The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its ‘phosphorylated’ and ‘dephosphorylated’ states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer’s disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains , with S367 being critical, is responsible for the PS1 pathogenic ‘closed’ conformation, and resulting increase in the Aβ42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment.DOI:http://dx.doi.org/10.7554/eLife.19720.001 Alzheimer’s disease is a widely recognised disorder caused by the progressive deterioration and death of brain cells. A key feature of the disease is the formation of structures called plaques in the brain. Plaques occur when many copies of a molecule known as amyloid beta stick together outside of the brain cells. Healthy brains also produce amyloid beta but it is in a different form, which cannot form plaques.One in twenty people with Alzheimer’s disease have a family history of the disease. Of these, many are linked to changes in a gene that produces a protein called Presenilin 1 (or PS1 for short). Cells need PS1 to make amyloid beta and the altered versions of PS1 produce the type of amyloid beta that causes Alzheimer’s disease. Yet, in cases that do not run in families, the gene for PS1 is unchanged but the PS1 protein still produces the form of amyloid beta that is linked to Alzheimer’s disease.Maesako, Horlacher et al. wanted to find out how seemingly healthy PS1 proteins can be made to produce plaque-forming amyloid betas. Studies of PS1 from mice revealed that small chemical modifications, called phosphate groups, could be attached to PS1 in a process called phosphorylation. Modified PS1 proteins produce harmful amyloid betas and removing the modifications was enough to make PS1 behave normally again. Maesako, Horlacher et al. found three points in the PS1 protein where phosphorylation could change the behaviour of the protein, the most important one is a site called Ser367.Further investigation showed that an enzyme called Protein Kinase A (PKA) phosphorylates PS1; this enzyme is also able to attach phosphate groups to many different proteins. Maesako, Horlacher et al. went on to show that PS1 is phosphorylated in samples from people with Alzheimer’s disease, suggesting that this is a plausible cause for some cases of the disease. Finding a way to prevent phosphorylation or remove phosphate groups from PS1 could be the first step towards treating these cases of Alzheimer’s disease.DOI:http://dx.doi.org/10.7554/eLife.19720.002 Senile plaques, comprised primarily of β-amyloid (Aβ), are the major pathological hallmark of Alzheimer’s disease (AD). Presenilin 1 (PS1) is the catalytic component of γ-secretase , which iUsing Förster resonance energy transfer (FRET)-based imaging techniques we have previously shown that familial AD mutations in PS1 increase the proximity of PS1 N-terminus (NT) to C-terminus (CT) or to the large cytoplasmic loop domain, causing the so called ‘closed’ pathogenic PS1 conformation . On the 2+ levels and Aβ pathology. Notably, Aβ deposition is observed in brain regions exhibiting a high basal rate of neuronal activity in humans  inhibitors prevent the Ca2+-induced PS1 ‘closed’ conformational change whereas PKA activators trigger it. Therefore, we conclude that the Ca2+-increased PKA activity leads to phosphorylation of PS1 at the three domains, inducing the PS1 pathogenic ‘closed’ conformation. These results provide strong evidence that PS1 phosphorylation at certain residues can potentially be molecular targets for AD prevention.Phosphorylation, one of the crucial post-translational modifications, is a rapid and dynamic event in cells, and represents a common mechanism of regulating protein conformation and/or activity. In PS1, fifteen phosphorylation sites have been identified . Althoug2+. Specifically, we found that KCl or glutamate-induced increases of intracellular Ca2+ levels led to PS1 adopting a pathogenic ‘closed’ conformation  , and theormation . Now, byormation , indicat2+ influx may induce phosphorylation of PS1, and whether PS1 phosphorylation is responsible for the PS1 pathogenic conformational change. For this, the amount of phosphorylated PS1 was measured by immunoprecipitation/Western blotting in primary neurons treated with KCl. We found that KCl treatment significantly increased the level of PS1 phosphorylation at its serine residues  or mutant PS1 constructs, in which serine (S) or threonine (T) previously reported as phosphorylation sites were substituted by alanine (A)  and anti-PS1 CT antibodies to selectively detect the conformational change of ‘exogenous’ PS1 using the antibody-based FRET assay. Significant increases in Ca2+ levels were verified by Indo-1 Ca2+ imaging  . Then, t imaging   in single domains , in double domains , or in triple domains 1 + 2 + 3. The conformation of G-PS1-R with domain 1, or domain 2 only, or combined domain 1 and 2 mutants was not different from that of WT G-PS1-R . HoweverDomain 3 of PS1 consists of three consecutive serine residues: S365, S366 and S367. To identify specific residues in domain 3 responsible for the pathogenic conformational change of PS1, we next generated single phosphorylation-mimicking G-PS1-R mutants. The spectral FRET analysis revealed significantly higher FRET efficiency in S367D G-PS1-R but not in S365D G-PS1-R or S366D G-PS1-R, as compared to WT G-PS1-R . This in2+-triggered ‘closed’ PS1 conformation, whereas JNK, PKC and GSK3β inhibitors did not , or domain 1 and domain 2 combined had no effect on PS1 conformation . Thus, tSince allosteric changes in PS1 conformation underlie changes in the Aβ42/40 ratio, with the ‘closed’ conformation linked to the increased Aβ42/40 ratio , we nextCollectively, these data demonstrate that phosphorylation of domain 1 or 2 is not sufficient for the PS1 conformational change, but is required for PKA-mediated phosphorylation of domain 3, leading to the pathogenic conformational change in PS1/γ-secretase.2+ influx may trigger PS1 domain 3 phosphorylation and the pathogenic ‘closed’ conformation in vivo, we optimized our spectral FRET assay  promoter . pAAV8-h2+ levels in the somatosensory cortex neurons by employing Yellow Cameleon 3.6 (YC3.6)-based -based  in vivo  imaging . The acteviously , was inceviously , indicat2+ triggers the ‘closed’ conformational shift in PS1/γ-secretase via PS1 domain 3 phosphorylation, we expressed the phosphorylation-inhibited mutant S365A/S366A/S367A G-PS1-R in mouse brain prior to KCl stimulation. Notably, no change in the R/G ratio was detected in neurons expressing this mutant G-PS1-R after KCl application  immunoreactivity was detected by confocal microscopy  antibody to detect Cacroscopy . We showcroscopy . Increascroscopy . In addicroscopy , suggestcroscopy . Correlacroscopy .10.7554/2+ overload and Aβ in the pathogenesis of AD. Although it has been suggested that aberrant Ca2+ homeostasis affects Aβ production, the effect of Ca2+ elevations on PS1/γ-secretase function is unknown. We have recently reported that increases in Ca2+ levels induce the pathogenic change in PS1 conformation and increase the Aβ42/40 ratio in cultured neurons , domain 2  and domain 3  is responsible for the PS1/γ-secretase pathogenic conformational change in vitro and in vivo.Increasing evidence suggests interactions between Ca neurons . Conside2+-triggered PS1 pathogenic, ‘closed’ conformation linked to increased Aβ42/40 ratio , domain 2 (S310-S313) and domain 3 (S365-S366-S367) is important for PS1 adopting the ‘closed’ conformation. On the other hand, the results using phosphorylation-mimicking mutants revealed that phosphorylation of domain 1 and domain 2, though necessary, is not sufficient for the structural change of PS1. Whereas domain 3, specifically S367, is critical for PS1 achieving the ‘closed’ conformation. These results beg the questions of how domain 3 is phosphorylated and whether phosphorylation of domain 1 and domain 2 is involved in the Ca2+-triggered PS1 pathogenic conformational change. Although the domain 1 and domain two phosphorylation-mimicking PS1 isoform (T74D/S310D/S313D G-PS1-R) shows the same conformation as WT G-PS1-R, PKA activators were able to induce the ‘closed’ conformation of this mutant. Therefore, we conclude that PKA is involved in phosphorylation of domain 3. We propose the following model: PKA first phosphorylates PS1 at domain 2, which leads to spatial rearrangements around domain 1, and allows its phosphorylation. This subsequently changes the local conformation around domain 3, enabling its phosphorylation by PKA. This final phosphorylation of S365-S366-S367, with S367 being most crucial, drives the pathogenic conformational alteration of the PS1/γ-secretase . However, regardless of which assay we use, increased Ca2+  in WT mice brains, a concentration