NEUROGUIDE had two main objectives:
WP1: Study of neuronal migration and guidance processes in vivo, in wild-type (WT) (physiological condition) and in Ube3a-mutant mouse models (intellectual disability pathological conditions)
During the project, we performed in utero electroporation experiments by deleting or overexpressing UBE3A or its isoforms in subsets of neurons in the cerebral cortex. With these experiments we quantify the migration of neurons to the outer layers of the cortex, thus investigating the role of UBE3A in the migration process (Fig.2).
WP2: Study of the neuronal environmental sensing and guidance dynamics by micro/nano-engineered platforms in vitro, in WT and Ube3a-mutant neuronal models.
We developed micro-structured substrates with gratings patterns (i.e. alternating lines of ridges and grooves) (GRs) with different dimensionality (ridge size 1-10 µm). We mainly used GRs with ridge size of 1 µm in thermoplastic material (Masciullo C, Nanomaterials 2018). We also developed elastomeric GRs (ridge size 2-10 µm) in polydimethylsiloxane (PDMS) by soft lithography, and used them to develop immobilized biochemical GRs of neurotrophic/repulsive factors by micro-contact printing.
Neuronal responses (migration, guidance, growth cones) to physical and chemical stimuli have been investigated in WT, AS and Ube3a-overexpressing (15dup autism model) neurons. Thanks to microfluidic chambers we also investigated neuronal chemotactic responses to selected chemical gradients (i.e. BDNF, semap3A).
Results overview
1)We find that, during cortical development, the removal of UBE3A protein (by using different UBE3A silencing-RNAs) induces a migration delay in those cortical neurons in vivo. The migration delay is higher at early postnatal stage (P1 vs. P21). Finally, the presence of an excess of UBE3A does not impact cortical migration.
2)During the project, we developed thermoplastic and elastomeric GRs with different directional patterns (1-10 µm lines), able to induce directional stimuli to neurons. We set up a replica molding protocol to develop biochemical GR patterns (e.g. 10 µm lines of NGF and laminin) on standard glass coverslips by using PMDS-GRs membranes. Moreover, our substrates are coupled easily with standard PDMS chambers to perform collective migration assays in stimulated conditions.
3)We show that there is a specific loss of axonal guidance in AS neurons cultured on GRs while UBE3A overexpression does not affect neuronal directional polarization. Deficits at the level of growth cone orientation and actin fiber content, focal adhesion pathway activation and actin fiber-binding proteins are present in AS neurons. We further test different rescue strategies for restoring correct topographical guidance in AS neurons in vitro, by applying genetic and pharmacological treatments. Nocodazole, a drug that increases cytoskeleton contractility, rescues AS axonal alignment to GRs (by restoring, to a certain extent, FA pathway activation), while UBE3A reinstatement only partially does it (Fig.3).
Dissemination
NEUROGUIDE results have been accepted as an oral presentation (by I. Tonazzini) at:
1)NanoMedicine Int. Conference 2018, Venice IT, 23-25 October 2018
2)Materials.it 2018, Bologna IT, 22-26 October 2018
3)4th Int. symposium on Nanoengineering for Mechanobiology, Camogli IT, 24-27 March 2019
and as a poster presentation at:
1)11th Forum of European Neuroscience, Berlin DE, 7-11 July 2018
2)5th Int. Angelman Syndrome Scientific Conference, Hamburg-DE, 12 October 2018
3)4BIO Summit Europe, 4th Microfluidics Congress, Rotterdam NL, 27-28 November 2018
•Invited talk at the Biannual meeting of Istituto Nanoscienze CNR, at Scuola Normale Superiore, 28-29 October 2018, Pisa IT
Outreach and press activity
•Invited seminar (Angelman Syndrome explanation for parents) at the Annual meeting of the Italian Organization of Angelman Syndrome families, Assisi-IT, 6 October 2018
•Divulgative Neuroscience lesson at the De Blijberg Primary School – Int. Department in Rotterdam, during their Brain week, October 2018
•Participation at the Dutch events for the Researchers’ Night 2018, organized by Erasmus University: pitch at the Marie Curie corner at the event Science Hotel (28-09-2018, Nhow Hotel, Rotterdam) and poster at the event Science OPEN (29-09-2018)
