Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue.FMO1. Additionally, phase II conjugation genes including UDP-glucuronosyltransferases UGT1A5, UGT2A1 and UGT2B15, sulfotransferase STE, N-acetyltransferase NAT12 and glutathione S-transferase were identified. Phase I and phase II metabolic reactions increase the water solubility of steroids and play a key role in their elimination. Differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases  and plasma proteins alpha-1-acid glycoprotein (AGP) and orosomucoid (ORM1). 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. Differences in the expression of FMO1, NAT12, HSD17B2 and HSD17B13 were verified by quantitative real competitive PCR.Liver tissue samples from 58 boars of the two breeds Duroc and Norwegian Landrace, 29 with extreme high and 29 with extreme low levels of androstenone, were selected from more than 2500 individuals. The samples were hybridised to porcine cDNA microarrays and the 1% most significant differentially expressed genes were considered significant. Among the differentially expressed genes were metabolic phase I related genes belonging to the cytochrome P450 family and the flavin-containing monooxygenase A number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase I oxidation metabolism, phase II conjugation metabolism, and regulation of steroid availability. The study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint. Boar taint is an off-odour and off-flavour in pig carcasses that is primarily caused by high levels of 16-androstene steroids  in adipoet al. [Androstenone (5α-androst-16-en-3-one) is produced in the testis and is transported by blood to the salivary gland where it functions as a pheromone to stimulate mating stance in gilts , and defet al.  identifiet al. ,10.Enzymes responsible for metabolism of androstenone and skatole in liver have been identified but relatively few genes have been investigated. Several studies have shown that 3β-hydroxysteroid dehydrogenase (3β-HSD) is involved in androstenone metabolism, with low mRNA, protein and enzyme expression levels correlated to high androstenone levels in adipose tissue ,11,12. CAlthough both androstenone and skatole are responsible for boar taint, androstenone seems to be the biggest problem in the Duroc and Norwegian Landrace populations . Levels Porcine cDNA microarrays were used to obtain global gene expression profiles in liver tissues sampled from 58 D and 58 NL boars with extreme high and low levels of androstenone. Average levels of androstenone were 11.57 ± 3.2 ppm for D high (DH) and 0.37 ± 0.17 ppm for D low (DL) boars. Average levels of androstenone were 5.95 ± 2.04 ppm for NL high (NLH) and 0.14 ± 0.04 ppm for NL low (NLL) boars. Linear models were used to identify significantly differentially expressed genes and the top 1% (269) most significant genes were subsequently inspected as possible candidate genes affecting androstenone levels  terms among the 269 affected genes was performed relative to the global representation of GO terms on the microarray. A majority of the significant genes were related to oxidoreductase activities, glucuronosyltransferase activity and the binding of iron ion, haem, DNA and oxygen Figure . AdditioFMO1), transcription factor GATA-4 (GATA4), 17β-hydroxysteroid dehydrogenase 13 (HSD17B13), 17β-hydroxysteroid dehydrogenase 2 (HSD17B2) and N-acetyltransferase 12 (NAT12). Their expression levels were normalised to the housekeeping gene transferrin receptor (TFRC). Differential expression of FMO1, HSD17B13, HSD17B2 and NAT12 was confirmed in both D and NL breeds (p < 0.05) while altered expression of GATA4 was not found in either of the two breeds  approach was applied to five selected genes: Flavin-containing monooxygenase 1 . This gene is a member of the CYP2C family [CYP2C49 transcripts were shown to have a significant up-regulation in boars with high levels of androstenone in both D and NL lines (DH and NLH) but differences were clearly most prominent in the D breed.The most significantly differentially expressed gene involved in phase I oxidation reactions was cytochrome P450 2C49 (C family  and geneC family . In our CYP2E1). In contrast to expression of the CYP2C49 gene, CYP2E1 showed a down-regulation in DH and NLH boars. CYP2E1 encodes an enzyme that metabolises many endogenous and exogenous substrates such as alcohols, ketones and drugs [CYP2E1 in relation to androstenone.Another member of the cytochrome P450 superfamily, that was differentially expressed in our study, is cytochrome P450 2E1 , which is the pig ortholog of human CYP2A6, catalyses 7-hydroxylation of coumarin in pigs [CYP2A19 was significantly down-regulated in DH boars. Gene expression differences in animals with high and low androstenone levels have not previously been reported for porcine CYP2A19, however its protein expression has been shown to be inhibited by androstenone [et al. [Cytochrome P450 member 2A19  was significantly up-regulated in DH in the microarray results. The expression pattern in DH boars was confirmed by rcPCR and the rcPCR analysis also revealed that FMO1 was up-regulated in NLH boars. As shown in figure Our study implicates another class of genes involved in phase I oxidation reactions, namely the flavin-containing monooxygenases (FMOs). The FMO family of enzymes converts lipophilic compounds into more polar metabolites and decreases activity of the compounds, a similar activity to that of the cytochrome P450s . MicroarSTE (SULT2E1) was found to be up-regulated in DH boars and down-regulated in NLH boars. A previous microarray study performed by our group showed an up-regulation of CYP19 gene expression in the testis of D and NL boars with high androstenone levels [Phase I oxidation reactions of androstenone by enzymes including cytochrome P450s and FMOs are often followed by phase II conjugation reactions that are catalysed by glucuronosyltransferases, sulfotransferases, acetyltransferases and glutathione S-transferases . Throughe levels . These ge levels , and bote levels ,37.UGT1A5 was found to be up-regulated in DH boars. Furthermore, a transcript similar to UGT2B15 was up-regulated in D and a transcript similar to UGT2A1 was down-regulated in NLH. UGT2B15 has been shown to conjugate several androgens in humans [Phase II conjugation by glucuronidation is another major pathway of liver elimination of endogenous and exogenous compounds, and in D this molecular function was significantly overrepresented. It is catalysed by uridine diphospho-glucuronosyltransferases (UGTs) which transfer glucuronic acid to substrates to increase solubility . The UGTn humans , whereasn humans . UGT2A1 n humans . The UGTn humans ,42.NAT12) was found to be up-regulated in NLH based on the microarray results and this was verified by rcPCR. The rcPCR results also revealed an up-regulation in DH boars. Androgens have been shown to increase expression of NAT type 1 in human cancer cells [N-acetyltransferases (NATs) are another family of conjugation enzymes and act by transferring the acetyl group of acetyl coenzyme A to aromatic amines to increase their water solubility . NAT typer cells , but theLOC396850. We have previously described up-regulation of two GST genes, GSTO1 and MGST1, in association with testicular androstenone levels [LOC396850) in NL but not D boars, up-regulation of STE in DH but down-regulation in NLH, and up-regulation of two UGT genes in DH but down-regulation of another family member in NLH. Breed specific phase II mechanisms for these breeds are in agreement with our previous finding for the protein SULT2B1 [Glutathione S-transferases (GSTs) are functionally diverse enzymes mostly known to catalyse conjugation reactions of endogenous substances, haem, fatty acids, xenobiotics and products of oxidative processes . They hae levels , and her SULT2B1 .17β-HSD in porcine liver has been shown to be negatively associated with levels of androstenone in adipose tissue [HSD17B4 gene was significantly up-regulated in testicle samples from DH and NLH [HSD17B4 was up-regulated in the liver of DH boars. Additionally, we found that HSD17B2 was down-regulated in DH and that the isoform HSD17B13 was down-regulated in both DH and NLH. Differential expression of both HSD17B2 and HSD17B13 was verified by rcPCR, which also showed significant down-regulation of HSD17B2 in NLH boars. The HSD17B2 enzyme catalyses the interconversion of testosterone and androstenedione, as well as estradiol and estrone [HSD17B13, also known as short-chain dehydrogenase/reductase 9, has yet to be described but its expression has been characterised in human liver [DHRS8), also known as HSD17B11, was found to be down-regulated in NLH boars. HSD17B11 is involved in androgen metabolism [AKR1D1) was down-regulated in NLH animals. AKR1D1 is a liver specific enzyme that regulates the hormone levels of several steroids [AKR1C4 in the testis of DH and NLH boars [AKR1D1 has not formerly been associated with porcine androstenone levels.17β-hydroxysteroid dehydrogenases (17β-HSDs) regulate the availability of androgens and estrogens in tissues by catalysing interconvertion of active and inactive forms of steroids . They doe tissue . In a pr estrone . The funan liver . The shotabolism  and we ptabolism . The fousteroids . The enzsteroids . Aldostesteroids . A role AGP and human ORM1 were significantly down-regulated in DH and NLH boars. Significant expression of these genes might explain the inflammatory response ontology term in figure Another class of enzymes that regulate the availability of steroids is the plasma proteins. Binding of plasma protein is a reversible reaction that has no physiological effect, but it controls the amount of free drugs and hormones available to tissues by protecting them from metabolism . Alpha-1GATA4) was found to be down-regulated based on the microarray data, but we were not able to confirm this result with rcPCR. Other transcription factors identified in this study should be further investigated, including RAR-related orphan receptor A (RORA), transcription elongation factor B3 (TCEB3), nuclear factor I/X (NFIX), transcription factor 8 (TCF8) and heat shock transcription factor 1 (HSF1). We have previously identified iron ion binding, ferric