of Ca2+ consistent with that observed in AD mouse models displaying Ca2+ overload in vivo   phosphorylation in AD brains, demonstrating that Ca2+-driven PS1 phosphorylation at certain residues may be important factors contributing to the pathogenesis of AD. Of note, a wide distribution of the phosphorylated PS1 levels observed in AD cases did not correlate with either age or PMI. Since we have selected a relatively homogenous group of AD brains in terms of pathology, Braak and CERAD scores could not be used to explain the wide distribution. Further investigation is necessary to determine if other factors, such as medications taken, disease duration, ApoE genotype, etc. could have affected the p-PS1 levels.We have also established and validated novel in vivo PS1 conformation assay. Topical KCl application significantly increased intracellular Ca1535 nM) . We veri2+ overload, domains 1, 2 and 3 of PS1 can be hyper-phosphorylated by PKA. This induces the pathogenic ‘closed’ PS1 conformation, and leads to subsequent elevation of the Aβ42/40 ratio, followed by accelerated Aβ oligomerization or deposition. In line with these findings, we have previously reported that PS1 shows a ‘closed’ conformation around Aβ plaques in the sporadic AD brain  PS1 and the N-terminally FLAG-tagged phosphorylation-inhibited PS1 mutants: S28A PS1, T74A PS1, S310A/S313A PS1, S313A PS1, S324A PS1, S337A PS1, S365A PS1, S366A PS1, S367A PS1, S365A/S367A PS1 and S366A/S367A PS1, were kind gifts from Dr. Patrick C Fraering  . The N-tAB_301867), rabbit monoclonal anti-PS1 loop (RRID:AB_1310605), rabbit monoclonal anti-phosphorylated PS1 at S310 (RRID:AB_1267317), and mouse monoclonal anti-phosphoserine (RRID:AB_305611) antibodies were purchased from Abcam . The rabbit polyclonal anti-PS1 CT (RRID:AB_261178) and mouse monoclonal anti-FLAG (RRID:AB_259529) antibodies were from Sigma-Aldrich . The mouse monoclonal anti-PS1 loop (RRID:AB_95175), rabbit polyclonal anti-phosphoserine (RRID:AB_390205) and rabbit polyclonal anti-phosphorylated CREB at S133 (RRID:AB_310153) antibodies were from Millipore . Rabbit monoclonal CREB antibody (RRID:AB_309979) was from Upstate Biotechnology . Mouse (RRID:AB_737182) and rabbit (RRID:AB_737197) normal IgG were from Santa Cruz Biotechnology, Inc. . Alexa Fluor 488 (A488), and Cy3-labeled corresponding secondary antibodies were from Life Technologies  and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Pierce . The Ca2+ ionophore A23187, PKA inhibitors H-89 and KT5720, PKA activators forskolin and 8-Bromoadenosine 3’:5’-cyclic monophosphate (8-Bromo-cAMP), JNK inhibitor SP600125, PKC inhibitor Ro 31–8220, and GSK3β inhibitor TDZD-8 were from Sigma-Aldrich .The mouse monoclonal anti-PS1 NT (RRID:CHO cell lines stably overexpressing WT APP (7 W cells)  and stab12 GC/ml to 1.5 × 1013 GC/ml. The construction of pAAV2-CBA-Yellow Cameleon 3.6 (YC3.6) was described previously -WT G-PS1-R), S365A/S366A/S367A G-PS1-R (pAAV8-hSyn1-S365A/S366A/S367A G-PS1-R), G-PS1 (pAAV8-hSyn1-G-PS1), PS1-R (pAAV8-hSyn1-PS1-R) and RFP-GFP fusion (pAAV8-hSyn1-R-G). These plasmids were constructed by cloning corresponding PS1 constructs into an AAV8 vector containing the hSyn1 promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequences, using NheI/HindIII or HindIII/EcoRI restriction sites. All plasmids were packaged in AAV serotype 8 at the University of Pennsylvania Vector Core. Viral titers for all constructs were between 4.9 × 10eviously .Experimental procedures for viral injection have been described previously . BrieflyOne month after viral injection, mice were anesthetized with 1–3% isoflurane and placed in a stereotaxic apparatus. A circular area of skull was carefully drilled, removed, and then dura was carefully removed. A circular cranial window soaked in PBS was implanted to the brain surface, and fixed to the remaining skull with dental cement mixed with Krazy glue. For topical KCl application, a burr hole was created in the fixed dental cement to inject KCl with a Hamilton syringe into the space between the cranial window and brain surface.2 asphyxiation, perfused intracardially with PBS followed by 4% PFA. For the rescue experiments, 1 µl of 100 µM KT5720 (right hemisphere) or vehicle (left hemisphere) was injected 75 min prior to 8-Bromo-cAMP treatment.Five to seven months old male C57BL/6 mice were anesthetized as described above, placed into the stereotax, and injected with 1 µl of 100 mM 8-Bromo-cAMP (right hemisphere) or vehicle (left hemisphere) into the somatosensory cortex. 5 min post-injection, mice were sacrificed using COThe brains were dissected, post-fixed by immersion in 4% paraformaldehyde (PFA) with 15% glycerol  in PBS, and cryoprotected using 30% glycerol in PBS. Prior to immunostaining, the brains were cut on a freezing sliding microtome  into 35 μm-thick coronal sections. Brain tissue sections were permeabilized using 0.4% TX-100. For 4% PFA fixed 7 W cells and primary neurons 0.1% TX-100 was used. Non-specific binding of the antibodies was blocked by incubation of the cultured cells or free-floating brain sections with 1.5% normal donkey serum . The primary antibodies were applied overnight at 4°C, followed by 1 hr incubation at room temperature with corresponding Alexa Fluor 488- or Cy3-conjugated secondary antibodies. The samples were mounted in VectaShield mounting medium .t1) of the donor fluorophore was measured in the absence of the acceptor fluorophore . In the presence of the acceptor fluorophore (RFP or Cy3), excitation of the donor fluorophore results in reduced donor emission energy if the donor and acceptor are less than 5–10 nm apart (FRET present). This yields characteristic shortening of the donor fluorophore lifetime (t2). The acquired FLIM data were analyzed using SPC Image software  by fitting the data to one (negative control) or two (acceptor present) exponential decay curves. In the latter case, t1 of the non-FRETing population was ‘fixed’ and thus excluded from the analysis, and only shorter, t2, values were analyzed. The FRET efficiency (%EFRET) was calculated using the following equation: %EFRET = 100*(t1-t2)/t1. Higher %EFRET reflects closer proximity between fluorophores labeling the PS1 domains.The FLIM analysis of PS1 conformation was performed as described previously . BrieflyThe spectral FRET assay with single photon excitation for the experiments using cultured cells and immunostained mouse brain sections was conducted as described previously . BrieflyThe spectral FRET assay for monitoring PS1 conformation in living mouse brain using two-photon excitation is newly established. First, to determine the excitation wavelength that preferentially excites GFP, the G-PS1-R probe was excited at different wavelengths from 750 nm to 975 nm with a mode-locked titanium/sapphire laser . The 900 nm wavelength was chosen to selectively excite GFP, and emitted fluorescence was detected by two emission channels: 495–540 nm range for channel 1 (for GFP) and 575–630 nm for channel 2 (for RFP), on an Olympus Fluoview 1000 MPE microscope  . Time-lapse images were obtained every 10 s for a duration of 2 min. The average pixel fluorescence intensity after subtraction of the background fluorescence for the whole cell was measured using ImageJ in each channel. The R/G ratio was used as readout of the FRET efficiency. Pseudo-colored images were generated in MATLAB.2+ levels in 7 W cells were determined using the ratiometric Ca2+-sensitive dye Indo-1 . A Chameleon Ti:Sapphire laser was used for excitation at 750 nm, and the emitted fluorescence was detected in two channels: 390–465 nm and 500–550 nm.Intracellular Cae Indo-1 . Briefly2+ levels in the somatosensory cortex of living mice was measured using the FRET-based ratiometric probe, Yellow Cameleon 3.6 (YC3.6)  , as desc (YC3.6) . Brieflyeviously .The levels of secreted Aβ40 and Aβ42 were measured using ELISA kits according to the manufacturer’s instructions .Primary neurons were lysed in radio-immunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitor cocktail . The cells were homogenized and incubated for 30 min on ice. Each sample was then centrifuged, and the supernatants were collected. Protein concentrations were determined using a Pierce BCA Protein Assay Kit . Aliquots with equal amount of total protein were treated with protein G-Sepharose  for 1 hr. After removing protein G-Sepharose by centrifugation, primary antibodies were added to the supernatants. Each sample was rotated for 1 hr and then treated with protein G-Sepharose for 1 hr. The immunoprecipitates were washed with RIPA buffer and resuspended in 2 x LDS sample buffer (Life Technologies). After boiling and centrifugation, the supernatants were subjected to SDS-PAGE on NuPAGE Bis-Tris gels (Life Technologies).Frontal cortex of frozen