The flow of the Greenland Ice Sheet is controlled by subglacial processes and conditions that depend on the geological provenance and temperature of the crust beneath it, neither of which are adequately known. Here we present a seismic velocity model of the uppermost 5 km of the Greenlandic crust. We show that slow velocities in the upper crust tend to be associated with major outlet glaciers along the ice-sheet margin, and elevated geothermal heat flux along the Iceland hotspot track inland. Outlet glaciers particularly susceptible to basal slip over deformable subglacial sediments include Jakobshavn, Helheim and Kangerdlussuaq, while geothermal warming and softening of basal ice may affect the onset of faster ice flow at Petermann Glacier and the Northeast Greenland Ice Stream. Interactions with the solid earth therefore control the past, present and future dynamics of the Greenland Ice Sheet and must be adequately explored and implemented in ice sheet models. Anomalously slow seismic velocities in the upper Greenlandic crust reveal soft sedimentary substrates beneath major outlet glaciers. This, together with elevated geothermal heat flux observed at the onset of fast ice flow, has major implications for ice-sheet dynamics. Ice mass loss has increased over six fold from 34 Gt per yr in the period 1991–2001 to 215 Gt per yr in 2002–2011 and shows no sign of slowing down1. The basal geological conditions beneath an ice sheet or glacier are a fundamental control on ice flow with the substrate and the presence of liquid water being a key prerequisite for fast ice flow.The Greenland Ice Sheet (GrIS) is the second largest reservoir of freshwater on Earth. Accelerated ice mass loss of the GrIS as a result of climatic forcing from the early 1990’s accounts for ~10% of mean global sea level rise2. Subsequent geological evolution has been restricted to the shield’s margins with the formation of extensive sedimentary basins to the north and north-east dating from the Precambrian to early Devonian. The sedimentary basins to the north and east were affected by late Palaeozoic orogenic events culminating in the east-west trending Ellesmerian Fold Belts to the north and the extensive north-south trending Caledonian-Appalachian orogenic belt along the east of Greenland2. Successive rifting events during the late Devonian to earliest Carboniferous resulted in the development of sedimentary basins to the west and east of Greenland and culminated in the opening of the Labrador Sea in ~ 62 Ma and the North Atlantic in ~ 56 Ma2. Atlantic sea floor spreading was concurrent with the passing of the Icelandic plume beneath Greenland at ~80 to 50 Ma4, resulting in the formation of Eastern and Western flood basalt provinces2.The Greenlandic crust primarily comprises an Archaean and early Proterozoic crystalline basement Fig. , which w7. Basal till layers are generated by the erosion of the soft rock, usually sediments, by the overlying ice6. For example, the fast ice streams of the West Antarctic Ice Sheet are typically underlain by wet till and sedimentary basins hundreds of metres to kilometres thick11. Borehole and seismic surveys have been used to infer the basal conditions at various locations along the west of Greenland. Till layers have been identified beneath the ablation zone14, which are accompanied by sedimentary rock layers on the order of tens to a hundred metres15. In addition, till has also been observed beneath the North-East Greenland Ice Stream, Greenland’s only ice stream, but the sedimentary source rocks have yet to be identified16. However, Harper et al.17 drilled 32 boreholes in the Kangerlussuaq region of Greenland and identified predominantly hard bed conditions. Where any till was observed, it was entrained in the ice and limited to a few decimetres17.Subglacial geology plays a critical role in ice flow dynamics and ice-bedrock coupling where (i) a hard bedrock can result in the overpressure of subglacial water decoupling the ice and substrate; or, (ii) soft, wet till layers reduce basal friction encouraging basal sliding18. Fast ice flow and wet subglacial basal conditions are concurrent with regions of high geothermal heat flux22. These regions of elevated geothermal heat flux beneath the ice sheet are the result of the Iceland plume, which traversed the island ~80 to 50 Ma23. Several hotspot tracks have been proposed based on tectonic reconstructions, mantle dynamics and rock outcrops on both east and west coasts, but differ significantly with increasing age towards the west3. Constraining the effect of the Icelandic plume track is therefore important in testing model simulations of the Greenland Ice Sheet.In addition to the mechanical properties of the basal substrate, the thermal condition of the subglacial environment plays a fundamental role in the generation of water from geothermal and frictional heating of the ice from beneath24. However, a series of temporary seismic networks were deployed from the turn of the century and included both on and off-ice stations, culminating in the permanent installation of the Greenland Ice Sheet Monitoring Network (GLISN)25 in 2009. Receiver functions have been used to characterise the Moho depth ( ~ 40–50 km), which displayed little lateral variation and is consistent with cratonic regions27. Walter et al.15 used receiver functions from teleseismic events to identify sedimentary rock layers up to ~ 160 m thick beneath the ablation zone of West Greenland. Tomographic imaging using both regional and global earthquake data32, as well as ambient noise data34 have been used to image the lithospheric structure and potential Icelandic hotspot track beneath the GrIS and wider Arctic. Unfortunately, the resolution of these seismic studies has been limited to depths of > 5 km due to a lack of local seismicity and the large station distances29, and so offer limited insight into the subglacial conditions beneath the GrIS.Until the early 2000s the study of the crustal structure of Greenland had been mostly limited to coastal regions due to the inaccessibility of Greenland’s interiorVs) structure of the upper 5 km beneath the Greenland ice sheet. Inverting Rayleigh wave ellipticity is particularly well suited to determining the crustal structure beneath the seismic stations in regions of uneven or sparse station coverage35. In particular, there are many methods available to measure horizontal-to-vertical ratio and ellipticity from seismic ambient noise38. In this study, we use the degree-of-polarisation-ellipticity (DOP-E) method39, which allows the extraction of short period Rayleigh wave ellipticity from ambient noise measurements and enables characterisation of the upper 5 km of the crust. To our knowledge this is the first attempt to characterise the upper crustal structure of Greenland.In this study, we measure and invert Rayleigh wave ellipticity measurements  for the 1-D shear wave velocity . The main source region of Rayleigh waves is the Denmark Strait in the north Atlantic between Greenland and Iceland , which is dependent on ice thickness , with measurements with SD > 0.2 being rejected.We apply the inversion scheme explained in the Methods section to data from each station to generate 1-D Vs profiles show little variance in Vs with the layer thickness showing the greatest uncertainty , Greenland’s only ice stream, discharges more than 10% of the Greenland Ice Sheets area with fast velocities observed up to the ice divide48. Major outlet glaciers Jakobshavn, Helheim and Kangerdlugssuaq, which are located on the central east and west coasts, are some of the fastest-flowing glaciers in the world, together draining ~15.6% of the Greenland Ice Sheet area49.Our analysis highlights a heterogeneous crust beneath Greenland. The range of our calculated shear wave velocities 52. Disco Bay comprises Upper Cretaceous and Palaeogene sediments with thickness on the order of 100s of metres to 1–2 km draped above the Precambrian basement52. Our 1-D Vs profiles obtained for station ILULI, located close to the town of Ilulissat, comprise two layers: a shallow 480 ± 190 m thick layer with Vs = 1.6 ± 0.19 km s−1, which overlies a 2.95 ± 0.53 km thick layer with Vs = 2.95 ± 0.12 km s−1  used by Chalmers et al.50. The classification of the second layer (Vp = 4.65 km s−1) is more challenging, with both basalts and Precambrian basement rocks being suitable candidates based on the local geology and seismic velocities (Vp = 4.5 km s−1)50. Previous reflection seismic studies50 have been unable to distinguish between these two rock types. We note that this layer is seismically slower (δVs ~ –2%) at depths of up to 3km relative to other stations on the West of Greenland, making the interpretation difficult  show positive δVs < -5 % up to ~3 km depth. At 1 km depth the low velocities along the east coast closely match the Caledonian fold belt . Bell et al.54 identified large units several hundred metres thick of warm, soft ice produced by the refreezing of basal water and attributed to the onset of fast ice flow at the Petermann Glacier. Along the ice divide of the GrIS, geothermal heat flux plays a leading role in the generation of basal melt18. The source of this elevated heat flux is believed to be related to the Icelandic mantle plume that traversed beneath Greenland ~80 to 50 Ma20. Although a variety of plume and geothermal heat flux models have been proposed, the thermal effect on the ice-sheet bed has so far remained poorly constrained due to limited borehole measurements and significant discrepancies between models derived from seismic55 and magnetic3 data. Seismic derived geothermal heat flux models use tomographic models of the crust and upper mantle, correlating regions of low Vs with regions of elevated heat flux55. The location of these low Vs regions varies between models, with robust measurements limited to depths greater than 10 km33. Magnetic derived geothermal heat flux models are also depth limited because airborne or satellite data are used to compute the Curie temperature to constrain the model56. Both types of models are therefore reliant on observations at depth to simulate the geothermal heat distribution at the ice-bedrock interface. These uncertainties in the geothermal heat flux models have been shown to have a significant effect on ice-sheet models, with NEGIS discharge uncertainties of 2.10 Gt per yr57. Finally, topography has also been shown to play a role in the distribution of heat flow particularly at the scale of individual glaciers and catchments58.Ice-penetrating radar measurements have identified extensive regions along the coast and ice divide where basal melt is present and attributed to the onset of fast ice flow of the NEGISVs < 3.4 km s−1 and regions with elevated geothermal heat flux ( > 60 mW m−2) in the model of Martos et al.3. In particular, the south-east to north-west linear low Vs feature that traverses Greenland can be clearly seen in the geothermal heat flux model  have identified a frozen bed. However, ice cores from NEEM found inverted ice strata with old ice from the Last Glacial Maximum, which is typically found at the ice-sheet bed, close to the ice surface59. Ice from the surrounding area of the drill site also displays evidence of enhanced deformation54. Moreover, studies of ice structure at SUMG identified strong seismic anisotropy generated by the partial melting and deformation and alignment of ice crystals61. The low Vs in the region at depths of 2 km in the crust  approach65, which is briefly described below.Rayleigh waves are surface waves with particle displacements polarised along a vertical ellipse and typically have retrograde motions at the Earth’s surface. Rayleigh wave ellipticity is the ratio of the horizontal to vertical axis of the elliptical particle motion, and under ray theory assumptions, it depends only on the subsurface directly below the station65 using the S-transform, and collated in a spectral matrix. The S-transform has properties similar to the Short Term Fourier Transform, but employs a window length, which is scaled to the periods of interest permitting multiresolution signal analysis66. A moving window eigen-decomposition of the spectral matrix permits the calculation of the semi-major and semi-minor axes of the best-fitting ellipse to the data to be computed as a function of time and period66. The planarity vector of the signal is defined as the vector product of the semi-major and semi-minor axes and is perpendicular to the best-fitting ellipse. For elliptical signals the unit planarity vector is expected to be constant and as such is used for the definition of the DOP for Rayleigh waves66. DOP is calculated as the projection of the instantaneous unit planarity vector on the mean planarity vector for the data window and ranges between 0 and 1, where 0 indicates randomly changing polarisation throughout the data window and 1 is a stable polarisation measurement66. To further improve Rayleigh wave detection, the DOP estimates are down-weighted based on the deviation of the best-fitting ellipse from the vertical. Finally, once the DOP has been estimated and is above a given threshold, the instantaneous semi-major and semi-minor vectors are used to compute the period-dependent ellipticity measurements39.Three-component seismograms recorded in the north, east and vertical directions are transformed into the time-frequency domain39. For each period only the best measurements are retained by selecting those with a DOP ≥ 0.95 67, which is a self-adaptive Monte Carlo approach that efficiently samples the parameter space. The NA search is tuned by three parameters: (i) the number of random Vs models sampled in the first iteration; (ii) the number of models created at each iteration; and, (iii) the number of best-fitting models around which the algorithm will continue to the next iteration67. Having tested different parameters, we obtained stable inversion results when performing an initial search of 1000 models, followed by 20 models for each subsequent iteration where 5 best-fitting models were selected for the next iteration. The NA outputs an ensemble of models along with their data misfit that can be used to empirically assess model uncertainties. Following these previous studies, the uncertainties in Vs are defined by the range of models with misfit not exceeding 20%, the lowest misfit value obtained in the inversion.We follow the ellipticity inversion scheme of Ferreira et al.42. In addition to Vs, the input 1-D Earth model also includes seismic P-wave velocity (Vp) and density (ρ) profiles, which are estimated empirically from Vs using the generalised Brocher relations68. Finally, the misfit function m minimised during the inversion is given by:d is the measured ellipticity, g(x) is the predicted ellipticity for the sampled model x, σD is the uncertainty in the measurements described previously and N is the number of measurements. Selecting an appropriate parametrisation is fundamental to obtain an accurate representation of the subsurface. We tested different layered parametrisations in the inversions and considered models with up to 3 subsurface layers with variable thickness and Vs ranges. Berbellini et al.39 showed that ellipticity data with periods between 2 and 10 s are sensitive to the upper 10 km of the subsurface, which is corroborated by sensitivity kernels 69. We found that with this Vs search range the inversion did not hit any bounds and hence we did not need to consider a wider Vs interval.Assuming that the ellipticity measurements include only fundamental mode Rayleigh waves, we compute theoretical ellipticity curves using a normal mode approach as implemented in the software package of Herrmannmentclasspt{minima70. The gradual accumulation and compaction of snow gives rise to changes in ice properties with depth18. Ice crystals are anisotropic with hexagonal symmetry, where the symmetry axis is parallel to the crystal c-axis18. Ice crystals are randomly orientated following deposition, which gives rise to isotropic aggregate behaviour. With increasing burial air is removed from the snow until it becomes ice, this transition stage between snow and ice is known as the firn layer18. The depth and properties of the firn layer is highly variable  with ~ 80 m being the upper firn thickness observed in Greenland along the North-East Greenland Ice Stream71. As the ice continues to undergo burial, the c-axis of the individual crystals rotate vertically towards the direction of maximum compressive stress, giving rise to anisotropic behaviour. Seismically this manifests as VTI at depths > 1.7 km on the Greenland Ice Sheet61. We find that ellipticity measurements calculated for ice models with VTI at depths > 1.7 km do not show a significant difference from the isotropic ice , intermediate ice-thickness of 1.7 km (DY2G) and thick ice of 2.77 km (ICESG) conditions. The subsurface layer to be recovered has Vs = 2.5 km s−1 and Vp and ρ estimated from Vs by the Brocher relations68  for which we model a single 2 km thick layer below the surface. Figure T < 4 s. Concurrent with these deviations from the predictions, the standard deviation of the ellipticity measurements also increases, which is not observed at off-ice stations. These deviations may be due to a number of effects such as, e.g., firn structure or overtone contamination, which will be investigated in detail in future work. In order to circumvent this issue, our final synthetic inversion tests examine the capability of accurately recovering the subsurface layer by excluding ellipticity measurements, which deviate strongly from the theoretical predictions. We classify as anomalous measurements those with standard deviations > 0.2 and that show a substantial deviation from synthetic ellipticity curves . We find that by excluding these anomalous measurements we can still recover the subsurface structure well (Supplementary Figs. Vs is overall well determined, the layer depth estimates show some variability (Supplementary Figs. Comparing the synthetic ellipticity curves Fig.  with theSupplementary Information


Protein kinases (PKs) have been recognized as central nervous system (CNS)-disease-relevant targets due to their master regulatory role in different signal transduction cascades in the neuroscience space. Among them, GSK-3β, FYN, and DYRK1A play a crucial role in the neurodegeneration context, and the deregulation of all three PKs has been linked to different CNS disorders with unmet medical needs, including Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal lobar degeneration (FTLD), and several neuromuscular disorders. The multifactorial nature of these diseases, along with the failure of many advanced CNS clinical trials, and the lengthy approval process of a novel CNS drug have strongly limited the CNS drug discovery. However, in the near-decade from 2010 to 2020, several computer-assisted drug design strategies have been combined with synthetic efforts to develop potent and selective GSK-3β, FYN, and DYRK1A inhibitors as disease-modifying agents. In this review, we described both structural and functional aspects of GSK-3β, FYN, and DYRK1A and their involvement and crosstalk in different CNS pathological signaling pathways. Moreover, we outlined attractive medicinal chemistry approaches including multi-target drug design strategies applied to overcome some limitations of known PKs inhibitors and discover improved modulators with suitable blood–brain barrier (BBB) permeability and drug-like properties.  The human kinome plays a crucial role in many physiological events, and its dysregulation is associated with a large portion of multifactorial disorders, including cancer and neurodegenerative diseases. Among these latter ones, neurodegeneration has a dramatic impact on the aging population. Protein kinases (PKs) represent very attractive and challenging drug targets for industry and academia to tackle complex disorders affecting peripheral and central tissues ,2. HowevCNS drug discovery places unique challenges linked to the blood–brain barrier (BBB) and the paucity of translational animal models to test new drug candidates ,6. The BCNS drugs must penetrate the BBB, reaching a brain concentration adequate for target engagement and modulation. Therefore, particular attention must be devoted to the factors that hinder the passive BBB penetration of a molecule in the CNS drug discovery, including large size, high topological polar surface area, and a high degree of hydrogen bonding. Size and lipophilicity are two crucial properties influencing the brain exposure and efficacy of a CNS drug candidate. Often, a balance must be found between decreasing size and increasing lipophilicity to make a drug more penetrating while simultaneously avoiding both efflux mechanisms  and drug sequestration elsewhere in the body  .2 (25–60 Å2), at least one nitrogen , fewer than seven (two to four) linear chains outside of rings, less than three (preferred zero or one) polar hydrogen atoms, the volume of 740–970 Å3, the solvent accessible surface area of 460–580 Å2, and a positive QikProp (QP) CNS parameter  ..N-terminIn general, the phosphorylation of specific amino acid residues such as Tyr216 within the activation loop of GSK-3β induces a conformational change and consequent increase of the enzyme activity. However, this PK can also achieve a catalytically active conformation without a specific phosphorylation ,34. ConcAbnormal regulation of GSK-3β has been linked to both the onset and progression of different chronic conditions such as diabetes, cancer, neurodegenerative , and behavioral diseases , major depression (MD), schizophrenia). In CNS-related disorders, aberrant GSK-3β activity is associated with the dysregulation of different proteins, namely microtubule-associated protein τ, presenilins, amyloid precursor protein (APP), collapsin response mediator proteins, components of the Wnt signaling pathway, β-catenin, and heat shock proteins . MoreoveFYN is a non-receptor Tyr kinase (TK) identified and characterized in 1988 . It beloC-terminal regulatory region , and FYN-Delta7 is mainly expressed in peripheral