THE PROTEOSTASIS OF THE ENDOPLASMIC RETICULUM AND THE ACTIVATION OF THE UNFOLDED PROTEIN RESPONSE PATHWAY IN VIVO

ABSTRACT Background. The protein components of eukaryotic cells face acute and chronic challenges to normal folding, refolding and function owing to a constant barrage of physical, metabolic and environmental stresses. Eukaryotic protein homeostasis, or proteostasis, enables healthy cell adaptation during development and protects against aging and diseases. Proteostasis refers to controlling the concentration, conformation, binding interaction and location of individual proteins making up the proteome by readapting the innate biology of the cell, often through transcriptional and translational changes. The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways, cumulatively called the unfolded protein response (UPR). The UPR activation triggers an extensive transcriptional and translational response, which adjusts the ER protein folding capacity according to needs. As such, the UPR constitutes one of the signaling pathways that regulates the capacity and composition of the proteostasis network according to the changing of the ER folding capacity. Previous work. My initial work, described in chapter 1.2.1 shows that the level of cellular energy is important for protein folding and disaggregation and thus can affect folding and repair processes. The proteostasis network is not only highly adaptable, enabled by the influence of multiple cell stress signaling pathways, but also can be quite distinct in each cell type. In chapter 1.2.2, by using as a cellular model the plasma cells differentiation, I could underline the role of the UPR signaling and proteasomal degradation in orchestrating the architectural and functional changes of the cells and balancing the proteostasis capacity. Recent studies suggest that lack/decrease of O2 perturbs the ER homeostasis. Indeed the ER has emerged as a cellular compartment that depends on O2 for oxidative folding of secretory and transmembrane proteins and that mediates the O2 signaling that is important for the survival and function of hypoxic cells. In vitro studies have shown that hypoxia triggers the UPR. However, in vivo and in vitro situations are indeed likely to diverge substantially with respect to parameters such as metabolic activity, O2 utilization and cell division rates, features that are predicted to vary the cell sensitivity to ER stress. Thus, despite a recognized role for hypoxia on UPR, few data exist on the effects of hypoxia in various organs in vivo. Aims. My study includes three aims: First, we will test if the hypoxic stress in vivo acts as a modifier that affects the activation of specific branches of the UPR in different tissues. Second, to get a better insight into the role of the UPR during low oxygen availability in tissues, we will test whether the UPR activation depends from the severity of the hypoxic stress. Third, we aim at delineating signaling circuits that control the capacity and composition of the proteostasis network through transcriptional and post transcriptional mechanisms to balance the ER homeostasis Results. I analyzed the effect of the hypoxic stress on the proteostasis network. Changes in O2 levels alter the ability of the cells to handle the proteostasis load with some differences between the cells type studied. Hepatocytes and myocytes respond to hypoxia by increasing their degradation activity as to increase the proteostasis capacity. While the hepatocytes activate an UPR-dependent apoptosis and are able to balance between apoptotic death and protein synthesis, in the myocytes the protein synthesis remains sustained under low oxygen availability while the UPR \u2013dependent apoptosis could not be detected. Conclusion. This studies underlined several features of the ER- proteostasis. First, the proteostasis network is adaptable and able to fine tune the UPR signaling pathway in response to stress. Second, different cells have varying proteostasis capacities reflected in the composition and concentrations of their proteostasis components. Third, within a given cell type, the proteostasis does not possess significant excess capability, rater it is finely tuned and offers just enough facility for the protein folding load. Therefore, by setting the proteostasis boundary as a threshold for generating folded and functional proteins, the proteostasis network can create and maintain functional proteins in response to the local environment

Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90346/1/j.1538-7836.2011.04545.x.pd

Expression of Mutant or Cytosolic PrP in Transgenic Mice and Cells Is Not Associated with Endoplasmic Reticulum Stress or Proteasome Dysfunction

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the UbG76V-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases

Gabapentin as add-on to morphine for severe neuropathic or mixed pain in children from age 3 months to 18 years - Evaluation of the safety, pharmacokinetics, and efficacy of a new gabapentin liquid formulation: Study protocol for a randomized controlled trial

Background: Gabapentin has shown efficacy in the treatment of chronic neuropathic or mixed pain in adults. Although pediatric pain specialists have extensive experience with gabapentin for the treatment of neuropathic pain, its use is off-label. Its efficacy and safety in this context have never been shown. The aim of this trial is to compare gabapentin with placebo as add-on to morphine for the treatment of severe chronic mixed or neuropathic pain in children. This trial is part of the European Union Seventh Framework Programme project Gabapentin in Paediatric Pain (GAPP) to develop a pediatric use marketing authorization for a new gabapentin suspension. Methods/design: The GAPP-2 study is a randomized, double-blind, placebo-controlled, multicenter superiority phase II study in children with severe chronic neuropathic or mixed pain. Its primary objective is to evaluate the efficacy of a gabapentin liquid formulation as adjunctive therapy to morphine. Sixty-six eligible children 3 months to 18 years of age with severe pain (pain scores ≥ 7), stratified in three age groups, will be randomized to receive gabapentin (to an accumulating dose of 45 to 63 mg/kg/day, dependent on age) or placebo, both in addition to morphine, for 12 weeks. Randomization will be preceded by a short washout period, and treatment will be initiated by a titration period of 3 weeks. After the treatment period, medication will be tapered during 4 weeks. The primary endpoint is the average pain scores in the two treatment groups (average of two measures each day for 3 days before the end-of-study visit [V10] assessed by age-appropriate pain scales (Face, Legs, Activity, Cry, Consolability scale; Faces Pain Scale-Revised; Numeric Rating Scale). Secondary outcomes include percentage responders to treatment (subjects with 30% reduction in pain scale), number of episodes of breakthrough pain, number of rescue interventions, number of pain-free days, participant dropouts, quality of life (Pediatric Quality of Life Inventory), and acceptability of treatment. Outcomes will be measured at the end-of-study visit after 12 weeks of treatment at the optimal gabapentin dose. Groups will be compared on an intention-to-treat basis. Discussion: We hope to provide evidence that the combination of morphine and gabapentin will provide better analgesia than morphine alone and will be safe. We also aim to obtain confirmation of the recommended pediatric dose. Trial registration: EudractCT, 2014-004897-40. Registered on 7 September 2017. ClinicalTrials.gov, NCT03275012. Registered on 7 September 2017

Diagnosis and Treatment of Chronic Neuropathic and Mixed Pain in Children and Adolescents: Results of a Survey Study amongst Practitioners

Validated diagnostic tools to diagnose chronic neuropathic and mixed pain in children are missing. Therapeutic options are often derived from therapeutics for adults. To investigate the international practice amongst practitioners for the diagnosis and treatment of chronic, neuropathic pain in children and adolescents, we performed a survey study among members of learned societies or groups whose members are known to treat pediatric pain. The survey included questions concerning practitioners and practice characteristics, assessment and diagnosis, treatment and medication. We analyzed 117 returned questionnaires, of which 41 (35%) were fully completed and 76 (65%) were partially completed. Most respondents based the diagnosis of neuropathic pain on physical examination (68 (58.1%)), patient history (67 (57.3%)), and underlying disease (59 (50.4%)) combined. Gabapentin, amitriptyline, and pregabalin were the first-choice treatments for moderate neuropathic pain. Tramadol, ibuprofen, amitriptyline, and paracetamol were the first-choice treatments for moderate mixed pain. Consensus on the diagnostic process of neuropathic pain in children and adolescents is lacking. Drug treatment varies widely for moderate, severe neuropathic, and mixed pain. Hence, diagnostic tools and therapy need to be harmonized and validated for use in children

The research gap in chronic paediatric pain: A systematic review of randomised controlled trials

Background and Objective: Chronic pain is associated with significant functional and social impairment. The objective of this review was to assess the characteristics and quality of randomized controlled trials (RCTs) evaluating pain management interventions in children and adolescents with chronic pain. Methods: We performed a systematic search of PubMed, Embase and the Cochrane Library up to July 2017. We included RCTs that involved children and adolescents (3 months-18 years) and evaluated the use of pharmacological or non-pharmacological intervention(s) in the context of pain persisting or re-occurring for more than 3 months. Methodological quality was evaluated using the Cochrane Risk of Bias (ROB) Tool. Results: A total of 58 RCTs were identified and numbers steadily increased over time. The majority were conducted in single hospital institutions, with no information on study funding. Median sample size was 47.5 participants (Q1,Q3: 32, 70). Forty-five percent of RCTs included both adults and children and the median of the mean ages at inclusion was 12.9 years (Q1,Q3: 11, 15). Testing of non-pharmacological interventions was predominant and only 5 RCTs evaluated analgesics or co-analgesics. Abdominal pain, headache/migraine and musculoskeletal pain were the most common types of chronic pain among participants. Methodological quality was poor with 90% of RCTs presenting a high or unclear ROB. Conclusions: Evaluation of analgesics targeting chronic pain relief in children and adolescents through RCTs is marginal. Infants and children with long-lasting painful conditions are insufficiently represented in RCTs. We discuss possible research constraints and challenges as well as methodologies to circumvent them. Significance: There is a substantial research gap regarding analgesic interventions for children and adolescents with chronic pain. Most clinical trials in the field focus on the evaluation of non-pharmacological interventions and are of low methodological quality. There is also a specific lack of trials involving infants and children and adolescents with long-lasting diseases

In vivo up-regulation of the unfolded protein response after hypoxia

BACKGROUND: Low oxygen (O(2)) availability, a condition called hypoxia, has different and profound consequences in tissues and organs. Besides the hypoxia-inducible response, mammalian cells induce a coordinated cytoprotective pathway called Unfolded Protein Response (UPR). We studied the molecular basis of UPR and apoptosis in animal models exposed to different hypoxic stresses and assessed the ability of liver and myocardium to respond to low oxygen by activating different arms of the UPR according to the severity of the insults in a tissue specific manner. METHODS: We assessed the levels of several UPR markers in hypoxic animals by Real Time PCR and Western blotting. RESULTS: While the hepatocytes activate the apoptotic pathway mediated, in part, by CHOP and p-JNK, we could not detect an UPR-dependent apoptosis in myocytes. Moreover, severe hypoxia results in ATF4 translation, and induction of CHOP and GADD34 transcripts in liver, by contrast in the myocardium, the ATF4-CHOP-GADD34 signaling pathway is not detectably activated. GENERAL SIGNIFICANCE: Comparison of several UPR markers in liver and myocardium enabled to underscore the ability of hepatocytes and myocites to selectively activate and fine tune the UPR signaling pathway during hypoxia in vivo