ion binding and electron transport as being associated with levels of androstenone [CYB5 to be down-regulated in NLH boars, however we previously identified CYB5 as being up-regulated in the testis of both DH and NLH [CYP2E1 and CYP2A6 have been found to be activated by CYB5 in humans [CYB5 together with CYP2E1 and CYP2A6 in this study. Up-regulation in testis and down-regulation in liver might also support a key regulatory role of CYB5 in both tissues.One of the most significant molecular function terms in NL was nucleic acid binding. This term includes transcription factor binding and several transcription factors were found to be differentially expressed in this study. The GATA factor 4 , which oxidises tryptophan, was differentially expressed in DH. A gene of the FMO family, kynurenine 3-monooxygenase (KMO), was found to be down-regulated in NLH. KMO is involved in tryptophan degradation and might therefore be interesting in regards to skatole [AOX1) gene was significantly down-regulated in high androstenone animals of both breeds and this gene has not previously been associated with androstenone levels. It has, however, been shown to play an important role in skatole metabolism in several species including pigs [Pigs in this study were selected based on extreme androstenone values. However, levels of androstenone and skatole have previously been found to be highly correlated, showing genetic correlations of 0.62 for D and 0.36 for NL . Some of skatole . The alding pigs -60.CYP2E1, CYP2A19, CYP2C49, CYP27A1 and CYP2C33) and the flavin-containing monooxygenase family (FMO1 and KMO) were significantly differentially expressed. Furthermore, genes involved in conjugation reactions, including the UDP-glucuronosyltransferases , sulfotransferases (STE), N-acetyltransferases (NAT12) and glutathione S-transferase were significant, in addition to genes of the 17β-hydroxysteroid dehydrogenase family , which are known to regulate availability of active steroids. We suggest a novel role for plasma proteins including AGP and ORM1 in regulating availability of androstenone in pigs. This is the first published microarray experiment describing liver metabolism of androstenone. A number of new candidate genes have been identified, both from phase I and phase II metabolism as well as pathways regulating steroid availability.In this study we compared global gene expression profiles from the livers of boars with extreme high and low levels of androstenone from two breeds, Duroc and Norwegian Landrace. Breed differences are evident for molecular functions and biological processes involved in metabolism of androstenone, however many of the same genes are differentially expressed in the two breeds as well. Genes encoding different oxidising enzymes including the cytochrome P450 family  [Animals used in this study were Duroc (D) and Norwegian Landrace (NL) boars from NORSVIN's three boar testing stations. The D boars were on average 156 days old at 100 kg live weight compared to the NL boars that were on average 143 days old at 100 kg live weight. The boars were slaughtered on average 14 days later. Tissue samples from liver were frozen in liquid Nce (NVH)  using ance (NVH) . The andce (NVH) , with 16The present work utilises and extends methods described in our previous microarray experiment . The por® Kit on 2100 Bioanalyzer . The microarray experiment was conducted as a common reference design using RNA purified from liver tissue sampled from an unrelated Danish Landrace × Hampshire pig as a reference. Aminoallyl-cDNA was synthesised from 15 μg of total RNA using the SuperScript indirect cDNA labelling system  and labelled using ARES Alexa Fluor labelling kits . Amino-modified and fluorescently labelled cDNA was purified using NucleoSpin 96 Extract II PCR Clean-up kits . The individual samples were labelled with Alexa Fluor 647 and the reference was labelled with Alexa Fluor 555. "Green" spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the individuals and "red" spike-in RNA was added to the reference. Hybridisation was performed in a Discovery XT hybridisation station , followed by manual washing and drying by centrifugation. The microarrays were scanned using a ScanArray Express scanner  and image analyses were conducted using GenePix Pro 6 software . Statistical analyses were carried out in R version 2.3.1 [2594/log2488, A = (log2594 + log2488)/2), image plots and box plots were constructed using the Limma tools for visualisation both before and after normalisation . RNA quantity was measured on a NanoDrop ND-1000 Spectrophotometer  and RNA quality was evaluated by the 28S:18S rRNA ratio using a RNA 6000 Nano LabChipon 2.3.1  using thon 2.3.1 -69 whichon 2.3.1 . Log-tra- Reverse Transcriptase . Assays for the genes included in this study , and p-values and confidence intervals for fold changes were calculated.A real competitive PCR (rcPCR) gene expression analysis was used to verify a subset of the results from the microarray study. Quantitative Gene Expression (QGE) was performed using MassARRAY methodology and the iPLEX protocol . Total RNA was isolated from liver using an automatic DNA/RNA extractor . Total RNA was treated with TURBO DNA-free™  for removal of contaminating DNA and first strand cDNA synthesis was conducted on 0.5 μg total RNA using SuperScript™-II Rnase Hructions . Paralleructions ,76 that D: Duroc; NL: Norwegian Landrace; DH: Duroc high androstenone; NLH: Norwegian Landrace high androstenone; DL: Duroc low androstenone; NLL: Norwegian Landrace high androstenone; 17β-HSD: 17β-hydroxysteroid dehydrogenase; 3β-HSD: 3β-hydroxysteroid dehydrogenase; AGP: alpha-1-acid glycoprotein; AKR: aldo-keto reductase; AKR1C4: aldo-keto reductase family member 1C4; AKR1D1: aldo-keto reductase family member 1D1; AOX1: aldehyde oxidase; CYB5: cytochrome b5; CYP17: cytochrome P450 family 17; CYP19: cytochrome P450 family 19; CYP27A1: cytochrome P450 family member 27A1; CYP2A: cytochrome P450 family 2A; CYP2A19: cytochrome P450 family member 2A19; CYP2A6: cytochrome P450 family member 2A6; CYP2C19: cytochrome P450 family member 2C19; CYP2C33: cytochrome P450 family member 2C33; CYP2C49: cytochrome P450 family member 2C49; CYP2E1: cytochrome P450 family member 2E1; CYP3A: cytochrome P450 family member 3A; DHRS8: short-chain dehydrogenase/reductase 8; FMO: flavin-containing monooxygenase; FMO1: flavin-containing monooxygenase; GATA4: transcription factor GATA-4; GO: gene ontology; GST: glutathione S-transferase; GSTO1: glutathione S-transferase omega; HSDs: hydroxysteroid dehydrogenases; HSD17B2: hydroxysteroid 17β dehydrogenase 2; HSD17B4: hydroxysteroid 17β dehydrogenase 4; HSD17B11: hydroxysteroid 17β dehydrogenase 11; HSD17B13: hydroxysteroid 17β dehydrogenase 13; HSF1: heat shock transcription factor 1; KMO: kynurenine 3-monooxygenase; MGST1: glutathione S-transferase; NAT: N-acetyltransferase; NAT12: N-acetyltransferase family member 12; NFIX: nuclear factor I/X; ORM1: orosomucoid 1; ORM2: orosomucoid 2; QGE: quantitative gene expression; RORA: RAR-related orphan receptor A; rcPCR: real competitive PCR; SDR: short-chain dehydrogenase/reductase; STE: estrogen sulfotransferase; SULT1A1: sulfotransferase family member 1A1; SULT2E1: sulfotransferase family member 1E1; SULT2A1: sulfotransferase family member 2A1; SULT2B1: sulfotransferase family member 2B1; TCEB3: transcription elongation factor B3; TCF8: transcription factor 8; TDO2: tryptophan 2,3-dioxygenase; TFRC: transferrin receptor; UGT: uridine diphospho-glucuronosyltransferase; UGT1A5: UDP-glucuronosyltransferase family member 1A5; UGT2A1: UDP-glucuronosyltransferase family member 2A1; UGT2B15: UDP-glucuronosyltransferase family member 2B15MM carried out the microarray experimental work, performed statistical analysis and drafted the paper. SL was involved in planning the project, provided laboratory facilities for rcPCR work and took part in writing the paper. CB was involved in planning the project and was in charge of lab facilities for microarray studies. JH was involved in microarray experimental work and took part in writing the paper. HH carried out bioinformatics work. IB was involved in statistical analysis. TM was involved in power calculations and statistical supervision. EG coordinated the study, was involved in planning the project, carried out rcPCR molecular work and took part in writing the paper.Microarray results for Duroc. Gene expression profiling was performed using 58 arrays and the 1% most differentially expressed genes were considered significant. The clone names are sequences with a hit to pig, human, mouse or bovine genes. Some genes are represented by several different clones on the array and may therefore show up more than once in the table, while some have no hits to the abovementioned species. The ID column gives the feature ID on the microarray, M value indicates fold change, t gives the t-statistics, P.value is the nominal p-value and adj.P.value is the FDR corrected p-value.Click here for fileMicroarray results for Norwegian Landrace. Gene expression profiling was performed using 58 arrays and the 1% most differentially expressed genes were considered significant. The clone names are sequences with a hit to pig, human, mouse or bovine genes. Some genes are represented by several different clones on the array and may therefore show up more than once in the table, while some have no hits to the abovementioned species. The ID column gives the feature ID on the microarray, M value indicates fold change, t gives the t-statistics, P.value is the nominal p-value and adj.P.value is the FDR corrected p-value.Click here for fileGene ontology (GO) results for the cellular component ontology in Duroc. The top 1% differentially expressed genes in Duroc were analysed for over-represented (p < 0.01) GO terms in the cellular component ontology.Click here for fileGene ontology (GO) results for the cellular component ontology in Norwegian Landrace. The top 1% differentially expressed genes in Norwegian Landrace were analysed for over-represented (p < 0.01) GO terms in the cellular component ontology.Click here for fileAndrostenone values. The androstenone values (ppm) in Duroc high (DH), Duroc low (DL), Landrace high (NLH) and Landrace low (LL) animals used in this study.Click here for fileBoxplots. Boxplots of normalised arrays for Duroc (D) and Norwegian Landrace (L).Click here for fileGene transcripts included in the rcPCR analyses.Click here for file


Hepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.We examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.Collectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy. HCV infects 200 million people worldwide. Current treatment options for HCV include pegylated interferon α and ribavirin. However this treatment is not always effective or tolerated well It was initially proposed that in vitro inhibition of HCV by CsA results from disruption of the interaction between the HCV polymerase, NS5B, and Cyclophilin B (CypB) We previously isolated HCV 1bN replicon sequences more resistant to CsA treatment In this study we present data supporting a direct role of NS5A in CsA resistance. The NS5A mutations from the 1bN replicon also confer CsA resistance for the canonical Con 1b replicon. The tyrosine residue at position 321 of NS5A is a particularly critical determinant of CsA resistance. This residue is within the ‘277-334’ NS5B binding region of NS5A The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.EN5.3 and Huh7.5 cells were used for all studies. Huh7.5 cells and the Con1 HCV replicon were generous gifts from Dr. Charles Rice. For these studies, the neomycin resistance gene was replaced with a renilla luciferase-neo fusion gene amplified from a replicon kindly provided by Drs. Ikeda and Kato The CsA-3s NS5A (acc. No. EF115302) cloning was performed as previously described Replicon DNA was linearized with XbaI  and transcribed using a T7 Ampliscribe Flash transcription kit . RNA was transfected into EN5.3 or Huh7.5 cells using the Transit mRNA transfection kit . Three weeks after the establishment of stable replicon cell lines using G418 selection, equal numbers of replicon cells were seeded into 6 well plates with or without CsA. The Secreted Alkaline Phosphatase (SEAP) assay was used to quantitate the Ntat replicon RNA as described previously Following the establishment of stable replicon cell lines, RNA was isolated using the Trizol method. Total RNA was used for cDNA synthesis using a primer located in N-terminal region of NS5B. PCR was performed using this reverse primer and a forward primer located within NS5A region. PCR product of the expected size was excised from gel, purified and cloned into the TOPO vector. Two clones from each mutant replicon were sequenced with M13 forward and M13 reverse primers. The results were analyzed and specific mutations were confirmed by checking the chromatogram.6 Huh7.5 cells. Following RNA electroporation, one tenth of the cells were set aside to check the luciferase activity at 4 hrs as an indicator of RNA electroporation efficiency. The rest of the electroporated cells were equally divided and plated into 24 wells so that each electroporation had 3 sets of 8 wells. Four wells of each set were left untreated while wells 5–8 were treated with 1.0 µg/ml of CsA until the assay was performed. The cells were collected at 4 time points  post RNA electroporation from both CsA treated and untreated wells. Luciferase activity was monitored as above.In vitro transcribed Con 1b RNA along with Chimeric 3s were electroporated into ∼4×10To monitor the replication assay in Huh7.5 cells, the cells were pretreated with 1.0 µg/ml CsA for one week. These cells were electroporated with either Con 1b or Chimeric 3s replicon RNA and luciferase activity was monitored at 24, 48, 72 and 96 hrs. The electroporations were performed in triplicate and average luciferase activity for both Con1b and Chimeric 3s is presented.Infectious HCV particles were produced as described before For northern blot assay, the same amount of RNA from JFH1-wt and a JFH1-Y325N mutant was electroporated into normal Huh7.5 cells and cells already treated with 1.0 µg/ml of cyclosporine for one week. The CsA-treated and electroporated cells were further incubated in presence of CsA while normal cells were grown in complete growth media. After 72 hrs post RNA electroporation, cells were lysed in Trizol  and total RNA was isolated following manufacturer's protocol. The northern blot was performed using standard molecular biology protocols The wt NS5A-His (1bN-SI) and mt NS5A-His (CsA-3s) constructs were made as follows. The NS5A genes were amplified from the Ntat/SI and CsA-3s replicon by RT-PCR. Forward primers were designed with an XhoI site and an in-frame start codon ATG 5′ to the NS5A sequence. The reverse primers encoded a 6XHis tag in frame with the NS5A sequence, a stop codon followed by Pac1 restriction site. The resulting PCR product was cloned into XhoI-PacI digested pIRES/GFP (Green Fluorescent Protein) mammalian expression vector (kindly provided by Dr. Kouacou Konan). The HA-wt NS5A and HA-mt NS5A constructs were made by incorporating the HA tag (MYPYDVPDYA) in the forward primer. The GFP gene in these constructs was removed and the plasmid recircularized with T4 DNA ligase. For the N terminal truncated NS5A constructs lacking domain 1 (D23NS5A), forward primers were designed to encode a XhoI site, Kozak sequence, HA, the ATG start codon, and NS5A sequence starting from amino acids 148 for both the 1bN and the CsA-3s replicon DNA templates. The reverse primers contained an in-frame stop codon and a Pac1 site. The GFP gene in these constructs was also removed and religated with T4 DNA ligase. The Con1b NS5B was generated by using standard PCR approach. The forward primer was designed to contain a FLAG epitope (MDYKDDDDK) and cloned in a CMV-based mammalian expression vector. The CypB mammalian expression vector was generated with forward primers containing XhoI, ATG 5′ to the start of the CypB sequence and reverse primers containing an HA tag, stop codon and a PacI site. The PCR fragment was then cloned into the pIRES/GFP vector.HA-wt NS5A and HA-mutant (mt) NS5A expression constructs were co-transfected with a FLAG-tagged Con 1b NS5B expression construct in 293T cells using transit LT1 reagent . Forty-eight hours after transfection, cells were harvested and immunoprecipitated with HA-specific antibody (Biodesign). Immunoprecipitates were separated using SDS-10% Poly Acrylamide Gel Electropheresis (PAGE), and western blotting was performed. Blots were probed with a monoclonal NS5A-specific antibody (Biodesign) and a monoclonal FLAG antibody against the FLAG tag of NS5B (Sigma). An HRP-linked mouse secondary antibody specific for antibody light chains  was used and the signal was developed using Immun-star HRP chemiluminescent substrate . For NS5A and CypB transfections, wt or mt NS5A-His and HA tagged CypB were immunoprecipitated with His-specific monoclonal antibody (Qiagen) bound to protein G agarose beads, washed, and loaded onto a gel. The blots were probed with a NS5A monoclonal (Biodesign) and a rabbit polyclonal to the HA tag. For the NS5A and NS5B cotransfections, HA-wt NS5A and HA-mt NS5A were cotransfected in 293T cells with FLAG Con1b NS5B. The lysates were immunoprecipitated with HA-specific antibody and probed with NS5A-, FLAG-, and CypB-specific antibodies to check levels of NS5A, NS5B and CypB respectively.35S methionine/Cysteine  using the TNT Quick coupled Transcription/Translation Systems  following the manufacturer's protocol. The assay for GST-CypB binding to NS5A or NS5B was performed as previously described Recombinant CypB was obtained as previously described ++ column following manufacturers protocol (Qiagen). Approximately 2.0 µg of CFP and NS5A proteins were incubated with equal amounts of purified GST or GST-CypA proteins and binding was performed as described before To further confirm the specificity of CypA NS5A binding, an irrelevant protein, HIS tagged Cyan fluorescent protein (CFP) was expressed in E. coli and purified using Ni35S methionine/cysteine and incubated with purified GST-CypA. The binding was performed as above. The signal was developed after fluorography and intensity of individual pull down signal was quantified using image quant following Storm. To further map the binding region within CypA, the two active site residues (R55 and F60) were mutated to Alanine. The 35S radiolabelled NS5A was again incubated with either wt GST-CypA or mt GST-CypA (carrying the two active site mutations) and binding was performed as described above.To map the region within NS5A responsible for binding, a series of in-frame 90 amino acids deletion mutants were generated in the HA-wt NS5A expressing plasmid. These constructs were in vitro translated in presence of The Student's t test was used to calculate p values.In previously published work The CsA-3s contains six point mutations in NS5A. It is unclear if a single mutation accounts for resistance or if the mutations act synergistically with each other. Despite the heterogeneity in NS5A sequence none of these mutations are commonly present in the extensive sequence banks of HCV isolated from patients see . We replTransient replication assays were also performed to determine the significance of NS5A mutations for CsA susceptibility. Replicon RNA was electroporated into cells that were untreated or treated with CsA and harvested at different time intervals to examine the luciferase activity. The Chimeric 3s and Con1b replicon replicate to similar levels . When CsTo further evaluate the role of the 321 mutation by itself, we engineered the 321 mutation in the background of the JFH1 infectious clone. The JFH1-Y325N and JFH1-wt produce a similar number of infectious viral particles in the absence of CsA . HoweverNS5A binding to NS5B maps to two regions of NS5A, amino acids 105–162 and 277–334 35S methionine/cysteine and proteins were incubated with GST-CypB or GST alone. The in vitro translated NS5B was used as a positive control for CypB binding. Both wt NS5A and mt NS5A interact with CypB  activity of CypA catalyzes the cis-trans isomerization of peptide bonds that connect prolines. To examine the role of the catalytic domain of CypA in HCV NS5A binding we converted the two amino acids (R55 and F60) in the catalytic domain to alanine. The mutant protein was then tested for its ability to pull down NS5A. Using the GST