We demonstrate substantial lowering of tau phosphorylation at multiple sites implicated in disease, suggesting that TTBK1 inhibitors may represent an exciting new approach in the search for neurodegenerative disease therapies.Hyperphosphorylated tau protein is a pathological hallmark of numerous neurodegenerative diseases and the level of tau pathology is correlated with the degree of cognitive impairment. Tau hyper-phosphorylation is thought to be an early initiating event in the cascade leading to tau toxicity and neuronal death. Inhibition of tau phosphorylation therefore represents an attractive therapeutic strategy. However, the widespread expression of most kinases and promiscuity of their substrates, along with poor selectivity of most kinase inhibitors, have resulted in systemic toxicities that have limited the advancement of tau kinase inhibitors into the clinic. We therefore focused on the CNS-specific tau kinase, TTBK1, and investigated whether selective inhibition of this kinase could represent a viable approach to targeting tau phosphorylation in disease. In the current study, we demonstrate that TTBK1 regulates tau phosphorylation using overexpression or knockdown of this kinase in heterologous cells and primary neurons. Importantly, we find that TTBK1-specific phosphorylation of tau leads to a loss of normal protein function including a decrease in tau-tubulin binding and deficits in tubulin polymerization. We then describe the use of a novel, selective small molecule antagonist, BIIB-TTBK1i, to study the acute effects of TTBK1 inhibition on tau phosphorylation  In addition, our data implicates TTBK1 as the major kinase responsible for the phosphorylation of tau at S422 in the brain, a modification almost completely absent from normal adults but significantly elevated across a multitude of neurodegenerative diseases. Therefore, we believe that due to the CNS restricted expression of TTBK1, pharmacological inhibition of this kinase represents a promising therapeutic approach in the treatment of tauopathies.The results described here are of significance because they represent the first demonstration, to our knowledge, that acute inhibition of TTBK1 with a small molecule inhibitor can lead to significant reductions in tau phosphorylation Tau is a neuron-specific, microtubule-associated-protein (MAP) that plays a key role in regulating tubulin polymerization. Tau-tubulin binding arises from ionic interactions between basic and acidic regions on the tau and tubulin molecules, respectively ,2. This Identifying kinases involved in the pathological phosphorylation of tau may lead to promising clinical targets. However, the ubiquitous tissue expression of most kinases and the promiscuity of their substrates represent a hurdle for drug discovery. Tau-tubulin kinase (TTBK) is a dual serine/threonine and tyrosine kinase originally identified in a screen for proteins enriched in the human brain . TTBK haC. elegans [Drosophila melanogaster [Accumulating evidence has linked the expression or activity of the tau kinase TTBK1 with neurodegenerative diseases. (1) Two independent studies in small cohorts of Spanish and Han-Chinese populations have identified multiple single nucleotide polymorphisms (SNPs) in the TTBK1 gene that are proposed to reduce protein expression and are associated with a decreased risk of late onset Alzheimer’s disease ,20. (2)  elegans , and Dronogaster . Therefoin vivo. These results represent the first demonstration that acute TTBK1 inhibition is a viable therapeutic approach for lowering tau phosphorylation.In the current studies, we set out to determine whether acute inhibition of TTBK1 could represent a viable strategy for lowering tau phosphorylation in disease. First, we demonstrate in both HEK293 cells and primary neuron cultures that the overexpression or knockdown of TTBK1 regulates the phosphorylation of tau at disease relevant sites. Importantly, we show that the TTBK1-specific phosphorylation of tau leads to a decrease in tau-tubulin binding and subsequent deficits in tubulin polymerization. We demonstrate that acute treatment with a newly identified TTBK1 inhibitor, BIIB-TTBK1i, results in a dose dependent decrease in the phosphorylation of tau at several different sites in mice. By using chemical proteomics, we were able to show both TTBK1 target engagement and the exquisite kinome selectivity of BIIB-TTBK1i 50; Previous work using phospho-peptide mapping indicates that TTBK1 can phosphorylate tau at serine residues 198, 199, 202, 422 and at tyrosine 197; all sites enriched in paired helical filaments which characterize tauopathies –30. In aTau is a neuronal, microtubule associated protein that plays a key role during microtubule assembly . PreviouSince the binding of tau to microtubules is essential for promoting microtubule polymerization , we inveCTACTTCACCAAGCCCGATTA; TTBK1 shRNA sequence 2: ACATCAAGCCGTCCAACTTTG) was efficient in completely knocking down the TTBK1 transcript with no significant effect on the TTBK2 isoform . or oral (P.O.) dosing at 35 mg/kg. Despite a rapid clearance rate in mouse, our data shows that we were able to achieve free drug concentrations of BIIB-TTBKi above the cellular IC50 for the first hour after dosing . After dosing BIIB-TTBK1i at 25, 50, and 75 mg/kg IP in 2-month-old, male C57Bl/6 mice, mice were euthanized one hour following injection and homogenized brain lysates were labeled with Desthiobiotin-ADP-acyl phosphate probe (ADP probe). Lysates were trypsin digested and desthiobiotinylated peptides were captured on streptavidin beads. A list of 269 unique peptides were quantified through LC-MS/MS parallel reaction monitoring method. Target engagement was calculated as the percentage of peptide signal lost after addition of BIIB-TTBKi when compared to the vehicle treated lysate. Using the ADP probe, we were able to detect a total of 150 kinases in the mouse cortical brain lysates . We did not see an increase in tau phosphorylation at S422 after the overexpression of the potential off-target kinases of BIIB-TTBK1i . To further solidify that any effects of BIIB-TTBK1i on tau phosphorylation were specific to TTBK1 inhibition, we co-transfected HEK293 cells with human tau plus any of the identified off-target kinases previously identified to phosphorylate tau  was projected to achieve an IC80 of TTBK1 inhibition for the one hour of hypothermia treatment . This further confirms that S422, a site implicated in the early stages of neurofibrillary tangle formation in disease [Under basal conditions, tau phosphorylation is tightly regulated by opposing kinase and phosphatase activities. In mice, isoflurane induced hypothermia causes a dramatic increase in tau phosphorylation through the temperature dependent inhibition of PP2A . In the reatment . Similarreatment . Acute tof TTBK1 . These r disease , is likein vivo.Following isoflurane induced hypothermia, one cortical hemisphere was taken for western blot analysis and one cortical hemisphere was used to determine the levels of BIIB-TTBK1i in the brain. Our results demonstrate an inverse correlation between the amount of free drug detected in the brain and levels of tau phosphorylation at S422 . Based oin vivo. Our results show that TTBK1 can phosphorylate tau both directly and indirectly leading to a reduction in the affinity of tau for microtubules thereby preventing the enhancement of tubulin polymerization by tau. The restriction of TTBK1 expression to the CNS and its ability to phosphorylate tau at S422, a phosphorylation site previously shown to be increased in a variety of neurodegenerative diseases, makes TTBK1 an exciting target for the treatment of tauopathies.The reduction of tau phosphorylation through the inhibition of tissue specific kinases is an attractive therapeutic approach for the treatment of Alzheimer’s disease and other tauopathies. The data presented here represent the first proof of biology that acute inhibition of TTBK1, a CNS specific tau kinase, can lower tau phosphorylation at disease relevant sites 50 > 30μM) we were able to identify both direct and indirect phosphorylation sites of TTBK1 on tau. Our data are the first to identify several novel phosphorylation sites on tau that are either directly  or indirectly  regulated by TTBK1 activity. Importantly, we have confirmed in primary neuron cultures that TTBK1 knockdown can reduce tau phosphorylation levels at S422 when both tau and TTBK1 are present at endogenous levels. The indirect phosphorylation of tau which results from TTBK1 overexpression may occur through the activation of other kinases by TTBK1 or by the priming of tau through phosphorylation at other sites. Previously, Sato et al., [http://cnr.iop.kcl.ac.uk/hangerlab/tautable). Further work is needed to determine if TTBK1 overexpression in this cell system also initiates aberrant CDK5 activity driving indirect tau phosphorylation.In a cell free system, numerous kinases have been reported to phosphorylate tau; however, these interactions may not reflect physiological kinase-substrate interactions which rely on factors such as cell compartment localization, kinase phosphorylation state, and substrate priming. Our results show that in HEK293 cells, co-expression of human full-length TTBK1 and human Tau (2N4R) leads to a significant increase in tau phosphorylation at numerous sites spanning the entire tau protein sequence. Making use of the fact that the pan kinase inhibitor staurosporine has almost no activity on TTBK1 catalytic activity  are known to co-segregate with the disease of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Preclinically, a number of MAPT mutations have been shown to effect tau-tubulin binding [The main physiological role of tau appears to be in the promotion of microtubule assembly  and prev binding  and prom binding . These o binding , nuclear binding , pre-syn binding  and post binding . Therefoin vivo target engagement and the off-target profile of BIIB-TTBK1i, we used the chemoproteomics platform—KiNativ [The presence of intraneuronal tangles composed of hyperphosphorylated tau are a pathological hallmark of many neurodegenerative diseases grouped as tauopathies ,7. Many —KiNativ ,51. ThisMouse models of Alzheimer’s disease commonly use the transgenic overexpression of genes involved in the production of amyloid β  and/or Tau (MAPT) and many of these models have been shown to recapitulate the hyperphosphorylated tau tangles characteristic of disease ,53. HowePrevious experiments have indicated that TTBK1 protein expression is significantly elevated in brains of both AD  and FTLDin vitro and in vivo data indicate that S422 is a direct phosphorylation site of TTBK1 on tau. Previous studies have shown that phosphorylation of tau at S422 is almost completely absent in cognitively normal adults and in the fetal brain [in vitro and in vivo models, TTBK1 inhibition almost completely abolished phosphorylation at this site. Therefore, our data implicates TTBK1 as the major kinase responsible for the phosphorylation of tau at S422 in the brain.Together, our al brain ,65). In al brain ,67 and tal brain . Intrigual brain –70. Veryal brain ,72 and iin vivo can lead to significant reductions in tau phosphorylation. Therefore, we believe that due to the CNS restricted expression of TTBK1, pharmacological inhibition of this kinase represents a promising therapeutic approach in the treatment of tauopathies.In conclusion, although both the tau species and the cellular mechanisms that lead to tau toxicity are unknown, it is clear that tau pathology correlates with disease progression and that the hyper-phosphorylation of tau is an early event in this disease