We further show that these sites are phosphorylated in vivo and provide evidence revealing that LATS1,2 participate in the phosphorylation of the most C-terminal S814 site, present on both isoforms. This S814 phosphosite was previously reported to constitute a 14-3-3 binding site, which plays a role in Kif23 clustering during cytokinesis. Surprisingly, we found that phosphorylation of the upstream S716 NDR/LATS consensus site, present only in the longest Kif23 isoform, is required for efficient phosphorylation at S814, thus revealing sequential phosphorylation at these two sites, and differential regulation of Kif23-14-3-3 interaction for the two Kif23 isoforms. Finally, we provide evidence that Kif23 is largely unphosphorylated on S814 in post-abscission midbodies, making this Kif23 post-translational modification a potential marker to probe these structures.Kif23 kinesin is an essential actor of cytokinesis in animals. It exists as two major isoforms, known as MKLP1 and CHO1, the longest of which, CHO1, contains two HXRXXS/T NDR/LATS kinase consensus sites. We demonstrate that these two sites are readily phosphorylated by NDR and LATS kinases  NDR/LATS kinases form a specific subgroup in the AGC kinase family and are present throughout the eukaryotic domain, including protists. NDR/LATS are characterized by their activation through binding to MOB proteins and phosphorylation by a member of the MST or YSK subgroups of the STE20 kinase family. The NDR/LATS clade itself comprises two distinct members which are called NDR and LATS in animals. These two subgroups are duplicated as NDR1,2 and LATS1,2 in vertebrates.S. cerevisiae and Sid2 in S. pombe are key players respectively of the MEN (Mitotic Exit Network) and SIN (Septum Initiation Network) pathways, whose activations are essential for cytokinesis . I. IS. cerokinesis 13], and, andS. cude Chs2 ) and Hofude Chs2 . Howeverude Chs2 , 17]. S. SS. cerude Chs2 . On the ude Chs2 , 20]. A. AS. cerude Chs2 21]. Thi. ThiS. cude Chs2 ,22. It iin vivo. We also identify a new in vivo phosphorylation site (pS716) present specifically on the longer isoform, and which is necessary for phosphorylation at S814 and 14–3–3 binding. Finally, we show that dephosphorylation of pS710/pS814 is a post-abscission process that is uncoupled from Kif23 degradation.In the present work, we have focused on kinesin Kif23 as a candidate target of NDR/LATS kinases, as it contains consensus HXRXXS/T phosphorylation sites. Kif23 is a member of the kinesin-6 subgroup and forms a heterotetramer complex with MgcRacGAP. This complex, also called centralspindlin, is involved in stabilization of the mitotic spindle anti-parallel microtubules, from anaphase onset to abscission . Then, AAffinity purified anti-Kif23 was prepared from rabbit serum immune to human GST-Kif23-iso1(682–907). Antibodies directed against pS716 (ab pS716) and pS814 (ab pS814) of Kif23-iso1 were prepared as follows. Rabbits were immunized against peptides RRSNpSCSSISVAC and RRSRpSAGDRWVDC, where pS denotes phospho-serine and C at the C-terminus was used for crosslinking to thyroblobulin. Rabbits were handled in the animal house of the Institut Universitaire de Technologie de Montpellier which has an institutional agreement (number D34–172–8) from Direction Départementale de la Protection des Populations (DDPP) de l’Hérault , which operates under the supervision of the Ministry of Agriculture and is dedicated to rabbit immunization and blood sampling. The protocol was not further submitted to the approval of an ethics committee. Such approval was not necessary for those experiments under the French and European legislation at the time they were conducted. At the end of the immunization protocol, rabbits were anaesthetized with pentobarbital (30mg/kg) and then sacrificed by injection of dolethal . Rabbit sera were first purified on the peptides above immobilized as BSA conjugates on CNBr-activated sepharose, and the eluates where rapidly passed over unphosphorylated peptide-coupled resin. Anti-myc antibodies where prepared from mouse 9E10 hybridoma. Anti-human LATS1 clone C66B5 was from Cell Signalling Technology, anti-human MgcRacGAP from abcam (ab2270), anti-human cyclin B1 from Santa Cruz (sc-752), anti-Flag clone M2 and anti-tubulin clone DMA1 from Sigma.E. coli, fragments of human Kif23-iso1 , human PARD3 , human CYLD , human TSG101  and mouse MTSS1  were cloned by PCR C-ter of 6xhis tag in pRSETA. Kif23-iso1  was also cloned in pGEX-4T1 for expression of GST-Kif23. For expression of Nter-tagged fusion proteins in human cells, complete open reading frames of human Kif23-iso1 (BC071566.1), X. laevis NDR1 (NM_001086949.1) and X. laevis MOB1A (NM_001089248.1) were cloned in pCMV10–3xFlag, pRK5HAGST and pRK5myc, respectively. Human 14–3–3, Kif23iso1 and LATS2 were also expressed from vectors pEF6-myc-14–3–3γ . W. WR), wh signals . To bettor pS814 . This suin vitro, and that the presence of histidine at position-5 is essential for these phosphorylations. A majority of the identified in vitro and in vivo NDR/LATS phosphorylated sites comply with the HXRXXS/T signature [in vivo was dependant on the upstream histidine [in vivo, we obtained different outcomes. While S716 phosphorylation was unaffected by its absence, that of S814 was strongly reduced. As no other basophilic kinase has been reported to rely on a histidine at-5 for substrate recognition, this suggests that S814 is phosphorylated by a NDR/LATS kinase in vivo. On the other hand, the neutral outcome of the H711A mutation rather suggests that NDR/LATS are not involved in S716 phosphorylation in vivo, in contrast to what we observed in vitro. It cannot be excluded though, that NDR/LATS could be less stringent for this site in vivo. As compared to mutating the upstream histidine, which resulted in a 4 fold decrease in S814 phosphorylation, a more modest effect emerged when we performed LATS depletion. LATS1,2 depletion resulted in a 56% decrease in S814–phosphorylated Kif23. While it is conceivable that this modest impact may arise from the potentially high redundancy of the four NDR/LATS kinases or incomplete depletion by siRNA, it is also possible that other kinases play a role in this phosphorylation. In that respect, YAP and p21, two in vivo NDR/LATS substrates, are also known to be phosphorylated by other kinases [in vivo phosphorylation of S814 on both Kif23 isoforms.During our search of mitotic substrates for NDR/LATS kinases, we noticed that Kif23 displayed two consensus HXRXXS phosphorylation sites. One of these sites, pS814, was previously shown to act as a 14–3–3 binding site, but the kinase responsible for this phosphorylation was not identified. In this work, we could show that LATS1/MOB1A and NDR1/MOB1A kinases phosphorylate both consensus sites istidine . Studiesistidine ,20. When kinases –37. We cin vitro NDR/LATS kinase assay using a ~20 kDa Kif23 fragment. Phosphorylation at S710 of Kif23-iso2 (S814 in Kif23-iso1) was previously shown to be important for binding to 14–3–3 [per se for 14–3–3. Such a hierarchical phosphorylation system regulating 14–3–3 binding was reported for the FOXO3/FKHR transcription factor [While monitoring the phosphorylation status at both S716 and S814 on non-phosphorylatable Kif23-iso1 mutants, we could uncover an interplay between these two sites. Namely, mutation of S716 drastically reduced phosphorylation of the distant S814, but not the opposite. This would imply that phosphorylations at S716 and S814 obey to a hierarchical order in Kif23-iso1, whereby S716 phosphorylation would occur first and be necessary for efficient phosphorylation at S814. We note that this cross-talk between these two phosphorylation events was not observed in our o 14–3–3 . We founo 14–3–3 , and secn factor . The undBoth isoforms of Kif23 reside at the spindle midzone and at the MB, and each is sufficient to achieve proper cytokinesis ,39. HoweR). It was first thought that MBRs did not fulfill any specific function and were merely destined to degradation, but recent studies have demonstrated their involvement in post mitotic functions. While in cancer and stem cells, MBRs remain in the cytoplasm during multiple cell divisions, in differentiated or differentiating cells, MBRs are either degraded by the ubiquitin and autophagosome pathways or expulsed in the culture medium [Rs remaining inside the cell might play a role in cell fate [R is segregated specifically in the daughter cell [R repositions the mitotic spindle in the P1 cell, a crucial step for the dorso-ventral patterning of the embryo [Rs are rarely released and are mainly found as intracellular organelles with an average life of 11 hrs [R are Cep55, MgcRacGAP and Kif23. In this work, we have shown that Kif23 is largely unphosphorylated on S814 in MBRs, in contrast to what is observed for cytokinetic MBs. Since a non insignificant portion of MBRs do have relative pS814 levels comparable to those of MBs, it is probable that dephosphorylation of pS814 takes place after abscission, but rather early during the lifespan of the MBRs. It will be interesting to determine whether this dephosphorylation occurring in the next cell cycle is the result of lower LATS1,2 kinase activity or higher phosphatase activity, or both. While regulation of LATS1,2 kinases during the cell cycle is still elusive, control of its activity as part of the hippo pathway during control of cell proliferation is well documented. It is worth noting that cell proliferation is correlated with low LATS kinase activity and a high number of MBRs. We could show that in the numerous MBRs of HeLa cells, phosphorylation of Kif23 on S814, that we suggest to be driven by LATS, is low. On the opposite, we would expect this phosphorylation to be stronger in cells containing fewer MBRs. This could merely be the consequence of the expected shorter half-life of MBRs in these low-population MBRs, the new MBRs just arising from abscission being fully phosphorylated on S814. On the other hand, those MBRs which escape degradation for longer times would be less phosphorylated on S814, residing in a context of low LATS kinase activity. However, it is tempting to speculate that the phosphorylation state of S814 on Kif23 could itself behave as a determinant of MBRs’ longevity. It will be interesting to establish the phosphorylation status of S814 of Kif23 on MBRs in other cell types containing different number of MBRs and to verify if altering this phosphorylation could modulate the MBRs per cell ratio. Alternatively, it could be worth monitoring pS814 to probe MBRs during differentiation or embryogenesis.At the end of mitosis, the two future daughter cells are connected by the MB, a thin intercellular bridge containing a microtubule rich zone . The MB e medium ,49–51. Tell fate . In diffter cell . In the e embryo ,55. In Hf 11 hrs ,56. AmonS1 FigGST-NDR1, GFP-LATS2 kinases and kinase-dead (KD) versions were purified from HEK293T cells as described in Materials and Methods and analysed by Coomassie blue staining on polyacrylamide gels. Asterisks denote recombinant GST-GFP-trap and its degradation products.