DISSECTING THE ROLE OF ACID SPHINGOMYELINASE INHIBITION IN GAUCHER DISEASE AND GBA-DEPENDENT PARKINSON¿S DISEASE IN VITRO MODELS

GBA gene encodes for the lysosomal enzyme β-glucocerebrosidase (GCase), which is involved in the catabolism of glucosylceramide (GlcCer) into glucose and ceramide. Biallelic mutations in GBA gene result in the GCase loss of function, leading to the accumulation of GlcCer and causing Gaucher Disease (GD). GD is a lysosomal storage disorder that manifests in various forms, ranging from the severe neuropathic type II to the less severe non-neurological type I. Interestingly, monoallelic mutations in GBA are now recognized as a major recessive genetic risk factor for Parkinson's Disease (PD), known as GBA-dependent PD. PD is a progressive neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons and by the presence of aggregates of α-synuclein in the substancia nigra pars compacta of the brain. Up to now, the molecular mechanism linking GCase deficiency to the onset of neurodegeneration is still unknown. This is largely due to the lack of suitable in vitro models that accurately replicate the complexities of the human pathology associated with these conditions. To increase the complexity, it is important to consider that not all the individuals with GBA mutations develop PD. To understand this variability, the scientific community has explored the presence of other genetic variants in lysosomal enzymes that could influence the penetrance of GBA mutations. Our collaborators at Humanitas university have analysed a panel of 50 lysosomal genes, involved in the onset of lysosomal storage disorders when mutated, in a cohort composed by GBA asymptomatic carriers and GBA-mutated PD patients. They found that SMPD1 gene, coding for the enzyme acid sphingomyelinase (ASM), was more frequently mutated in controls than in GBA-PD, suggesting a possible protective effect of SMPD1 mutation in GBA carriers. In addition, a recent study has found that in a zebrafish GD model the inhibition of ASM, ameliorates the pathological phenotype and the survival, rescuing the mitochondrial chain function. Based on these considerations, the aim of my PhD project was to investigate whether the inhibition of ASM could have a beneficial role in GBA-related pathologies exploiting 2D and 3D in vitro neuronal models. To conduct this research, I generated two experimental models: a pharmacological one, using hiPSCs-derived dopaminergic neurons (hiPSCs-DANs), and a patient-derived one, using human midbrain organoids (hMOs). For the pharmacological model, I treated DANs for 30 days with conduritol-B- epoxide (CBE), a specific inhibitor of GCase, to induce the GD-PD phenotype. This is an experimental model that we have already demonstrated to show GCase deficiency, neurodegeneration and GlcCer accumulation. For the patient-derived one, I generate hMOs from iPSCs of a GD type I patient carrying the biallelic mutation N370S/F213I in GBA gene, who developed parkinsonism. I characterized this model and I demonstrated that it recapitulates the pathological features observed in the 2D model, with the addition of α-synuclein aggregation. These results point out that both dopaminergic neurons and organoids partially recapitulate the pathological phenotype observed in vivo. Thus, they constitute a good model for further investigations related to GD-PD pathology. Furthermore, I treated both models with amitriptyline (AMI), a functional inhibitor of ASM, to verify the effect of ASM inhibition in GD-PD pathology. I treated DANs 24h and 7 days before the collection at day 60 of differentiation. In hMOs instead the administration was longer, lasting 30 days and 70 days. Surprisingly, I found opposite results in the two models. Indeed, the acute (24h) and chronic (7 days) treatment with AMI has ameliorated the neurodegenerative phenotype in CBE-treated DANs, reducing GlcCer accumulation by increasing the non-lysosomal glucosylceramidase (NLGase) activity. Conversely, in hMOs the prolonged drug administration was detrimental for GD-PD organoids, increasing neurodegeneration and lysosomal impairment, without changes in GlcCer accumulation and promoting α-synuclein aggregation. This study introduces two reliable in vitro models for studying GCase deficiency in neurons, providing valuable insights into GBA-related neurodegenerative disorders. However, midbrain organoids emerge as a preferable choice for these investigations since they closely mirror the physiological conditions of the brain. And according to the results obtained, it become evident that ASM inhibition has a damaging effect on GBA-dependent Parkinson’s disease in vitro models.