Michael Wilhelm

Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model.

Nix, a pro-apoptotic BH3-only protein, promotes apoptosis of non-neuronal cells, although the mechanisms involved remain incompletely understood. Using a yeast two-hybrid screen with POSH (plenty of SH3 domains, a scaffold involved in activation of the apoptotic JNK/c-Jun pathway) as the bait, we identified an interaction between POSH and Nix. Co-immunoprecipitation and in vitro binding studies confirmed a direct interaction between POSH and Nix in mammalian cells. When overexpressed in HEK293 cells, Nix promotes apoptosis along with enhanced phosphorylation/activation of JNKs and their target c-Jun. These effects appear to be dependent on POSH because Nix does not promote either JNK/c-Jun phosphorylation or apoptosis of 293 cells that do not express POSH. Nix and POSH appear to mutually stabilize one another and this effect could contribute to their promotion of death. Past work showed induction of Nix transcripts in a cellular model of Parkinson disease based on neuronal PC12 cells exposed to 6-hydroxydopamine. Here, we confirm elevation of Nix protein in this model and that Nix over-expression causes apoptotic death of PC12 cells by a mechanism dependent on c-Jun activation. Expression of s-Nix, a dominant-negative form of Nix, protects neuronal PC12 cells from 6-hydroxydopamine but not from nerve growth factor deprivation. These results indicate that Nix promotes cell death via interaction with POSH and activation of the JNK/c-Jun pathway and that Nix protein is induced and contributes to cell death in a cellular model of Parkinson disease.

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein.

Background: Scaffold proteins, such as the pro-apoptotic scaffold POSH (Plenty of SH3s), organize MAP kinase pathways into functional modules. Results: Sh3rf2 promotes the degradation of POSH and prevents apoptosis in multiple cell types. Conclusion: Sh3rf2 antagonizes POSH-JNK signaling under basal conditions and provides a “brake” on apoptosis. Significance: Sh3rf2 may provide a target in neoplasia and apoptosis involving POSH such as trophic factor deprivation. We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.

Cbl negatively regulates JNK activation and cell death

Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apoptosis — nerve growth factor (NGF) deprivation and DNA damage — cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activation) of c-Cbl. Targeting c-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase (JNK) signaling. We demonstrate that Cbl proteins interact with the JNK pathway components mixed lineage kinase (MLK) 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK activation and on cell death.

Identification of POSH2, a Novel Homologue of the c-Jun N-Terminal Kinase Scaffold Protein POSH

The c-Jun N-terminal kinase (JNK) pathway plays an important role in neuronal apoptosis both during normal CNS development and following stroke in adult animals. As with other MAP kinase pathways, scaffold proteins regulate JNK signaling. The scaffold protein POSH (Plenty of SH3s) enhances JNK activation and apoptosis. We identified a POSH homologue, POSH2, which was cloned from rat brain and is present in cortical neurons in vitro. POSH2 mRNA is expressed in a variety of tissues including brain, and this distribution partially overlaps with that of POSH. POSH2 overexpression promotes JNK activation in HEK293 cells and promotes apoptosis in neuronal PC12 cells, which is blocked by a dominant-negative c-Jun. Finally POSH2 contains a functional RING domain and enhances the stability of coexpressed mixed-lineage kinases. These results indicate that POSH2 may regulate JNK activation and consequent apoptosis under conditions of increased expression.

c-Jun N-terminal kinase regulates mGluR-dependent expression of post-synaptic FMRP target proteins