blood mononuclear cells . As all y region . All FYNC-terminal domain Tyr residue (Tyr531 for FYN-B and Tyr528 for FYN-T) induces a global close protein conformation due to the SH2 domain engagement, leading to the non-accessibility of the catalytic domain. On the other hand, the phosphorylation of a distinct Tyr residue in the catalytic domain (Tyr420 for FYN-B and Tyr417 for FYN-T) improves the enzyme activity , which is a region known as the Down syndrome critical region (DSCR). Due to its location, triplication of the DYRK1A locus in Down syndrome (DS) results in a 1.5-fold increase of DYRK1A mRNA and protein levels in the fetal and adult brain. Under-/over-expression in mammals of DYRK1A gene or mutations in the orthologous gene minibrain (mnb) of Drosophila have been associated with severe retardation of CNS development and maturation ,49. MoreN-terminal motif DYRK-characteristic known as DYRK homology (DH) box. It rapidly autoactivates during folding by phosphorylation on Tyr321, the second Tyr residue of the conserved activation loop YxY motif . Ad. Ad61]. DYRK1A phosphorylates APP at Thr668, enhancing its cleavage by both β- and γ-secretases , and incc), and FYN at the plasma membrane, resulting in τ missorting and hyperphosphorylation at Tyr18 , and a loss of function of many antioxidant defense enzymes, resulting in a disequilibrium between the formation of cellular oxidants and the antioxidative processes ,74.Nrf2 is one of the major regulators of cytoprotective responses to endogenous and exogenous stresses caused by ROS and electrophiles. In basal conditions, it is bound to its endogenous inhibitor Kelchlike ECH-associated protein 1 (Keap1), a cysteine-rich zinc-metalloprotein, that promotes Nrf2 degradation. In response to stress insults, such as ROS, this factor is released from Keap1, and upon translocation to the nucleus, it binds to the antioxidant response element (ARE), promoting the expression of some phase II detoxifying enzymes and antioxidant stress genes, namely NQO1, heme oxygenase-1, glutathione S-transferase, and aldo-keto reductase. Moreover, Nrf2 ameliorates the inflammation response by inhibiting the translocation of the nuclear factor-κB (NF-κB) and activating anti-inflammatory genes.A linkage between GSK-3β/Nrf2 signaling pathway dysregulation and the reduction of oxidative stress defenses in both AD and PD has been demonstrated. Activated GSK-3β plays a pivotal role in the downregulation of Nrf2 through direct phosphorylation at Ser338 and Ser335 of Neh6 domain of Nrf2 and consequent proteasomal degradation of this latter in a Keap1-independent manner ,75,76. NIt is noteworthy that activated GSK-3β phosphorylates Src A subfamily members, including FYN, that enter the nucleus, leading to the phosphorylation of Nrf2 at Tyr568, nuclear export, and subsequent degradation of Nrf2  77,78,7,80,81,82PD is neuropathologically characterized by the presence of α-synuclein (α-syn)-containing Lewy bodies and loss of dopaminergic neurons in the substantia nigra, manifesting as reduced facilitation of voluntary movements. In this scenario, different investigations suggested that α-syn neurotoxicity in PD and related synucleinopathies may result from an imbalance between the detrimental oligomer-promoting effect of α-syn phosphorylation at Ser129 and the neuroprotective action of α-syn phosphorylation at Tyr125, which inhibits toxic oligomer formation ,84.In addition to τ protein, GSK-3β phosphorylates α-syn at Ser129. Interesting cooperation between α-syn and τ in increasing the magnitude or rate of phosphorylation of the other by GSK-3β has been demonstrated, establishing a novel upstream role for GSK-3β as one of several PKs associated with aberrant post-translational modifications (PTMs) of key proteins known to be causal in PD  85]..85].Several in vitro and in vivo evidence have supported a potential neuroprotective activity of FYN due to its capability to phosphorylate α-syn at Tyr125 . HoweverDYRK1A also plays an important role in PD and additional synucleinopathies. Its capability to bind to α-syn and phosphorylate the same protein at the Ser87 residue facilitates intracellular inclusion formation  [87].N-methyl-d-Aspartate receptors (NMDARs) has been recently implicated in AD and related to synaptic dysfunction. While synaptic NMDARs are neuroprotective, overactivation of those located outside of the synapse cause a loss of mitochondrial membrane potential and cell death. The activation of 2+ signals in hippocampal neurons and contribute to the expression of long-term potentiation (LTP) and long-term depression (LTD), which are two major forms of long-lasting synaptic plasticity, by employing both NR2A and NR2B subunits . In. In89]. C-terminal tail of NMDARs have been identified as FYN phosphorylation sites: Tyr1252, Tyr1336, and Tyr1472, among which the latter is the most prominently phosphorylated site in vitro  represents a wide and heterogeneous group of neuromuscular disorders, including ALS, SMA, and DM1, resulting in the loss of motor neurons and progressive muscle wasting. The most common hereditary forms of SMA are caused by large deletions that inactivate the SMN1 gene, leading to low levels of the ubiquitously expressed protein survival of motor neuron (SMN) with a predominant function in neuronal development and synapse formation ,96. In sN-terminal kinase). However, the same inhibitor produced neurodegenerative-like effects and behavior deficits in healthy mice, suggesting how over-inhibition of GSK-3 may result in conditions that prevent neurons from operating normally pyrimidine core structurally related with that of well-known non-selective Src inhibitors PP1 (41) and PP2 . Tintori et al. identified by a virtual screening three micromolar ATP-competitive FYN inhibitors 38–40,  as promi PP2 42, , a hit-t PP2 42,  as the mAmong the latter ones, derivative 43 did not significantly inhibit at 10 µM any of the Ser/Thr kinases tested , showing selectivity for Src family members over Ser/Thr kinases, including DYRK1A and GSK-3β, which were also implicated in AD pathology. Further studies evidenced the ability of both 43 and 44 to reduce Tyr18-τ phosphorylation mediated by FYN. In detail, in SH-SY5Y cells treated with Aβ 1–42 (Aβ42) oligomer/protofibril to induce AD-like neurotoxicity, both compounds reduced τ phosphorylation at Tyr18 in a dose-dependent manner. Although solubility issues were observed for both 43 and 44 inhibitors in ADME in vitro studies, good values of metabolic stability in human liver microsomes, passive membrane permeability, and BBB permeability in PAMPA assays suggested the great potential of these derivatives as promising agents to tackle tauopathies such as AD .a]pyrazine, 3,6-disubtituted imidazopyridazine, N- and 5-disubtituted imidazothiadiazol-2-yl)-amine, and 3,4-disubstituted 1H-pyrazolopyridine heterocycles , which is one of the most potent and selective ATP-competitive DYRK1A inhibitors presently available quinazoline-2-carbimidates derivatives identified by Chaikuad et al., present subnanomolar and selective DYRK1A/B inhibitory activity with a noncanonical binding mode at the ATP pocket of both enzymes pyrazoles as the first dual GSK-3β inhibitors/Nrf2 inducers considering the inverse correlation between the aberrant activation of GSK-3β and the decrease of antioxidant gene expression and cell defense effects due to Nrf2 downregulation. Among all derivatives, 70 (50 = 3.77 µM) and one of the most potent Nrf-2 inducers. In a Nrf2-dependent luciferase reporter assay, 70 was able to increase luciferase activity in AREc32 cells with a CD  value of 9.37 µM. Interestingly, cellular experiments in which GSK-3β was inhibited by a lithium salt (10 mM) and was silenced using siRNA demonstrated the independence of Nrf2 induction properties of compound 70 from its GSK-3β inhibitory activity. Moreover, fluorescence polarization and differential scanning fluorimetry assays indicated that the same compound was not able to inhibit the Nrf2-Keap1 interaction. Remarkably, in SH-SY5Y cells treated with okadaic acid  to induce τ-hyperphosphorylation and aggregation through the selective inhibition of protein phosphatases PP1 and PP2A and cell death for oxidative stress increase (an in vitro AD model), 70 showed at 1 µM concentration a neuroprotection percentage over 50% -annulated indole 74 (50 (CLK1) = 0.17 µM; IC50 (DYRK1A) = 0.20 µM) [Lechner et al. starting from KuFal194 73, , a potenndole 74 , which e0.20 µM) .50 = 1.6 nM for DYRK1A and 10.8 nM for GSK-3β) in a kinase panel screen. Notably, this multi-target compound showed a reduction of phosphorylation of multiple τ epitopes, especially Thr12 site (EC50 = 16 nM) in cell-based assays, and in an anesthesia-induced transient τ hyperphosphorylation mouse hypothermia model, it showed reduced τ phosphorylation by 47% with the lowest dose of 1.25 mg/kg. Moreover, compared to the vehicle, a significant reduction of τ phosphorylation and aggregation was observed with alternative dose regimen of 75 leading to significantly lower numbers of τ-positive inclusions in brain stem and spinal cord samples [Melchior et al. identified SM07883 75,  as an in samples . The saf samples . 50 = 5 µM for DYRK1A) with Aβ40 inhibition . The same compound proved to inhibit DYRK1A-catalyzed τ phosphorylation in stably transfected HEK293 cells and due to its favorable physicochemical properties might be potentially applicable to in vivo AD models [Ultimately, Mariano et al. reported the application of a focused multi-target approach to develop a novel class of selective dual inhibitors of DYRK1A and Aβ aggregation based on the bis(hydroxphenyl)thiophene scaffold. Among all derivatives, compound 76  exhibiteD models . Developing drugs for CNS remains the most challenging area in drug discovery, which is accompanied with the long timelines and high attrition rates. Nowadays, there is an ever-growing need to find drugs able to reach the brain in an adequate concentration for engaging CNS targets and modulating complex and interconnected signaling pathways linked to different neurological disorders. There is a plethora of CNS-related diseases with more complex pathological mechanisms, for which rationally designed multi-target compounds could offer higher efficacy and safety compared with single-target small-molecule ligands and overcome the same limits of mono-target drugs. In this scenario, GSK-3β, FYN, and DYRK1A are three attractive closely related PKs widely investigated within the neurokinome context due to their involvement in the pathophysiology of both neurodegenerative and neuromuscular disorders. Herein, we reported an overview of the most common neuronal pathological events and the crucial roles of all three PKs in different neurodegeneration pathways. We also included a deep analysis of the last decade from 2010 to 2020 literature focused on the development of novel mono- and multi-target GSK-3β, FYN, and DYRK1A inhibitors to overcome some limitations of known inhibitors and discover improved brain permeable modulators with drug-like properties.