human brains from neuropathologically verified AD cases were obtained from the brain bank of the Alzheimer’s Disease Research Centre (ADRC) at Massachusetts General Hospital, and were lysed by RIPA buffer with protease and phosphatase inhibitor cocktail (Fisher Scientific) and calyculin A . Control cases were non-demented individuals who did not meet pathological diagnostic criteria of AD or any other neurodegenerative diseases . All the2+ concentration, two-way repeated-measures ANOVA and Bonferroni post-hoc analysis were used. For the correlation analysis in the human brain-based experiments, Spearman's nonparametric correlation analysis was used. An appropriate sample size was computed when the study was being designed and each experiment was performed using at least three independent trials. p<0.05 was considered to indicate a significant difference.Statistical analysis was performed using StatView for Windows, Version 5.0.1 or GraphPad Prism 5. All values are given as means ± SEM. Comparisons were performed using One sample t-test, Student’s t-test, or one-way factorial ANOVA followed by Bonferroni’s post-hoc analysis. For the in vivo time course experiments monitoring PS1 conformation and Ca In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by a Senior Editor and two reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Pathogenic PS1 phosphorylation at Ser367" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The manuscript by Maesako et al. reports on a very interesting set of studies of the link between calcium increases, PS1 phosphorylation, enhancing the population of its closed conformation, along with pathogenic increases in the Abeta42 to Abeta40 ratio.As the authors can see from consulting both reviews, the reviewers want to see the raw FRET data central to your main conclusions and better understand your longer wavelength antibody-based FRET assay in fixed tissue. In the traditional FRET assay the changes are small and both reviewers want to be convinced that these changes are significant. In addition, reviewer 1 wants to see your pharmacologic line of evidence expanded into at least two cell lines and ideally in mice to provide a line of evidence to support your hypothesis in addition to your PS1 Ser/Thr to Ala mutations and Ser/Thr to Asp mutational data.Please also consider the reviews pasted below in full in terms of revising your manuscript.Reviewer #1:This manuscript is generally well written and the experimental execution is earnest.This is a complicated paper experimentally in the sense that it is assumed that the PS1 Ser/Thr to Ala mutations and Ser/Thr to Asp mutations have no influence other than preventing and simulating phosphorylation, respectively –the desired interpretation. Of course, these mutations can change conformational ensemble for other reasons as well. Collectively, considering the data as a whole in this paper, it is supportive of the hypothesis reflected by the title.That said, the changes observed for individual dataplots are small and in many cases only significant in some cases because of smaller than typical error bars on for example the control measurements . If this inhibitor is selective, it seems that this is the most unassailable data in the paper to show via the suite of experiments in several cell lines and ideally in mouse brain that inhibitor pre-treatment prevents pathogenic collapse of PS1 and prevents altering the Abeta ratio associated with pathology, in the presence of triggered calcium increases. As presented this section is intriguing but not compelling.There are modern mass spectrometry methods that also should prove useful in affinity purifying PS1 to ascertain its phosphorylation state quantitatively, not by reliance solely on the PS1 Ser/Thr to Ala mutations and Ser/Thr to Asp mutations. If the pharmacologic data is not possible to generate, then this type of data becomes central as a second line of evidence.Reviewer #2:2+ triggered PKA activity results in sequential phosphorylation of domains 2, 1 and 3, with S367 phosphorylation being critical for change to a closed conformation. An exciting additional set of experiments directly probe FRET in vivo, supporting the developed model. Finally, an important link to disease was uncovered by the observation that the phosphorylation at S310 (domain 2) increased on average in AD brains. Overall, the work represents a strong combination of interesting tools with many important control experiments to develop a mechanistic model for how a PKA phosphorylation cascade results in PS1 conformational changes including in vivo. The obtained novel mechanistic understanding combined with the observed links of phosphorylation levels in AD suggests potential venues for therapeutic strategies. Therefore, I believe the paper would be of interest to the broad readership of eLife. Some technical issues are noted below that should be addressed by the authors.The manuscript by Maesako et al. reports on a very interesting set of studies of the link between PS1 phosphorylation and its closed conformation, with links to AD. The authors use a series of experiments, including with biochemical and FRET experiments to systematically dissect the relationship of different phosphorylation states of the protein and its conformational properties in cells and neurons. The model that emerges is a complex one where Ca1) The antibody based FRET experiments need more description and discussion, at least in the Methods section.How large are the antibodies and how are they labeled? What range of distance changes (at least approximately) are being detected? Are factors other than distance affecting the rather small changes in the reported changes in FRET efficiency ? FactorsThe changes in average reported FRET efficiencies are small. However, from the images in 2) Some of the above points are more generally relevant for the FRET studies in the paper, and authors should discuss these issues in the paper.3) It would be useful to have a few additional images/time traces (corresponding to 4) The authors observe the interesting result that phosphorylation at domain 2 (S310) is increased on average in AD brains. However, in addition,  […]Reviewer #1:This manuscript is generally well written and the experimental execution is earnest.This is a complicated paper experimentally in the sense that it is assumed that the PS1 Ser/Thr to Ala mutations and Ser/Thr to Asp mutations have no influence other than preventing and simulating phosphorylation, respectively –the desired interpretation. Of course, these mutations can change conformational ensemble for other reasons as well. Collectively, considering the data as a whole in this paper, it is supportive of the hypothesis reflected by the title.That said, the changes observed for individual dataplots are small and in many cases only significant in some cases because of smaller than typical error bars on for example the control measurements , 100% was assigned to the vehicle treated sample, and the percent change in phospho-PS1 for KCl treated neurons was analyzed in each experiment. Phosphorylation is a very dynamic process and its detection can be influenced by a number of factors. However, even though the average increase in phospho-PS1 after KCl treatment was relatively small, the increase was consistent and reproducible. For the Aβ ELISA measurements , whereas the PKA inhibitor KT5720 blocks this effect new . This reI want to know more about KT5720, the pharmacologic PKA inhibitor-its selectivity, potency, etc. This is critical for me to get comfortable with publishing this paper. I want to see a second line of evidence to support the authors hypothesis (i.e. in addition to the PS1 Ser/Thr to Ala mutations and Ser/Thr to Asp mutations). If this inhibitor is selective, it seems that this is the most unassailable data in the paper to show via the suite of experiments in several cell lines and ideally in mouse brain that inhibitor pre-treatment prevents pathogenic collapse of PS1 and prevents altering the Abeta ratio associated with pathology, in the presence of triggered calcium increases. As presented this section is intriguing but not compelling.There are modern mass spectrometry methods that also should prove useful in affinity purifying PS1 to ascertain its phosphorylation state quantitatively, not by reliance solely on the PS1 Ser/Thr to Ala mutations and Ser/Thr to Asp mutations. If the pharmacologic data is not possible to generate, then this type of data becomes central as a second line of evidence.JNK, PKC, and GSK3β) and found that unlike KT5720 inhibition of PKA, inhibition of these kinases was not able to prevent the Ca2+ influx-induced PS1 conformational change(We agree with the reviewer that having PKA-selective inhibitor(s) would provide truly unassailable data. Unfortunately, it is practically impossible to have a pharmacological reagent that is 100% selective. KT5720 has been reported/used in almost 1000 studies as a selective PKA inhibitor. Still, according to the review by Andrew Murry, , KT5720 may inhibit some other kinases as well, although to a lesser degree. Thus, we have tested several reported inhibitors of other kinases , and show that both PKA inhibitors, KT5720 and H-89, inhibit the Caange new . This isange new .Furthermore, to verify the physiological relevance of the PKA-inhibition in mouse brain and to determine if KT5720 pre-treatment would prevent the cAMP-induced “pathogenic collapse of PS1”, we injected 1µl of 100 µM KT5720 inhibitor or vehicle control into the somatosensory cortex. 