cascade. Our data demonstrates that TTBK1, a CNS specific kinase, phosphorylates tau at several sites upregulated in disease including S422. These results are the first demonstration that acute inhibition of TTBK1 with a small molecule 2/air environment at 37°C. When the cells reached 50–80% confluence, they were transfected with a complex consisting of a 3:1 ratio of FuGENE 6 Transfection Reagent  and plasmid DNA. Plasmid DNA was balanced out across reactions using an empty plasmid so that the same amount of DNA was added per well. This transfection complex was prepared following the manufacturers protocol and added directly to the cell media for 24 or 48 hours prior to lysis.HEK293 cells obtained from American Type Culture Collection  were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 units/ml penicillin, and 10μg/ml streptomycin . Cell cultures were maintained in a humidified 5% (v/v) COSamples were lysed in Pierce RIPA lysis and extraction buffer  supplemented with 1% Halt protease and phosphatase inhibitor cocktail  and centrifuged at 14000 x g for 20 min at 4°C to clear the lysate. Protein concentrations were determined using the Direct Detect Infrared Spectrometer  and 10μg of each sample was denatured in 6X SDS sample buffer  for 7 minutes at 90°C. Proteins were loaded into a Criterion 7.5% tris-glycine gel  and separated by SDS-PAGE at 120V for 120 minutes. The gel was transferred to an IBlot2 nitrocellulose membrane , blocked with TBST blocking buffer  for 1 hour, and washed three times with TBST. The membrane was probed with primary antibodies (1:1000 dilution) in antibody dilution buffer (1:1 TBST blocking buffer and 1X TBST) overnight at 4°C. The following antibodies were used for immunoblotting: TTBK1 , Tau-5 , phospho-Tau S422 , S198 , AT8 S202/T205 , S214 , AT180 T231 , S262 , S356 , S396 , GAPDH , and β-actin . The blot was then washed in triplicate with TBST and incubated for 1 hour with secondary antibody  in antibody dilution buffer. After a final triplicate wash with TBST, the blot was visualized using the Odyssey CLx imaging system .Samples were lysed in RLT buffer  + 0.1% β-mercaptoethanol, transferred to individual QIAshredders  and homogenized via centrifugation at 17000 x g for 15 minutes. The supernatant was collected, and total RNA was extracted using the RNeasy mini kit  and RNase-free DNase set  following the manufacturers protocol. Total RNA concentration was determined using the Nanodrop 8000 spectrophotometer  and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit  using up to 2μg of RNA as a template. 50ng of the resulting cDNA product was subjected to duplex PCR reactions using Gene Expression Master Mix  containing Taqman primers for TTBK1 (Cat# Mm01269698), TTBK2 (Cat# Mm00453709), and housekeeping gene GAPDH . Real-time PCR reactions were performed on the Via7 Real-time PCR system  using the following thermocycling conditions: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Relative gene expression levels of TTBK1 and TTBK2 were calculated using the ΔΔCt method.All steps of the microtubule sedimentation assay were performed at 37°C to maintain the microtubule network of the samples. Samples were lysed at 37°C for 10 min in warm 1:1 Lysis and Microtubule Stabilization Buffer  and water, supplemented with 20μM taxol, 1mM ATP, 100μM GTP, and 1% protease inhibitor cocktail . The lysate was syringe sheared ten times and spun at 2000 x g at 37°C for 5 min to clear out debris. The pre-cleared supernatant was then subjected to ultracentrifugation at 100,000 x g for 1 hour at 37°C to separate the soluble fraction and the microtubule pellet. These fractions were denatured and analyzed for relevant proteins using SDS-PAGE.Co-transfected HEK293 cells or recombinant proteins were lysed in cold General Tubulin Buffer  with 0.1% Triton X100, 0.1% Tween20, and 1% Halt protease and phosphatase inhibitor cocktail  for 30 min at 4°C before being centrifuged at 17,000 x g for 15 min at 4°C to pellet debris. Protein concentrations were determined using a Direct Detect Infrared Spectrometer  and 10μl of protein at 1.5μg/μl were loaded into each well of a 96-well black coated assay plate . 50μl of tubulin polymerization mix, consisting of 205μl fluorescent reporter Buffer 1, 150μl Tubulin Glycerol buffer, 4.4μl 100mM GTP, and 85μl 10 mg/ml tubulin . The plate was transferred to a Spectramax M2 microplate reader  set at 37°C and the rate of microtubule polymerization was determined by measuring fluorescence  every minute for 60 minutes.HEK293 cells were transfected with plasmids encoding Tau and TTBK1 were seeded 24 hours later into 384-well plates at 10,000 cells per well. After an additional 24 hours, the transfected cells were treated with compounds or DMSO and incubated at 37°C for an hour. Cell were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors . Phosphorylation of Tau at residue S422 was measured by FRET assay using the anti-Tau and anti-phospho S442 Tau antibodies conjugated with d2 (acceptor) and Tb cryptate (donor), respectively . The fluorescence level was measured using an EnVision plate reader .Guide for the Care and Use of Laboratory Animals. For in vivo experiments, subjects were anesthetized with isoflurane to induce hypothermia and were sacrificed after an hour. Subjects were kept under anesthesia the entire duration of hypothermia to ensure no pain or distress. These protocols were approved by the Biogen IACUC committee.All experiments were conducted in compliance with the rules set forth by the Biogen Institutional Animal Use and Care Committee in accordance with the guidelines established in the National Institutes of Health 3, and 1mM Hepes . The meninges were removed, and the cerebral cortices were dissected from the rest of the brain. The cortices were finely chopped, washed in triplicate with ice-cold HBS, and enzymatically digested with 5 mls of digestion buffer , plus 4mg of trypsin  per cortex and 4.5KU Dnase I  for 10 min at 37°C. The digested neurons were washed three times with HBS + 20% FBS, followed by three washes with HBS. The cells were dissociated in 5mls of trituration buffer (HBS with 2.95g/L MgSO47H20) plus 4.5KU DNase I  using 60 strokes of a 1ml pipette tip and spun at 300 x g for 10 min at 4C. The trituration buffer was aspirated, and the pellet was resuspended in culture media, consisting of Minimum Essential Media  with 5g/L glucose, 0.2g/L NaHCO3, 0.1g/L transferrin , 5% FBS, 500μM L-glutamine , 2% B-27 , 10 units/ml penicillin and 10μg/ml streptomycin . After filtering out cell debris using a 40μM filter , 175,000 live cells were plated into each well of a 12-well pre-coated poly-D-lysine plate  and incubated at 37°C in a humidified 5% (v/v) CO2/air environment. Every subsequent two days, the media was supplemented with an additional quarter volume of fresh culture media for maintenance. For lentiviral knockdown experiments, two TTBK1 shRNA sequences were cloned into the pLKO1 backbone . Lentivirus was made in HEK293T cells and added onto primary neurons at DIV2.Primary cultures of mouse neurons were prepared using the brains of E18, CD-1 mouse embryos. Following decapitation, the embryo brains were extracted and transferred to a dish with ice-cold 20% FBS  and HBS, consisting of Hank’s Balanced Salts , 0.35g/L NaHCOSeven-week-old male C576Bl/6 mice were used for all hypothermia experiments . Hypothermia was induced by exposure to vaporized isoflurane in an anesthetic induction chamber attached to a V-1 Tabletop Anesthesia System . The mice remained in the chamber for 60 minutes with a constant isoflurane concentration of 2%. Temperature measurements were obtained via rectal probe  immediately before and after anesthesia to ensure successful induction of hypothermia. Normally the animals had a body temperature drop to a mean of 27.6°C over the one hour of anesthesia.2EDTA-treated BD Microtainers . Mice were sacrificed via decapitation and brains were quickly removed and bisected sagitally. The right cortices, were isolated, placed into 2 mL microtubes along with a single 5 mm stainless steel bead and the left cortices were placed into 2 mL Lysing Matrix A tubes . Both tubes were immediately snap frozen in liquid nitrogen. The whole blood-containing microtainers were centrifuged  and plasma was transferred to 96-well plates for pharmacokinetic determination. Microtubes containing the right cortices and beads were placed into a TissueLyser II  with Pierce RIPA buffer  + Halt Protease and Phosphatase Inhibitor Cocktail  + PhosSTOP  + cOmplete Mini  + PMSF  in a 5:1 ratio of buffer volume to tissue weight. The samples were shaken at 30 Hz for 2 minutes. Upon cycle completion, lysates were transferred to individual QIAshredders  and centrifuged at 17000 x g for 15 minutes. Following centrifugation, the supernatant was carefully transferred to a set of fresh 1.5 mL microcentrifuge tubes. The protein content of each sample was quantified using a Direct Detect Infrared Spectrometer .Following hypothermia induction, 200–300 μL of blood was collected via cardiac punch and transferred to KLinear regression was performed to investigate that relationships between free drug concentration (nM) and either target engagement (measured by the percentage of ATP/ADP peptide probe inhibition) or the phosphorylation level at S422 (represented as a percentage of the vehicle treated control). In the hypothermia model the level of tau phosphorylation at each epitope was normalized to the amount of total tau within a sample. An ordinary one-way ANOVA was used to test for a drug treatment effect on the level of tau phosphorylation at each epitope separately. A Tukey’s multiple comparisons test was used to look for effects between groups–using the vehicle treated hypothermia group as the control.S1 Methods(DOCX)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


Ursus maritimus) in Hudson Bay to examine their sea ice habitat using Advanced Microwave Scanning Radiometer 2 data during sea ice breakup in May through July from 2013–2018. Spatial autocorrelation resulted in better model fits across 64% of individuals, although both metrics were more effective in describing movement patterns than habitat selection. Variation in local spatial autocorrelation allows for the visualization of sea ice habitat at complex spatial and temporal scales, condensing a targeted time period of habitat that would otherwise have to be analyzed daily.Habitat fragmentation occurs when continuous habitat gets broken up as a result of ecosystem change. While commonly studied in terrestrial ecosystems, Arctic sea ice ecosystems also experience fragmentation, but are rarely studied in this context. Most fragmentation analyses are conducted using patch‐based metrics, which are potentially less suitable for sea ice that has gradual changes between sea ice cover, than distinct “long‐term” patches. Using an integrated step selection analysis, we compared the descriptive power of a patch‐based metric to a more novel metric, the variation in local spatial autocorrelation over time. We used satellite telemetry data from 39 adult female polar bears ( Sea ice fragmentation does not create distinct patches in the same way terrestrial fragmentation does. We compared traditional patch‐based metrics to our novel spatial autocorrelation metric using integrated step selection analyses to determine which metric was better for predicting polar bear movement and habitat selection. Spatial autocorrelation provided a better model fit for 64% of individuals. As the For ice‐associated species, variation in sea ice cover  and open water  are comparable to patches in fragmented terrestrial landscapes  are strongly associated