The sympathetic nervous system regulates skeletal muscle motor innervation and acetylcholine receptor stability

Aim: Symptoms of autonomic failure are frequently the presentation of advanced age and neurodegenerative diseases that impair adaptation to common physiologic stressors. The aim of this work was to examine the interaction between the sympathetic and motor nervous system, the involvement of the sympathetic nervous system (SNS) in neuromuscular junction (NMJ) presynaptic motor function, the stability of postsynaptic molecular organization, and the skeletal muscle composition and function. Methods: Since muscle weakness is a symptom of diseases characterized by autonomic dysfunction, we studied the impact of regional sympathetic ablation on muscle motor innervation by using transcriptome analysis, retrograde tracing of the sympathetic outflow to the skeletal muscle, confocal and electron microscopy, NMJ transmission by electrophysiological methods, protein analysis, and state of the art microsurgical techniques, in C57BL6, MuRF1KO and Thy-1 mice. Results: We found that the SNS regulates motor nerve synaptic vesicle release, skeletal muscle transcriptome, muscle force generated by motor nerve activity, axonal neurofilament phosphorylation, myelin thickness, and myofibre subtype composition and CSA. The SNS also modulates the levels of postsynaptic membrane acetylcholine receptor by regulating the Gα i2 -Hdac4-Myogenin-MuRF1pathway, which is prevented by the overexpression of the guanine nucleotide-binding protein Gα i2 (Q205L), a constitutively active mutant G protein subunit. Conclusion: The SNS regulates NMJ transmission, maintains optimal Gα i2 expression, and prevents any increase in Hdac4, myogenin, MuRF1, and miR-206. SNS ablation leads to upregulation of MuRF1, muscle atrophy, and downregulation of postsynaptic AChR. Our findings are relevant to clinical conditions characterized by progressive decline of sympathetic innervation, such as neurodegenerative diseases and aging.Fil: Rodrigues, Anna C. Zaia. Wake Forest School of Medicine; Estados UnidosFil: Messi, Maria Laura. Wake Forest School of Medicine; Estados UnidosFil: Wang, Zhong Min. Wake Forest School of Medicine; Estados UnidosFil: Abba, Martín Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; ArgentinaFil: Pereyra, Andrea Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Wake Forest School of Medicine; Estados UnidosFil: Birbrair, Alexander. Wake Forest School of Medicine; Estados UnidosFil: Zhang, Tan. Wake Forest School of Medicine; Estados UnidosFil: O´Meara, Meaghan. Wake Forest School of Medicine; Estados UnidosFil: Kwan, Ping. Wake Forest School of Medicine; Estados UnidosFil: Lopez, Elsa I. S.. Wake Forest School of Medicine; Estados UnidosFil: Willis, Monte S.. University of North Carolina; Estados UnidosFil: Mintz, Akiva. Wake Forest School of Medicine; Estados UnidosFil: Files, D. Clark. University of North Carolina; Estados UnidosFil: Furdui, Cristina. Wake Forest School of Medicine; Estados UnidosFil: Oppenheim, Ronald W.. Wake Forest School of Medicine; Estados UnidosFil: Delbono, Osvaldo. Wake Forest School of Medicine; Estados Unido

Initial biological evaluations of 18F-KS1, a novel ascorbate derivative to image oxidative stress in cancer

Background Reactive oxygen species (ROS)-induced oxidative stress damages many cellular components such as fatty acids, DNA, and proteins. This damage is implicated in many disease pathologies including cancer and neurodegenerative and cardiovascular diseases. Antioxidants like ascorbate (vitamin C, ascorbic acid) have been shown to protect against the deleterious effects of oxidative stress in patients with cancer. In contrast, other data indicate potential tumor-promoting activity of antioxidants, demonstrating a potential temporal benefit of ROS. However, quantifying real-time tumor ROS is currently not feasible, since there is no way to directly probe global tumor ROS. In order to study this ROS-induced damage and design novel therapeutics to prevent its sequelae, the quantitative nature of positron emission tomography (PET) can be harnessed to measure in vivo concentrations of ROS. Therefore, our goal is to develop a novel translational ascorbate-based probe to image ROS in cancer in vivo using noninvasive PET imaging of tumor tissue. The real-time evaluations of ROS state can prove critical in developing new therapies and stratifying patients to therapies that are affected by tumor ROS. Methods We designed, synthesized, and characterized a novel ascorbate derivative (E)-5-(2-chloroethylidene)-3-((4-(2-fluoroethoxy)benzyl)oxy)-4-hydroxyfuran-2(5H)-one (KS1). We used KS1 in an in vitro ROS MitoSOX-based assay in two different head and neck squamous cancer cells (HNSCC) that express different ROS levels, with ascorbate as reference standard. We radiolabeled 18F-KS1 following 18F-based nucleophilic substitution reactions and determined in vitro reactivity and specificity of 18F-KS1 in HNSCC and prostate cancer (PCa) cells. MicroPET imaging and standard biodistribution studies of 18F-KS1 were performed in mice bearing PCa cells. To further demonstrate specificity, we performed microPET blocking experiments using nonradioactive KS1 as a blocker. Results KS1 was synthesized and characterized using 1H NMR spectra. MitoSOX assay demonstrated good correlations between increasing concentrations of KS1 and ascorbate and increased reactivity in SCC-61 cells (with high ROS levels) versus rSCC-61cells (with low ROS levels). 18F-KS1 was radiolabeled with high radiochemical purity (> 94%) and specific activity (~ 100 GBq/μmol) at end of synthesis (EOS). Cell uptake of 18F-KS1 was high in both types of cancer cells, and the uptake was significantly blocked by nonradioactive KS1, and the ROS blocker, superoxide dismutase (SOD) demonstrating specificity. Furthermore, 18F-KS1 uptake was increased in PCa cells under hypoxic conditions, which have been shown to generate high ROS. Initial in vivo tumor uptake studies in PCa tumor-bearing mice demonstrated that 18F-KS1 specifically bound to tumor, which was significantly blocked (threefold) by pre-injecting unlabeled KS1. Furthermore, biodistribution studies in the same tumor-bearing mice showed high tumor to muscle (target to nontarget) ratios. Conclusion This work demonstrates the strong preliminary support of 18F-KS1, both in vitro and in vivo for imaging ROS in cancer. If successful, this work will provide a new paradigm to directly probe real-time oxidative stress levels in vivo. Our work could enhance precision medicine approaches to treat cancer, as well as neurodegenerative and cardiovascular diseases affected by ROS

Blood-Based Bioenergetic Profiling Reflects Differences in Brain Bioenergetics and Metabolism

Blood-based bioenergetic profiling provides a minimally invasive assessment of mitochondrial health shown to be related to key features of aging. Previous studies show that blood cells recapitulate mitochondrial alterations in the central nervous system under pathological conditions, including the development of Alzheimer’s disease. In this study of nonhuman primates, we focus on mitochondrial function and bioenergetic capacity assessed by the respirometric profiling of monocytes, platelets, and frontal cortex mitochondria. Our data indicate that differences in the maximal respiratory capacity of brain mitochondria are reflected by CD14+ monocyte maximal respiratory capacity and platelet and monocyte bioenergetic health index. A subset of nonhuman primates also underwent [18F] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging to assess brain glucose metabolism. Our results indicate that platelet respiratory capacity positively correlates to measures of glucose metabolism in multiple brain regions. Altogether, the results of this study provide early evidence that blood-based bioenergetic profiling is related to brain mitochondrial metabolism. While these measures cannot substitute for direct measures of brain metabolism, provided by measures such as FDG-PET, they may have utility as a metabolic biomarker and screening tool to identify individuals exhibiting systemic bioenergetic decline who may therefore be at risk for the development of neurodegenerative diseases

Metabolic networking in Brunfelsia calycina petals after flower opening

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography–quadrupole time of flight–tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species

Antiviral therapy: Valacyclovir Treatment of Alzheimer’s Disease (VALAD) Trial: protocol for a randomised, double-blind, placebo-controlled, treatment trial

Mechanisms underlying resilience in ageing Y Stern, G Chételat, C Habeck, EM Arenaza-Urquijo, P Vemuri, A Estanga, ...Nature Reviews Neuroscience 20 (4), 246-24

The sympathetic nervous system regulates skeletal muscle motor innervation and acetylcholine receptor stability

Aim: Symptoms of autonomic failure are frequently the presentation of advanced age and neurodegenerative diseases that impair adaptation to common physiologic stressors. The aim of this work was to examine the interaction between the sympathetic and motor nervous system, the involvement of the sympathetic nervous system (SNS) in neuromuscular junction (NMJ) presynaptic motor function, the stability of postsynaptic molecular organization, and the skeletal muscle composition and function. Methods: Since muscle weakness is a symptom of diseases characterized by autonomic dysfunction, we studied the impact of regional sympathetic ablation on muscle motor innervation by using transcriptome analysis, retrograde tracing of the sympathetic outflow to the skeletal muscle, confocal and electron microscopy, NMJ transmission by electrophysiological methods, protein analysis, and state of the art microsurgical techniques, in C57BL6, MuRF1KO and Thy-1 mice. Results: We found that the SNS regulates motor nerve synaptic vesicle release, skeletal muscle transcriptome, muscle force generated by motor nerve activity, axonal neurofilament phosphorylation, myelin thickness, and myofibre subtype composition and CSA. The SNS also modulates the levels of postsynaptic membrane acetylcholine receptor by regulating the Gαi2 -Hdac4-Myogenin-MuRF1pathway, which is prevented by the overexpression of the guanine nucleotide-binding protein Gαi2 (Q205L), a constitutively active mutant G protein subunit. Conclusion: The SNS regulates NMJ transmission, maintains optimal Gαi2 expression, and prevents any increase in Hdac4, myogenin, MuRF1, and miR-206. SNS ablation leads to upregulation of MuRF1, muscle atrophy, and downregulation of postsynaptic AChR. Our findings are relevant to clinical conditions characterized by progressive decline of sympathetic innervation, such as neurodegenerative diseases and aging.Centro de Investigaciones Inmunológicas Básicas y Aplicada

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Interleukin 13 Mutants of Enhanced Avidity Toward the Glioma-Associated Receptor, IL13Rα2

Interleukin 13 (IL13) binds a receptor that is highly overexpressed in malignant gliomas, IL13Rα2. IL13 protein is composed of four helices: α-helix A, B, C, and D, and we found a new “hot spot” in α-helix D that is crucial for the binding of IL13 to IL13Rα2. Lys-105 plus Lys-106 and Arg-109 represent this hot spot. In the current study, we have made substitutions at these three positions in IL13. We examined both neutralization of an IL13-based cytotoxin's glioma cell killing and direct receptor binding of the new IL13 mutants. We observed that Lys-105 and Arg-109 are critical for IL13 binding to IL13Rα2, indeed. However, new mutants of important properties were identified with regard to tumor targeting. IL13.K105R mutant, in which lysine was substituted by arginine, neutralized the killing of IL13Rα2-positive cells by IL13-based cytotoxin more efficiently than wild-type IL13. However, IL13.K105L or IL13.K105A was deprived of any such activity. Furthermore, IL13.K105R and IL13.R109K competed 77- and 27-fold better, respectively, with the binding of [(125)I]IL13 to the IL13Rα2 binding sites when compared with wild-type IL13. Thus, we have uncovered the first forms of IL13 of higher avidity toward IL13Rα2. These mutants should prove useful in the further design of anticancer diagnostics/therapeutics