There is a paucity of randomized data regarding the safety and efficacy of the use of intracardiac echocardiography (ICE) from the left atrium (LA) to guide left atrial appendage occlusion (LAAO) procedures under local anesthesia using either of the available devices.The aim of this study was to compare the efficacy and safety of ICE from the LA with transesophageal echocardiography (TEE) for guidance during transcatheter LAAO procedures.n = 175) or TEE under general anesthesia (n = 49). Efficacy outcomes were procedural success and peri‐device leaks 6 weeks after LAAO. The safety outcome was a composite of procedure‐related complications.Single‐center, cohort study of patients undergoing LAAO with the Amplatzer Cardiac Plug or Watchman device. Procedures were guided by ICE from the LA with local anesthesia (p = 0.590). The rate and degree of peri‐device leaks or presence of a thrombus on the device did not differ between groups on follow‐up CT. Turnover time in the catheter laboratory and use of contrast agent were reduced with ICE.Procedural success was similar between groups: 100% in the TEE‐guided group, and 98% in the ICE‐guided group. Procedure‐related complications such as death, embolism, migration, or major vascular complications occurred similarly between groups (ICE in the left atrium to guide LAAO procedures appears to be as effective and safe as TEE. There was no increase in procedure‐related complications, whatever the device used. ICE resulted in similar procedural success while decreasing procedure time and requiring only local anesthesia. This study complied with the Declaration of Helsinki and was approved by the ethics committee of the University Hospital of Dijon. All patients gave written consent before participation.The participants in this non‐randomized study were prospectively recruited between January 2014 and April 2019. Initially, all patients underwent LAAO with TEE guidance (TEE cohort). In March 2015, after 10 procedures had been performed with a dual imaging modality (ICE and TEE), the ICE technique was used alone for all subsequent LAAO (ICE cohort) to increase patient comfort and facilitate procedural logistics. The dual technique imaging procedure patients were integrated into the TEE cohort. Patients eligible for LAAO were known to have non‐valvular AF , an increased risk of stroke  with the Zonare ViewMate Ultrasound Console . All procedures were performed by two experienced physicians  as follows Figure . (1) Sin2.32DS2‐VASC and the HAS‐BLED risk scores were calculated with the standard admission variablesWe collected data relative to comorbidities, including type of AF, prior congestive heart failure, history of CAD, CABG, or TAVR, cardiovascular risk factors such as history of hypertension or treated hypertension, and diabetes. The CHA2.4t test or non‐parametric Mann–Whitney U‐test, as appropriate. Continuous variables were expressed as means ± SD, or medians with interquartile range (IQR), and were compared using the Student's t test or Mann–Whitney U‐test, as appropriate. Categorical variables were reported as absolute numbers and proportions, and compared using the Chi‐square or Fisher exact test. A 2‐tailed p‐value <0.05 was considered statistically significant.All analyses were performed using Sigmastat Software (Jandel Inc.). The results are expressed as means ± SD or median values (25‐75th percentile) for continuous variables, or as percentages for qualitative variables. The normality of distribution for each variable was analyzed using the Kolmogorov–Smirnov test. Comparisons between the two groups were performed either with an unpaired Student's 33.1A total of 224 consecutive patients undergoing LAAO were included in the study (175 in the ICE group and 49 patients in the TEE group). Mean age was 76 years, and both groups were predominantly male. The primary indication for LAAO was prior major bleeding events in 91% and 84% of the ICE and TEE groups, respectively. Baseline characteristics were comparable between groups (Table p = 0.895)  Table . There wp = 0.566). Though the fluoroscopy time was similar between groups, the overall time spent in the catheter laboratory was significantly reduced with ICE guidance (p < 0.001), facilitating the execution of more LAAO procedures. Finally, the quantity of iodinated contrast media used was significantly decreased with ICE (p = 0.028). The length of hospital stay was similar between groups, except for the fact that patients from the TEE group spent time in the post‐anesthesia care unit before returning to the ward, whereas the ICE group returned directly to the ward. The complete exclusion of the left appendage, evaluated either by CT or FU TEE was also comparable between groups during post‐procedure evaluation.Cardiac CT interpretation, which was used to select the best device for to the anatomy and diameter of the LA, improved over time. This explains why the first device selected was implanted in 88% of TEE‐guided procedures and 96% of ICE‐guided procedures (3.2p = 0.532).Procedural serious adverse events (SAE) are described in Table 3.3p = 0.053).During the clinical follow‐up, rates of stroke/TIA, major bleeding or device migration were similar between groups. In addition, we found no significant difference in mortality at 30 days of follow‐up in both groups (1.5% vs 1.8%). Finally, we found a trend toward a higher rate of heart failure symptoms in the TEE group compared to the ICE group (16.7% vs. 6%, 4In most cases and centers, the evaluation of the LAAO procedures have been done using TEE imaging. The key to successful and safe procedures is an understanding of the transseptal puncture, LAAO size selection for the device‐specific landing zone, and post‐deployment evaluation for leaks and complications. While some operators have developed expertise in using intracardiac echocardiographic imaging for LAAO, this imaging method remains underused. Given the limited randomized data on the topic, we describe here one of the largest studies in the field with 175 patients. There was a recently published meta‐analysis including 391 ICE cases.Going forward, the challenge is undoubtedly the learning curve of the LAAO technique under ICE. The use of this approach will require teams to develop a real long‐term policy in order to successfully ensure that training is completed for all the key stages, and especially the transseptal puncture, which remains the riskiest part of the procedure.Our experience with ICE imaging from the left atrial position using a single transseptal puncture offered excellent guidance for the length of the left appendage, and we were easily able to discriminate thrombus versus sludge in the appendage, and obtain reliable assessment of peri‐device leaks and complications such as thrombus occurrence. Only few data are available regarding left atrium navigation of the ICE probe. The largest studyFinally, LAAO procedures under ICE imaging guidance have the additional advantages of avoiding the risks of general anesthesia or sedation, and of increasing the potential number of procedures performed in the lab. Although an ICE catheter cannot be re‐sterilized, this procedure can reduce direct and indirect expenses related to medications and staff costs.4.1Our study has several limitations. First, we described a single center experience, so the efficacy of our approach needs to be proven in larger and multicenter cohorts. Furthermore, we excluded patients with a history of atrial septal defect or PFO closure because the manipulation of catheters through the implanted device could be dangerous and induce migration. Imaging guidance was not randomly chosen, but the cohort consisted of two consecutive groups. When learning a new procedure, performance tends to improve with experience, which explains why the results are slightly different for the first procedures with any approach. As the first group underwent TEE guidance, a learning curve may be in favor of the ICE group. On the other hand, a learning curve associated with introduction and handling of the ICE probe is also expected. Nevertheless, from the introduction of ICE approach, it was used almost exclusively in our center. Consequently, our two groups have unequal numbers with potential biases linked in particular to the small number of the TEE group. Because the device sizing was based on preprocedural CT analysis, TEE and/or intraprocedural angiography and not on ICE imaging, no comparisons between ICE and TEE could be made for LAA measurements. Finally, the results are currently limited to procedural, in‐hospital outcomes and one‐year follow‐up, but long‐term follow‐up is ongoing.5Our study provides additional data suggesting that ICE guidance for LAAO, whatever the device used, is feasible and comparable to TEE guidance. Furthermore, ICE guidance precludes the need for general anesthesia or sedation and has the potential to become a preferred imaging modality for LAAO. Left atrial navigation of the ICE probe may also potentially be used to assist other technique such as transcatheter mitral valve replacement.The authors declare no potential conflict of interest.


Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes  was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca Heart failure (HF) is a clinical syndrome where the ability of the heart to pump blood to the rest of the body is compromised. HF arises either alone or as a combination of dysfunctional pericardium, myocardium, endocardium, valves or major vessels . PatientThe most common cause of HF is myocardial infarction (MI), which occurs as a result of severe localised ischaemia due to blockage in the coronary artery . FollowiAligned with the above hypothesis, a study using an MI rat model showed that the contractile twitch force of the cardiac trabeculae correlates with the myosin heavy chain content in end-stage HF , while tIn this study, we focused on myosin regulatory light chains (RLCs), as they play an important role in cardiac physiology regulation ,15. RLC A study reported by Stull et al., showed that RLC phosphorylation potentially inhibits pathological cardiac hypertrophy . Other sIn this study, we used an MI mouse model to study the time course of changes in maximal isometric force, calcium sensitivity (EC50) and RLC phosphorylation modifications between 2D and 28D post-MI. We also manipulated RLC phosphorylation levels in permeabilized diseased papillary fibres by exchanging native RLCs with phosphorylated recombinant RLCs and measured the corresponding muscle contraction to observe their recovery. Finally, to gain mechanistic insights, we quantified the expression levels of key enzymes  facilitating cardiac RLC and other regulatory sarcomeric proteins’  phosphorylation levels during HF progression.When the longitudinal profiles of maximal isometric force and RLC phosphorylation were superimposed, they indicate that changes in RLC phosphorylation immediately affect force generation, in a way that promotes contractility of diseased hearts. Additionally, we showed that enhancing RLC phosphorylation levels reverses compromised contractility in decompensated papillary muscle segments. The longitudinal enzyme quantification unveils the possibility of enzymes such as MYPT2 (other than MLCK and ZIPK) being involved in regulating RLC phosphorylation levels during HF progression. The quantification of sarcomeric protein phosphorylation and enzyme levels suggests that the resultant cardiac functionality is a complex interplay of varying sarcomeric proteins. Bringing these results together, we link mechanical, biochemical and physiological changes in the heart tissue during the progression of HF. These results demonstrate that RLC phosphorylation is a key sarcomeric protein modification that influences cardiac function during HF progression and that RLCs and their associated enzymes have potential in the treatment and management of HF.2 when ligated at LAD (H) and 7.63 ± 0.3 kN/m2 when ligated at LAD (L). Similarly, the force at 8 weeks post-MI was lower when ligated at LAD (H) than LAD (L). This shows that the rate of functional deterioration is higher when ligated at LAD (H) as compared to LAD (L). Since we aim to study HF progression over a short period of 28 days, ligating at LAD (H) will be extensive and appropriate to capture the advancement. Thus, this position was ligated to create an MI mouse model throughout the study.The ligature position was optimised by experimenting at two positions, just above the bifurcation LAD (H) or just below the bifurcation (LAD (L)) A. The lo2 and the average contraction force of the sham group was 7.11 ± 0.83 kN/m2. There was no significant difference between these two groups across the 28 day experimental period. Thus, we concluded that the changes that occurred following LAD ligation were not a consequence of ageing, open chest surgery or the recovery from it.To confirm that open chest surgery and ageing during the time course of the experiments do not affect cardiac muscle contractile force, the contractile force of the permeabilised papillary muscle in two control groups (age-matched control and age-matched sham surgery groups) was studied . The aveTo validate HF progression, key physiological and biological markers such as hypertrophic indices and brain natriuretic peptide (BNP) expression levels were monitored. Hypertrophy is the thickening of the heart due to the enlargement of cells and other alterations, such as extracellular matrix deposition . Thus, tThe brain natriuretic peptide (BNP) is a cardiac neurohormone biomarker predominantly secreted by the ventricular myocardium in response to increased wall stress ,24,25,26Besides hypertrophic index and BNP gene expression levels, HF progression was evaluated on a structural front. The event of hypertrophy is evident from the obvious ventricular septal hypertrophy at 7D and 14D post-MI in histology sections E 19]. O. O19]. OOn a microstructural front, the accumulation of inflammatory molecules (in between cardiac muscle fibres) is evident at both 7D and 14D post-MI, as highlighted by red arrows F. GranulThe results collectively showed that the hearts concurrently displayed characteristics of hypertrophic and dilated cardiomyopathy and that the hearts failed with time (with dilation dominating in the later stages).2+ sensitivity and cooperativity of myofilaments contribute to the characteristics of cardiac contraction. EC50 is the calcium concentration at which half-maximal force is achieved, which indicates calcium sensitivity of the contractile machinery. A lower EC50 indicates an increased Ca2+ sensitivity, as a given steady-state isometric force is reached at a lower concentration of free calcium . Br. Br12]. 2 in relaxing solution by adjusting the length of the preparation. In practice, the muscle segments were allowed to stabilise in relaxed buffer for 3 min before being transferred to the pre-active buffer. The muscle segments were transferred to the pre-activating buffer by rotating the stage so that the length remained and was incubated for 3 min to prime the muscles for active contraction. For the measurement of maximal isometric force, the muscle segments were transferred to an experimental trough containing activating solution with 5 mM Mg2+ ATP and saturating calcium concentration (32 μM) and then incubated for 3 min . By then, the plateau of the maximal isometric force was reached and recorded using LabVIEW software . The average force values from three contractions were calculated for each papillary muscle segment. The detailed buffer formulations are provided in the 2+ sensitivity is also described in the Prior to force measurements, the permeabilised papillary segments were attached to the experimental rig by hooking the ends to a customised force transducer  and a servomotor in a 6-trough stage . The iniv/v) glycerol, 50 mM dithiothreitol (DTT), 6.7 M Urea, pH 6.8) at a ratio of 20 µL/mg. The lysate was mixed and quickly heated at 100 °C for 5 min to denature proteases and phosphoproteases. Prior to conducting gel electrophoresis, the concentration of the lysate proteins was determined using a Micro BCA Protein Assay Kit . Phos-tag polyacrylamide  gel was used to quantify sarcomeric protein phosphorylation levels. The resolving and stacking gels (v/v) methanol, pH 8.3) containing 10 mM EDTA for a total of 20 min (two sets of 10 min treatment with a change of buffer in between) to improve transfer efficiency. After this, the gel was equilibrated in EDTA free-transfer buffer for a total of 20 min (two sets of 10 min treatment and change of buffer in between). A pore-sized PVDF membrane measuring 0.2 µm was used for transferred proteins to adhere to. PVDF membranes were soaked in 100% methanol for activation. Proteins were transferred at 400 mA for an hour at 4 °C. Blots containing transferred proteins were blocked in 5% BSA in TBST for an hour and then incubated with 1° antibodies according to the recommended dilutions in the product specifications. The incubation was carried out overnight at 4 °C. The next day, excess antibodies were removed by washing the blots with TBST for 30 min (change of buffer every 10 min). The blot was then incubated in 2° antibodies conjugated with HRP, for an hour at room temperature. After this, the blots were exposed to chemiluminescence reagents . The oxidation of the reagents was catalysed by HRP, resulting in a chemiluminescence signal (light at 425 nm) being emitted [During cardiac dissection for papillary muscle isolation, the left ventricles were excised and snap frozen in liquid nitrogen prior to storing them at −80 °C. The frozen ventricles were mechanically pulverised using a mortar and pestle and added to Laemmeli sample buffer , 10 µg myosin light chain kinase  and 10 µg zipper-interacting protein kinase  at a volume ratio of 800:200:1:8 (RLC:PKB:MLCK:ZIPK). The mixture was then incubated at 37 °C for two hours, with MLCK and ZIPK being replenished with the same volume every half hour. The phosphorylated RLCs were resolved in 10% polyacrylamide gel containing 25 µM phos-tag molecules for 45 min at 180 V under room temperature, followed by the above-mentioned Western blotting procedure. RLC phosphorylation levels were calculated using ImageJ, showing that 51.7% of the recombinant RLCs were phosphorylated (figure not shown). After this, they were aliquoted into small fractions and stored at −80 °C for long-term use.Concentrated RLCs (1 mg/mL) were mixed with phosphorylation kinase buffer (PKB) , phosphorylated rRLC and TnC  were required for the exchange procedure. The use of the above-mentioned chemicals, primarily involving trifluoperazine, induced a conformational change in native RLCs, resulting in it dissociating from the myosin heavy chains [Exchange buffer . The normality of the sample populations was determined using Shapiro–Wilk’s test and homogeneity was determined using the F-test, prior to conducting Student’s Ordinary one-way ANOVA was used to determine statistical differences among the means of more than two sample populations that were parametric and homogenous. Brown–Forsythe or Bartlett’s test were performed to verify homogeneity of results prior to ANOVA test. If the sample populations were nonparametric, Kruskal-Wallis test was employed. If the samples were parametric but not homogenous, Brown–Forsythe or Welch’s ANOVA test was employed. All data are shown as means ± S.E.This study suggests that RLC phosphorylation changes transiently to bring about a compensatory effect of cardiac function. RLC phosphorylation also has the potential to reverse contractile impairment stemming from cardiac disease. There are likely other key players altering muscle contraction during HF progression. Thus, the interactions between RLCs and the other players need to be elucidated to design an effective treatment for HF.