with ice fragmentation as they only stay in the marine environment when sea ice is present  female polar bears were immobilized via remote injection of Zoletil®  following standard protocols  on the GCOM‐W1 platform with a resolution of 6.25 km × 6.25 km grid cells from 2013 to 2018  as a radius to define an area of potential daily use. This resulted in an area of 1,385 km2, which we translated into ~35 6.25 × 6.25 km pixels and rounded to a 25 pixel (5 pixel × 5 pixel) moving window, because local analyses required a central pixel.To create our local habitat fragmentation metrics for comparison we used a moving window around each pixel to scale fragmentation to the local potential habitat an individual bear could experience in one day. We used the mean daily distance travelled by collared bears in the study during breakup , for each pixel in the study area across early breakup was calculated and the resulting values were plotted. SASD was calculated for late breakup, resulting in two SASD raster files per year. Pixels with high SASD reflect locations where sea ice cover is highly variable and fragmentation most dynamic.To assess the temporal dynamics of fragmentation we used the variation in spatial autocorrelation at each pixel in space over time. In each year, the standard deviation of the Geary's landscapemetrics package in R  and percent habitat (PHAB), using FRAGSTATS metrics TE and PLAND via the 2.4Integrated step selection analysis (iSSA) furthers habitat selection analysis by incorporating movement parameters and defining availability by the distribution of the used movement metrics , and SASD for early and late breakup were extracted at the end of both used and random steps. We also included the square of daily ice cover to test for a nonlinear relationship. We included the distance from each point to Cape Churchill on the west coast of Hudson Bay at the end of each step as a proxy for summer refuge (refuge) as a candidate covariate Figure . Cape ChCandidate environmental covariates for patch‐based models included daily sea ice cover (ice), PHAB, and TE, and were extracted at the end of used and random steps. Similar to above, the distance to summer refuge (refuge) was also a candidate covariate extracted at the end of each step. We also included PHAB and TE interacting with the natural log of step length, as well as PHAB and TE interacting with the cosine of turning angle, using PHAB and TE extracted at the start of each step to analyze movement. We examined all daily covariates for collinearity and excluded any with Pearson correlation values >|0.6| from the same models. Collinear covariates were tested in separate models and the one with better fit, determined by AIC, was retained. Individuals with all locations in regions of solely optimal or suboptimal habitat locally resulted in no variation in PHAB and were removed from further analysis.In each of the four model groups, SASD early, SASD late, PHAB/TE early, and PHAB/TE late, all combinations of all corresponding candidate covariates and interactions outlined above were tested for each individual. Akaike information criteria (AIC) was used for each individual in each group to determine model of best fit, where the top‐ranked model was >2 AIC lower than the next ranked model. Polar bears are individually variable . Mean breakup period lasted 63 days  and ranged from 44 days in 2014 to 81 days in 2015. The mean date for start of breakup was May 12 . The mean date for end of breakup was July 14 . The breakup period based on global PHAB was May 2 to July 23. The start of the second half of breakup was June 24, June 23, June 23, June 30, June 17, and June 26, in 2013–2018, respectively.The global percent of optimal sea ice habitat (PHAB) was similar across all years, with a significant sigmoidal trend in mean global PHAB during breakup  resulted in better model fits than patch‐based metrics. The iSSA models we tested were better at describing polar bear movement in response to sea ice fragmentation, reflected by the significant interaction between environmental covariate and turning angle, than habitat selection in response to sea ice fragmentation, reflected by the lack of significance in environmental covariates alone.Using variation in spatial autocorrelation over time to analyze sea ice fragmentation allows for a synthetic, yet complex spatial and temporal visualization of sea ice. SASD allowed for the summarization of habitat over time that would otherwise have to be considered on a daily scale, while still being an effective movement descriptor. Breakup is often defined as a single time span ; Formal analysis (lead); Methodology (lead); Writing‐original draft (lead); Writing‐review & editing . Erin Bayne: Conceptualization (supporting); Methodology (supporting); Writing‐review & editing . Nicholas J. Lunn: Funding acquisition ; Resources ; Writing‐review & editing (supporting). David McGeachy: Resources ; Writing‐review & editing (supporting). Andrew Edward Derocher: Conceptualization (supporting); Funding acquisition ; Methodology (supporting); Resources ; Supervision (lead); Writing‐review & editing . Supplementary MaterialClick here for additional data file.


PIK3CA with the neuronal stem/progenitor marker NES was associated with poor prognosis in PN GBM patients, indicating a unique role for PI3Kα in PN GSCs. Alpelisib inhibited GSC neurosphere growth and these effects were more pronounced in GSCs of the PN subtype. The antineoplastic effects of alpelisib were substantially enhanced when combined with pharmacologic mTOR inhibition. These findings identify the alpha catalytic PI3K isoform as a unique therapeutic target in proneural GBM and suggest that pharmacological mTOR inhibition may sensitize GSCs to selective PI3Kα inhibition.Glioblastoma (GBM) is the most common and lethal primary intrinsic tumour of the adult brain and evidence indicates disease progression is driven by glioma stem cells (GSCs). Extensive advances in the molecular characterization of GBM allowed classification into proneural, mesenchymal and classical subtypes, and have raised expectations these insights may predict response to targeted therapies. We utilized GBM neurospheres that display GSC characteristics and found activation of the PI3K/AKT pathway in sphere-forming cells. The PI3Kα selective inhibitor alpelisib blocked PI3K/AKT activation and inhibited spheroid growth, suggesting an essential role for the PI3Kα catalytic isoform. p110α expression was highest in the proneural subtype and this was associated with increased phosphorylation of AKT. Further, employing the GBM BioDP, we found co-expression of  Currently, maximal surgical resection followed by chemoradiation and adjuvant temozolomide treatment is the standard-of-care, resulting in a median overall survival of 14.6 months2. Because of the very poor outcomes and aggressive behaviour of these tumours, considerable efforts have been made to extensively characterize GBM at the molecular level. Initially, four different GBM subtypes were identified through comprehensive patient sample analyses4. However, single-cell transcriptome analysis revealed the existence of only three GBM subtypes designated as proneural (PN), mesenchymal (MES), and classical (CL) GBM5. Additionally, a report using epigenomic profiling allowed the sub-classification of GBM into six categories, based on distinct DNA methylation profiles6. Although these efforts have advanced our overall molecular understanding of GBM, they have not yet resulted in effective targeted approaches that might improve clinical outcomes.Glioblastoma (GBM), classified as WHO grade IV glioma, is the most prevalent and malignant primary brain tumour and is essentially incurable9. Brain CSCs play key roles in GBM progression because of their enormous capacity for proliferation, self-renewal, and multilineage differentiation; characteristics that are important for tumour-initiation in serial transplantation experiments11. In recent years, numerous studies provided strong evidence for glioma stem cells (GSCs) being associated with intratumoural cellular heterogeneity16 and plasticity20, both major factors contributing to the poor prognosis and recurrence of GBM patients. These characteristics have fuelled the concept that therapeutic approaches must include strategies tailored to target the GSC population to block GBM growth and prevent recurrence.GBM tumours are not homogeneous neoplasms but rather represent ecosystems that contain diverse neoplastic populations, including cancer stem cells (CSCs). CSCs are believed to contribute to recurrence in multiple cancer types, but the exact mechanisms underlying such recurrence are unclear21, while activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway is associated with poor prognosis in GBM patients22. Heterodimeric Class IA PI3Ks are composed of a catalytic subunit  and a p85-type regulatory subunit23. PI3Ks are antagonized by the tumour suppressor phosphatase and tensin homolog (PTEN)24. Hence, alterations in the PI3K and PTEN pathways can be frequently found in different human malignancies, including GBM. For instance, 89.6% of GBM tumours have at least one alteration in the PI3K pathway and 39% have two or more3. Specifically, 59.4% of GBM show mutations in PI3K genes or PTEN mutations/deletions with the majority affecting p110α and/or p85α subunits3. Therefore, alterations in the PI3K and PTEN network resulting in aberrant signalling activity may provide unique therapeutic opportunities for treatment interventions in GBM.Increasing evidence indicates that key aspects of CSC function are dependent on phosphatidylinositol-3 kinase (PI3K) signalling25. Also, in the perivascular niche, nestin expressing cells exhibit increased PI3K/AKT activation, indicating a role for PI3K/AKT in neural progenitor cells residing in their stem cell niche26. Hence, in mouse medulloblastoma models, PI3K/AKT promotes survival and radioresistance of CSCs in the perivascular niche27. Importantly, in GSCs the regulatory PI3K subunit p85 directly interacts with CD133, a marker for both neural stem cells and brain CSCs, and this CD133 association promotes activation of the PI3K/AKT pathway, providing further evidence for a distinct and crucial role for PI3K in GSCs28. Given that CD133 expressing cells contribute to glioma radioresistance17, inhibition of PI3Kα might be an effective approach for specific targeting of therapy resistant GSCs.There is evidence indicating that PI3K/AKT signalling stimulates gliomagenesis specifically in brain CSCs because neural progenitors expressing the progenitor/stem cell marker nestin are particularly prone to AKT-driven oncogenic transformation29. Studies investigating distinct roles of PI3K isoforms suggest that isoform-selective PI3K inhibitors might show better target selectivity, resulting in reduced toxicities23. Particularly, inhibitors targeting the alpha catalytic PI3K isoform have emerged as potentially effective but less toxic30. In fact, PI3Kα specific inhibitors have demonstrated encouraging efficacy in GBM31. Alpelisib (BYL719) is such a PI3Kα selective inhibitor that has demonstrated a favourable safety profile and encouraging activity in patients with solid tumours32. Additionally, in initial studies, alpelisib has shown antineoplastic effects in GBM and GSCs33. However, there is evidence suggesting that selective PI3Kα inhibition requires simultaneous targeting of additional pathways, such as mTOR signalling, to efficiently block compensatory survival signalling36. Cancer cell growth is largely dependent on lipid metabolic processes, which are tightly controlled by mTOR signalling, a pathway that