Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndrome-related kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.Phosphorylation of Munc18-1 ( Presynaptic proteins  are phosphorylated by abundant (not neuro-specific) kinases like protein kinase C (PKC) and protein kinase A (PKA) and these modifications impact on synaptic transmission in a variety of ways (see review5). However, many more  phosphorylation sites in synaptic proteins are reported, for which the impact is unknown.Post-translational modification of synaptic proteins is a powerful way to regulate synaptic strength. Phosphorylation is probably the most well-studied mechanism of regulation of synaptic protein function. Many synaptic proteins have been identified in large-scale phospho-proteomics screensin vivo6 and in vitro7. Munc18-1 null neurons survive for 4 days in culture but are subsequently unable to support viability7. The MUNC-18 gene in human is STXBP1, and exonic mutations in STXBP1 have been reported to cause STXBP1-encephalopathy, characterized by severe intellectual disability (ID), spasms, and epilepsy11. Many of these clinical symptoms are also observed in Stxbp1 mouse models of STXBP1-encephalopathy12.Munc18-1 is a major regulator of synaptic transmission and among the best validated presynaptic phosphorylation targets. Loss of Munc18-1 in neurons leads to a loss of neurotransmitter release and cell death 1 or predicted13. To date, all confirmed phosphorylation sites have major impacts on Munc18-1 function: protein kinase C (PKC)16, Src17 and ERK18. Munc18-1 is also reported as a target for Dyrk1a, a kinase involved in Down syndrome which phosphorylates Munc18-1 at threonine 479 (T479)19. The functional impact of this modification is unknown.Several potential phosphorylation sites of Munc18-1 have been found20). Dyrk1a is located in the nucleus and cytoplasm of neurons in the mouse cerebellar cortex21, plasma membrane in synaptosomal fractions22, and in the human brain23, and is involved in neuronal differentiation26, regulating the balance between proliferation and apoptosis27, dendrite and spine outgrowth28, and tau phosphorylation30. The human DYRK1A gene is located in the Down syndrome critical region and its levels are important in the pathogenic mechanism33. Furthermore, mutations in Dyrk1a are linked to other forms of ID9, similar to mutations in STXBP1/Munc18-111. Mouse models for Dyrk1a and STXBP1 haploinsufficiency show similar autistic-like features and seizures34, and full deletion of both Dyrk1a and Munc18-1 is embryonically lethal35. Dyrk1a knockout mice are lethal because phosphorylation of Hip-1 blocks neuronal cell death in neural progenitor cells and promotes neurite outgrowth36. Hence, these similarities between Dyrk1a and Munc18-1 strengthen the idea that the phosphorylation of Munc18-1 by Dyrk1a might be important for synapse function and contributes to an explanation for the severe ID caused by mutations in the two genes.The dual-specificity tyrosine phosphorylation-regulate kinase (Dyrk) family are serine/threonine kinases that regulate many cellular processes 38 we identified exonic regions that were markedly intolerant to variation across the whole exome. Genetic variation can be expected to occur virtually everywhere in the genome: if genetic variation is not observed in an exon-sized region in adult humans, this region is constrained and likely subject to strong purifying selection. Given the known criticality of STXBP1 to synaptic function, we wished to prioritize phosphorylation sites that were highly intolerant to exonic mutation: 12 of 18 Stxpb1 phosphorylation sites were found to be highly intolerant to variation  or alanine  in munc18 null neurons. Wildtype Munc18-1 (M18WT) served as a positive control. Munc18 null neurons die within days in vivo and in vitro7. Survival of munc18 null neurons expressing M18T479D was comparable to M18WT expressing neurons, but higher variability was observed in neurons expressing M18T479D. Compared to neurons expressing M18WT, survival of M18T479A expressing neurons was reduced by 15% , we investigated the effects of M18ncy Fig.  and amplncy Fig.  as neuro79D Fig. . Paired 8WT Fig. .Figure 3T479D showed significantly enhanced recovery after depletion during the 40 Hz train stimulation compared to neurons expressing M18T479A or M18WT  241 and tyrosine (Y) 473, are in close proximity to the Dyrk1a phosphorylation site in the three dimensional crystal structure45, suggesting a regulatory hub. Thus, surprisingly, spontaneous, evoked synaptic transmission and most short-term plasticity parameters were unaltered by replacing wildtype Munc18-1 for one of the T479 mutants.This study shows that T479 is among several phosphorylation sites in Munc18-1 that are constrained and can be phosphorylated et al. hypothesized a Dyrk1a-dependent regulation of the interaction between Munc18-1 and syntaxin, despite T479 being localized further away from the interaction site19. T479 is located at the edge of domain 3b of Mun18-1. This region is not associated with binding to synaptobrevin/VAMP2 or syntaxin45, the canonical interactors of Munc18-1, which are essential for its function. A phosphorylation site that critically influences Munc18-1 function, the Src kinase phosphorylation site (Y473)17, is located close to T479. Furthermore, Y473 is a predicted binding site of synaptobrevin/VAMP245. Despite being 6 amino acids away from Y473, mimicking or preventing phosphorylation at T479 does not have a strong regulatory effect on synaptic transmission and is therefore likely to be located away from where synaptobrevin/VAMP2 and Munc18-1 interact.Park 19. In contrast to the full length Dyrk1a protein used in this study, we used a truncated form of Dyrk1a that comprises the n-terminal amino acid residues 1 to 499 including the kinase domain. The construct used was comprised primarily of the kinase domain and might therefore be missing important interaction sites in the C-terminal end that makes the interaction sufficiently stable to show using co-immunoprecipitation. However, Dyrk1a was not identified as an interactor of Munc18-1 in a previously conducted pulldown experiment either (data not shown). The interaction between Munc18-1 and Dyrk1a is therefore probably transient, sufficient for phosphorylation to occur but not stable enough to be picked up using pulldown experiments.Using co-immunoprecipitation, we did not replicate the interaction between Munc18-1 and Dyrk1a reported previously47 or transport from the reserve pool to the RRP48); the number or vesicles at the active zone49; or the availability of Munc18-1 at the active zone43. It is difficult to distinguish which process contributes to the observed phenotype but increased synaptic levels of Munc18-1 after high frequency stimulation might be the most plausible seeing as the baseline Munc18-1 levels at the synapse were not affected. Moreover, acutely overexpressing Munc18-1 using Semliki virus shows a similar increase in EPSC size after a 40 Hz train stimulation41. Altered local levels of Munc18-1 at the synapse could change synaptic transmission by increasing the number of available fusion-ready vesicles. Dyrk1a-dependent phosphorylation is the first known phosphorylation site of Munc18-1 that does not regulate the role of Munc18-1 in synaptic transmission but mildly influences recovery after high frequency stimulation. In a neuronal network this could make the network more vulnerable or resistant after activity, which could be relevant in epilepsy.In neurons expressing the phosphorylation mimicking Munc18-1 mutant, high frequency stimulation was followed by increased EPSC size, consistent with either increased recovery of the RRP or increased release probability. This observation can be attributed to at least three mechanisms: the speed at which the RRP is refilled . DYRK1A and STXBP1 associated disorders show symptomatic overlap. Core symptoms of DYRK1A-deficiency disorders include ID, microencephaly, developmental delay, seizures, and autistic feature 53. and STXBP1 encephalopathy patients show severe ID, autistic features, and in 95% of the cases occurrence of epilepsy11. Hippocampal neurons expressing the phosphorylation mimicking or preventing mutants of Munc18-1 show normal synaptic transmission and only after high frequency stimulation does Dyk1a-dependent phosphorylation of Munc18-1 have a minor effect, increasing synaptic response. This stands in contrast to the similarities of the clinical phenotypes and they are therefore unlikely to be caused by the same mechanism and phosphorylation of Munc18-1 by Dyrk1a is not underlying the common symptoms observed in DYRK1A and STXBP1 associated disorders.Munc18-1 and Dyrk1a play an important role in brain development54. Munc18 null mutant mice are stillborn and can be easily distinguished from wild-type or heterozygous littermates. E18 embryos were obtained by cesarean section of pregnant females from timed mating of heterozygous mice. Munc18-1 lox mice were generated as described previously55. Newborn Munc18-1 lox mice were obtained at P0-1 from a homozygous litter. Newborn P0-P1 pups from pregnant female Wistar rats were used for glia preparations.Animal experiments were approved by the animal ethical committee of the VU University/VU University Medical Centre (license number: FGA 11-03) and are in accordance with Dutch governmental guidelines and regulations. Animals were housed and bred according to institutional, Dutch, and U.S. governmental guidelines. Munc18-1 deficient mice were generated as described previously56. Transduction efficiencies of lentivirus containing Munc18-1 were assessed on HEK293T cells using a concentration range, and were taken into account when viruses were applied to neuronal cultures. For this purpose, Human embryo kidney 293 T (HEK293T) cells were infected with lentivirus 1 day after plating in DMEM containing 10% fetal calf serum  and 1% penicillin/streptomycin (Gibco) and number of GFP positive cells were counted after 1 day.Single amino acid substitutions on T479 in Munc18-1 were generated using Quikchange and verified by sequencing. The Munc18-1 constructs were connected with a T2 linker to Cre-EGFP and cloned into pLenti vectors, ensuring that expression levels of Cre-EGFP and Munc18-1 were similar. Viral particles were produced as described2, 5.0 mM EDTA, 100 mM NaCl). Lysates were centrifuged 10 min at 4 °C 16000 g and supernatants were collected. Antibodies against myc or munc18 were added to the supernatant. ProteinA Agarose beads were added (Vector Laboratories) and the samples were tumbled 2 hours at 4 degrees. After the immunoprecipitations were washed 5 times with IP-buffer, the samples were eluted from the beads with Laemmli sample buffer  and analysed with SDS-PAGE.HEK293T cells were cultured in DMEM supplemented with 10% FCS, pen/strep(1:100) and non-essential amino acids  and plated in 6-wells dishes to 30% confluency on the day of transfection. Cells were transfected with Munc18-1 and myc tagged active Dyrk1a constructs  using calcium phosphate precipitation. The cells were washed 16 hr after transfection (DMEM). Immunoprecipitations were done 36 hours after transfections. Cells were lysed in IP-buffer . Blots were stained for myc (Genetex) and Munc18-1 . Secondary antibodies were conjugated with Alkaline Phosphatase (Jackson lab) and Attophos (Promega) was used as substrate. The blots were imaged with the Image Reader FLA-5000 (Fuji)2, 5.0 mM EDTA, 100 mM NaCl). Munc18-1 was immune-precipitated with polyclonal Munc18-1 Antibody . ProteinA agarose beads were added and the samples were tumbled for 2 hours at 4 degrees. After 2 hours the precipitations were washed 2x with IP-buffer and 3x with 1x kinase buffer (New England Biolabs). Kinase mixes with either active Dyrk1a (Millipore) or ERK (Sigma Aldrich) were added to the samples (400 ng kinase for each sample). 