Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - NEUROGUIDE (Study of neuronal sensing and migration/guidance dynamics in neurodevelopmental disorders by nano-engineered chips)

Teaser

Defects in neuronal migration have a severe impact on normal brain function. Although deficits in neuronal micro-connectivity are recently emerging as crucial in neurodevelopmental disorders and intellectual disabilities (e.g. autism), the role of neuronal migration processes...

Summary

Defects in neuronal migration have a severe impact on normal brain function. Although deficits in neuronal micro-connectivity are recently emerging as crucial in neurodevelopmental disorders and intellectual disabilities (e.g. autism), the role of neuronal migration processes during brain development is still poorly understood in these pathological conditions. NEUROGUIDE project aims to study neuronal migration/guidance dynamics in neurodevelopmental disorders, with a multidisciplinary approach that combines nano-engineered devices (i.e. nano-structured substrates and microfluidic chips), molecular neuroscience and state-of-the-art fluorescence microscopy.
As a pathological model, the project focus on the unbalanced levels of ubiquitin ligase E3a (UBE3A), which leads to several neurodevelopmental disorders. UBE3A has a key role in the brain. In fact, the lack of UBE3A leads to Angelman Syndrome (AS), while its increase is the most prevalent genetic origin of autism (≈ 3%). However, the specific role of UBE3A in brain development is still not known and there is no cure for AS or autistic disorders currently available. These disorders do not only affect the patients but are also a great burden on families and on the healthcare system.
NEUROGUIDE project aims to study the neuronal responses to guidance stimuli bi-directionally: 1) in vivo, in Ube3a-mutant mice (using in utero electroporation), to identify if the UBE3A loss/increase impacts neuronal migration and guidance during brain development; 2) in vitro, in Ube3a-mutant neurons by using nano-engineered platforms to apply specific biomimetic environmental physical and/or chemical stimuli to neurons: this approach allows studying the biochemical mechanisms involved in the neuronal guidance processes, at the molecular level (Fig.1).
The objectives of NEUROGUIDE project are: - to clarify the role of UBE3A (and its isoforms) in neurodevelopment; - to establish the value of nano-engineered platforms as advanced devices for investigating neuronal guidance dynamics in vitro. These bio-mimicking platforms can be exploited as read-out systems for in vitro testing of novel potential drugs, thus helping in the application of the 3Rs principles.

Work performed

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

Final results

Progresses
• Preliminary results from in vivo experiments show the involvement of UBE3A in neuronal cortical migration
• NEUROGUIDE demonstrates the presence of a specific deficit in axonal guidance in AS neurons thanks to GRs substrates. Conversely, it is still not possible, at our present knowledge, to identify specific morphological differences between WT and AS neurons in vitro on standard substrates. We further show two rescue strategies for restoring correct topographical guidance in AS neurons, by genetic and pharmacological approaches

Potential impacts
• NEUROGUIDE microstructured substrates are optimal in vitro read-out systems for neuronal migration studies in neurodevelopmental disorders, for fundamental research and for drug screening in vitro
• Nano-engineered scaffolds allow us reducing, refining and also partially replacing (in the longer-term) animal testing

Website & more info

More info: https://www.facebook.com/neuroguide2018/.