is commonly perturbed in cancers37. In response to targeted intervention, multiple mechanisms can converge on mTOR to rewire these processes, thereby promoting cancer cell survival and therapy resistance38. This suggests mTOR represents a key target for combinatorial anti-cancer strategies.Given the essential contributions of PI3K/AKT for GSCs and GBM development, pharmacological PI3K inhibition might represent a promising strategy for this disease. However, pan-PI3K inhibitors have limited potential as they exhibit only a narrow therapeutic windowPIK3CA in the PN GBM subtype as compared to CL and MES subtypes. Moreover, elevated coexpression of PIK3CA and NES (the gene encoding nestin) was associated with poor prognosis in this GBM subtype. We employed PN and MES GSC lines and found greatly elevated phosphorylation of AKT in PN GSCs, indicative of increased PI3K activity. Concomitantly, neurosphere growth of PN GSCs was potently inhibited by alpelisib and these effects were significantly enhanced when combined with pharmacological mTOR inhibition. Altogether, we provide evidence for a distinct role for PIK3CA in PN GSCs that suggests increased vulnerability of this GBM subtype to PI3Kα targeted combinatorial approaches.In the present study we explored the roles of PI3Kα and mTOR in GBM and GSCs. Analysis of gene expression data indicated elevated expression of 32. Alpelisib inhibited PI3Kα signalling in a dose dependent manner, as reflected by the reduced phosphorylation of AKT on Ser-473 in U87  in U87  in GBM spheres as compared to their 2-D counterparts, suggesting activation of the PI3K/AKT pathway in GBM spheroids  spheroid cultures may more accurately reflect the complexity of solid tumours than simple 2-D cell monolayers45. Hence, curative approaches must include strategies tailored to target GSCs. To study the effects of selective PI3Kα inhibition in GSCs, we employed the GSC neurosphere model system. These neurosphere cultures propagated in serum-free medium under stem cell-permissive conditions produce multipotent neurospheres capable of self-renewal47 that are enriched for cells competent to initiate tumours48. All established patient-derived GSC lines cluster in groups representing the PN or MES subtypes only18. We used patient-derived GSC lines 83Mes, 157PN, AC17PN19 and JK1650 to determine the effects of alpelisib on GSC lines. Initial experiments determined the dose response effects on GSCs grown as neurospheres. We found that GSC lines of the MES subtype were less responsive with half maximal inhibitory concentration (IC50) values of over 10 µM for 83Mes 5. Using the GBM-BioDP portal (http://gbm-biodp.nci.nih.gov/)52 , we found that expression of PIK3CA, the gene encoding p110α, was significantly increased in the PN subtype , mesenchymal (MES), and classical (CL) GBMrms Fig. A. This e53. We found that concurrent overexpression of PIK3CA with the neural stem cell marker NES was significantly associated with poor prognosis in the PN subtype, but not in the CL or MES subtypes  lines as compared to their MES  counterparts  phosphorylation in the PN subtype as compared to CL and MES patient samples  was greatly reduced in JK16 and 83Mes GSC lines Fig. C, corrobrts Fig. C. To elurts Fig. 73 phosphrts Fig. 73 phosphrts Fig. 73 phosph3. Early reports indicated enhanced PI3K signalling is associated with poor prognosis in GBM22 and also described increased AKT signalling as one hallmark of poor outcome in the disease54. This raises the possibility of therapeutic targeting of PI3K for the treatment of GBM. As pan-PI3K inhibitors have limited potential due to a narrow therapeutic window29, we explored the effects of alpelisib, a PI3Kα selective inhibitor with a favourable safety profile that has shown promise in patients with a variety of solid tumours32. We found alpelisib inhibited phosphorylation of AKT(Ser473) and exhibited antineoplastic effects in GBM cell lines. These effects were enhanced when alpelisib was combined with pharmacological mTOR inhibition. This is in line with earlier studies, indicating mTOR activity can sustain survival after selective PI3Kα inhibition55. In order to study stem-like cancer cells in vitro, we propagated U87 and LN18 cell lines as spheroids in 3-D under stem cell conditions, as described before56. Resulting spheres exhibited substantially increased nestin expression, suggesting they adopted stem-like characteristics, consistent with previous reports57. Under these conditions, spheres exhibited greatly enhanced phosphorylation of AKT(Ser473), indicating increased PI3K/AKT pathway activation in this spheroid model. Importantly, this AKT phosphorylation on Ser-473 was efficiently blocked by alpelisib, indicating the alpha catalytic PI3K isoform is responsible for this increased AKT phosphorylation in GBM 3-D spheroids. This raises the possibility that stem-like cancer cells grown as 3-D spheroids are highly dependent on increased PI3K signalling for growth and survival. In line with this, alpelisib potently blocked spheroid growth and this effect was significantly enhanced when combined with pharmacological mTOR inhibition.Accumulating evidence suggests key roles for the PI3K/AKT/mTOR pathway in GBM, and this is underscored by the finding that roughly 90% of GBM exhibit at least one alteration in the RTK/PI3K/PTEN pathwayPIK3CA is highest in the PN subtype  with pluripotency markers PROM1, CD44 and the neural stem/progenitor marker NES exclusively in the PN subtype. This suggests a discrete role for the alpha catalytic PI3K isoform in the GSC population of this subtype. In line with this, RPPA analysis revealed significantly elevated phosphorylation of AKT on Ser-473 in patients with the PN subtype, as compared to MES and CL subtypes, which is consistent with previous reports3. Concomitantly, there was a trend towards poor prognosis associated with p-AKT(Ser473) in PN, but not in MES or CL patient samples. Together, these data indicate a distinct dependency of PN GBM on PI3Kα signalling, which may result in greatly enhanced vulnerability of GSCs of the PN subtype to selective PI3Kα inhibition  signalling in GBM59 and, thus, targeting GSCs might be important for improving GBM clinical outcomes45. Besides characterizing PI3Kα as a promising target in PN GBM, we additionally provide further evidence for key roles of PI3Kα signalling in GSCs. There has already been some evidence for alterations in PI3K or PTEN signalling contributing to brain CSC function. For instance, previous studies using Pten conditional knockout mice demonstrated increased self-renewal ability of PTEN-deficient neural stem/progenitor cells61. Reciprocally, PI3K activation is required to promote self-renewal in embryonic stem cells62, and neural progenitors are prone to AKT-driven oncogenic transformation25. Additionally, increased PI3K/AKT activation in brain CSCs27 and activation of PI3K in CD133+ GSCs28 stimulate radioresistance. Together, these findings suggest key roles of PI3K signalling in GSCs that may promote self-renewal and therapy resistance. Here, we define this key role of PI3K signalling in GSCs is exceptionally important in GSCs of the PN subtype. PN GSCs depict greatly increased PI3Kα activation as judged by enhanced p110α expression and substantially increased phosphorylation of AKT. In line with this, PN GSC lines show increased sensitivity to specific PI3Kα inhibition, as compared to MES GSCs  models may have important clinical-translational implications for the treatment of GBM.The antineoplastic effects of alpelisib can be enhanced by pharmacological mTOR inhibition in breast cancerPTEN mutated), LN18 (PTEN wild-type) and LN443 (PTEN mutated) GBM cells were grown in DMEM supplemented with 10% FBS (Thermo Fisher) and gentamycin (1 mg/ml). U87 and LN18 3-D spheroid cultures were propagated in cancer stem cell (CSC) medium consisting of DMEM/F12 supplemented with B27 (2%), EGF (20 ng/ml), bFGF (20 ng/ml), heparin (5 µg/ml) and gentamycin (1 mg/ml), as described previously67. Glioma stem cell (GSC) lines 83Mes, 157PN and AC17PN were subtyped and described before19. Also, the JK16 GSC line was described previously50. GSC lines were propagated as 3-D neurospheres in CSC medium. All cell lines were regularly tested for mycoplasma contamination and subjected to short-tandem repeat (STR) analysis (Genetica DNA Laboratories) to ensure genetic stability. U87, LN18 and LN443 were authenticated using published reference STR profiles. The most recent STR analysis was done in March/April 2020. The PI3Kα specific inhibitor alpelisib (BYL719) and the mTOR inhibitor OSI-027 were purchased from ChemieTek and both were dissolved in DMSO as a vehicle.For conventional 2-D adherent culture, U87 (68. The CytoSelect 96-Well Cell Transformation Assay (Cell Biolabs) was used to determine anchorage-independent growth as described68. For confocal laserscanning microscopy, U87 and LN18 cells and spheroids were processed and imaged as described before69.To determine cell viability, the WST-1 assay kit (Roche) was used as described69. ELDA was done as described before69 with the exception that GSCs were allowed to grow for 12 days. The Cytation 3 software (Gen5 v 2.09) was used to measure neurosphere diameters and only neurospheres with a diameter of ≥ 300 µm (83Mes), ≥ 50 µm (JK16), ≥ 75 µm (157PN), ≥ 75 µm (AC17PN), were scored positive for ELDA analysis (http://bioinf.wehi.edu.au/software/elda/)51.For neurosphere assays, cells were seeded into Ultra-Low Attachment Round Bottom 96-Well Plates (Thermo Fisher/Corning) at 500 cells per well and processed as described previouslyPIK3CA, PIK3CB and PIK3CD gene expression data were downloaded from the GBM Bio Discovery Portal (GBM-BioDP) (http://gbm-biodp.nci.nih.gov/) using the Extended Verhaak dataset52 and analysed using GraphPad Prism 8. Similarly, protein Reverse Phase Protein Microarray (RPPA) data of the Extended Verhaak dataset4 for p-AKT(S473) were downloaded and analysed using GraphPad Prism 8. Survival analysis of TCGA gene expression data for patients from the Verhaak Core4 was performed using the multigene prognostic index from the GBM-BioDP. For survival analysis interrogating the phosphorylation status of AKT on Ser-473, RPPA data from the Extended Verhaak dataset4 were analysed using the GBM-BioDP software. GraphPad Prism 8 was used for statistical analysis including calculating IC50 values. One-way analysis of variance (ANOVA) was used to compare more than two groups followed by Tukey test.2, 0.5% Triton, 10% glycerol, 0.5% sodium deoxycholate, pH 7.9) supplemented freshly with 1 mM 1,4-Dithiothreitol (DTT), phosphatase and protease inhibitors (Roche). Cell extracts were processed as described before69. Antibodies against phospho-AKT(Ser473), phospho-AKT(Thr308), AKT, p110α, and p110β were purchased from Cell Signalling Technology. Antibody for p110δ was from Abcam. Antibodies against GAPDH were purchased from Millipore. Antibodies against HSP90 and NF1 were purchased from Santa Cruz. Following incubation with primary antibodies, membranes were incubated with anti-mouse horseradish peroxidase (HRP)-conjugated antibody (BioRad), or anti-rabbit HRP-conjugated antibody  and anti-mouse-AF488 antibody (Thermo Fisher) simultaneously, and visualized in a ChemiDoc MP Imaging System (BioRad). Uncropped blots as well as a comprehensive list of antibodies used in this study can be found in Supplementary Information Cells or neurospheres were treated with alpelisib or OSI-027 at the indicated concentrations for 90 min. Cells or neurospheres were lysed in phosphorylation lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgClSupplementary Figures.Supplementary Information.


