In neurons, the presence and functions of SOCE are still in question. Here, we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase, which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs), being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions, but the underlying mechanism and, specifically, the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons, we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD, pointing out a key function of calcium and SOCE in synaptic plasticity.Store-operated calcium entry (SOCE) is a Calcium (Ca All efforts were made to minimize animal suffering.Wild-type mice with a mixed C57BL6/Sv129 genetic background were used. Mice were housed in a humidity- and temperature-controlled room on a 12 h light/dark cycle, receiving water and food 5 cells/cm2 on poly-L-lysine and laminin-coated pretreated glass coverslips in Neurobasal medium supplemented with 2% B27, 1% glutamax  and 100 mg/ml penicillin–streptomycin. On the fifth day in vitro (DIV) half of the plating medium was removed from each well and replenished with BrainPhys medium  in cortical neurons cultured in both BrainPhys and Neurobasal medium.Cortical neuronal cultures were prepared from E15 to E16 mouse embryos as described earlier , for 30 min at 37 °C, and rinsed with HCSS 2 mM CaCl2, for 30 min. Fluorescence (emission 510 nm) ratio of Ca2+-free (F380) to Ca2+-bound probe (F340) was analyzed using Aquacosmos 2.5 software (Hamamatsu) and Metafluor for Leica developed by Metamorph . Regions of interest (ROIs) were selected covering single cells. SOCE analysis was performed using a 2 mM CaCl2 and 1 μM tetrodotoxin  medium, ER-Ca2+ was depleted using 1 μM of Tg . Drugs used to study the influence of other neuronal Ca2+ channels were: 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 μM MK-801 –(+)-5-Methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine hydrogen maleate), 50 μM NiCl2  and 5 μM MPEP (2-Methyl-6-(phenylethynyl) pyridine) . mGluR analysis was performed in a 2 mM CaCl2 and 1 μM TTX medium, where 200 μM DHPG  was added in absence or presence of the SOCE blockers 10 μM YM-58483  or 50 μM 2-Aminoethoxydiphenyl borate .Cytosolic calcium imaging with Fura-2 was performed as described by Ruiz et al. . NeuronsLevels of messenger RNA (mRNA) in neurons were determined by quantitative real time PCR (qRT-PCR). Specific intron-spanning primers for PCR amplification of the SOCE genes: Orai1, 2 and 3, Stim1 and 2, Trpc1, 2, 3, 4, 5, 6 and 7, were designed using Universal Probe Library web® Green, Biorad, Hercules, CA, USA). The parameter analyzed was the quantification cycle (Cq). A standard curve of each amplicon (from 10 to 108 copies) was used and the number of copies of SOCE genes was obtained by extrapolation. The PCR efficiency was calculated for each pair of specific primers and was applied where appropriate.For qPCR, 2.5 ng of the cDNA synthesized was amplified using the ABI Prism 7900HT real-time PCR System . The amplification protocol was: hot start (10 min 95°C) and 40 amplification cycles . All reactions were carried out in triplicate. Primer sequences for cDNA analysis by qRT-PCR were as follows (5′-3′): Orai1 forward tacttaagccgcgccaag; Orai1 reverse acttccaccatcgctacca; Orai2 forward cacagacgctagccacgag; Orai2 reverse atgggcacattgagctctg; Orai3 forward cacatctgctctgctgtcg; Orai3 reverse aggcctggtgggtattcat; Stim1 forward cagggactgtactgaagatgaca; Stim1 reverse aggtgattatgccgagtcaag; Stim2 forward gagggcgcagagtgtgag; Stim2 reverse tttagagccatgcggacct; Trpc1 forward tgtggttgtgattgtgctga; Trpc1 reverse tccattctttatcctcatgatttg; Trpc2 forward cccatcgggacctttacc; Trpc2 reverse tcgaaggcggtaggacac; Trpc3 forward ttaattatggtctgggttcttgg; Trpc3 reverse tccacaactgcacgatgtact; Trpc4 forward aaggaagccagaaagcttcg; Trpc4 reverse ccaggttcctcatcacctct; Trpc5 forward ggcgatgcattactctacgc; Trpc5 reverse atcatcagcgtgggaacct; Trpc6 forward tactggtgtgctccttgcag; Trpc6 reverse caaacttcatgaacggtcctc; Trpc7 forward aacgatgaagtcaatgaaggtg; Trpc7 reverse ccagctctcctgtagcctga. We performed an absolute quantification using linearized plasmids containing the cDNA fragments and the standard curve method. For quantification of the PCR products, we used the fluorescent dye SYBR-Green  and adjusted to pH 7.4). The samples were homogenized by sonication, and aliquots of 30 μg protein (Bradford assay) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies used were α-ORAI1 (1:200) rabbit polyclonal, α-ORAI2 (1:200) rabbit polyclonal, α-STIM2 (1:500) rabbit polyclonal, α-TRPC1 (1:500) rabbit polyclonal, α-TRPC4 (1:500) rabbit polyclonal,  and α−βActin (1:5000) mouse monoclonal .Cortical neurons were plated at a density of 1 × 102, 5.4 KCl, 25 HEPES, 2.5 D-glucose, 2 CaCl2 and supplemented with 1 μM TTX to prevent action potential-evoked responses; pH was adjusted to 7.4. Patch electrodes with resistances between 4–6 MΩ were filled with an internal solution containing (in mM) 115 K gluconate, 20 KCl, 10 HEPES, 2 MgCl2, 4 Na2-ATP, 0.3 Na3-GTP, pH adjusted to 7.3 and osmolarity ~290 mOsm. Neurons were voltage-clamped at −60 mV. Electrophysiological recordings and data acquisition were carried out with a Multiclamp 700B amplifier and Clampfit 10.7 software . Miniature excitatory postsynaptic currents (mEPSC) were detected and analyzed using the Clampfit EventDetection module. Only events with an amplitude >8 pA, an exponential decay and a monotonic rising phase, which could be clearly discriminated from the background noise, were considered as mEPSCs. mEPSC amplitude and frequency were compared during a 2-min baseline period and in 2-min windows 15 min after 200 μM DHPG application (5 min). Decay time constants (τ) were calculated from single exponential fits of averaged mEPSC traces.Voltage-clamp whole-cell recordings were performed in DIV 10–11 cortical neurons. Neurons at 37°C were continuously perfused with a recording medium containing (in mM) 120 NaCl, 0.8 MgClpost hoc Bonferroni test were applied. In non-normal distributions (mEPSC parameters) Wilcoxon matched paired test was applied. Significance was *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as the mean ± SEM.Statistical analyses were performed using STATISTICA software, version 7 (StatSoft). Shapiro-Wilk test was applied to determine the distribution of the data. In normal distributions, one-way or two-way ANOVA test, following of a 2+ ATPase (SERCA) inhibitor, in a medium with 1 μM TTX, in order to prevent neuronal activity. Tg application resulted in a sustained increase in cytosolic calcium , 10 μM MK-801 (NMDA receptor inhibitor) and 5 μM MPEP (a mGluR inhibitor) to block excitatory action of ionotropic and metabotropic glutamate receptors, 10 μM TTX to block voltage-gated sodium channels, and 50 μM NiCl2 to block voltage-activated T-type calcium channels (the inhibition cocktail was present during the whole experiment). Figure 2+ increase triggered by emptying ER-Ca2+ was similar both in the absence and the presence of the inhibitory cocktail. This result contrasts with those recently reported in rat cortical neurons using a different SOCE induction protocol, in which AMPA receptors appeared to contribute to SOC mediated Ca2+ entry , formed by mGluR1 and 5, is coupled to a variety of signaling pathways. The Gαq/Gα11 protein/PLC/inositol-3,4,5-triphosphate (IP3) signal cascades have been considered as the canonical pathway, which leads to Ca2+ release from ER and activation of PKC. The decrease of ER-Ca2+ levels could activate SOCE in the PM and play a role in mGluR-driven Ca2+ signaling.After determining that ER-Ca2+ followed by a more moderate but sustained Ca2+ signal that did not reach baseline during the recording period  caused a long lasting reduction of the mEPSC amplitude at 15 min after mGluR stimulation  and protein interacting with C kinase (PICK1) in a Ca2+-dependent manner (Jo et al., 3 pathway and postsynaptic Ca2+ (Schnabel et al., 3 mediated ER-Ca2+ release from dendritic spines and calmodulin activation for group I mGluR LTD (Holbro et al., In cortical neurons, the strong impairment of DHPG-LTD and the failure to maintain DHPG-driven cytosolic Ca2+ influx through SOCs channels has been implicated in several functions in dendritic spines (Brini et al., 2+ entry through SOC channels themselves in DHPG-LTD.Although Ca2+ signals. Blocking SOCE activation upon mGluR stimulation prevented DHPG-LTD. Altogether the results suggest that, contrary to what has been observed in hippocampus, in cortical neurons SOCE-mediated Ca2+ signal could be relevant in generating downstream effects to evoke mGluR-LTD.In conclusion, our findings reveal a close relationship between mGluRs and SOCE in cortical neurons, and show that SOCE activation is a necessary step in the development of DHPG-driven cytosolic CaPG-S: designed and performed the experiments, analyzed the data and wrote the manuscript. AdA: designed and performed the experiments. JAE and JS: designed the study and wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. β2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.Intracellular Ca Curr. Curr2-acholine [.2+ concentration ([Ca2+]i) stimulates contraction of ASM, but additional mechanisms regulate the Ca2+-sensitivity of the contractile machinery, notably through RhoA and inhibition of myosin light chain (MLC) phosphatase i evoked by receptors that stimulate PLC . Hence, there is a need to determine in human ASM the interplay between the different G protein-coupled receptors (GPCRs) that stimulate Ca2+ and cAMP signals.Concern about long-term use of long-acting β-agonists has prompted interest in alternative therapies for asthma and COPD. These include prostaglandin Eeceptors . High cory tract  and theyry tract . PGE2 re airways ; EP2 andpatients ; and inhonchitis . However express ,26. Evenpress adenine were from Perkin Elmer . Ionomycin was from Apollo Scientific . Smooth muscle growth medium 2 (SMGM-2) and supplement were from Promocell . Pertussis toxin (PTX) was from List Biological Laboratories . Histamine dihydrochloride, carbamylcholine chloride (carbachol), (−)-isoproterenol hydrochloride, PGE2, 3-isobutyl-1-methylxanthine (IBMX), bradykinin acetate, Dowex 50WX4-400, alumina, imidazole, probenecid, anhydrous dimethyl sulfoxide (DMSO), pluronic F127, triton-X-100, poly-l-lysine, 8-Br-cAMP, 8-Br-cGMP, dibutyryl cAMP, KT5720 -2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolopyrrolobenzodiazocine-10-carboxylic acid, hexyl ester), GdCl3, nimodipine, 2-aminoethoxyphenylborane (2-APB), ATP and acetonitrile were from Sigma . 