Background <p>Parkinson’s disease (PD) involves progressive loss of midbrain dopaminergic (mDA) neurons in the substantia nigra. No disease-modifying treatments exist, only symptomatic relief. Our lab reported an unbiased screen in larval zebrafish identifying renin–angiotensin–aldosterone system (RAAS) inhibitors, including clinically used AGTR1 inhibitors for hypertension, as potent neuroprotective agents. This study aims to investigate the effects of AGTR1 inhibition on human mDA neuron survival using inducible neurodegenerative 2D and 3D models for human mDA neuron degeneration.</p> Methods <p>We report a scalable high-content platform, using CRISPR-engineered human induced pluripotent stem cell (hiPSC)-derived mDA neurons expressing a tyrosine hydroxylase (TH) fluorescent reporter, allowing for tracking mDA neuron survival live “in a dish”. We developed chemically inducible 2D and 3D neurodegenerative models of human mDA neurons, enabling us to recapitulate PD pathology in human cells in vitro<i>.</i></p> Results <p>Our model establishes scalable human cellular models of PD well-suited for therapeutic discovery. Using 2D and 3D mono and co-cultures, this study demonstrates that inhibition of AGTR1, via chemical or genetic means, protects against chemically induced mDA neuron degeneration. Transcriptomic analyses show AGTR1 inhibition lowers synuclein transcription by reducing <i>SNCA</i> and <i>SNCB</i> gene expression. In 3D neuron-glia assembloids, AGTR1 inhibition protects against the accumulation of the phosphorylated (p129α-Syn), aggregated, and thioflavinS + forms of α-synuclein.</p> Conclusions <p>We uncover AGTR1 as a key regulator of α-synuclein transcription and aggregation in human iPSC-derived mDA neurons, and AGTR1 inhibition as pro-survival in human iPSC-derived 2D and 3D neurodegenerative models of mDA neurons. These findings position inhibition of AGTR1 as a promising therapeutic strategy for PD neuroprotection.</p> Graphical abstract <p></p>