(TIF)Click here for additional data file.S2 Fig6His-tagged candidate substrate domains, as indicated, were incubated with NDR1-MOB1A kinase (WT or kinase dead (kd)) and analysed by SDS-PAGE and autoradiography.(TIF)Click here for additional data file.S3 FigPhosphorylated and unphosphorylated peptides lining S716 and S814 were deposited on a nitrocellulose membrane as two-fold serial dilutions and revealed with the corresponding affinity purified ab pS716 and ab pS814.(TIF)Click here for additional data file.S4 FigUnsynchronised HeLa cells were fixed and stained with ab pS814, anti-Kif23 and anti-tubulin antibodies.(TIF)Click here for additional data file.S5 FigHeLa cells were released from a double thymidine block and analyzed for pS814, Kif23 and cyclin B1 content by Western blot. Cyclin B1 was used as a marker of cell cycle progression. A longer exposure (right panel) allowed monitoring of S814 phosphorylation level for the minor isoform 1.(TIF)Click here for additional data file.S6 FigHeLa cells were transfected with myc-LATS2 and control or LATS2 siRNAs (set 1) and analyzed for the amount of myc-LATS2 by Western blot.(TIF)Click here for additional data file.S7 FigMyc-14–3–3  and WT or mutant GFP-Kif23-iso1, iso2 (A) were expressed in HEK293T cells and immunoprecipitated with anti-myc antibody. Whole cell extracts as well as immunoprecipitated materials were analyzed by Western blot.(TIF)Click here for additional data file.S8 FigWT and mutant Flag-tagged Kif23-iso1 were expressed with myc-tagged 14–3–3 in HEK293T cells. Material immunoprecipitated with anti-myc antibodies was analyzed by Western blot for the presence of Kif23.(TIF)Click here for additional data file.S9 FigUnsynchronized HeLa cells were fixed and stained with anti-Kif23, anti-MgcRacGAP and anti-tubulin antibodies and DAPI. Yellow arrow points to MBs in cytokinetic cells.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


A-receptor complex α4β3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors.Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABA Stroke is the major cause of an acquired lifelong physical disabilityMotor impairments, including sensorimotor failures, hemiparesis, ataxia and spasticity, are the most common deficits after stroke and affect up to 80% of patients578A receptors, which are composed of heterogeneous combinations of receptor subunits, to fine-tune fast synaptic inhibition and to control overall network excitability by tonic inhibition. In the cerebrum, the GABAA receptor subunits δ and α5 have been identified as specific subunits incorporated into extrasynaptic GABAA receptors that mediate tonic inhibition.The neural basis for motor recovery following stroke depends on brain plasticity, which defines the ability of the brain to structurally reorganize and adapt its function. Brain plasticity-dependent motor learning is mainly controlled by GABA mediated inhibition12A receptor subunit α5  has been suggested to reduce excessive tonic inhibition found in the peri-infarct cortex following photothrombotic injury52The potential pharmacological modulation of tonic inhibition has attracted considerable attention in stroke research. As a novel therapy, the administration of a benzodiazepine inverse agonist specific for the GABAunit α5 L55,708 haA receptor subunit δ following stroke in miceIndeed, by screening of transcriptome data, we found a significant decrease of the extrasynaptically localized GABAN-methyl-D-aspartate) receptors by stroke-induced excitotoxicity.Our data indicate that following stroke the brain itself down-regulates tonic GABAergic inhibition and produces an environment, which supports brain plasticity and thereby facilitates motor improvements. In agreement with this, a decrease in tonic inhibition was identified as a reason for post-stroke epileptic seizures. As mechanism for the impaired tonic inhibition, we identified the activation of NMDA . Beside their unique morphological specificities, the input resistance  was in the typical range for pyramidal cellsWe examined GABAA receptor subunit δ transcripts (Gabrd) and protein (GABRD) in peri-infarct tissue 7 days after stroke in mice 20A receptor subunits  following mild MCAO . The impact of diminished tonic inhibition on post-stroke functional motor performance was subsequently analyzed using an accelerated RotaRod test25GABAA receptors. Our finding that post-stroke down-regulation of δ-GABAARs is responsible for the improved motor performance could further be corroborated by the better RotaRod performance of GABRD+/− vs. wild-type mice [GABRD+/+: 82.63 ± 8.81 sec, GABRD+/−: 121.12 ± 5.10 sec (n = 11 each), p ≤ 0.01; Next, we analyzed the impact of the NMDA-receptor blockade on motor performance after stroke by employing the RotaRod test. MK-801 administration to MCAO mice prevented the improvement of motor performance following stroke [3 days after stroke: 69.92 ± 7.03% and 7 days after stroke: 114.05 ± 11.41 (n = 12), ARs, delayed the onset of seizures in response to PTZ in MCAO mice [92.26 ± 8.83 sec, (n = 10), p ≤ 0.05; A receptors on the seizure threshold was corroborated by experiments on transgenic mice with reduced expression of the δ subunit: GABRD+/− mice displayed a higher seizure susceptibility than control mice [GABRD+/+: 119.78 ± 13.20 sec (n = 4); GABRD+/−: 82.46 ± 7.35 sec (n = 9), p ≤ 0.05; ARs as a cause of post-stroke seizures.Post-stroke reduction of tonic inhibition may be expected to enhance seizure susceptibility. To test this hypothesis, we injected pentylenetetrazole  and determined the interval after which epileptic seizures appearedA receptor subunit δ as well as by a decrease of its α4 and β3 co-subunits. We considered glutamate-driven excitotoxicity, which plays a key role in ischemic brain injury, as a potential mechanism of down-regulation36+ Cl− co-transporter KCC2, which in turn is associated with a decreased expression of δ-containing GABAA receptorsARs via a KCC2-mediated mechanism in a cell culture model. Glutamate application to cortical neurons markedly decreased transcript expression of Kcc2 and Gabrd. Because administration of the NMDA-receptor antagonist MK-801 to MCAO mice significantly attenuated the post-ischemic down-regulation of δ-GABAARs, an excitotoxicity-driven mechanism is corroborated in vivo.Here, we identified a decrease of tonic GABAergic inhibition following MCAO in adult mice by patch-clamp recordings in the motor cortex (M1). This reduction was paralleled by a down-regulation of the extrasynaptically localized GABA−-extrusion in overall GABAergic inhibition has been extensively described, but less is known concerning KCC2 function and tonic inhibition. A recent study investigated KCC2-mediated intracellular chloride levels as possible regulators of the developmental switch in GABAA receptor subunits. They found that tonic current amplitudes and the surface expression of the δ subunit decreased following KCC2 shRNA transfectionARs remains elusive.Several cerebral pathophysiologies that are associated with excitotoxicity e.g., ischemia38Gabrd and Gabra5 was unchanged following photothrombosis in several peri-lesional and remote brain regions in adult mice ARs. Following a unilateral lesion of the anteromedial cortex, ipsilateral muscimol infusions disrupted the recovery from somatosensory asymmetryARs with increased efficacy compared to GABAWhile several studies point to an advantage of a decrease in GABAergic inhibition to facilitate motor skills, the specific relevance of tonic inhibition in this context remains uncertain. Blocking of excessive tonic inhibition was beneficial for the recovery of motor functions following photothrombotic strokeGabrd following stroke in aged vs. young miceThese studies revealed the tight homeostatic control of motor functions by GABAergic tonic inhibition because variations above and below trigger motor dysfunctions. In our model, decrease of tonic inhibition facilitates motor improvements. We therefore suggest that higher cortical excitability induces a homeostatic plasticity that can be used for rehabilitation. Whereas this is in agreement with the accepted role of GABA in controlling excitability, the specific impact of GABAergic tonic inhibition on the emerging post-stroke motor plasticity is new. Moreover, we suggest that the regulation of tonic inhibition following stroke might be a reason for the inter-individual differences in behavioral performance and recovery. This is supported by our results that reveal an attenuated decrease of Patients surviving brain ischemia often develop enhanced brain excitability and epileptic seizures+/− mice, which is consistent with the findings of previous studies on GABRD−/− miceWe therefore studied tonic inhibition as a possible mediator of post-stroke brain hyperexcitability. PTZ administration promoted the onset of epileptic seizures in mice after MCAO. This effect was abolished by the post-ischemic application of gaboxadol (THIP). We also confirmed the impact of lower tonic inhibition on seizure thresholds in GABRDAR function and are found to be associated with generalized idiopathic epilepsies5859ARs are targets of anticonvulsant drugsMissense mutations within the human GABRD gene tend to decrease GABAHere, we identified a decrease in GABAergic tonic inhibition as a cause of epileptic seizures following stroke. This is important because it opens the possibility of specifically treating severe post-stroke seizures and thereby omitting the side effects that often emerge from anticonvulsants. Gaboxadol (THIP), a muscimol derivative, is an attractive candidate in this context.Survivors of stroke often recover from motor deficits, either spontaneously or during early rehabilitative training478A receptor subunit δ - deficient mice; GABRD+/− All studies were performed on 2–3-month-old male Wistar rats and C57Bl/6J mice. All animal care and experimental procedures were performed in accordance with EU animal welfare protection laws and regulations. We confirm that all experimental protocols were approved by a licensing committee from the local government . GABAMCAO was performed as described in detail for ratsPhotothrombosis was induced in anaesthetized mice by fixing the head in a stereotactic frame. A fiberoptic bundle (diameter 1.3 mm) connected to a cold light source  was positioned in the middle between the bregma and lambda, 1.5 mm lateral to the mid-sagittal suture. Following Bengal Rosa administration into the tail vein (50 mg/kg body weight), the skull was illuminated for 15 min. The controls underwent the same procedure but without illumination. The brains were removed after cervical dislocation 7 days following injury induction. Specific brain