18:1 lysophosphatidic acid (LPA) was from Avanti Polar Lipids . 8-pCPT-2′-O-Me-cAMP, ESI-05 , Rp-8-CPT-cAMPs and 6-Bnz-cAMP were from Biolog . TCS2510 ((5R)-5-[(3S)-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5-yl)hexyl]-2-pyrrolidinone), H89 dihydrochloride (N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride), myristoylated protein kinase inhibitor 14–22 amide (PKI-myr), ryanodine, trans-Ned-19 and edelfosine were from Tocris/Biotechne . Sulprostone was from Enzo Life Sciences . R-butaprost (free acid), NKH477 -5-(acetyloxy)-3-ethenyldodecahydro-10,10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-1H-naphthopyran-6-yl ester β-alanine hydrochloride) and forskolin were from Cayman Chemicals . When DMSO or ethanol was used as a solvent, all related assays included solvent at the same final concentration; neither solvent, at the highest concentrations used, affected biological responses.FLIPR calcium 4 assay kit was from Molecular Devices . Fluo-4-AM, fura-2-AM and Hank's balanced salt solution (HBSS) with Ca2.2−1), basic fibroblast growth factor (2 ng·mL−1) and insulin (5 μg mL−1) . Cells were grown at 37 °C in humidified air containing 5% CO2, and passaged when they were 80–90% confluent. Cells from passages 4–10 were used for experiments. There were no obvious changes in morphology, growth rate or signaling responses within this range of passages.Human bronchial ASM cells  from three male donors  were from Lonza . The cells had been isolated from the major bronchi of undiseased tissue and shown to stain for α-smooth muscle actin, but not for von Willebrand Factor VIII. Cells were grown in SMGM-2 (Lonza) supplemented with fetal calf serum , and recombinant human forms of epidermal growth factor , hBASMCs were seeded into 96-well plates (104 cells per well). After about 4 days, when the cells were confluent, the medium was replaced with SMGM-2 without serum or growth factor supplements, and the cells were used after a further 24 h. This period in serum-free medium increased by about 2-fold the amplitude of the increases in [Ca2+]i evoked by histamine (results not shown). Cells were washed with HEPES-buffered saline , and loaded with fluo-4 by incubation with fluo-4-AM (2 μM) in HBS (100 μL per well) containing pluronic F127  and probenecid (2.5 mM). After 1 h at 20 °C, the medium was replaced with HBS (100 μL per well) containing probenecid (2.5 mM). After a further incubation at 20 °C for 1 h, the medium was replaced with HBS (60–80 μL per well) or nominally Ca2+-free HBS, and the cells were used immediately for experiments at 20 °C. In some experiments, BAPTA (2.5 mM) was added to HBS during the recording; this reduced the free [Ca2+] of the HBS to ∼120 nM without affecting the pH. Drug additions  were added automatically. Fluorescence from fluo-4  was recorded at 1.44-s intervals using Softmax Pro 5.4 (Molecular Devices). Fluorescence was calibrated to [Ca2+]i from:2+ (345 nM) i using an FDSS 7000 FLIPR (Hamamatsu), hBASMCs were seeded into 384-well plates (8000 cells per well) in 20 μL of SMGM-2 containing 5% serum. After 24 h, the medium was replaced with 15 μL of serum-free SMGM-2, and after a further 6 h the cells were loaded with Ca2+ indicator by addition of FLIPR calcium 4 assay kit (Molecular Devices) supplemented with probenecid (2.5 mM). The exact composition of this ‘no-wash’ indicator kit is not disclosed by the manufacturer, but it contains fluo-4-AM and components that reduce background fluorescence. The manufacture’s stock solution was diluted 10-fold into HBSS containing BSA  and HEPES (20 mM); 5 μL of this solution was then added to each well (containing 15 μL of serum-free SMGM-2). After 2 h at 37 °C in humidified air containing 5% CO2, the plate was used for experiments at 20 °C. Most additions (5 μL) were prepared in HBSS supplemented with HEPES (20 mM) and BSA . For more prolonged incubations, drugs were diluted in the initial loading medium (to avoid changes in dye-loading during the ‘no wash’ protocol). Fluorescence signals  were calibrated to [Ca2+]i after measurement of Fmin and Fmax uniquely for each well, using a For measurements of -adenine . After 2 h at 37 °C in humidified air with 5% CO2, the medium was removed, and the cells were washed twice with HBS. The cells were stimulated at 20 °C in HBS. Incubations were terminated by removal of the medium, addition of ice-cold trichloroacetic acid  and rapid freezing. This protocol ensured that only intracellular [3H]-cAMP was detected. [3H]-adenine nucleotides were separated by column chromatography [3H]-cAMP activity as a percentage of the sum of the activities of the fractions containing [3H]-cAMP, [3H]-ATP, [3H]-ADP and [3H]-AMP; henceforth, reported as [3H]-cAMP (%).hBASMCs in 24-well plates  were grown to confluence. The medium was then replaced with serum-free SMGM-2, and after 24 h this was supplemented with i in 29 of 44 cells (from a single experiment). This response was reversibly inhibited by nimodipine : the peak increase in [Ca2+]i recorded from all 44 cells was 50 ± 11 nM and 20 ± 14 nM in the absence and presence of nimodipine, respectively . Neither ryanodine to inhibit ryanodine receptors (RyR) (trans Ned-19 to inhibit two-pore channels (TPC)  A lack of response to caffeine (data not shown) and the insensitivity of most responses to ryanodine  D nor trals (TPC)  , bradykinin (1 nM) and carbachol (10 μM) were 4.64 ± 0.13, 4.47 ± 0.06 and 4.40 ± 0.04, respectively (n = 3). There are no selective and effective membrane-permeant inhibitors of IP3Rs i, that both EP2 and EP4 receptors stimulate accumulation of cAMP i (results not shown). The differential susceptibility of the Ca2+ signals evoked by histamine and bradykinin to inhibition by cAMP is not therefore due to differential distribution of their receptors between cells.2 receptors by carbachol might, via Gi, have counteracted the increases in cAMP evoked by forskolin or the Gs-coupled GPCRs [2+ signals evoked by carbachol or bradykinin remained insensitive to isoproterenol after treatment with pertussis toxin  or with some contribution from Gi   Figs. A. These gs. q/11 , althouggs. q/11 .2+ signals evoked by histamine were attenuated by stimulation of β2-adrenoceptors, consistent with results from human lung slices where formoterol caused a long-lasting inhibition of histamine-evoked Ca2+ oscillations [2 or EP4 receptors, 8-Br-cAMP, direct activation of AC, or by inhibition of cyclic nucleotide PDEs (2+ signals by maximally effective concentrations of forskolin and isoproterenol (2+ signals by β2-adrenoceptors is entirely mediated by cAMP. The inhibition is not mediated by activation of epacs (2+ signals by isoproterenol was shown to be prevented by viral infection with a peptide inhibitor of PKA [2+ signals by β-adrenoceptors is entirely mediated by cAMP and PKA (The Callations . In our ide PDEs , Fig. 6.oterenol C establiof epacs F and 5A,of epacs A[for fur and PKA A.Fig. 7S2+ signals by cAMP was selective for histamine. The Ca2+ signals evoked by bradykinin and carbachol were insensitive to cAMP, while cAMP caused only a modest and inconsistent inhibition of responses to LPA (Figs. 2-adrenoceptors than the contractions evoked by histamine [2+ by cAMP may reduce contractions evoked by all contractile stimuli [2+ signals. Selective inhibition of histamine-evoked Ca2+ signals by PKA suggests a target close to the histamine H1 receptor, and perhaps the receptor itself [see discussion in reference The inhibition of GPCR-evoked CaPA Figs. A. Isoproistamine . These r stimuli , while t2+ signals by β2-adrenoceptors, there is no consistent relationship between intracellular cAMP and inhibition of Ca2+ signals under different stimulation conditions (2-adrenoceptors most effectively inhibits Ca2+ signals, but the sensitivity to isoproterenol is unchanged during sustained stimulation despite further accumulation of cAMP. This suggests that local regulation of histamine responses must continue throughout the sustained stimulation, but this is accompanied by diffusion of cAMP, which then accumulates in cytoplasmic regions where it does not effectively inhibit histamine responses (2+ signals nor their sensitivity to isoproterenol increases (2-adrenoceptors (2+ signals by β2-adrenoceptors is associated with higher overall levels of intracellular cAMP than comparable inhibition with IBMX (2-adrenoceptors, PKA and histamine responses (Although cAMP entirely mediates the inhibition of histamine-evoked Canditions C. This sesponses B. This sncreases B. But whoceptors C? We sugoceptors B. These,oceptors B. Hence,ith IBMX C, we sugesponses B.2+ signals evoked by histamine in hBASMCs. Communication between the GPCRs that stimulate AC and the PKA that mediates the inhibition occurs within hyperactive signaling junctions. These junctions, which may be a general feature of cAMP signaling, allow rapid, robust and specific communication between receptors and effectors [We conclude that cAMP selectively inhibits the Caffectors ,54,55.PD performed and analysed most experiments. VH completed the MS analysis of cAMP samples, with input from MRD. CWT supervised the project and contributed to analysis. CWT. CWT and PD wrote the paper. All authors reviewed the paper.VH and MRD are employees of Novartis, which manufactures drugs used to treat respiratory diseases. CWT and PD declare that they have no competing financial interests.


