Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro

Abstract

Vav proteins activate GTPases of the RhoA subfamily that regulate the cytoskeleton and are involved in adhesion, migration, differentiation, polarity and the cell cycle. While the importance of RhoA GTPases for neuronal morphology is undisputed, their regulation is less well understood. In this perspective, we studied the consequences of the deletion of Vav2, Vav3 and Vav2 and 3 (Vav2−/−, Vav3−/−, Vav2−/−/3−/−) for the development of embryonic hippocampal neurons in vitro. Using an indirect co-culture system of hippocampal neurons with primary wild-type (WT) cortical astrocytes, we analysed axonal and dendritic parameters, structural synapse numbers and the spontaneous network activity via immunocytochemistry and multielectrode array analysis (MEA). Here, we observed a higher process complexity in Vav3−/−, but not in Vav2−/− neurons after three and five days in vitro (DIV). Furthermore, an enhanced synapse formation was observed in Vav3−/− after 14 days in culture. Remarkably, Vav2−/−/3−/− double knockout neurons did not display synergistic effects. Interestingly, these differences were transient and compensated after a cultivation period of 21 days. Network analysis revealed a diminished number of spontaneously occurring action potentials in Vav3−/− neurons after 21 DIV. Based on these results, it appears that Vav3 participates in key events of neuronal differentiation.

Vav3-Deficient Astrocytes Enhance the Dendritic Development of Hippocampal Neurons in an Indirect Co-culture System

“…Furthermore, wildtype neurons were cultured in an indirect contact with Vav3 –/– astrocytes. Finally, Vav3 –/– neurons were kept in culture with wildtype astrocytes since it is known that a deficiency of Vav3 –/– influences the early development of axons and dendrites in vitro ( Wegrzyn et al, 2020 ). The neurons of all three culture combinations formed highly branched and complex networks after a cultivation time of 12 and 17 days in vitro ( Figures 1B–D,1B’–D’ ).…”

“…All experiments of this study were performed in accordance with the Society for Neuroscience and the European Council Directive of September 22, 2010 (2010/63/EU), for care of laboratory animals and approved by the animal care committee of North Rhine-Westphalia, Germany, based at the LANUV (Landesamt für Umweltschutz, Naturschutz und Verbraucherschutz, North Rhine-Westphalia, D-45659 Recklinghausen, Germany). Wildtype (WT) SV129 and Vav3 knockout animals of both sexes were used and originated from the mouse colony of the Department for Cell Morphology and Molecular Neurobiology of the Ruhr-University Bochum ( Luft et al, 2015 ; Wegrzyn et al, 2020 ). The animals were kept under standardized conditions with a regulated temperature (21°C), humidity (60–70%) and a 12 h dark/light circle.…”

“…Since RhoA, Rac-1 and Cdc42 are strong modulators of the cytoskeleton, Vav3 functions as a mediator of intracellular reorganizations of the cytoskeleton ( Tapon and Hall, 1997 ). In this way, Vav3 regulates key events of CNS development like migration ( Khodosevich et al, 2009 ), proliferation ( Fujikawa et al, 2002 ), differentiation ( Ulc et al, 2019 ) and axo-dendritic maturation ( Quevedo et al, 2010 ; Sauzeau et al, 2010 ; Ulc et al, 2017 ; Wegrzyn et al, 2020 ).…”

“…The altered axon guidance of GABAergic neurons in the brainstem of mice was consequently linked to hypertension, tachycardia, and renocardiovascular dysfunctions ( Sauzeau et al, 2010 ). Additionally, primary hippocampal neurons derived from Vav3-deficient embryonic animals displayed a significant raise in their axonal lengths and complexities as well as in their structural synapse numbers in vitro ( Wegrzyn et al, 2020 ). In schizophrenia patients VAV3 could be furthermore identified as a risk gene and polymorphisms were discussed to possibly be responsible for reduced volumes of the left superior and middle temporal gyri ( Aleksic et al, 2013 ).…”

“…

Wildtype (WT) SV129 and Vav3 knockout animals of both sexes were used and originated from the mouse colony of the Department for Cell Morphology and Molecular Neurobiology of the Ruhr-University Bochum (Luft et al, 2015;Wegrzyn et al, 2020). The animals were kept under standardized conditions with a regulated temperature (21 • C), humidity (60-70%) and a 12 h dark/light circle.

…”