pull down assay, the CypA mutant protein displays complete loss of binding to NS5A indicating crucial involvement of the active site residues in NS5A binding .To determine if the interaction between NS5A and CypB occurs in vivo, 293T cells were co-transfected with His-tagged wt-NS5A or mt- NS5A along with CypB-HA. The cells were lysed and immunoprecipitated with anti-His antibody to purify NS5A-containing complexes. Western blots were probed with both NS5A-specific antibodies and HA-specific antibody to detect CypB. Both wt NS5A and mt NS5A interact with CypB-HA . HoweverCyclophilins have now been implicated in the life cycle of several different DNA and RNA viruses The barrier to resistance for CsA is relatively high compared to other antivirals in which a single mutation confers substantial resistance. The different mutant NS5A sequences localize to domains 2 and 3. Here, we provide data that shows mutations from one strain (1bN) also affects other genotype 1b strains (Con1b) and even a genotype 2a (JFH) . The affA Chimeric 3s N321Y replicon was constructed restoring the asparagine mutation at amino acid position 321 to the wild-type tyrosine residue. This mutant replicon, contains all of the other CsA-3s mutations, but lacks the mutation at 321. Chimeric 3s N321Y is more sensitive to CsA treatment than Chimeric 3s suggesting a tyrosine residue at position 321 contributes to some but not all of the CsA sensitivity. This mutation is in a region of NS5A that binds NS5B The interaction between NS5B and cyclophilins has been demonstrated by several groups. In one study, CsA was used to select mutations in NS5B using a HCV replicon with GFP inserted into NS5A Previous functional studies have investigated the interaction of NS5B alone and cyclophilins. Here, we show a novel interaction between NS5A and both CypA and CypB. Mutant NS5A binds CypB to a greater extent than wild-type in cells  and S4. We made several attempts to immunoprecipitated endogenous CypA and CypB with NS5A (data not shown). Neither endogenous CypA nor CypB could be pulled down by NS5A expressed alone in cells. It is possible the NS5A: cyclophilin interaction cannot stably compete with other NS5A partners in the absence of other viral proteins such as NS5B. NS5A could pull down endogenous CypB only in the presence of NS5B . NS5A diIt is unclear if cyclophilins assist in folding NS5A and NS5B or, alternatively, form part of the HCV replication complex. It is possible that cyclophilins allow both NS5A and NS5B to access important conformations. Alternately, binding of both NS5A and NS5B to cyclophilins may facilitate incorporation of cyclophilins into the replication complex. Either way the catalytic portion of cyclophilins is critically involved in NS5A  just as Figure S1The absolute value of data presented in (0.15 MB TIF)Click here for additional data file.Figure S2The absolute value of data presented in (0.25 MB TIF)Click here for additional data file.Figure S3NS5A constructs without domain1  were translated in vitro and incubated with either GST alone or GST-CypB as described in (0.80 MB TIF)Click here for additional data file.Figure S4Mutant NS5A binds CypB better than wild-type NS5A. Huh7.5 cells were co-transfected with either HA-wt NS5A or HA-mt and wt NS5B (Con 1b) in the presence of CypB-FLAG tagged plasmid. Forty eight hours after transfections, cell lysates were immunoprecipitated with anti-HA antibody and Western blotted with anti-FLAG (bottom panel). The top panel shows probing the total cell extract with anti-FLAG antibodies to demonstrate equivalent CypB expression.(0.38 MB TIF)Click here for additional data file.Figure S5HCV NS5A binds CypA A) Coomassie staining of HIS-tagged E. coli purified proteins B) Western blot analysis using anti-HIS monoclonal antibody demonstrates NS5A in the GSTCypA complex but not in GST, and no CFP binds either GST or GSTCypA.(3.00 MB TIF)Click here for additional data file.


Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize βPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages.C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The  The ability of a cell to accurately respond to external signals in different developmental contexts relies on the integration of multiple sets of signaling pathways, which in many cases are highly conserved through evolution. A key event of such cellular signaling is the ligand-mediated activation of receptor tyrosine kinase (RTK) family proteins, leading to the recruitment of a multitude of proteins that function to transmit signals to the proper downstream targets. Essential players in this process include protein and lipid kinases, adaptor and scaffolding molecules, as well as members of the small GTPase superfamily. Small GTPases are monomeric GTP-binding proteins of 20–25kDa, which act as molecular switches during diverse cellular and developmental events, including proliferation, differentiation, apoptosis and control of the cytoskeleton in vitroThe Rap family of GTPases, and thus their associated regulators, are primarily involved in regulating cell-cell junction formation, cell adhesion to extracellular matrix and polarity In vivo studies in mouse models have shown that C3G is essential during early embryogenesis and that C3G null mutant embryos die around day 5.0 of gestation, thereby creating difficulties in the study of C3G function during development gtC3G, which produces less than 5% of normal C3G protein levels, allows the survival of gt/gtC3G mutants to embryonic day 14 (E14.5) gt/gtC3G mutant mice die of hemorrhaging due to vascular defects, suggesting a role for C3G in vascular myogenesis, in keeping with the lack of correctly developed supporting cells in gt/gtC3G animals. Moreover, C3G has recently been implicated in the regulation of the size of the cerebral cortex neural precursor population, and mice lacking C3G exhibit excessive proliferation in the cortical neuroepithelium Drosophila is unknown Drosophila C3G has been shown to have a genetic interaction with Rap1 and with components of the Ras-MAPK pathway Drosophila C3G GEF activity and specificity is lacking. In addition, the absence of C3G mutants has precluded an evaluation of the importance of C3G protein function in the fly.in vivo, we have generated Drosophila C3G null mutants and concluded that loss of C3G results in semi-lethality, with escaping adults characterized by a shorter life span and reduced general fitness. While in situ hybridization depicts C3G expression in embryonic CNS, somatic and visceral muscles, our analysis of Drosophila C3G mutant embryos has failed to reveal any major embryonic developmental defects in any of these tissues. However, when investigating the larval development of Drosophila C3G mutants, we detected substantial disorganization of the larval muscle architecture, as well as defects in integrin localization at muscle attachment sites. In support of a role of C3G in somatic muscles, we have also observed that misexpression of an activated form of C3G results in defective muscle structure and muscle-muscle detachment. To study the function of C3G in detail at the molecular level, we have performed guanine nucleotide exchange assays, providing conclusive evidence that Drosophila C3G acts as a GEF for Drosophila Rap1 in vitro. Moreover, Drosophila C3G also exhibits GEF activity towards human Rap1 but not H-Ras. In conclusion, we propose that C3G is an accessory component of the Drosophila musculature, essential for the proper localization of integrins at muscle-muscle and muscle-epidermis attachment sites and important for maintaining muscle integrity during larval stages.In order to increase the understanding of C3G Drosophila husbandry procedures were applied. Drosophila strains were maintained on standard potato-meal medium, and raised and crossed at room temperature unless otherwise stated. The PiggyBac elements used in the study were from the Harvard Exelixis stock collection UAS-C3G transgenic flies  were kindly provided by Dr U. Gaul Standard Drosophila C3G deletion mutant (ΔMSC3G) was generated by heat shock FLP-induced FRT-mediated recombination of the transposable elements RBe03301 and XPd00064 MSC3G deletion mutants were identified and verified by genomic PCR using the primers, AACAAATTGTTGTTATGCGT for the 5′ side and AGAATGCGGTGTGCCGTAAG for the 3′ side. Drosophila Miple2 was used as a control for the genomic PCR using the primers CGAAATAAAACACTCTACC for the 5′ side and CAAATCGCCATTGGAAACTC for the 3′ side. Mutants were additionally confirmed by Southern Blot analysis.The Drosophila C3G specific DNA probes, which were detected by the DIG detection chemiluminescent assay (Roche). Primers used for making the probe were CTCCATCCACGCCCGGCACCTGTT for the 5′ side and ATTGGAACTGCAGTCCAGGTCCGA for the 3′ side.Genomic DNA was prepared using standard techniques. Genomic DNA, digested with HindIII, EcoRI (New England Biolabs) was electrophoresed on 1% agarose gel and blotted onto Hybond N+ filter . The filter was subsequently analyzed using Digoxigenin-labeled (DIG) Drosophila using RNA isolation kit (Bio-Rad). Reverse transcription was performed using the Superscript II reverse transcriptase (Invitrogen) and primers for shifted mRNA. The primers used were GCGGGATTTTTCAAGTGTCGAAAC for the 5′ side and CCTTTTGTCGCTGTTGCTGTTGTT for the 3′ side.RNA was isolated from adult Drosophila C3G cDNA  as template. In situ hybridization of whole-mount wild type Drosophila embryos was carried out as described A DIG RNA labeling kit (Roche) was applied to generate DIG-labeled RNA probes, using full-length Drosophila C3G antiserum, DNA encoding C3G amino acids 1- 155 was generated by PCR and subcloned into pETM-11. The reading frame of the His-C3G sequence was subsequently confirmed by sequence analysis. Recombinant His-C3G fusion protein was purified form Echerichia coli (BL21 (DE3)) bacterial lysates by standard protocols using Ni-NTA agarose (Qiagen) and subsequently used for immunization of guinea pigs.For the preparation of Drosophila flies of the indicated genotypes were ground in Lysis Buffer consisting of 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X-100, 10% Glycerol, 25 mM NAF, 10 µM ZnCl2 and a protease inhibitor cocktail . Lysates were cleared by centrifugation and protein concentrations determined using the Bio-Rad protein assay. Protein samples were separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked in 5% BSA for 1 hour, prior to incubation with either guinea pig anti-C3G, or mouse anti-α-Tubulin (Sigma) antibodies and detection by ECL .Adult For life span experiments, flies were collected during 24 hours and aged at the temperatures indicated. Flies were transferred daily onto fresh food and counted. The lethality determination was performed by placing 100 embryos of the indicated genotypes onto apple juice plates. Embryos developing into larvae and larvae enclosing into adult flies were counted.Embryos were fixed and immunostained as previously described −1 in 50 mM Tris-HCl, pH 7.5, 1 g l−1 BSA) for 10 minutes at room temperature to induce DNA strand breaks. As negative control permeabilized cells were incubated in TUNEL reaction mixture without addition of the terminal transferase.TUNEL assay was performed according to the manufacturer's protocol . Larval muscles were dissected and fixed in 4% paraformaldehyde, before being washed and permeabilized in PBS-T (0.1% Triton X-100 in PBS). Muscles were subsequently stained with the TUNEL reaction mixture for 1 hour at 37°C in the dark. Samples were rinsed three times with PBS and analyzed directly using a fluorescence microscope. As positive control permeabilized cells were incubated with DNase I  were obtained from DGRC (Drosophila Genomics Resource Center). Drosophila C3G RNA was isolated (Bio-Rad) and cloned by RT-PCR (Invitrogen). Fragments were cloned using BamHI/EcoRI for (C3GGEF) and EcoRI/NotI for (Drosophila Rap1 and Rap2L). The following constructs were generated; pGEX4T3:Rap1 (aa 1–167), pGEX4T3-1:Rap2L (aa 1-167), pGEX4T-1:C3GGEF (aa 1332–1565). Rap1B  and H-Ras  were expressed from ptac; Rap2B  was expressed from pGEX4T3, C3G  expressed from pET15 and Sos  from pET28.Full-length Drosophila C3G, human C3G and Sos on Drosophila Rap1 and Rap2, human Rap1B, human Rap2B and human H-Ras purified GTPases were preloaded with fluorescent mant-GDP (2′-/3′-O-(N'-Methylanthraniloyl)guanosine-5′-O-diphosphate) by incubating the GTPase in the presence of EDTA and an excess of mant GDP. The EDTA and free nucleotides were removed by gelfiltration Drosophila Rap2, Drosophila Rap2 was loaded by incubating 200nM mant-GDP with 200 nM Drosophila Rap2 until a stable fluorescent signal was obtained. The reaction was started by adding an excess of GDP and GEF and the fluorescence signal was measured over time (excitation wavelength is 366 nm and emission 450 nm). The buffers used are , 5mM MgCl2, 5mM DTT, 5% glycerol). The reaction mixture consisted of 200nM of GTPase, 150nM RapGEF6 or 1500nM Drosophila C3G, human C3G or Sos, and 27 µM of GDP.To test the GEF activity of Confocal imaging was performed by using a Nikon confocal microscope C1,  fitted with Ar, He/Ne, and blue diode lasers. Digitized confocal images were assigned red and green pseudocolors for Cy3/Alexa Fluor 594 and FITC/Alexa Fluor 488 respectively. 40X and 60X objectives were used. Captured images were exported as TIFF format files and further processed using Adobe Photoshop 8.0 for figure mounting and labeling purposes. Larval muscle pictures at high magnification were taken using a Leica confocal microscope (TCS-SPE), images were assigned red (532), green (488) and Cy5 (647). At low magnification, images of larval body wall muscles were captured using an Axia Imager.Z2 system (Zeiss).Drosophila C3G protein contains a catalytic CDC25-type GEF domain, in tandem with a conserved GEFN/REM domain, showing 67% and 39% identity to its human counterparts, respectively. Strong homology is also displayed within the N-terminal SH3 CRK binding proline-rich regions, suggesting a potential evolutionary conservation of the CRK-C3G interaction (Drosophila C3G loss-of-function mutant (MSΔC3G) was generated by FLP/FRT-mediated recombination of two independent PiggyBac elements flanking the Drosophila C3G locus C3G coding region  by performing RT-PCR on mRNA extracted from MSΔC3G flies. Indeed, shifted mRNA can still be detected in MSΔC3G mutants . Taken together, this data indicates that formation of the gut musculature proceeds normally in MSΔC3G mutant animals, and by extension that Drosophila C3G is not required for gross visceral muscle development.During embryonic stages  the CNS , suggestmesoderm . Founder process . In addicle type . Furtherembryos  . Thus, bigration . ImportaMSC3G mutants. To do this, body wall muscles from third instar larvae were stained with phalloidin to visualize the actin cytoskeleton, together with βPS integrin, marking muscle attachment sites. A significant proportion  of ΔMSC3G mutant specimens analyzed showed abnormal muscle architecture of the ventral longitudinal muscles. These experiments were repeated in a double-blind manner with similar results. The ΔMSC3G mutants most commonly displayed phenotypes including deformations and detachment of the ventral longitudinal muscles  under control of the muscle specific Mef2-Gal4 driver in ΔMSC3G mutants   resulted in an intriguing accumulation of C3G both in a punctuate manner and on one side of the muscle attachment site (DN) was localized in a punctuate pattern, spread throughout the muscle fibers with accumulation in perinuclear regions , which when expressed at 25°C resulted in embryonic lethality. Examination of somatic muscles of Mef2-Gal4>UAS-C3GCA animals at embryonic stages revealed severe muscle defects  resulted in a clear accumulation of β-integrin together with C3G  or constitutively activated C3G (a membrane-targeted version of C3G) in somatic muscles nization . The str defects , in contc muscle . Detachmc muscle , C″. Givwith C3G , suggestDrosophila contains two Rap proteins, both of which are putative targets for the GEF activities of C3G. To test if Drosophila C3G functions as a GEF for the Drosophila small GTPases Rap1 and Rap2, we performed in vitro GDP release assays, using the RasGEF domain of C3G together with Rap1 or Rap2. These assays establish that Drosophila C3G stimulates guanine nucleotide release from Drosophila Rap1, but not Rap2  and Rap2 (hRap2B)  . Drosoph control . Thus, aDrosophila causes semi-lethality, and that C3G null mutants (ΔMSC3G) display defects in larval muscle architecture and decreased survival during both larval and adult phases. Both the muscle defects and lethality observed in ΔMSC3G animals can be significantly rescued by the ectopic expression of wild type C3G protein in muscles with the Mef2-Gal4 driver, suggesting that the C3G GEF protein performs important functions in maintaining somatic muscle integrity. Our detailed analysis of the ΔMSC3G mutants resulted in several important findings. Firstly, Drosophila C3G is crucial for the formation of normal larval body wall muscle structure, in particular the morphology and attachment of the ventral longitudinal muscles. During larval movement, it is the contraction of these longitudinal muscles, which alternately shorten the length of these segments uni- and bilaterally, that drives locomotion. The deformed morphology of these muscles may be the underlying explanation for the requirement of C3G for larval fitness. One possibility is that the altered integrin localization in ΔMSC3G mutants arises as a result of distorted muscle activity due to aberrant sarcomeric muscle structure. Indeed, in both Drosophila melanogaster and Caenorhabditis elegans integrins are required for proper sarcomere assembly and Z band formation Drosophila absence of integrin function causes muscle detachment with no defects in the initial specification and fusion of muscle cells Drosophila α-actinin mutant flies exhibit flight muscle paralysis -actinin in anchoring and stabilizing the thin filaments of the muscles. Furthermore, a recent study in Drosophila has shown the PDZ-LIM domain containing protein Zasp to interact genetically with integrins, which is interesting given that zasp mutants do not form Z-bands and fail to recruit α-actinin to the Z-band, resulting in muscle detachment at the onset of contractility MSΔC3G mutants, the Z-band itself is irregular, raising the possibility that the integrin mislocalization observed in MSΔC3G mutant animals may partially be the result of defective anchorage of actin filaments to the Z bands. Other LIM domain adaptor proteins such as Paxillin and PINCH have also been proposed to strengthen integrin linkage to the actin cytoskeleton MSC3G mutants has not been analyzed.In this paper we report that loss of the guanine nucleotide exchange factor C3G in Drosophila C3G at sites of cell-cell contact. The C3G-induced activation of Rap1 in response to the mechanical stress emanating from cell stretching MSC3G mutants, together with C3G overexpression studies, suggests that C3G acts as one such stabilizing protein required for preserving muscle integrity.In mammalian cells, endogenous C3G has been shown to interact with E-cadherin, a key component of cell-cell junctions, a binding that in the yeast-two-hybrid system was further mapped to the intracellular tail of E-cadherin Drosophila C3G specifically exerts GEF activity towards Drosophila Rap1, but not markedly towards Rap2. Furthermore, both Drosophila and human C3G display an ability to stimulate nucleotide exchange of both human Rap1 and, to a lesser extent, human Rap2B. Human C3G was previously shown not to act on human Rap2A in vitro evidence showing that Drosophila C3G selectively functions as a GEF for Rap1. The Drosophila genome contains one annotated Rap1 protein (roughened) and one Rap2 family member (rap2L) Drosophila PDZ-GEF (gef26) has also been reported as a GEF for Drosophila Rap1, and has been implicated in vivo in photoreceptor development as well as the regulation of cell migration and morphogenesis in wing disc epithelia, dorsal closure and hemocyte migration Drosophila Rap2L has not been extensively studied in vivo and no mutants have been described at this point. Furthermore, it is not known which GEF proteins are responsible for the activation of Drosophila Rap2L, although our results indicate that it is unlikely to be activated directly by Drosophila C3G. In the GEF assays performed here we