Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Institut Pasteur

Université Paris Diderot, Sorbonne Paris Cité

The neural cell-adhesion molecules contactin 4, contactin 5 and contactin 6 are involved in brain development, and disruptions in contactin genes may confer increased risk for autism spectrum disorders (ASD). We describe a co-culture of rat cortical neurons and HEK293 cells overexpressing and delivering the secreted forms of rat contactin 4-6. We quantified their effects on the length and branching of neurites. Contactin 4-6 effects were different depending on the contactin member and duration of co-culture. At 4 days in culture, contactin 4 and -6 increased the length of neurites, while contactin 5 increased the number of roots. Up to 8 days in culture, contactin 6 progressively increased the length of neurites while contactin 5 was more efficient on neurite branching. We studied the molecular sites of interaction between human contactin 4, -5 or -6 and the human Protein Tyrosine Phosphatase Receptor Gamma (PTPRG), a contactin partner, by modeling their 3D structures. As compared to contactin 4, we observed differences in the Ig2 and Ig3 domains of contactin 5 and -6 with the appearance of an omega loop that could adopt three distinct conformations. However, interactive residues between human contactin 4-6 and PTPRG were strictly conserved. We did not observe any differences in PTPRG binding on contactin 5 and -6 either. Our data suggest that the differential contactin effects on neurite outgrowth do not result from distinct interactions with PTPRG. A better understanding of the contactin cellular properties should help elucidate their roles in ASD.

Contactin 4, -5 and -6 differentially regulate neuritogenesis while they display identical PTPRG binding sites.

Les mol&eacute;cules d&#39;adh&eacute;sion-cellulaire neuronal contactin 4, contactin 5 et 6 de contactin sont impliqu&eacute;s dans le d&eacute;veloppement du cerveau, et les perturbations dans la contactin de g&egrave;nes peuvent conf&eacute;rer &agrave; un risque accru de troubles du spectre autistique (TSA). Nous d&eacute;crivons une co-culture de neurones corticaux de rat et les cellules HEK293 surexprimant et livrer les formes s&eacute;cr&eacute;t&eacute;es de rat contactin 4-6. Nous avons quantifi&eacute; leurs effets sur la longueur et la ramification des neurites. Contactin 4-6 effets ont &eacute;t&eacute; diff&eacute;rents selon la dur&eacute;e de co-culture et le membre contactin. &Agrave; 4 jours de culture, contactin 4 et -6 a augment&eacute; la longueur des neurites, tandis que 5 contactin a augment&eacute; le nombre de racines. Jusqu&#39;&agrave; 8 jours de culture, contactin 6 progressivement augment&eacute; la longueur des neurites tout en contactin 5 &eacute;tait plus efficace sur la ramification des neurites. Nous avons &eacute;tudi&eacute; les sites mol&eacute;culaires de l&#39;interaction humaine 4 contactin, -5 ou -6 et l&#39;humain prot&eacute;ine Tyrosine Phosphatase Receptor Gamma (PTPRG), partenaire de contactin, par leurs structures 3D de mod&eacute;lisation. Par rapport &agrave; contactin 4, nous avons observ&eacute; des diff&eacute;rences dans les domaines Ig2 et Ig3 de contactin 5 et -6 avec l&#39;apparition d&#39;une boucle om&eacute;ga qui pourrait adopter trois conformations distinctes. Toutefois, les r&eacute;sidus interactives entre humains contactin 4-6 et PTPRG &eacute;taient strictement conserv&eacute;s. On n&#39;a pas observ&eacute; de diff&eacute;rences quant au lie PTPRG contactin 5 et -6 non plus. Nos r&eacute;sultats sugg&egrave;rent que les effets diff&eacute;rentiels de contactin sur la croissance des neurites ne r&eacute;sultent pas d&#39;interactions distinctes avec PTPRG. Une meilleure compr&eacute;hension des propri&eacute;t&eacute;s cellulaires contactin devrait aider &agrave; &eacute;lucider leur r&ocirc;le dans l&#39;ASD.

Contactin 4, -5 et -6 diff&eacute;rentiellement r&eacute;glementent neuritogenesis alors qu&#39;ils affichent des sites identiques de liaison PTPRG.

Le molecole di adesione cellulare neurale anche 4, anche 5 e anche 6 sono coinvolti nello sviluppo del cervello, e interruzioni nel anche geni possono conferire aumento del rischio di disturbi dello spettro autistico (ASD). Descriviamo un co-culture dei neuroni corticali di topo e cellule HEK293 con sovraesprimono relativa e trasportando le forme secrete di ratto anche 4-6. Abbiamo quantificato gli effetti sulla lunghezza e la ramificazione dei neuriti. Anche 4-6 effetti erano diverse a seconda del membro contactin e la durata del co-culture. A 4 giorni in coltura, contactin 4 e -6 aumentato la lunghezza dei neuriti, mentre 5 contactin aumentato il numero di radici. Fino a 8 giorni in coltura, anche 6 progressivamente aumentato la lunghezza dei neuriti mentre anche 5 era pi&ugrave; efficiente sulla diramazione dei neuriti. Abbiamo studiato i siti molecolari dell&#39;interazione tra umano contactin 4, -5 o -6 e l&#39;umano proteina tirosina fosfatasi del recettore Gamma (PTPRG), un partner contactin, modellando le strutture 3D. Rispetto al anche 4, abbiamo osservato le differenze nei domini Ig2 e Ig3 contactin 5 e -6 con l&#39;aspetto di un ciclo di omega che potrebbe adottare tre distinte conformazioni. Tuttavia, residui interattivi tra 4-6 contactin umano e PTPRG erano rigorosamente conservati. Non ha fatto osservare eventuali differenze nell&#39;associazione PTPRG contactin 5 e -6 o. I nostri dati suggeriscono che gli effetti contactin differenziali su neuriti escrescenza non derivano da interazioni distinte con PTPRG. Una migliore comprensione delle propriet&agrave; cellulari contactin dovrebbe aiutare a chiarire i loro ruoli in ASD.

Laura Gouder

Human Genetics and Cognitive Functions,
CNRS URA 2182 'Genes,
synapses and cognition',
CNRS URA 2182 'Genes, synapses and cognition'

Institut Pasteur

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Laura Gouder

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Human Genetics and Cognitive Functions,CNRS URA 2182 'Genes,synapses and cognition',CNRS URA 2182 'Genes, synapses and cognition'

Institut Pasteur

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Human Genetics and Cognitive Functions,
CNRS URA 2182 'Genes,
synapses and cognition',
CNRS URA 2182 'Genes, synapses and cognition'