25 μM [γ-32P]-ATP was added and samples were incubated at 37 °C for 30 minutes. The kinase reaction was stopped by adding 5ul 5x Laemmli sample buffer and samples were boiled for 5 minutes before analysing them with SDS-PAGE. The gels were imaged with the Image Reader FLA-5000 (Fuji).HEK293T cells were transfected with Munc18-1. 36 hours after transfection cells were lysed in 1x Laemmli sample buffer . After denaturing (boiling for 5 min) the samples were diluted 15x in IP-buffer  buffered with 7 mM HEPES (Invitrogen). After removal of the meninges, hippocampi were incubated in Hanks-HEPES containing 0.25% trypsin  for 20 minutes at 37 °C. After washing, neurons were triturated in DMEM, supplemented with 10% FCS, 1% nonessential amino acids (NAA) using a fire-polished Pasteur pipette and counted in a Fuchs-Rosenthal chamber. Neurons were plated in pre-warmed Neurobasal medium supplemented with 2% B-27, 1.8% HEPES, 0.25% glutamax, and 0.1% Pen/Strep  and infected with lentiviral particles encoding Munc18-1 variants after 3 days in vitro (DIV).Hippocampi from Munc18-1 lox neurons were plated on micro-islands of rat glia at a density of 1.5 k per well in a 12-well plate to achieve autaptic cultures. To generate micro-islands, glass coverslips (Menzel) were etched in 1 M HCl for at least 2 hours and neutralized with 1 M NaOH for maximum 1 hour, washed thoroughly with MiliQ water, and washed once with 70% ethanol. Coverslips were stored in 96% ethanol and coated with agarose type II-A . Coating was done by spreading a thin layer of agarose solution (heated in a microwave and kept at 55 °C during use) with a cotton swab over the entire coverslip. Microdots were created using a custom-made rubber stamp (dot diameter 250 µm) to apply a solution consisting of 0.1 mg/ml poly-D-lysine (Sigma), 0.7 mg/ml rat tail collagen (BD Biosciences), and 10 mM acetic acid (Sigma) by stamping from a wet filter paper . Coverslips were UV-sterilized for 20 minutes before further use. Astrocytes were plated at 6–8 k/well in prewarmed DMEM (Invitrogen) supplemented with 10% FCS, 1% nonessential amino acids (NAA), and 1% penicillin/streptomycin .Hippocampal Munc18-1 lox neurons were grown for 12–15 days before electrophysiological measurements were performed. Whole-cell voltage-clamp recordings (Vm = −70 mV) were performed at room temperature. Borosilicate glass pipettes (2.5–4.5 mOhm) filled with 125 mM K+-gluconic acid, 10 mM NaCl, 4.6 mM MgCl, 4 mM K2-ATP, 15 mM creatine phosphate, 10 U/ml phosphocreatine kinase, and 1 mM EGTA . External solution was made up of 10 mM HEPES, 10 mM glucose, 140 mM NaCl, 2.4 mM KCl, 4 mM MgCl2, and 2 mM CaCl2 . Only excitatory neurons, identified by decay of the postsynaptic currents, were included. Recordings were acquired with an Axopatch 200B amplifier, Digidata 1440 A, and ClampX 10.2 software (Molecular Devices). After whole-cell mode was established, only cells with an access resistance of <15 mΩ and leak current of <500 pA were included in the analysis. Evoked EPSCs were elicited by a 1 ms long depolarization to 30 mV. RRP size was estimated using back-extrapolation by making a linear fit through the last 20 pulses of a 40 Hz stimulation train, where intracellular calcium levels are high and synchronous release has been mostly abolished. At this point newly-recruited fusion-capable vesicles are released immediately. This is considered to be a steady-state situation, at which a linear back-extrapolation procedure can be used to estimate the size of the initial RRP (y-axis intercept) corrected for vesicle recruitment. Offline analysis was performed using custom-written software routines in Matlab R2017b (Mathworks).Autaptic cultures of 57) antibodies. After washing, cells were incubated for 2 hours at room temperature with secondary antibodies conjugated to Alexa dyes  and washed again. Coverslips were mounted with DABCO-Mowiol (Invitrogen).Cover slips from the same 12-well plates used for electrophysiology were used each week to control Munc18-1 levels as well as assessment of morphology. Cultures were fixed with 3.7% formaldehyde (Electron Microscopy Sciences). After washing with PBS, cells were permeated with 0.5% Triton X-100 for 5 minutes and incubated in 2% normal goat serum and 0.1% Triton X-100 for 20 minutes to block non-specific binding. Cells were incubated for 2 hours at room temperature with a primary antibody mixture of polyclonal guinea pig anti-Synaptophysin-1 , polyclonal chicken anti-MAP2 , and polyclonal rabbit anti-Munc18-1  with a 10x Plan Apo λ objective (NA = 0.45) at 1024 × 1024 pixels and averaged over two scans. Z-stacks were acquired with an interval of 3.7 µm, individual images were stitched together and maximally projected for analysis and display.Images of single cells were acquired with a confocal microscope  using a 40x oil immersion objective (NA = 1.3) with 0.7x zoom at 1024 × 1024 pixels and averaged over four scans. Neuronal morphology and protein levels were analysed using automated image analysis routinen referring to the number of cells from each group. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software). Data was tested for normality with the Kolmogorov and Smirnov test. As the data was not normally distributed the nonparametric Kruskal-Wallis test and Dunn’s multiple comparison test were used. P-values below 0.05 are considered significant.Data is presented as mean values ± SEM, with Supplementary Figure S1 - Original uncropped gels and blot.


When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca  •KCl-depolarization induces phosphoproteome changes in isolated nerve terminals (synaptosomes).•Botulinum neurotoxin affects protein phosphorylation in depolarized synaptosomes.•BoNT treatment reveals phosphorylation sites that depend on SV-cycling activity.•SV-cycling-dependent sites on Vamp2, Stx1a, and Cnr1 affect exo- and endocytosis. C. botulinum neurotoxins (BoNT) to inhibit synaptic vesicle (SV) cycling reveals phosphorylation events that are primarily dependent on depolarization-induced Ca2+ influx and those that also require active SV-cycling machinery. In particular, SV-cycling-dependent phosphorylation sites on synaptobrevin (Vamp2), syntaxin-1 (Stx1a), and cannabinoid receptor-1 (Cnr1) are capable of changing the rate of exo- and endocytosis in cultured hippocampal neurons.Analysis of protein phosphorylation in isolated nerve terminals (synaptosomes) treated with  N-ethylmaleimide-sensitive factor-attachment protein receptors (SNARE)  , 4, 5, 6 (SNARE) , 8.i.e., refilling of the neurotransmitter, SV priming, and docking).In order to sustain continuous neurotransmitter release and to maintain the structure and composition of the plasma membrane, fused SVs are retrieved by endocytosis, followed by rapid regeneration of SV precursors that are filled with neurotransmitters and prepared for the next round of neurotransmitter release. It is still debated which mechanisms are primarily responsible for endocytosis and recycling, with several occurring in parallel , 15, 16.An increasing body of data suggests that the basic exo–endocytotic machinery is fine-tuned by regulatory control mechanisms that mainly operate by phosphorylations and dephosphorylations of proteins involved in the SV cycle. In particular, calcium plays an important role not only as a trigger of exocytosis, but also as a modulator of kinase and phosphatase activity in the presynapse . CalciumSnap25-S187, Stx1a-S14, S188, Vamp2-T35, S75), as well as of the SNARE-interacting protein Munc18 (Stxbp1-S313), can indeed interfere with the formation of the SNARE complex, which results in altered exocytosis (Cnr1-S317) that is controlled by PKC , synaptobrevin (Vamp2-S75), and cannabinoid receptor-1 . Finally, we demonstrated that phosphorylation of these sites elicits a pronounced effect on exo- and endocytosis in cultured hippocampal neurons by expressing phosphomimetic and nonphosphorylatable variants of these proteins.However, it remains to be established which of the vast amount of phosphosite changes observed in recent phosphoproteomic studies are dependent on the membrane trafficking steps of the SV cycle and which are, while being targeted by the activated network of kinases and phosphatases, not directly related to exo–endocytotic cycling. Changes in the protein conformation that accompany assembly and disassembly of multimolecular protein complexes during SV cycling may alter the accessibility of phosphorylation sites as well as the availability of docking sites for kinases and phosphatases. To address this issue, we conducted a quantitative phosphoproteome analysis using synaptosomes as a functional model of the synapse. Synaptosomes contain the complete SV-cycling machinery, can maintain the membrane potential and ATP at physiological levels, and respond to depolarization by Ca release , 52, 53.P levels , 55. We  release , 57. By All experiments involving animals complied with the regulations as designated in the section 4 of the Animal Welfare Law of the Federal Republic of Germany . All experiments were conducted in the animal facility at the Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany, and registered accordingly to the section 11 Abs. 1 TierSchG as documented by 39 20 00_2a Si/rö, dated Dec 11, 2013  by the Veterinär-und Verbraucherschutzamt für den Landkreis und die Stadt Göttingen and examined regularly by the supervisory veterinary authority of the Landkreis Göttingen. All procedures were supervised by the animal welfare officer and the animal welfare committee of the Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany, established in accordance with the TierSchG and the regulation of animal experiments, dated Aug 31, 2015 .4OH, CaCl2, KCl, KH2PO4, MgCl2, NaCl, NaHCO3, NaHPO4, sucrose, and glucose from Merck, Darmstadt, Germany. Trifluoroethanol (TFE), triethylammonium bicarbonate buffer (TEAB), formic acid (FA), EGTA, PM400-Ficoll (Ficoll), NADP, L-Glutamic dehydrogenase from bovine liver (GluDH), guanidine hydrochloride, tris(2-carboxyethyl)phosphine (TCEP), chloroacetamide (CAA), Triton-X100, and MS-grade trypsin were obtained from Sigma-Aldrich, Taufkirchen, Germany. HEPES was purchased from VWR Chemicals, Darmstadt, Germany. SDS was purchased from Serva Electrophoresis GmbH, Heidelberg, Germany. TFA was obtained from Roth, Karlsruhe, Germany. Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F) was obtained from Roche, Mannheim, Germany. Rapigest was obtained from Waters, Milford, USA. Pierce 660 nm protein assay, Halt Protease and phosphatase inhibitor cocktail, and TMTsixplex isobaric labeling reagents (TMT6) were purchased from Thermo Fisher Scientific, Bleiswijk, Netherlands. C. botulinum strain A–D cell culture supernatants  were obtained from Miprolab, Göttingen, Germany.LC/MS-grade water, methanol, and acetonitrile (ACN) were used in this study if not otherwise stated and were purchased together with chloroform, 25% (v/v) NHg in an SS-34 fixed angle rotor . The supernatant was collected and recentrifuged for 12 min at 14,462×g. The synaptosomal pellet was resuspended in the homogenization buffer and loaded onto discontinuous Ficoll gradient (6%/9%/13% (wt/v) Ficoll in the homogenization buffer) and centrifuged for 35 min at 86,575×g in an SW-41 swinging bucket rotor . The synaptosomal band at the interface between 9% and 13% Ficoll was collected and washed with the homogenization buffer. Synaptosomal protein concentration was estimated using Pierce 660 nm protein assay according to manufacturer’s instructions. The viability of synaptosomes was confirmed by glutamate release assay  to achieve a final concentration of 1 mg/ml of synaptosomal proteins. CaCl2 was added to a final concentration of 1.3 mM, and synaptosomes were incubated for 5 min at 37 °C before adding BoNT. For Ca versus EGTA experiments, synaptosomes were preincubated with 1.3 mM CaCl2 or 0.5 mM EGTA for 15 min at 37 °C before stimulation with KCl (50 mM). To block SNARE-assisted neurotransmitter release, 20 μl of each BoNT was added per 1 mg of estimated synaptosomal protein . The following combinations of C. botulinum cell culture supernatants were used: (i) C. botulinum A and D or (ii) C. botulinum C1 and B. Alternatively, synaptosomes were treated with the same amount of respective cell culture supernatants inactivated by heating at 95 °C for 1 h (mock control). BoNT- and mock-treated synaptosomes were incubated at 37 °C for 90 min and then directly subjected to the glutamate release assay. In brief, synaptosomal suspension was transferred into a quartz glass cuvette  and kept stirred at 37 °C during the measurements. In total, 1 mM NADP and 50 U per 1 mg of synaptosomal protein of GluDH were stepwise added to the suspension. Synaptosomes were depolarized by adding KCl to a final concentration of 50 mM. NADPH-fluorescence at 440 nm was monitored at each step using Fluorolog-3 fluorometer . After 2 min of stimulation, the reaction was quenched with an equal volume of lysis buffer . The synaptosomal sample was incubated for 5 min at 95 °C, chilled on ice, and then sonicated for 10 min using 30 s on/30 s off cycles at the maximum output of Bioruptor ultrasonication device . The sample was repeatedly incubated for 5 min at 95 °C, and proteins were precipitated following methanol/chloroform protein precipitation method , respectively. Peptides were incubated with TiO2 beads  at 1:10 protein-to-beads ratio for 20 min at 40 °C. Unbound peptide fraction (nonphosphorylated peptides) was collected, and TiO2 beads were subjected to four washing steps using washing buffer (60% (v/v) ACN, 1% (v/v) TFA in water). Bound phosphopeptides were eluted using 3.75% (v/v) NH4OH 40% (v/v) ACN in water, snap-frozen in liquid nitrogen, and dried in a centrifugal Savant SpeedVac vacuum concentrator . Dried phosphopeptides and peptides in the unbound fraction were subjected to desalting using prepacked C18 spin columns . Desalted and dried peptides were redissolved in 50 mM TEAB 50% (v/v) ACN in water, and TMT6-labeling reaction was conducted according to manufacturer’s instructions. After the labeling, peptide samples were accordingly combined, cleaned using the prepacked C18 spin columns, and concentrated in a SpeedVac.Precipitated synaptosomal proteins (1–2 mg per condition) were redissolved in a digestion buffer  and sonicated in the Bioruptor for 10 min at maximum intensity using 30 s on/30 s off cycles. Trypsin was added at 1:40 trypsin-to-protein ratio (wt/wt), and proteins were digested overnight at 37 °C. Next day, the sample was treated with 2.5 U of PNGase F per 1 mg of the initial protein amount for 1 h. Afterward, ACN was added to a final concentration of 45% (v/v), and the sample was incubated with a second portion of trypsin  for 1 h. The sample was centrifuged for 15 min at 17,000×ications . Briefly4OH in water, pH ∼10; buffer B: 10 mM NH4OH and 80% (v/v) ACN in water, pH ∼10). The column was equilibrated with the 95% buffer A and 5% buffer B mixture. Peptides were resolved using a linear gradient ranging from 10% to 35% buffer B for 35 min followed by a linear increase to 60% over 5 min and a washing step at 90% buffer B for 5 min. One-minute fractions were collected and concatenated into 24 (for Mock/BoNT phosphorylated peptides) or 12 (for Mock/BoNT nonphosphorylated peptides and Ca/EGTA phosphorylated peptides) final fractions as suggested elsewhere  equipped with a C18-X-Bridge column . The HPLC was operated at the flow rate of 60 μl/min under basic pH (buffer A: 10 mM NHlsewhere . BRP fraDried peptides were redissolved in 2% (v/v) ACN 0.1% (v/v) TFA in water. Each concatenated BRP fraction was analyzed in triplicates (phosphorylated peptides) or as a single injection (nonphosphorylated peptides). Dissolved peptides were injected onto a C18 PepMap100-trapping column  connected to an in-house packed C18 analytical column . The columns were pre-equilibrated using a mixture of 98% buffer A (0.1% (v/v) FA in water), 2% buffer B (80% (v/v) ACN, 0.1% (v/v) FA in water), or 95% buffer A, 5% buffer B (nonphosphorylated peptides). Liquid chromatography was operated on an UltiMate-3000 RSLC nanosystem . Phosphopeptides were eluted using a 60 min-linear gradient ranging from 10% to 25% buffer B (80% (v/v) ACN, 0.1% (v/v) FA in water) followed by a linear increase to 45% buffer B over 10 min and a washing step at 90% of buffer B for 5 min. For nonphosphorylated peptides, a steeper gradient was applied ranging from 10% to 45% buffer B followed by an increase to 60% buffer B and a washing step at 90% buffer B. Eluting peptides were sprayed into a QExactive HF-X or Orbitrap Exploris 480 . For the analysis of phosphorylated peptides, MS1 scans (350–1600 m/z) were acquired with a resolution of 120,000 at 200 m/z, 3e6 automatic gain control (AGC) target, and 50 ms maximum injection time. Precursor ions were isolated using a 0.8 m/z isolation window, and a normalized collision energy of 33% was applied to obtain fragment spectra. Only charge states from 2+ to 6+ were considered, and the dynamic exclusion was set to 25 s. The fragment spectra were acquired with a resolution of 30,000, 1e5 AGC target, and 130 ms maximum injection time. For the analysis of nonphosphorylated peptides, following parameters were set differently: MS1 scans were acquired with a resolution of 60,000 at 200 m/z, 300% normalized AGC target. Fragment spectra were acquired with the resolution of 15,000 at 200 m/z, 50% of the normalized AGC target and 54 ms maximum injection time.Rattus norvegicus proteins were retrieved from Uniprot  containing canonical protein sequences  was supplemented with sequences of BoNT B-D . Peptide-spectrum matches (PSM) and protein FDR threshold were kept at 0.01. Following steps in data analysis were conducted in R statistical programming language using custom scripts. The scripts are available at GitHub . For each phosphorylation site, a leading protein was selected based on the list of potential candidate proteins for this site reported by MaxQuant. Proteins were ranked based on (listed from the most to the least significant) (i) the number of the unique phosphorylation sites assigned to the protein in the data set; (ii) the number of all phosphorylation sites per protein in the data set; (iii) “reviewed” status and Uniprot-annotation score of the protein sequence; (iv) isoform status of the protein sequence. The official gene name and Uniprot accession of the leading protein were used in the subsequent analyses. Impurity-corrected reporter ion intensities for each phosphorylation site identified at one of the three multiplicity levels were extracted from MaxQuant “Phospho.txt” output table. Potential contaminants, reversed sequences, and phosphorylation sites identified with localization probability <0.75 as determined by MaxQuant . The choice for this number of replicates was dictated by the constraints of the TMT6 labeling and the previous study  and q-value < 0.01  < 1%).In total, 216 .raw files were analyzed in order to quantify changes in phosphorylation site intensities in BoNT- or mock-treated synaptosomes or synaptosomes stimulated in the presence of calcium or EGTA. Out of the 216 .raw files, 144 .raw files were obtained from two TMT6-labeling sets of mock- versus BoNT-treated synaptosomes. These files correspond to 24 concatenated BRP-fractions and three injection replicates per fraction. Remaining 72 .raw files were obtained from another two TMT6-labeling experiments of synaptosomes stimulated in the presence of Caus study . The phous study . We used package  to test p-values  were subp-values . Phospho2+-dependent” if they show absolute log2 (Ca/EGTA) > 0.263 and absolute log2 (Mock/BoNT) < 0.263 or were not quantified in the Mock/BoNT experiment. Conversely, events showing absolute log2 (Mock/BoNT) > 0.263 were termed as “SV-cycling-dependent.” Phosphorylation events that exhibited only small changes in both experiments (absolute log2 (Ca/EGTA) < 0.263 and absolute log2 (Mock/BoNT) < 0.263) were considered as being “not-affected.” In the following analyses, only phosphorylation events that surpassed an FDR-threshold of <1% were considered. Specifically, “primary Ca2+-dependent” phosphorylation events had to satisfy a q-value threshold of <0.01 in the Ca/EGTA experiment, whereas “SV-cycling-dependent” sites had to meet a q-value threshold of <0.01 in Mock/BoNT experiment or a q-value-threshold of <0.01 and an absolute log2 FC threshold of >0.263 in Ca/EGTA experiment. Phosphorylation events that did not match these criteria were considered as being “not-affected” as well. If a phosphorylation site was quantified at different multiplicities (phosphorylation events), whereby one of which was classified as being “SV-cycling-dependent,” the site was considered as being “SV-cycling-dependent.” To define regulation groups at protein level, number of significantly regulated phosphorylation sites were counted per protein (gene). Proteins were distributed in the following groups (i) “mostly primary Ca2+-dependent” (>60% of sites being categorized as “primary Ca2+-dependent”); (ii) “mostly SV-cycling-dependent” (>60% of sites being categorized as “SV-cycling-dependent”); (iii) “mixed” (none of the groups was dominating).Phophorylation events were defined as “primary CaR. norvegicus proteome as a background  “neuronal system” categories were selected  GO-biological function terms  and Calcineurin docking motifs  intensity of the phosphorylated