75 minutes post-injection, 1µl of 100 mM 8-Bromo-cAMP, a direct PKA activator, was delivered to the same brain region for 5 minutes. Mice were quickly euthanized, the brain was removed, fixed, sectioned, and ex-vivospectral FRET analysis of endogenous PS1 conformation was performed. We found that KT5720 prevents the 8-Bromo-cAMP-induced PS1 “closed” conformational change in mouse brain in vivo new . The inhReviewer #2: […] 1) The antibody based FRET experiments need more description and discussion, at least in the Methods section.How large are the antibodies and how are they labeled? What range of distance changes (at least approximately) are being detected? Are factors other than distance affecting the rather small changes in the reported changes in FRET efficiency ? FactorsThe changes in average reported FRET efficiencies are small. However, from the images in 2) Some of the above points are more generally relevant for the FRET studies in the paper, and authors should discuss these issues in the paper.Per reviewer’s request we provided more details for the antibody-based FRET assays in the Material and methods of the revised manuscript. We appreciate and agree with the reviewer’s concern about large complexes of the primary + fluorescently labeled secondary antibodies. However, the great advantage of using the antibody-based FRET assay is the ability to monitor changes in the proximity between endogenous PS1/γ-secretase domains. We are aware that all FRET-based assays only report relative changes in the proximity between fluorophores tagging these domains, which does not directly translate into comparable changes in the distance between the protein epitopes . Nevertheless, both spectral FRET and FLIM assays we used have proven to be highly reliable and reproducible in reporting consistent changes in the FRET efficiency  after experimental treatments.However, to further substantiate the findings we have also used our previously validated GFP-PS1-RFP FRET reporter probe  to monitor PS1 conformational change in real time in live cells in vitro and in vivo. In the G-PS1-R probe, donor and acceptor fluorophores are fused to the PS1 NT and cytosolic loop, respectively, reporting relative change in the GFP-NT to RFP-loop proximity.2+/PKA activation consistently increases the proximity between donor and acceptor fluorophores tagging PS1 NT and CT in the antibody-based FRET . Even though the change in the proximity between fluorophores may be a relatively small, it could signal substantial conformational change on the molecular level that would affect PS1/γ-secretase function.2+ stimulation  leads to the average per neuron change in the ~3~4.5 Å range.In regard to the range of the distance changes: Since in our GFP-PS1-RFP conformational reporter probe donor and acceptor are in the 1:1 ratio, and the Förster radius of the RFP-GFP pair is known (5.7 Å) , we could translate the FRET signal into the change in the distance between GFP and RFP by using the following equation: d= 5.7 x [1/(1-t2/t1) -1]^(1/6) . The GFP-NT/RFP-loop distance is approximately 70 Å in the vehicle-treated condition. Ca2+ concentration or in PKA distribution. A combination of the PS1 conformational assay and Ca2+ imaging or PKA staining in the same cell could provide more detailed mechanistic insight. However, since both rely on the fluorescent labeling/detection, our experience shows that the presence of the third fluorophore may interfere with the reliability of the FRET detection in cells.We fully agree with the reviewer that some areas in neurons show higher FRET efficiency than others, which could be due to local differences in the Ca3) It would be useful to have a few additional images/time traces  The authors observe the interesting result that phosphorylation at domain 2 (S310) is increased on average in AD brains. However, in addition, Indeed, a wide distribution in the level of PS1 S310 phosphorylation was revealed in AD brains. It is possible thatbroad phospho-PS1distribution could be result of the phosphorylation dyshomeostasis in the distressed AD brains (i.e. over activation/inhibition of kinases/phosphatases). There are so many factors that are difficult to control or monitor  when analyze human brain samples, such as individual progress of the disease, past medical history, medications, or even lifestyles, etc. All of these could contribute to broader variability in AD brain.All AD and control cases we used in this study were matched as best as possible by the subject’s age and post-mortem interval (PMI). We have also selected a relatively homogenous group of AD patients in terms of pathology . Since the original submission of the manuscript we have analyzed more AD and Control cases, bringing the total number to n=20 Controls and n=23 AD. The wide distribution of phospho-PS1 among AD, but not Control, cases still remains. Having higher “n” allowed us to do correlation analysis with the available data. We found that neither age nor PMI affect the level of PS1 phosphorylation updated .The reviewer raised the interesting question that warrants further investigation of the different influencing factors  that may shed light on variations in the PS1 S310 phosphorylation level between different AD subjects. Unfortunately, such data were only available for a very few of the AD cases used in this study, and thus cannot be included in this manuscript. Since the main focus of the current study is on the molecular mechanism of the pathogenic PS1 conformational change, a different follow up epidemiological study that will select a different set and significantly higher number of AD and Control cases with more complete clinical data would be needed.


While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.PSY1R is a leucine-rich repeat (LRR) receptor-like kinase (RLK) previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1) and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H As multicellular organisms, plants rely on a fine-tuned cell-to-cell communication system to coordinate growth responses. At the core of this system are members of the receptor-like kinase (RLK) superfamily. This massive family of plant proteins, which is implicated in both plant development  and plan+-ATPase (AHA2) and thereby promoting proton pumping  and undergo post-translational modification followed by secretion via the secretory pathway, are emerging as important players in this process . Plant p pumping . Proton  pumping . Interes-ATPases  and PSKR-ATPases , but the-ATPases .S-locus Receptor Kinase (SRK)  , Brassinse (SRK) , Flagellse (SRK) , and these (SRK) .However, about a decade ago, an interaction partner of BRI1 was identified and named BRI1-Associated Kinase 1 (BAK1) . SequentAdditionally it has been found that SERKs acts as co-receptors for a range of LRR-RLKs. Each ligand-binding receptor seems to only interact with a limited number of SERK proteins . For exain vitro autophosphorylation sites. By homology modeling to known structures of BRI1 and SIRK1 we propose how these residues are involved in stabilization of the activation loop in the active and inactive state, respectively. We furthermore showed that PSY1R interacted with members of the SERK family in planta.In this study, we investigated the transactivation mechanism of PSY1R and identified its SERK2, SERK4, and SERK5 cDNAs were amplified from an Arabidopsis thaliana Col-0 cDNA preparation. A cDNA clone of SERK1 was kindly provided by Professor S. C. de Vries (University of Wageningen). The PCR fragments were subcloned into Gateway pENTR/D-TOPO vector (Invitrogen Life Technologies). The BAK1 cDNA clone in the pCR8/GW/TOPO vector was obtained from Arabidopsis Biological Resource Center (ABRC) (clone CIW00115). The SERK, and BAK1 genes were cloned into Gateway-compatible BiFC vectors by LR recombination using LR Clonase II Enzyme Mix (Invitrogen Life Technologies) to yield C-terminal fusions to cCFP or nYFP expressed from the 35S promoter. The PSY1R BiFC constructs were used in a previous study . Mutations and stop codons