noted a high intrinsic exchange activity of Drosophila Rap2L, a feature which potentially could be explained by the slight sequence difference in the GTP-binding (NKXD) motif displayed by Drosophila Rap2L, as compared to the other Drosophila and human Rap proteins as well as most other Ras proteins.Our nucleotide exchange activity assays revealed that The identification here of a function for C3G in the somatic musculature adds another GEF to the list of GTPase modulators with described roles in muscle development. Previousely identified GEFs include Myoblast city (MBC) which functions as a GEF for Rac1, playing a critical role in the myoblast fusion process Drosophila muscles.The punctuate and perinuclear localization of C3G upon overexpression is intriguing, although this pattern and its relevance for muscle integrity and attachment is unclear. In addition to their sarcomeric architecture, muscles contain tubular plasma membrane invaginations (transverse tubules or T-tubules) that are important for the connection between neuronal depolarization and muscle contraction. A number of proteins have been reported as critical for the biogenesis of these tubular structures. Two of these are Amphiphysin and Caveolin, which both have established functions in membrane dynamics in vivo evidence for a requirement of the guanine nucleotide exchange factor C3G in the preservation of muscle integrity during larval development. Further studies are clearly needed to fully understand the function of C3G, explain the phenotypes observed in C3G deficient animals, and uncover the molecular machinery that together with C3G regulate muscle attachment, integrity, contractility and muscle maintenance.Taken together, the data we have presented here provide the first Figure S1shifted is expressed in ΔMSC3G mutants. RT-Reverse transcription PCR was performed on RNA extracts from wild type flies, flies carrying the starting transposable elements (RBe03301 and XPd00064) and ΔMSC3G mutants. shf (shifted) is expressed in ΔMSC3G mutants.(0.96 MB TIF)Click here for additional data file.Figure S2MSC3G mutant embryos undergo normal fusion and migration of somatic and visceral muscles. To visualize the somatic muscle attachment sites, wild type and ΔMSC3G mutant embryos were stained with βPS integrin (red) and β3-tubulin (green) antibodies. βPS integrin localization appears to be normal in ΔMSC3G mutant embryos .  Wild type and ΔMSC3G mutant embryos were stained with anti-Alk antibodies (green) to visualize the visceral muscles and anti-Connectin antibodies (red) as a cytoskeletal marker in the visceral mesoderm. The founder cells and fusion competent myoblasts fuse normally in ΔMSC3G mutant embryos (D).Δ(4.02 MB TIF)Click here for additional data file.Figure S3MSC3G mutant larvae display muscle abnormalities already at the 2nd instar stage of development.  Wild type and ΔMSC3G mutant larvae were dissected at the 2nd instar stage and stained with Phalloidin to visualize the body wall musculature. Whereas wild type animals (A) at this stage display regular and robust longitudinal muscle fibers, the ΔMSC3G mutants (B) are characterized by thin longitudinal muscle fibers that tend to be both mistargeted and in some cases detached (right panel).Δ(2.22 MB TIF)Click here for additional data file.Figure S4MSC3G mutant muscles do not display increased apoptosis. Third instar wild type and ΔMSC3G mutant larval muscles were dissected and subjected to a TUNEL assay. DNA fragmentation, characteristic for apoptosis, was analyzed by fluorescent labeling (Green). Samples treated with DNAse I were used as positive controls (A'). As negative control samples were treated with the TUNEL reaction mixture, but without addition of the terminal transferase. No apoptosis was observed in ΔMSC3G mutant larva, similar to wild type muscles (D'-C').Δ(7.43 MB TIF)Click here for additional data file.Figure S5CA, but not C3G WT or C3G DN misexpression. (A–D) Mef2-GAL4 was used to drive expression of wild type (C3G WT), dominant negative (C3G DN) or activated (C3G CA) specifically in muscles. Late stages embryos were collected and stained with anti-β3-tubulin (green) and anti-βPS (red) antibodies. The integrity of the embryonic somatic musculature and the targeting of integrins to muscle attachment sites was not affected by misexpression of either C3G WT (B) or C3G DN (C). However, misexpression of C3G CA resulted in muscle defects of variable magnitudes (D).Embryonic muscle development is affected by C3G (4.03 MB TIF)Click here for additional data file.


In sensory systems with broad bandwidths, polarized receptor cells utilize highly specialized organelles in their apical and basolateral compartments to transduce and ultimately transmit signals to the rest of the nervous system. While progress has been made in elucidating the assembly of the transduction apparatus, the development of synaptic ribbon-containing terminals remains poorly understood. To begin to delineate the targeting of the exocytotic machinery specifically in ribbon-containing neurons, we have examined the expression of complexins 3 and 4 in the zebrafish visual and acousticolateral systems during the first week of development.We have identified five members of the complexin 3/4 subfamily in zebrafish that show 50 to 75% amino acid identity with mammalian complexins 3 and 4. Utilizing a polyclonal antibody that recognizes all five orthologs, we demonstrate that these proteins are enriched in ribbon-containing sensory neurons. Complexin 3/4 is rapidly targeted to presynaptic terminals in the pineal organ and retina concomitantly with RIBEYE b, a component of ribbons. In hair cells of the inner ear and lateral line, however, complexin 3/4 immunoreactivity clusters on the apical surfaces of hair cells, among their stereocilia, rather than along the basolateral plasma membrane with RIBEYE b. A complexin 4a-specific antibody selectively labels the presynaptic terminals of visual system ribbon-containing neurons.These results provide evidence for the concurrent transport and/or assembly of multiple components of the active zone in developing ribbon terminals. Members of the complexin 3/4 subfamily are enriched in these terminals in the visual system and in hair bundles of the acousticolateral system, suggesting that these proteins are differentially targeted and may have multiple roles in ribbon-containing sensory neurons. Photosensitive and non-cutaneous mechanosensitive neurons transduce stimuli, via specialized sensory cilia and microvilli on their apical surfaces, into graded membrane potentials. A rod or cone photoreceptor elaborates, from a primary cilium, vast quantities of plasma membrane that contain opsins and the rest of the transduction machinery (reviewed in ). Less wRIBEYE genes, RIBEYE a and RIBEYE b, that are differentially expressed in zebrafish , human complexin 3 [GenBank:AY286501], mouse complexin 4 [GenBank:AY264291], and human complexin 4 [GenBank:AY286502] [The nucleotide sequences of mouse complexin 3 [GenBank:Y286502]  were useTaq DNA polymerase (Invitrogen), 1 μl of reverse-transcribed cDNA (or non-reverse transcription control), and 10 pmol of primers. The PCR protocol consisted of 95°C for 5 minutes, followed by 30 cycles of 95°C, 45°C, and 72°C for 1 minute each, ending with 72°C for 4 minutes. PCR products of approximate expected sizes, obtained with all five primer pairs, were gel-purified, subcloned in pGEM-T Easy, and sequenced. Comparison of these cDNA clones with the aforementioned annotated genes in the zebrafish ENSEMBL database via BLAST revealed >95% nucleotide identity for four of the genes (ENSDARG00000067826 was the exception).Reverse transcription of adult zebrafish retina poly(A) + RNA was carried out with Superscript II reverse transcriptase . PCR was performed with Platinum cplx 3a, ENSDARG00000067826 was named cplx 3b, ENSDARG00000059978 was named cplx 4a, ENSDARG00000059486 was named cplx 4b, and ENSDARG00000069963 was named cplx 4c. The sequences of the isolated cDNA clones have been deposited in GenBank:  for zebrafish complexins 3a, 3b, 4a, 4b, and 4c, respectively.Alignment of the cDNA clones and the aforementioned mammalian complexins 3 and 4 was performed with ClustalW in Molecular Biology Workbench . Based oIn silico analysis of the following genes was performed based on sequence in the ENSEMBL database (Zv8): human cplx 3 (ENSG00000213578), human cplx 4 (ENSG00000166569), mouse cplx 3 (ENSMUSG00000039714), mouse cplx 4 (ENSMUSG00000024519), zebrafish cplx 3a (ENSDARG00000062508), and zebrafish cplx 4a (ENSDARG00000059978). Proteomic analysis of the cplx3/4 subfamily was performed in Molecular Biology Workbench with the PROSEARCH and PFSCAN algorithms.Complexins 3a, 3b, 4a, 4b, and 4c were each fused in-frame with the myc tag from pCMV-Tag3b using the In-Fusion Dry-Down PCR Cloning Kit  according to the manufacturer's instructions. We used 50 pmol of the following primers to amplify 100 ng full-length complexins for eventual fusion with linearized pCMV-Tag3b: (complexin 3a forward) 5'-GAGCCCGGGCGGATCCATGGCTTTTATGTTGAAACACATG-3'; (complexin 3a reverse) 5'-GCAGCCCGGGGGATCCCTACATGACATCACACTTCTCGGC-3'; (complexin 3b forward) 5'-GAGCCCGGGCGGATCCATGGCTTTTATGGTGAAACACGTA-3'; (complexin 3b reverse) 5'-GCAGCCCGGGGGATCCTCACATGACGCAGCACTTCTCA-3'; (complexin 4a forward) 5'-GAGCCCGGGCGGATCCATGGCGTTTTTAATCAAAAGTATG-3'; (complexin 4a reverse) 5'-GCAGCCCGGGGGATCCCTACATGACAGAACATTTCTCCTC-3'; (complexin 4b forward) 5'-GAGCCCGGGCGGATCCATGTCTCATGATGGGATGTCC-3'; (complexin 4b reverse) 5'-GCAGCCCGGGGGATCCTCACATGACGGTGCATTTCTC-3'; (complexin 4c forward) 5'-GAGCCCGGGCGGATCCATGGCGTTCCTTCTGCAGCAG-3'; (complexin 4c reverse) 5'-GCAGCCCGGGGGATCCTCACATCAAGACACATTTTTCTTCC-3'.2 and 95% air at 37°C. Cells on 100 mm dishes were transfected with Lipofectamine (Invitrogen) and 8 μg of a complexin-myc construct or pCMV-Tag3b. After 24 hours, the cells were trypsinized, plated onto poly-L-lysine-coated glass coverslips, and maintained for an additional 24 hours before processing for immunocytochemistry.HEK 293T cells were grown in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum  and glutamine (Invitrogen) in a humidified incubator containing 5% COHEK 293T cells were washed with PBS for 5 