peptide; Prot is the normalized protein intensity, and NPh is the normalized intensity of nonphosphorylated peptide. The model was fit by using intensity data from Mock/BoNT experiments. Phosphorylation occupancy was then calculated using the following formula: Occupancy = a/(1+a), where a = m2∙Ph/NPh. “Illegal” stoichiometry values, i.e., values outside the interval  were excluded from the analysis. Only m2 values with a p value below 0.1 were considered.Gene enrichment analyses were conducted using gene names of proteins carrying significantly regulated phosphorylation sites in Ca/EGTA or Mock/BoNT experiments. Gene ontology enrichment analysis was performed using David web interface and whole ckground . Direct selected . Synapseon terms . Experimon terms . In ordeon terms . Only exon terms , 74. Speon terms  databaseon terms . The secg motifs . To demo  glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA) and was a generous gift of Prof. Dr Lührmann laboratory. The extract was supplemented with 1.3 mM CaCl2 and 20 μl of each C. botulinum cell culture supernatant (A and D or C1 and B), heat-inactivated cell culture supernatants, or 40 μl of the sodium buffer per 1 mg of nuclear proteins. Each treatment condition was performed in triplicate, resulting in a total of nine samples. The samples were incubated for 1.5 h at 37 °C. Afterward, urea in 100 mM HEPES pH 8 was added to the final concentration of 1 M and proteins were digested overnight at 30 °C using MS-grade trypsin at a trypsin-to-protein ration (wt/wt) of 1:50. Next day, peptides were subjected to phosphopeptide enrichment as described above excluding the deglycosylation and second endoproteinase digestion steps. Enriched phosphopeptides were cleaned using the prepacked C18 spin columns, redissolved in 2% (v/v) ACN, 0.1% (v/v) TFA in water, and analyzed on UltiMate 3000 RSLC nanosystem connected to Orbitrap Exploris 480 mass spectrometer. Raw phosphorylation site intensities as reported by MaxQuant were analyzed as described before using R scripts and the limma package for data normalization, visualization, and statistical testing.To test for unspecific phosphatase activity, escribed  in Roede2 and additional 40 μl of the sodium-containing buffer or 1.3 mM CaCl2 and 20 μl of each C. botulinum cell culture supernatants in two combinations (A and D or C1 and B). Synaptosomes were then incubated for 1 h at 37 °C under mild agitation. Afterward, synaptosome suspensions were supplemented with 5 μM of Fura-2AM dye  and incubated for additional 30 min at 37 °C. Following steps were conducted as previously described  SDS, 1 mM EGTA were added stepwise and the signal was acquired for 3 min at each step. Fluorescence intensity at 340 nm excitation was divided by the intensity at 380 nm excitation and then by the maximum fluorescence ratio following SDS lysis. Median fluorescence ratio before stimulation with KCl was subtracted to obtain proportional changes in fluorescence intensity relatively to its maximum.Synaptosome suspensions containing 1 mg of synaptosomal proteins were prepared as described for glutamate release assay. Suspensions were supplemented with 1.3 mM CaClescribed . BrieflyPrimary hippocampal neuronal cultures were prepared from P2 rats as previously described . HEK293 in silico based on rat genes with the appropriate restriction enzymes for viral cloning and ordered at GenScript (USA). The synaptobrevin (Vamp2) mutants were based on rat Vamp2 (Uniprot: P63045) fused to the optimized version of superecliptic GFP (pHluorin) (Vamp2-pHluorin and the respective mutants (S75D pseudo-phosphorylated and S75A non-phosphorylatable) in the viral vector  and cannabinoid receptor 1  and their mutants  were inserted in the WT Vamp2-pHluorin viral vector by cutting with the enzymes SgsI (AscI) and SbfI (SdaI). Their expression was driven by mean of a second human synapsin-1 promoter from the same viral plasmid to allow consistent coexpression. To distinguish these proteins from the endogenous, an ALFA-tag was added (All the sequences (including mutants) were designed Hluorin)  by mean scribed, ) allowinas added . Also inStx1a and Cnr1 in all viruses and mutants was confirmed by staining with the NbALFA  and 0.1% (v/v) Triton-X 100 for 15 min at RT. Fluorescently labeled NbALFA  was diluted 1:200 in blocking solution for 1 h at RT and subsequently washed three times for 5 min with PBS. Coverslips were mounted on glass slides using Mowiol solution, dried, and imaged. Note that superecliptic GFP (pHluorin) keeps its fluorescence in Mowiol solution since its pH is ∼7.5 and can thus be imaged with no additional staining.The expression of e NbALFA . Briefly2, 1 mM MgCl2, pH 7.4), passed through 0.22 μm syringe filters, and viral titer was adjusted by serial dilution on primary hippocampal neurons monitoring pHluorin levels .Recombinant AAV particles were prepared as previously described . BrieflyStx1a and Cnr1 coexpression was confirmed by imaging. Live experiments were performed in Tyrode’s solution  supplemented with 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione  and 50 μM 2-amino-5-phosphonopentanoic acid  to avoid spontaneous network activation. At the end of the imaging, 50 mM NH4Cl was applied to evaluate total SV content. Live imaging was performed with an inverted Nikon Ti epifluorescence microscope  equipped with a Plan Apochromat 60x 1.4 NA oil-immersion objective, an HBO-100W Lamp, an IXON X3897 Andor camera , and an OKOLab cage incubator system  to maintain a constant temperature of 37 °C. ND2 Images were imported using the Bio-Formats (OME) plug-in. For quantifications, the fluorescence intensity was measured with custom-made Matlab (USA) code that can be provided on an individual basis if requested. Briefly, multiple regions containing synaptic boutons were manually selected, in 5–8 independent experiments. Between 140 and 345 regions were selected for each condition, over all independent experiments. The Vamp2-pHluorin signal was then measured in the respective regions, and the background intensity  was subtracted. The Vamp2-pHluorin signal curves were then normalized to the maximum intensity obtained during the addition of NH4Cl. To compare easily different conditions and mutant variants, all curves were furthermore normalized to their initial (prestimulus) baselines. Statistical differences were tested in Matlab, relying on Kruskal–Wallis tests, followed by Tukey–Kramer post-hoc tests.SV exo/endocytosis assays were performed analogously to what previously described , 85. Bri2+-deprived and free Ca2+ conditions; for the latter, depolarization of the plasma membrane of synaptosomes was induced by KCl in buffer containing EGTA or CaCl2 (A). We applied depolarization for 2 min; this duration was chosen in order (i) to achieve comparability with our previous studies  studies . In the ive BoNT . PhosphoA, left panel).Our analysis shows that phosphorylation is a prominent modification of synaptosomal proteins. Specifically, we identified and quantified phosphorylation under stimulated and nonstimulated conditions on 12,021 sites belonging to more than 2600 proteins (unique gene names) with a localization probability of >75% as determined by MaxQuant , 64. ThiFig. 2B). Using the PhosphositePlus database , we detected substantial changes in protein phosphorylation. Specifically, there are 1363 phosphorylated sites mapped to approximately 470 proteins that show intensity changes of at least 20% at an FDR of 1% B. Using database  to annotdatabase , 74 to pdatabase . ImportaBsn), piccolo (Pclo), synapsin-1 (Syn1), and dynamin-1 (Dnm1), as well as the microtubule-associated protein Tau (Mapt) and the dihydropyrimidinase-related proteins 2 and 5 (Dpysl2 and Dpysl5), and others (D). Overall, the overlap of identified and quantified sites with the results of previous studies is approximately 60% as determined by the overlap in protein sequences flanking phosphorylation sites (sequence windows).We also observed changes of phosphorylated sites in response to KCl treatment that were similar to changes reported in earlier studies  for bassd others D. OveralA). A similar picture  was obtained when using the synapse-specific SynGo database in which a large set of synaptic proteins was annotated by experts . For example, the number of sites predicted to be substrates for GSK3 and CDK is proportionally higher among proteins involved into the GO term “axonogenesis” as compared with other GO terms. Similarly, many phosphorylated sites on proteins that are involved in the “calcium-dependent regulation of synaptic functions” were also predicted as CaMKII substrates. The proportion of putative MAPK substrates is higher for proteins associated with “synapse assembly” and “cytoskeleton organization” as compared with other GO terms.Enrichment analysis of the biological function of proteins carrying regulated phosphorylation sites (DAVID ) revealeFig. 3 experts . It show experts . Interesi.e., for SV cycling. However, the observed effects can be a result of both Ca2+ influx and active SV cycling.This analysis reflects, in accordance with recent studies , 42, theAs indicated above, our first quantitative analysis does not allow for differentiating phosphorylation changes that are directly dependent on active membrane trafficking in the synapse rather than being caused by the activation of calcium-dependent kinases and phosphatases such as CaMKII , calcine2+ ions (B) but did not compromise Ca2+ influx (C). It led to significant changes in the phosphoproteome of the synaptosomes: 1497 sites showed significant difference with a minimum of 20% intensity changes at 1% FDR that can be mapped to approximately 700 proteins. Strikingly, BoNT treatment reduced the intensity of many phosphorylation sites (C). The phosphorylation status of these sites and their corresponding proteins is apparently dependent on the Ca2+-induced SV cycling activity in the presynapse.We suppressed SV cycling by incubating synaptosomes with BoNT that block exocytosis by cleaving SNARE proteins. Toxin-treated synaptosomes and (as controls) synaptosomes incubated with inactivated toxins were stimulated with the high-potassium buffer in the presence of Ca2+ ions . The tox2+ ions B but did+ influx C. It ledon sites C. The php < 0.01) of phosphorylation sites that can be attributed to MAPK, PKC, and GSK3 kinase groups (P ≅ 1.0 × 10-5) regulated phosphorylation sites could be ascribed to being CaMKII substrates, whereas putative sites for MAPK (1), CLK (2), and PAK (3) were slightly over-  or under- (3) represented, respectively . The balance of up- and downregulated phosphorylation sites was also significantly altered (p < 0.01) for CLK, CK1, and PAK kinases.The enrichment analyses for GO terms did not show substantial differences when compared with proteins carrying regulated sites in Ca versus EGTA experiments (not shown). The impact of BoNT treatment became apparent when we investigated possible substrate–kinase relationships. The enrichment analysis showed significant overrepresentation phosphorylated by Ca2+-dependent protein kinases and phosphatases upon