KOs at baseline and up to 1 h post-injury. CCI injury increased cortical/hippocampal SCOP/PHLPP1 levels in WTs 7d and 14d post-injury. Injured KOs had higher brain tissue levels of phosphorylated AKT (pAKT) in cortex (14d post-injury), and higher levels of phosphorylated MEK (pMEK) in hippocampus (7d and 14d post-injury) and in cortex (7d post-injury). Consistent with an important role of SCOP/PHLPP1 on memory function, injured-KOs had near normal performance on the probe trial of the Morris water maze, whereas injured-WTs were impaired. CA1/CA3 hippocampal survival was lower in KOs vs. WTs 24 h post-injury but equivalent by 7d. No difference in 21d cortical lesion volume was detected. SCOP/PHLPP1 overexpression in cultured rat cortical neurons had no effect on 24 h cell death after a mechanical stretch-injury.Suprachiasmatic nucleus circadian oscillatory protein (SCOP) (a.k.a. PHLPP1) regulates long-term memory consolidation in the brain. Using a mouse model of controlled cortical impact (CCI) we tested if (1) brain tissue levels of SCOP/PHLPP1 increase after a traumatic brain injury (TBI), and (2) if SCOP/PHLPP1 gene knockout (KO) mice have improved (or worse) neurologic outcomes. Blood chemistry (pH, pCO New therapies are desperately needed to improve neurologic outcomes in surviving patients with moderate-severe TBI2. An ideal therapy (1) limits acute secondary injury and (2) ameliorates chronic behavioral deficits. In this study we tested if gene deletion of suprachiasmatic nucleus circadian oscillatory protein (SCOP)  in mice improves recovery following a controlled cortical impact (CCI) injury.Traumatic brain injury (TBI) is a leading cause of death and disability in the young3. MEK is a vital modulator of memory function in the brain; bilateral injection of the MEK inhibitor U0126 into the medial prefrontal cortex impairs memory consolidation as well as long-term retrieval on the MWM task but does not affect acquisition (i.e. learning)4. Thus, MEK activation in rodents links the cell signaling pathways involved in hippocampal spatial memory processing with consolidation of the engram in the frontal cortex.Neuronal SCOP/PHLPP1 blocks membrane localized K-Ras , which in turn inhibits the downstream kinase, mitogen activated protein kinase (MEK)5. Furthermore, in naïve WT mice, training on novel object recognition resulted in endogenous calpain-mediated degradation of hippocampal SCOP/PHLPP1 and increased MEK activation5. Thus, initial studies suggested that therapeutic inhibition of SCOP/PHLPP1 might be a useful strategy to enhance memory functions. However, it was later reported that conditional KO (gene deletion) of SCOP/PHLPP1 in the mouse neocortex/hippocampus also impaired long-term memory on a novel object recognition task6. Finally, crossbreeding whole body SCOP/PHLPP1 KOs with calpain 1 KOs, reversed memory deficits normally seen in the latter on a fear-conditioning paradigm7. Thus, SCOP/PHLPP1 levels that are too high or low lead to behavioral impairments. To the best of our knowledge it has not been tested if (1) SCOP/PHLPP1 levels are disturbed after a TBI, (2) if memory deficits induced by acute brain injury are improved or exacerbated in SCOP/PHLPP1 KOs, and (3) if naïve KOs have normal or altered memory function as assessed by the MWM learning and memory paradigm.Disrupting SCOP/PHLPP1 signaling in the brain alters MEK activation and impairs memory formation. Transgenic mice overexpressing SCOP/PHLPP1 in the forebrain had impaired long-term memory consolidation on a novel object recognition task  but normal acquisition (i.e. overexpression did not affect learning)9. In vitro studies in primary cortical neurons show that normal synaptic activity leads to calpain mediated SCOP/PHLPP1 degradation, which in turn activates AKT, and protects against subsequent apoptotic mechanisms induced by starvation or oxidative stress9. Furthermore, cerebral infarct volume in a model of stroke is decreased in whole body SCOP/PHLPP1 KOs 24 h post-injury vs. WTs, and neuroprotection is reversed by co-administration of an AKT inhibitor10. Thus, SCOP/PHLPP1 KOs may also have improved histological outcomes after a TBI; however, the evolution of secondary cell death pathways after a TBI vs. stroke differs, which could influence the therapeutic efficacy of AKT activation in the setting of trauma11.Protein kinase B (AKT) survival signaling is a second major target of SCOP/PHLPP1 in neurons. SCOP/PHLPP1 inhibits AKT which promotes apoptotic cell deathIn this study we report several novel findings: (1) we identified differences in baseline and post-injury physiology in isoflurane anesthetized KOs vs. WTs, (2) baseline MWM performance is similar in naïve KOs vs. WTs, (3) after a CCI, hippocampal/cortical pMEK is increased in injured KOs vs. WTs and is associated with improved performance on the probe trial (memory component) of the MWM, and (4) after a CCI, long-term histological outcomes are equivalent in KOs vs. WTs. Our findings add to a growing body of evidence which supports SCOP/PHLPP1 targeting as a novel therapeutic strategy to improve neurological recovery after brain injury.14. Baseline physiology has not been reported for any of the three currently available SCOP/PHLPP1 transgenic mice10. Also, whole body SCOP/PHLPP1 KOs are the most widely studied of the various strains and have been investigated in the setting of cancer, immunity, cardiovascular disease, and in stroke18.Acute physiology after a CCI was assessed using our standard protocol2 was increased in KOs vs. WTs. Conversely, PaO2 was increased in WTs vs. KOs  under isoflurane/nitrous oxide anesthesia. Bicarbonate and PaCOKOs Fig. . Whole bKOs Fig. .Figure 113, MAP immediately decreased following insult in WTs. Lower pressures were maintained in WTs up to 1 h post-injury , base excess (BE), and blood osmolarity vs. WTs after CCI  physiological differences presented after a CCI, whereas others observed at baseline resolved during the post-injury period. Pre-injury mean arterial pressure (MAP) did not differ between genotypes. Consistent with the effect of a CCI on arterial pressure in miceury Fig. . In contury Fig. . This avury Fig. . InjuredCCI Fig. . In contCCI Fig. . RespiraCCI Fig. .19. Similarly, hippocampal SCOP/PHLPP1 levels increased ~2 fold in WTs after a CCI at 7d and 14d post-injury  was significantly increased in KOs vs WTs 7d post-injury Fig. . No diffWe next tested if (1) naïve or (2) CCI-injured KOs vs. WTs had altered learning and memory function. There were no genotype differences in naïve or CCI-injured mice on escape latencies (learning component) across training days Fig. . Further20, whereas protein phosphatase 2A (PP2A) inhibits pAKT308 phosphorylation21. In the cortex, as expected, pAKT473 levels were increased in KOs vs. WTs 14d post-injury ury Fig.  but not ury Fig. . Corticaury Fig. . In the ury Fig.  but equiury Fig. . Hippocaury Fig. .Figure 612. As expected, a CCI injury significantly decreased the number of live cells in the CA1 stratum pyramidale vs. uninjured shams across genotypes  which yielded robust neuronal death 24 h later  after a TBI is influenced by extra-cerebral factors such as MAP, which was increased in KOs vs. WTs after a CCI. To investigate SCOP/PHLPP1 mediated neuronal cell death in a pure system we used our ter Fig. . A 75% bter Fig. . Neuronster Fig. . SCOP ovter Fig. . Howeverter Fig. .Figure 918. A key unanticipated finding in this study is that SCOP/PHLPP1 KOs exhibit alterations in baseline and post-injury physiology which may be important. This includes alterations in MAP, arterial blood gases, pH, bicarbonate, and osmolarity  and duration (~45–50 min) of the difference in MAP seen in our study, we expected WTs to have worse histological outcomes. Contrary to that prediction, 24 h CA1/CA3 cell death was less severe in WTs vs. KOs. Also, there was no genotype difference in 21d lesion volume. Therefore, our histological assessment does not suggest that WTs had greater secondary injury vs. KOs. Moreover, it is unclear why 24 h hippocampal injury is slightly worse in KOs given that they had a normal MAP post-injury. One possible explanation is that inhibition of the SCOP–regulated mechanism(s) that in turn mediate a stable blood pressure (refractory to TBI), may also be associated with a broader (unidentified) pathology which negates the potential benefits of raised MAP to reduce injury.Interestingly, KOs resisted the acute reduction in MAP that is seen after a CCI26. Thus, additional physiological parameters that need to be assessed in WTs vs. KOs after a CCI include cerebral blood flow 13, intracranial pressure (via ICP monitoring)27, and analysis of pressure autoregulation28. Furthermore, a comparison of total body vs. conditional (brain specific) SCOP/PHLPP1 KOs would help to address if cognitive benefits seen here were due to central and/or peripheral mechanisms. Using an in vitro neuronal stretch-injury model we found that SCOP/PHLPP1 overexpression had no effect on 24 h cell death, which supports the idea that altered physiology in KOs may partly mediate cognitive benefits. Finally, studies using remote telemetry may unmask small differences in MAP at various points during the circadian cycle of KOs, and also to track the time at which injury induced changes in MAP returns to baseline in WTs.More experiments outside the scope of this study are needed to investigate the nature of MAP changes in KOs, and to address if/how they affect neurological outcomes after a CCI. The difference in MAP in KOs vs. WTs would be expected to yield clinically meaningful changes in cerebral perfusion pressure (CPP)2, PaO2, pH, bicarbonate, base excess, and osmolality were also notable, and the effect of each on outcomes post-injury remain undefined. Genotype differences in PaCO2 and PaO2 would represent clinically meaningful differences, while the difference in osmolality was quite small. Elevated PaCO2 causes vasodilation of the cerebral vasculature, which increases cerebral blood flow/CBF29. Normal baseline PaCO2 levels in isoflurane anesthetized mice range from ~25–28 mmHG30. High PaCO2 levels in KOs were below the threshold of hypercapnia (i.e. <45 mmHG)31. However, in the compromised brain, even borderline hypercapnia may be detrimental. The main concern, particularly in the setting of severe TBI, is that hypercapnia may contribute to increased cerebral blood volume, hyperemia, and promote intracranial hypertension33. Thus, it is possible that secondary injury mechanisms related to high PaCO2 exacerbated hippocampal damage in KOs. However, additional studies are needed to critically evaluate these assumptions, and also to consider the impact of high PaCO2 in concert with other overlapping physiological changes such as MAP. We also recognize that the specific mechanism(s) underlying the observed physiological differences remain to be identified.In addition to MAP, differences in PaCO2 and increase in pH observed in the WT after preparatory surgery (vascular catheter placement and craniotomy) and prior to TBI. This may have resulted from either a blunted stress response, and/or an alteration in anesthetic MAC in the KO vs. WT. The other blood gas findings  could logically represent secondary and compensatory effects of the respiratory alkalosis seen in the WT vs. KO. Curiously, these findings were evident at baseline prior to TBI despite no difference in MAP and no difference in respiratory rate between genotypes. However, even if differences in the response to anesthesia or surgical stress precipitated these effects, the differential response is an important finding that merits consideration. It strongly indicates that physiological parameters need to be carefully assessed in studies investigating either SCOP/PHLPP1 mutant strains or drugs targeting this pathway, which would act systemically much like whole body KO here and potentially under conditions of anesthesia in brain injured patients. (2) The genetic background of our mice (B6129SF1/J) is slightly different vs. past studies that used similar whole body KOs on a B6X129 background15. Thus, we cannot rule out the possibility that some of the new findings reported here are related to the interaction between genotype and genetic background of our mice. However, SCOP/PHLPP1 inhibitors, if developed for human clinical trials, would ultimately face much greater biological diversity in patient backgrounds.A few limitations must be addressed. (1) Physiology was measured under isoflurane/nitrous oxide anesthesia after preparatory surgery. Thus, physiological differences in KOs may not manifest, or be as severe, under normal awake conditions (which was not investigated here). It was interesting that the KO phenotype appeared to blunt the reduction in PaCO3. Also, MEK inhibitors applied to the brain specifically impair memory function but do not impede learning35. Here we found that SCOP/PHLPP1 levels increased in the hippocampus and cortex after a CCI in WTs at 7d and 14d post-injury. Therefore, we anticipated that (a) KOs would have higher MEK activation in the injured brain at these time points, and (b) have improved memory performance after a CCI but no improvements in learning. We confirmed these predictions but more studies are needed to verify that increased MEK was responsible for cognitive benefits. Specifically, future studies involving injection of MEK inhibitors directly into the KO brain  are needed to clarify if improved memory performance after a CCI in KOs was due to increased MEK activation in the cortex, the hippocampus, or possibly due to upregulation in both regions. Alternatively, other signaling pathways regulated by SCOP may play a key role in mediating cognitive improvements.MEK activation promotes memory function, and SCOP/PHLPP1 inhibits MEK6. Specifically, (1) overexpression of SCOP/PHLPP1 in the mouse forebrain impaired memory on a novel object recognition task5. Alternately, (2) conditional KO of SCOP/PHLPP1 in the mouse neocortex/hippocampus also impaired memory on a novel object recognition task6. (3) In whole body SCOP/PHLPP1 KOs, baseline memory performance is impaired on an object location memory (OLM) and recognition memory (ORM) task7, but (4) is unimpaired on a fear-conditioning based memory task7. Thus, the type of memory task used to assess cognition in SCOP/PHLPP1 KOs is an important factor. Here we report the first analysis of SCOP/PHLPP1 KOs on the MWM. Baseline learning and memory performance did not differ in naïve WTs vs. KOs using our standard protocol36. After a CCI, memory function was improved in KOs. We chose the MWM because it is the gold standard in pre-clinical TBI research - due to high correlation between functional deficits detected by this paradigm and histological lesion volume37. Furthermore, MEK showed the largest fold change in KOs compared to other signaling targets examined, and the MWM can detect MEK disturbances with high sensitivity (even on a 1d training protocol)4. In future studies, a battery of behavioral tests 39 may uncover additional benefits or disturbances induced by SCOP/PHLPP1 KO after a CCI.Prior work suggests that SCOP/PHLPP1 disturbances in either direction (overexpression or knockdown) in the hippocampus can impair memory functionin vitro. Together these findings suggest that increased SCOP/PHLPP1 levels observed in the cortex after a TBI may play a minor role in neuronal survival. Furthermore, deletion of SCOP/PHLPP1 in the hippocampus worsened outcome. KOs had decreased CA1 cell survival 24 h post-injury. Levels of pAKT473 were also slightly decreased in the hippocampus of injured KOs. These findings suggest that, contrary to our hypothesis, SCOP/PHLPP1 may promote hippocampal survival in the acute post-injury period. Our prior reports offer some insight as to a possible mechanism; selectively inhibiting SCOP by lentivirus mediated RNAi in primary hippocampal neurons decreased pAKT473/pAKT308 levels and increased pro-apoptotic FOXO3a levels40. However, more studies are needed to elucidate potential signaling mechanisms involved in SCOP mediated neuroprotection – which seems to occur selectively in the hippocampus. Several limitations of AKT studies in this report must be acknowledged. (1) AKT activation increases necroptosis mediated cell death after a TBI43. Thus, future studies are needed to test if RIPK pathways are increased in KOs after a TBI, and if it contributed to the lack of neuroprotection seen here. (2) AKT activation is also regulated by pAKTSer477/pAKTThr479 phosphorylation, and by binding to carboxyl-terminal modulator protein (CTMP), which were not studied here but could also play an important role in KOs45. Notably, CTMP which blocks pAKT473/pAKT308 phosphorylation (i.e. the sites investigated here) is increased in the brain after CCI-TBI in mice46.We expected to observe widespread brain tissue sparing effects in KOs after a TBI. In the cortex, lesion volume was equivalent in injured KOs vs. WTs (despite increased pAKT473 levels in KOs). Also, rat cortical neurons overexpressing recombinant SCOP had similar survival outcomes after a stretch-injury 47. In contrast, AKT2 protein expression is low in the CA1/CA347. Importantly, AKT2 is the major isoform dephosphorylated by SCOP/PHLPP148. Our anti-pAKT473 antibody does not distinguish AKT1 vs. AKT2 vs. AKT3 . Thus, hippocampal vs. cortical AKT2 levels may differ before and after CCI injury, and have contributed to the unique regional and temporal pAKT473 expression patterns which we observed in SCOP/PHLPP1 KOs.The effect of genotype on pAKT473 was region and time -dependent. Hippocampal pAKT473 levels were significantly decreased 7d post-injury, whereas cortical pAKT473 levels were significantly increased 14d post-injury. Additional studies are needed to elucidate the molecular underpinnings to explain this discrepancy. We speculate that regional differences in the distribution of AKT isoforms may play a role. It was recently demonstrated that protein levels of AKT1 and AKT3 isoforms are abundant across hippocampal sub-regionsIn summary, here we tested if SCOP/PHLPP1 gene deletion improves behavioral outcomes in mice given a TBI. Specifically, we observed no evidence on tissue sparing effects in KOs vs. WTs. However, we did observe marked benefits on the memory component of the MWM behavioral test. The latter finding is in agreement with observations of increased MEK phosphorylation. More studies are warranted to investigate if SCOP/PHLPP1 inhibition is a robust and/or translatable strategy to enhance cognitive recovery in the setting of acute brain injury. Future studies are also warranted to expand upon our initial analysis of acute physiology in KOs, and to determine if any of the observed differences also occur chronically in awake mice. The underpinnings of physiological changes in KOs remains unknown but our results on MAP and blood chemistry raise concerns germane to the pleotropic roles of SCOP/PHLPP1 and its regulation of biological processes in other organs that have yet to be discovered.Anti-SCOP/PHLPP1 , anti-pAKT473, anti-pAKT308, anti-AKT Total, anti-pMEK, anti-MEK Total, anti-α-Tubulin .15. Mice were re-derived by The Jackson Laboratories  as we reported49. Breeding/genotyping was performed at Safar Center for Resuscitation Research. Male WT/KO mice were injured at ~12 wk of age, and littermates were used in all experiments. Timed pregnant (E16–17) Sprague Dawley rats (Charles River) were used for in vitro primary cortical neuron cultures studies.Animal work was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh and by the Animal Care and Use Review Office (ACURO) of the US Army/department of defense. All experiments were performed in accordance with relevant guidelines and regulations. Euthanasia protocols follow guidelines established by the America Medical Veterinary Association Guideline for Euthanasia. Heterozygous (+/−) SCOP/PHLPP1 wt and KO mice were provided as a generous gift from Dr. Tianyan Gao at University of Kentucky. The SCOP/PHLPP1 targeting strategy and PCR genotyping was described previously2O)/30%O2. Mice were given moderate CCI using our standard protocol50. Temperature was maintained at 37 °C. The head was shaved and prepped with betadine. Skin above the target injury site was opened and a small 5 mm craniotomy introduced for access to the left parietal cortex. Mice were given CCI using a pneumatic impactor device, at an injury level previously demonstrated by our group to induce hippocampal damage 50. The incision site was sutured, and mice were immediately weaned from anesthesia then allowed to recover on supplemental oxygen for 30 min to mimic clinical care before returning to the housing facility. Animals were randomized by the age at which they became available to enter the study.Briefly, all CCI-injured or sham mice were anesthetized with 4% isoflurane (induction) and 1–2% (maintenance) in 70% Nitrous Oxide (N2O)/30%O2, and switched to room air prior to a CCI. Mice were maintained on 1–2% anesthesia throughout the 1 h post-injury observation period. Arterial blood was collected at baseline and after CCI via femoral arterial catheter . Baseline samples were collected 15 min before CCI (under 1–2% isoflurane), 5 min pre-CCI, 10 min post-CCI, and 60 min post-CCI. Whole blood was immediately analyzed on an ABL90 Radiometer Blood Gas Machine . Respiratory Rate: A separate cohort of mice (n = 5/genotype) were used to measure respiratory rate. Naive isoflurane anesthetized mice were placed in a MouseOx apparatus  and respiratory rate recorded for 20 min under FiO2 of 33% 66%N2O)/O2 balance) immediately followed by 65 min in room air. Mice were returned to standard housing upon conclusion of these measurements.A separate cohort of mice (n = 5/genotype) were used to assess genotype differences in mean arterial blood pressure . The same mice were also used for 21d contusion volume analysis as described below. Briefly, training was done 14–18d post-TBI in a 100 cm diameter pool (60 cm high) and water temperature set to ~24 °C. Stationary extra-maze cues are visible around the 2.5 × 2.5 diameter room. Swim path was recorded via a video tracking system . Each training trial to find the hidden platform (10 cm diameter/clear plastic) consisted of four randomized placements in the pool. Mice were allowed a maximum of 120 sec to find the submerged platform and given a 4 min rest in a 37 °C incubator between trials. MWM performance was quantified as latency to find the hidden platform. On post-injury day 19 a single 60 s visible platform test was done (latency to find the platform) and 60 s probe trial (platform removed). Probe trial is calculated as percent time spent in target quadrant. Investigators were blinded to genotype/injury groups for behavioral assessment.MWM testing was performed as previously described by our group12. A total of 40 mice were used for H&E CA1/CA3 cell counts (i.e. n = 5/group). Briefly, mice were perfused with heparinized cold saline followed by 10% formalin fix under anesthesia. Brains were post-fixed for an additional 72 h in 10% formalin. 3 mm coronal brain sections were made and embedded in paraffin wax. Embedded brains were cut using a microtome to make 5 µM sections. Sections were mounted on glass slides and stained with Hematoxylin and Eosin  to quantify CA1/CA3 cell death in hippocampus. Images were captured on a Nikon Eclipse 90i microscope . For volumetric analysis of contusion size, mice were perfusion fixed as described above and entire brains cryosectioned; 10 µm sections were made every 0.5 mm. Brain area and lesion volume were analyzed using the program morphometric image analysis . The technician quantifying CA1/CA3 cell counts and contusion volume was blind to genotype/injury groups.Histology and contusion volume were assessed as previously described49. Briefly, brains were collected from E16–17 rat embryos and dissected in ice-cold hanks balanced salt solution (HBSS). Cortices were trypsinized, triturated, and cell pellet resuspended in Neurocult®/SM1 supplement (containing 25 µM glutamic acid and 0.5 mM L glutamine). Neurons were counted on a Cellometer and seeded onto Bioflex plates , coated with 0.5 mg/mL Matrigel. Cells were plated at a density of ~1.5 × 106 per well. Cultures were maintained by ½ media exchange with BrainPhys®/SM1 media on DIV 5 and 8.Neurons were cultured using our standard protocol as previously described52. BSL-2 work was approved by the University of Pittsburgh Institutional Biosafety Committee. Expression plasmids were purchased from GeneCopoeia . The control vector is an EGFP expression transcript in the pReceiver-Lv156 lentiviral plasmid system (Cat# Ex-EGFP-Lv156). The experimental vector is the mouse-specific SCOP/PHLPP1 open reading frame sequence in the Lv156 lentiviral plasmid system . In brief, purchased expression vectors were maxi-prepped, and concentrations of high-purity plasmids measured on a UV spectrophotometer. Expression plasmids and packaging plasmids (purchased from GeneCopoeia) were mixed in basal Opti-MEM culture media, combined with EndoFectin, diluted into Opti-MEM/10%FBS/Pen-Strep, and transduced on HEK293ta cells in T225 culture flasks. 12 h after transduction, the medium was removed and replaced with ~18 mL fresh maintenance media per flask. Virus containing supernatant was collected at ~36 h and ~48 h post-transduction, pooled, passed through a 0.45 µM filter top system, concentrated in Centricon® Plus-70 Centrifugal Filter Units, further concentrated by ultra-centrifugation , the pellets resuspended in 200 µL basal Opti-MEM, and left overnight in a 4 °C refrigerator. The following day concentrated virus was aliquoted into sterile screw-cap tubes and stored at −80 °C. Viral titer was measured using the Quick Titer™ Lentivirus Titer Kit . Neurons were transduced on the day of plating (DIV0) with a multiplicity of infection of 30 (MOI 30).Lentivirus production was done as described by our group53. In brief, on DIV9, ~750 µL of conditioned media (C.M.) was collected from each well, pooled but separated by viral manipulation, and diluted with an equal volume of fresh BrainPhys®/SM1 to generate treatment media (i.e. 50% C.M.). The remaining media was aspirated off wells, replaced with 1 mL 50% C.M., subjected to a stretch-injury using the CIC-II device , and culture plates returned to a 37 °C 5% CO2 incubator for 24 h. After collection of medium for LDH analysis, cells were washed 1X with PBS, and left on ice for ~1 h in Cell Recovery Solution® to dissolve Matrigel. Neurons were scraped off the Silastic membranes, transferred into a 1.5 mL tube, centrifuged for 5 min/4 °C/200 g, resuspended 1X in PBS, centrifuged again for 5 min/4 °C/200 g, and proteins extracts made by re-suspending cell pellet in ~65 µL RIPA buffer (with EDTA/Protease/Phosphatase Inhibitors). The % biaxial stretch of the Silastic membrane is indirectly measured by assessing the peak pressure achieved inside the well during the 50 ms nitrogen burst (measured by a pressure transducer connected to the CIC-II device). The average peak pressures (n = 3/group) in Fig. Mechanical stretch-injury was done as described previously54. Treatment media (50% C.M.) was saved as the negative control. 400 µL of media was collected from neurons 24 h after mechanical-stretch injury, from uninjured controls, and from uninjured controls lysed 24 later to obtain maximum LDH values. Media was centrifuged ~10,000 g/5 mins/4 °C to remove debris. Samples were run in triplicate. LDH levels were analyzed using the LDH Cytotoxicity Assay Kit II . The percent cytotoxicity was calculated by the equation:LDH analysis was performed as described by our group19. 24 mice were used for ex vivo brain tissue Western blot studies (n = 3/group). Briefly, cells/tissues were pulse-sonicated 20–30 s on ice in RIPA buffer plus inhibitors. Homogenates were spun in an ultracentrifuge at 4 °C/10 min/16,000 g. Total protein extracts (supernatants) were analyzed by BCA  to estimate protein concentrations. Samples (10–20 µg/well) were loaded onto pre-cast 4–15% gradient SDS-PAGE gels . Protein was transferred to PVDF membrane . Primary antibodies were diluted in tris-buffered saline plus tween-20 (TBS-T) with 7.5% milk, and incubated on membranes overnight. Membranes were washed with TBS, incubated 2 h with secondary antibodies, washed with TBS, treated with ECL-II Western blot detection reagent , exposed to films in a dark room, developed, and images collected via a scanner. Blots were stripped and re-probed with subsequent antibodies using methods described above. The order in which targets were detected is indicated in the Supplementary data. Images for figures were compiled in Photoshop .Proteins were analyzed as previously described by our groupin vitro LDH release  were analyzed by One-Way ANOVA. Densitometic analysis of AKT/MEK, analysis of ipsilateral total lesion volume, and % contralateral side were analyzed by unpaired T-test in GraphPad Prism Software . Post-hoc differences for Two-way Repeat Measures ANOVA were analyzed by Bonferroni. Post-hoc differences for One-Way ANOVA were analyzed by Newman-Keuls. Data were significant at p < 0.05. Graphs show mean + SEM.Physiology data were analyzed by Two-Way Repeated Measures ANOVA and MWM latency by three factors GLM ANOVA in NSCC Statistical Software. Densitometic analysis of SCOP/PHLPP1 western blot data, CA1/CA3 Cell death, Day 19 MWM probe trial, path length, swim speed, visible platform, and All data generated or analyzed during this study are included in this published article. Images of the original Western blots used in this study are available in the Supplementary data; antibody detection characteristics across the full molecular range are available for membranes which were not pre-cut prior to immunodetection.Supplementary Western blot Images


Long non-coding RNA (lncRNA) expression has been implicated in a range of molecular mechanisms that are central in cancer. However, lncRNA expression has not yet been comprehensively characterized in acute myeloid leukemia (AML). Here, we assess to what extent lncRNA expression is prognostic of AML patient overall survival (OS) and determine if there are indications of lncRNA-based molecular subtypes of AML.We performed RNA sequencing of 274 intensively treated AML patients in a Swedish cohort and quantified lncRNA expression. Univariate and multivariate time-to-event analysis was applied to determine association between individual lncRNAs with OS. Unsupervised statistical learning was applied to ascertain if lncRNA-based molecular subtypes exist and are prognostic.p value < 0.05). We established four distinct molecular subtypes based on lncRNA expression using a consensus clustering approach. LncRNA-based subtypes were found to stratify patients into groups with prognostic information . Subsequently, lncRNA expression-based subtypes were validated in an independent patient cohort (TCGA-AML). LncRNA subtypes could not be directly explained by any of the recurrent cytogenetic or mutational aberrations, although associations with some of the established genetic and clinical factors were found, including mutations in NPM1, TP53, and FLT3.Thirty-three individual lncRNAs were found to be associated with OS  contains supplementary material, which is available to authorized users. NPM1, CEBPA, and FLT3 internal tandem duplications (FLT3-ITD) form the basis of the European LeukemiaNet (ELN) risk classification system [Acute myeloid leukemia (AML) is a heterogeneous disease on both the molecular- and phenotypic level, caused by malignant transformation of hematopoietic progenitor cells. During pre-leukemic evolution and disease progression, affected hematopoietic cells gradually accumulate a range of molecular alterations, including somatic mutations, cytogenetic abnormalities, epigenetic alterations, and transcriptomic changes , 2. Numen system , which pLncRNAs are defined as RNA molecules longer than 200 nucleotides that are transcribed while not protein coding. It has been estimated that more than 58,000 lncRNAs are encoded in the human genome . LncRNAsHOX transcript antisense intergenic RNA (HOTAIR) is known to act as an epigenetic regulator in breast and colorectal cancer [IGF1R expression [IGF1R locus, directly interacts with the IGF1R promotor [NPM1, CEBPA, IDH2, ASXL1, and RUNX1, and FLT3-ITD [Most large-scale genomic analyses of cancer patient data have focused on the protein coding region of the genome. However, estimates from the ENCODE study suggest that up to 75% of the human genome gets transcribed into RNA, whereas only about 3% of the human genome is protein coding , 10. Lncl cancer –16. Sevel cancer , 17. Mulpression . The IRApromotor . IRAIN iFLT3-ITD . LncRNA FLT3-ITD –23.Despite growing evidence for the potential importance of lncRNAs as prognostic and diagnostic markers across a multitude of cancers, including AML, lncRNA expression in AML has not been comprehensively characterized to date with a focus on ascertaining the potential presence of prognostic lncRNA-based AML subtypes. In this study, we applied whole-transcriptome RNA-sequencing (RNA-seq) with the aim to identify prognostic lncRNAs, to define novel lncRNA-based AML subtypes and to ascertain their prognostic value and relevance for risk stratification of AML patients. Furthermore, novel lncRNA expression-based subtypes were validated in independent patient cohort.We applied RNA sequencing to characterize lncRNA expression in 274 intensively treated AML patients from the Clinseq-AML cohort see the “” sectionp value < 0.05, Fig. p value (< 0.05). A possible reason might be the small sample size of the TCGA-AML dataset.First, we investigated to what extent individual lncRNAs were associated with overall survival in the Clinseq-AML cohort. Individual Cox proportional hazards regression models were fitted for each lncRNA using time-on-study as the time scale, adjusting for age, sex, ELN risk score, mutation status of CEBPA, NPM1, TP53, WT1, TET2, ASXL1, DNMT3A, RUNX1, IDH1, IDH2, and FLT3-ITD, and chromosomal abnormalities as covariates in the models. We found 33 prognostic  lncRNAs ). Among patients in cluster G1 (N = 65), the mean (±SE) overall survival at 60 months (5 years) was 61 ± 7%. Patients in clusters G2 and G3 had an intermediate rate of overall survival of 36 ± 8% and 26 ± 5% respectively. The cluster G4 has the worst survival outcome with an overall survival at 60 months of 18 ± 5%. The prognostic performance was also evaluated in the subset of cytogenetically normal patients (N = 130), which confirmed that the lncRNA-based subtypes were significantly associated with overall survival also in this subpopulation . Event-free survival for the lncRNA-based subtypes ).We assessed