regions were dissected.2, 10HEPES, 2EGTA, 2Mg-ATP, 0.5Na-GTP, pH adjusted to 7.3 with CsOH in the whole-cell modus at a holding potential of −70 mV. DL-APV (30 μM), CNQX (10 μM) and GABA (5 μM) were added to aCSF. Total GABAergic currents consisting of both phasic and tonic components were blocked by the application of 100 μM gabazine. The size of the tonic inhibitory current was determined by subtracting the holding current under baseline conditions from the holding current in the presence of gabazine. Holding currents were estimated by plotting 20 sec periods of raw data in an all-points histogram . A Gaussian line was fitted to the right part of the smoothed curve , which was not skewed by synaptic events. The mean of the fitted Gaussian line was considered the mean holding currentPatch-clamp recordings were performed as described previously® II or III SYBR Green QPCR Master Mix , cDNA  and primers . Specific transcripts were amplified with Rotor Gene 6000 . Expression of each GABAA receptor subunit was normalized to Gapdh as well as to Tubb3. The relative expression levels (MCAO ipsi vs. contra and MCAO contra vs. sham contra) were calculated using the Pfaffl equationTotal RNA was isolated using the RNeasy Lipid Tissue Mini Kit  or using the phenol/chloroform extraction method. qPCR was performed as described previouslyProtein isolation and Western blotting were performed as described previously+/−1day) were removed and transferred into fresh 1× Hanks’ balanced salt solution on ice . Dissected cortices were rinsed with HBSS and were trypsinized with 0.25% trypsin at 37 °C for 30 min. The supernatant was removed, and the cortices were washed with HBSS and passed through Pasteur pipettes of decreasing diameter to dissociate the tissue. Approximately 250,000 cells were plated onto poly-L-lysine (Sigma-Aldrich)-coated coverslips (ø 30 mm). Cells were allowed to settle for 120 min at 37 °C and 5% CO2 in the incubator. Finally, coverslips were transferred to 6 cm culture dishes containing Neurobasal medium (Invitrogen) supplemented with 1 mM L-glutamine, B27 supplement (Gibco), and penicillin and streptomycin (Gibco). Medium was added once a week, and the cultures were maintained for 3 weeks before the experiments. At DIV17–18, cells were incubated with 20 μM glutamate for 10 min. Twenty-four hours later, cells were harvested in Qiazol (3 coverslips each were pooled as one RNA sample) and processed for RNA isolation and qPCR as described above.Cortical neurons were dissected from mouse embryos as described previously66A receptor agonist gaboxadol pyridin-3-ol THIP, Tocris Bioscience, UK). THIP was administered i.p. at a dose of 5 mg/kg BW 30 min before starting the RotaRod tests and 30 min before epilepsy induction. MK-801, a non-competitive antagonist of NMDA- receptors, was administered i.p at a dose of 1 mg/kg BW at the beginning of reperfusion. Control groups received physiological saline.Tonic inhibition was modulated by the GABAA walking task was performed to assess the locomotor activity of mice 3 and 7 days following MCAO. The walking apparatus consisted of a Plexiglas box equipped with a 12-mm square wire mesh with a grid area of 40 × 40 cm, 10 cm above the ground. A Panasonic Color CCTV Camera (Model No. WV-CP 450)/G) was fixed above the walking grid. Each mouse was placed individually on top of the grid and allowed to walk for 5 min. To assess the total distance, the moving time and the velocity, the Ethovision XT 6.1.326 software  was used. Motor performance was tested by an accelerated RotaRod procedure 3 and 7 days after MCAO. The RotaRod apparatus  consisted of a striated rod providing a good grip (diameter: 3 cm), separated in four compartments (width: 8.5 cm) and located 27.2 cm above the floor grid. One day before MCAO induction, mice were placed on the rod for 30 sec without rotation followed by 120 sec of low speed rotation at 4 rpm. Subsequently, the mice were tested in 3 trials for 5 min (4–40 rpm). After MCAO, the mice were tested under the same conditions at 3 and 7 days of reperfusion. At each trial, the latency to fall was recorded.Post-ischemic seizures were induced in mice 7 days after MCAO using PTZ . PTZ was administered i.p. at 60 mg/kg BW. Immediately after PTZ injection, the mice were placed into a video-monitored Plexiglas cage, and the convulsive behavior was observed for a period of 15 min. Native and sham-operated mice underwent the same procedure. Tonic inhibition was modulated by gaboxadol (THIP), administered i.p. at a dose of 5 mg/kg BW 30 min before PTZ injection. The control groups received physiological saline injections.qPCR data and the parameters of the behavioral studies were analyzed with one- or two-way analysis of variance (ANOVA) with post-hoc Tukey’s test or with Wilcoxon’s test. For statistical analysis of the patch-clamp recordings, the Mann-Whitney test, the Kolmogorov-Smirnov test or Student’s t-test was used. The onset of seizures was analyzed using the Mann-Whitney test. For Western-blot analysis, the paired Student’s t-test was used. All data are expressed as the mean or geomean ± s.e.m. The levels of significance were set at *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.How to cite this article: Jaenisch, N. et al. Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures. Sci. Rep.6, 26173; doi: 10.1038/srep26173 (2016).


TGR5 is a widely expressed and highly conserved G protein coupled receptor. Its activity and functionality is commonly modulated by bile acids, especially by lithocholic acid. As true for all ligand activated G protein coupled receptors a G protein subunit is released from TGR5 after ligand binding and initiates a signaling cascade resulting in a cell type specific response. Current investigations suggest an involvement of TGR5 in bile homeostasis, inflammatory responses and hepatobiliary diseases. Therefore a targeted therapy involving site specific inhibition of TGR5 is of immense interest. However, up to date no solved structure of TGR5 exists and oligomerization properties are largely unknown. To determine structural changes and oligomerization properties of TGR5 we designed a three way strategy including TGR5 plasmids coupled with (I) fluorescent proteins (FPs) at the C-terminus or (II) a N-terminal peptide tag (ACP) for subsequent labeling with a fluorescent dye and (III) unnatural amino acids (UAAs) for site specific extracellular labeling.Oligomerization properties were studied by high-precision Förster Resonance Energy Transfer (FRET) measured by multi-parameter fluorescence imaging spectroscopy (MFIS) using the labeling strategies (I) and (II) in live cells ,2. FRET For strategy (I) cells were co-transfected with TGR5-GFP and TGR5-mCherry and analyzed with MFIS. Successfully transfected Hep2 cells clearly presented TGR5 coupled with fluorescent proteins at the cell membrane. An overlay of TGR5-GFP and TGR5-mCherry figures suggests co-localization of GFP and mCherry. Further analysis of these cells showed a reduced donor fluorescence lifetime of GFP compared to those only transfected with TGR5-GFP. This effect could be increased by acceptor titration of TGR5-mCherry (EGFP-mCherry from ratio 1:1 to 1:20). The shortened donor lifetime indicates that FRET occurs between TGR5-GFP and TGR5-mCherry probably due to TGR5 oligomerization. Consistent results were obtained with labeling method (II). For this purpose Hep2 cells were transfected with ACP-TGR5. The ACP-tag fused N-terminal to TGR5 was located extracellular and labeling was possible under live cell conditions by simply adding ACP synthase and CoA tagged dyes to the normal medium with further incubation for 1 hour at 37°C. The ACP-tag was labeled with CoA-Cy3 or CoA-Atto488 alone or in combination with CoA-Cy5. Live cell imaging suggests co-localization of ACP-tagged Cy3 and Cy5. The analysis of the extracellular labels clearly indicated a reduced donor fluorescence lifetime of Cy3 as compared to Hep2 cells labeled with CoA-Cy3 only or CoA-Atto488. This suggests intermolecular, extracellular FRET between ACP-TGR5 labeled with Cy3 and Cy5. To show, whether intramolecular FRET might occur, we used a combination of labeling strategy (I) and (II) and transfected cells with TGR5 N-terminally tagged with ACP and C-terminally fused to YFP. After 24h cells expressing ACP-TGR5-YFP were subsequently labeled with CoA-Cy3 or CoA-Cy5. However, intra-molecular FRET from the C-terminal YFP to the N-terminally labeled ACP tag could not be observed. As a control for oligomerization ACP-tagged ADRbeta2 was measured in parallel. CoA-Atto488 or CoA-Cy3 and Cy5-labeled ADRbeta2 co-localized at the cell membrane and energy transfer occurred from Cy3 to Cy5 and CoA-Atto488 to CoA-Cy5, respectively. We applied a quantitative analysis method on the MFIS-FRET data taking account of the double exponential decay characteristic of EGFP in live cells. The results showed that more than one FRET population exist in all the measurements, which suggest that TGR5 may form a higher-order complex than dimer. Also, the interacting pattern of TGR5 is distinctive comparing with 14 other receptor protein interactions we have previously measured and analyzed using the same method.With our labeling strategies (I) TGR5-FPs and (II) ACP-tagged TGR5 we showed that FRET proved TGR5 oligomerization. However, the stoichiometry remains to be determined. The structural properties  and structural changes induced by ligand binding will be probed using the third labeling strategy. Based on homology models several amino acid positions in the extracellular loops were selected for site specific labeling. From overlap extension mutagenesis derived TGR5 mutants carrying an Amber stop codon will be used for incorporation of unnatural amino acids and further site specific extracellular labeling with tetrazine-dyes via copper free click chemistry. Preliminary results with a control GFP39TAG mutant showed successful incorporation of UAAs in live cells as verified by green fluorescence of the completely translated protein, which will otherwise not be fluorescent. TGR5, like other GPCRs, may form higher-order complexes which further complicates the analysis. Our goal is to utilize the high-precision FRET screening method to filter out the possible complex conformations of TGR5 [


