INVESTIGATING THE INVOLVEMENT OF BETA-GLUCOCEREBROSIDASE DEFICIENCY IN THE ONSET OF NEURODEGENERATION

β-glucocerebrosidase (GCase) is the enzyme responsible for the catabolism of glucosylceramide (GlcCer) mainly at lysosomal level. The deficiency of this enzyme causes the onset of so called GCase-related pathologies such as Gaucher (GD) and Parkinson’s Disease (PD), which share neurodegenerative features. GD is a lysosomal storage disorder characterized by a broad spectrum of phenotypes, ranging from the lethal neuropathic type-II form to type-I, which is characterized by a more favorable prognosis. Interestingly, patients with GD type-I have a 10- fold increased risk of developing PD. On the other hand, PD is a progressive neurodegenerative disorder characterized by the primary selective and progressive loss of dopaminergic neurons. Nowadays, mutations affecting GBA1, the gene coding for GCase, represent the major inherited recessive risk factor for the development of PD. Unfortunately, the molecular mechanism leading from GCase deficiency to the onset of neuronal damage in GD and PD is still unclear. One of the main drawbacks in the study of neurodegenerative disorders in general and in particular in studies involving GD and PD is the lack of suitable in vitro models. Based on these considerations, for my PhD project I have focused on i) performing: the biochemical characterization of different brain areas obtained from healthy subjects and from a patient affected by GCase- dependent PD and ii) developing suitable in vitro models able to recapitulate the pathological features, with the aim to dissect the molecular mechanisms relating GCase deficiency to the onset of neurodegeneration. Related to the first part I found that in the frontal and parietal cortex, substantia nigra, cerebellum and hippocampus of GCase- PD patients, GCase activity was decreased of about 50% and in case of the parietal cortex and the hippocampus the reduction was even of greater entity. On the other hand, in almost all areas I observed an increased activity of the non- lysosomal β- glucoceramidase when compared to healthy areas, which could be a compensatory effect that hinders GlcCer accumulation in the GCase- PD brain areas. To figure out the possible molecular mechanism linking GCase loss of function with the onset of neuronal damage I developed two in vitro models of the neuronal form of GD, which are represented by mouse cerebellar granule neurons and iPSCs-derived dopaminergic neurons differentiated from healthy subject’s fibroblasts. Both models were treated with conduritol B epoxide to suppress GCase activity. These models correctly recapitulate the pathological phenotype by presenting a time- dependent accumulation of GlcCer and onset of neuronal damage. I demonstrated that the aberrant accumulation of GlcCer induces alterations of the endolysosomal compartment, with consequent accumulation of GlcCer at the plasma membrane, where it alters PM architecture and functioning. Furthermore, GCase deficiency induces several alterations in terms of cell metabolites, in particular involving amino acids, but seems to have no effect on the overall energetic balance of the neurons. These in vitro models help to identify the existence of new possible mechanisms involving the PM and metabolism in the onset of the neuronal degeneration occurring in GD and GCase-PD. Taken together the obtained data allow a better characterization of the effects of a partial reduction of GCase activity in PD brain, while the developed in vitro models help to shed light on some molecular mechanisms involved in the neuronal degeneration occurring upon the almost complete deficiency of GCase activity as it occurs in GD patients.