Titolo della tesi: Impact of Atmospheric Heavy Metals on Synaptic Development via hiPSC-Based 2D and 3D Brain Models
The World Health Organization (WHO) estimates that 8.9 million persons die each year of diseases caused by pollution and 8.4 million (94%) of them in poor countries (WHO 2014a,b). Focusing attention on air pollution and specifically particulate matter PM2.5, a link between the exposure to these compounds and an increased risk of neurodegenerative disease such as Alzheimer's, Amyotrophic Lateral Sclerosis and Parkinson's, has already been identified. Moreover, PM2.5 and polycyclic aromatic hydrocarbons (PAHs) exert neurotoxic effects, especially during critical periods of brain development, resulting in long-term changes in brain structure and function. Cumulative exposure over time exacerbates these effects, with early-life contact with heavy metals and pollutants linked to later learning and behavioral issues. Manganese (Mn) is also included in PM2.5 and, although the metal is normally present in the environment, today, due to the large quantity released into the atmosphere by industrial production processes, it has been found to be very harmful to human health. Alterations in body Manganese status are associated with changed neuronal physiology and cognition in humans, and its bioaccumulation, in the basal ganglia, can cause an irreversible neurological syndrome similar to Parkinson's disease (Manganism). This situation is aggravated by the fact that there are inconsistencies in standards and guidelines levels of air Manganese exposure both for allowed occupational exposure and for public settings.
For all these reasons, the main proposal of my PhD was to investigate the impact of Manganese at the neuronal and synaptic cellular level. To carry out this goal, I utilized bidimensional and tridimensional cell cultures derived from hiPSCs developed using three different control hiPSCs lines (WT#1, DS2U, and iPS28); cultures were untreated or treated with two doses of Manganese (MnCl2), 1 μM and 20 μM, chosen in relation to the state of the art of scientific literature and after a MTS viability assay. Treatments started from day 40 until the end of differentiation (day 70). We collected data about cortical populations of both control and treated groups performing real-time PCR analysis and immunofluorescence analysis at different time points. Furthermore, we used markers to identify astrocytes maturation, cytoskeletal proteins, synaptic proteins and amino acid transporters. Also, as Mn impact on synaptic neurotransmission is one of the main hallmarks of its neurotoxicity, functional calcium imaging studies were performed to analyze spontaneous intracellular calcium dynamics at network level. Moreover, patch clamp experiments have been conducted to record electrical activity from single cells, providing high-resolution data that can reveal detailed information about cellular mechanisms. Finally, to analyze the impact of the treatment in a more complex model that better recapitulates the full three-dimensional cytoarchitecture of the human brain, we setted up iPSC-derived organoids using the protocol adapted and modified from Sloan SA, et al., 2018. The organoids were treated with the aforementioned doses (1 μM and 20 μM of MnCl2) from day 60 until the end of differentiation at day 90. Tissue clearing and staining were performed at this time point.