Fragile X syndrome (FXS) is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of FMRP results in an elevated basal protein expression profile of FMRP targeted mRNAs, a loss of local metabotropic glutamate receptor (mGluR)‐regulated protein synthesis, exaggerated long‐term depression and corresponding learning and behavioral deficits. Evidence shows that blocking mGluR signaling in FXS models ameliorates these deficits. Therefore, understanding the signaling mechanisms downstream of mGluR stimulation may provide additional therapeutic targets for FXS. Kinase cascades are an integral mechanism regulating mGluR‐dependent protein translation. The c‐Jun N‐terminal kinase (JNK) pathway has been shown to regulate mGluR‐dependent nuclear transcription; however, the involvement of JNK in local, synaptic signaling has not been explored. Here, we show that JNK is both necessary and sufficient for mGluR‐dependent expression of a subset of FMRP target proteins. In addition, JNK activity is basally elevated in fmr1 knockout mouse synapses, and blocking JNK activity reduces the over‐expression of post‐synaptic proteins in these mice. Together, these data suggest that JNK may be an important signaling mechanism downstream of mGluR stimulation, regulating FMRP‐dependent protein synthesis. Furthermore, local, post‐synaptic dysregulation of JNK activity may provide a viable target to ameliorate the deficits involved in FXS.

Developing Accelerated Learning Models in GIFT for Medical Military and Civilian Training

This paper will discuss the protocol of an inter-institutional study between the Army Research Laboratory (ARL) and Columbia University Medical Center that seeks to identify pedagogical models that can be employed in the Generalized Intelligent Framework for Tutoring system (GIFT) to support the transfer of skills from training to operations in individual Soldiers within the domain of critical care, addressing topics in hemorrhage, airway compromise, and/or tension Pneumothorax. The scientific approach will include two studies. The first correlational study aims to examine the effect of human variability on learning, performance, retention, and transfer by using individual differences (e.g., personality traits, cognitive abilities, and motivation) as criteria to tailor individual training for Soldier learning needs. The second study will be an experiment to examine how the priming of analogical reasoning tasks effects the problem-solving outcomes of increasingly complex critical care case study content. The authors intend to incorporate the findings of these two studies to support the development of accelerating expert-level reasoning skills and strategies to achieve cognitive flexibility, one of two paths that has been identified as a way to accelerate proficiency.

Acid-Base Disorders.

1. Benson S. Hsu, MD, MBA* 2. Saquib A. Lakhani, MD† 3. Michael Wilhelm, MD‡ 1. *Pediatric Critical Care, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD. 2. †Pediatric Critical Care, Yale School of Medicine, New Haven, CT. 3. ‡Pediatric Critical Care, University of Wisconsin School of Medicine and Public Health, Madison, WI. To treat critically ill children, a physician must have a clear understanding of acid-base balance. After completing this article, readers should be able to: 1. Describe the mechanisms regulating acid-base physiology and identify blood gas abnormalities associated with an acid-base imbalance. 2. Recognize the differential diagnosis and clinical and laboratory features associated with metabolic acidosis and metabolic alkalosis as well as how to manage each appropriately. 3. Calculate an anion gap and formulate a differential diagnosis associated with various anion gap values. 4. Identify factors contributing to compensatory changes associated with primary metabolic and respiratory acidoses and alkaloses. The body’s ability to maintain acid-base homeostasis is based on a complex set of interactions between the respiratory and metabolic systems. This article reviews normal acid-base physiology and examines disorders of acid-base imbalances, first within a primary metabolic cause and then within a primary respiratory cause. Covering the complex nuances of acid-base control within a limited-scope review article is impossible. Thus, this article focuses on the traditional model based on the Henderson-Hasselbalch equation rather than the strong ion (or Stewart) model, which explores the difference between all the dissociated cations and anions. Using the traditional model, the authors explore the various metabolic and respiratory disturbances while addressing the implications of the anion gap on metabolic acidoses. ### The Henderson-Hasselbalch Equation Homeostatic control of acid-base balance is critical for all metabolic and physiologic functions of the human body. The Henderson-Hasselbalch equation describes the relationship between pH and the bicarbonate buffering system (the predominant buffering system in plasma) to establish this homeostasis:![Formula][1] ![Formula][2] When accounting for H2CO3, … [1]: /embed/graphic-1.gif [2]: /embed/graphic-2.gif

A normal capillary refill time predicts adequate superior vena cava oxygen saturation.

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