148 | Lysosomal and synuclein pathology in IPSC-derived dopaminergic neurons from GBA1-mutant Parkinson’s disease patients

Gaia Pinna¹, Giuseppe Uras^1|2, Veronica Lentini¹, Luca Miuzzo¹, Sara Del Pozo³, Federico Fierli³, Anthony Schapira³, Antonella Pantaleo¹ | ¹Department of Biomedical Science, University of Sassari, Italy; ²Department of Haematology, UCL, London, United Kingdom; ³Department of Clinical and Movement Neurosciences, UCL, London, UK.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is marked by a progressive decline in motor function resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of α-synuclein. Genetic studies have identified mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase), as the most frequent genetic risk factor for PD. Heterozygous GBA1 variants are present in approximately 1% of the general population and confer an age-dependent increase in PD risk. While reduced GCase activity has been linked to lysosomal impairment and altered α-synuclein homeostasis, the cellular mechanisms through which distinct GBA1 mutations drive neuronal damage are still not fully elucidated. The work presented here aims to investigate subcellular protein organization in dopaminergic neurons derived from PD patients carrying GBA1 mutations. Midbrain dopaminergic (mDA) neurons were generated from induced pluripotent stem cells (iPSCs) obtained from healthy controls (WT/WT) and from PD patients harboring heterozygous GBA1 mutations (L483P/WT and N409S/WT). A high-content screening (HCS) strategy was established to quantitatively assess lysosomal markers, α-synuclein aggregates, and neuronal network morphology. Neurons derived from GBA1-mutant PD patients displayed an altered distribution of lysosomal markers together with an increased density and enlargement of α-synuclein species, as revealed by confocal microscopy. Both components showed abnormal subcellular localization associated with synaptic dysregulation. Overall, these findings demonstrate that GBA1 mutations induce distinct alterations in lysosomal positioning, α-synuclein trafficking, and synaptic organization in human dopaminergic neurons. Such cellular phenotypes may contribute to PD pathogenesis and provide insight into the mechanistic link between lysosomal dysfunction and α-synuclein pathology.