Phosphoprotein Research Articles

Relationship between expression of golgi phosphoprotein 3 and angiogenesis in colorectal cancer tissues

Objective To study the relationship between the expression of golgi phosphoprotein 3 (GOLPH3) and angiogenesis in colorectal carncer tissues.Methods The expression of GOLPH3 was examined by Western blotting in colorectal cancer tissues,and that of GOLPH3,vascular endothelial growth factor (VEGF) and CD34 in the paraffin-embedded sections of colorectal cancer from 62 cases was detected by using the standard streptavidin-peroxidase (SP) technique.CD34 was used as a marker to account microvessel density (MVD) in colorectal cancer tissues.The correlation between GOLPH3 expression with VEGF expression and MVD was analyzed.Results The positive expression rate of GOLPH3 was 53.2％ in colorectal cancer tissues.The expression of VEGF and MVD in colorectal cancer tissues positive for the GOLPH3 expression was significantly higher than in those negative for the GOLPH3 expression.The protein level of GOLPH3 expression with VEGF negative-expression in colorectal cancer tissues was significantly lower than that with VEGF positive-expression.Pearson correlation analysis revealed that the protein level of GOLPH3 expression was positively correlated with MVD (r =0.335,P ＜ 0.01).Conclusion The positive expression of GOLPH3 is related with angiogenesis in colorectal carncer tissues. Key words: Colorectal neoplasms; Golgi phosphoprotein 3; Angiogenesis

Oligomerization state of the multimerization domain of Nipah virus phosphoprotein

Oligomerization of the phospho (P) proteins of paramyxoviruses plays important roles in the viral genome replication and transcription. The multimerization domain of the P proteins (PMD) was found to be responsible and sufficient for the oligomerization process. However, the oligomeric status of Nipah virus (NiV) P protein is still unknown. Therefore, the aim of this study was to produce the PMD region of NiV in bacteria in order to determine the oligomeric status of the P protein. The NiV PMD region was cloned into pTrcHis2-TOPO vector and expressed in Escherichia coli. SDS-PAGE and Western blot analysis confirmed the protein expression upon IPTG induction at 37°C for 5h. Immobilized metal affinity chromatography (IMAC) purification was developed to purify the recombinant histidine-tagged NiV PMD which yielded a purity of 97%. Chemical cross-linking revealed that the PMD of NiV formed tetramer. The stability of the oligomer was studied with dynamic light scattering while mass spectrometry confirmed the exact molecular masses of the cross-linked products. Circular dichroism analysis revealed that the NiV PMD contained high α-helical content (55%) suggesting the presence of a coiled coil structure.

Oxidative Versus Thrombotic Stimulation of Platelets Differentially activates Signalling Pathways.

Atherosclerosis is one of the inflammatory underlying disease associated by oxidative stress and thrombotic agents. This study aimed to evaluate the potential role of cupper oxidized low-density lipoprotein (OxLDL) and thrombin for inducing mitogen activated protein kinases (MAPKs) in platelets. Phosphorylation of P38MAPK, Jun N-terminal Kinase (JNK), and Extracellular signal-regulated kinases (ERK1/2) and P-selectin expression were determined in lysates of washed human platelets pretreated with low doses of thrombin and cu2+-OxLDL By Enzyme-linked immunosorbent assay (ELISA). Pharmacological inhibition was performed by SB203580, PD980559 and SP6000125 for P38MAPK, ERK1/2 and JNK activity, respectively. The ratio of phosphorylated to total protein was used for normalizing the phospho proteins contents of cells. OxLDL and thrombin significantly and differentially increased P-selectin expression (P<0.05), P38MAPK (P<0.05) and c-JNK (P<0.05) and ERK1/2 (P<0.05) phosphorylation in platelets. SB 203580 and SP6000125 significantly decreased P-selectin expression in both oxidative (P<0.05) and thrombotic (P<0.05) activated platelets. Our results indicated that MAPK inhibitors can reduce atherothrombotic events via alterations in P-selectin expression suggesting that these inhibitors may be useful in the inhibition of atheroma development.

Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress

Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447–56; Qin, Drug Dev. Res. 68 (2007) 213–225).Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613–21).To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions.Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPKα1 (S485), acetyl-CoA carboxylase (S79), LDHA).Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell–cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.

Abstract 2408: Comparison of ERK, AKT and MEK isoform phosphorylations between primary and corresponding metastatic colorectal carcinoma lesions using the NanoPro1000 technology.

Abstract Proteomic analysis of cellular signaling networks has strongly gained in importance in the field of cancer research and treatment. In particular, there is a focus on the activation status of growth factor receptor related signaling cascades represented by phosphoproteins, as key mediators of cellular signaling. For example, the inhibition of the EGF-receptor pathway has been widely integrated into clinical practice for different tumor entities, e.g. non small lung cancer and colorectal cancer. Although, predictive genetic markers of response to targeted therapy such as KRAS testing are already in use, heterogeneity in responses to treatment has been clinically observed. In order to understand this heterogeneity on the proteome level, the activation status of cell signaling proteins has to be analyzed. The in-depth analysis of isoform phosphorylation can potentially reveal significant biological differences in activation patterns and might result in predictive signatures. In this study, we differentially analyzed basal isoform phosphorylations of selected key proteins of two EGF-receptor downstream pathways, in four primary colorectal tumors and their corresponding metastases using the NanoPro1000 technology. Patient's biospecimen were collected according to standard operating procedures, ensuring a high quality of tissues characterized by ischemia times below 10 minutes, comprehensive clinical data and corresponding sets of body fluids. The detailed characterization of isoform phosphorylations was conducted using the recently introduced NanoPro1000 technology. This assay provides the separation of phospho protein isoforms by high-resolution isoelectric focusing (IEF) combined with a specific, antibody based detection. In addition to these data, the mutation status of selected, clinical relevant genes (KRAS, BRAF and PIK3CA) was determined in primary and metastatic tumor samples using Sanger sequencing. Among the analyzed primary tumors, there were two KRAS mutants, one BRAF mutant and one wild type tumor. Identical results were found in the matched metastasis. NanoPro1000 analysis revealed that isoform phosphorylation of ERK1/2, AKT and MEK1/2 differed among patients, as well as primary tumors and metastases. For example, ERK1 showed a tendency to be higher mono- and dual-phosphorylated in the primary tumors, whereas mono-phosphorylated ERK2 was elevated in metastases. However, a correlation to the mutation status was not found. These differences may explain the heterogeneity of individual responses to target anticancer treatment among patients, independent of their mutation status. Therefore, the NanoPro1000 technology can potentially be useful to identify predictive biomarker for the development of resistance to anti-EGF-receptor treatment and further improve personalized medicine. Citation Format: Florian T. Unger, Jana Krueger, Janina Schaller, Cordula Dede, Alexandra Samsen, Hartmut Juhl, Kerstin A. David. Comparison of ERK, AKT and MEK isoform phosphorylations between primary and corresponding metastatic colorectal carcinoma lesions using the NanoPro1000 technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2408. doi:10.1158/1538-7445.AM2013-2408

Isorhamnetin modulates the γ‐aminobutyric acid transporter GAT‐3 in mesencephalic and cortical astrocytes exposed to neurotoxic manganese

Excessive manganese (Mn) exposure leads to a Parkinson's‐like disorder characterized by neurochemical imbalances. A disruption in γ‐aminobutyric acid (GABA) is an early event of Mn‐toxicity, and evidence suggests that Mn impairs the function of GABA transporters leading to elevated synaptic GABA. We suspect that Mn inhibits GABA uptake by altering the density of GABA transporters on the plasma membrane (PM). The purpose of this study was to characterize the effect of Mn on the cellular localization of the GABA transporter GAT‐3 in astrocytes, and to identify whether the blood‐brain permeable quercetin metabolite, isorhamnetin (ISO), preserves membrane GAT‐3 protein levels and function. Primary astrocytes were isolated from the cortex and mesencephalon of Sprague‐Dawley rats at postnatal day two. Astrocytes were exposed to 500μM Mn for 1, 6, or 24hrs with or without 72hr ISO (10μM) pre‐treatment. Western blot analysis of GAT‐3 and phospho protein kinase C‐δ (PKC‐δ) were conducted on PM and cytosolic fractions. Mn decreased total GAT‐3 protein in astrocytes from both regions after 24hr exposure. ISO pre‐treatment did not improve total GAT‐3 levels, but shifted GAT‐3 from the cytosol to the PM in astrocytes from both regions, which corresponded with elevated phospho‐PKCδ at the PM. ISO also enhanced 3H‐GABA uptake in astrocytes. These data suggest that ISO may protect against early events in Mn‐toxicity.

