Ausilio, Chiara (2025) Interconnected human brain spheroids for investigating neuronal circuitry in health and disease. [Tesi di dottorato]

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Abstract

Neurodegenerative disorders are characterized by the progressive loss of neuronal connectivity and synaptic function, leading to network disintegration long before overt cell death occurs. Understanding how early molecular alterations translate into circuit-level dysfunction remains one of the major challenges in neuroscience. Traditional two-dimensional (2D) cultures and animal models have provided valuable insights but often fail to reproduce the structural and functional complexity of the human brain. Therefore, the demand for reliable and physiologically relevant in vitro systems has markedly increased, particularly those enabling the dynamic study of neuronal connectivity. This PhD thesis aimed to develop a human-based microengineered platform to model the structural connectivity and to assess the functional circuitry of human neuronal network, with a particular focus on dopaminergic network dynamics and their impairment under Parkinsonian stress. Human induced pluripotent stem cell (hiPSC)-derived neurospheroids were integrated within a customised Polydimethylsiloxane (PDMS)-based microfluidic chip. This configuration enabled the formation of bidirectional axonal fascicles connecting spatially separated spheroids, reproducing long-range neuronal projection. Furthermore, the system was coupled with high-density microelectrode arrays (HD-MEAs) to monitor functional neuronal activity. Electrophysiological analyses revealed progressive maturation of spontaneous firing, bursting behavior, and synchronization in both single and double interconnected spheroids. Lastly, upon exposure to rotenone, a mitochondrial complex I inhibitor and environmental neurotoxin epidemiologically linked to Parkinson’s disease (PD), a significant reduction in neuronal activity and network connectivity strength was observed. These findings recapitulate key early-stage pathological hallmarks of PD, establishing the model as a sensitive tool for investigating neuronal network vulnerability. In conclusion, this work presents a novel brain-on-chip model that bridges the gap between reductionist cell cultures and the complex organization of the human brain, by enabling the real-time study of neuronal structural connectivity and functional circuitry under both physiological and Parkinsonian-like conditions.

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