were introduced through QuikChange Site Directed Mutagenesis. The constructs were transferred into pDEST15 and pDEST17 (Invitrogen Life Technologies) through the LR reaction. All constructs were sequenced by Eurofins MWG Operon.To generate constructs for expression in Agrobacterium tumefaciens strain C58C1 was grown overnight in liquid YEP medium containing 25 μg/mL gentamicin and 50 μg/mL spectinomycin. Cells were washed and resuspended in infiltration solution , and diluted to an OD600 of 0.05, before mixing the transformed cells in the combinations to be tested. N. benthamiana leaves were infiltrated with the A. tumefaciens mix using a needleless syringe. Fluorescence was monitored approximately 48 h after infiltration.Transformed A Leica SP5 confocal laser-scanning microscope with a 20 × 0.7 numerical aperture water-immersion objective was used to examine the lower epidermis of the infiltrated tobacco leaves. The complemented YFP/CFP fluorescence was excited at 448 nm and emission was detected at 515–540 nm. The gain was fixed in all samples to ensure that the emission intensity was comparable. Interaction was tested using both combinations of fusion proteins with similar results.600 of 0.1 and grown at 37°C until the OD600 reached 0.5–0.7. Expression from the pDEST15 plasmid in BL21-AI cells was induced by the addition of L-(+)-arabinose to a final concentration of 0.2% (w/v) and the culture was incubated at 20°C overnight. Expression from the pGEX-4T-1 vector in BL21(DE3) cells was induced by the addition of 100 μM IPTG and the culture was incubated at 28°C for 3–4 h. In both cases, the cells were harvested at 14,000×g for 10 min, washed in cold H2O, and pelleted again. The cells were resuspended in P-buffer  containing 1 mM PMSF, 0.01% (w/v) DNase I, and 0.01% (w/v) lysozyme and lysed by sonication. The cell debris was collected by centrifugation at 15,000×g for 30 min and the lysate was incubated with Glutathione Sepharose 4B  for 2 h at 4°C. The resin was washed three times with P-buffer before eluting the protein with 50 mM L-glutathione in P-buffer, adjusted to pH 8.0. His-tagged proteins were expressed from the pDEST17 plasmid in BL21-AI cells as described above. The cells were harvested as described above, resuspended in lysis buffer  containing 1 mM PMSF, 0.01% (w/v) DNase I, and 0.01% (w/v) lysozyme and opened by sonication. The cell lysate was collected as described above and incubated with Ni-NTA agarose (Qiagen) for 2 h at 4°C. The resin was washed three times with wash buffer  before eluting the protein with elution buffer .GST-tagged proteins were expressed either from the pGEX-4T-1 vector  in BL21(DE3) cells or from the pDEST15 vector in BL21-AI cells (Invitrogen Life Technologies). Briefly, LB medium was inoculated with an overnight LB + 100 μg/mL ampicillin culture to a start OD2, 1 mM DTT) with 50 μM ATP and 5 μCi γ-32P-ATP (PerkinElmer) in a volume of 50 μL. The reaction was stopped by the addition of 10 μL 100% (w/v) TCA. The TCA-precipitated protein was dissolved in twofold concentrated Laemmli buffer and proteins were separated on a 10% SDS-PAGE gel. After the gel was stained and dried, it was exposed to a phosphor screen for 3 days and phosphorylation was detected using a Storm 860 scanner (Molecular Dynamics).In a radiometric assay, purified kinase was incubated for 30 min at 30°C in kinase assay buffer  BSA. Rabbit polyclonal phosphoserine  and phosphothreonine  antibodies were used as primary antibodies and polyclonal goat anti-rabbit alkaline phosphatase-coupled immunoglobulin  were used as secondary antibody. The immunoblot was developed using NBT/BCIP substrate .1 . Full-length recombinant proteins were digested in-solution and/or in-gel with sequencing grade trypsin . Freeze-dried tryptic peptides were dissolved by adding 40 μL of 0.1% formic acid and subjected to MS analysis. Ten microliters of each sample was resolved using a Finnigan Surveyor liquid chromatography system interfaced with the mass spectrometer. Tryptic peptides were fragmented using either collision-induced dissociation (CID) or “decision tree” methods that utilize both CID and electron-transfer dissociation .The MS RAW files were searched against the TAIR10 database combined with a decoy database containing the randomized sequences of the original database. The search parameters, described in detail previously , were brIdentification data were evaluated using the XCorr function of SEQUEST, and phosphorylation-site localization was accomplished using phosphoRS . The XCorr values for each charge state were set to default, and no decoy hits were allowed. Peptide mass deviation was 10 ppm and two peptides/protein were used to further filter the data. Phosphopeptides with a pRS score of ≥50 and a pRS site probability of ≥50% were considered as high-confidence phosphosites. For final validation, each spectrum was inspected manually and accepted only when the phosphopeptide had the highest pRS site probability, pRS score, XCorr value, and site-determining fragment ions allowed for unambiguous localization of the phosphorylation site.E. coli as GST-fused protein . An inactive variant of the kinase were generated by mutating the lysine residue in the catalytic domain to an alanine (kPSY1R K831A). Recombinant kPSY1R were incubated in an in vitro kinase assay in the absence or presence of ATP and phosphorylated amino acids were detected with phosphoamino acid-specific antibodies. As seen in Figure 1, phosphoserine (pSer) and phosphothreonine (pThr) antibodies reacted with the active kPSY1R protein, but weakly or not at all with the inactive kPSY1R K831A. This implies that PSY1R autophosphorylates on Ser and Thr residues. The addition of ATP to the kinase reaction assay increased the phosphorylation level especially of Thr residues. The presence of phosphorylated residues in samples without ATP indicates the kinase is active and autophosphorylated when expressed in E. coli. The detected phosphorylation of kPSY1R is, however, true autophosphorylation, since phosphorylation of the inactive kPSY1R K831A was nearly undetectable.We set out to investigate the autophosphorylation mechanism of PSY1R. The cytosolic KD of PSY1R  were expressed in Figure 2A. These spectra presented evidence for phosphorylation of the peptides AKHENLVALQGYCVHDsAR and DIKSsNILLDGNFK (lowercase letters denote phosphorylated residues), which contain the Ser870 and Ser933 phosphosites, respectively. We unambiguously identified seven total autophosphorylation sites , including three threonine and four serine residues, each of which were located within the KD. Detailed information on the identified phosphopeptides is listed in Table 1. The PSY1R intracellular domain contains 12 possible phosphorylation targets within the juxtamembrane domain (JMD) , 35 possible targets within the KD , and 1 possible target within the C-terminal domain (1 Thr). However, autophosphorylation of JMD or C-terminal domain residues was not detected in our MS analysis. Spectral count analyses revealed that the addition of ATP during the in vitro kinase assay enhanced autophosphorylation of Ser870, whereas the remaining six phosphosites were also identified in the control lacking ATP .We then mapped the autophosphorylation sites of the intracellular domain of PSY1R. We incubated kPSY1R in the absence or presence of ATP to allow autophosphorylation. Afterward the proteins were digested with trypsin. The tryptic peptides were analyzed by LTQ-Orbitrap high-resolution mass spectrometry (MS/MS) to map phosphorylation events. Examples of MS/MS spectra for two of the determined autophosphorylation sites are shown in trans- or cis-phosphorylation. To investigate this, we constructed a hexahistidine-tagged inactive variant of the intracellular domain of PSY1R  and incubated it with the active kPSY1R protein. Interestingly, the inactive intracellular domain was phosphorylated in the presence of the active kPSY1R, but not when it was incubated alone , which demonstrates that in vitro autophosphorylation of the intracellular domain occurs in trans. Transphosphorylation of the inactive H6-kPSY1R K831A by the active kPSY1R was detected when using the pThr antibody, but not with the pSer antibody .Since the full-length LRR RLK PSY1R protein was previously shown to form homodimers, we were prompted to ask whether the intracellular kinase activation mechanism involved inter- or intramolecular phosphorylation, i.e., 6-kPSY1R K831A with ATP in the presence or absence of active kPSY1R (GST-tagged) to allow for transphosphorylation. MS analysis identified three phosphorylated sites, Ser951, Thr959, and Thr963, within the KD of H6-kPSY1R K831A after incubation with kPSY1R . No phosphorylated sites were detected in H6-kPSY1R K831A incubated without kPSY1R. Detailed data for