the prognostic information of the lncRNA-based subtypes in respect to overall survival . However, for cytogenetically normal patients in the TCGA-AML cohort, the prognostic performance is not significant  which potentially might have occurred due to the low sample size (n = 78). Details of validation using the TCGA-AML cohort are provided in the following section.We validated the lncRNA expression-based subtypes in the independent TCGA-AML cohort Fig. . In the p value = 7.0 × 10−7). In particular, cluster G3 in this model was significantly different in overall survival compared with the reference group G1 . These results indicate that the lncRNA subtypes, prediction model, and the prognostic value of the subtypes are robust.To determine consistency of the subtype discovery, we implemented a nested cross-validation procedure that is analogous to repeatedly splitting our cohort into a training set  and an independent subset of patients for model evaluation in respect to prognostic value (test set). The misclassification rate of test set samples  was low, with overall classification accuracy in the nested cross-validation procedure of 85% . In concordance with Clinseq-AML cohort, in TCGA-AML cohort, subtype G1 (n = 30) has the best survival outcome with mean (SE) overall survival at 60 months (5 years) which is 40 ± 12%. Similarly, subtypes G2 (n = 43) and G3 (n = 69) show intermediate survival with mean (SE) overall survival at 60 months which are 31 ± 8% and 22 ± 87% respectively. Similar to Cliniseq, subtype G4 (n = 30) has the worst survival outcome in TCGA cohort, where no patient survive at 60 months . When compared with the reference group G1, in this model, subtype G4 was significantly different in overall survival . We also evaluated the prognostic performance in the subset of cytogenetically normal patients in the TCGA-AML cohort. In this subset of patients, the association to overall survival was not significant . However, this might be due to the low sample size (n = 78).Next, we assessed the reproducibility of newly discovered AML subtypes using independent TCGA-AML cohort. To handle intrinsic batch differences between the Clinseq and TCGA studies, we applied batch correction on Clinseq and TCGA lncRNA expression data . We traiths Fig. . We alsoths Fig. . PrognosTo determine if the lncRNA-based subtypes were associated with known cytogenetic or mutational aberrations, we applied association tests between subtypes and key genetic aberrations and clinical phenotypes Table . NeitherPatients belonging to group G1 are enriched for CEBPA mutations (2.56 and 1.46% single and double mutation respectively). CEBPA double mutations have been associated with favorable outcome in AML , 28. Clup value is 1.09 × 10−5 and 2.99 × 10−3 respectively, see Additional file We found that lncRNA expression-based subtypes were independent from the European LeukemiaNet (ELN) risk classification system  and the p value = 0.01), “chromosome organization” , “mRNA processing” , and “transmembrane receptor protein tyrosine kinase signaling pathway” . List of all pathways and differentially expressed genes in four clusters can be found in Additional files LncRNAs have very limited functional assignments. In order to gain some overview of potential molecular mechanism related to the lncRNAs that define the lncRNA-based subtypes, we performed pathway analysis. First, we determined which mRNA transcripts were associated with the lncRNA-based subtypes, and subsequently, we utilized this set of mRNAs for pathway enrichment analysis see the “ include p value = 2.56 × 10−68; Additional file Since lncRNA expression levels can in some cases be correlated to the expression levels of cis-located mRNAs  and poteThe present study is the most comprehensive lncRNA expression study in AML to date. We characterized lncRNA expression using RNA sequencing in a cohort of 274 AML patients  and del5 , have very similar distribution in both the cohorts. However, frequency of recurrent genetic abnormalities such as inv(16)  and inv(3)  are not similar. Interestingly, the Clinseq-AML cohort contains both de novo and non-de novo AML patients; however, the TCGA-AML cohort is completely comprised of de novo AML cases. We performed differential gene expression analysis between de novo and non-de novo samples in the Clinseq-AML cohort . The mRNA subtypes were generated using the same methodology as lncRNA expression-based subtypes , since there would be too few principal examples present in this cohort. Furthermore, the RNAseq-based lncRNA profiling method applied in this study has limitations in quantifying lncRNA molecules at very low abundances. These limitations can be overcome by using a larger sample size and deeper sequencing technology.Expression profiles of lncRNAs have previously been studied in several cancer types, including proposed lncRNA subtypes –33. HoweFor detailed material and method, refer to the supplementary information provided in Additional file https://gdc.cancer.gov) and TCGA-AML study publication [We used Clinseq-AML cohort, consist of 274 AML patients, treated according to the national guidelines in Sweden. The study was approved by the regional ethical review board in Stockholm, Sweden. All samples from the Clinseq-AML cohort were collected prior to the initiation of treatment. For detail characteristics of patients in Clinseq-AML cohort, see Table lication . DetaileTranscriptomic RNA and somatic mutation panel of genes were sequenced using the Illumina HiSeq-2500 platform. Ribosomal RNA depletion was performed using the Ribo-Zero gold kit. HTSeq count version 0.6.1  was usedk = 4) was determined using weighted silhouette index. For validation, first, we performed 10-fold cross-validation on Cliniseq-AML data. At each cross-validation round, data was randomly divided into train and test set. Unsupervised learning was performed on training set, and labels were used to train random forest model [Consensus clustering-based unsupervised learning was applied for subtype discovery . Optimalst model . Labels For independent validation, common lncRNA in Clinseq and TCGA dataset were selected as features and batch correction was applied . We traiFor association analyses, Chi-square test was used. Overall survival was measured from the date of diagnosis to the date of death. Kaplan-Meier curve and non-parametric log-rank statistic were used for comparison. Uni-variable and multivariable Cox’s proportional hazards regression models were fitted to the survival data. In multivariate analysis, we adjusted for age, sex, etiology, ELN score, and mutational status of genes. Analysis was carried out using R (version 3.1.1).Additional file 1:Supplementary figures and tables. Description of data: Figures and tables. (PDF 2723 kb)Additional file 2:List of lncRNAs. Description of data: A list of lncRNAs selected using random-forest method. (XLSX 5 kb)Additional file 3:p-values and FDR adjusted p-values. (XLSX 2434 kb)List of pathways. Description of data: A list of pathways, corresponding Additional file 4:List of genes. Description of data: A list of differentially expressed genes in lncRNA subtypes. (XLSX 2264 kb)Additional file 5:Supplementary methods. Description of data: Detailed description of the methods used in the analysis. (DOCX 66 kb)Additional file 6:LncRNA expression data. Description of data: LncRNA expression data and corresponding AML subtypes. (CSV 8427 kb)Additional file 7:Description of TCGA-AML cohort. Description of data: Clinical characteristics of the TCGA-AML cohort used in this study. (PDF 46 kb)Additional file 8:lncRNA in de novo vs. non-de novo AML. Description of data: Differentially expressed lncRNA in de novo vs. non-de novo AML patients. (XLSX 13 kb)


This commentary provides an update on the current understanding of sound encoding in the cochlea with a focus on presynaptic mechanisms.The inner ear uses specialized synapses to indefatigably transmit sound information from hair cells to spiral ganglion neurons at high rates with submillisecond precision. The emerging view is that hair cell synapses achieve their demanding function by employing an unconventional presynaptic molecular composition. Hair cell active zones hold the synaptic ribbon, an electron-dense projection made primarily of RIBEYE, which tethers a halo of synaptic vesicles and is thought to enable a large readily releasable pool of vesicles and to contribute to its rapid replenishment. Another important presynaptic player is otoferlin, coded by a deafness gene, which assumes a multi-faceted role in vesicular exocytosis and, when disrupted, causes auditory synaptopathy. A functional peculiarity of hair cell synapses is the massive heterogeneity in the sizes and shapes of excitatory postsynaptic currents. Currently, there is controversy as to whether this reflects multiquantal release with a variable extent of synchronization or uniquantal release through a dynamic fusion pore. Another important question in the field has been the precise mechanisms of coupling presynaptic Ca In the mammalian cochlea, inner hair cells (IHCs)—the genuine sensory cells of the cochlea transform sound-induced mechanical signals into a neural code at their ribbon synapses. Upon hair bundle deflection, mechanotransducer channels, located in the stereociliar tips, provide hair cell depolarization. This process leads to presynaptic glutamate release from IHCs onto spiral ganglion neurons (SGNs), and ultimately activates the auditory pathway. Coding of sound at IHC ribbon synapses achieves impressive performance: each glutamatergic presynaptic active zone (AZ) of an IHC provides the sole excitatory input to a postsynaptic SGN. Yet, each single AZ drives SGN spike rates at sound onset in the kilohertz range and supports firing at hundreds of hertz during ongoing stimulation. Moreover, these synapses are capable of transmitting information on the timing of the stimulus with submillisecond precision.2+ nanodomain hypothesis of Ca2+ influx-exocytosis coupling at IHC ribbon synapses. Finally, we will provide a brief overview of the current state of the debate on the mode of exocytosis at the hair cell AZ, which remains a hot topic of current research.The underlying mechanisms that mediate this performance have remained enigmatic but likely relate to the molecular and structural specializations of the IHC ribbon-type AZ. Here, we briefly review the latest progress on the molecular anatomy and physiology of the IHC ribbon synapse, with a focus on the presynaptic AZ  to act as the putative Ca2+ sensor in IHCs26, (ii) to facilitate vesicular priming and replenishment27, and (iii) to participate in exocytosis-endocytosis coupling through direct interaction with the adaptor protein 2 (AP-2) complex29. It is tempting to speculate that IHCs evolved this unconventional molecular machinery in order to achieve the utmost performance. One possible requirement could be a rapid and low-affinity engagement of synaptic vesicles with release sites with molecular links to a nearby Ca2+ channel, followed by rapid clearance of vesicular lipid and proteins from that site for it to be quickly reloaded. Clearly, more work is required to elucidate the molecular fusion machinery of IHCs.In addition to unexpectedly finding that IHC ribbon synapses appear to operate without neuronal SNAREs2+ channel clusters, which localize underneath the presynaptic density30 and consist predominantly of pore-forming L-type CaV1.3 subunits32, auxiliary CaVβ233, and likely yet-to-be-identified CaVα2δ subunits34. Ca2+ channel clustering depends on multiple