A SAGE-based screen for genes expressed in sub-populations of neurons in the mouse dorsal root ganglion

<p>Abstract</p> <p>Background</p> <p>The different sensory modalities temperature, pain, touch and muscle proprioception are carried by somatosensory neurons of the dorsal root ganglia. Study of this system is hampered by the lack of molecular markers for many of these neuronal sub-types. In order to detect genes expressed in sub-populations of somatosensory neurons, gene profiling was carried out on wild-type and TrkA mutant neonatal dorsal root ganglia (DRG) using SAGE (serial analysis of gene expression) methodology. Thermo-nociceptors constitute up to 80 % of the neurons in the DRG. In TrkA mutant DRGs, the nociceptor sub-class of sensory neurons is lost due to absence of nerve growth factor survival signaling through its receptor TrkA. Thus, comparison of wild-type and TrkA mutants allows the identification of transcripts preferentially expressed in the nociceptor or mechano-proprioceptor subclasses, respectively.</p> <p>Results</p> <p>Our comparison revealed 240 genes differentially expressed between the two tissues (P < 0.01). Some of these genes, CGRP, Scn10a are known markers of sensory neuron sub-types. Several potential markers of sub-populations, Dok4, Crip2 and Grik1/GluR5 were further analyzed by quantitative RT-PCR and double labeling with TrkA,-B,-C, c-ret, parvalbumin and isolectin B4, known markers of DRG neuron sub-types. Expression of Grik1/GluR5 was restricted to the isolectin B4+ nociceptive population, while Dok4 and Crip2 had broader expression profiles. Crip2 expression was however excluded from the proprioceptor sub-population.</p> <p>Conclusion</p> <p>We have identified and characterized the detailed expression patterns of three genes in the developing DRG, placing them in the context of the known major neuronal sub-types defined by molecular markers. Further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities.</p

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants

Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). GL is supported by the Agence Nationale de la Recherche (Paris, France) Program Grant CHOPIN (CHolesterol Personalized Innovation) ANR-16-RHUS-0007 and Project Grant KRINGLE2 ANR-20-CE14-0009 as well as by La Fondation De France (FDF-00096274). U.G-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019-2020. A.L.-S. was supported by a grant PIF (2019–2020), Gobierno Vasco, and partially supported by Fundación Biofísica Bizkaia. KC and AKJ received a scholarship from the European Union (European Regional Development Fund INTERREG V) and the Région Réunion (Saint-Denis, Réunion, France)

Regulation of the Na,K-ATPase Gamma-Subunit FXYD2 by Runx1 and Ret Signaling in Normal and Injured Non-Peptidergic Nociceptive Sensory Neurons

Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons which relay nociceptive, thermoceptive, mechanoceptive and proprioceptive information from peripheral tissues toward the central nervous system. These neurons establish constant communication with their targets which insures correct maturation and functioning of the somato-sensory nervous system. Interfering with this two-way communication leads to cellular, electrophysiological and molecular modifications that can eventually cause neuropathic conditions. In this study we reveal that FXYD2, which encodes the gamma-subunit of the Na,K-ATPase reported so far to be mainly expressed in the kidney, is induced in the mouse DRGs at postnatal stages where it is restricted specifically to the TrkB-expressing mechanoceptive and Ret-positive/IB4-binding non-peptidergic nociceptive neurons. In non-peptidergic nociceptors, we show that the transcription factor Runx1 controls FXYD2 expression during the maturation of the somato-sensory system, partly through regulation of the tyrosine kinase receptor Ret. Moreover, Ret signaling maintains FXYD2 expression in adults as demonstrated by the axotomy-induced down-regulation of the gene that can be reverted by in vivo delivery of GDNF family ligands. Altogether, these results establish FXYD2 as a specific marker of defined sensory neuron subtypes and a new target of the Ret signaling pathway during normal maturation of the non-peptidergic nociceptive neurons and after sciatic nerve injury

Identification et caractérisation de gènes exprimés dans les sous-populations de neurones sensoriels des ganglions rachidiens dorsaux

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions

Introduction: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. Methods: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood–brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. Results: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity

Gene expression determined by real-time PCR on P0 and P0 TrkA mutant mouse lumbar DRG

<p><b>Copyright information:</b></p><p>Taken from "A SAGE-based screen for genes expressed in sub-populations of neurons in the mouse dorsal root ganglion"</p><p>http://www.biomedcentral.com/1471-2202/8/97</p><p>BMC Neuroscience 2007;8():97-97.</p><p>Published online 19 Nov 2007</p><p>PMCID:PMC2241628.</p><p></p> TrkA and Ube2e3 (ubiquitin-conjugating enzyme E2E 3) were used as controls. Data (means ± SEM) were calculated by the delta-CT method [37] on three independent experimental replicates. The arithmetic means of the expression levels of two genes (Polr2j, Ddx48) whose expression do not change in the course of development and in TrkA-/- DRG were used to normalize the expression levels. Data were analyzed using the Mann Whitney U-test (*P < 0.05). ND: Not detected

Double in situ hybridization was carried out by using fluorescein/fast red detection for TrkA (A, E), TrkB (B, F), TrkC (C, G), c-ret (D, H) and DIG/NBT-BCIP for Dok4 (A, B, C, D) and Crip2 (E, F, G, H)

<p><b>Copyright information:</b></p><p>Taken from "A SAGE-based screen for genes expressed in sub-populations of neurons in the mouse dorsal root ganglion"</p><p>http://www.biomedcentral.com/1471-2202/8/97</p><p>BMC Neuroscience 2007;8():97-97.</p><p>Published online 19 Nov 2007</p><p>PMCID:PMC2241628.</p><p></p> In situ signals were converted into pseudo colors and images were superimposed to show co-labelling of cells. Dok4 co-localised with all major subtypes of DRG neurons, Crip2 was specifically excluded from TrkC population. Scale bar 50 μm