Ser473, glycogen synthase kinase (GSK)-3βSer9, forkhead transcription factor (FKHR)Ser256, mitogen activated protein kinases (MAPKs), and protein kinase C (PKC) isoenzymes were monitored. The elevated blood pressure in SHRs was not influenced by PARP-inhibitor treatment. Systolic left ventricular function and BNP activity did not differ among the three groups. L-2286 treatment decreased the marked left ventricular (LV) hypertrophy which was developed in SHRs. Interstitial collagen deposition was also decreased by L-2286 treatment. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2Thr183-Tyr185, Akt-1Ser473, GSK-3βSer9, FKHRSer256, and PKC εSer729 and the level of Hsp90 were increased, while the activity of PKC α/βIIThr638/641, ζ/λ410/403 were mitigated by L-2286 administration. We could detect signs of LV hypertrophy without congestive heart failure in SHR groups. This alteration was prevented by PARP inhibition. Our results suggest that PARP-inhibitor treatment has protective effect already in the early stage of hypertensive myocardial remodeling.Spontaneously hypertensive rat (SHR) is a suitable model for studies of the complications of hypertension. It is known that activation of poly(ADP-ribose) polymerase enzyme (PARP) plays an important role in the development of postinfarction as well as long-term hypertension induced heart failure. In this study, we examined whether PARP-inhibitor (L-2286) treatment could prevent the development of hypertensive cardiopathy in SHRs. 6-week-old SHR animals were treated with L-2286 (SHR-L group) or placebo (SHR-C group) for 24 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY group). Echocardiography was performed, brain-derived natriuretic peptide (BNP) activity and blood pressure were determined at the end of the study. We detected the extent of fibrotic areas. The amount of heat-shock proteins (Hsps) and the phosphorylation state of Akt-1 Left ventricular hypertrophy (LVH) represents the heart's response to increased biomechanical stress such as arterial hypertension or valvular heart disease. Cardiac hypertrophy has traditionally been considered a compensatory mechanism required to normalize wall tension and to maintain cardiac output. However, recent clinical studies as well as several animal models have shown that cardiac hypertrophy is rather a maladaptive process, ultimately leading to heart failure (HF) and sudden cardiac death independent of the underlying cause of hypertrophy 2-terminal kinases (JNK) and several protein kinase C (PKC) isoforms Both physiologic and pathologic stimulation-induced cellular adaptations of the heart are typically initiated by stress-responsive signaling pathways, which serve as central transducers of cardiac hypertrophic growth and/or ventricular dilation. These signaling pathways include extracellular signal-regulated protein kinases (ERK), p38 mitogen-activated protein kinases (p38-MAPK), c-Jun NH+ and ATP levels decrease remarkably resulting in cell dysfunction and cell death via the necrotic route. Therefore, PARP-activation contributes to the pathogenesis of various cardiovascular diseases including endothelial dysfunction, ischemia-reperfusion injury and myocardial infarction, as well as HF. Several studies reported that endothelial dysfunction associated with hypertension also depends on PARP activity and can be prevented by its pharmacological inhibition The poly(ADP-ribose) polymerase (PARP) enzyme becomes activated in response to DNA single-strand breaks that can be excessive as a response to free radicals and oxidative cell damage. PARP is an energy-consuming enzyme that transfers ADP-ribose to nuclear proteins. As a result of this process, the intracellular NADIt has been shown previously that our experimental agent, an isoquinoline derivative PARP-inhibitor, L-2286  had a beHypertension in spontaneously hypertensive rat (SHR) is similar to that of human in numerous ways such as the occurence of long-term, stable LVH followed by a transition to HF The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Insitutes of Health , and was approved by the Animal Research Review Committee of the University of Pecs, Medical School.H)-one), a water-soluble PARP-inhibitor , while SHR-C group received only placebo  p. os for 24 weeks Six weeks old male WKY-strain Wistar Kyoto and spontaneously hypertensive rats  were used. Animals were kept under standard conditions throughout the experiment; 12 h light-dark cycle, water and rat chow provided ad libitum. SHRs were randomly divided into two groups; SHR-L and SHR-C. SHR-L group was treated with L-2286 (2-[(2-piperidin-1-ylethyl)thio]quinazolin-4(3Blood samples were collected into Lavender Vacutainer tubes containing EDTA and aprotinin (0.6 IU/ml of blood), and were centrifuged at 1600 g for 15 minutes at 4°C to separate the plasma. Supernatants were collected and kept at −70°C. BNP-45 were determined by enzyme immunoassay method as the manufacturer proposed .Ventricles fixed in formalin were embedded in paraffin, and 5 µm thick sections were cut from base to apex. Sectiones were stained with Masson's trichrome staining to detect the interstitial fibrosis, and quantified by the NIH ImageJ image processing program as described previously 473 (1∶1000), Actin (1∶10000), phospho-specific glycogen synthase kinase (GSK)-3β Ser9 (1∶1000), phospho-specific extracellular signal-regulated kinase (ERK 1/2) Thr202-Tyr204 (1∶1000), phospho-specific p38 mitogen-activated protein kinase (p38-MAPK) Thr180-Gly-Tyr182 (1∶1000), phospho-specific c-Jun N-terminal kinase (JNK) Thr183-Tyr185 (1∶1000), phospho-specific protein kinase C (PKC) (pan) βII Ser660 (1∶1000), phospho-specific protein kinase C α/βII (PKC α/βII) Thr638/641 (1∶1000), phospho-specific protein kinase C δ (PKC δ) Thr505 (1∶1000), phospho-specific protein kinase C ζ/λ (PKC ζ/λ) Thr410/403 (1∶1000), phospho-specific protein kinase C ε (PKC ε) Ser729 (1∶1000), anti-poly(ADP-ribose) , phospho-Foxo1A , Heat shock protein 72 , Heat shock protein 90 . Antibodies were purchased from Cell Signaling Technology  except from actin, which was bought from Sigma-Aldrich Co, , phospho-specific PKC ε, which was purchased from Upstate , anti-PAR, which was purchased from Alexis Biotechnology , Hsp90, which was bought from Santa Cruz Biotechnology , Hsp72, which was purchased from StressGene Biomol GmbH . Membranes were washed six times for 5 min in Tris-buffered saline, pH 7.5 containing 0.2% Tween before addition of goat anti-rabbit horseradish peroxidase-conjugated secondary antibody . The antibody-antigen complexes were visualized by means of enhanced chemiluminescence. After scanning, results were quantified by NIH ImageJ program. Pixel densities of bands were normalized to that of the loading controls.Fifty milligrams of heart samples were homogenized in ice-cold 50 mM Tris buffer, pH 8.0 containing protease inhibitor cocktail 1∶100, and 50 mM sodium vanadate , and were harvested in 2x concentrated sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis sample buffer. Proteins were separated on 10% or 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking , membranes were probed overnight at 4°C with primary antibodies recognizing the following antigenes: phospho-specific Akt-1/protein kinase B-α Serd-LVIDs)/LVIDd) , EF (%) was calculated by 100x((LVEDV-LVESV)/LVEDV), relative wall thickness (RWT) was calculated by /LVIDd.At the start of the experiment, all animals were examined by echocardiography to exclude rats with any heart abnormalities. Transthoracic two-dimensional echocardiography was performed under inhalation anesthesia at the beginning of the experiment and on the day of sacrifice. Rats were lightly anesthetized with a mixture of 1.5% isoflurane  and 98.5% oxygen. The chest of animals was shaved, acoustic coupling gel was applied and warming pad was used to maintain normothermia. Rats were imaged in the left lateral decubitus position. Cardiac dimensions and functions were measured from short- and long-axis views at the mid-papillary level by a VEVO 770 high-resolution ultrasound imaging system  equipped with a 25 MHz transducer. LV fractional shortening (FS), ejection fraction (EF), LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and the thickness of septum and posterior wall were determined. FS (%) was calculated by 100x. There were no significant differences among the groups. Comparisons among groups were made using one-way ANOVA (SPSS for Windows 11.0). For post hoc comparison Bonferroni test was chosen. Values of p<0.05 were considered statistically significant.Effect of L-2286 on normotensive WKY rats was also examined, but the investigated parameters did not differ significantly from the non-treated WKY animals. Therefore, data of L-2286 treated WKY rats were not shown to avoid unnecessary redundancies.Body weights did not differ significantly among the three groups  at the beginning of our study. However, at the end of the 24-week-long treatment period, body weights of WKY group were significantly higher than those of SHR-C and SHR-L groups . The degree of myocardial hypertrophy was determined by ventricular weight to body weight ratio . This parameter was significantly increased in SHR groups compared to the WKY group . Similar results were obtained in case of weights of ventricles . The WV and WV/BW ratios were significantly decreased by L-2286 treatment (p<0.05 SHR-L vs. SHR-C). The lung wet weight-to-dry weight ratio was not elevated significantly in SHR-C and SHR-L compared to WKY groups . All theSlightly elevated plasma BNP levels were found both in SHR-C and SHR-L groups (not significant vs. WKY group). Although plasma BNP level was a little higher in SHR-C group than in SHR-L group, this difference was also not statistically significant . In bothHistological analysis revealed slight interstitial collagen deposition in the WKY group. Chronic high blood pressure caused significantly higher collagen deposition in SHR-C rats that was significantly diminished (p<0.05) in the SHR-L group .At the beginning of the study the echocardiographic parameters of the three groups did not differ significantly from each other . At the Ser473 was moderately phosphorylated in WKY group. In SHR-C group, the phosphorylation of Akt-1Ser473 was more pronounced (p<0.01 vs. WKY). Moreover, in SHR-L rats the L-2286 treatment caused further elevation in Akt-1Ser473 phosphorylation (p<0.01 vs. WKY and SHR-C groups) (Ser9 phosphorylation (Akt-1 groups) . The samrylation .Ser473 (besides GSK3βSer9) is FKHRSer256. Consistently with the result of Akt-1Ser473 phosphorylation, the strongest phosphorylation (therefore inhibition) could be observed in SHR-L group (p<0.01 vs. SHR-C and WKY). The lowest phosphorylation and therefore the highest activity of FKHR was seen in SHR-C group , and the lowest amount of this protein was present in WKY samples .Thr180-Gly-Tyr182, ERK 1/2Thr183-Tyr185 and JNK was the lowest in the WKY group compared to SHR-C and SHR-L groups . In the case of p38-MAPKThr180-Gly-Tyr182, ERK 1/2Thr183-Tyr185 and JNK, their phosphorylation was elevated in both SHR-C and SHR-L groups, but there were no significant differences between the two SHR groups (Phosphorylation of p38-MAPKSer660) was low in the WKY group and became significantly higher in SHR-C and SHR-L groups (p<0.01 WKY vs. SHR groups). Administration of L-2286 could not affect the phosphorylation state of PKC pan βII Ser660 in SHR-L group compared to the SHR-C group  phosphorylation of PKC . As PKC