Characterization of Pseudophosphatase MK-STYX-Induced Neuronal Differentiation in PC12 Cells

MK-STYX [MAPK (mitogen activated protein kinase) phosphoserine/threonine/tyrosine binding protein] is a pseudophosphatase member of the MAPK phosphatase family. Though structurally related to the MAPK phosphatases, MK-STYX lacks the nucleophilic cysteine and histidine residues essential for catalysis. Despite its lack of catalytic activity, MK-STYX maintains its ability to bind to phosphorylated proteins, but not dephosphorylating them. This thesis focuses on further characterizing the role of MK-STYX in PC12 neuronal differentiation. The PC12 cell line is widely used as a model to study neuronal differentiation. Our previous data demonstrated that MK-STYX induces neuronal differentiation in PC12 cells. The results presented here also show that MK-STYX induces neuronal differentiation in PC12 cells. We further investigated whether MK-STYX induced a unique branching pattern in PC12 cells when they differentiate. In the presence and absence of nerve growth factor (NGF), both primary and secondary neurite distributions are changed, suggesting that MK-STYX changes neurite branching patterns. To investigate what caused this pattern, we turned to the cytoskeleton. Actin polymerization drives the protrusion of lamellipodia and filopodia in growth cones, while tubulin is responsible for making up the ‘core’ of a developing neurite. In the presence of NGF, PC12 cells overexpressing MK-STYX caused an increase in actin protrusions commonly associated with growth cones, dendritic spines, and future dendritic branches. In addition, MK-STYX induced neurites exhibit presynaptic and post-synaptic qualities, as shown through immunostaining with anti-Tau and MAP2 antibodies, respectively. Cofilin, an actin binding and severing protein, has been shown to play a role in regulating neurite outgrowth and branching. MK-STYX further regulates actin dynamics, and thus branching through a possible temporal regulation of cofilin phosphorylation and dephosphorylation. A transient decrease in cofilin phosphorylation at 72 hours of NGF stimulation allows MK-STYX-induced neurites to branch. This strongly supports a model in which the pseudophosphatase MK-STYX has a critical role as a regulator in PC12 neuronal differentiation and morphology

The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X-5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

MK-STYX Alters the Morphology of Primary Neurons, and Outgrowths in MK-STYX Overexpressing PC-12 Cells Develop a Neuronal Phenotype

We previously reported that the pseudophosphatase MK-STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein) dramatically increases the number of what appeared to be primary neurites in rat pheochromocytoma (PC-12) cells; however, the question remained whether these MK-STYX-induced outgrowths were bona fide neurites, and formed synapses. Here, we report that microtubules and microfilaments, components of the cytoskeleton that are involved in the formation of neurites, are present in MK-STYX-induced outgrowths. In addition, in response to nerve growth factor (NGF), MK-STYX-expressing cells produced more growth cones than non-MK-STYX-expressing cells, further supporting a model in which MK-STYX has a role in actin signaling. Furthermore, immunoblot analysis demonstrates that MK-STYX modulates actin expression. Transmission electron microscopy confirmed that MK-STYX-induced neurites form synapses. To determine whether these MK-STYX-induced neurites have pre-synaptic or post-synaptic properties, we used classical markers for axons and dendrites, Tau-1 and MAP2 (microtubule associated protein 2), respectively. MK-STYX induced neurites were dopaminergic and expression of both Tau-1 and MAP2 suggests that they have both axonal and dendritic properties. Further studies in rat hippocampal primary neurons demonstrated that MK-STYX altered their morphology. A significant number of primary neurons in the presence of MK-STYX had more than the normal number of primary neurites. Our data illustrate the novel findings that MK-STYX induces outgrowths in PC-12 cells that fit the criteria for neurites, have a greater number of growth cones, form synapses, and have pre-synaptic and post-synaptic properties. It also highlights that the pseudophosphatase MK-STYX significantly alters the morphology of primary neurons

Map of a part of Louisiana and Mississippi, illustrating the operations of the U.S. Forces in the Department of the Gulf /

Relief shown by hachures.Covers the area from west to east, Alexandria, La. to Pearl River, and from north to south, Vidalia, La. to Atchafalaya Bay."Track of Gen. Banks" shown in red.LC Civil War maps (2nd ed.) 232From the Nathaniel Prentice Banks papers in LC Manuscript Division. DL

Pseudophosphatase MK-STYX induces neurite extensions in PC12 cells.

<p>(<b>A</b>) Representative examples are presented to illustrate neurite outgrowths of PC12 cells over-expressing MK-STYX and GFP, MK-STYX_active, or pMT2 control plasmid and GFP. Cells were incubated 5 days. (<b>B</b>) Cells transfected with pEGFP, pMT2, or pMT2-FLAG-MK-STYX-FLAG plasmids were scored for neurite extensions ≥20 µm with a phase objective. Three replicate experiments were performed (n = 100 cells per experiment); the results are ± SEM. Statistical analysis was performed using ANOVA (F_2,8 = 76.10, ***p<0.0001).</p

MK-STYX induces neurites in the presence of MEK inhibitor (PD98059).

<p>Representative examples of PC12 cells over-expressing pMT2 and GFP, or MK-STYX and GFP, that were stimulated with 100 ng/ml NGF and (<b>A</b>) treated with, or (<b>B</b>) not treated with 50 µM PD98059. (<b>C</b>) Cells were scored for neurite extensions ≧20 µm. Statistical analysis was performed with t-tests for control group (pMT2 and pEGFP; **p<0.01) and experimental group (MK-STYX and pEGFP; **p<0.01).</p

Knockdown of MK-STYX prevents NGF stimulated PC12 neurite extensions.

<p>(<b>A</b>) PC12 cells were transfected with pMT2-FLAG-MK-STYX-FLAG. Transfected and non-transfected PC12 cells were lysed and immunoblots performed. Blots were probed with anti-STYXL, to detect endogenous MK-STYX, and anti-FLAG to detect over-expressed MK-STYX. (<b>B</b>) PC12 cells were transfected with shRNA against MK-STYX. Quantitative RT-PCR analysis of MK-STYX mRNA levels after knockdown with three specific shRNAs targeting unique regions of MK-STYX. MK-STYX shRNA-A provided the best knockdown of endogenous MK-STYX at 42%, compared to Clone B (MK-STYX-shRNA-B) (∼15%) or Clone C (MK-STYX-shRNA-C) (∼32%). (<b>C</b>) PC12 cells transfected with control, MK-STYX shRNA-A, or MK-STYX shRNA-C were lysed and immunoblotted. Anti-STYXL1 antibody showed that endogenous MK-STYX was down-regulated by both MK-STYX shRNA-A and MK-STYX shRNA-C relative to the scrambled negative control. The blot was stripped and probed with anti-ß tubulin as a loading control. Replicate experiments were performed. (<b>D</b>) Cells expressing negative control, MK-STYX shRNA-A, or MK-STYX shRNA-C were scored for neurite extensions ≥20 µm. Three replicate experiments were performed (n = 100 cells per experiment); error bars indicate ± SEM. Statistical analysis was performed using ANOVA (F_2,8 = 357.85, ***p<0.0001). (<b>E</b>) Representative examples of PC12 cells over-expressing shRNAs against MK-STYX (MK-STYX shRNA-A, MK-STYX shRNA-C, or scrambled negative control. The shRNA expression plasmids co-express GFP, which allows visualization of successful transfection. 24 hr post-transfection cells were stimulated with 100 ng/ml NGF, and 72 hr post-stimulation images were taken with phase contrast or fluorescence microscopy. Untransfected cells treated with NGF served as a positive control for neurite outgrowth.</p

MK-STYX decreases RhoA activation.

<p>(<b>A</b>) PC12 cells were transfected with pMT2 or pMT2-FLAG-MK-STYX-FLAG. 24 hr post-transfection cells were stimulated with 100 ng/ml NGF, and lysed at the indicated time points. Activation of RhoA was quantified by RhoA G-LISA small G-protein assay. Total RhoA was normalized by RhoA ELISA. Three replicate experiments were performed. Error bars indicate ± SEM. Statistical analysis was performed using multiple t-tests, and there was a significant increase in the percentage of active RhoA in pMT2-expressing control cells within 30 minutes (p<0.05) (<b>B</b>) PC12 cells were transfected with pMT2-FLAG-MK-STYX-FLAG, MK-STYX shRNA-A, or scrambled shRNA. 24 hr post-transfection cell were stimulated with 100 ng/ml NGF, lysed, and RhoA activation was quantified by RhoA G-LISA small G-protein assay. Total RhoA was normalized by RhoA ELISA. Three replicate experiments were performed. Error bars indicate ± SEM. Statistical analysis was performed using ANOVA (F_3.9 = 5.066, **p<0.01; *p<0.05). (<b>C</b>) PC12 cells transfected with MK-STYX, scrambled shRNA, or MK-STYX shRNA-A. 24 hr post-transfection cells were stimulated with NGF or not, and were lysed 24 hr thereafter and immunoblotted. Anti-phospho-cofilin antibody showed that MK-STYX decreased cofilin phosphorylation in non-stimulated cells relative to the MK-STYX shRNA-A. However, MK-STYX increased cofilin phosphorylation in cells stimulated with NGF relative to the MK-STYX shRNA-A. These blots were stripped and probed for cofilin as a loading control. Anti-STYXL1 antibody showed over-expressed MK-STYX relative to the scrambled control, and that endogenous MK-STYX was down-regulated by MK-STYX shRNA-A relative to the scrambled negative control. The blot was stripped and probed with anti-FLAG to detect over-expressed MK-STYX, and probed for anti-ß tubulin as a loading control. Three replicate experiments were performed.</p