Injection-site reactions have been reported with biologicals. In this post hoc analysis of Phase 3 studies in participants with migraine, we provide a comprehensive overview and detailed summary of injection-site reaction with galcanezumab.Data were obtained from two randomised clinical studies in participants with episodic migraine (EVOLVE-1 and EVOLVE-2), one randomised study in participants with chronic migraine (REGAIN) and one open-label study (Study CGAJ) in participants with episodic or chronic migraine. The injection-site reactions were measured for two different cohorts: 1) six-month double-blind treatment phase in the EVOLVE-1 and EVOLVE-2 studies and three-month double-blind treatment phase in the REGAIN study, where participants received placebo and galcanezumab ; 2) three month double-blind  + 9 months open-label extension phase (Month 3 to Month 12) of REGAIN and twelve month open-label phase of Study CGAJ, where participants received only galcanezumab .P < 0.001) higher in participant receiving galcanezumab versus placebo. In the galcanezumab exposure analysis set participants received up to 12 doses and the frequency of injection-site reactions reported for both doses combined was 21.8%. The reporting of injection-site reactions did not increase with the number of doses received. No ISR-related serious adverse events were reported in both the placebo-controlled and galcanezumab exposure analysis sets.A total of 477 participants in the placebo-controlled analysis set  reported at least one injection-site reaction. Most of the injection-site reactions were reported as injection-site pain, unspecified injection-site reaction, injection-site erythema, and injection-site pruritus. The incidence of injection-site pain was highest among all reported injection-site reactions and were reported with similar frequency by participants receiving galcanezumab  and placebo (9.5%) and was the most common injection-site reaction reported within 60 min of injection (~ 86% of participants). The frequency of unspecified injection-site reaction, injection-site erythema and injection-site pruritus was significantly . People with migraine have been shown to have elevated blood levels of calcitonin gene-related peptide (CGRP) and targeting the CGRP pathway using antibodies has been demonstrated to be effective in preventing migraine attacks –4. ThreeEfficacy of galcanezumab was demonstrated in pivotal phase 3 studies in patients with episodic or chronic migraine , 11–13. Therefore, in this post hoc analysis of Phase 3 migraine studies of galcanezumab, we provide a comprehensive overview and detailed summary of injection-site reactions.clinicaltrials.gov identifier: NCT02614183, https://clinicaltrials.gov/ct2/show/NCT02614183; EVOLVE-2, clinicaltrials.gov identifier: NCT02614196, https://clinicaltrials.gov/ct2/show/NCT02614196), one randomised study in participants with chronic migraine  and one open-label study  in participants with episodic or chronic migraine. All clinical trials were first posted on 25 November 2015, and before enrolling the first patient. The EVOLVE and REGAIN studies were designed to study efficacy and safety of galcanezumab, and the open-label Study CGAJ was designed primarily to assess long-term safety. The study designs of all four studies have been previously published [Data were obtained from two randomised clinical studies in participants with episodic migraine  treatment phase wherein participants were randomised 2:1:1 to receive monthly placebo, galcanezumab 120 mg or galcanezumab 240 mg. The REGAIN study had a 3-month DB treatment phase, wherein participants were randomised 2:1:1 to receive monthly placebo, galcanezumab 120 mg, or galcanezumab 240 mg. The DB phase was followed by an optional nine month open-label extension (OLE) phase wherein either galcanezumab 120 mg or galcanezumab 240 mg was administered based on the investigator’s discretion. Study CGAJ comprised a 12-month open-label (OL) phase wherein participants were randomised 1:1 to receive galcanezumab 120 mg or galcanezumab 240 mg. All four studies had a four-month safety follow-up period immediately after the treatment phase.In the EVOLVE and REGAIN studies, study site personnel administered the injections using prefilled syringes. In Study CGAJ, injections were self-administered by participant or caregiver using prefilled syringes for up to 9 months, and then participants were switched to self-administer galcanezumab using an autoinjector . NotablyTwo cohorts from the integrated analysis sets of four studies  were analysed. These studies included: 1) all participants from the six-month DB treatment phase in the EVOLVE-1 and EVOLVE-2 studies and three-month DB treatment phase in the REGAIN study ; 2) galcanezumab-treated participants from the DB + OLE phase of REGAIN, Month 0 to Month 12, and OL phase of Study CGAJ, Month 0 to Month 12, where participants received galcanezumab . Of note, participants who were randomized to placebo in the REGAIN study received up to 9 monthly doses of galcanezumab and participants who were randomized to galcanezumab received up to 12 monthly doses of galcanezumab.https://www.meddra.org/). The injection-site reactions  refer to the high-level grouping of different preferred terms (PT) used to describe various injection-site reactions. The reported verbatim terms collected during the study was mapped to a PT that best characterised the reported adverse event at the injection-site, such as “pain”, “erythema”, “rash” and “swelling”. When the site recorded the event as “injection-site reaction” the event was mapped to an unspecified PT of injection-site reaction (singular). An injection-site adverse event follow-up form was used to further characterise the reported unspecified event of injection-site reaction.The injection-site reactions are treatment-emergent adverse events (TEAEs) that occurred or worsened at any time after treatment initiation and were coded based on the Medical Dictionary for Regulatory Activities  were summarized. For the placebo-controlled analysis set, inferential statistics was provided. Treatment comparisons were evaluated using the Cochran-Mantel-Haenszel test stratified by study. In addition, patients with injection-site reactions were summarized by maximum severity as “mild”, “moderate” or “severe”. The reaction timings from the last injection to the occurrence of injection-site reactions were summarised as follows: immediate reaction (< 60 min); acute reaction (1 to 6 h); delayed reaction (classified as injection-site reactions post 6 h on the day of the injection up to 14 days after the day of injection); and reactions ≥14 days. The duration of injection-site reactions were also summarized.For the galcanezumab exposure data set, to determine if injection-site reactions excluding pain were reported more frequently with repeated doses of galcanezumab, the number of TEAEs were assessed by the total number of doses received.n = 1451; galcanezumab 120 mg, n = 840; galcanezumab 240 mg, n = 865). In this integrated analysis set of EVOLVE-1, EVOLVE-2 and REGAIN studies, participants were predominantly women , with a mean age of ~ 41 years , with a mean age of ~ 42 years  and a diagnosis of migraine of ~ 20 years prior to study enrolment . The detailed demographics are published elsewhere .P ≤ 0.001) with galcanezumab 240 mg or 120 mg compared with placebo  participants reported at least one injection-site reaction . The injection-site reactions were significantly higher , all participants completed at least one follow-up form which was used to further characterise the reported unspecified injection site reaction. Of these 67 participants, 59.1% (13/22) participants on galcanezumab 120 mg reported itching, rash or redness, and injection-site hardening. Itching, rash or redness, and injection-site hardening were reported by 60.0% (27/45), 84.4% (38/45), and 44.4% (20/45) participants on galcanezumab 240 mg, respectively.n = 2; galcanezumab 240 mg, n = 5). Among these participants, four participants discontinued due to moderate unspecified injection-site reaction . The remaining three patients discontinued due to moderate injection-site pain , severe injection-site erythema , and moderate injection-site swelling  Table Among patients who reported injection-site reactions, most reported injection-site reactions of mild-to-moderate severity Table . No injeInjection-site pain was the most common immediate injection-site reaction reported within 60 min of injection) and was observed in approximately 86% of participants reporting injection-site pain Table . Majoritn = 5; REGAIN, n = 4). All discontinuations were observed following multiple doses of galcanezumab .During the long-term exposure of galcanezumab, 289  participants reported AEs related to injection-sites. The most frequently reported injection-site reactions were injection-site pain , unspecified injection-site reaction , injection-site erythema , and injection-site pruritus  over consecutive monthly injections, the number of TEAEs related to injection-sites (excluding pain) by total number of doses is provided in Table P < 0.001) higher in participants being treated with galcanezumab compared with placebo. Overall, the incidence of injection-site reactions were higher in the galcanezumab dose groups (240 mg > 120 mg) compared with placebo-treated participants and appear to be driven by AEs related to injection-sites including unspecified injection-site reaction, injection-site erythema, and injection-site pruritus. These reactions are commonly observed with other approved monoclonal antibodies for subcutaneous use, including adalimumab, denosumab, ixekizumab, and canakinumab [In Phase 3 studies demonstrating efficacy and safety of galcanezumab as a treatment option for management of migraine, injection-site reactions were the most commonly reported AEs. The incidence of injection-site pain was highest among all reported injection-site reactions and was reported equally by participants receiving galcanezumab and placebo. Unspecified injection-site reaction, injection-site erythema and injection-site pruritus were significantly  were observed in the galcanezumab exposure analysis set. Most participants reported onset of injection-site pain within 60 min of injection, and approximately 50% of injection-site pain was reported as injection-site burning. Of interest, 17.5% of patients reported a history of hypersensitivity, however only 5.2% of those patients reported injection-site related AEs other than pain. During exposure to galcanezumab up to 12 months of treatment, the number of injection-site reactions reported by the same patient does not increase with more doses received.In this post hoc analysis of four Phase 3 studies, we provide a more comprehensive summary of injection-site reactions with short-term and long-term exposure to galcanezumab. Of the most commonly reported injection-site reactions in galcanezumab-treated participants, 70 to 100% were reported on the day of injection, were generally mild-to-moderate in severity, non-serious, and resolved spontaneously. In total 16/1705 (< 1%) treatment discontinuations were observed due to injection-site reactions; seven were observed in placebo-controlled analysis set and nine , including galcanezumab, have the potential to have anti-drug antibody (ADA) formation, which in turn can block the active mAb site. The formation of ADA can be associated with changes to pharmacokinetic and pharmacodynamic parameters, resulting in a range of effects from no clinically important effects to reduced drug efficacy and/or increased risk of adverse events (AEs) , 22. A rTherapeutic proteins are typically administered in the subcutaneous tissue, especially when the treatment is required frequently, on a long-term basis and requires self-administration . InjectiThis post hoc analysis, which was not pre-specified at the time of design of the EVOLVE-1, EVOLVE-2, REGAIN and Study CGAJ, limits its ability to make definitive conclusions. There could also be some under-reporting possibly as all AEs were reported spontaneously by the study participants, and use of analgesic, and histamine creams, and potential comfort measures were not excluded. Lastly, galcanezumab injections were administered by study site personnel in the galcanezumab Phase 3 studies, with the exception of Study CGAJ. In Study CGAJ, patients self-administered galcanezumab either with the prefilled syringe or the autoinjector devices. The injection experience and tolerability of self-administration with both devices has been published elsewhere .In conclusion, galcanezumab-treated participants reported a significantly higher frequency of injection-site reactions versus placebo. Most of these events were self-limiting; no SAEs related to injection-site were reported in any of the Phase 3 migraine studies. Future post-marketing study evidence is warranted to augment these findings.Additional file 1. List of ethics committee.


Fusarium oxysporum. The results demonstrated that Trichoderma spp. present a high self-inhibitory activity in vitro, being affected in both their growth rate and the macroscopic structure of their colonies. These effects were highly similar to those exerted against F. oxysporum in the same conditions, showing no significant differences in most cases. Consequently, membrane assays may not be very informative by themselves to assess putative biocontrol capabilities. Therefore, different methods, or a combination of antifungal and self-inhibitory experiments, could be a better approach to evaluate the potential biocontrol activity of microbial strains in order to pre-select them for further in vivo trials.Self-inhibitory processes are a common feature shared by different organisms. One of the main mechanisms involved in these interactions regarding microorganisms is the release of toxic diffusible substances into the environment. These metabolites can exert both antimicrobial effects against other organisms as well as self-inhibitory ones. The in vitro evaluation of these effects against other organisms has been widely used to identify potential biocontrol agents against phytopathogenic microorganisms. In the present study, we performed membrane assays to compare the self-inhibitory effects of soluble metabolites produced by several Trichoderma isolates and their antifungal activity against a phytopathogenic strain of  Autotoxicity and self-inhibition have been described as common traits shown by different species, genera, and kingdoms . Some geTrichoderma harzianum mediated by extracellular self-DNA [Trichoderma spp. or any other biocontrol agent. Trichoderma is a ubiquitous genus of filamentous fungi, widely used as a biocontrol agent against many phytopathogenic microorganisms [Regarding filamentous fungi, some studies have been conducted to evaluate self-inhibitory processes, allowing the identification of different autotoxic compounds ,4. Nonetself-DNA . Besidesrganisms ,8, as werganisms ,10 and arganisms .Fusarium oxysporum is a common endophyte and phytopathogenic filamentous fungus that thrives in numerous important crops [F. oxysporum f. sp. phaseoli being one of the forms that mainly affects bean plants (Phaseolus vulgaris L.) [Trichoderma spp. against different Fusarium species.On the other hand, nt crops , F. oxysaris L.) . Furtheraris L.) ,14,15, aaris L.) , produceTrichoderma spp. produce both self-inhibitory effects and antifungal effects against F. oxysporum. The main objective of the present study is to evaluate the self-inhibitory effects of soluble metabolites produced by several Trichoderma isolates and to compare them to the antifungal activity exerted by the same fungal strains against a phytopathogenic strain of F. oxysporum.The hypothesis of this work is that soluble metabolites released in vitro by T. harzianum, one T. citrinoviride, one T. velutinum, and one T. gamsii. Out of the ten strains, eight of them were isolated from bean (P. vulgaris L.) fields belonging to de Protected Geographical Indication (PGI) “Alubia de La Bañeza-León”, while the other two were isolated from sugar beet crops growing in the same area [Ten Trichoderma strains were used to evaluate the activity of their diffusible metabolites: seven ame area . TrichodF. oxysporum (F3 from now onwards) was isolated from bean (P. vulgaris) fields belonging to the same PGI and was selected for its high virulence against this crop.A strain of All fungal strains were preserved in 50% glycerol spore suspension at −80 °C and stored in the “Pathogens and Antagonists Collection” at the “Pest and Diseases Diagnosis Laboratory” . All cultures were activated on PDA  at 25 °C.A membrane assay was performed, as described by Mayo et al. (2015) , by placF. oxysporum was performed as described above, but inoculating a 6 mm plug from the edge of a 5 day-old F. oxysporum culture on the center of the plates after removing the membranes. The diameters of the colonies were measured in the same way, but 3 and 7 days post-inoculation (dpi)  b, as theTrichoderma spp. over the cellophane membrane. The 1st round of data collection was performed when fungal colonies in the controls reached 1/3 of the plate (24 hpi for Trichoderma spp. and 3 dpi for F. oxysporum). The 2nd round of data collection was performed when fungal colonies in the controls reached the edge of the plate (48 hpi for the fastest Trichoderma spp. and 7 dpi for F. oxysporum). Diameters were measured with a ruler in three different directions. Four replicates per treatment were performed. All cultures were performed on PDA  at 25 °C.Controls were performed in the same manner but without growing p ≤ 0.05) after confirmation of normality and equality of variances. Subsequently, treatments were contrasted between them and with their controls using Tukey’s post hoc test (p ≤ 0.05). Statistical analyses were performed separately for self-inhibition and antifungal activity of the ten Trichoderma spp.  by the diameter of the plugs, as shown by Mutawila et al. 2016) . PIs wer . PIs werma spp. , as wellrma spp. . Four reT. harzianum isolates consistently demonstrated a higher inhibitory activity than the other three Trichoderma species, with PIs ranging from 99.51% to 86.92% at day 3 post-inoculation, and from 95.25% to 89.40% in day 7 post-inoculation. In contrast, T. citrinoviride T008 showed lower PI values (70.73% in day 3 and 59.38% in day 7), while T. velutinum T028 exerted the lowest inhibition of all strains, with 34.15% in day 3 and a mere 15.89% in day 7. Finally, T. gamsii T057 showed similar PI to those of some T. harzianum in day 3 (92.90%) with a significant decrease in day 7 (76.27%). All treatments showed significant differences (p ≤ 0.05) compared to the growth of F3 controls. These results are in accordance with previous studies regarding the antifungal activity of Trichoderma spp. on F. oxysporum [Under the referred conditions, all Trichoderma isolates showed important antifungal effects on F3 growth . T. harzxysporum ,19.T. harzianum tested) in the second round of data collection (p ≤ 0.05) than those of the T. harzianum strains  to 87.66% . However, T008, T028, and T057 showed, respectively, self-inhibitory PIs of 74.51%, 42.29%, and 82.53% after 24 h, and 71.02%, 23.57% and 68.74% after 48 h (p ≤ 0.05)  showed no significant differences between self-inhibitory and antifungal activities during the first round of data collection a. Moreovllection b. The re strains . Self-inter 48 h . Additio ≤ 0.05) .Trichoderma spp. are closely related and might be general traits shared for both heterologous competition with other fungal species in the environment, as well as ecological adaptations within the same genus, species, and strain. The latter likely being related to directional centrifugal growth of filamentous fungi [T. harzianum, mediated by conspecific DNA [These results seem to indicate that the antifungal and self-inhibitory effects exerted in vitro by us fungi , competius fungi . The selific DNA . These hific DNA . The higThe results also point out that self-inhibition varies between Trichoderma strains and species, suggesting that either they produce higher quantity and/or stronger autotoxic metabolites, or they possess different detoxification capabilities to confront these compounds. We propose that, taking into account the similarities presented here between self and heterologous antifungal inhibitory effects within the same strains, the differences seen between strains are mostly due to different antifungal activity and/or the amount rather than different detoxification capabilities. Besides, the high PI values, especially regarding those referred to self-inhibition, are likely due to an extremely high accumulation of soluble metabolites in the PDA medium, derived from the unnatural conditions of the in vitro design. Nevertheless, with the available data, it is not possible to determine whether the observed effects are produced by toxic subproducts of fungal metabolism or secondary metabolites that are purposefully released for antifungal activity. It also is not possible to assure that both the self-inhibitory and the heterologous antifungal activities are produced by the same metabolites, even though the very similar PI values between them seem to point in this direction. New studies should be directed to elucidate these aspects.In addition, as can be seen in F. oxysporum, we believe that their high in vitro inhibitory activity alone is not enough to claim a potential biocontrol activity, being likely biased by the unnatural concentrations reached in the plates. Thus, different experimental laboratory designs could be more adequate to evaluate these traits before transitioning to in vivo and field trials. For example, Kron et al. (2020) [Pseudomonas orientalis mutants against Erwinia amylovora were more informative than in vitro assays.It is well known that in vitro assays are only an approximation of more complex natural contexts  and that. (2020)  demonstrIn addition, we suggest that if membrane assays are used to identify potential biocontrol strains, autotoxic studies, as the here presented using the same strains, should be conducted in identical conditions in order to compare and select those microbial agents showing both high antimicrobial activity alongside a lower self-inhibitory one. For example, in our case, while T002, T007, T015, and T021 show very high antifungal effects on the first round of data collection, we may consider discarding T015 as a potential biocontrol strain, as its antifungal activity against F3 is not significantly different from its own self-inhibitory one a. HoweveFinally, a deeper understanding of self-inhibitory mechanisms within biocontrol strains, species, and genera might be a key factor to develop commercial pesticides based on a mixture of similar biological control agents. Additionally, this knowledge would be very useful to determine the ideal concentration of the agent when applying it on the field in order to avoid self-inhibitory effects, as well as to select the adequate growth conditions for its industrial production.Trichoderma spp. show, in vitro, a high self-inhibitory activity mediated by secreted soluble metabolites. This activity varied among different strains and species, with T. harzianum showing the highest self-inhibitory effects. Trichoderma strains are affected both in their growth rate and the macroscopic structure of their colonies, arguably reflecting microscopic structural and physiological abnormalities in the fungal hyphae. These growth-inhibitory effects are highly similar to those exerted against Fusarium oxysporum in the same conditions, especially for T. harzianum isolates, indicating a likely comparable mechanism in both cases. In this regard, the great inhibition observed in both cases for most Trichoderma strains is possibly derived from the extremely high concentration of metabolites accumulated in the culture medium. As a result, membrane assays seem to not be very informative by themselves, in regards the direct biocontrol capabilities of the tested strains. Therefore, different methods or a combination of antifungal and self-inhibitory experiments could be a better approach to preliminarily evaluate the potential biocontrol activity of microbial strains in order to pre-select them in vitro for further in vivo trials. This strategy may serve to optimize the process of pre-selection, helping to save time and resources in the subsequent phases of the research. Nevertheless, these conclusions need to be further investigated and confirmed by new assays, involving both in vivo studies and a wider variety of biocontrol and phytopathogenic microbial strains.To summarize, the results presented demonstrate that the majority of the tested


Bietti crystalline dystrophy (BCD) is a distinct entity of retinitis pigmentosa with a wide range of genotypic and phenotypic variabilities. The goal of the present study was to investigate the morphological, functional and genetic features of BCD.A full series of multimodal imaging was performed in four Chinese patients with BCD, including fundus photography, fundus autofluorescence, fundus fluorescein angiography (FFA), indocyanine green (ICG) angiography, optical coherence tomography (OCT) and microperimetry. Electrophysiological tests including full-field electroretinography (ERG) and multifocal ERG were employed. CYP4V2 gene sequencing was performed.Intraretinal crystalline deposits were observed in fundus photographs in all patients. The crystals were better appreciated in infrared images. Autofluorescence imaging demonstrated multifocal patchy hypofluorescence, suggesting massive RPE atrophy. FFA and ICG angiography further confirmed atrophy of the RPE and the underlying choroidal vessels. OCT revealed disruption of the photoreceptors, RPE and the choroid. Outer retinal tubulations (ORTs) confining to the outer nuclear layer were detected in three out of four patients. Full-field ERG showed markedly diminished responses. Multifocal ERG displayed reduced central and peripheral responses in a patient with normal vision. Gene sequencing identified two deletion mutations in CYP4V2, c.802_807del and c.810delT. BCD complicated by choroidal neovascularization (CNV) was diagnosed in one patient, and intravitreal anti-vascular endothelial growth factor (VEGF) injection was given with favorable response.Multimodal imaging features and electrophysiological findings of BCD patients were comprehensively discussed. A novel deletion mutation, c.802_807del, in the CYP4V2 gene was reported. ORTs are important changes in the outer retina of BCD patients, further investigation of this structure may provide insights into pathology of BCD. Intravitreal anti-VEGF therapy was effective for treatment of BCD complicated by CNV. CYP4V2 has been identified as the main causative gene for BCD [Bietti crystalline dystrophy BCD, MIM 210370) is an autosomal recessive inherited chorioretinal degenerative disease [10370 is  for BCD . In affeCYP4V2 encodes a member of the cytochromes p-450 protein family which is responsible for oxidation of substrates during fatty acid metabolism. CYP4V2 is widely expressed in various tissues. In the eye, it is highly expressed in the choroid and the RPE while relatively less expressed in the cornea [e cornea . In patie cornea , 7. Celle cornea .The diagnosis of BCD relies mainly on the detection of characteristic intraretinal crystalline deposits together with RPE/choroidal dystrophies. Multimodal imaging has been shown to be useful in the diagnosis of BCD and differentiation from other chorioretinal degenerative diseases . The aimFour Chinese patients Table  diagnoseA comprehensive ophthalmologic examination was performed using standard protocols, including best-corrected Snellen visual acuity (BCVA), Ishihara color vision test, slit-lamp examination, fundus examination, fundus photography, autofluorescence imaging, microperimetry, fundus fluorescein angiography  and indocyanine green angiography . Optical coherence tomography (OCT) images were acquired using a spectral-domain system  as described previously . HorizonCYP4V2 gene in our patients. Blood samples were obtained and sent to an external service to be sequenced. Signed informed consent were obtained from the patients. Direct sequencing of the entire coding regions was performed and flanking sequences of the CYP4V2 gene was determined.Next-generation sequencing was performed to characterize mutations in the Slit-lamp examination of the anterior segment was unremarkable in all four subjects. No corneal crystalline deposit or dystrophy was observed Table . No coloOCT examination displayed numerous hyperreflective spots of various configurations, disruption of RPE, macular thinning and choroidal hyperreflective lesions Fig. . The briFull-field ERG showed non-recordable responses in patient 1 and 3. Rod responses were significantly reduced and the photopic wave amplitudes were severely attenuated Fig. . In caseMolecular analysis of the CYP4V2 gene revealed two homozygous deletion mutations in patient 1: c.802_807del (p.268_269del) and c.810delT (p.A270fs). The c.802_807del mutation has not been published in the Human Gene Mutation Database (HGMD). This is the first report of this deletion mutation in BCD patient. c.810delT deletion is a known frameshift mutation and has been reported in BCD patients as a disease-causing mutation .The diagnosis of BCD was made in our patients based on the characteristic presentation, including the presence of yellowish-white reflective intraretinal crystals, widespread retinal and choroidal thinning, and choriocapillary atrophy. It is noted that none of our patients showed corneal changes such as crystalline deposits or atrophy. This is consistent with previous report of high prevalence of pure retinal involvement in Asians , 15. BCDThe origin and nature of the crystalline deposits are unclear, yet their role in RPE and retinal degeneration remains to be determined. In our study, intraretinal crystalline deposits with a range of severity were detected by fundus examination. In our observation, the crystalline deposits in the retina were only present between the atrophic areas. In other words, the crystals were located selectively in the relatively “healthy” areas of the retina as reported , 18. GocOCT imaging is a useful method in specifying the anatomical location of the crystals in the retinal layers. By analyzing the crystals on fundus photographs/infrared images and the hyperreflective spots in OCT images, many authors proposed that the crystals might be located in the RPE/Bruch’s membrane complex , 20. ThiElectrophysiological test is helpful to determine the extent of retinal function loss in BCD patients. Full-field ERG, which reflects function of the entire retina, can show varying degrees of rod and cone dysfunction depending on the stages of disease progression , 26. HowCYP4V2 have been widely studied in patients with BCD, including missense, nonsense, insertion or deletion mutations etc. Most mutations reported in BCD patients are missense mutations. In the present study, we identified two homozygous deletion mutations in the CYP4V2 gene, c.802-807del and c.810delT in patient 1. The c.802-807del deletion has not been reported before. Although no explicit genotype-phenotype correlation has been determined in BCD patients, studies have suggested that genetic deletions might be associated with a more severe disease and worse visual acuity when compared with relatively mild mutations [CYP4V2 gene mutation may have a causal effect on choroidal vascular damage thus contributing to visual impairment in BCD patient [Disease-causing mutations in utations , 28. Fur patient . MoreoveIn addition to the classical features described by conventional methods, more characteristics of BCD have been reported recently with the utilization of optical coherence tomography angiography (OCTA). Miyata et al. reported a significant reduction in choriocapillaris blood flow in BCD patients when compared with that in the healthy subjects by using OCTA . SimilarCNV is a complication rarely reported in BCD patients. Although breakdown of Bruch’s membrane is known to be present, the explicit pathogenesis of CNV in BCD patients is unclear. In common with other reported BCD cases complicated by CNV, the CNV lesion observed in our patient appeared to confine within the foveal/parafoveal retina. Therefore, the development of CNV is not likely a direct consequence of the intraretinal crystals, as the latter tend to be scattered throughout the posterior pole. Fuerst et al. demonstrated that CNV in BCD patients may be attributed to the dysfunction of RPE and abnormalities of photoreceptor outer segment-RPE interface at the fovea and parafovea . Anti-VEBCD is a distinct entity of retinitis pigmentosa with a wide range of genotypic and phenotypic variabilities. Our study provided comprehensive information regarding this rare condition from multimodal imaging, electrophysiological test and genetic analysis. We identified two homozygous mutations, c.802_807del and c.810delT, in our patient. This is the first report of c.802_807del mutation in patient with BCD. ORTs are important morphological changes frequently detected in BCD patients, further investigation of this structure may provide insights into pathology of BCD. Intravitreal anti-VEGF therapy is effective for treatment of BCD complicated by CNV.


Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known. Activation-dependent phosphorylation of rhodopsin was discovered in the early 1970s , long beRhodopsin phosphorylation was shown to increase the binding of a 48-kDa protein  , norepinephrine , and dopamine (with lower affinity). The three β-adrenergic receptors, β1, β2 (β2AR), and β3, couple to Gs, whereas alpha-adrenergic α1 receptors predominantly couple to Gq/11 and α2 receptors preferentially couple to members of the Gi/o subfamily of G proteins. The role of GRKs in the regulation of non-visual GPCRs was first discovered using β2AR as a model receptor . It was Both subfamilies of α-adrenergic receptors are regulated by GRKs and arrestins, but α1 and α2 are structurally different. While α1 receptors resemble β-adrenergic receptors, with a relatively short third cytoplasmic loop and a C-tail that contains GRK phosphorylation sites, α2 receptors have very long third cytoplasmic loops that contain serine (Ser) and threonine (Thr) residues targeted by GRKs . InteresMammals express five dopamine receptors, D1, D2, D3, D4, and D5. Both D1 and D5 couple to the adenylyl cyclase-activating Gs, whereas D2, D3, and D4 couple to the Gi/o subfamily of cyclase-inhibiting G proteins. Structurally, both Gs-coupled D1 and D5 have a relatively short third cytoplasmic loop and a fairly long C-terminus with numerous potential GRK phosphorylation sites, whereas Gi/o-coupled D2, D3, and D4 have a much larger third cytoplasmic loop with potential phosphorylation sites and a very short C-terminus . D1 and The rat D1 receptor has eight potential GRK phosphorylation sites in the third loop and 18 in the C-terminus . MutatioThe second main dopamine receptor subtype, the D2 receptor, has a very short C-terminus but an extra-long third loop . It has The D3 dopamine receptor belongs to the same subfamily as D2 but displays unique signaling properties. It has a significantly higher affinity for dopamine than D2, a minimal agonist-induced shift to the high-affinity state, and a weak ability to activate Gi/o-dependent signaling ,65. WhenAll five members of the muscarinic receptor subfamily are GPCRs  subject Phosphorylation and arrestin binding have been extensively studied for the M2 muscarinic receptor. The M2 receptor has a very long third cytosolic loop that possesses multiple Ser/Thr sites (44 in the human M2) and a very short C-terminus with no potential GRK phosphorylation sites . EarlierCuriously, while GRKs play a clear role in the regulation of M2 signaling , GRK-med284Ser-Glu292 in the third loop leads to a defect in internalization [A member of another subfamily of muscarinic receptors, the M1 receptor, which is a major postsynaptic muscarinic receptor throughout the brain, is phosphorylated in an agonist-dependent manner by GRK2 at sites located in the middle of the third loop . Differelization . Howeverlization ) contriblization ,94. This331SSS333 and 348SASS351, are the key regions phosphorylated by GRK2: mutation to alanines of either of the clusters reduces phosphorylation, and mutation of both clusters reduces the phosphorylation level by 75% [289Cys-His330 region, which partially covers the first cluster, was shown to be deficient in agonist-induced internalization. The authors attributed this effect to the loss of the Gβγ binding site, which they localized to this region. An earlier study identified 286ESLTSSE292 as critical for M3 internalization [The pattern of agonist-dependent phosphorylation by GRKs has been studied in more detail for the M3 muscarinic receptor, a relative of the M1 receptor. The rat M3 possesses 53 Ser/Thr residues on the third cytosolic loop, clustered in six Ser/Thr-rich regions. Two of these clusters located in the N-terminal part of the loop, l by 75% . It was lization . Althouglization , contrarlization . Mice lalization , whereaslization . The M3 lization ,100. Thulization . ArrestiAmong other things, the large number of potential phosphorylation sites means that they are unlikely to be