Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

International audienceIn conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia

Étude morphologique et fonctionnelle d'un modèle de dysconnexion synaptique

La schizophrénie est une maladie psychiatrique qui touche 1% de la population et qui se déclenche tardivement à l adolescence. On considère qu elle résulte d une vulnérabilité d origine génétique associée à des facteurs déclencheurs environnementaux. L hypothèse physiopathologique actuelle suppose des dysconnexions cortico-corticales, où la synapse serait l élément principalement perturbé. Nous avons recherché quelles modifications morpho-fonctionnelles auraient pu être engendrées par de telles perturbations. Pour cela, nous avons choisi comme modèle une souris transgénique, la souris KO-STOP, qui présente des anomalies synaptiques (au moins dans l hippocampe) et des troubles comportementaux lui conférant un phénotype adapté à l étude de la schizophrénie. Nous avons établi la cartographie d expression spatiale et temporelle de la protéine mutée afin de connaître la localisation et l évolution des perturbations synaptiques chez cette souris. Nos résultats montrent des expressions localisées dans des régions cérébrales qui, pour la plupart, pourraient avoir un lien avec la schizophrénie. Du fait d une expression robuste et précoce dans le système olfactif, nous nous sommes focalisés sur le glomérule, zone synaptique par excellence et de grande plasticité. Nous avons montré que des anomalies synaptiques y sont également présentes : l expression en GAP43 est réduite et des accumulations de membranes sont trouvées au niveau de l élément pré-synaptique. Ce travail a donc permis de caractériser de manière globale les conséquences des perturbations synaptiques d un microcircuit neuronal, la synapse olfactive principale, sur son microenvironnement. Ces résultats ouvrent des perspectives d études des circuits neuronaux corticaux plus complexes afin de permettre l élaboration de thérapies innovantes.Schizophrenia is a severe psychiatric illness with a lifetime prevalence of 1% and a late adolescence onset. Schizophrenia probably results from the combination of genetic vulnerability and environmental factors. The main hypothesis supposes cortico-cortical dysconnections where the synapse is the main disturbed element. We looked for the consequences of these modifications both from morphologic and functional aspects. We chose to study the transgenic KO-STOP mouse. Synaptic defects (at least in the hippocampus) and behavioral troubles confer to this mouse a good phenotype for the study of schizophrenia. We established the spatial and temporal STOP protein expression map in order to find the localization and the evolution of the synaptic perturbations. We found expressions in well delimitated cerebral regions with, for most of them, a potential link with schizophrenia. A robust and precocious expression of STOP in the olfactory system leads us to study the olfactory glomerulus. The glomerulus is a synaptic zone with a large plasticity. We also found synaptic defects in this region: GAP43 expression was reduced and membranes were abnormally accumulated in the pre-synaptic part of the synapse. Thus, in this work, we characterized the consequences of the synaptic defects on the micro-environment of the main olfactory synapse, which could be considered as a neuronal micro-circuit. These results offer research perspectives on more complex cortical neuronal circuits in order to find out new therapeutic strategies.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Glomerular structure after regeneration in 10-month-old mice.

<p>Localisation of Vglut2 (A–D) and OMP (E–H) immunolabelling in olfactory bulb glomeruli of WT (A, C, E, G) and STOP null (B, D, F H) mice in controls (A, B, E, F) and after regeneration (C, D, G, H). Note the characteristic feature of a mosaicism between Vglut2 or OMP positive fibers and Vglut2 or OMP negative dentrites in WT mice either in controls or after regeneration (A, E and C, G respectively). In STOP null mice, a clumped aspect of either Vglut2 (D) or OMP (H) positive fibers was observed. Scale bar: 20 µm.</p

Neurogenesis in the vomeronasal epithelium.

