortex. In the analysis of Richards et al. [3, respectively.Mineral density of the trabecular bone, on the transverse section of the sacral bone, is highest on the line spanning between the facet joint and the anterio-medial cortex, just laterally to the spinal canal. Its density in different places starting from the posterior cortex has BMD subsequent values of 128.14, 75.12, 185.79 and 167.86 g/cms et al. , this is3 and is not as much affected by osteopenia or osteoporosis as in the middle and medial region. In the medial region, the mostly affected area is closest to the posterior cortex. Regular BMD of 128.14 g/cm3 drops down to 125.43 g/cm3 in osteopenia and is as low as 45.42 g/cm3 in case of osteoporosis.In the lateral region of the sacral ala, the BMD is 132.62 g/cmIn concluding the above analysis, we may state that1. the posterio-lateral margin of the facet joint falls within the potential and safe area for transpedicular screw placement,2. the angles of both facet joints and transpedicular screw trajectory, in S1 vertebra, are very similar,3. both BMD and cortex thickness is highest in the area close to the midline anteriorly and to the facet joint posteriorly. Also, as for the quality of bone in this area, it is relatively good even in osteopenia and osteoporosis cases,4. degenerative changes, both in the anterior cortex of the S1 body and in the area of the facet joint, should be taken into account in screw placement technique.The comparison of the classical and evaluated techniques is summarized in the Table The better stability of the medially introduced screws can be achieved by perforation apex of the sacral promontory  or even Some similarities to the evaluated technique modification, in the lumbar region, have been mentioned by Fuentes et al. in the mid-1980s; they wrote about ‘…small 5 millimeters resection of the inferior articular process allows a more cranial drill-hole which is more steady in the pedicle of the vertebra as confirmed by X rays controls and anatomic specimens’ , though According to the data summarized above, cranial and medial shift of the starting point for the S1 screw introduction is safe because it still falls within the pedicle projection with no significantly greater risk for collision with neural structures. More medial position of the screw makes its trajectory through the highest density of the trabecular bone and region where bone loss is significantly lower in osteoporotic patients, compared to more lateral regions. Additionally, changes related to osteoarthritis of the facet can be used to augment the posterior cortex supporting point, and the saddle-like shape of the lower pole of the opening in the posterior cortex should give better support than with flat cortex perforation.From the clinical point of view, comprehension of the angular configuration of the facet may help in taking the decision for ultimate screw trajectory for medial placement. From the practical point of view, compared to the classical technique, the modified technique gives a well-defined starting point for screw insertion, decreasing variability between surgeons and improving teaching procedure. To confirm the above data correlation, biomechanical and clinical studies have to be performed to prove its superiority to classical technique.Modification of the starting point for S1 transpedicular screw placement is justified by the analysis of available literature data. The upward and medial shift of the entry point from ‘below and lateral to the inferior tip of the superior S1 facet’ to the ‘lateral rim of the upper facet’ does not impose greater neurological risk, and the screw passes through the bone of higher durability. Additionally, BMD loss in osteoporotic patient may be compensated by osteoarthritic changes of the facets.The authors declare that they have no competing interests.LK initiated the concept of the study, made literature review and analysis, and prepared the paper. AN analysed the literature and wrote and approved the final manuscript. JK accepted the concept, analysed the data and did the preliminary manuscript approval. All authors read and approved the final manuscript.