Mtor S2159A knock-in allele (MtorA/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promotes mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 coimmunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal cross talk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Although TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an  The insferation , 30, 31.feration , 33, witGrowth factors and hormones also activate mTORC2 in a manner that requires PI3K , 31, 34.in vivo, with either mTORC1 or Akt suggested as downstream mediators of TBK1 action  and primary macrophages from these mice, we find that TBK1-mediated mTOR phosphorylation increases mTORC2 signaling to Akt in primary macrophages and in vivo in response to the TLR3-activating agonist poly(I:C), which mimics viral dsRNA. This work not only elucidates the poorly defined upstream activation of mTORC2 but also improves our understanding of the contribution of TBK1 and mTORCs to physiology and pathologic conditions such as tumorigenesis and obesity-linked metabolic disorders.To better understand how TBK1 contributes to health and disease, we investigated the molecular mechanism by which TBK1 promotes the phosphorylation of Akt. We find that TBK1 phosphorylates mTOR to activate mTORC2 directly, resulting in increased Akt phosphorylation during cellular treatment with EGF and other growth factors. Moreover, by studying +/+) and knockout (TBK1−/−) MEFs. TBK1−/− MEFs displayed significantly reduced phosphorylation of Akt S473 (A) across an EGF time course, consistent with prior work . To confirm that reduced EGF-stimulated Akt phosphorylation in TBK1−/− MEFs results from loss of TBK1, we stably expressed vector control or Flag-tagged TBK1 in TBK1−/− MEFs by lentiviral transduction followed by puromycin selection. We selected several independent clones in which expression of exogenous Flag-TBK1 matched endogenous TBK1, as our prior work found that overexpression of TBK1 functions in a dominant negative manner to inhibit mTORC1 signaling . Moreover, stable expression of kinase-dead Flag-TBK1 failed to rescue P-Akt S473 . These results indicate that the kinase activity of TBK1 rather than its scaffolding function promotes Akt phosphorylation. Consistent with this conclusion, the TBK1/IKKε inhibitor amlexanox significantly reduced Akt P-S473 in response to EGF in MEFs  and HEK293 cells . Taken together, these results indicate that TBK1 kinase activity promotes mTOR-dependent phosphorylation of Akt S473 and T308 during EGF stimulation.To elucidate the mechanism by which TBK1 positively controls Akt phosphorylation, we first analyzed TBK1 wildtype . TBK1 knockout also reduced Akt T308 phosphorylation. Pretreatment with Torin1 ablated Akt P-S473 and P-T308 at each time point in response to EGF in both TBK1+/+ and TBK1−/− MEFs . Consistent with our prior work, TBK1 knockout reduced S6K1 P-T389  and mTOR autophosphorylation on S2481 . Torin1 reduced Akt P-S473 at low to mid doses of Flag-TBK1, indicating mTOR dependency, whereas higher doses displayed less dependence on mTOR activity. We next cotransfected Flag-TBK1 together with Flag-HA-Akt. The double Flag-HA tag allowed resolution of exogenous Akt (Flag-HA tagged) (upper band) from endogenous Akt (lower band), enabling distinct assessment of phosphorylation on each of these two Akt populations. As before, TBK1 overexpression increased phosphorylation of endogenous Akt in a Torin1-sensitive manner at low doses. Upon coexpression of Flag-TBK1 with Flag-HA-Akt, however, Akt P-S473 became Torin1 resistant at even the lowest dose of Flag-TBK1 . These data indicate that, in the context of TBK1 and Akt overexpression, TBK1 phosphorylates Akt S473 independently of mTOR activity, as reported in prior work , consistent with other groups , consistent with our prior work . Thus, TBK1 phosphorylates diverse substrates with dissimilar consensus phosphorylation motifs, at least in vitro, particularly at elevated levels of expression of kinase and/or substrate.We next investigated whether elevated levels of TBK1 and/or Akt enables TBK1 to directly engage and phosphorylate Akt. We therefore overexpressed increasing amounts of Flag-TBK1 in HEK293 cells. Consistent with prior work , 17, 18,Fig. 2ior work , 17. At r groups , 17, 18,ior work . Phospho−/− MEFs expressing vector control displayed extremely low Akt S473 phosphorylation in response to EGF, while rescue with HA-Rictor rescued this phenotype in a manner sensitive to the mTOR inhibitor Ku-0063794 (A). Consistent with D and E, amlexanox reduced Akt P-S473 in the rescued Rictor−/− MEFs, indicating dependence on TBK1 activity (A). Upon long blot exposure time, however, EGF increased Akt P-S473 in the Rictor−/− MEFs in a Ku-0063794-sensitive but amlexanox-resistant manner (A). Although somewhat unexpected, this Ku-0063794 sensitivity suggests that MEFs lacking Rictor, and thus expressing crippled mTORC2, still retain a low level of activity toward Akt S473. In agreement, Xie et al.  mediates Akt S473 phosphorylation in Rictor−/− MEFs. The amlexanox resistance of Rictor−/− MEFs suggests that TBK1 activity contributes negligibly to Akt phosphorylation in the context of crippled mTORC2. Curiously, shRNA-mediated knockdown of TBK1 in Rictor−/− MEFs reduced Akt P-S473 (B), consistent with Xie et al.  and S2159A knock-in mice (MtorA/A), we demonstrated that mTOR S2159 phosphorylation is required for mTORC1 signaling and IFNβ production in macrophages stimulated with innate immune agonists ; LPS) (Mtor+/+ and MtorA/A mice (pair #1 MEFs), subjected them to spontaneous immortalization, and analyzed their response to EGF following serum deprivation. Relative to MEFs from Mtor+/+ mice , MEFs from MtorA/A mice  displayed significantly reduced Akt P-S473 (A) and Akt P-T308 (A) across an EGF time course. mTORA/A MEFs also displayed reduced S6K1 P-T389 and mTOR S2481 autophosphorylation (A). Activation of the MAPK/ERK pathway in response to EGF remained unperturbed in the mTORA/A MEFs (as monitored by the phosphorylation of ERK1/2 on T202/Y204), indicating intact activation of EGF-receptor signaling to MAPK/ERK in mTORA/A MEFs (A). Of importance, we observed reduced Akt P-S473 and S6K1 P-T389 in response to EGF in a second, independently derived pair of mTOR+/+ and mTORA/A MEFs (pair #2 MEFs) (B). These data demonstrate that mTOR S2159 phosphorylation increases mTOR catalytic activity and promotes mTORC2 and 1 signaling upon cellular stimulation with EGF.In prior work we generated genome-edited mice bearing an alanine knock-in substitution at chnology . By studC); LPS) . Therefot P-S473 A and Aktt P-T308 A across rylation A. ActivaA/A MEFs A. Of imp#2 MEFs) B. These C) or TBK1 MEFs (+/+ and −/−) (B). We analyzed LPS treatment of RAW264.7 macrophages as a positive control. As expected, LPS strongly increased TBK1 P-S172 (C). Note that EGF also failed to increase P-TBK1 in HEK293 cells (see (We next asked whether EGF activates TBK1 by monitoring TBK1 phosphorylation on its activation loop site (S172). We found that EGF failed to increase TBK1 P-S172 in either mTOR MEFs (+/+ and A/A) C or TBK1and −/−) B. We ana1 P-S172 C. Note tlls (see ). These in vitro kinase assay, we found that recombinant active TBK1 increased mTOR P-S2159 on Rictor-associated mTOR immunoprecipitated from cells, and inclusion of the TBK1/IKKε inhibitor BX-795 in vitro blocked this increase (A). Moreover, we confirmed that TBK1 phosphorylates wildtype but not S2159A Myc-mTOR immunoprecipitated from transfected cells (B), confirming the site specificity of the mTOR P-S2159 antibody, as demonstrated previously (+/+ but not TBK1−/− MEFs (C). Together, these data support a mechanism whereby TBK1 interacts with mTORC2 and subsequently mediates the direct phosphorylation of mTOR S2159 within mTORC2. We next asked whether EGF increases mTOR S2159 phosphorylation and whether TBK1 promotes mTOR S2159 phosphorylation in intact cells. EGF failed to increase P-S2159 on mTOR immunoprecipitated from wildtype TBK1+/+ or mTOR+/+ MEFs (D and E). Of importance, TBK1 knockout reduced mTOR P-S2159 (D), and mTOR P-S2159 was undetectable in mTORA/A MEFs (E), confirming Mtor S2159A knock-in. We speculate that the remaining mTOR P-S2159 found in TBK1−/− MEFs results from IKKε-mediated phosphorylation of mTOR. Indeed, although IKKε expression is generally tissue restricted and extremely low in nonimmune cells, MEFs express detectable levels of IKKe (D and E). These data demonstrate that TBK1 mediates mTOR S2159 phosphorylation in vitro and in intact cells and support the conclusion that basal rather than EGF stimulated TBK1 kinase activity mediates mTOR P-S2159 to promote mTORC2 signaling.To further define the mechanism by which TBK1 promotes mTORC2 signaling, we asked whether recombinant TBK1 phosphorylates mTOR S2159 within mTORC2 directly. It is important to note that our prior work demonstrated that TBK1 phosphorylates mTOR S2159 within mTORC1 . By in vincrease A. Moreoved cells B, confireviously . We next−/− MEFs C. Togeth+/+ MEFs , D and E P-S2159 D, and mTA/A MEFs E, confir of IKKe , D and Ein vitro kinase assays. To do so, we immunoprecipitated Rictor from TBK1+/+versus TBK1−/− MEFs and from mTOR+/+versus mTORA/A MEFs after EGF stimulation of serum-deprived cells. The Rictor immunoprecipitates were washed and incubated in kinase buffer with ATP and recombinant His-Akt1 as substrate, and the ability of Rictor-associated mTOR to phosphorylate Akt S473 in vitro was monitored by Western blot. In both TBK1+/+ MEFs (A) and mTOR+/+ MEFs (B), EGF increased mTORC2 catalytic activity in a Torin1-sensitive manner, as expected. The fold increase in mTORC2 catalytic activity mediated by EGF, however, was reduced in TBK1−/− MEFs (A) and mTORA/A MEFs (B). To assess the role of TBK1 and mTOR S2159 phosphorylation in control of mTORC2 catalytic activity by an independent approach, we monitored S2481 autophosphorylation on Rictor-associated mTOR . Like results obtained with mTORC2 in vitro kinase assays, the fold increase in mTOR S2481 autophosphorylation mediated by EGF was reduced in TBK1−/− (C) and mTORA/A MEFs (D). Taken together, these results indicate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 catalytic activity.We next asked whether TBK1 and mTOR S2159 phosphorylation increase the intrinsic catalytic activity of mTORC2 by +/+ MEFs A and mTO+/+ MEFs B, EGF in−/− MEFs A and mTOA/A MEFs B. To ass TBK1−/− C and mTOA/A MEFs D. Taken +/+versus mTORA/A MEFs in response to several growth factors known to activate mTORC2 and 1 signaling. We first assessed the role of mTOR P-S2159 in control of mTORC2 and 1 signaling in MEFs cultured in complete media  containing a full repertoire of serum growth factors. mTORA/A MEFs as well as TBK1−/− MEFs displayed reduced Akt S473 and S6K1 T389 phosphorylation (A). Treatment of cells with Torin1 ablated these phosphorylation events, indicating that mTOR activity is required for mTORC2 and 1 signaling in complete media (A). We next assessed the role of mTOR P-S2159 in control of mTORC2 and 1 signaling in response to stimulation of MEFs with fetal bovine serum (FBS), PDGF (a major constituent of FBS), and insulin (which acts through IGF-1 receptors in MEFs) following serum deprivation. Like their response to EGF, mTORA/A MEFs displayed reduced Akt P-S473 and S6K1 P-T389 in response to all three growth factors with varying dynamics across a time course (B–D). In addition, all three growth factors failed to increase TBK1 S172 phosphorylation  for 10 min. We found that overall Akt P-S473 and S6K1 P-T389 was reduced in mTORA/A and TBK1−/− MEFs in both the absence and presence of amino acids (E). As expected, amino acid stimulation increased S6K1 P-T389 but not Akt P-S473 in wildtype, mTORA/A, and TBK1−/− MEFs (E), confirming that amino acid stimulation increases mTORC1 but not mTORC2 signaling. These results indicate that TBK1 and mTOR P-S2159 support both mTORC1 and 2 signaling in parallel to growth factor and amino acid signaling pathways.Considerable evidence indicates that amino acids increase mTORC1 but not mTORC2 signaling through a mechanism involving recruitment of mTORC1 to the surface of lysosomal membranes, where Rheb resides , 65, 66.no acids E. As exp−/− MEFs E, confirMtor S2159A knock-in modulated mTORC2 signaling to Akt in response to TLR3 activation with poly(I:C) . In cultured RAW264.7 macrophages, poly(I:C) activated TBK1 (as monitored by increased TBK1 P-S172) and PI3K-dependent mTORC2 signaling  increased TBK1 P-S172 and mTOR-dependent Akt P-S473 in wildtype BMDMs (B). As in RAW264.7 macrophages, amlexanox suppressed Akt P-473 to a basal level (B). Of importance, BMDMs from MtorA/A mice displayed reduced poly(I:C)-induced Akt P-S473 (B), thus demonstrating a required role for mTOR S2159 phosphorylation in TLR3-mediated activation of mTORC2 signaling. Consistent with our prior work . These results demonstrate that TBK1 activity, mTOR activity, and mTOR S2159 phosphorylation are required for mTORC2 signaling to Akt upon activation of TLR3 in macrophages. Finally, to demonstrate a role for mTOR S2159 phosphorylation in activation of mTORC2 signaling by TLR3 in vivo, we injected Mtor+/+ and MtorA/A mice with poly(I:C) and harvested macrophage-rich spleen tissue. We found that spleen tissue from MtorA/A mice displayed reduced Akt S473 phosphorylation in response to poly(I:C) (C).We next isolated primary bone marrow derived macrophages (BMDMs) from pe BMDMs B. As in al level B. Of impt P-S473 B, thus dior work , amlexannd BMDMs , A and Boly(I:C) C.Figure in vivo (D). Moreover, they reveal that mTORC2 represents an essential link between TBK1 and Akt phosphorylation at physiological levels of protein expression. We find that, in MEFs, basal TBK1 kinase activity signals in parallel to growth factors and amino acids to augment mTORC2 (and mTORC1) activity, as EGF and other growth factors increased mTORC1/2 signaling in a TBK1- and mTOR P-S2159-dependent manner without increasing TBK1 S172 or mTOR S2159 phosphorylation. The relationship between growth factor signaling and TBK1 activity appears to be complex and context dependent, however, as growth factors were shown recently to activate TBK1  in lung cancer cells (D). It is important to note that our prior work demonstrated that poly (I:C) increases mTOR-S2159 phosphorylation in RAW264.7 macrophages and primary BMDMs in a manner sensitive to the TBK1/IKKε inhibitor BX-795  led to correspondingly similar changes in Akt S473 phosphorylation  phosphorylates a substrate  directly in intact cells rather than indirectly within a complex, several lines of evidence considered together support our model that TBK1 phosphorylates mTOR S2159 directly to increase mTORC2 activity and signaling to Akt (D): TBK1 phosphorylates mTOR S2159 within mTORC2 in vitro; TBK1 and mTORC2 coimmunoprecipitate; TBK1−/− MEFs display reduced mTOR P-S2159 in intact cells; TBK1−/− MEFs and mTORA/A MEFs (lacking mTOR P-S2159) display reduced mTOR-dependent Akt P-S473 and Akt P-T308 in response to EGF; TBK1 overexpression increases Akt P-S473 in a largely Torin1-sensitive manner at endogenous levels of Akt; and finally, both TBK1−/− MEFs and mTORA/A MEFs display reduced mTORC2 intrinsic catalytic activity in response to EGF, as measured by mTORC2 IVK assay and by Rictor-associated mTOR S2481 autophosphorylation. In support of these results in cell lines, we also demonstrate that, in primary macrophages in culture  and spleen tissue in vivo, mTOR S2159 phosphorylation is required for Akt S473 phosphorylation in response to TBK1 activation with poly(I:C). In addition, Zhao et al.  , 68, 69.et al.  to searcet al. . Therefoi.e., TBK1 activity) or mTOR S2159 phosphorylation. We therefore conclude that, in this context, the basal kinase activity of TBK1 promotes mTORC2 signaling in parallel to growth factors. It is important to note, however, that stimulation of human A549 lung cancer cells and primary mouse lung cancer cells with several different growth factors  increased TBK1 S172 phosphorylation, which required TBKBP1 (TBK1 binding protein 1), a TBK1 adaptor protein  was generated as described -dimethylammonio]-1-propanesulfonate) detergents were from Pierce; cOmplete Protease Inhibitor Cocktail (EDTA-free) tablets were from Millipore Sigma (#11836170001); Immobilon-P polyvinylidene difluoride (PVDF) membrane (0.45 μM) was from Millipore; Bradford Reagent for protein assays was from Bio Rad (#5000001); reagents for enhanced chemiluminescence were from either Alkali Scientific (Bright Star) or Advansta (Western Bright Sirius HRP substrate). Recombinant active GST-TBK1 was from Thermo Fisher/Life Technologies (#PV3504); recombinant His-Akt1 was from EMD Millipore (#14-279); recombinant GST-mTORescribed .The following antibodies from Cell Signaling Technology (CST) were used in this study : Akt #9272); Akt P-S473 ; Akt P-T308 ; TBK1 ; TBK1 P-S172 ; S6K1 (#9202); S6K1 P-T389 ; mTOR (#2972); MAPK (#9102); MAPK P-T202/Y204 ; GST ; IgG-conjugated Sepharose beads (#3423); Rictor-conjugated Sepharose beads (#5379). The following antibodies were from other commercial vendors: mTOR P-S2481 (Millipore Sigma #09-343); Myc ; HA.11 . The following custom, anti-peptide polyclonal antibodies were generated by us in-house with assistance from a commercial vendor: Rictor . pRK5/Myc-mTOR S2159A was generated via site-directed mutagenesis as described (pcDNA3/Flag-TBK1 wildtype (WT) and pcDNA3/Flag-TBK1kinase dead (K38M) were obtained from Dr A. Saltiel . pcDNA3/Flag-HA-Akt was obtained from Addgene (#9021). pRK5/Myc-mTOR WT was originally from Dr D. Sabatini (MIT and the Whitehead Institute) and obtained escribed . pCI/HA-i.e., MEFs, HEK293, RAW264.7 murine macrophages, and primary mouse BMDMs) were cultured in DMEM that contained high glucose (4.5 g/l), glutamine (584 mg/l), and sodium pyruvate (110 mg/l) (Thermo Fisher/Life Technologies) supplemented with 10% FBS (Gibco/Invitrogen). Note that heat-inactivated FBS was used to culture the RAW264.7 macrophages and BMDMs. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. HEK293 cells (from ATCC) were transfected using Mirus Trans-It LT1 in accordance with manufacturer’s instructions and lysed ∼24 to 48 h post transfection. To stimulate cells with growth factors, MEFs and HEK293 cells were first serum starved via incubation in DMEM containing 20 mM Hepes pH 7.2 overnight, ∼20 h. The cells were then stimulated with EGF (25 or 50 ng/ml) (Sigma Aldrich #E9644 and #E4127), FBS , PDGF (10 ng/ml) (EMD Millipore #GF149), or insulin (100 nM) (Thermo Fisher/Life Technologies #12585) for various amounts of time (0–60 min). To stimulate macrophages with innate immune agonists, RAW264.7 macrophages and BMDMs cultured in complete DMEM  were treated with poly(I:C) (30 μg/ml) (Sigma Aldrich #P1530) or ultrapure LPS (100 ng/ml) (InVivo Gen #tlrl-3pelps). Cells were treated with the following drugs: Torin1  (shared by Dr D. Sabatini), Ku-0063794  (Tocris #3725); amlexanox  (EMD Millipore #SML0517); BX-795   (30 min); BYL-719  . To amino acid deprive and stimulate cells, MEFs were first cultured for 50 min in DMEM lacking all amino acids  but containing dialyzed FBS (10%) (Life Technologies #A33820-01). The cells were then acutely stimulated with amino acids for 10 min using a 50× solution of total amino acids (RPMI 1640 Amino Acid Solution)  supplemented with L-glutamine (Sigma # G8540) to a final concentration of [1×]. Note that the pH of this amino acid solution is quite basic (∼pH 10), and thus the pH of this solution was first normalized to pH 7.4 prior to addition to cells in order to maintain physiological pH.All cell lines used in this study  containing NP-40 (0.5%) and Brij35 (0.1%), as described  were generated by lentiviral transduction. Flag-TBK1 was subcloned into a modified lentiviral vector, pHAGE-Puro-MCS (pPPM)  Flag-TBK1 were selected in puromycin and pooled for analysis. Rictor −/− MEFs  stably expressing vector control or HA-Rictor were generated by lentiviral transduction and stable selection, as described   and then selected in puromycin (8 μg/ml) for 4 days.TBK1S (pPPM)  (modifieescribed . These rIn vitro kinase (IVK) assays were performed by incubating recombinant His-Akt1 (50 ng) or GST-mTORf (50 ng) substrates with recombinant active GST-TBK1 (50 ng) in kinase buffer containing 25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, and 200 μM ATP. Reactions were incubated at 30 °C for 30 min and stopped by addition of sample buffer followed by incubation at 95 °C for 5 min. Samples were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted. The phosphorylation of His-Akt1 was measured using anti-Akt P-S473 antibodies, and the phosphorylation of GST-mTORf was monitored using anti-mTOR P-S2159 antibodies. For certain IVK reactions, recombinant kinases were preincubated with BX-795 (10 μM) in kinase buffer on ice for 30 min.HEK293 cells were transfected with Myc-mTOR wildtype or mutant S2159A, and lysates were immunoprecipitated with anti-Myc antibodies. Myc-mTOR S2159A was generated by QuikChange mutagenesis, as described . Alterna2 at 30 °C for 30 min. Certain reactions were also pretreated with Torin1 (10 μM) in kinase buffer on ice for 30 min prior to initiating the IVK reaction.mTORC2 IVK assays were performed as described , 75. Bri+/+ or TBK1−/− MEFs were lysed in buffer A containing CHAPS (0.3%) detergent. Protein levels in each cell type were normalized after performing protein assays with the Bradford assay. IgG control Sepharose beads (CST) or Rictor-conjugated Sepharose beads (CST) were washed 2x with PBS, blocked ∼1 h in PBS containing 2% bovine serum albumin to reduce nonspecific binding, and then washed in PBS 2× more. Whole-cell lysates were then added