Year: 2026
Type: dissertation
Abstract: Parkinson’s disease (PD) is a neurodegenerative disease representing a major burden for society and is expected to increase in prevalence due to the population ageing, population growth and industrialization. One of the main hallmarks of the disease is the selective degeneration of midbrain dopaminergic neurons (mDANs) in the Substantia Nigra pars compacta (SNpc), leading to dopamine depletion in the striatum and consequently resulting in different motor and non-motor symptoms. Several mechanisms underlying this multifactorial disease have been described, including the accumulation of α-synuclein in Lewy bodies, neuroinflammation, oxidative stress, and mitochondrial dysfunction. However, reliable methods for detecting the disease prior to symptom onset, as well as effective therapeutic strategies that could significantly improve the lifespan and quality of life of patients with PD, are still lacking. Cell-replacement therapies based on the transplant of induced pluripotent stem cell (iPSC)-derived mDAN precursors have shown promising results. However, further improvements of the in vitro models are required to increase their long-term efficacy and ability to alleviate symptoms. Studying mDAN development and identifying novel regulatory factors is therefore essential to better recapitulate mDAN development in vitro and to advance multiple areas of PD research, including disease modelling, the study of genetic variation and gene–environment interactions, and the development of improved therapeutic strategies. We have previously performed integrative analysis of epigenomic and transcriptomic data of mDAN differentiation, which enabled the identification of novel regulators of mDAN development. LBX1, NHLH1, and NR2F1/2 were prioritized based on their novelty and dynamic induction during mDAN differentiation. In the present study, depletion of these transcription factors (TFs) resulted in reduced maturation of mDANs, indicating their essential role during mDAN development. Furthermore, their depletion impaired neuronal activity, as assessed by electrophysiological recordings, supporting their involvement in functional maturation. Notably, NR2F1/2 was found to specifically regulate mDAN neuronal activity. Building on this, we further interrogated the previously generated multi-omic data to identify the SE-associated TFs in mDANs, highlighting ZFHX4 as a promising candidate key regulator of mDAN development. ZFHX4 has previously been implicated in neurodevelopment across species and shows reduced expression in the PD midbrain. Here we show that ZFHX4 is a SE-controlled TF induced during mDAN specification. Importantly, ZFHX4 is necessary but not sufficient for mDAN differentiation. Genome-wide profiling of ZFHX4 binding revealed targeting to active promoters and transcriptomic profiling after ZFHX4 depletion identified primary target genes to be enriched for cell-cycle regulation. Consistently, ZFHX4-depleted cells showed reduced proliferation and accumulated in G2 phase, impairing cell cycle progression. LIN28A, an RNA-binding protein involved in stem-cell maintenance and microRNA maturation, is among the strongest upregulated genes upon ZFHX4 depletion, with direct ZFHX4 binding at its locus. Our findings indicate ZFHX4 regulates mDAN maturation through a mechanism involving the LIN28A–miR-9 axis. These findings highlight the importance of multi-omic analyses in prioritizing TFs with key roles in developmental pathways. Using this approach, this study identified and characterized novel TFs that play selective roles in human mDAN development. Furthermore, it provides deeper insight into the functional mechanisms of these TFs, particularly ZFHX4, within the context of human mDAN development. These insights may serve as a foundation for refining the characterization of mDAN identity, and improving in vitro differentiation protocols, ultimately supporting the development of more effective cell-based therapies.
Author: VALCESCHINI, Elena
Institution: University of Luxembourg
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7167860931