ζ antibody, we used a combined antibody (i.e. PKC ζ/λ Thr410/403), which did not discriminate between PKC ζ and λ; PKC λ being structurally highly homologous to PKC ζ in the COOH-terminal end of the molecule Thr638/641 and ζ, while it could increase the phosphorylation of εSer729  (Thr505 there was no significant difference between the SHR groups (The lowest phosphorylation could be observed in the WKY group in case of PKC α/βII. SHR-C) ,7. In thR groups .The major findings of this study are that chronic inhibition of nuclear PARP enzyme reduces excessive ADP-ribosylation of nuclear proteins, beneficially influences the intracellular signaling pathways and thus prevents the development of cardiac hypertrophy, which is an early consequence of hypertension. We used the SHR model that is a relevant animal model of essential hypertension in humans Previously, we have proved that PARP-inhibition could inhibit the transition of hypertensive cardiopathy to end-stage heart failure Hypertension is a major risk factor for cardiovascular mortality and morbidity, and it is associated with left ventricular hypertrophy and diastolic dysfunction and later with systolic dysfunction and it can lead to heart failure. There is a strong correlation between left ventricular mass and the development of cardiovascular pathogies Significant LV hypertrophy develops by the age of 3 months in SHR animals but it is more often studied closer to 6 months of age Chronic hypertension leads to excessive collagen deposition (fibrosis) as part of the process of cardiovascular remodeling. In our previous studies, when SHR or postinfarcted animals exhibited overt heart failure, L-2286 also prevented interstitial fibrosis and adverse structural remodeling In our experiment the systolic LV function was not decreased in SHR rats during the 24-week-long treatment. It is in accordance with several other studies Ser473 was far the lowest in WKY group and the highest in SHR-L group. Phosphorylation and therefore the inhibition of GSK-3βSer9 and Foxo1 (FKHR) (downstream targets of Akt-1) Previous works demonstrated that PARP inhibitors can induce the phosphorylation and activation of Akt-1 in reperfused myocardium, thus raising the possibility that the protective effect of PARP inhibition can be mediated through the activation of the prosurvival phosphatidylinositol-3-kinase (PI3-kinase)/Akt-1 pathway Ser473, this can contribute to the cell survival in L-2286 treated rats. The level of Hsp72 was not influenced significantly by L-2286 administration in our investigation.Cellular stress leads to the expression of Hsp's Thr180-Gly-Tyr182, JNK and ERK 1/2Thr183-Tyr185 was elevated both in SHR-C and SHR-L groups. Our results are consistent with the results of Kacimi et Gerdes Previous works demonstrated that PARP inhibitors have a moderate effect on MAPKs in acute phase of myocardial infarction and in postinfarction heart failure The elevated blood pressure may induced ERK activation PARP inhibitors were found to affect PKC isoenzymes Ser660 and δThr505 were not altered by L-2286 treatment, while activation of α/βIIThr638/641 and ζ/λ Thr410/403 were attenuated and activation of εSer729 was augmented by L-2286. These alterations can mediate – at least partly – the favourable cardiovascular effects of L-2286, similarly as it was found in previous works Activation of PKC pan βIISer473 and PKCεSer729 were elevated by L-2286 administration.PKC α is the most extensively expressed among the myocardial PKC isoforms, and it is a key regulator of cardiomyocyte hypertrophic growth In our study, we examined the effect of a PARP inhibitor (L-2286) in SHR at the stage of LV hypertrophy. L-2286 exerted a beneficial effect on the progression of myocardial hypertrophy  and myocardial fibrosis. In the background of these changes, we did not observe any blood pressure lowering effect of PARP-inhibition. According to our results, PARP-inhibition can exert this antihypertrophic effect due to the activation of several prosurvival  and the inhibition of prohypertrophic  protein kinases .


Rearing coral fragments in nurseries and subsequent transplantation onto a degraded reef is a common approach for coral reef restoration. However, if barnacles and other biofouling organisms are not removed prior to transplantation, fish will dislodge newly cemented corals when feeding on biofouling organisms. This behavior can lead to an increase in diver time due to the need to reattach the corals. Thus, cleaning nurseries to remove biofouling organisms such as algae and invertebrates is necessary prior to transplantation, and this cleaning constitutes a significant time investment in a restoration project. We tested a novel biomimicry technique of animal-assisted cleaning on nursery corals prior to transplantation at a coral reef restoration site in Seychelles, Indian Ocean. To determine whether animal-assisted cleaning was possible, preliminary visual underwater surveys were performed to quantify the fish community at the study site. Then, cleaning stations consisting of nursery ropes carrying corals and biofouling organisms, set at 0.3 m, 2 m, 4 m, 6 m and 8 m from the seabed, were placed at both the transplantation (treatment) site and the nursery (control) site. Remote GoPro video cameras recorded fish feeding at the nursery ropes without human disturbance. A reef fish assemblage of 32 species from 4 trophic levels  consumed 95% of the barnacles on the coral nursery ropes placed 0.3 m above the seabed. Using this cleaning station, we reduced coral dislodgement from 16% to zero. This cleaning station technique could be included as a step prior to coral transplantation worldwide on the basis of location-specific fish assemblages and during the early nursery phase of sexually produced juvenile corals. Active coral reef restoration is increasingly being seen as a new tool for conservation biology  as coralThe cleaning of algae and sessile invertebrates  in nurseries is essential to avoid the space competition with corals, which leads to shading and coral death. Nursery cleaning consumes a significant portion of the time invested in restoration projects . Prior tin situ) nurseries, polyps of Acropora tenuis have been co-cultured with hatchery-supplied juveniles of the top shell Trochus niloticus (ex situ) nurseries, polyps of Pocillopora damicornis have been co-cultured with juveniles of the sea urchin Salmacis spaheroides and the gastropod Trochus maculatus collected from the wild  diver hours (range 12–36 diver hours) per month. Biofouling cleaning was repeated every 2–4 months. When we used a different type of midwater coral nursery (6 × 6 m PVC pipe frames layered with 5.5 cm-mesh tuna nets), reef fishes recruited at the nurseries consumed the biofouling organisms and reduced the required cleaning time , we deployed 9 mid-water rope nurseries, following methodology modified from ing time . Net nurLethrinus mahsena), Tripletail Wrasse (Cheilinus trilobatus), Titan Triggerfish  and flagtail triggerfish , we searched for a biomimicry solution, i.e., a solution inspired by nature, to develop an innovative and sustainable technique . Our insThis animal-assisted cleaning technique was developed as a result of two experiments conducted between 3 and 18 December 2013 at a coral reef restoration project within the marine protected area of Cousin Island Special Reserve, Seychelles, Indian Ocean , 600 × 64 mm in size, placed approximately 4 m apart, to which 20 m-long ropes were perpendicularly attached. Each rope held 80–150 corals, totaling approximately 5,000 corals in each rope nursery. The nurseries were attached to the 17 m-deep sandy seabed by anchor lines and maintained at a depth of 8 m below the sea surface by using recycled plastic jerrycans as buoys. The reef transplantation site, located on the south-west side of the island, consisted of a degraded coral reef affected by the mass coral bleaching event of the 1998, due to the coupling of the El Niño and the Indian Ocean Dipole  as well A field permit was not required to conduct the experiments described herein at the marine reserve within the Cousin Island Special Reserve. The Special Reserve is managed by Nature Seychelles. As Nature Seychelles employees, we were able to perform underwater observations without the issuing of a specific permit at the no-take marine reserve, as long as we complied with the demands for no damage, harassment or taking of fish.To test the animal-assisted cleaning of the nursery corals, we first assessed fish diversity at the transplantation site to determine whether the fish community could provide animal-assisted cleaning; then, we performed field experiments to quantify animal-assisted cleaning. To assess fish diversity at the transplantation site, we conducted visual underwater surveys via the standard point count method . BrieflyAcropora bruegemanni, A. formosa, A. abrotanoides, A. nobilis and A. robusta. The corals grew at the nursery for 17 months, and their size was 15.6 ± 4.51 cm (mean ± SD) at their maximum dimension . Therefore, the experimental units allowed the concept of a cleaning station to be tested by depth and site.To investigate animal-assisted cleaning, we developed an experimental unit resembling a ladder . Angle bhttp://www.fishbase.org) and the Encyclopedia of Life (http://www.eol.org). In FishBase, the mean trophic position is calculated based on all the food items consumed by a species, weighted by their relative abundance. The trophic level is obtained by adding 1 to the mean trophic position  were placed next to the experimental units to remotely document the fish interacting with the nursery ropes without human disturbance. To analyze the fish assemblages at the transplantation site using underwater visual surveys and video recordings performed at the experimental units (both the transplantation and nursery sites), we generated an inventory of fish species and compared the video-recorded feeding behavior of each species to known trophic levels and food items reported in FishBase . The experimental units were then left Due to the variable number of barnacles per rope, the count data were converted into the percentage of barnacles eaten per rope. To meet the assumptions of parametric statistical analysis, the percentages were ArcSin transformed . We testThe fish community at the transplantation site was diverse (51 species) and was dominated by wrasses  was the only species that was video recorded interacting with the experimental setup at the nursery site. This species feeds on benthic invertebrates and has a trophic level of 3.0 ± 0.0 (http://www.fishbase.org). Four additional species were video recorded at the nursery site but did not interact with the experimental setup: emperors , goatfishes , razorfishes (Xyrichtys sp. Labridae) and porcupinefishes . However, at the transplantation site, 32 fish species were observed