to the washed beads, rotated at 4 °C overnight, washed 3× in lysis buffer, and resuspended in 1x SDS-PAGE sample buffer.TBK1MtorA/A) were generated using CRISPR-Cas9 genome editing technology and genotyped, as described . The resuspended cells were transferred to a 10-cm culture dish with fresh complete medium and incubated at 37 °C in a humidified atmosphere containing 5% CO2. The MEFs were washed 1× with PBS pH 7.4, detached with 0.05% trypsin-EDTA, centrifuged 4 min at 300g, and transferred to a 15-cm culture dish. At confluency, the MEFs were trypsinized and aliquoted to multiple cryovials for long-term storage in liquid N2. MEFs were immortalized spontaneously through serial passaging. As they reached confluency in 15-cm culture dishes following isolation, primary MEFs were plated into 10-cm culture dishes at a 1:9 split ratio into 10 ml complete medium (passage #2). Upon reaching confluency, the MEFs were again passaged into 10-cm culture dishes with a split ratio of 1:3. This process was repeated every 3 to 4 days until senescence was reached (∼passage 5–7). The culture medium was replaced every 3 to 4 days, and the cells were trypsinized and transferred into a new culture dish once a week. Once the MEFs began proliferating, they were passaged with a 1:3 split ratio every 3 to 4 days until the cell number doubled every ∼24 to 30 h. MEFs were genotyped as described  and plated into 6-well tissue culture plates. Cells were incubated at 37 °C in 5% CO2, and the medium was replaced every other day until day 5, at which point the monocytes had differentiated into macrophages. Macrophages were studied at ∼80% confluency.Bone marrow from 8- to 14-week-old Mtor+/+ and MtorA/A mice  fed a normal chow diet were fasted (5 h) and injected with saline or poly (I:C) . Spleen tissue was isolated, homogenized, and analyzed by Western blotting. A motorized tissue homogenizer  was used to homogenize whole spleen in 1 ml ice-cold RIPA buffer that contained protease and phosphatase inhibitors. The lysate was incubated on ice (10 min) and centrifuged at 13,200 rpm , and the supernatant was collected. DC (Detergent Compatible) protein assay  was used to standardize protein amount for immunoblot analysis.t test assuming equal variances. Error bars represent either standard deviation (SD) or standard error of the mean (SEM), as indicated in the figure legend.Western blot images were prepared for publication using Adobe Photoshop. Only the parameters levels, brightness, and contrast were employed to fine-tune exposure time and band sharpness. Of importance, these parameters were adjusted equivalently across the entire blot, and the final image shown reflects the raw image. In certain panels, thin dotted lines indicate excision of an irrelevant lane(s) from a Western blot. Western blot signals were quantitated using FIJI to measure integrated densities of protein bands. The ratios of phosphorylated proteins over cognate total protein were calculated and normalized as indicated in the figure legends. Statistical significance was tested using paired Student’s All data are contained in this article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Due to the characteristics of neurofibromatosis type I (NF-1) scoliosis, the precise placement of pedicle screws still remains to be a challenge. Triggered screw electromyography (t-EMG) has been proved to exhibit high sensitivity to identify mal-positioned pedicle screws, but no previous study assessed the combination of t-EMG with O-arm-assisted pedicle screw placement in NF-1 scoliosis surgery.To evaluate efficacy and safety for combination of t-EMG with O-arm-assisted pedicle screw placement in NF-1 scoliosis surgery.From March 2018 to April 2020, sixty-five NF-1 scoliosis patients underwent t-EMG and O-arm-assisted pedicle screw fixation were retrospectively reviewed. The channel classification system was applied to classify the pedicle morphology based on pedicle width measurement by preoperative computed tomography scans. The minimal t-EMG threshold for screw path inspection was used as 8 mA, and operative screw redirection was also recorded. All pedicle screws were verified using a second intraoperative O-arm scan. The correlation between demographic and clinical data with amplitude of t-EMG were also analyzed.A total of 652 pedicle screws (T10-S1) in 65 patients were analyzed. The incidence of an absent pedicle (channel classification type C or D morphology) was 150 (23%). Overall, abnormal t-EMG threshold was identified in 26 patients with 48 screws (7.4%), while 16 out of the 48 screws were classified as G0, 14 out of the 48 screws were classified as G1, and 18 out of the 48 screws were classified as G2. The screw redirection rate was 2.8% (18/652). It showed that t-EMG stimulation detected 3 unacceptable mal-positioned screws in 2 patients (G2) which were missed by O-arm scan. No screw-related neurological or vascular complications were observed.Combination of t-EMG with O-arm-assisted pedicle screw placement was demonstrated to be a safe and effective method in NF-1 scoliosis surgery. The t-EMG could contribute to detecting the rupture of the medial wall which might be missed by O-arm scan. Combination of t-EMG with O-arm could be recommended for routine use of screw insertion in NF-1 scoliosis surgery. Scoliosis resulting from neurofibromatosis type I NF-1) accounts for 2% of pediatric scoliosis  accounts. Mal-posIntra-operative O-arm-based spinal navigation can improve accuracy of initial pedicle screws position, and further help detect and reposition the mal-positioned screws , 7. HoweThus, the trend to maximize the safety of pedicle screw placement encouraged us to combine t-EMG with O-arm scan. We conduct the current retrospective study to verify efficacy and safety of t-EMG in combination with O-arm-assisted pedicle screw placement in NF-1 scoliosis surgery. The hypothesis of current study is that combination of t-EMG with O-arm-assisted pedicle screw placement was a safe and effective method in NF-1 scoliosis surgery.After obtaining approval from our institutional review board, consecutive eligible patients diagnosed with NF-1 scoliosis were retrospectively reviewed. All the patients underwent posterior spinal fusion involving thoracolumbar spine (T10–S1) using t-EMG and O-arm-assisted pedicle screw placement. No exclusion criteria were applied for gender, age, clinical or medical condition. A preoperative low dose 64-slice CT scan with slice thickness of 2 mm was applied to classify the pedicle morphology according to the channel classification system . Type C After midline exposure, the reference frame was fixed to the spinous process, and the binocular infrared camera were adjusted to receive the reflector ball. The first 3D scan was acquired in 13 s, and the intraoperative 3D images were then automatically obtained and visualized on O-arm viewing station . With the guidance of virtual navigation images, pedicle screws were routinely placed after verifying the integrity of medial wall. Once all screws have been placed, a second intraoperative 3D scan with O-arm were conducted to confirm whether they were in the target position.Abdominal muscles were used to verify the position of thoracic screws, and lower extremity muscles  were selected to verify the position of lumbar screws. The target muscles tested in t-EMG were as follows: lower rectus abdominis for T10, T11; inferior rectus abdominis for T12, L1; vastus medialis and adductor magnus for L1-L4; vastus lateralis and tibialis anterior for L4, L5; proneous longus and gastrocnemius for L5, S1. A pulse width of 200 us and a repetition rate of 3.0 Hz were set for pedicle screw stimulation, and a monopolar electrode was used for t-EMG. A total intravenous anesthesia technique with no neuromuscular blockade was used throughout the procedures.Pedicle screw violations were categorized into four grades according to Laine’s classification: grade 0 (G0), screws were completely within the pedicle; grade 1(G1), penetration less than 2 mm; grade 2 (G2), penetration between 2 and 4 mm; and grade 3 (G3), penetration over 4 mm . Screws p value < 0.05 was defined as statistically significant. Data were presented as mean ± standard deviation. Logistic regression analysis was performed between demographic or clinical parameters  and t-EMG amplitude.SPSS 17.0 for Windows  was used for statistical verification, and Sixty-five patients  with mean age of 16.2 ± 7.3 years (range 13–24 years) were enrolled  in 2 patients detected by t-EMG stimulation but missed by O-arm scan. All of the 3 screws were located in the periapical area of the curve. When used in combination with O-arm, t-EMG was found to obtain a sensitivity of 100%, specificity of 96.2%, and a positive predictive value of 66.7%. Logistic regression analysis showed there was positive correlation between t-EMG amplitude and pedicle width (Table  in 2 patFor complications, dural tear occurred in 1 patient during operation due to bony spur from broken pedicle medial wall. No additional screw-related neurological or vascular complications were observed.Although pedicle screws provide numerous advantages, placement of pedicle screws in NF-1 scoliosis still remains a challenge since the vertebrae and pedicle are always seriously destroyed, especially in the apical region , 17. MisSilbermann et al. noted that higher accurate rate of pedicle screw could be obtained in O-arm group than that in free-hand group . It was The t-EMG was developed as a method to electrically stimulate the positioned pedicle screw to assess its proximity to nearby nerve roots. However, t-EMG was criticized for its false negatives and positives, and thresholds utilized for safety assessment differed in the thoracic and lumbar regions according to previous published literature , 14. SamIn current study, the combination of O-arm and t-EMG was applied to assist pedicle screw placement in NF-1 scoliosis. All the broken medial wall could be effectively recognized during operation, and no neurological complications caused by screw misplacement occurred. Although limitations exist in individual technique, we should acknowledge that combination of two techniques greatly contributes to the accurate pedicle screw placement and avoiding the occurrence of neurological complications in IF-1 scoliosis. However, due to the limitation of sample size, the results of this study still need to be verified with a larger sample.Combination of t-EMG with O-arm-assisted pedicle screw placement was demonstrated to be a safe and effective method in NF-1 scoliosis surgery. The t-EMG could contribute to detecting the rupture of the medial wall which might be missed by O-arm scan. Combination of t-EMG with O-arm could be recommended for routine use of screw insertion in NF-1 scoliosis surgery.