the three phosphopeptides AYVADFGLsR, tHVTTELVGTLGYIPPEYGQAWVATLR, and LILPYRTHVTtELVGTLGYIPPEYGQAWVATLR harboring Ser951, Thr959, and Thr963, respectively, are provided in Table 2. Interestingly, all three transphosphorylation sites are located within the kinase activation loop, which was previously shown to carry phosphosites important for catalytic kinase activity in other kinases . This explains why transphosphorylated Ser951 is not detected by the pSer antibody , as this residue has low phosphorylation levels compared to Thr959 and Thr963. Alignment of the activation loops from a number of plant LRR RLKs, showed that residues corresponding to Thr959 and Thr963 are conserved within in the activation loop whereas the Ser951 residue is unique for PSY1R .To examine the transphosphorylation mechanism of kPSY1R in detail, we mapped the transphosphorylation sites of kPSY1R by MS. We incubated the inactive H kinases . Spectra6-kPSY1R K831A-3x) in which all three proposed transphosphorylation sites  were mutated to alanine. We then asked whether the active kPSY1R could transphosphorylate H6-kPSY1R K831A-3x. As shown in Figure 3B, transphosphorylation of threonine residues was completely abolished in H6-kPSY1R K831A-3x. This indicates that Thr959 and Thr963 are exclusive first-phase transphosphorylation sites.To verify the transphosphorylation sites identified by MS, we created a new construct .To evaluate the Figure 5). PSY1R was modeled in the active conformation using the crystal structure of phosphorylated BRI1 KD as template   . As seenFigure 6A, the intracellular domains of SERK1, SERK2, BAK1, and SERK4 were all able to transphosphorylate H6-kPSY1R K831A, whereas kSERK5 exhibited weak autophosphorylation activity and did not transphosphorylate H6-kPSY1R K831A. In the Col-0 accession, SERK5 harbors an R401L mutation in the otherwise conserved HRD motif of the KD, which could explain the lack of autophosphorylation activity observed in our assay. To test the ability of kPSY1R to transphosphorylate a SERK protein, we constructed an inactive H6-tagged version of the intracellular domain of BAK1, H6-mBAK1 K317A. As seen in Figure 6B, kPSY1R was able to transphosphorylate H6-BAK1 K317A. Taken together, these results suggest that one or more of the SERK proteins act as co-receptors for PSY1R via a trans-activation mechanism.BAK1 and other members of the SERK family were previously shown to act as co-receptors for LRR-RLKs such as BRI1 and FLS2. To test whether SERK proteins could regulate PSY1R activity through phosphorylation, we created GST-tagged constructs of the intracellular domain of the SERK proteins (kSERK and kBAK) and tested whether these were able to transphosphorylate the inactive H6-kPSY1R K831A protein. In this case, we used the pThr antibody to detect transphosphorylation. As shown in in planta. As seen in Figure 6C, full-length PSY1R forms homodimers in planta, as we reported previously (psy1rK831A) resulting in loss of kinase activity, also forms homodimers in planta .Using a BiFC assay, we evaluated the ability of full-length PSY1R and PSY1R mutants to dimerize eviously . PSYR1 pFigure 6D, PSY1R interacts with SERK5, even though SERK5 seems inactive, and to a lesser extent with BAK1, SERK1, and SERK2, whereas PSY1R barely interacted with SERK4 in the employed BiFC assay. All the interactions take place at the plasma membrane as expected. We did not detect homodimerization of any of the SERK members. Furthermore, PSY1R K831A forms a strong complex with BAK1.We further investigated whether PSY1R also forms complexes with SERK proteins. As shown in Taken together, we identified three residues within the activation loop of PSY1R, which are part of the first phase transphosphorylation mechanism. By homology modeling to known structures of BRI1 and SIRK1 we demonstrated how these residues are involved in stabilization of the activation loop in the active and inactive state, respectively. Finally we demonstrated that PSY1R interacts with co-receptors from the SERK family and that kPSY1R are target for SERK kinases.in vivo phosphorylation site database PhosPhAt 4.0  within the activation loop. It is surprising that we did not detect any phosphorylation sites within the JMD previously reported to be important for the function of plant RLKs . The in PhAt 4.0  containsFigure 3D), the Ser951 residue is not conserved among the aligned RLKs and is unique for PSY1R. Based on homology modeling we propose that the phosphorylated Ser951 stabilizes the inactive conformation of PSY1R. In addition to Ser951, PSY1R contains four threonine residues, Thr959, Thr962, Thr963, and Thr968, which are conserved among the plant RLKs shown here. The residues within the activation loop of plant RLKs are key phosphorylation sites that are important for kinase activity (Here we have demonstrated that PSY1R first-phase transphosphorylation occurs within the activation loop, specifically at residues Ser951, Thr959, and Thr963. Phosphorylation of Thr959 and Thr963 were only found in the transphosphorylation experiments and not in the initial mapping of phosphosites. This indicates that these sites are only transiently phosphorylated during the catalytic cycle and therefore caught in the inactive mutant; alternatively a full-length receptor protein is required in order to maintain the conformation with these sites phosphorylated. As seen from an alignment of the activation loops of a number of plant LRR RLKs (activity . In the activity . Structuactivity . Interestrans-phosphorylation or intramolecular cis-phosphorylation. Members of the former group are able to transphosphorylate an inactive version of the KD and show second order kinetics with respect to the kinase concentration and include BRI1 (Plant RLKs are categorized into two groups based on whether they are activated through intermolecular ude BRI1 , CLV1 (Wude BRI1 , SERK1 (ude BRI1 , and HAEude BRI1 . Membersude BRI1 , CrRLK1 ude BRI1 , and Xa2ude BRI1 . In our ude BRI1 . BRI1 isude BRI1 .in vitro assay applied for MS studies. In the crystal structure of the closely related PSK receptor PSKR1, a co-receptor is not required for ligand binding but the potential involvement of a co-receptor for the complex activation was suggested and it was found that PSK induces the heterodimerization of PSKR1 with SERK1, SERK2, and SERK3 in planta (It is tempting to speculate that PSY1R is fully activated by SERK phosphorylation, analogous to BRI1. The requirement for a co-receptor would also explain the lack of detected phosphosites in the PSY1R JMD, as this would require the presence of a co-receptor in the n planta .in planta interactions between PSY1R and members of the SERK family, which act as co-receptors and regulate distinct signaling pathways (in vivo.Additionally, we demonstrated pathways . SERK prpathways . Also itin vivo function of the identified autophosphorylation sites and how the interaction with the SERK co-receptor proteins fine-tunes PSY1R signaling.Our study has revealed details of the regulation of the kinase activity of PSY1R. Future studies will reveal the CO and AF designed the study. CO, LG, and TS performed the kinase phosphorylation analysis. CO and AK performed the BiFC analysis. NA performed the MS analysis. JP performed the structural analysis. CO, JT, and AF supervised the project.CO and AF wrote the manuscript. All authors reviewed the results and approved the final version of 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.


Using a double immunofluorescent technique, we observed that both IBV Massachusetts-type 41 (M41) and Connecticut A5968 (Conn A5968) strains replicate in avian macrophages at a low level in vivo. This in vivo observation was substantiated by demonstrating IBV antigens in macrophages following in vitro IBV infection. Further, IBV productive infection in macrophages was confirmed by demonstrating corona viral particles in macrophages and IBV ribonucleic acid (RNA) in culture supernatants. Evaluation of the functions of macrophages following infection of macrophages with IBV M41 and Conn A5968 strains revealed that the production of antimicrobial molecule, nitric oxide (NO) is inhibited. It was also noted that replication of IBV M41 and Conn A5968 strains in macrophages does not interfere with the induction of type 1 IFN activity by macrophages. In conclusion, both M41 and Con A5968 IBV strains infect macrophages in vivo and in vitro resulting productive replications. During the replication of IBV in macrophages, their ability to produce NO can be affected without affecting the ability to induce type 1 IFN activity. Further studies are warranted to uncover the significance of macrophage infection of IBV in the pathogenesis of IBV infection in chickens.Infectious bronchitis virus (IBV) causes respiratory disease leading to loss of egg and meat production in chickens. Although it is known that macrophage numbers are elevated in the respiratory tract of IBV infected chickens, the role played by macrophages in IBV infection, particularly as a target cell for viral replication, is unknown. In this study, first, we investigated the ability of IBV to establish productive replication in macrophages in lungs and trachea  Infectious bronchitis (IB) is primarily a respiratory disease of chickens but with potential to cause more widespread infection in the urinary and reproductive tracts in chicken leading to significant production losses in commercial broiler and layer flocks worldwide . The cauIBV is known to replicate in the respiratory tract leading to changes in the muco-cilliary clearance mechanism, as such, expose the IBV infected birds to secondary bacterial infections. Additionally, IBV has tropisms for a variety of tissues. However, the mode of dissemination from the common route of entry, i.e. the respiratory route, to the rest of the body systems could potentially be due to the initial viremia . Once diin vitro studies indicated that IBV, particularly nonpathogenic Beaudette and Massachusetts type 82822 strains do not replicate in avian macrophages [Macrophages play roles in innate immune responses, as well as in mounting adaptive immune responses by functioning as antigen presenting cells, as such they are critical in protecting animals from microbial infections. Although it is known that macrophage numbers are elevated in the respiratory tract in response to IBV infection , the rolrophages .in vivo and macrophage cell cultures in vitro using two IBV strains, Connecticut A5968 (Conn A5968) and Massachusetts-type 41 (M41) which are known to induce clinical disease and pathological lesions in chickens. As implicated in some other viruses, we hypothesized that these two strains of IBV replicate within avian macrophages leading to productive replication and interfering with selected macrophage functions in the process.Therefore, in this study we investigated the interaction of IBV with macrophages in lungs and trachea The Veterinary Science Animal Care Committee (VSACC) and Health Science Animal Care Committee (HSACC) have approved the use of specific pathogen free (SPF) chickens used in all our experimental procedures. One day old unsexed SPF layer chickens (White Leghorn) were obtained from the Canadian Food Inspection Agency (CFIA), Ottawa, and raised to six days of age for use in these experiments. These chickens were not immunized and were housed in high containment poultry isolators at the University of Calgary’s Spyhill campus or at the Foothills campus, with ample access to a standard food ration and water.2 [Conn A5968 and M41 strains of IBV as well as avian fibroblast cells line (DF-1) were purchased from American Type Culture Collection . The macrophage cell line, Muquarrab Qureshi-North Carolina State University (MQ-NCSU), which is derived from mononuclear cells harvested from the spleen of a chicken  was kind2 . The ves50). Briefly, ten-fold serial dilutions of virus were prepared in PBS   and 100 μL of the dilutions were inoculated into the allantoic cavity of eggs. The inoculated eggs were incubated at 37.2°C and relative humidity of 60% for another 6 days. On 6 dpi, the embryos were euthanised and examined for the presence of embryo dwarfing and curling, which is characteristic for IBV infection. Based on these observations, end points were determined [50/mL.IBV M41 and Conn A5968 strains were propagated in 9-day old SPF embryonated eggs and allantoic fluid harvested at 3 days post-infection (dpi). Viral titers were determined using 9 days old SPF embryonated chicken eggs and expressed as fifty percent embryo infectious dose  or Conn A5968 (n = 5) strains of IBV at the dose rate of 2.75 x 10 or Conn in vitro:Infectability of avian macrophages with IBV  Macrophages (MQ-NCSU) were cultured on coverslips placed in 6-well plates (1.5x106 viable cells per well). After 24 hours cells were washed twice with Hank’s balanced salt solution (HBSS) and infected with 0.1 multiplicity of infection (MOI) of IBV M41 or Conn A5968 strains. After one hour, virus inoculum was removed, cells were washed twice with HBSS and supplied with fresh medium. At 24 hours post-infection (hpi), each section was subjected to two-color immunostaining to co-localize macrophages and IBV antigens. Each experiment was performed in triplicate including uninfected controls.in vitro leading to productive infection:Infectability of avian macrophages with IBV  Macrophages (MQ-NCSU cells) were suspended in LM-HAHN media and seeded in 6-well plates at a density of 1.5x106 cells per well and incubated at 37°C in 5% CO2 for 24 hours. The confluent monolayers were washed twice with HBSS and infected with IBV M41 or Conn A5968 strains at an MOI of 0.1 or mock infected in triplicate. At one hour, the inoculum was completely removed, the cells were washed twice with HBSS in order to remove potential residual inoculum, and incubated with serum-free 2X MEM media  at 37°C, 5% CO2. At 6, 12, 18, 24 and 48 hpi, cell culture supernatants derived RNA was used to quantify IBV RNA following IBV infection of macrophages. The experiment was repeated three times.Detection of IBV particles in avian macrophages: Macrophages (MQ-NCSU cells) were cultured on T25 culture flasks until 90% confluence was achieved. After 24 hours, the cells were washed twice with HBSS and infected at an MOI of 0.1 with IBV M41 or Conn A5968 strains or mock infected. Each experiment was performed in triplicate. After one hour, the plates were washed twice with HBSS and incubated with serum-free 2X MEM media for 24 hours at 37°C and 5% CO2 before harvesting cells for transmission electron microscopy (TEM) imaging.IBV interference with nitric oxide (NO) production and type 1 interferon (IFN) activity during the infection of macrophages: To determine whether type 1 IFN activity or NO production is induced in macrophages following IBV infection in vitro, MQ-NCSU cells were seeded in 12-well plates at 0.75 x 106 cells per well and incubated for 24 hours. Subsequently, they were either infected or treated with 0.1 MOI of IBV M41 strain, 0.1 MOI of IBV Conn A5968 strain, 1μg/ml lipopolysaccharide (LPS)  as a positive control for NO induction or double stranded (ds) RNA  50μg/ml as a positive control for type 1 IFN induction. RPMI media was used as the negative control. All experiments were done in triplicates and repeated three times. Twenty-four hpi or treatment, culture supernatants were preserved at -80°C for subsequent quantification of NO production or type 1 IFN activity.The Diagnostic Services Unit of the University of Calgary Faculty of Veterinary Medicine processed the tissues kept in 10% formalin and provided with us with stained hematoxylin and eosin (H&E) sections for evaluation.Trizol LS reagent  was used for RNA extraction from 250 μl of macrophage-culture supernatants following manufacturer’s instructions. The RNA pellet was re-suspended in 20 μl of RNase-free water. The RNA concentration was quantified using the Nanodrop 1000 spectrophotometer at 260 nm wavelength . The RNA (2000 ng) was transcribed using 10X RT random primers  as has been instructed by the manufacturer.5’GACGGAGGACCTGATGGTAA3’ and reverse primer, IBV-N Re: 5’CCCTTCTTCTGCTGATCCTG3’ [® Green Master Mix  with 5 nM of each of the gene-specific primers, 200 ng of cDNA derived from each sample and RNAse-free water were used in the reaction in a final reaction volume of 20 μL. A positive control (plasmid) and negative template control (NTC) (RNAse-free water) and no reverse transcriptase control (NRT), and the dilution series of the N-gene plasmid as previous described [10 copies/reaction to 1 log10 copies/reaction in the standard curve.A quantitative PCR (qPCR) protocol was used for IBV RNA determination in the cell culture supernatants using forward primer, IBV N Fw: Double immunostaining of cryosections: Frozen tissue sections (5 μm) from OCT preserved tissues were adhered on to positively charged slides, fixed with acetone (4°C) and stained for macrophages and IBV antigens sequentially. After protein blocking with 5% goat serum, the cryosections were incubated for 30 minutes with mouse monoclonal anti-chicken macrophage (KUL01) antibodies  (1:200 in PBS containing 5% goat serum) and washed three times after incubation. These sections were then stained with the secondary antibody, DyLight® 550 conjugated goat anti-mouse IgG (H+L)  (1:500 in PBS containing 5% goat serum). Before being stained for IBV, the sections were blocked for endogenous avidin /biotin . A solution of 5% goat serum was used for 30 minutes for blocking followed by 30 minutes incubation using anti-IBV rabbit polyclonal serum (1:3000) . Rabbit IgG was used as the isotype control  (1:500). Goat biotinylated anti-rabbit IgG (H+L) (1:400)  was used as the secondary antibody. Avidin conjugated with Dylight® 488 (Streptavidin)  was used for visualization. In the final step, the sections were mounted on Vectashield® antifade mounting medium with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) nuclear stain . All incubations were carried out at room temperature in a humidifying chamber to prevent drying the sections while on incubation.in vitro:IBV antigen quantification in macrophages  First, macrophages grown on coverslips were subjected to cell wall staining with wheat germ agglutinin conjugated with Alexa Fluor® 594  as has been instructed by the manufacturer. Then, the cells were fixed with 4% paraformaldehyde before being treated with 0.2% Triton X-100  for permeabilization. Avidin, biotin and protein blocking was followed by incubation with mouse anti-N monoclonal antibody  for 30 minutes (1:400) followed by goat anti-mouse IgG (H+L), which was conjugated to biotin  (1:400) for 20 minutes and by avidin conjugated with Dylight® 488 (Streptavidin) (15 μg/ml)  for 20 minutes. Finally, coverslips were mounted on glass slides with Vectashield® antifade mounting medium with DAPI nuclear stain  and sealed with lacquer.via an acetone gradient. Sections of 60 nm were cut in a Leica EM UC7  using a diamond knife. These sections were stained with 4% uranyl acetate and Reynolds’s lead citrate and analyzed with a Hitachi H7650 TEM at 80 kV. Images were obtained using an AMT 16000 digital camera. For obtaining high-resolution images, an EFI Tecnai F20 electron microscope was used.Macrophages (MQ-NCSU cells) infected with M41 or Conn A5968 strains of IBV were harvested by scraping, centrifuged at 1000 g at 4°C for 10 minutes and submitted to the imaging facility of the University of Calgary for transmission electron microscope (TEM) imaging. For TEM, the cell pellets were immersed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer at pH 7.4 at 4°C for 2 hours. Then cells were embedded in Epon 812 mixture following treating the cells with 1% osmium tetroxide buffered with cacodylate and dehydrating The manufacturer’s instructions were followed for quantification of NO concentrations using the Griess assay reagent system . Briefly, cell culture supernatants were mixed with an equal volume of Griess assay reagent (1% sulfanilamide and 0.1% N-1-naftyletylendiamin dihydrochloride) in 96-well plates and incubated at room temperature for 10 minutes. The mean absorbance at 548nm was determined using SpectraMax M2 microplate reader . Nitrite concentration of each sample was estimated based on sodium nitrate standard curve.Culture supernatants of macrophages collected following IBV infection or treatment with dsRNA and LPS (250μl) were transferred to DF-1 cells grown for 24 hours. At 24 hours of culture supernatant transfer, these DF-1 cells were infected with VSV-GFP at an MOI of 0.1 and at 24 hpi the GFP signal was quantified under epifluorescent microscope following fixation with 4% paraformaldehyde and nuclear staining with Hoechst 33342 .Following collection of MQ-NCSU culture supernatants, the cells were scraped into fresh media. The cell suspensions were centrifuged at 1000 rpm for 10 minutes at 4°C. The cell pellet was re-suspended in 100 μl of growth media and maintained in ice until counted. The number of live and dead cells in each of the suspension was enumerated using a hemocytometer after staining with Trypan blue. The number of dead cells in each well was presented as a percent of the total number of cells in the respective well.IBV RNA copies per 200 ng of cDNA were calculated following performing qPCR assay. A standard curve based on a serial dilution of plasmids was used for the calculation of IBV copies.® 550 signals were captured along with applicable nuclear stained (DAPI) areas. Similarly, for the quantification of IBV antigens, 5 areas (x40 magnification) with DyLight® 488 fluorescent signals were captured along with applicable nuclear stained areas. The signals were captured using an Olympus IX51 inverted epifluorescent microscope and quantified using Image-J® software .For the quantification of macrophage numbers, 5 areas (x40 magnification) with highest DyLightStudent’s t- test or one-way analysis of variance (ANOVA) test followed by Tukey’s were done using the Graphpad software  for the confirmation of group differences. The differences were considered significant at P<0.05.Chickens infected with M41 or Conn A5968 strains of IBV showed transient and nonspecific clinical signs starting at 3 dpi that included huddling under the lamp, increased respiration and ruffled feathers. All the tracheal and lung sections of IBV Conn A5968 and M41 infected chickens collected on 4 dpi were positive for IBV antigen expression . HistoloThe macrophage quantification data and representative immunofluorescent images of trachea and lung of IBV infected and control chickens are illustrated in Double immunofluorescent assay revealed that IBV antigens were detectable in 3.6% (±0.8) and 3.3% (±0.7) of trachea and lung macrophages respectively in IBV M41 strain infected chickens . In IBV in vitro study 13.6% (± 1.6) and 21.97% (± 4.3) of the macrophages were infected when these cells were infected with IBV M41 and Conn A5968 strains respectively  increase in IBV RNA in culture supernatants of MQ-NCSU cells peaking around 18 hours. Although it is possible that higher MOI of IBV may lead to higher increase in IBV RNA in culture supernatants [As evidenced by the With the finding of replication of M41 and Conn A5968 strains in macrophages, further studies are required in order to see whether the IBV established infection in macrophages is strain dependent. In general, many members of the family Coronaviridae target macrophages as a site of viral replication. However, as shown before this was not the case for Beaudette and Massachusetts type 82822 strains of IBV . Althougin vitro. This observation is in agreement with the observation made in murine macrophages using H5N1 and H1N1 influenza viruses [in vitro although inhibition of type 1 IFN production has been shown by other corona viruses such as porcine epidemic diarrhea virus [via the inhibition of signal transducer and activator of transcription (STAT)1 in epithelial and fibroblast cells [Avian macrophages are known to produce antiviral molecules such as NO and type 1 IFN , 29. The viruses , swine m viruses  and avia viruses  infectioea virus , porcineea virus , SARS-Coea virus  and muriea virus . Althougea virus –45. Althst cells , 45, furin vivo. However, we could not extend our observations further to elucidate the consequences of IBV-macrophage interaction in chickens. Although M41 and Conn A5968 strains of IBV infections first establish infections in the respiratory tract, these two strains of IBV are known to replicate in a variety of tissues leading to pathology in these tissues [In the current study, we observed that M41 and Conn A5968 strains of IBV replicate in macrophages  tissues , 46. Sim tissues , demyeli tissues  and HIV  tissues  and MERS tissues . SimilarAs illustrated in the in vivo observations of co-localization of IBV and macrophage antigens in vitro using IBV infected monolayers of macrophages. Use of macrophage monolayers precluded the possibility of false positive co-localization of IBV and macrophage antigens due to overlapping cells in vitro.It is important to note that most of the IBV-stained cells were epithelial cells and only 3–4% of the macrophages contained IBV antigen as indicated by overlapping fluorescent signals. In lung and trachea sections, there is a possibility of false positive co-localization due to fluorescent signals originating from two or more overlapping cells rather than fluorescent signals originating from a single cell. This potential false positive co-localization of fluorescent signals was minimized in a number of ways. First, lung and trachea sections were cut at 5μm thickness which is substantially smaller than the size of macrophage and epithelia cells. Second, we used a double immunostaining technique to stain both macrophages and IBV antigens sequentially in a single section. These double immunostained sections were observed in two separate channels and then merged to visualize the co-localization. Third, we confirmed the in vivo and in vitro. It was also noted that the process of IBV replication in macrophages inhibits the NO production but not the type 1 IFN activity of macrophages. Further studies are required to determine the significance of avian macrophage-IBV interactions in the pathogenesis of IBV infection in chickens.In conclusion, our data show evidence for the fact that avian macrophages support IBV replication