feeding at the experimental setup , a coral predator, was not video-recorded at the nursery or transplantation sites; however, it was a regular visitor feeding on biofouling fragments falling to the seabed when divers cleaned the nurseries. After the 48 h-long experiment, predation of corals (indicated by scarring or bite marks) was absent at both the transplantation and nursery sites.The only species that was observed breaking and feeding on the barnacles attached to the ropes was the Titan Triggerfish  and between the number of dead and live corals per rung . Therefore, each independent replicate provided the same feeding substrate under the experimental field conditions.Each 1.8 m-long rung in the experimental units harbored a similar number of corals: 13.4 ± 1.68 (mean ± SD) corals per rung at the transplantation site  and 13.5 ± 1.84 corals per rung at the nursery site . Despite these similar numbers of corals per rung, the average number of live and dead corals per rung varied. For instance, there were 7.9 ± 3.93 (mean ± SD) live corals per rung  and 5.4 ± 4.22 dead corals per rung  at the transplantation site, whereas there were 7.2 ± 3.43 live corals per rung  and 6.2 ± 4.16 dead corals per rung  at the nursery site. However, the differences were not significant (two-way ANOVA model I) between the transplantation and nursery sites . The depth of placement was critical. The highest barnacle predation was observed at the transplantation site on the 0.3 m ropes (94.8% ± 2.7 S.E.) and the 2 m ropes . Post hoc comparisons of the interaction term using Tukey’s HSD test revealed that barnacle predation at the transplantation site was similar on the 0.3 and 2 m ropes (p = 0.99), but it was 5–94 times higher compared with all other combinations of depths and sites  site was 3.25 times higher overall than at the nursery (control) site . We used the cleaning station technique for 5 months (January–May 2014), and the rate of coral detachment due to fish attacks fell from an initial 16% to zero.Based on the results obtained from both experiments, we set up a dedicated cleaning station at the edge of the restoration site, away from the transplanted corals, marked by rebars hammered onto the hard substrate at 5 m intervals. We eliminated diver-assisted cleaning prior to coral transplantation; instead, we attached a nursery rope at the cleaning station, which was set at 0.3 m above the seabed, resembling the bottom rope shown in Balistoides viridescens) was the key species because it is capable of crushing the calcareous exoskeletons of the barnacles. The other fish species observed either fed on half-consumed barnacles left behind by B. viridescens or fed directly on the biofouling organisms found on the ropes and at the coral/rope interface. In contrast, the absence of a reef-associated fish community at the nursery site (only 1 species was observed feeding) explains the lack of animal-assisted cleaning of the experimental setup at this location.Fish fed on barnacles and other biofouling organisms from the coral nursery ropes located at the transplantation site within 48 h of deployment of the experimental setup. The diverse fish community at the transplantation site, where 32 species from 4 trophic levels were recorded feeding at the experimental setup, ensured effective animal-assisted cleaning. The Titan Triggerfish  on the coral nursery ropes prior to transplantation. The recorded barnacle predation rates were 95% and 83% on the ropes placed at 0.3 and 2 m from the seabed, respectively, 48 h after placement. The fish community at the transplantation site that provided the animal-assisted cleaning service included 32 species from 4 trophic levels: herbivores consumed filamentous, coralline and benthic algae; omnivores and secondary consumers consumed sessile and vagile benthic invertebrates; and carnivores consumed vagile benthic invertebrates. The animal-assisted cleaning technique derived from the experiments described herein consisted of a 48 h deployment of a coral nursery rope at the transplantation site within 0.3 m of the seabed (the “cleaning station”). Using the cleaning station, we reduced the dislodgement of newly cemented corals by fish from 16% to zero.Lachnolaimus maximus), a large wrasse (Labridae), and the Batfish  has revealed the presence of crushed barnacles  and carefully designed experiments can provide innovative and sustainable solutions. We recommend such an approach to advance the emerging field of coral reef restoration.10.7717/peerj.1287/supp-1Video S1This video provides an overview of the experimental setup at the transplantation (treatment) site and fish species feeding on sessile and mobile biofouling attached to the coral nursery ropes.Click here for additional data file.10.7717/peerj.1287/supp-2Supplemental Information 1This Excel file contains the raw data obtained during the baseline fish surveys conducted at the transplantation site, November 2013.Click here for additional data file.10.7717/peerj.1287/supp-3Supplemental Information 2This Excel file contains the raw data of species found feeding at the experimental setup at the transplantation (treatment) and nursery (control) sites, based on video recordings with GoPro cameras.Click here for additional data file.10.7717/peerj.1287/supp-4Supplemental Information 3This Excel file contains raw data of the barnacle predation quantified at the experimental setup at the transplantation (treatment) and nursery (control) sites. Data are shown by site and depth.Click here for additional data file.


N-methyl-d-aspartate type of glutamate receptors (NMDARs). Failures in neurotrophic support and glutamate signaling are involved in pathologies related to excitotoxicity and/or neurodegeneration, where different components of these dynamic protein complexes result altered by a combination of mechanisms. In the case of Kidins220/ARMS, overactivation of NMDARs in excitotoxicity and cerebral ischemia triggers its downregulation, which contributes to neuronal death. This key role in neuronal life/death decisions encouraged us to investigate Kidins220/ARMS as a novel therapeutic target for neuroprotection. As the main mechanism of Kidins220/ARMS downregulation in excitotoxicity is proteolysis by calpain, we decided to develop cell-penetrating peptides (CPPs) that could result in neuroprotection by interference of this processing. To this aim, we first analyzed in detail Kidins220/ARMS cleavage produced in vitro and in vivo, identifying a major calpain processing site in its C-terminal region (between amino acids 1669 and 1670) within a sequence motif highly conserved in vertebrates. Then, we designed a 25-amino acids CPP (Tat-K) containing a short Kidins220/ARMS sequence enclosing the identified calpain site (amino acids 1668–1681) fused to the HIV-1 Tat protein basic domain, able to confer membrane permeability to attached cargoes. Transduction of cortical neurons with Tat-K reduced Kidins220/ARMS calpain processing in a dose- and time-dependent manner upon excitotoxic damage and allowed preservation of the activity of pERK1/2 and pCREB, signaling molecules central to neuronal survival and functioning. Importantly, these effects were associated to a significant increase in neuronal viability. This Kidins220/ARMS-derived peptide merits further research to develop novel neuroprotective therapies for excitotoxicity-associated pathologies.Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), has a central role in the coordination of receptor crosstalk and the integration of signaling pathways essential for neuronal differentiation, survival and function. This protein is a shared downstream effector for neurotrophin- and ephrin-receptors signaling that also interacts with the  N-methyl-d-aspartate type of glutamate receptors (NMDARs) induces neuronal survival through extracellular signal-regulated kinases (ERKs) activation,1 antioxidant defenses induction,2 and cAMP response element-binding protein (CREB) phosphorylation.3 In turn, CREB increases expression of BDNF5 and its receptor tropomyosin-related kinase B (TrkB).7 Neurotrophin receptors enhance neuronal survival via signaling cascades involving PI3K-Akt, ERK,9 CREB10 and nuclear factor kappa-B (NF-κB).11 Signaling requires large complexes formed at postsynaptic membranes by receptors and effectors. For instance, NMDAR association with ephrin receptor (Eph)B is critical for synaptic function,13 while EphB activation by ephrin-B modulates NMDAR-dependent calcium influx and receptor expression.14 Likewise, TrkB interacts with ephrin-A7 and EphA.16 In addition, Fyn tyrosine-kinase associates to TrkB and NMDAR-GluN2B,18 while neural Shc (N-Shc), a neurotrophin signaling adaptor, also regulates NMDAR function.19 Coordination of these receptors crosstalk requires their shared effector Kinase D-interacting substrate of 220 kDa (Kidins220),20 also known as ankyrin repeat-rich membrane spanning (ARMS).21 Kidins220 bears twelve ankyrin repeats, four trans-membrane (TM) segments forming a KAP-NTPase domain,22 a proline-rich region, a sterile alpha domain (SAM) and a PDZ ligand (PDZ-L) at the C-terminus.21 It interacts with Trk receptors21 and is obligatory for neurotrophin-sustained ERK activation and neuronal differentiation.25 In addition, Kidins220 associates with NMDARs and modulates their ERK signaling and neuronal survival.26Neurotransmitters and neurotrophins regulate nervous system development, and preservation and remodeling of adult neural circuits. Prominent roles are played by glutamate, major excitatory neurotransmitter, and brain-derived neurotrophic factor (BDNF). By activating their receptors and signaling pathways, these ligands regulate multiple neuronal processes, including survival. Physiological stimulation of 27 Defective neurotrophic support and signaling are also involved in neurodegeneration.29 Altogether, these conditions cause high mortality and/or neurological impairment representing a social and health challenge. As there are no satisfactory treatments, it is capital to characterize excitotoxic mechanisms and how they affect proteins fundamental to survival/death choices30 in order to develop novel therapies. We discovered decreases in NMDAR32 and TrkB33 signaling during excitotoxicity due to activation of the Ca2+-dependent protease calpain and transcriptional inhibition. Similar mechanisms control Kidins220 downregulation that contributes to neuronal death after NMDAR overactivation.26 Indeed, Kidins220 is crucial for neuronal survival as its knockdown decreases ERK activation and neuronal viability and enhances excitotoxic death.26 This key role in neuronal life/death decisions points to Kidins220 as a novel therapeutic target for neuroprotection. As calpain processing is the major mechanism of Kidins220 downregulation in excitotoxicity, we approach here the design of a neuroprotective peptide able to prevent Kidins220 calpain-dependent loss. First, we analyzed Kidins220 processing and identified a major calpain cleavage site within its C-terminus. We then designed a cell-penetrating peptide (CPP) containing the identified calpain site fused to a HIV-1 Tat protein basic domain, which confers membrane permeability and capability of crossing the blood–brain barrier (BBB).35 Transduction of this peptide reduced Kidins220 calpain processing upon excitotoxicity and significantly increased neuronal viability.Excitotoxicity is a specific neuronal-death process due to NMDAR overstimulation that contributes to neurodegeneration in acute disorders  or chronic neurodegenerative diseases .in vitro (DIVs) with high concentrations of NMDAR co-agonists, NMDA and glycine, and analyzed Kidins220 by immunoblot using antibodies recognizing a C-terminal peptide (Kid-Ct)20 or the N-terminal region (Kid-Nt)36 (26 Kid-Ct showed a rapid decrease of full-length (FL) Kidins220 but no fragments, suggesting that those were very small and/or unstable. However, Kid-Nt exposed a slower FL-Kidins220 decay, suggesting a prominent calpain target close to the C-terminus 36 . As desc site 0, . Early c site 0, . Thereaf site 0, . Absence site 0, . Require site 0, , which sin vivo excitotoxicity. We used a mice model of focal cerebral ischemia produced by distal occlusion of middle cerebral artery (dMCAO) where NMDAR overactivation is the major mechanism of neurodegeneration.27 MCA compression for 1 h followed by 24 h reperfusion induced large infarcts in cortical areas of this artery territory  observing an important decrease in FL-Kidins220  entered neurons in a dose-dependent manner, the best results obtained for 10–25 μM (μM) before NMDA addition (μM) and NMDA treated for different times . The neuroprotective effect of Tat-K after NMDAR overstimulation was also analyzed by MTT assays  in Tat-K-treated neurons. These peptides had no toxicity as no significant differences in viability were found in transduced versus non-transduced neurons , respectively . In neurT assays . The via neurons . Our res26 Therefore, calpain-induced Kidins220 downregulation might significantly contribute to excitotoxic neuronal death affecting this pathway. We analyzed ERK1/2 activation in cultures pre-incubated with Tat-K or Tat-S and treated with NMDA for 2 h, time of maximum kinase activation,26 or 4 h   . NMDAR ove pCREB . Importaive pAkt .In conclusion, our results unveil a novel strategy for neuroprotection based on the use of Tat-K, and strongly support that this peptide, by hindering Kidins220 calpain processing induced by excitotoxicity, is able to decrease neuronal death and specifically enhance ERK1/2 and CREB activity, preserving signaling pathways central to neuronal survival.in vitro digestion of neuronal lysates with calpain I or II. The lower stability of this fragment in the cellular model of excitotoxicity might be simply related to a higher activity, compared with cells in vivo, of additional proteases further degrading the 15-kDa fragment. Anyway, appearance of this fragment in ischemic brain supported the relevance of pursuing the identification of that specific calpain-cleavage site within Kidins220 to develop a neuroprotective strategy.Herein we have discovered a major Kidins220 calpain cleavage site highly conserved in vertebrates. On the basis of this sequence, we have developed a neuroprotective peptide that decreases Kidins220 calpain processing and excitotoxic death in cortical neurons, establishing this sequence motif as a novel target for neuroprotection in excitotoxicity-associated pathologies. Analysis of Kidins220 cleavage in excitotoxic neurons using a N-terminal antibody (Kid-Nt) allowed prediction of at least nine calpain targets downstream ankyrin repeats. Progressive accumulation of shorter intermediates along NMDA treatment suggested a similar processing efficiency for all of them. However, an antibody recognizing Kidins220 last 17 amino acids (Kid-Ct) could not detect fragments in excitotoxic neurons suggesting a nearby and prominent calpain-cleavage site, and the production of a low-stability fragment. Supporting this hypothesis, brain tissue from ischemic mice rendered a 15-kDa C-terminal Kidins220 fragment, which could be also observed by α-syntrophin, a PDZ protein enhancing EphA4 signaling in a Kidins220-dependent manner40 and known to associate with NMDAR-GluN2A-C subunits.41 In addition, Kidins220 PDZ-L interacts with the PDZ protein S-SCAM forming a tetrameric complex with PDZ-GEF1 and Trk,25 a major component for neurotrophin-induced sustained ERK stimulation leading to differentiation. Therefore, Kidins220 PDZ-L cleavage will rapidly uncouple this effector from these and other yet unknown downstream signaling pathways and hamper functions mediated by this motif. Some minor Kidins220 calpain sites might be located at KAP-NTPase domain bearing the four TM segments.20 Other NTPases in this KAP family have been suggested to participate in assembling/disassembling of signaling complexes mostly associated to the cytosolic side of cell membranes in a NTPase-dependent way.22 Independently of Kidins220 participation in similar functions, processing of KAP-NTPase domain would primarily affect Kidins220 putative intrinsic enzymatic activity. In addition, as TM4 mediates Kidins220/Trk interaction,23 calpain cleavage at this domain might impact its role as neurotrophin effector. The remaining processing sites could occur in Kidins220 interdomain sequences, a frequent trend among calpain substrates.42The pre-eminence of this calpain site at the very C-terminal region indicates that Kidins220 PDZ-L will be rapidly lost in excitotoxicity having important functional consequences. This PDZ-L is responsible for Kidins220 localization at the neuromuscular junction through association with in vitro excitotoxicity produced a highly unstable Kidins220 C-terminal fragment, we obtained a more stable GFP-fused fragment by in vitro calpain processing of Kidins220-GFP-Ct. By purifying this fragment, we identified the precise calpain cleavage site between amino acids 1669 and 1670 within rat Kidins220 sequence. Mechanisms of calpain substrate recognition and cleavage are largely unknown38 and there is no consensus recognition sequence. Different bioinformatic tools allow in silico predictions based on empirically derived rules for position-based residues preference , Support Vector Machine (SVM) algorithms for machine learning ,43 or a Group-Based Prediction System (GPS) algorithm.44 We analyzed Kidins220 C-terminal 1000 amino acids using these tools  and found a coincident hit between amino acids 1669–1670, the site identified by Edman sequencing. The resulting 93-amino-acids polypeptide bears a destabilizing N-terminal arginine that might be a target for the Arg/N-end rule pathway and explain its short life.45 Interestingly, seven amino acids contained within this processing site are completely conserved in six different vertebrate species, including humans, supporting the importance of this sequence for Kidins220 downregulation in excitotoxicity.Because 47 and, therefore, pharmacological inhibitors have been considered as potential neuroprotective drugs.49 However, as this protease is likewise required for normal neuronal function, calpain inhibition promotes neuronal survival after injury but also causes deficits in spine density and dendritic branching complexity associated with impaired LTP and spatial memory.50 The use of peptides to interfere calpain pathological activities on specific substrates is emerging as an alternative to generic inhibitors. It has already succeeded in blocking NMDA-induced truncation of mGluR1α51 or transient-receptor potential canonical 6 (TRPC6) channel,52 proteins having neuroprotective roles. We have designed a peptide that inhibits Kidins220 excitotoxic calpain downregulation and tested if stabilization of this protein, central to neuronal physiology , resulted in neuroprotection. This peptide, Tat-K, bearing the identified calpain-recognition sequence (amino acids 1668–1681), efficiently inhibits early NMDA-induced Kidins220 processing and exhibits neuroprotection against in vitro excitotoxicity. The decrease of Tat-K efficacy after 4 h of NMDAR overactivation is probably consequence of intrinsic peptide instability and/or inactivation by calpain processing once inside neurons. Anyway, these neuroprotective effects could be still sufficient in vivo to rescue neurons of the ischemic penumbra from secondary death induced by dying cells in the infarct core.Calpain activation regulates neuronal death in brain damage and neurodegeneration26 which is probably induced by the synaptic pool of receptors, followed by a gradual shut-off possibly derived from NMDAR downregulation.32 Kidins220 silencing hampers sustained neurotrophin-induced ERK1/2 activation,24 decreases basal and NMDA-dependent ERK1/2 activity and neuronal viability, and enhances excitotoxic death.26 Thus, inhibition of Kidins220 processing by Tat-K might help to preserve ERK1/2 signaling, which mediates synaptic NMDAR-dependent neuronal plasticity and survival.1 In addition, CREB activity preservation in excitotoxicity by Tat-K would be highly relevant to neuronal viability and function because this transcription factor is involved in activity-dependent3 or neurotrophin-mediated10 neuronal plasticity and survival. A secondary mechanism induced by Tat-K might also have a minor contribution to maintenance of ERK1/2 and CREB activities. Unlike generic calpain inhibitors, Tat-K did not have a general effect on excitotoxicity-induced processing of alternative calpain substrates such as spectrin. However, a small Tat-K effect was observed on stability of other Kidins220-associated proteins undergoing calpain cleavage during excitotoxicity such as NMDAR-GluN2-subunits26 or PSD95. This fact supports the existance of an indirect mechanism, probably derived from Kidins220 preservation, responsible of partial stabilization of NMDAR complexes. As these complexes are central to neuronal survival and function, this secondary mechanism of Tat-K action could be also important for neuroprotection.Tat-K-induced neuroprotection correlated with specific preservation of ERK1/2 and CREB activities, both central to neuronal survival. Excitotoxic regulation of ERK activity highly depends on experimental conditions. In our model, where both synaptic and extrasynaptic NMDARs are overstimulated and downregulated, we demonstrated an early activation of ERK peaking at 2 h,54 mostly due to their low toxicity and capability to cross cell membranes and BBB.35 Results of a phase-2 trial in patients undergoing endovascular aneurysm repair showed a decrease in the number of ischemic lesions typically induced by surgery after administration of Tat-NR2B9c.55 This CPP contains NMDAR–GluN2B–PDZ-L and dissociates a ternary complex formed with PSD95 