Advances of GOLPH2 in tumors

As a novel Golgi protein,GOLPH2 expression is associated with maintaining the structural integrity of the Golgi apparatus.As an oncoprotein,GOLPH2 expression is associated with malignant biological behavior of cancer cells.Important roles of GOLPH2 in the tumorigenesis and metastasis make it become a new target for tumor diagnosis and treatment. Key words: Golgi phosphoprotein 2; Neoplasms

Establishment of an in vitro transcription system for Peste des petits ruminant virus

BackgroundPeste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells.ResultsRNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3’ end to 5’ end of the genome similar to that exhibited by the virus in infected cells.ConclusionThis report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

Résistance des Paramyxoviridae aux interférons de type I: mécanismes d'échappement et interactions virus-hôte.

Host immunity against viruses integrates both innate and adaptive responses. Innate immunity represents the first line of defense triggered by viral infections whereas adaptive immunity allows for the establishment of long-term memory. Initially described for their antiviral activity, type I interferons are also involved in the induction of all immune responses. Since these cytokines are crucial for the establishment of an efficient antiviral response, viruses have developped mechanisms to block their expression and signals. The family of Paramyxoviridae presents a wonderful example of how viruses escape the antiviral response. Evasion strategies mainly involve the non structural proteins encoded by the gene of the phosphoprotein (P). These virulence factors can simultaneously or sequentially interact with cellular proteins through multiple interfaces and sophisticated mechanisms. This review describes the different mechanisms developped by paramyxoviruses to hijack the type I IFN response and molecular details, in particular protein-protein interactions, will be highlighted.

The induction of systemic lupus erythematosus by human cytomegalovirus pp65

Objective To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65 (HCMV pp65) in murine systemic lupus erythematosus (SLE).Methods The prokaryotic vector pET-28b and eukaryotic vector pcDNA 3.0 were constructed to express the HCMV pp65 protein.All the C57BL/6 mice were inoculated with pp65 eukaryotic vector intramuscularly five times at 2-week intervals and then were bled via the retro-orbital vein.Subsequently,indirect ELISA was used to evaluate the concentration of anti-pp65 IgG,anti-dsDNA and ANA.At the same time,IL-1 b,IL-6,and TNF-α were determined by competitive ELISA.Results The early onset of autoantibodies and an overexpression of IL-6 were observed in immunized male C57BL/6 mice.Conclusion HCMV pp65 triggers the deregulation of humoral immunity in C57BL/6 mice,which indicates that the immune responses induced by HCMV pp65 may be involved in the development of SLE. Key words: Autoimmune diseases(AD) ; Human cytomegalovirus(HCMV) ; Systemic lupus erythematosus(SLE) ; Lower matrix phosphoprotein 65 (pp65)

A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.

La protéine L des Mononegavirales.

The order Mononegavirales comprises viruses representing a large variety of human, animal and plant pathogens. These viruses have a non-segmented negative stranded RNA genome encoding their own RNA dependent RNA polymerase, the large protein L used to transcribe and replicate their genome. Due to the absence of methods to produce purified recombinant L proteins, these later were poorly characterized. Recently, the vesicular stomatitis virus L protein has been purified and used for structural and functional analysis. Electron microscopy images of L provided the first structural insight into the molecular architecture of L, and its conserved regions were mapped. Moreover, a new in vitro assay was developed and the mechanism by which L initiates RNA synthesis has been characterized, as well as the effects of its co-factor, the phosphoprotein P, on its own activity. This system constitutes a powerful tool to screen inhibitors directed specifically against the polymerase activity of L. These data have led to significant progress into the understanding of how the replicative machinery of these viruses function.

The Greatwall kinase: a new pathway in the control of the cell cycle

New data have recently established that protein phosphorylation during mitosis is the result of a controlled balance between kinase and phosphatase activities and that, as for mitotic kinases, phosphatases are also regulated during cell division. This regulation is at least in part induced by the activation of the Greatwall (Gwl) kinase at mitotic entry. Activated Gwl phosphorylates its substrates cAMP-regulated phospho protein 19 (Arpp19) and α-endosulfine (ENSA), promoting their binding to and the inhibition of PP2A. Interestingly, besides the role of the Gwl-Arpp19/ENSA in the control of mitotic division, new data in yeast support the involvement of this pathway in mRNA stabilization during G(0) program initiation, although in this case the phosphatase PP2A appears not to be implicated. Finally, Gwl activity has been shown to be required for DNA checkpoint recovery. These new findings support the view that Gwl, Arpp19 and ENSA could function as the core of a new signalization pathway that, by targeting different final substrates, could participate in a variety of physiological functions.

PERANAN KASEIN DALAM PENCEGAHAN KARIES GIGI: ROLES OF CASEIN IN DENTAL CARIES PREVENTION

Dental caries is the highest prevalence disease in many countries, it is shown by many studies have been performed to solve dental caries problem. Dental caries process had develop since the teeth are going to erupt in the mouth. However, it has been proved that not all food cause dental caries. Milk product which has high milk protein such casein and calcium, could prevent demineralization. By its ion calcium penetration in soluble enamel, the remineralization process will happen. It make a biofilm coated with calcium phosphoprotein and amorph calcium phosphate formation that formed along milk digestion. So it has a possibility to become an alternative food to prevent dental caries, as anticariogenic and cariostatic agent. As examples, human breast milk, bovine milk, cheese, and yogurt that would be explained in this paper. In conclusion, milk product consumption without any sugar can inhibit caries development.

Production and purification of the phosphoprotein of Nipah virus in Escherichia coli for use in diagnostic assays

Nipah Virus (NiV) is an emerging zoonotic paramyxovirus that can be fatal in humans and various types of animals. The phospho (P) protein of NiV plays an important role in RNA synthesis, replication, and genome synthesis. In this study, the NiV P gene was cloned into a pTrcHis2-TOPO vector and the recombinant protein containing a His-tag was produced in Escherichia coli. SDS-PAGE and Western blot analysis using the anti-His antibody confirmed the protein expression. An optimization study of E. coli fermentation showed that the optimal cultivation temperature was 37°C, while the optimal induction time for P protein expression was at 9 h with 1 mM IPTG. Solubility analysis showed that E. coli cultivated at 37°C produced the highest fraction (70%) of soluble P protein. The recombinant P protein was purified from clarified E. coli lysate using an immobilized metal affinity chromatography (IMAC) technique to a purity of 92.67%, with a purification factor of 11.58. The purified P protein strongly reacted with the anti-NiV swine sera collected during a NiV outbreak, suggesting its potential as a diagnostic reagent.

Synergistic effect of p38MAPK and ERK1/2 pathways on regulation of osteoblastic differentiation

