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

Cytotoxic CD8⁺ T lymphocytes expressing ALS-causing SOD1 mutant selectively trigger death of spinal motoneurons.

Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis (ALS). CD4 &lt;sup&gt;+&lt;/sup&gt; T cells that regulate a protective immunity during the neurodegenerative process have received the most attention. CD8 &lt;sup&gt;+&lt;/sup&gt; T cells are also observed in the spinal cord of patients and ALS mice although their contribution to the disease still remains elusive. Here, we found that activated CD8 &lt;sup&gt;+&lt;/sup&gt; T lymphocytes infiltrate the central nervous system (CNS) of a mouse model of ALS at the symptomatic stage. Selective ablation of CD8 &lt;sup&gt;+&lt;/sup&gt; T cells in mice expressing the ALS-associated superoxide dismutase-1 (SOD1) &lt;sup&gt;G93A&lt;/sup&gt; mutant decreased spinal motoneuron loss. Using motoneuron-CD8 &lt;sup&gt;+&lt;/sup&gt; T cell coculture systems, we found that mutant SOD1-expressing CD8 &lt;sup&gt;+&lt;/sup&gt; T lymphocytes selectively kill motoneurons. This cytotoxicity activity requires the recognition of the peptide-MHC-I complex (where MHC-I represents major histocompatibility complex class I). Measurement of interaction strength by atomic force microscopy-based single-cell force spectroscopy demonstrated a specific MHC-I-dependent interaction between motoneuron and SOD1 &lt;sup&gt; G93A &lt;/sup&gt; CD8 &lt;sup&gt;+&lt;/sup&gt; T cells. Activated mutant SOD1 CD8 &lt;sup&gt;+&lt;/sup&gt; T cells produce interferon-γ, which elicits the expression of the MHC-I complex in motoneurons and exerts their cytotoxic function through Fas and granzyme pathways. In addition, analysis of the clonal diversity of CD8 &lt;sup&gt;+&lt;/sup&gt; T cells in the periphery and CNS of ALS mice identified an antigen-restricted repertoire of their T cell receptor in the CNS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS

Odontodes mineralization in chondrichthyan fishes: insights in the development of early vertebrate oral and dermal skeleton

International audienc

Odontodes mineralization in chondrichthyan fishes: insights in the development of early vertebrate oral and dermal skeleton

International audienc

Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates

International audienceIn order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton

Oxidative Stress Plays an Important Role in Glutamatergic Excitotoxicity-Induced Cochlear Synaptopathy: Implication for Therapeutic Molecules Screening

International audienceThe disruption of the synaptic connection between the sensory inner hair cells (IHCs) and the auditory nerve fiber terminals of the type I spiral ganglion neurons (SGN) has been observed early in several auditory pathologies (e.g., noise-induced or ototoxic drug-induced or age-related hearing loss). It has been suggested that glutamate excitotoxicity may be an inciting element in the degenerative cascade observed in these pathological cochlear conditions. Moreover, oxidative damage induced by free hydroxyl radicals and nitric oxide may dramatically enhance cochlear damage induced by glutamate excitotoxicity. To investigate the underlying molecular mechanisms involved in cochlear excitotoxicity, we examined the molecular basis responsible for kainic acid (KA, a full agonist of AMPA/KA-preferring glutamate receptors)-induced IHC synapse loss and degeneration of the terminals of the type I spiral ganglion afferent neurons using a cochlear explant culture from P3 mouse pups. Our results demonstrated that disruption of the synaptic connection between IHCs and SGNs induced increased levels of oxidative stress, as well as altered both mitochondrial function and neurotrophin signaling pathways. Additionally, the application of exogenous antioxidants and neurotrophins (NT3, BDNF, and small molecule TrkB agonists) clearly increases synaptogenesis. These results suggest that understanding the molecular pathways involved in cochlear excitotoxicity is of crucial importance for the future clinical trials of drug interventions for auditory synaptopathies

Oxidative Stress Plays an Important Role in Glutamatergic Excitotoxicity-Induced Cochlear Synaptopathy: Implication for Therapeutic Molecules Screening

The disruption of the synaptic connection between the sensory inner hair cells (IHCs) and the auditory nerve fiber terminals of the type I spiral ganglion neurons (SGN) has been observed early in several auditory pathologies (e.g., noise-induced or ototoxic drug-induced or age-related hearing loss). It has been suggested that glutamate excitotoxicity may be an inciting element in the degenerative cascade observed in these pathological cochlear conditions. Moreover, oxidative damage induced by free hydroxyl radicals and nitric oxide may dramatically enhance cochlear damage induced by glutamate excitotoxicity. To investigate the underlying molecular mechanisms involved in cochlear excitotoxicity, we examined the molecular basis responsible for kainic acid (KA, a full agonist of AMPA/KA-preferring glutamate receptors)-induced IHC synapse loss and degeneration of the terminals of the type I spiral ganglion afferent neurons using a cochlear explant culture from P3 mouse pups. Our results demonstrated that disruption of the synaptic connection between IHCs and SGNs induced increased levels of oxidative stress, as well as altered both mitochondrial function and neurotrophin signaling pathways. Additionally, the application of exogenous antioxidants and neurotrophins (NT3, BDNF, and small molecule TrkB agonists) clearly increases synaptogenesis. These results suggest that understanding the molecular pathways involved in cochlear excitotoxicity is of crucial importance for the future clinical trials of drug interventions for auditory synaptopathies

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