minutes, fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed with PBS, permeabilized and blocked with PBS plus 0.3% Triton X-100 plus 6% normal goat serum  at room temperature for 2 hours. The coverslips were then incubated overnight at room temperature with mouse monoclonal anti-myc  and either rabbit polyclonal anti-complexin 3 or rabbit polyclonal anti-complexin 4  diluted in the blocking solution. In parallel, coverslips from each cell line were incubated solely with anti-myc, anti-complexin 3, anti-complexin 4, or the blocking solution. All of the coverslips were then washed three times with blocking solution and incubated for 1 hour at room temperature with affinity-purified, Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 546-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) diluted 1:200 in the blocking solution. Finally, the coverslips were washed three times in PBS, once in distilled water, and mounted in Vectashield .Sections and wholemounts were washed with PBS and blocked for 2 hours at room temperature as described for the HEK293T cells. Primary antibodies Table  were addFor labeling of neuromasts, live zebrafish larvae were bathed in 3.0 μM AM1-43 in embryo medium for 30 to 45 seconds . After sImages were acquired with either a FluoView 300 laser scanning confocal microscope running Olympus FluoView 5.0 or an Olympus FluoView FV1000 laser scanning confocal microscope running Olympus10-ASW software . For the Olympus FluoView FV300 confocal, images were collected with an Olympus UPlan-Apochromat 60 × NA 1.20 objective or a Zeiss Fluar 10 × NA 0.5 objective. For the Olympus FluoView FV1000 confocal, images were collected with an Olympus Plan-Apochromat 60 × NA 1.42 objective or an Olympus UPlan-S Apochromat 10 × NA 0.4 objective.For all double-labeling experiments, sequential scans were taken to reduce crosstalk. For most experiments, a series of confocal optical sections (with a step size of 0.5 to 2.0 μm) was taken through the entire tissue section and a planar projection was generated. Images were passed through a median filter, and exported to ImageJ64  and Photoshop CS3 , where levels were increased to reflect the original captures in FluoView.t-tests were performed with Microsoft Excel 2008, with P < 0.05 considered to be statistically significant.To quantify the zebrafish complexin 3/4 reactivity profiles of the rabbit polyclonal anti-complexin 3 and anti-complexin 4 antibodies, 50 random fields of transfected HEK 293T cells were photographed from a representative of triplicate experiments for each cell line. All images were taken within the dynamic range of the photomultiplier, and identical gain and offset settings were used when imaging complexin (and myc) antibody immunoreactivity between cell lines. For each field, confocal z-stacks were taken through the entire cell with a step size of 2 μm, and the middle three optical sections were chosen to make a z-projection for each cell. These projections were encircled in ImageJ, and the mean pixel intensities were measured for both channels. Background fluorescence, which was obtained by measuring the mean pixel intensity for both channels in a region outside of a cell, was subtracted from the pixel intensity measurements. To compare the immunoreactivity of a complexin antibody directly, we calculated mean fluorescence ratios for each complexin paralog by dividing the corrected mean complexin antibody fluorescence intensity by the corrected mean myc fluorescence intensity. Student's Total protein extracts were prepared from freshly isolated adult mouse and zebrafish retinae. Zebrafish extract was made in hypotonic lysis buffer (with protease inhibitors) and loaded at 200 μg, while mouse extract was loaded at 100 μg in SDS-sample buffer (with protease inhibitors). Proteins were size-fractionated by 15% SDS-PAGE and transferred to nitrocellulose membranes. The blot was probed with rabbit polyclonal anti-complexin 3 antibody  in tris-buffered saline tween-20 (TBST) plus 2.5% milk overnight, followed by DyLight 800 conjugated anti-rabbit IgG . Fluorescence was detected with the Odyssey Imaging System .To knock down complexin 4a protein expression, 5 ng of an antisense morpholino oligonucleotide complementary to the translation start site of the complexin 4a gene  was pressure-injected in a volume of 0.5 nl into one-cell-stage, dechorionated zebrafish embryos. A control morpholino oligonucleotide (5 ng/0.5 nl) directed against human beta-globin (Gene Tools) was used as a negative control. Morpholino solutions were diluted to a final concentration of 10 ng/nl with 0.2 M KCl and 2 mg/ml phenol red. An additional negative control condition consisted of dechorionated zebrafish embryos that were not injected. All zebrafish embryos were then raised at 25.5°C in embryo medium ).cplx: complexin; CSK: c-src kinase; dpf: days post-fertilization; ERGIC-53: ER Golgi intermediate compartment 53-kDa; hpf: hours post-fertilization; IPL: inner plexiform layer; lman1: lectin mannose-binding 1; OPL: outer plexiform layer; PBS: phosphate-buffered saline; RAX: retina and anterior neural fold homeobox gene.The authors declare that they have no competing interests.GZ conceived the study, carried out the experiments, prepared the figures, and wrote the initial manuscript. GM also conceived the study, revised the manuscript, and supervised the work, which was done in his laboratory.Identification of a pan-immunoreactive polyclonal antibody that recognizes zebrafish complexins 3 and 4.(A-J) HEK 293T cells were transfected with myc-tagged, full-length zebrafish complexin 3a , 3b , 4a , 4b , or 4c . After 48 hours, cells were fixed with 4% paraformaldehyde and double-stained with a rabbit polyclonal antibody directed against mammalian complexin 3 (Synaptic Systems antibody 122302)  and a mouse monoclonal anti-myc antibody . This experiment was done in triplicate with a complexin antibody dilution of 1:10,000, and 50 random cells from one experiment were randomly selected for quantification. The mean green fluorescence intensity for each cell was divided by the mean red fluorescence intensity. (K) The mean fluorescence ratios and SEM for each cell line are shown, revealing that this antibody recognizes all five isoforms. Scale bar = 25 μm.Click here for fileThe complexin 3/4 antibody predominantly recognizes an approximately 20 kDa band on western blots.Adult mouse and zebrafish retinae were lysed in sample buffer containing protease inhibitors. Mouse extract (100 μg) and zebrafish extract (200 μg) were fractionated by SDS-PAGE (15% gel), blotted onto nitrocellulose, and probed with the complexin 3/4 polyclonal antibody . An approximately 20 kDa band is apparent in both mouse and zebrafish lysates. A minor band of approximately 17 kDa can also be observed in the zebrafish extract.Click here for fileIdentification of a polyclonal antibody that preferentially recognizes zebrafish complexin 4a. (A-J) HEK 293T cells were transfected with myc-tagged, full-length zebrafish complexin 3a , 3b , 4a , 4b , or 4c . These transiently transfected cells were stained with the mouse monoclonal anti-myc antibody  and a rabbit polyclonal antibody directed against mammalian complexin 4 (Synaptic Systems antibody 122402) .  Complexin and myc immunoreactivity, respectively, in a representative cell transfected with zf cplx4a-myc. This experiment was done in triplicate with a complexin antibody dilution of 1:10,000, and 50 random cells from one experiment were randomly selected for quantification. The mean green fluorescence intensity for each cell was divided by the mean red fluorescence intensity. (K) This antibody preferentially recognizes zebrafish complexin 4a . Scale bar = 25 μm.Click here for fileComplementary expression of complexin 3 and complexin 4 in adult mouse retina. Adult mouse retinal sections were labeled with anti-carboxy-terminal binding protein 2  and either anti-complexin 3  or anti-complexin 4 . Since CtBP 2 and RIBEYE are transcribed from the same gene [ame gene , anti-CtClick here for file


However, the ability of S100B inhibitors to prevent/reverse AD histopathology remains controversial. This study examines the effect of S100B ablation on in vivo plaque load, gliosis and dystrophic neurons.Numerous studies have reported that increased expression of S100B, an intracellular Ca-/- line was generated by crossing PSAPP double transgenic males with S100B-/- females and maintained as PSAPP/S100B+/- crosses. Congo red staining was used to quantify plaque load, plaque number and plaque size in 6 month old PSAPP and PSAPP/S100B-/- littermates. The microglial marker Iba1 and astrocytic marker glial fibrillary acidic protein (GFAP) were used to quantify gliosis. Dystrophic neurons were detected with the phospho-tau antibody AT8. S100B immunohistochemistry was used to assess the spatial distribution of S100B in the PSAPP line.Because S100B-specific inhibitors are not available, genetic ablation was used to inhibit S100B function in the PSAPP AD mouse model. The PSAPP/S100B-/- mice exhibited a regionally selective decrease in cortical but not hippocampal plaque load when compared to PSAPP littermates. This regionally selective reduction in plaque load was accompanied by decreases in plaque number, GFAP-positive astrocytes, Iba1-positive microglia and phospho-tau positive dystrophic neurons. These effects were not attributable to regional variability in the distribution of S100B. Hippocampal and cortical S100B immunoreactivity in PSAPP mice was associated with plaques and co-localized with astrocytes and microglia.PSAPP/S100BCollectively, these data support S100B inhibition as a novel strategy for reducing cortical plaque load, gliosis and neuronal dysfunction in AD and suggest that both extracellular as well as intracellular S100B contribute to AD histopathology. The extracellular effects of S100B are concentration dependent; nanomolar S100B levels beneficially promote neuronal survival while micromolar levels detrimentally promote apoptosis [S100B, a member of the S100 protein family, is expressed predominantly in astrocytes and functions as both an intracellular Caopeptide . The teropeptide . S100 pring loop . Upon bi effects ,6. Over poptosis -9. S100Bpoptosis ,8. S100Bpoptosis . Increaspoptosis ,17.2+ homeostasis followed by inflammation, neurodegeneration, senile plaques comprised of aggregated amyloid β (Aβ) peptide, intracellular neurofibrillary tangles comprised of aggregated hyperphosphorylated tau, and ultimately cognitive dysfunction [Altered S100B function is associated with AD pathobiology. The clinical presentation and pathology of early- and late-onset AD include early disturbances in Cafunction -21. In hfunction ,22,23. Sfunction . In addifunction . While tfunction -28. In tfunction  resultinfunction ,30.In vivo studies in genetically modified mouse models have yielded conflicting results regarding the contribution of increased S100B expression to AD pathology. Transgenic TghuS100B mice express 4-5 fold more S100B protein [ protein  and exhi protein . However protein . The mec protein . Surpris protein . Thus, tin vivo genetic approach which recapitulates the entire spectrum of S100 function  to ascertain the net effect of S100B ablation on AD histopathology in the PSAPP AD mouse line. Although no AD mouse model exhibits all aspects of the human disease, the PSAPP double transgenic (APPK670NM671L/PS-1M146L) line has a rapid disease onset and mimics many facets of the human disease including plaque deposition, dystrophic neurites, glial activation, and memory deficits [While specific inhibitors that block the interaction of S100B with its target proteins are under development, currently available compounds do not cross the blood-brain barrier and cannot be used to inhibit CNS S100B . Therefodeficits -38. PSAPK670N/M671L mutation) with the 6.2 line (PS-1M146L) [-/- line has been described previously [-/- line was generated by crossing PSAPP double transgenic males with S100B-/- females and subsequent interbreeding of the PSAPP/S100B+/- heterozygous offspring (PSAPP/S100B+/- X PSAPP/S100B+/-). To control for changes in genetic background, all experiments used PSAPP/S100B+/+ and PSAPP/S100B-/- littermates. Procedures involving animals were approved by the Texas A & M University Institutional Animal Care and Use Committee and comply with the NIH Guide for the Care and Use of Laboratory Animals.The PSAPP double transgenic line was generated by crossing the Tg2576 line ("Swedish" APP-1M146L) -38. The eviously . The PSAFor genotyping, amplification of a 500 bp product using PCR primers for the mouse β-casein gene (forward primer 5' GAT GTG CTC CAG GCT AAA GTT 3' and reverse primer 5' AGA AAC GGA ATG TTG TGG AGT 3') was used to assess genomic DNA quality. The PS-1 and APP transgenes were detected as previously described . Amplifi2, 0.01% (wt/vol) sodium deoxycholate, 0.02% (vol/vol) Nonidet P-40 in 100 mM sodium phosphate buffer pH 7.5 for 36-48 hours. After post-fixation in 10% buffered formalin for 16 hours, tissues were embedded in paraffin and 5 micron sagittal sections were mounted on glass slides for subsequent staining. This processing procedure, originally developed for visualization of β-galactosidase reporter gene activity in transgenic mouse tissues, provides optimum S100B antibody specificity/sensitivity without compromising the detection of other antigens.Brains were removed from anesthetized animals, rinsed in phosphate buffered saline (PBS) and fixed in 4% (wt/vol) paraformaldehyde in PBS for 30 minutes. Sagittal slices, 2 mm in thickness, were prepared using an acrylic brain matrix  and post-fixed for an additional 30 minutes. Slices were then permeabilized in 2 mM MgClTo minimize variability, sections from experimental and control groups were processed simultaneously. Consecutive slides  each containing sections at Allen Brain Atlas Sagittal Levels 8 and 17 were depariffinized and rehydrated to distilled water. For Congo red staining, slides were incubated in 0.02 M NaOH in 80% ethanol saturated with NaCl for 20 minutes followed by a 30 minute incubation in 0.2% (wt/vol) Congo Red  in 0.02 M NaOH in 80% ethanol saturated with NaCl, dehydration and mounting. Immunostaining was performed on a DAKO autostainer  using a biotin-free polymer detection kit  and conditions recommended by the primary antibody manufacturer. Primary antibodies for immunohistochemistry included a mouse monoclonal S100B antibody (1-1000 dilution of Z0311 Dako); rabbit polyclonal GFAP antibody ; mouse monoclonal Iba1antibody ; and mouse monoclonal Ser202/Thr205 phosphorylated tau antibody . For immunofluorescence microscopy, the anti-Iba1 antibody was diluted 1-10, the anti-GFAP antibody 1-100, and the anti-S100B antibody 1-50 . Secondary antibodies included an Alexa Fluor 546 donkey anti-rabbit ; Alexa Fluor 488 rabbit anti-mouse ; and Alexa Fluor 546 donkey anti-mouse .-/-). An independent samples t-test  was used to determine the significance (p < 0.05) of measured differences between the two genotypes. Pearson's Correlation Coefficient and scatter plots of the mean hippocampal and cortical GFAP/Iba1 burden versus plaque load for each animal were used to determine the relationship between plaque load and astrocytosis/microgliosis.For quantification, digital images were captured at 10× magnification on an Olympus IX70 Imaging System using a single exposure setting as follows: the entire hippocampus (2 images); the visual (1 image), somatosensory (1 image) and somatomotor (1 image) cortex as well as representative areas of the cerebellum and olfactory bulb. Images were converted to gray scale and the threshold intensity was set to the intensity observed in areas without tissue. Image J software  was used to quantify positive pixels, plaque size, plaque number and total area. Plaque load and immunoreactivity were defined as the % area, i.e. the area of positive pixels/total pixels × 100. The data were expressed as the mean ± SEM  and colocalization (overlap coefficient) of GFAP/S100B and Iba1/S100B determined using the LSM software.-/- and PSAPP mice  when compared to PSAPP mice (0.168 ± 0.016 percent area)   Figure . This de) Figure  and 1D. -/- mice  ,26,32,33e Figure  and 2B. e Figure . Howevere Figure . In bothe Figure  and olfae Figure . Hippocae Figure  and 3B ae Figure . In addie Figure  and olfae Figure . Collect-/- mice exhibited fewer cortical but similar numbers of hippocampal phospho-tau foci/plaques when compared to PSAPP mice  levels [This study definitively demonstrates that S100B ablation/inhibition reduces AD pathology. PSAPP/S100B knockout mice exhibited a regionally selective decrease in cortical but not hippocampal plaque load that was accompanied by reductions in astrocytosis, microgliosis and dystrophic neurons. These regionally selective effects were not attributable to variations in the S100B distribution; cortical and hippocampal S100B staining patterns were indistinguishable in PSAPP mice. Finally, in PSAPP mice S100B immunoreactivity was associated with plaques and colocalized with astrocytes as well as microglia suggesting that both intracellular and extracellular S100B contribute to AD histopathology. Interestingly, other studies have reported regionally selectively effects of S100B ablation on Ca) levels -47. Asce) levels -50.-/- mice hippocampal plaque load remains unchanged regardless of the mechanism used to induce plaque deposition, i.e. Aβ infusion [These findings clarify inconsistencies in the literature regarding S100B's contribution to AD histopathology. Genetic ablation (this study), pharmacological inhibition  and geneinfusion  or overeinfusion  and overinfusion . These dinfusion , upregul-/- mice will be instrumental in identifying S100B-regulated events that contribute to AD pathology and in discerning the relationship between plaques and inflammation. Behavioral data are not available for any of the S100B/AD mouse models. Strengthened synaptic plasticity and enhanced spatial memory in S100B-/- mice [-/- mice will exhibit improved cognitive function. This hypothesis is supported by the inverse correlation of serum/CSF S100B levels and direct correlation of the rs2300403 SNP in the S100B gene with low cognitive performance, dementia and AD [A consistent finding in this and previous studies is a direct correlation between changes in plaque load and gliosis/inflammation in response to alterations in S100B expression. It is unclear, however, whether changes in plaque load are the cause or the result of changes in gliosis. Furthermore, it is unclear how these histopathological changes impact cognitive function. Microglia are an essential component of the inflammatory response and exist in many forms ,53. They-/- mice  suggest a and AD ,25. Expe2+ handling, synaptic plasticity long-term potentiation, neuronal apoptosis and/or BDNF levels [2+ homeostasis with AD pathobiology and/or serve as a common upstream regulator of both tau phosphorylation/neurofibrillary tangles and Aβ production/plaque deposition [S100B's plaque association and co-localization with cells (microglia/astrocytes) in this and previous studies ,23,33 suF levels -47,57,58F levels ,59-64 coF levels -21. In fposition -21. Whilposition -67. DefiCollectively, these data definitively demonstrate that S100B ablation reduces plaque load, gliosis and dystrophic neurons in the cortex but not the hippocampus. If this reduction in histopathology can be demonstrated to positivity impact cognitive changes related to AD, then additional impetus to the search for new therapeutic interventions targeted at S100B will be provided. The development of effective pharmacological strategies for modulating S100B function in patients will also require quantifying the contribution of extracellular versus intracellular forms, identifying the S100B-regulated target proteins/cellular processes, and ascertaining the contribution of the five other S100 family members implicated in AD, S100A1, S100A6, S100A7, S100A9, and S100A12 -70. FinaThe authors declare that they have no competing interests.LH and KY provided the PSAPP mouse line. AM provided the S100B knockout mouse line. DZ and LH conceived the study and developed the PSAPP/S100B knockout mouse line. ER performed the experiments and participated in the writing of the manuscript as partial fulfillment of the requirements for the PhD degree. DZ supervised the data collection/analyses and drafted the manuscript. All authors read and approved the final manuscript.