Ca2+ influx. Conversely, a large group of phosphorylation events has shown changes when comparing mock- and BoNT-treated synaptosomes. Hence, we termed these sites “SV-cycling-dependent.” We noted with interest that the majority of “SV-cycling-dependent” sites did not show significant intensity changes in the Ca/EGTA experiment. We also noted that many proteins tend to carry regulated phosphorylation sites of one of the two groups introduced above, e.g., mostly “primary Ca2+-dependent” or “SV-cycling-dependent” sites, while only a few contain sites of various categories (B). Interestingly, the number of proteins that carry mostly “primarily Ca2+-dependent” sites is lower than the number of proteins that carry mostly “SV-cycling-dependent” sites (B). It indicates the tendency that “primarily Ca2+-dependent” phosphorylation sites appear clustered on a few proteins, whereas “SV-cycling-dependent” sites are often found as the only significantly regulated phosphorylation site in a single protein. The site occupancy assessment , sites predicted to be CaMKII substrates include significantly more “primary Ca2+-dependent” sites than sites classified as “SV-cycling dependent.” This harmonizes well with the strict Ca2+ dependence of CaMKII activity . The majority of phosphorylation sites on CAMKII, MAPK1, MAPK3, and PRKCB themselves show dependence on calcium ions. Increased phosphorylation of the regulatory sites of CaMKII, MAPK1, MAPK3, and PRKCB  points to the activation of those kinases following depolarization of synaptosmes by KCl  suggestn events . It shoun events C, sites activity . Converst” sites C. The mas by KCl  and S6.FA). Particularly, Neurabin-1 (Ppp1r9a), a PP1 regulator, showed increased phosphorylation in the Mock/BoNT experiment, suggesting the involvement of PP1 in regulating SV cycling. Although phosphatase–substrate interactions are more difficult to analyze due to the degenerated nature of phosphatase docking motifs, previous studies led to the proposal of docking motifs of PP1 and the Ca2+-calmodulin-dependent phosphatase calcineurin (B and C). Available interactome data suggest a partially overlapping set of proteins that can be regulated by the phosphatases , which does not allow confident assignment of a phosphatase to a particular phosphorylation event. When considering all potential targets, our results show a minor increase in the percentage of “SV-cycling-dependent” sites over “primary Ca2+-dependent” sites on proteins that can be regulated by PP1 .We noticed that numerous phosphorylation events on phosphatases and phosphatase regulators were significantly regulated in our study A. Particcineurin . We thercineurin , B and C2+-dependent” or “SV-cycling dependent” phosphorylations. Reactome pathway analysis (D). One obvious difference regarding the two groups is that proteins are connected to potassium channels (“Voltage-gated potassium channels”), which are more enriched in the “SV-cycling-dependent” group. Another striking difference is that phosphorylated proteins termed with “Synaptic adhesion-like molecules” and “Neurexins and neuroligins” are enriched in SV-cycling-dependent group. In contrast, terms associated with “Neurotransmitter release cycle”, e.g., glutamate, acetylcholine, etc., release, are barely enriched in phosphorylated proteins that carry “SV-cycling-dependent” sites . Reactome pathway annotation aligned well with the manual annotation of keywords describing protein function or location according to information available in the literature , amphiphysin (Amph), and AP180 (Snap91) showed a clear Ca2+-dependent character  and spectrin-binding proteins adducin alpha (Add1) and beta (Add2), as well as potassium channel subunits that are known constituents or modulators of Kv1 , Kv2 , Kv4.3 (Kcnd2), Kv7.2 (Kcnq2), K2p10.1 (Kcnk10), Hcn  potassium channels  and several other potassium channel subunits and modulators  have shown the “primary Ca2+-dependent” character of phosphorylation (2(Ca/EGTA) and log2(Mock/BoNT), respectively). These can be also visualized using a shiny app accessible at https://s1608-shiny.mpibpc.mpg.de. Because of the tendency of proteins to carry preferentially one of the two groups of significantly regulated phosphorylation sites, we set out to investigate the putative biological functions of these proteins in order to delineate processes that can be controlled by “primary Caanalysis  sheds lianalysis D. One obt” sites D, top. Rterature . On the haracter  and S10.P180 Snap showed aharacter , 94, 95 haracter . In contchannels   and syntaxin-1a (Stx1a), have a phenotype, which belongs to the “SV-cycling-dependent” group (Vamp2-S75) were previously described as being important for the interaction with Munc18-1, a protein that can exhibit a stimulatory effect on trans-SNARE zippering (Cnr1), T314 and T322, belong to the “SV-cycling-dependent” group as well. In order to test the effects of phosphorylation at these sites, we expressed the respective wild-type (WT) proteins, their phosphomimetic mutants, and phosphorylation-null mutants in cultured hippocampal neurons (A).We noted with interest that certain phosphorylation sites on SNARE proteins, including synaptobrevin . For these proteins we created adeno-associated viruses (AAVs) that allowed us to mimic the effects of phosphorylation, and we expressed them in neurons together with Vamp2-pHluorin, as a sensor for exo-/endocytosis (B and C).With this strategy, we addressed ocytosis , B and CVamp2-S75D reduced exocytosis following short and long stimulation; it also reduced endocytosis in response to long stimulation. Expression of nonphosphorylatable Vamp2-S75A reduced exo- and endocytosis following both long and short stimulations (D and E). Conversely, pseudo-phosphorylated Stx1a-T21E/T23E enhances exocytosis efficiency during short stimulation, while the nonphosphorylatable variant does the opposite (F). The effects on exocytosis become negligible during long stimulation, but both mutant variants result in an enhancement of endocytosis under these conditions (G).Our stimulation experiments demonstrated that both phosphomimetic and nonphosphorylatable amino-acid substitutions, in both proteins, influence exo- and endocytosis in the cultured neurons. Specifically, we stimulated neurons with either 60 action potentials (AP) at 20 Hz (short stimulation) targeting the readily releasable pool of SVs and thus reflecting the immediate exocytotic response, or using 600 AP at 20 Hz (long stimulation) to mobilize the more inert recycling SV pools, reaching a state in which the entire vesicle cycle is fully activated . The expulations , D and Eopposite F. The efnditions G.Cnr1-T314E/T322E reduced exocytosis (most evident during short stimulation), while the nonphosphorylatable variant Cnr1-T314A/T322A increased exocytosis in response to long or short stimulation. The effect on endocytosis was observed only for the pseudo-phosphorylatable Cnr1 variant after long stimulation (H and I).The cannabinoid receptor-1 is known for modulation of the neurotransmitter release , 99, 100mulation , H and IVamp2 both mutants led to a substantial reduction in exocytosis rates, its pseudo-phosphorylated form (Vamp2-S75D) showed a greater effect on exocytosis than the nonphosphorylatable form (Vamp2-S75A). Presumably, the absence of a charged residue did not fully compensate the steric effect caused by the amino acid substitution at position 75 of Vamp2, which has apparently a stronger impact on protein function than the double mutant at positions 21/23 of Stx1a. The S75 phosphorylation site is located within the R-SNARE motif required for forming a coiled-coil complex with Stx1a and Snap25 during exocytosis. In contrast, the T21/T23 positions of Stx1a are located in the regulatory N-terminal helical bundle, explaining why mutations show different effects on exocytosis. Overall, these findings support the concept that protein phosphorylation is a key regulatory mechanism in neurotransmitter release that modulates different steps in the SV cycle.These experiments tested only three of the synaptic phosphorylation sites that we found differentially phosphorylated in this work. Nevertheless, even if these experiments are based on overexpression in a background containing normal levels of WT proteins, all tested candidates resulted in significant modulations of exo- or endocytosis. Although in the case of In this study, we have used isolated nerve terminals as a model to differentiate phosphorylation changes that are induced by calcium influx alone from those that in addition require active endo–exocytotic SV cycling. Surprisingly, we found that inhibition of exocytosis by BoNT had a profound effect on the phosphoproteome despite unchanged calcium influx. These findings reveal complex signaling cascades during which regulatory changes in protein phosphorylation are not only dependent on the activation of kinase/phosphatase networks but also on the progression of vesicular trafficking.D) , thymosin beta , ADP-ribosylation factor GTPase activating protein 1 (Arfgap1-S247 2.57), calcium/calmodulin-dependent protein kinase type II subunit delta , protein bassoon , septin 5 , and RIM-binding protein 2 . We also confirm phosphorylation changes in response to stimulation that were reported earlier for synapsin-1, dynamin-1, Tau protein, as well as the multisite phosphorylations of the giant active zone proteins bassoon and piccolo  and stimulation (50 mM KCl for 2 min versus 75 mM KCl for 10 s), many of the regulated phosphorylation sites observed by us are in excellent agreement with earlier results from another laboratory D 42). O. OD) . It would be of interest to investigate whether these proteins are also present at measurable levels in the presynapse and whether they are affected by synaptic activity. Although synaptosomal preparations are known to contain fragments of the postsynaptic membrane  but also in other proteins that might take part in the Wnt pathway such as Apc, Amotl1, and Tnik. Given the connection between the Wnt pathway and the regulation of cell–cell contacts and cytoskeletal elements, it is not surprising that many cytoskeleton- and cell-adhesion-related proteins contain BoNT-responsive phosphorylation sites . It is tempting to speculate that these phosphorylation changes represent the activation of pathways directed at increasing synaptic strength (most importantly LTP) by mechanisms such as size increase of the synaptic contact zone, enlargement of the presynaptic bouton, and adaptations of the presynaptic cytoskeleton to increase the efficiency of SV cycling  can play a crucial role in this, by recruiting PP1 to the synapse by regulating its binding to actin filaments, which impacts PP1 activity  had an effect only on exocytosis in cultured hippocampal neurons. The effect can be also linked through the activation of MAPK to Munc-18 phosphorylation and regulation of SNARE assembly  format. The source data and code are available at GitHub: https://github.com/IvanSilbern/2021_Silbern_etal_ShinyApp. The app is deployed and accessible at: https://s1608-shiny.mpibpc.mpg.de.We also