PDGF-BB serum levels are decreased in adult onset Pompe patients

Adult onset Pompe disease is a genetic disorder characterized by slowly progressive skeletal and respiratory muscle weakness. Symptomatic patients are treated with enzymatic replacement therapy with human recombinant alfa glucosidase. Motor functional tests and spirometry are commonly used to follow patients up. However, a serological biomarker that correlates with the progression of the disease could improve follow-up. We studied serum concentrations of TGFβ, PDGF-BB, PDGF-AA and CTGF growth factors in 37 adult onset Pompe patients and 45 controls. Moreover, all patients performed several muscle function tests, conventional spirometry, and quantitative muscle MRI using 3-point Dixon. We observed a statistically significant change in the serum concentration of each growth factor in patients compared to controls. However, only PDGF-BB levels were able to differentiate between asymptomatic and symptomatic patients, suggesting its potential role in the follow-up of asymptomatic patients. Moreover, our results point to a dysregulation of muscle regeneration as an additional pathomechanism of Pompe disease

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Interaction between G proteins and accessory β subunits in the regulation of α1B calcium channels in Xenopus oocytes

The accessory β subunits of voltage-dependent Ca2+ channels (VDCCs) have been shown to regulate their biophysical properties and have also been suggested to antagonise the G protein inhibition of N-type (α1B), P/Q-type (α1A) and α1E channels. Here we have examined the voltage-dependent involvement of the four neuronal isoforms (β1b, β2a, β3 and β4) in the process of G protein modulation of α1B Ca2+ channels.All β subunits hyperpolarised α1B current activation, and all antagonised the G protein-mediated depolarisation of current activation. However, except in the case of β2a, there was no generalised reduction by β subunits in the maximal extent of receptor-mediated inhibition of α1B current.In addition, all VDCC β subunits enhanced the rate of current facilitation at +100 mV, for both receptor-mediated and tonic modulation. The rank order for enhancement of facilitation rate was β3 > β4 > β1b > β2a. In contrast, the amount of voltage-dependent facilitation during tonic modulation was reduced by β subunit co-expression, despite the fact that the apparent Gβγ dissociation rate at +100 mV was enhanced by β subunits to a similar level as for agonist-induced modulation.Our data provide evidence that G protein activation antagonises Ca2+-channel β subunit-induced hyperpolarisation of current activation. Conversely, co-expression of all β subunits increases the apparent Gβγ dimer dissociation rate during a depolarising prepulse. This latter feature suggests the co-existence of bound Ca2+-channel β subunits and Gβγ dimers on the α1B subunits. Future work will determine how the interaction between Gβγ dimers and Ca2+-channel β subunits with α1B results in a functional antagonism at the molecular level

Tetralol derivative NNC-55-0396 targets hypoxic cells in the glioblastoma microenvironment: an organ-on-chip approach

Abstract Glioblastoma (GBM) is a highly malignant brain tumour characterised by limited treatment options and poor prognosis. The tumour microenvironment, particularly the central hypoxic region of the tumour, is known to play a pivotal role in GBM progression. Cells within this region adapt to hypoxia by stabilising transcription factor HIF1-α, which promotes cell proliferation, dedifferentiation and chemoresistance. In this study we sought to examine the effects of NNC-55-0396, a tetralol compound which overactivates the unfolded protein response inducing apoptosis, using the organ-on-chip technology. We identified an increased sensitivity of the hypoxic core of the chip to NNC, which correlates with decreasing levels of HIF1-α in vitro. Moreover, NNC blocks the macroautophagic process that is unleashed by hypoxia as revealed by increased levels of autophagosomal constituent LC3-II and autophagy chaperone p62/SQSTM1. The specific effects of NNC in the hypoxic microenvironment unveil additional anti-cancer abilities of this compound and further support investigations on its use in combined therapies against GBM

The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α(2)δ subunits is key to trafficking voltage-gated Ca(2)+ channels

All auxiliary α(2)δ subunits of voltage-gated Ca(2+) (Ca(V)) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α(2)δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α(2)δ-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant α(2)δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α(2)δ-2, the MIDAS mutant α(2)δ-2 subunit did not enhance and, in some cases, further diminished Ca(V)1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba(2+) or Na(+) as a permeant ion. Furthermore, expression of the MIDAS mutant α(2)δ-2 reduced surface expression and strongly increased the perinuclear retention of Ca(V)α1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α(2)δ subunits, heterologous expression of α(2)δ-2 in differentiated NG108–15 cells further enhanced the endogenous high-threshold Ca(2+) currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that α(2)δ subunits normally interact with the Ca(V)α1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α(2)δ subunit is required to promote trafficking of the α1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains