Sustained synchronized neuronal network activity in a human astrocyte co-culture system.

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.

Age-dependent concomitant changes in synaptic dysfunction and GABAergic pathway in the APP/PS1 mouse model.

Synaptic dysfunction is a well-documented manifestation in animal models of Alzheimer's disease pathology. In this context, numerous studies have documented reduction in the functionality of synapses in various models. In addition, recent research has shed more light on increased excitability and its link to seizures and seizure-like activities in AD patients as well as in mouse models. These reports of hyperexcitability contradict the observed reduction in synaptic function and have been suggested to be as a result of the interplay between inhibitory and excitatory neuronal mechanism. The present study therefore investigates functional deficiency in the inhibitory system as complementary to the identified alterations in the glutamate excitatory pathway in AD. Since synaptic function deficit in AD is typically linked to progression/pathology of the disease, it is important to determine whether the deficits in the GABAergic system are functional and can be directly linked to the pattern of the disruption documented in the glutamate system. To build on previous research in this field, experiments were designed to determine if previously documented synaptic dysfunction in AD models is concomitantly observed with excitation/inhibition imbalance as suggested by observation of seizure and seizure-like pathology in such models. We report changes in synaptic function in aged APPPS1 mice not observable in the younger cohort. These changes in synaptic function are furthermore accompanied by alteration in the GABAergic neurotransmission. Thus, age-dependent alteration in the inhibitory/excitatory balance might underpin the symptomatic changes observed with the progression of Alzheimer's disease pathology including sleep disturbance and epileptic events.

Templated misfolding of Tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in Tau transgenic mice.

Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.

Using Human iPSC-Derived Neurons to Model TAU Aggregation.

Alzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.