Objective To study the synergistic effect of p38MAPK and ERK1/2 in bone marrow mesenchymal stem cells (BMSCs), and to explore their influence on osteogenic differentiation in BMSCs cultures. Methods Mouse BMSCs were cultured in phenol red-free α-MEM containing osteogenic supplements (OS) for inducing osteogenic differentiation. The temporal sequence of osteogenic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity (ALP) and calcium deposition gene expression. The activation of p38MAPK and ERK1/2 was detected by western blotting using phospho-specific MAP kinase antibody. BMSCs were treated with the inhibitor of p38MAPK pathway (SB203580) or ERK1/2 pathway (PD98059), and osteogenic differentiation was measured. BMSCs were treated with SB203580 or sodium arsenite (ARS), a strong activator of p38MAPK, and the phosphorylation of ERK 1/2 was measured. BMSCs were treated with PP2A inhibitor, Okadaic acid (OA), the phosphorylation of ERK1/2 and osteogenic differentiation were measured. lmmunoprecipitation was used to test the binding interaction between PP2A and ERK1/2, and the effect of SB203580 on the interaction. Results Treatment of BMSCs with osteogenic supplements resulted in activation of p38MAPK and ERK1/2 that coincided with osteogenic differentiation. Inhibition of p38MAPK activation by SB203580, blocked the osteogenic differentiation, whereas inhibition of ERK1/2 activation by PD98059, enhanced the osteogenic differentiation in a dose-dependent manner.SB203580 treatment resulted in increased ERK1/2 phosphorylation. By contrast, ARS treatment resulted in decreased ERK1/2 phosphorylation. Inhibition of PP2A by OA resulted in increased ERK1/2 phosphorylation. OS-induced osteogenic differentiation was also attenuated by PP2A inhibition. Immunoprecipitation confirmed the association of PP2A with ERK1/2 in BMSCs cultures, which was decreased by SB203580 treatment. Conclusion The present study demonstrates a synergistic effect between p38MAPK and ERK1/2 signaling pathways via PP2A in BMSCs cultures, which may regulate the osteogenic differentiation of BMSCs. Key words: Extracellular signal-regulated MAP kinases; Osteoblasts; Cell differentiation; Phosphoprotein phosphatase

Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome

Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.

Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling

To identify regulators of intracellular signaling we targeted 541 kinases and kinase-related molecules with siRNAs and determined their effects on signaling with a functional proteomics reverse phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and MAPK with 115 genes that when targeted by siRNAs demonstrated opposite effects on MAPK and AKT phosphorylation. Network based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on TSC2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.

An embryonic culture system for the investigation of striatal medium spiny neuron dendritic spine development and plasticity

Dendritic spines of striatal Medium Spiny Neurons (MSNs) receive converging dopaminergic and glutamatergic inputs. These spines undergo experience-dependent structural plasticity following repeated drug administration and during disease states like Huntington's and Parkinson's. Thus, understanding the molecular mechanisms leading to structural plasticity is an important step toward establishing a clear relationship between spine structure and function, and will ultimately contribute to understanding how changes in dendritic spine structure relate to behaviors or diseases. One major difficulty faced when studying MSN development is the lack of a detailed, standardized in vitro model system that produces MSNs with in vivo-like morphologies. For example, unlike cultured pyramidal neurons, MSNs grown in mono-cultures display stunted dendritic arborization and fail to develop a full cohort of mature dendritic spines. Here we report the generation of an embryonic mouse cortical–striatal co-culture that generates high cell yields from a single embryo. Unlike MSNs in striatal mono-culture, MSNs in co-culture develop in vivo-like morphologies and high densities of dendritic spines. Morphological identification of co-cultured MSNs expressing a soluble fluorescent protein can be confirmed by immunochemical detection of DARPP-32 ( Dopamine and cyclic AMP regulated phospho protein of 32 kDa). Additionally, co-cultured MSN spines contain PSD-95 puncta and are apposed to SV2 puncta, indicating the spines express synaptic machinery. Finally, whole-cell recordings of co-cultured MSNs exhibit higher mEPSC frequency compared to mono-cultured MSNs, suggesting that the spines are functionally mature. These studies establish that this co-culture system is suitable for studying the morphological and physiological development and function of MSN dendritic spines.

Inhibition of IL-17 Release by the Novel Anti-Inflammatory Drug Vidofludimus Involves Attenuation of STAT3 and NF-kappa B Signaling Pathways in Murine Splenocytes and Hapten-Induced Colitis

IL-17 Secretion: Murine Splenocytes Vidofludimus Inhibits the Nuclear Binding of STAT3 in a Dose Dependent Fashion: Murine Splenocytes Vidofludimus Does Not Affect the Expression of IκB-α: Murine Splenocytes Vidofludimus Attenuates Phospho-p65 Staining: Murine TNBS Colitis The STAT3 and NF-kappa B (NF-κB) pathways play a prominent role in IL-17 production and intestinal inflammation. We have shown that a novel drug named Vidofludimus (Vido, 4SC-101, SC12267) inhibited colonic IL-17 and murine colitis [Inflammatory Bowel Diseases, 2010]. Aims: The aims of our study were: 1) to determine if Vido inhibited IL-17 production by murine splenocytes, 2) to evaluate if Vido inhibited key components of the STAT3 and NF-κB signaling pathways in splenocytes and 3) to determine if Vido inhibited phosphorylated (phospho) STAT3 and NF-κB in the colons of mice with hapten (TNBS)-induced colitis. Methods: Splenocytes were collected from BALB/c mice. These cells were stimulated with IL-23 or IL-1β (10 ng/ml) alone, or in combination. Vido (25 to 100 μM), or vehicle, were added along with the cytokines. IL-17 secretion was determined by ELISA at Vidofludimus (4SC-101, SC12267) is a proprietary compound of 4SC AG Vidofludimus Inhibits Cytokine-Induced IL-17 Secretion: Murine Splenocytes Synergistic activation by IL-23 + IL-1β Vidofludimus Attenuates Phospho-AKT1 Expression: Murine Splenocytes Effects of Vidofludimus on Signaling Pathways: Murine Splenocytes 48 hours. Cytosolic extracts from splenocytes were used for western blots of phospho JAK2, STAT3 and AKT1 (STAT3 pathway proteins). Phospho I-κB and I-κB degradation were evaluated as indices of NF-κB pathway activation. Extracts were also collected from stimulated splenocytes for the evaluation of STAT3 and NF-κB nuclear binding by ELISA. Immunohistochemistry for phospho STAT3 and p65 was done on samples from BALB/c mice that underwent colitis induction with TNBS ± oral treatment for six days with Vido (50-200 mg/kg). Using coded slides, the staining for these proteins in the mucosa/submucosa was graded on a six point intensity scale. Results: IL-23 and IL-1β alone did not induce significant IL-17 production (< 30 pg/ml), but combined cytokine treatment induced a synergistic stimulation of IL-17 secretion (630 pg/ml). IL-17 production was blocked by Vido in a dose dependent fashion (IC50 = 58.1 μM). At 10 to 60 minutes after cytokine stimulation, Vido reduced the expression of phospho proteins (JAK2, STAT3, and AKT1) involved in STAT3 signaling, and also inhibited the nuclear binding of STAT3 (IC50 = 53.9 μM). Vido did not reduce the phosphorylation or degradation of I-κB, but at 100 μM inhibited the nuclear binding of p65 by 47%. Evidence of increased phospho STAT3 and p65 immunostaining was found in leukocytes within the colons of TNBS-treated mice. The staining was attenuated in mice treated with Vido. Phospho STAT3 staining scores (2-3 mice per group) were: 1.3 ± 0.8 (no TNBS), 4.3 ± 0.9 (Vehicle/TNBS), 1.1 ± 0.3 (Vido-50 mg/kg), 1.2 ± 0.7 (Vido-100 mg/kg) and 0.3 ± 0 (Vido-200 mg/kg/TNBS). Similar METHODS immuno-staining scores were found for phospho p65. Summary: Vidofludimus inhibited STAT3 and NF-κB signaling pathways in murine splenocytes. It also normalized the enhanced phospho STAT3 and p65 immunostaining associated with murine colitis. Conclusion: The inhibition of STAT3 and NF-κB pathways by Vidofludmus reduces IL-17 production and contributes to the established anti-colitis activity of this drug. Splenocyte Cultures: Spleens were rapidly harvested from BALB/c mice. Splenocytes were collected by standard techniques, and erythrocytes were removed by using RBC lysis buffer. For the IL-17 secretion studies, we plated 2 x 106 cells/ml in 4 ml of RPMI 1640 medium containing 10% FBS and 5 x 105 β-mercaptoethanol. Therefore, there were 8 x 106 cells per well in the standard six-well plates. We utilized two wells (1.6 x 107 cells) per treatment condition in these studies. Splenocytes were cultured for 48 hours at 37 degrees C. Then, the culture media was collected for the measurement of IL-17 by ELISA (R&D systems, Minneapolis, MN). In subsequent studies (western blots, transcription factor binding assays), splenocytes were plated at a density of 1 x 107 cells/ml in 4 ml of RPMI 1640 medium. Therefore,