Project: MOlecular DIAgnostic of brain disease mutations in human embryonic stem cells derived 2D- and 3D- neuronal cultures, using intracellular naNOparticle tracking, synapse naNOscopy, and microcircuit calcium imaging

Acronym	MoDiaNo (Reference Number: JTC2018-107)
Duration	01/06/2019 - 31/12/2022
Project Topic	Advances in genome sequencing technologies facilitate the detection of de novo mutations. Recently, LAC-ENS Paris-Saclay P of this project identified such point mutations in brain diseases from schizophrenia, autism spectrum disorders and intellectual disabilities cohorts. However, no validation of their functional impact is yet available, limiting their use for personalized medicine. In this project, we will develop three complementary screens of functional impacts of these de novo mutations, based on targeted nanotracers combined with advanced microscopies. These screens rely on intraneuronal transport quantification in 2D and 3D human neuronal cultures, nanoscale synaptic and microcircuits consequences. Mutations will be engineered by CRISPR-Cas9 technology into human embryonic stem cells from which neuronal 2D-cultures and 3D cerebral organoids will be derived (Max Planck group, Germany). Intradendritic molecular transport will be measured by tracking optically active nanoparticles with very high stability (LAC/ENS Paris Saclay): fluorescent nanodiamonds as small as 10 nm (FND Biotech, Taiwan) for 2D cultures and second-harmonic generating nanocrystals (nanoSiC) for deep imaging in organoids. Functionalization of these nanotracers (Academica Sinica, Taiwan) will allow targetting specific transport. AbbeLight SAS (France) will build an automatized 3D-nanoscope (˜15 nm resolution) to image key dendritic spine proteins and actin in order to assess synaptic consequences of the mutations. LaVision Biotec (Germany) will build a new horizontal 2-photon microscope with unrivalled sensitivity and volume imaging speed to track nanoSiC and measure intracellular transport in organoids. Concomitantly, microcircuit functional organization will be recorded based on calcium activity reporter (Max Planck group & LaVision). Altogether, these complementary readouts are expected to generate a molecular diagnosis of de novo mutations suitable for personalized medicine.
Network	EuroNanoMed III
Call	Joint Transnational Call (2018)

Project partner

Number	Name	Role	Country
1	Academia Sinica	Coordinator	Taiwan
2	FND Biotech, Inc.	Partner	Taiwan
3	LaVision Biotec GmbH	Partner	Germany
4	Max Planck Institute for Evolutionary Anthropology	Partner	Germany
5	Ecole Normale Supérieure de Paris-Saclay	Partner	France
6	AbbeLight S.A.S.	Partner	France