<p>Localisation and mean density of BrdU (A–C), Ki67 (D–F), cleaved caspase 3 (G–I), GAP 43 (J–L), doublecortin (M–O) and OMP (P–R) labelled cells in the vomeronasal epithelium of WT and STOP null mice. The x-axis refers to the three levels studied, from rostral (level 1) to caudal (level 3), where the vomeronasal organ was present. A statistically significant increase in proliferating (BrdU and Ki67 positive cells), apoptotic (caspase 3 positive cells) and immature neurons (GAP 43 and doublecortin positive cells), but not mature OMP positive neurons was observed in STOP null mice as compared to WT mice. All values are represented as mean +/− SEM, *p<0.05, **p<0.01. Scale bar: 100 µm.</p

Ultrastructure of olfactory bulb glomeruli.

<p>Electron microscopy micrographs of olfactory bulb glomeruli in WT (A) and STOP null (B–F) mice at 3 to 6 months of age. In STOP null mice, olfactory axon endings are filled with autophagic-like structures (B, arrow), tubulovesicular profiles (C, arrowhead), or both (D). When few autophagic structures were present (arrows) (E, F), olfactory axons endings could be identified by the presence of synaptic vesicles and postsynaptic densities (arrowheads) (E, F). De: dentrite; OA: olfactory axon. Scale bar: 0,5 µm (A, C, E, F); 1 µm (B, D).</p

Regeneration of the olfactory epithelium.

<p>Mean epithelial OMP positive area before and after regeneration at the three levels 4, 5, 6 in WT and STOP null mice at two different ages. The x-axis refers to the three levels studied, from rostral (level 4) to caudal (level 6), where turbinates are most developed and olfactory epithelium most abundant. There is no difference in the ability of olfactory epithelium to regenerate at the three levels studied between WT and STOP null mice in 3 month-old (A) and 10 month-old (B) animals. All values are represented as mean +/− SEM, *p<0.05. The photomicrographs in A and B illustrate OMP immunostaining in the olfactory epithelium of animals after regeneration. Scale bar: 500 µm.</p

Localisation of BrdU-labelled cells in the olfactory epithelium.

<p>Mean density of BrdU-labelled cells in cytokeratine 5 positive HBC layer (A), cytokeratine 5 negative GBC layer (B) and superficial layer (C) in the olfactory epithelium of WT and STOP null mice. The x-axis refers to the six levels studied, from rostral (level 1) to caudal (level 6). Double immunolabelling (arrows) for BrdU (brown) and cytokeratin 5 (green) in the olfactory epithelium of a STOP null mouse is illustrated in D. An increase in globose basal cells but not in horizontal basal and superficial cells was observed in STOP null mice as compared to WT mice. All values are represented as mean +/− SEM, **p<0.01. Scale bar: 30 µm.</p

Neurogenesis in the olfactory epithelium after regeneration.

<p>Mean density of Ki 67 (A, B), caspase 3 (C, D), GAP 43 (E, F) and OMP (G, H) positive cells in the olfactory epithelium of WT and STOP null mice at two different ages. In the 3-month-old groups (A, C, E, G), apoptotic, proliferating and immature, but not mature neurons are more numerous in STOP null mice as compared to WT mice, both in control animals and after regeneration. In the 10-month-old groups (B, D, F, H) only the number of caspase 3 positive neurons (D) was increased in STOP null mice as compared to WT mice, in controls and after regeneration. All values are represented as mean +/− SEM, *p<0.05, **p<0.01. The photomicrographs illustrate immunostaining in animals after regeneration. Scale bar: 25 µm.</p

Glomeruli ultrastructure after regeneration in 10-month-old mice.

<p>Semithin sections (A–D) and electron microscopy micrographs (E, F) of olfactory bulb glomeruli in WT (A, C) and STOP null (B, D, E, F) mice. In STOP null controls, some olfactory endings are densely packed (*) (B); after regeneration this aspect is more pronounced (D). E illustrates the ultrastructure of a densely packed terminal area in D (*); olfactory axons are densely packed and associated with few electron clear dendrites area. F illustrates concentrically organized axon terminals (F, arrow) in STOP null mice following regeneration. Scale bar: 20 µm (A–D); 5 µm (E); 3 µm (F).</p