Generation of two gene edited iPSC-lines carrying a DOX-inducible NGN2 expression cassette with and without GFP in the AAVS1 locus.

Neurog2 is the gene encoding the neuronal transcription factor NGN2, which can convert stem cells into functional neurons in a fast and efficient way. Here we report the generation of two iPS cell lines, where DOX inducible constructs of neurog2 either without or with T2A-eGFP were inserted into the safe-site locus AAVS1. These iPS cell lines, BIONi010-C-13 and BIONi010-C-15, respectively, stay pluripotent without DOX but differentiate to (GFP positive) neurons when DOX is added without the need of differentiation factors.

Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format.

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer's disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting ß-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

Alteration of the nuclear pore complex in Ca(2+)-mediated cell death.

Cell death requires coordinated intracellular signalling before disassembly of cell architecture by degradative enzymes. Although the death signalling cascades that involve the mitochondria, the ER and the plasma membrane have been extensively characterized, only a handful of studies have examined the functional and structural alterations of the nuclear pore complex (NPC) during neuronal death. Here, we show that during excitotoxic neuronal degeneration calpains redistributed across the nuclear envelope and mediated the degradation of NPC components causing altered permeability of the nuclear membrane. In primary dissociated neurons, simultaneous recording of cytosolic [Ca(2+)] and localization of fluorescent proteins showed that the onset of Ca(2+) overload signalled a progressive increase in the diffusion of small reporter molecules across the nuclear envelope. Later, calpain-mediated changes in nuclear pore permeability allowed accumulation of large proteins in the nucleus. Further, in a model of excitotoxic neuronal degeneration in Caenorhabditis elegans, we found similar nuclear changes and redistribution of fluorescent probes across the nuclear membrane in dying neurons. Our findings strongly suggest that increased leakiness of the nuclear barrier affects nucleocytoplasmic transport, alters the localization of proteins across the nuclear envelope and it is likely to be involved in Ca(2+)-dependent cell death, including ischemic neuronal demise.