targeted all at once. Two models are conceivable: the sequence of phosphorylation of the available serines and threonines can be random, or they can be modified in a certain order. This issue, like many in GPCR regulation, was first explored in rhodopsin. Using a bright flash that activates ~40% of rhodopsin in the mouse retina , it was shown that Ser343 is phosphorylated first, Ser338 is phosphorylated slower, and Ser334 even slower . As dephIn contrast to many other protein kinases, none of the GRKs has a consensus phosphorylation site, i.e., a particular sequence context of serines and threonines they preferentially target. This makes biological sense, as the intracellular elements of GPCRs demonstrate very low sequence conservation . HoweverIn some cases, the phosphorylation appears to be hierarchical, i.e., certain sites must be phosphorylated first, whereupon others can be modified. One interpretation of this phenomenon is that a receptor phosphorylated at certain sites becomes more “attractive” for some  GRKs. However, an alternative explanation is that phosphorylation might simply induce conformational rearrangement of the intracellular receptor elements, which results in the exposure of some sites inaccessible in the unphosphorylated receptor. The latter explanation appears correct for D1 dopamine receptor phosphorylation, where the C-terminal sites are likely phosphorylated first to give the kinase access to the sites on the third loop . A relatArguably, opioid receptors are the most clinically relevant peptide receptors. Synthetic ligands of these receptors are widely used to treat acute and chronic pain. Opioid agonists also cause addiction. These are the reasons that the function of opioid receptors has been studied more extensively than other neurotransmitter-binding GPCRs. Additionally, the opioid receptors happen to have a limited number of potential GRK phosphorylation sites, as compared to other important neurotransmitter/neuromodulator receptors such as muscarinic or dopamine (see above sections). This makes the work of identifying the sites and assessing their relative functional contribution in cultured cells and living animals feasible. Four opioid receptors (OR) have been cloned, the mu (MOR), kappa (KOR), delta (DOR), and nociceptin/orphanin FQ (NOR) receptors. Opioid receptors mostly couple to the pertussis toxin-sensitive Gi/o subfamily of G proteins and, to a lesser extent, to Gz (reviewed in ). While 370TREHPSTANT379 was required. High-efficacy opioid drugs generally have the ability to cause phosphorylation of these extra residues, albeit to a different extent. DAMGO promotes phosphorylation not only of Ser375 but also of T370, T376, and T379 in its vicinity [354TSST357, Ser356 and T357 are phosphorylated in an agonist-dependent manner, but without any detectable effect on receptor internalization [370TREHPSTANT379 motif [370TREHPSTANT379 stretch in the MOR C-terminus  and anandamide (AEA) (reviewed in ). CB1 isThe removal of the last 14 residues of the rat CB1 receptor (residues 460–473) prevented its internalization in AtT20 cells . HoweverThere are numerous reports of oligomerization of class A (rhodopsin-like) GPCRs  reconstituted into nanodiscs showed that GRKs 2 and 5 differentially phosphorylate it. The action of GRK2 was strictly agonist-dependent, whereas GRK5 phosphorylated NTSR1 independently of its activation. GRK2 phosphorylated only C-terminal Ser residues, whereas GRK5 phosphorylated Ser and Thr residues in both the third intracellular loop and the C-terminus . It appeIn the case of rhodopsin , β2AR 1, and D2RWhen discussing GRK-dependent regulation of the neurotransmitter receptors, yet another set of questions must be asked: whether or how distinct molecular events affect neural function. It is not difficult to imagine that altered availability and/or function of GRKs, leading to impaired receptor desensitization/trafficking, would result in enhanced or reduced responsiveness to agonists, as in cultured cells. However, chronic treatment with neurotropic drugs often results in long-term neural plasticity, altering the responsiveness to these drugs, which develops over hours or days, in contrast to the much faster timescale of receptor phosphorylation and arrestin recruitment, both of which occur within minutes in cultured cells. This long-lasting plasticity is initiated by agonist stimulation of the neurotransmitter receptors, which would initially engage GRKs, and it is a legitimate—and intriguing—question whether GRK-dependent phosphorylation of the neurotransmitter receptors plays a role in the development of long-term neural plasticity.Studies in mice lacking individual GRK isoforms have demonstrated supersensitivity to acute stimulation of select neurotransmitter receptors. The loss of GRK6 confers supersensitivity to dopaminergic stimulation . The dopLoss of GRK5 results in supersensitivity to muscarinic M2 receptor stimulation ,80. GRK5The exact relation between GRK-mediated receptor phosphorylation and in vivo adaptations to receptor stimulation has been studied in some detail in the case of opioid receptors. Physiologically, opioids induce analgesia in humans and animals, and classic opioid drugs exert their effects via MOR. If opioid analgesia is mediated by G protein activation via MOR, desensitization of the receptor would serve to limit the extent and duration of the analgesic response. Indeed, knockin mice expressing MOR with Ser 375 mutated to alanine (Ser375Ala), which cannot be phosphorylated, showed greater antinociceptive response to morphine and fentanyl . In knocGRK-arrestin-mediated homologous desensitization of MOR is a short-term adaptation that eventually results in re-sensitization or a longer-lasting adaptation such as receptor degradation/downregulation. Treatment with opioid drugs leads to a loss of responsiveness referred to as tolerance, which could be short-term (acute tolerance) or long-term (chronic tolerance). From a physiological perspective, it is important to understand the relationship between the opioid receptor desensitization and tolerance. This subject has been extensively reviewed previously . An intDOR agonists are efficacious in chronic pain but, similarly to MOR agonists, induce tolerance upon prolonged use. The role of GRK-dependent phosphorylation in tolerance to DOR agonists remains essentially unexplored. However, there is evidence that tolerance depends on the availability of arrestin-2. Thus, loss of arrestin-2 reduced tolerance to the DOR agonist SNC80, which induces strong DOR internalization, but not to ARM390, which does not . DOR phoGenerally speaking, chronic tolerance appears to involve GRK-dependent phosphorylation of opioid receptors enabling arrestin recruitment, and when these functions are compromised, tolerance is diminished. The mechanism of this effect is intriguing, since the timings of these events—GRK/arrestin-dependent homologous desensitization and tolerance—are so different (discussed here ). HoweveIn mice with the Ser426 and Ser430 phosphorylation sites in the C-terminus of the CB1 receptor mutated to alanines, dependence on Δ9-THC was increased, but tolerance delayed, which was accompanied by enhanced acute responses . ToleranAll therapeutically active drugs have side effects. Since the discovery of arrestin-mediated signaling by GPCRs, the idea of biased signaling, i.e., signaling engaging only one branch of the GPCR pathway, either G protein or arrestin, has gained popularity. For the biased signaling to be possible, the two branches, the G protein- and arrestin-mediated, should be independent, and originally they were believed to be. There are obvious limitations to this independence. The fact that GRKs 2 and 3 use two products of G protein activation, Gβγ ,61,62 an50 values for morphine analgesia and respiratory depression/constipation across lines with different phosphorylation levels are highly correlated (R2 > 0.9), suggesting that these effects are not independent. These experiments strongly suggest that all major physiological effects of MOR activation, both good and bad from our perspective, are largely mediated by G proteins.On the physiological side, the idea is based on the notion that the therapeutic action might be mediated by one signaling branch and the side effects by another. The attraction of this theory is obvious but the experimental basis is limited (discussed in ). ArguabExisting data show that most GPCRs, including neurotransmitter receptors, are regulated by GRKs. However, the relative role of the five distinct non-visual GRKs expressed in all vertebrates in the regulation of these receptors has not been experimentally established. The situation is further complicated by the possibility that the role of GRK subtypes in the regulation of the same receptor is not necessarily the same in different neurons, possibly even under different conditions in the same neuron. In most cases, GRK phosphorylation promotes the binding of one or both non-visual arrestins. The two ubiquitous non-visual arrestins are functionally distinct, as their duality persisted in vertebrate evolution for millions of years . There iThe generalized paradigm of GPCR desensitization  was deveExperimental testing of the role of individual phosphorylation sites by mutagenesis has serious caveats that must be kept in mind. Some GPCRs possess a huge number of potential phosphorylation sites, which makes the task of determining the key residues for GRK phosphorylation truly daunting. The replacement of Ser/Thr with alanines or valines to determine the sites phosphorylated by GRKs is a common approach. However, replacement of a serine with an alanine, in addition to preventing phosphorylation, changes the H-bonding capability of the residue. Replacement of threonine with alanine or valine also changes the H-bonding capability, as well as the size of the side chain. Thus, it is not always apparent whether these serines and/or threonines participate in arrestin binding as such or whether their phosphorylation by GRKs is needed for arrestin recruitment. Often, the “phosphomimetics” aspartate or glutamate are used in place of putative phosphorylation sites to determine whether phosphorylation is required. However, it has been experimentally shown that these “phosphomimetics” do not always mimic phosphorylated serine or threonine, likely because both the size and the charge at physiological near-neutral pH is quite different. Thus, if alanine substitution and knockout of a particular GRK yields the same result, while replacement of the residue with a phosphomimetic rescues the GRK knockout phenotype, we can conclude that the observed changes were due to lack of phosphorylation. If each change produces a unique phenotype and there is no observable rescue, we cannot conclude anything with confidence. We should keep in mind that the results of only one or even two of these three approaches are also inconclusive. The full battery of tests is almost never performed, even in cultured cells. Conclusions based on incomplete experimental testing should be always taken with a grain of salt. Unfortunately, collecting such complete sets of data in vivo is rarely feasible. However, as demonstrated with the mouse MOR knockin experiments , even a A recent structural study suggests that GPCRs must have a certain “phosphorylation code”, i.e., a particular spacing between phosphorylated and/or negatively charged residues, in order to bind arrestins with high affinity . Two codThe connection between fairly rapid (minutes to hours) molecular events involving GRKs and arrestins, which were mostly established in vitro and in cultured cells, and physiological mechanisms generating long-term (days to weeks) changes in vivo is one of the mysteries that needs to be resolved. This is further complicated by the known fact that behavioral changes involve circuit effects that cannot be reproduced in cell culture. Biologically relevant in vivo consequences of phosphorylation of particular residues in each GPCR can only be established using genetically modified animals. While these experiments take a lot of time, effort, and resources, they are necessary, as extrapolation from test tube and cell culture tends to be misleading.