Identification of the Drosophila Ortholog of HSPB8: IMPLICATION OF HSPB8 LOSS OF FUNCTION IN PROTEIN FOLDING DISEASES*

Protein aggregation is a hallmark of many neuronal disorders, including the polyglutamine disorder spinocerebellar ataxia 3 and peripheral neuropathies associated with the K141E and K141N mutations in the small heat shock protein HSPB8. In cells, HSPB8 cooperates with BAG3 to stimulate autophagy in an eIF2α-dependent manner and facilitates the clearance of aggregate-prone proteins (Carra, S., Seguin, S. J., Lambert, H., and Landry, J. (2008) J. Biol. Chem. 283, 1437–1444; Carra, S., Brunsting, J. F., Lambert, H., Landry, J., and Kampinga, H. H. (2009) J. Biol. Chem. 284, 5523–5532). Here, we first identified Drosophila melanogaster HSP67Bc (Dm-HSP67Bc) as the closest functional ortholog of human HSPB8 and demonstrated that, like human HSPB8, Dm-HSP67Bc induces autophagy via the eIF2α pathway. In vitro, both Dm-HSP67Bc and human HSPB8 protected against mutated ataxin-3-mediated toxicity and decreased the aggregation of a mutated form of HSPB1 (P182L-HSPB1) associated with peripheral neuropathy. Up-regulation of both Dm-HSP67Bc and human HSPB8 protected and down-regulation of endogenous Dm-HSP67Bc significantly worsened SCA3-mediated eye degeneration in flies. The K141E and K141N mutated forms of human HSPB8 that are associated with peripheral neuropathy were significantly less efficient than wild-type HSPB8 in decreasing the aggregation of both mutated ataxin 3 and P182L-HSPB1. Our current data further support the link between the HSPB8-BAG3 complex, autophagy, and folding diseases and demonstrate that impairment or loss of function of HSPB8 might accelerate the progression and/or severity of folding diseases.     

Scaffold hopping approach towards various AFQ-056 analogs as potent metabotropic glutamate receptor 5 negative allosteric modulators

The metabotropic glutamate receptor subtype 5 has evolved into a promising target for the treatment of various diseases of the central nervous system, such as Fragile X and l-DOPA induced dyskinesia. One of the most advanced clinical compound is Novartis’ AFQ-056 (Mavoglurant), which served us as a template for a scaffold hopping approach, generating a structurally diverse set of potent analogs. Both the limited aqueous solubility and the relatively poor metabolic stability of AFQ-056 were improved with hexahydrocyclopenta[c]pyrrole derivative 54a, which proved to be a valuable candidate for further development.     

Placental Expression of miR-517a/b and miR-517c Contributes to Trophoblast Dysfunction and Preeclampsia

Preeclampsia is a pregnancy specific hypertensive disease that confers significant maternal and fetal risks. While the exact pathophysiology of preeclampsia is unknown, it is widely accepted that placental dysfunction is mechanistically involved. Recent studies reported aberrant expression of placenta-specific microRNAs (miRNAs) in preeclampsia including miR-517a/b and miR-517c. Using placental biopsies from a preeclampsia case-control study, we found increased expression of miR-517a/b in term and preterm preeclampsia vs controls, while, miR-517c was increased only in preterm preeclampsia vs controls. To determine if miR-517a/b and miR-517c are regulated by hypoxia, we treated first trimester primary extravillous trophoblast cells (EVTs) with a hypoxia mimetic and found both were induced. To test for a mechanistic role in placental function, we overexpressed miR-517a/b or miR-517c in EVTs which resulted in decreased trophoblast invasion. Additionally, we found that miR-517a/b and miR-517c overexpression increased expression of the anti-angiogenic protein, sFLT1. The regulation of sFLT1 is mostly unknown, however, TNFSF15, a cytokine involved in FLT1 splicing, was also increased by miR-517a/b and miR-517c in EVTs. In summary, we demonstrate that miR-517a/b and miR-517c contribute to the development of preeclampsia and suggest that these miRNAs play a critical role in regulating trophoblast and placental function.     

Questions Left Unanswered: How the Brain Responds to Missing Information

It sometimes happens that when someone asks a question, the addressee does not give an adequate answer, for instance by leaving out part of the required information. The person who posed the question may wonder why the information was omitted, and engage in extensive processing to find out what the partial answer actually means. The present study looks at the neural correlates of the pragmatic processes invoked by partial answers to questions. Two experiments are presented in which participants read mini-dialogues while their Event-Related brain Potentials (ERPs) are being measured. In both experiments, violating the dependency between questions and answers was found to lead to an increase in the amplitude of the P600 component. We interpret these P600-effects as reflecting the increased effort in creating a coherent representation of what is communicated. This effortful processing might include the computation of what the dialogue participant meant to communicate by withholding information. Our study is one of few investigating language processing in conversation, be it that our participants were ‘eavesdroppers’ instead of real interactants. Our results contribute to the as of yet small range of pragmatic phenomena that modulate the processes underlying the P600 component, and suggest that people immediately attempt to regain cohesion if a question-answer dependency is violated in an ongoing conversation.     

Right atrial pressure affects the interaction between lung mechanics and right ventricular function in spontaneously breathing COPD patients

Introduction

It is generally known that positive pressure ventilation is associated with impaired venous return and decreased right ventricular output, in particular in patients with a low right atrial pressure and relative hypovolaemia. Altered lung mechanics have been suggested to impair right ventricular output in COPD, but this relation has never been firmly established in spontaneously breathing patients at rest or during exercise, nor has it been determined whether these cardiopulmonary interactions are influenced by right atrial pressure.

Methods

Twenty-one patients with COPD underwent simultaneous measurements of intrathoracic, right atrial and pulmonary artery pressures during spontaneous breathing at rest and during exercise. Intrathoracic pressure and right atrial pressure were used to calculate right atrial filling pressure. Dynamic changes in pulmonary artery pulse pressure during expiration were examined to evaluate changes in right ventricular output.

Results

Pulmonary artery pulse pressure decreased up to 40% during expiration reflecting a decrease in stroke volume. The decline in pulse pressure was most prominent in patients with a low right atrial filling pressure. During exercise, a similar decline in pulmonary artery pressure was observed. This could be explained by similar increases in intrathoracic pressure and right atrial pressure during exercise, resulting in an unchanged right atrial filling pressure.

Conclusions

We show that in spontaneously breathing COPD patients the pulmonary artery pulse pressure decreases during expiration and that the magnitude of the decline in pulmonary artery pulse pressure is not just a function of intrathoracic pressure, but also depends on right atrial pressure.     

Individual variations in maternal care early in life correlate with later life decision-making and c-fos expression in prefrontal subregions of rats

Early life adversity affects hypothalamus-pituitary-adrenal axis activity, alters cognitive functioning and in humans is thought to increase the vulnerability to psychopathology--e.g. depression, anxiety and schizophrenia--later in life. Here we investigated whether subtle natural variations among individual rat pups in the amount of maternal care received, i.e. differences in the amount of licking and grooming (LG), correlate with anxiety and prefrontal cortex-dependent behavior in young adulthood. Therefore, we examined the correlation between LG received during the first postnatal week and later behavior in the elevated plus maze and in decision-making processes using a rodent version of the Iowa Gambling Task (rIGT). In our cohort of male and female animals a high degree of LG correlated with less anxiety in the elevated plus maze and more advantageous choices during the last 10 trials of the rIGT. In tissue collected 2 hrs after completion of the task, the correlation between LG and c-fos expression (a marker of neuronal activity) was established in structures important for IGT performance. Negative correlations existed between rIGT performance and c-fos expression in the lateral orbitofrontal cortex, prelimbic cortex, infralimbic cortex and insular cortex. The insular cortex correlations between c-fos expression and decision-making performance depended on LG background; this was also true for the lateral orbitofrontal cortex in female rats. Dendritic complexity of insular or infralimbic pyramidal neurons did not or weakly correlate with LG background. We conclude that natural variations in maternal care received by pups may significantly contribute to later-life decision-making and activity of underlying brain structures.     

Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli

We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W 3110. While these proteins were distributed randomly at the growth temperature (37 °C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 °C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes.The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction.Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while d-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes.These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.     

Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise

This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise.