Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

SummaryTauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

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Functionally related neurons assemble into connected networks that process and transmit electrochemical information. To do this in a coordinated manner, the number and strength of synaptic connections is tightly regulated. Synapse function relies on the microtubule (MT) cytoskeleton, the dynamics of which are in turn controlled by a plethora of MT-associated proteins, including the MT-stabilizing protein Tau. Although mutations in the Tau-encoding MAPT gene underlie a set of neurodegenerative disorders, termed tauopathies, the exact contribution of MT dynamics and the perturbation thereof to neuronal network connectivity has not yet been scrutinized. Therefore, we investigated the impact of targeted perturbations of MT stability on morphological (e.g., neurite- and synapse density) and functional (e.g., synchronous calcium bursting) correlates of connectivity in networks of primary hippocampal neurons. We found that treatment with MT-stabilizing or -destabilizing compounds impaired morphofunctional connectivity in a reversible manner. We also discovered that overexpression of MAPT induced significant connectivity defects, which were accompanied by alterations in MT dynamics and increased resistance to pharmacological MT depolymerization. Overexpression of a MAPT variant harboring the P301L point mutation in the MT-binding domain did far less, directly linking neuronal connectivity with Tau's MT binding affinity. Our results show that MT stability is a vulnerable node in tauopathies and that its precise pharmacological tuning may positively affect neuronal network connectivity. However, a critical balance in MT turnover causes it to be a difficult therapeutic target with a narrow operating window.

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