molecular scaffolds, such as Bassoon or the ribbon (or both)35 as well as RIM2α and β36. The seamless interplay and correct localization of these proteins is not only required for establishing a normal Ca2+ channel complement36 but also critical to stabilize a large readily releasable pool of vesicles at the AZ36. Moreover, IHC AZs contain additional scaffolds such as Piccolino, a short splice variant of Piccolo37, and the Usher protein harmonin that directly interacts with presynaptic Ca2+ channels, regulates their gating, and likely promotes their proteasomal degradation39. Although the endocytic machinery is still largely uncharted, it was recently shown to include AP-229, dynamins41, amphiphysin, and clathrin heavy chain41.Besides the presence of otoferlin, IHC AZs are characterized by large Ca2+ influx couples to vesicle fusion critically determines the properties of synaptic transmission. Two limiting cases can be distinguished  “pure” Ca2+ microdomain control, where the amount of Ca2+ at the Ca2+ sensor is governed by a population of Ca2+ channels, with negligible impact of individual channels. Aside from the precise topography of the channels with respect to the vesicular Ca2+ sensors and their numbers and open probabilities, the Ca2+-binding properties of the vesicular Ca2+ sensor and the cytosolic Ca2+ buffering at the AZ govern the coupling45. Previous work has examined the Ca2+-binding properties of the Ca2+  sensor of fusion by combining whole-cell Ca2+ uncaging and membrane capacitance measurements in IHCs of mice right after hearing onset46. In these experiments, a requirement for four to five Ca2+ ions to bind cooperatively prior to fusion was indicated, and an overall low Ca2+ affinity of the sensor can be assumed. Note however, that this approach yielded massive exocytosis . Hence, it is unlikely to be entirely mediated by exocytosis at IHC AZs, but probably also involved extrasynaptic exocytosis. This calls for revisiting the intrinsic Ca2+ dependence of synaptic vesicle fusion by using refined approaches to exocytosis at AZs, ideally of more mature IHCs.The manner in which Ca47 and more recent48 work indicates that hair cell AZs harbor tens of Ca2+ channels on average. However, within a given IHC, regardless of its tonotopic position, the number of Ca2+ channels per AZ varies dramatically. This presynaptic heterogeneity is thought to be related to the requirements of wide dynamic range sound encoding51. Interestingly, IHCs display opposing gradients of their AZs for Ca2+-channel complement  and voltage-dependence of activation 52.Classic2+ channel open probability at IHC AZs vary between 0.248 and 0.430. To date, the exact topography of individual Ca2+ channels within the observed stripe-like clusters beneath the ribbon and their putative molecular linkers to vesicular release sites remains to be experimentally determined. Here, biophysically constrained modeling has proven to be a useful tool in exploring the consequences and feasibility of various scenarios . Recently, measurements of exocytic membrane capacitance changes in mutant IHCs lacking the three major cytosolic EF-hand Ca2+-binding proteins  were combined with Ca2+ buffer substitution—using different concentrations of synthetic Ca2+ chelators with either slow (EGTA) or fast (BAPTA) kinetics—and computational modeling of stimulus-secretion coupling53. With this combinatorial approach, the effective average coupling distance between the Ca2+ sensor of the release site and the nearest Ca2+ channels was estimated to equate to approximately 17 nm in mature IHCs and this is well in line with a Ca2+ nanodomain-like control of exocytosis and similar to previous estimates .Endogenous Ca2+ nanodomain-like control of exocytosis is supported by observations of a lower apparent Ca2+ cooperativity (approximately 1.4) of IHC exocytosis upon changes in the channel open probability, when compared with that found with changes in single-channel Ca2+ current . The interpretation of these discrepant apparent cooperativity estimates as evidence for Ca2+ nanodomain-like control of exocytosis was further substantiated by modeling30. There, 50 Ca2+ channels were distributed over an area of 80 × 400 nm, aiming to match the Ca2+-channel clusters, assuming different topographies of the Ca2+ channels to a dozen release sites that were placed at the rim of the Ca2+ channel cluster. Modeling of Ca2+-triggered exocytosis was constrained by experimental observations as much as possible. When channels were randomly positioned, the apparent Ca2+ cooperativity of exocytosis during changes in the number of Ca2+ channels was close to two, and hence higher than the experimentally observed value of 1.4. The physiological Ca2+ cooperativity was best matched when allocating one molecularly coupled channel to each release site, while the other channels were distributed randomly but respected an exclusion zone of 10 nm around the coupled channels. This was taken to support the notion of Ca2+ nanodomain-like control of exocytosis at the IHC AZs of mice after hearing onset, likely realized by molecular coupling of a “private” channel to the release site.The notion of a Ca2+ influx-exocytosis coupling tightens from Ca2+ microdomain-like control to Ca2+ nanodomain-like control30, as also found for other synapses60. At this point, Ca2+ microdomain control of exocytosis seems less likely for mature IHC synapses. Initially, this mechanism had been considered as an explanation for the low apparent Ca2+ cooperativity of exocytosis in mature IHCs, which was thought to employ a linear Ca2+ sensor (one Ca2+-binding step by synaptotagmin 4) after the onset of hearing61. Alternatively, a lower cooperativity, even in the presence of a sensor with several Ca2+-binding steps, was suggested to result in a linear apparent Ca2+ dependence in whole-cell membrane capacitance measurements, due to summing exocytosis from heterogeneous, but Ca2+ microdomain-governed AZs in IHC capacitance measurements of exocytosis62. However, whereas the former hypothesis seems hard to reconcile with the comparable and supralinear intrinsic Ca2+ dependence of fusion prior and after hearing onset30, the latter appears incompatible with the indications for Ca2+ nanodomain control of exocytosis at the single-synapse level59.During development, Ca2+ channels within the cluster and dissect putative linker proteins connecting release sites to the nearest Ca2+ channel(s). Moreover, further characterization of the molecular composition of the CaV1.3 Ca2+ channel complexes, also testing the presence and role of splice variants of the pore-forming CaV1.3α subunit, will assist in understanding (i) the molecular mechanisms that govern the heterogeneity of Ca2+ channel expression, (ii) the developmental tightening of excitation-secretion coupling, and (iii) the respective contributions of individual Ca2+ channel variants to shaping the efficiency of presynaptic Ca2+ influx at individual IHC AZs.Future experiments should elucidate the topography and mobility of individual Ca63.We will now focus on the mode of vesicular release at auditory ribbon synapses, a mechanism that remains only partially understood. The phenomena that raised uncertainty about this fundamental process of synaptic transmission are (i) a remarkable heterogeneity of AMPA receptor-mediated excitatory postsynaptic current (EPSC) amplitudes, that can range from about 20 to more than 800 pA at postsynaptic SGN terminals of rodents and (ii) the differences in release kinetics, as reflected in variable rise times and waveforms of the EPSCs64. At IHC synapses, large variance of EPSC amplitudes and waveforms is found in individual postsynaptic boutons even in complete absence of stimulation. Synchronized  release of multiple vesicles  was offered as a plausible mechanism explaining such EPSC heterogeneity65. Synchronized multiquantal release has also been indicated for hair cell synapses of turtle and bullfrog papillae69, and for other sensory synapses, such as those in retinal bipolar cells72.Conventionally, quanta of neurotransmitter are released spontaneously , thereby producing so-called miniature EPSCs (mEPSCs). These mEPSCs are uniform in size and have characteristic monoexponentially decaying waveforms. Such mEPSCs are thought to correspond to spontaneous and statistically independent fusion of individual vesicles, constituting the basis of the quantal hypothesis of transmitter release73. Potential mechanisms mediating synchronized multiquantal release include release site synchronization and compound or cumulative fusion of vesicles , or fuse to a vesicle that is in the process of release (cumulative exocytosis).Heterogeneity of the EPSC shape might result from varying degrees of synchronicity of multiquantal release76. The motivation came from considering the spike rates of hundreds of hertz over prolonged periods of time, which, according to the multiquantal hypothesis , would require at least sixfold-higher vesicle release rates, which seem very high considering measured rates of sustained exocytosis of about 700 vesicles per second27. Moreover, in conditions omitting the synchronizing effect of presynaptic Ca2+ entry, large monophasic, as well as multiphasic, EPSCs persisted76, seemingly arguing against a Ca2+-synchronized multiquantal release scenario at the IHC ribbon synapse. In addition, biophysically constrained mathematical modeling of compound exocytosis suggested the presence of large vesicles near the AZ membrane, which was not found in electron microscopy of stimulated samples. These latter findings are difficult to reconcile with a synchronized multiquantal release mode to take place at mammalian IHC ribbon synapses. So, could a uniquantal release model offer an explanation for the large heterogeneity of EPSC amplitudes?Recently, uniquantal release through a dynamic fusion pore has been suggested as an alternative hypothesis for IHC synapses of rodents78; however, for this scenario to be plausible at IHC synapses, two major questions arise: (i) could the glutamate content of a single synaptic vesicle elicit the observed large EPSCs  and (ii) could transmitter release be governed by a fusion pore that might regulate the extent and timing of release events? Using mathematical modeling—constrained by morphological estimates of the postsynaptic AMPA receptor clusters  or alternatively the formation of a transitory instable fusion pore prior to collapse, potentially flickering open and closed—a mechanism permitting progressive glutamate unloading that may explain the observation of multiphasic EPSCs. In addition, variable vesicle size that occurs also in the absence of homotypic vesicle-to-vesicle fusion and different filling states of vesicles79 might contribute to the EPSC heterogeneity at IHC synapses. Finally, other mechanisms such as spill-over of glutamate from neighboring synapses and postsynaptic receptor properties need to be considered when relying on postsynaptic recordings.Uniquantal release through a dynamic fusion pore has also been proposed for other synapseslusters  and assuThese different hypotheses might not be mutually exclusive, and appear to strongly depend on the chosen experimental model system. Further experiments, such as (i) membrane capacitance recordings of individual fusion events, (ii) electron tomography of synapses immobilized within milliseconds after stimulation, or (iii) super-resolution fluorescence live-cell imaging of vesicular exocytosis, will help in the future to pinpoint the mode of exocytosis at hair cell ribbon synapses.


Drosophila that may be non-Hebbian, and is critical for generating the precise connectivity observed in that system. In Drosophila, motoneurons use both glutamate and the biogenic amine octopamine for neurotransmission, and the muscle fibers receive multiple synaptic inputs. Motoneuron growth cones respond in a time-regulated fashion to multiple chemotropic signals arising from their postsynaptic partners. Central to this mechanism is a very low frequency (<0.03 Hz) oscillation of presynaptic cytoplasmic calcium, that regulates and coordinates the action of multiple downstream effectors involved in the withdrawal from off-target contacts. Low frequency calcium oscillations are widely observed in developing neural circuits in mammals, and have been shown to be critical for normal connectivity in a variety of neural systems. In Drosophila these mechanisms allow the growth cone to sample widely among possible synaptic partners, evaluate opponent chemotropic signals, and withdraw from off-target contacts. It is possible that the underlying molecular mechanisms are conserved widely among invertebrates and vertebrates.During development, neurons establish inappropriate connections as they seek out their synaptic partners, resulting in supernumerary synapses that must be pruned away. The removal of miswired synapses usually involves electrical activity, often through a Hebbian spike-timing mechanism. A novel form of activity-dependent refinement is used by  As a result, the mechanisms regulating synaptic connectivity generally involve Ca2+-dependent effectors. Ca2+-dependent signaling can influence early growth events, such as the motility and exploration of the growth cone  from internal stores  bursts of action potentials spaced every 2–3 min , the Ca(v)2.1 channel  are required for proper synaptic refinement.2+ through Ca2+ channels , and the Ca2+-dependent adenylyl cyclase Rutabaga  were identified in the same screen among the molecules that regulate AP1 function , are testing whether there is a direct correlation between growth cone motility and the underlying low frequency Ca2+ oscillation.At the s Figure . We viewFV and HK wrote the manuscript and designed the figures.This study was supported by the National Institutes of Health .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here using intravital imaging, we captured the dynamics of [Ca2+]i in mouse skin. [Ca2+]i was elevated in basal cells on the second time scale in three spatiotemporally distinct patterns. The transient elevation of [Ca2+]i also occurred at the most apical granular layer at a single cell level, and lasted for approximately 40 min. The transient elevation of [Ca2+]i at the granular layer was followed by cornification, which was completed within 10 min. This study demonstrates the tightly regulated elevation of [Ca2+]i preceding the cornification of epidermal keratinocytes, providing possible clues to the mechanisms of cornification.Epidermal keratinocytes achieve sequential differentiation from basal to granular layers, and undergo a specific programmed cell death, cornification, to form an indispensable barrier of the body. Although elevation of the cytoplasmic calcium ion concentration ([Ca During this differentiation, the morphology of epidermal keratinocytes changes markedly. On the basis of its morphological characteristics, the epidermis can be subdivided into four layers: basal, spinous, granular, and cornified layers. The granular layer is further subdivided into three layers designated as SG1, SG2, and SG3 from the apical to the basal side2.The body surface of mammalians is covered with skin, which protects them against infection, dehydration, chemicals, and mechanical stress. This barrier function is mainly provided by the stratified squamous epithelium of the skin, the epidermis. The epithelial cells  proliferate at the lowest layer of the epidermis and then commence differentiation and upward migration, which takes 40–56 or 8–10 days in humans and mice, respectively3. Although apoptosis and cornification share similarities, such as the loss of an intact nucleus and other organelles, the dead cell corpses of epidermal keratinocytes are not removed; instead, they are maintained to fulfill a barrier function4. The regulatory mechanisms of cornification remain elusive, and their clarification is important to furthering our understanding of skin homeostasis.At the border of the granular and cornified layers, epidermal keratinocytes (SG1 cells) undergo a programmed cell death called cornification, which is characterized by a rapid loss of cell volume2+]i) is one of the factors predicted to regulate cornification for several reasons6. First, it is known that the various forms of cell death  share molecular effectors and signaling routes, and the elevation of [Ca2+]i is involved in the process of apoptosis, which is assumed to have many similarities to cornification7. Second, in cultured keratinocytes, [Ca2+]i elevation following stimulation for differentiation has been identified8. Third, in both the human and murine epidermis, a number of experimental and theoretical analyses have shown the preferential distribution of calcium ions in the granular layer, which are indicated to localize to intracellular compartments14. Therefore, it has been suspected that the transient elevation of [Ca2+]i occurs during the process of differentiation and/or cornification. However, the spatio-temporal dynamics of [Ca2+]i in the sequential differentiation of epidermal keratinocytes remains largely unknown because of technical limitations.The concentration of cytoplasmic calcium ions 18. GCaMP3 is a modified green fluorescent protein (GFP) that increases its fluorescence in proportion to the level of [Ca2+]i19.To visualize i elevation or derives from uneven subcellular distribution of GCaMP3 protein, we immunostained GCaMP3 protein with anti-GFP antibody using sections of fingertip skin of GCaMP3 mice. In the basal cells, the protein of GCaMP3 distributed preferentially in the cytoplasm, whereas it was hardly detected in the nucleus  form a tight junction barrier20. Subsequently, granular cells in the most apical layer (SG1 cells) finally achieve cornification, and then become integrated into the cornified layer.Next, we investigated the precise location of GCaMP321 (ZO-1 EGFP/GCaMP3 mice). In ZO-1 EGFP mice, zonula occludens-1 (ZO-1), a constituent molecule of tight junctions, is fused with enhanced green fluorescent protein (eGFP). Using these mice, we can visualize the SG2 layer by exciting eGFP. In the ear and hind paw skin of ZO-1 EGFP/GCaMP3 mice, GCaMP3high cells were located above the tight-junction signal  was well visualized with the red fluorescence of tdTomato. In addition, the border of the granular and cornified layers was visualized by contrast with fluorescence intensity  mice. Tdtomato is one of the red fluorescent proteins, which was specifically designed for low aggregationity Fig. , which myer Fig. . During yer Fig. .Figure 52+]i.Taken together, these results demonstrate that a subset of SG1 cells residing immediately beneath the cornified layer showed the transient elevation of [Ca2+]i and the transition from granular to cornified cells using tdTomato/GCaMP3 mice.Next, we investigated the association between the transient elevation of i activates transglutaminases in SG1 cells before the transformation to cornified cells.The transient elevation of i. Because the elevated fluorescence of GCaMP3 disappears in skin sections for immunohistochemistry, establishment of an in vivo evaluation system to detect GCaMP3 fluorescence and keratinocyte differentiation markers or cell death markers would be necessary to clarify this issue.GCaMP3-protein was distributed exclusively in the cytoplasm in the basal cells, but distributed to both the nucleus and cytoplasm in the granular cells. This observation raises a hypothesis that GCaMP3 protein in the cytosol re-distributed to nucleus due to increased permeability of nuclear envelope in the process of cell death2+ homeostasis result in skin lesions, including aberrantly promoted cornification in congenital diseases such as keratitis-ichthyosis-deafness (KID) syndrome and Darier’s disease38. This implies a possible link between Ca2+ homeostasis and cornification. In KID syndrome, mutated connexin proteins were recently shown to form hyperactive hemi channels, connecting the inside and outside of the cells40. Therefore, Ca2+ influx through hyperactive hemi channels in the granular layer might promote cornification in the skin of patients with KID syndrome. Whereas, Darier’s disease is caused by a mutation in ATP2A2 coding type 2 isoform of SERCA (SERCA2)38. Because calcium ions in the granular layer are suggested to localize to intracellular compartments including endoplasmic reticulum (ER), it is possible that the source of calcium ions in the transient elevation of [Ca2+]i in SG1 cells are intracellular compartments14. Generally, the high concentration of calcium ions in these compartments is sustained by calcium pumps or channels including SERCA2. Therefore, mutated SERCA2 proteins might affect the concentration of calcium ions in ER and the dynamics of [Ca2+]i in SG1 cells, eventually resulting in aberrant cornification in the granular layer in Darier’s disease.Gene mutations related to Ca2+]i in SG1 cells preceding cornification. This finding significantly deepens our understanding of the mechanisms of cornification in epidermal keratinocytes and may lead to the development of specific treatment for skin diseases affecting the cornified layer.In conclusion, we identified the transient elevation of i elevation in the granular layerSupplemental Movie 7: Cornification of granular cells following the transient [Ca2+]i elevation