and nNOS, uncoupling NMDARs from neurotoxic signaling56 and reducing neuronal damage in ischemia models without affecting synaptic activity.57 Additional therapeutic strategies aimed to prevent downregulation of proteins that, like Kidins220, are important to neuronal survival have a great potential for neuroprotection in excitotoxicity-associated pathologies.CPP-based strategies similar to the one described here have shown a great potential for the treatment of acute and chronic disorders of the nervous system,ß-d-arabinofuranoside (AraC), poly-l-lysine, l-laminin and propidium iodide were acquired from Sigma Co. . Calpain I , calpain II , carbobenzoxy–valinyl–phenylalaninal (CiIII) and lactacystin (Lact) were from Calbiochem–Merck Bioscience , and antagonist 2-amino-phosphopentanoic acid (DL-AP5) from Tocris . Lipofectamine 2000, Glutamax and Calcein-AM were purchased from Invitrogen-Live Technologies . ECL Western Lighting Chemiluminisence Reagent Plus was from Perkin-Elmer Life Sciences  and BCA reagent was from Pierce Thermo Scientific . Tat-S, FITC-Tat-S and Tat-K peptides were obtained from Immunostep .NMDA, glycine, cytosine 36 (generously provided by Dr. G. Schiavo) or C-terminal (Kid-Ct)20 regions of Kidins220 were previously described. Rabbit polyclonal antibodies against active phospho-p44/p42 MAPK (Thr202/Tyr204), herein pERK1/2, and phospho-Akt kinase (Ser473) were from Cell Signaling Technology  while those for active phospho-CREB (Ser133) and NSE were from Millipore Corporation  and ICN Biomedicals , respectively. Mouse monoclonal antibodies against ß-actin, GluN2B and GluN1 were purchased from Sigma Co., BD Biosciences  and Pharmigen , respectively. Mouse monoclonal antibodies recognizing non-erythroid spectrin and PSD95 were from Chemicon . Mouse monoclonal and rabbit polyclonal antibodies against GFP were obtained from Invitrogen-Live Technologies. Goat and rabbit polyclonal antibodies recognizing, respectively, GluN2A C-terminus and p44/p42 MAPK  were purchased from Santa Cruz Biotechnology . Horseradish peroxidase-conjugated antibodies were from General Electric .Rabbit polyclonal antibodies recognizing N-terminal (Kid-Nt)37 Mice were anesthesized with isoflurane . Body temperature was maintained at 36.5–37 °C using a self-regulating heating blanket and a rectal probe. An incision was made between the left eye and ear under an operating microscope  and the temporal muscle divided, exposing the skull lateral aspect. The MCA was identified through the semi-translucent skull, and a small burr hole (2–3-mm diameter) was made with a microdrill at the level of the inferior cerebral vein to expose the M1 portion, leaving the dura intact. Drying injury of the tissue was prevented by continuous hydration via saline application to the area. Regional cerebral blood flow (CBF) was recorded from 5 min before MCAO to 5 min after reperfusion by laser Doppler flowmetry using a flexible fibreoptic  placed on the surface of the M1 parietal branch bifurcation. The MCA was directly compressed using a micromanipulator holding a 30G blunted needle (0.4-mm diameter). After 60 min of occlusion, the needle was carefully removed and the blood flow restored. Arterial occlusion was considered successful when CBF was maintained for 60 min below 20% of baseline value, and reperfusion if CBF recovered to at least a 75% of baseline values. Twenty-four hours after blood reperfusion, animals were killed and their brains sectioned into 1-mm-thick coronal slices and briefly stained with a 2% solution of triphenyltetrazolium chloride  in the cold to avoid endogenous post-mortem calpain activation. The unstained area of the cerebral cortex, defined as infarcted tissue, as well as the corresponding region in the contralateral hemisphere were dissected to prepare protein lysates, frozen and stored at −80 °C until use.All animal procedures were performed in compliance with Spanish legislation and European Union directives and approved by the local Animal Care Committee . Adult male Balb/c mice  were given free access to food and water before surgery, performed as previously described.31 Rats were obtained from the animal care facility at the Instituto de Investigaciones Biomedicas “Alberto Sols” . Animal procedures were approved by the CSIC ethical committee and performed in compliance with European Directive 2010/63/EU. Neurons were used after 14 days in vitro (DIVs) and pre-treated or treated for different times, as indicated, with the following concentrations of reactives: 100 μM NMDA, 10 μM glycine, 20 μM CiIII, 15 μM lactacystin and 5, 10 or 25 μM Tat-S or Tat-K. Excitotoxicity was induced by overstimulation of cortical neurons with NMDAR co-agonists NMDA and glycine (herein denoted NMDA). Unless otherwise stated, inhibitors and Tat-peptides were added 1 h before NMDA treatment and remained in the culture media for the duration of the experiment. When indicated, FITC-conjugated Tat-S was used, and its entrance into cultured neurons was followed by in vivo fluorescent microscopy using an Eclipse TE2000-U Nikon microscope and a Hamamatsu digital camera C10600. Pictures were processed with NIS-Elements BR 3.00, SP6  and Adobe CS3 Extended software .Neuronal cultures were prepared from cerebral cortex of 19-day-old Wistar rat embryos as we have previously described.32 and contains a hemagglutinin (HA) epitope between residues 51 and 52 of this protein. To obtain plasmid Syn-HA-GluN1, a BamHI fragment isolated from pME18S-HA-GluN1  was subcloned into the Syn-GFP lentiviral plasmid32 digested as before. This plasmid produces a GluN1 subunit with an HA epitope between amino acids 27 and 28. The vector Kidins220-GFP-Ct encoding rat Kidins220 fused at the C-terminal with GFP has been previously used,58 and was generated by subcloning a SmaI/NdeI fragment containing FL rat Kidins220 cDNA from pGEMT easy  into pEGFP-N2 SmaI site.Plasmid driving the expression of N-terminal tagged rat HA-GluN2A subunit has been previously described2. Transfection was performed in serum-free medium with Lipofectamine 2000 reagent and, 4 h after addition, liposomes were removed and cells fed with supplemented medium and maintained in culture for 48 h. In those experiments where HA-GluN1 and HA-GluN2A subunits were co-expressed with Kidins220-GFP-Ct, the NMDAR competitive antagonist DL-AP5 (2 mM) was added after transfection to prevent HEK293T excitotoxic death by NMDAR activation due to glutamate present in the culture medium.59 Removal of the antagonist was performed just before NMDA stimulation.Human embryonic kidney 293T cells (HEK293T) were cultured at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen-Live Technologies), supplemented with 10% fetal bovine serum, 2 mM glutamine and 100 U/ml of penicillin/streptomycin in an humidified atmosphere containing 5% CO26 Neurons grown for eight DIVs were transduced with lentivirus expressing Kidins220 shRNA (shK) or a control shRNA (shC) directly added to the growing media and infection proceeded for six additional days.Plasmids and lentivirus suspensions were prepared as previously described.26 Equal amounts of total lysates were resolved in SDS-PAGE and analyzed by immunoblot. Membranes were incubated with different primary and secondary antibodies and immunoreactive bands were detected by ECL.Protein extracts from primary cultures, HEK293T or brain tissue were prepared as described.in vitro with purified calpain I or calpain II as reported.26 When indicated, 20 μM CiIII or 0.5 μM Tat-peptides were added to lysates immediately before protease addition. Calpain activation was established by immunoblot analysis through the detection of full length brain spectrin (FL) and its BDPs. Analysis of the potential calpain cleavage sites in rat Kidins220 last C-terminal 1000 amino acids was performed using the online services provided by Calpain Modulatory Proteolysis Database  and GPS-Calpain Cleavage Detector . Predictions from five different algorithms  were run. The results were compared for the identification of common putative cleavage sites present in Kidins220 C-terminus.Cell extracts were digested in vitro proteolysis were immunoprecipitated using a rabbit polyclonal anti-GFP antibody, resolved on SDS-PAGE, transferred to PVDF membranes and stained with Coomasie Blue R-250. The band of interest was cut, washed twice and kept at 4 °C until Edman degradation analysis. Sample duplicates were run in parallel and analyzed by immunoblot with the GFP antibody in order to ensure that electrophoretic mobility of purified bands matched that previously detected in HEK293T digestions. Edman N-terminal sequencing was performed by the Proteomic Service of the Centro de Investigaciones Biológicas . Six rounds of protein degradation followed by HLPC identification of excised amino acids revealed the sequence of interest.Total lysates from HEK293T cells expressing Kidins220-GFP-Ct fusion protein were digested with 80 U/ml of purified calpain I, as above. The C-terminal fragments generated after 31 As we are using primary cultures containing cortical neurons and glial cells, we established the contribution of glia viability to total values by exposing sister cultures to 400 μM NMDA, 10 μM glycine for 24 h, conditions which induce nearly complete neuronal death and no glial damage. After subtracting this absorbance value, we obtained the viability of the neuronal subpopulation. For Calcein-AM/propidium iodide staining, neuronal cultures grown on Ibidi l-Slide 8-well chambers  were left untreated or stimulated with NMDA for 4 h. Then, culture media was replaced by serum-free Neurobasal medium (Invitrogen-Live Technologies) containing a mixture of 1 μM Calcein-AM, a highly permeable non-fluorescent molecule transformed to fluorescent calcein dye in viable cells, and 0.5 mM propidium iodide which can only reach the nucleus of dead cells. Incubation proceeded for 15 min at 37 °C and neuronal viability was assessed by time-lapse microscopy using the Cell Observer Z1 (Carl Zeiss MicroImaging) at 37 °C and 5% CO2/95% air, AxioVision 4.8 imaging software and a Cascade 1 k camera. A minimum of five random fields and at least 500 cells were counted for each sample. Only cells with a clear neuronal morphology were considered for further analysis. The percentage of viable (green) or death (red) neurons was expressed relative to the total number (green+red) of cells examined per condition.Measurement of neuronal survival by MTT reduction assay (Sigma Co.) was performed as previously described.t-test. A P-value smaller than 0.05 was considered statistically significant: *P<0.05, **P<0.01, *** P<0.001.Immunoblot signals were quantified by densitometric analysis (NIH Image), normalized using NSE and expressed relative to values obtained in their respective controls. Results are shown as mean±S.E.M. of 3–7 independent experiments. Statistical significance was determined by Student's