Redox homeostasis and age-related deficits in neuromuscular integrity and function

Skeletal muscle is a major site of metabolic activity and is the most abundant tissue in the human body. Age-related muscle atrophy (sarcopenia) and weakness, characterized by progressive loss of lean muscle mass and function, is a major contributor to morbidity and has a profound effect on the quality of life of older people. With a continuously growing older population (estimated 2 billion of people aged >60 by 2050), demand for medical and social care due to functional deficits, associated with neuromuscular ageing, will inevitably increase. Despite the importance of this ‘epidemic’ problem, the primary biochemical and molecular mechanisms underlying age-related deficits in neuromuscular integrity and function have not been fully determined. Skeletal muscle generates reactive oxygen and nitrogen species (RONS) from a variety of subcellular sources, and age-associated oxidative damage has been suggested to be a major factor contributing to the initiation and progression of muscle atrophy inherent with ageing. RONS can modulate a variety of intracellular signal transduction processes, and disruption of these events over time due to altered redox control has been proposed as an underlying mechanism of ageing. The role of oxidants in ageing has been extensively examined in different model organisms that have undergone genetic manipulations with inconsistent findings. Transgenic and knockout rodent studies have provided insight into the function of RONS regulatory systems in neuromuscular ageing. This review summarizes almost 30 years of research in the field of redox homeostasis and muscle ageing, providing a detailed discussion of the experimental approaches that have been undertaken in murine models to examine the role of redox regulation in age-related muscle atrophy and weakness

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.RCUK | Biotechnology and Biological Sciences Research Council (BBSRC): Lu Zhao, Cahir J O'Kane, BB/L021706/1; Wellcome: Martin Stofanko, Cahir J O'Kane, 08136; European Commission (EC): Lu Zhao, Niamh C O'Sullivan, Sophie Zaessinger, Olivier Blard, MCSA fellowships 220851,220874,236777,660516; Yousef Jameel Foundation: Belgin Yalçın; Singapore A*STAR Scholarship: Zi Han Kang, BM/RES/07/005; Cambridge Commonwealth, European and International Trust (Cambridge Commonwealth, European & International Trust): Belgin Yalçın, Anood Sohail; Pakistan Higher Education Council Scholarship: Anood Sohail; Motor Neurone Disease Association (MNDA): Alex L Patto, Studentship 861-792 The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

Regulation of Hemocytes in Drosophila Requires dappled Cytochrome b5

A major category of mutant hematopoietic phenotypes in Drosophila is melanotic tumors or nodules, which consist of abnormal and overproliferated blood cells, similar to granulomas. Our analyses of the melanotic mutant dappled have revealed a novel type of gene involved in blood cell regulation. The dappled gene is an essential gene that encodes cytochrome b5, a conserved hemoprotein that participates in electron transfer in multiple biochemical reactions and pathways. Viable mutations of dappled cause melanotic nodules and hemocyte misregulation during both hematopoietic waves of development. The sexes are similarly affected, but hemocyte number is different in females and males of both mutants and wild type. Additionally, initial tests show that curcumin enhances the dappled melanotic phenotype and establish screening of endogenous and xenobiotic compounds as a route for analysis of cytochrome b5 function. Overall, dappled provides a tractable genetic model for cytochrome b5, which has been difficult to study in higher organisms

Simple, Rapid and Inexpensive Quantitative Fluorescent PCR Method for Detection of Microdeletion and Microduplication Syndromes

<div><p>Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (<i>MECP2</i> duplication) syndrome and the 17q11.2 microduplication involving the <i>NF1</i> gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well.</p></div

MQF-PCR results for patients with microdeletion and microduplication syndromes.

<p>MQF-PCR results for patients with microdeletion and microduplication syndromes.</p

Research data supporting "Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins"

The supporting data include sequence analyses, microscopy, electron microscopy, quantitative analyses and reconstructions of images, and statistical analyses, for each figure presented in the paper.Listed in 10.7554/eLife.2388

Schematic overview of MQF-PCR primer selection for Williams-Beuren and Velocardiofacial syndromes critical regions (not drawn to scale).

<p>A) Williams-Beuren critical region at 7q11.23 contains centromeric (white arrow), middle (grey arrow), and telomeric (black arrow) blocks of low copy repeats. The minimal critical region in this study represents the region that is flanked by the inner block of low copy repeats. Sequence similarity search identified a region that can be amplified using the LIMK1-MQF primers (bold) and has significant similarity to homologous region at 18p11.32. B) Multiple sequence alignment of the LIMK1-MQF region with its homologous region on chromosome 18. A deletion of 2 bp (dashed box) differentiates the two fragments amplified using the same primer pair. C) Velocardiofacial critical region at 22q11.2 is delimited by 4 blocks of low copy repeats. The minimal critical region in this study represents the region that is flanked by two blocks of low copy repeats most proximal to the centromere (black boxes). Sequence similarity search identified a region within the critical region that can be amplified using the VCF-MQF primers (bold) with significant similarity to a homologous region at 3p11.1. D) Multiple sequence alignment of the VCF-MQF region with its homologous region on chromosome 3. A deletion of 5 bp (dashed box) differentiates the two fragments amplified using the same primer pair. Microsatellite markers (italicized) and Real-Time PCR primers used in molecular diagnosis of patients are shown in both panels.</p

Evaluation of MQF-PCR in detection of Williams-Beuren and Velocardiofacial syndromes.

<p>A–D) Representative electropherograms showing the peak areas corresponding to the syndrome-related chromosomes (black) that are reduced by about 50% in comparison with the peaks representing the control chromosomes (white) between controls and affected individuals. Electropherogram depicting change in peak area between chromosome 7 and its control chromosome in normal (A) and individual with WBS syndrome (B). Electropherogram depicting change of the peak area between chromosome 22 and its control chromosome in normal (C) and individual with VCF syndrome (D). E–F) Interactive dot diagrams of ROC curve analysis of Z scores in WBS (E), and PZ scores in VCF syndrome (F). Both diagnostic primers achieved 100% diagnostic sensitivity and 100% diagnostic specificity. The number of cases analyzed and the detection threshold values for both syndromes are given.</p

MQF-PCR primers used in detection of microdeletion and microduplication syndromes.

a<p>Universal M13-40 extension (5′-GTTTTCCCAGTCACGAC-3′) was added to the 5′ end of the primer to allow for cost-efficient fluorescent labelling of amplicons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061328#pone.0061328-Schuelke1" target="_blank">[31]</a>.</p>b<p>A PIG-tail extension (5′-GTTTCTT-3′) was added to the 5′ end of the primer to promote full adenylation of the 3′ end of the forward strand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061328#pone.0061328-Brownstein1" target="_blank">[32]</a>.</p

Detection of the Lubs (<i>MECP2</i> duplication) and 17q11.2 microduplication syndromes.

<p>Representative electropherograms showing changes in the peak area ratios between a control sample (A) and a patient (B) in diagnosis of the Lubs syndrome. The peak area corresponding to the duplicated region on chromosome Xq28 (black) has significantly increased in size compared to its control region on chromosome 5p13.3 (white). Electropherogram depicting change of the peak area between chromosome 17 and its control chromosome in normal (C) and individual with microduplication of 17q11.2 (D) The peak area corresponding to the duplicated region on chromosome 17 (black) has significantly increased in size compared to its control region on chromosome 13q12.11 (white). E–F) Interactive dot diagrams of ROC curve analysis of Z scores in Lubs (E), and 17q11.2 microduplication syndrome (F). Both diagnostic primers achieved 100% diagnostic sensitivity and 100% diagnostic specificity. The number of cases analyzed and the detection threshold values for both syndromes are given.</p