provide a visualization tool in a shiny-app (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Cr concentration of polyaluminum ferric chloride (PAFC), and NH4+-N was removed by calcium hypochlorite. Soil backfill was replaced, such that the informal landfill site was immobilized, which was perfectly suitable for this southern rainy area. This study proposes rules for a comprehensive improvement scheme for a landfill, and provides a reliable theoretical basis and practical experience for the treatment of similar informal landfills.A typical informal landfill in a rainy area of southern China was taken as an example in this study. The comprehensive control ideas and processes of the informal landfill site were systematically reviewed. The basic situation for the early stage of the government survey and investigation was provided, including a waste stock survey, water volume measurement, and a waste source survey. The main contents and key factors of a comprehensive investigation of the environmental quality status were briefly summarized. The water quality in the landfill, groundwater quality inside and outside of the site, and heavy metals in the bottom sediment were all determined. A low-cost practical landfill technology was explored to reduce the Chemical Oxygen Demand COD Informal landfills refer to sites that use natural terrain conditions for waste treatment and are not constructed and operated in accordance with relevant national standards and norms ,2. They Low-lying landfills form pools where waste is subject to long-term immersion, increasing the leaching efficiency of pollutants. Heavy seasonal rains and subsequent water flow causes surrounding surface water and soil environment pollution ,9,10.Given the continuous updating of national environmental protection policies and the improvement of public environmental protection awareness during recent years, the governance of informal landfills has become an imperative environmental protection task . BecauseAt present, China has accumulated a relative breadth of experience in the comprehensive treatment of informal landfills, but there is still a lack of systematic management ideas and complete technical schemes for informal landfills in rainy areas in southern China. The main reason for this is that for this type of landfill, during the process of treatment, in addition to considering the treatment and disposal of solid waste, there is also the problem of water environmental treatment as a result of rainfall. At the same time, the waste is degraded and converted to silt, which causes insufficient stability of its structure during the comprehensive treatment process and affects the implementation of the project. Silt pollutants result in questions of how to safely and effectively control or treat waste, which will influence the final determination of regulation schemes.There are few reports regarding the treatment of water accumulated at informal landfills, but research regarding leachate in sanitary fields is quite mature and has achieved good results. Leachate contains a large number of harmful organic matters, heavy metals, and inorganic salts . There aTreatment of leachate in general sanitary landfills is conducted in the field; however, this type of long process technology is not suitable for informal landfills. Leachate is treated only once, that is, after treatment, water is removed or connected to a municipal pipe network and no subsequent leachate is generated. At present, there is no effective integrated equipment to sufficiently treat leachate. Mature, low-cost, and high-efficiency leachate treatment technology is an effective means to solve the problem of water management at informal landfills. Regarding site investigations of informal landfills, most focus on the site risk assessment of the landfills, and those in arid areas of northern China are often taken as case studies .A survey of this type in the rainy areas in southern China has not been previously reported. Taking an informal landfill site in southern China as a typical case, this study aimed to (1) select the specific method of basic situation for the early stage of the government survey and investigation; (2) determine the main contents and key factors of comprehensive investigation of the environmental quality status; (3) explore a low-cost practical landfill technology to solve water pollution problem in site; and (4) summarize the processes of comprehensive treatment of the informal landfill site in a rainy area of southern China.The informal landfill is in southern China, with an average annual rainfall of 1034.4 mm, mainly from June to September, and an approximately 226-day frost-free period. The site was previously an abandoned quarry. As a result of years of mining, a natural pit formed. Beginning in the 1990s, waste began to accumulate in the pit. The changing process of the landfill is shown in 2, the stock of waste and mudstone is approximately 240,000 m3, and the leachate volume is approximately 30,000 m3. The water depth is up to 12 m.As the waste stock in the landfill is not clear, we used a method of geophysical exploration to determine the hydrology in the field. We drilled holes at positions 1–4 to determine water depth, garbage depth and silt depth. The range finder was used to measure the size of the site. The geophysical profile is shown in The investigation of the source of the waste is a necessary to determine the types of pollutants at the site. For this informal landfill, it is known that the waste in the site mainly consists of textile industrial waste and domestic waste through public inquiry and a data search. The classification and analysis of the industrial enterprises around the landfill during the past 30 years is shown in The purpose of the hydrogeological survey was to understand the stratigraphic structure of the site and the groundwater level and flow direction, with a focus on determining the possible means by which the site water could contaminate the groundwater.A total of seven drilling wells, G1, G2, G3, G4, G5, G6, and G7, were established in the landfill area. A summary of the measured water level is shown in Considering site subsequent reuse and environment risk analysis, the heavy metals in the sediment needed to be tested. Sediment samples, after being dry ground, were sifted through 100 mesh sieve. The samples (0.1g) were collected and put in PTFE test tube and then nitric acid (5 mL), hydrofluoric acid (2 mL), and perchloric acid (1 mL) were added. The samples were heated at 180℃ for 4h, and then the acid was evaporated. After cooling, the samples were filled to 25 mL with high purity water and detected by inductively coupled plasma atomic emission spectrometry . Organic matter content is among the important indicators. A stability assessment field detection method with reference to the determination methods of soil organic matter (NY/T 1121.6 2006) was determined.The sampling points were geophysical detection points, as shown in 4+-N and CODCr, and contain indexes that inhibit microbial growth. According to the depth of the landfill, two sampling areas were established in the site as shown in 4+-N, CODCr, TP, TN, and sulfide were tested and analyzed. Water quality testing methods are shown in The purpose of the water quality analysis was to understand the water quality of the site so as to determine the subsequent treatment and discharge of water. The main pollutants in the landfill leachate are NHTo determine the environmental quality of the groundwater at the landfill site and assess whether the surrounding groundwater environment had been affected by the landfill, groundwater sampling sites were established inside and outside the site for water quality analysis. The sampling sites were G3 (outside the site) and G2 (inside the site) as part of the geological condition detection sites.Because there is an industrial plant on the east side of a nearby factory, to avoid interference of monitoring point data caused by external factors and lack of conviction, the off-site groundwater monitoring point was set on the south side of the factory, and the testing indicators were analyzed by referring to representative indicators in the groundwater environmental quality standard .The bottom mud in the pit of the landfill site was collected for detection. The detection results are shown in Cr and NH4+-N concentration in the pond reached 3012 mg·L−1 and 1014 mg·L−1, respectively. After surface aeration treatment, the concentration of CODCr and NH4+-N in the pond significantly decreased. The results for conventional pollutants in the water are shown in Cr, NH4+-N, and TN were 238 mg·L−1, 129 mg·L−1, and 148 mg·L−1, respectively, exceeding the surface water environment quality standard (v class) [Through testing of the raw water quality of the site, it was found that the CODv class)  and the v class)  limit. Fv class)  but met v class)  limit.Cr, NH4+-N, and TN.It can be concluded that the water in the landfill should be removed or discharged after subsequent treatment to meet the relevant standards. The key pollutant indexes in the water are COD-, NH4+, Na+, and COD as parameters to measure the penetration of landfill leachate pollution. In this study, the excessive turbidity was up to 1049 times the limit; NH4+-N exceeded the limit by approximately 9 times, and volatile phenol and oxygen consumption exceeded the limit by 7.1 and 1.9, respectively. The three indicators at G3, smell and position, visible objects, and turbidity, exceeded the standard; the excess multiple of turbidity was 2.1. According to the analysis of the G2 data, the groundwater at the site is affected by landfill leachate. By comparing and analyzing the detection data of G2 and G3, it was found that the number of indicators and pollutants exceeding the limits outside the site were far lower than those inside the site, indicating that the environmental quality of the off-site groundwater is less affected by the landfill leachate. The reasons for this may be that the flow direction of the groundwater varies in a certain area because of the microgeomorphic units in southern China and there is no connectivity between G3 and the landfill. This field shows such results, but if the water outside the site is also polluted, then the subsequent site treatment measures will increase the groundwater remediation. For example, vertical curtain or cutoff wall should be adopted to cut off the migration of pollution source to the downstream direction of groundwater flow [The results were compared to the IV grade standard for groundwater quality (signed GW-standard) shown in . It was ter flow .Because all the pollutants at the site originate from the site waste, the primary task to reduce the environmental risk of the landfill is to eliminate the source of pollution. The removal of waste can effectively control the continuous release of pollutants and reduce the environmental risk.Cr could be increased to 35.94% when the ratio of PAFC was 1‰, as shown in According to the water quality test results, the main concept of water treatment is to determine a low-cost and efficient water-treatment technology and discharge after meeting the relevant requirements or connect to off-site treatment facilities. Water treatment is the key to the management of this type of landfill. Compared to biochemical treatment, physical-chemical treatment is simple and feasible; thus, it has some advantages in the treatment of water in an informal landfill. At the same time, sewage treatment can be combined with local actual conditions. Sewage can be treated to meet the standards and then removed via a nearby sewage pipe network that can not only reduce the treatment cost but also reduce the environmental risk caused by sewage outflow. Polyaluminum ferric chloride (PAFC) is an inorganic polymer flocculant developed on the basis of the coagulation and hydrolysis mechanism of aluminum salt and iron salt with a good pollutant removal effect. The mixing ratio of the three gradients of 0.4‰, 0.7‰, and 1‰ was selected for a coagulation and precipitation experiment. The results showed that the removal rate of CODCr effluent could meet the requirements of the connection to a sewage pipe network (≤500 mg·L−1), but the NH4+-N concentration could not be met. Thus, calcium hypochlorite (CH) was considered to reduce the NH4+-N concentration.Under the condition of this proportion, the COD4+-N removal effect than adding CH first and then adding PAFC. The difference in the effect is shown in 4+-N concentration is shown in The proportion of addition was set as 1‰, 2‰, 3.0‰, and 4.0‰ (the effective rate of CH was 50%). Through experimental comparison, it was found that adding PAFC first and then adding CH had a better NH4+-N reaches an optimal state. However, the removal effect of NH4+-N was relatively poor when the reagent was added in the opposite order. It can be seen that the sequence of adding agents is different, resulting in the difference of reaction process. Under the condition that the same treatment effect is achieved, the amount of adding agents will also change accordingly.According to the experimental comparison between By adopting the treatment method of adding PAFC first and CH later, the sewage in the pond can be treated to the standard and then connected to the downstream sewage plant for further treatment, thus solving the problem of sewage treatment.After the sewage is discharged, the silt moisture content at the bottom of the pond is very high, which will lead to further expansion of the silt and the formation of surface runoff given the rainwater during the rainy season, endangering the surrounding environment.It is necessary to immobilize the silt in the field and then stabilize the landfill. In a backfill site with low moisture content, the silt is concentrated into several internal sections of the site and lime is solidified on the surface. Compared to full-field curing, this method can greatly improve the infrastructure stability and lower costs. Through the implementation of this solidification project by backfilling the guest soil, the landfill can meet follow-up stability requirements.The stability of the landfill was greatly improved after the backfilling of the guest soil and lime solidification in the field. The landfill covering was conducted in accordance with the Technical Specification for Sanitary Landfill Covering . This prThrough the investigation and analysis of the current situation of a typical informal landfill in southern China and the detection of the environmental quality of the site, the landfill stock and water volume were determined. The source and properties of incoming waste were determined, and the hydrogeological conditions, water quality, heavy metal content in the bottom mud, and extent of pollution were obtained. It was found that the number of indicators and pollutants exceeding the limits outside the site were far lower than those inside the site, indicating that the environmental quality of the off-site groundwater is less affected by the landfill leachate. However, if the water outside the site is also polluted, then the subsequent site treatment measures will increase the groundwater remediation. Adopting the treatment method of adding PAFC first and CH later results in higher clarity and lower color, better appearance of the treated water, and a faster removal rate of ammonia nitrogen. The sewage in the pond was treated to the standard and then connected to a downstream sewage plant for further treatment, thus solving the problem of sewage treatment. The project of backfilling guest soil and landfill covering fully achieved the comprehensive regulation of the landfill and reduced the risk of the site. The research and application of this project can provide a theoretical basis and practical experience for the comprehensive regulation of informal landfills in rainy areas in southern China.


The use of aftermarket computer-aided design/computer-assisted manufacturing (CAD/CAM) prosthesis components in dental implants has become popular. This study aimed to (1) compare the accuracy of aftermarket CAD/CAM screws with that of original equipment manufacturer (OEM) abutment screws and (2) examine the biomechanical effects of different abutment screws used with zirconia abutment in an implant fixture by using three-dimensional finite element analysis (FEA). Significantly different measurements were obtained for the aftermarket CAD/CAM and OEM screws. The FEA results indicated that under the same loading condition, the maximum stress of the aftermarket CAD/CAM screws was 15.9% higher than that of the OEM screws. Moreover, the maximum stress position occurred in a wide section of the OEM screws but in the narrowest section of the aftermarket screws. The stress of the OEM zirconia abutment was 14.9% higher when using the aftermarket screws than when using the OEM screws. The effect of the manufacturing differences between aftermarket and OEM screws on the clinical effect of aftermarket screws is unpredictable. Therefore, aftermarket screws should be cautiously used clinically. Replacing a missing tooth with a dental implant is a popular dental treatment. Therefore, achieving long-term success and avoiding complications in dental implantation is important. The implant assembly is divided into several parts with close interlocking links. The implant assembly includes the crown, abutment, and screw and implant fixture. The screw is a critical and fragile link that affects the success or failure of the entire implant system.1, clinicians may encounter various biological and mechanical complications2. Biological complications include the loss of osseointegration or peri-implantitis3, and mechanical complications include crown fracture, abutment fracture, and loosening or fracture of abutment screws4. The fracture of the abutment screw can be a serious problem. A fixture and an abutment are connected through a screw, and the incidence of abutment screw fracture ranges between 2%5 and 3.9%6. The removal of a fractured screw is always challenging, and sometimes, the entire implant fixture must be removed if the fragment of screw remaining inside the implant prevents the implant from functioning efficiently8. Thus, broken screws may cause irreversible damage and affect the trust involved in the doctor–patient relationship.Despite the high success and survival rates of dental implants9. Several studies have reported that CAD/CAM technology can provide results comparable to those provided by conventional techniques in terms of implant survival, prosthesis survival, and technical and biologic complications13. Aftermarket manufacturers have begun to replicate the design of prefabricated OEM prosthetic implant components and produce aftermarket CAD/CAM engineered ones, such as abutments and screws, for clinical use. Clinicians can choose alternatively engineered implant components with high efficiency and low cost. However, the biomechanical effect of aftermarket prosthetic components on the entire implant system is still unclear.With advancements in computer-aided design/computer-assisted manufacturing (CAD/CAM) techniques in dentistry, clinicians can now choose prosthetic components, including abutments and screws, from the same manufacturer, which is known as the original equipment manufacturer (OEM)16. Although Zr abutments have good aesthetic outcomes and offer several clinical advantages over metal abutments, clinicians still have doubts regarding their cracking risk, especially under the high internal stress that may be caused by the screw mechanism.Zirconia (Zr) abutments are a restorative alternative to metal abutmentsBecause studies on OEM and aftermarket screw mechanisms have provided unclear results, the present study aimed to (1) compare the accuracy of aftermarket CAD/CAM screws with that of OEM abutment screws and (2) examine the biomechanical effects of different abutment screws used with Zr abutment in an implant fixture by using three-dimensional (3D) finite element analysis (FEA). To the best of our knowledge, this study is the first to compare OEM and aftermarket titanium screws in an OEM Zr abutment.A NobelReplace Conical Connection PMC dental implant  and a NobelProcera zirconia abutment  with a full zirconia crown were used to create a morphology resembling the right central incisor. Two models were used in the analysis: (1) model I (control group) was based on the use of OEM abutment screws and (2) model II (test group) was based on the use of aftermarket CAD/CAM abutment screws. Three samples of the aftermarket abutment screws were obtained from the manufacturer , who manufactured the screws based on the data of OEM abutment screws which got from the optical scanning machine, then the manufacturer input the information to their own CAD system and made adjustment based on experience, finally milled the screw with CAM system  using 3D microscope measurements and cloud data extraction  was performed. The OEM and aftermarket CAD/CAM screws were confirmed to be made of TiAl6V4, and the dental implant was confirmed to be made of pure titanium.In this study, we assumed that the implant, abutment, and screws were homogeneous and had isotropic elastic propertiests Table  were col23. The interface between the implant and bone was also set as a contact. The frictional coefficients for the surface contacts of the rough implant surface with the cortical bone and trabecular bone were assumed to be 0.6524 and 0.7725, respectively.The implant–abutment, implant–screw, and abutment–screw interfaces were set as contacts and had a frictional coefficient of 0.326.For the screw tightening torque, 35 N·cm torque was applied and the axial force was created and pointing down the screw’s axis which was calculated to be 511 N with the Eq. (T is the tightening torque (N·m), K is the torque coefficient, D is the screw diameter (m), and F is the axial force (N).In Eq. , T is th27. The loading period was 0.8 s. The amplitude of the loading varied from 0 to F in accordance with a semisinusoidal pattern  was installed on a desktop computer with a Pentium 4 processor, 16 GB of memory, and a Windows 7 operating system to analyse model data and perform stress analysis of the implant system subject to an oblique periodical loadingA 3D microscope was used to measure the sizes of the model I (OEM) and model II (aftermarket CAD/CAM) screws. Eight sets of measurements were conducted of parameters such as the screw length  and screw angle (hex and thread angle) Fig. . The resUnder the same loading conditions, the stress distributions of model I (OEM) and model II (aftermarket CAD/CAM) were similar. The stress was concentrated at the screw, abutment neck (the connection section where the abutment was inserted deep into the implant), and also the collar, first, second, and third threads of the implant Fig. . The maxIn the cross-sectional analysis of the screws Fig. , the ove28. If excessive force causes catastrophic damage to the implant–abutment–screw complex (such as screw fracture), physicians may need to remove or replace the complex surgically, which usually leads to extensive damage to the alveolar bone13. Therefore, in patients with the aforementioned risks, aftermarket screws should be cautiously used. We hope that manufacturers can enhance the accuracy of CAD/CAM for the clinical long-term success of implant prosthesis13.We compared the sizes of the OEM and aftermarket CAD/CAM abutment screws. The results indicated that statistical differences existed in the eight measurements, including the different screw lengths and angles. The measurement error caused by the manufacturer preprocessing problem in 3D optical scanning procedure, CAD and the postprocessing error in the accuracy of CAM are possible reasons for the differences in the sizes of the CAD/CAM screws fabricated by different manufacturers. The size of the aftermarket screw was different from that of the OEM screw; thus, the aftermarket screw exhibited a misfit with the OEM abutment. The FEA test indicated that the maximum von Mises stress of the aftermarket screw was 15.9% higher than that of the OEM screw under the same loading conditions. The system performance problems occurring with the aftermarket screw may not be visible to the naked eye in clinical use. Moreover, no immediate destructive failure would occur; thus, the aftermarket screw can still be used clinically. However, in some cases, such as trauma-related dental injuries or patients with parafunction habits , the incisive force can be considerably higher than the physiological range31. The microstructure and mechanical properties of the titanium alloys are affected by various manufacturing processes, such as thermomechanical and cold rolling processes29. The clinical performance of CAD/CAM titanium screws can be negatively affected if aftermarket manufacturers use raw materials that differ from those used by original manufacturers. Moreover, titanium machining is difficult, and that must be followed the machining guidelines include cutting at low speed, using sharp instruments, and never interrupting the cut procedure30. The mechanical performance of titanium screws can be significantly affected if aftermarket manufacturers fail to follow the machining guidelines. For example, poor processing can cause the discontinuity of the surface and reduce the ductility and fatigue properties of the metal. Surface overheating can result in the interstitial pickup of oxygen and nitrogen, which causes the machined titanium to be hard and brittle31.In addition to the CAD/CAM error, the differences in the mechanical properties of the titanium alloys used in model II (aftermarket) and model I (OEM) may also cause problems in clinical use. These differences may cause complications in long-term implant use16. Zr has a modulus of elasticity of 200 GPa and flexural strength of 900 MPa. Moreover, the high fracture resistance of Zr improves the clinical success of aesthetic abutments33. Several studies have reported good survival rates and limited biological and technical challenges for Zr abutments37. Although Zr abutments offer several clinical advantages over metal abutments, clinicians still have doubts regarding the risk of abutment crack, especially when the screw material is different from the Zr abutment. The higher wear at the interface between titanium alloy and Zr39 as well as the internal stress concentration and friction properties for different materials should be examined.In this study, the Zr abutment was used for FEA. Various materials, including titanium (Ti), Zr, and various metal alloys, are currently used for implant abutment fabrication. Due to their high strength and biocompatibility, Ti and metal alloy abutments are considered the standard choice for implant-supported restorations. However, Ti and metal abutments may show through the peri-implant soft tissue, resulting in greying of the marginal tissue and poor aesthetic outcomes. Hence, the Zr abutment was introduced in 1995 as a restorative alternative to metal abutments41. The differences in the maximum von Mises stress between model I and model II may have resulted from a misfit between the screw and the abutment. In this study, the maximum stress of the aftermarket screw was 15.9% higher than that of the OEM screw. Moreover, the maximum stress at the OEM abutment was 14.3% higher when using the aftermarket screw than when using the OEM screw. Screw misfit had a small effect on the maximum stresses of the implant and bone. Unsuitable connection between the screw and abutment may cause the compression of the implant screw, which results in torque loss, loosening of the screw, or fracture of the screw45. The highest stress was 526.97 MPa in model I and 611.52 MPa in model II. The aforementioned two stress values are lower than the yield strength of titanium alloy (760 MPa). This finding indicates that screw failure did not occur when a load of 170 N was applied obliquely on the implant system; however, model II, which had the highest stress in its narrowest part, was more susceptible to fatigue failure during clinical practice than model I was17.In this study, the highest stress of the overall implant system was observed in the fixation screw for both the models. This finding is in agreement with those of previous FEA studies46. Moreover, with an external hex implant, screw fracture at the third thread region which presence of a lever around the screw40. In this study, with an internal hex implant, the maximum stress occurred on the palatal side of section B in model I and on the palatal side of section D in model II. Section B is the first step in the screw neck region, which lies in the suprabony area and is in contact with the corresponding abutment. However, clinical fracture does not occur easily at section B due to its large cross-sectional area. Removing broken screw fragments is easy due to their suprabony location. However, section D is the junction of the screw neck and shank. In the adopted screw configuration, section D is the narrowest region and is located in the infrabony area. Therefore, the maximum stress after loading was 15.9% higher for the aftermarket screw than it was for the OEM screw. Moreover, the position of maximum stress changed from section B (wide section) for the OEM screw to section D (narrowest section) for the aftermarket screw. In addition, the stress in sections D and E (narrowest sections) were 30.1% and 69.5% higher when using the aftermarket screw than when using the OEM screw. Therefore, we must be mindful of the irreversible complications that can occur during the clinical use of aftermarket CAD/CAM screws.The fracture point of the screw sometimes is located on the palatal side in the narrowest region (shank area) below the implant platform of the internal hex implant6, the incidence of abutment screw fracture over a 5-year period was 3.9% and that of abutment screw loosening over the same period was 6.7%6. Although the screw loosening is not a critical complication in itself, it creates difficulties for both the dentist and patient. Screw loosening occurs when the compressive occlusal loadings are higher than the clamping force, which is the tension that holds the screw-abutment-implant together47. Another possible reason for screw loosening is screw defects. For example, the screw may be over-torqued when insertion or the misfit of the abutment when screw preload. The aforementioned defects result in increased stress on the screw, and unrecognised screw loosening may finally culminate in fracture of the screw8.Problems in the abutment screw can cause serious complications. The screw is the smallest component in the implant system, and it connects the implant fixture with the abutment. In the study of Kreissl et al.48 indicated that aftermarket CAD/CAM Zr abutments with OEM abutment screws may have low static load strength; thus, the suitability of these abutments for clinical use is questionable. Chang et al.13 investigated the mean maximum load capacity of OEM and aftermarket titanium abutments under static load and reported that OEM titanium abutments had significantly higher static load strength than the aftermarket abutments did. The failure mode involved deformation of the implant and abutment as well as fracture of the abutment and screw. Jarman et al.9 confirmed that the maximum load capacity of aftermarket Zr abutments was significantly lower than that of OEM Zr abutments. Kelly and Rungruanganunt49 reported that OEM abutments (Straumann) outperformed aftermarket abutments (Atlantis) under cyclic loading, and they concluded that the differences in abutment design and fabrication between aftermarket and OEM manufacturers can significantly influence the clinical performance of the abutments. The aforementioned results raise concerns regarding the use of aftermarket CAD/CAM prosthesis components. To the best of our knowledge, this study is the first to compare OEM and aftermarket titanium screws in an OEM zirconia abutment. The FEA results indicated that the maximum von Mises stress was higher in the aftermarket screws than it was in the OEM screws. This finding is in agreement with those of previous studies regarding aftermarket abutments49. Therefore, additional research and long-term clinical studies on the performance of aftermarket CAD/CAM screws are required to verify their reliability in clinical use.Few studies have examined aftermarket prosthetic components, and most of these studies have focused on aftermarket abutments with OEM screws. Gigandet et al.Significantly different measurements were obtained for the aftermarket CAD/CAM and OEM screws under 3D microscope. Therefore, the effect of the manufacturing differences between aftermarket and OEM screws on the clinical effect of aftermarket screws is unpredictable.The FEA results indicated that under the same loading condition, the maximum stress of the aftermarket CAD/CAM screw was 15.9% higher than that of the OEM screw. Moreover, the maximum stress position occurred in a wide section of the OEM screw but the narrowest section of the aftermarket screw. The stress of the OEM Zr abutment was 14.9% higher when using the aftermarket screw than when using the OEM screw.The following conclusions can be drawn from this study: