Understanding bone alterations in Gaucher disease using the zebrafish animal model: development of a novel pathogenetic paradigm for lysosomal storage disorders

Lysosomal storage disorders (LSDs) are rare inherited metabolic disorders due to a deficiency of specific lysosomal enzymes or transporters. Almost 50 genetic disorders caused by deficiencies of lysosomal and non-lysosomal proteins are known nowadays, collectively with an incidence of ~1/7000 newborns in the worldwide population. Dysfunctions of such lysosomal enzymes or transporters can lead to several consequences that include incomplete degradation and/or recycling of intracellular molecules. Despite lysosomal proteins are present in almost all type of cells, the accumulation of undegraded substrates in LSDs affected patients is generally limited to cells, tissues and organs in which substrate turnover is high, leading to a wide spectrum of phenotypes and affected organs for different LSDs. Among these LSDs, the most common is the Gaucher disease (GD) with an incidence of 1 in 200.000 newborn in the worldwide population, a rate that increases to 1 in 850 in the population of Ashkenazi Jewish (Eastern Europen). This disorder is due to a deficiency of the lysosomal enzyme β-glucocerebrosidase (GBA) that in pathological conditions is not able to degrade its substrate, glucosylceramide, into glucose and ceramide. A hallmark of the disease is the presence of “Gaucher cells”, which are lipid laden-macrophages, in different tissues. Together with this characteristic, GD patients manifest hepatosplenomegaly, anemia, thrombocytopenia and severe bone disfunction such as osteonecrosis, osteopenia, bone crisis and fracture of long bones. GD patients can be classified in three different clinical subtype, based upon the presence and severity of neurological defects. Type I GD, also referred to as non-neuronophatic form, is the most frequent subtype, the symptoms manifest in adulthood and patients do not present neurological involvement. Type II GD patients, manifest severe neurological defects since early life stages and death occurs in childhood. Type III GD patients manifest less severe neurological defects when compared to type II GD and generally symptoms occur during childhood. This classification of the disease based on the absence or presence and severity of neurological defects is oversimplified. Nowadays, the broad spectrum of phenotypes and the recognition of overlap among these GD subtypes has led to the concept that this disorder is a continuum of phenotypes, ranging from the less severe (GD 1) to the more severe forms (GD2 and GD3). The most effective and well tolerated treatment available for this disorders is the enzyme replacement therapy (ERT), that consists in the administration of a recombinant enzyme able to restore the β-glucocerebrosidase functions. Despite its effectiveness on hepatosplenomegaly, anemia and thrombocytopenia symptoms, this therapy has a very limited effect on the recovery of the skeletal and neurological defects. Different GD murine model have been developed through the years to understand the pathogenetic mechanism behind these broad spectrum of phenotype. Despite the availability of all mice models mimicking differents Gaucher disease phenotypes, a completely reliable animal model does not exist and the pathogenic alterations occuring during early life stages can not be explored and elucidated yet. The aim of my PhD project was to investigate the bone pathogenetic mechanisms of Gaucher disease using a new animal model. To address this purpose, I’ve used a zebrafish model due to its easy manipulation and the transparency of the embryos that allow to follow all the early developmental stages. Using a morpholino-mediated knockdown approach and a stable genetic mutant line, I could investigate what was the effect of deficiency of the enzyme Gba1 during early stages of embryonic development. Moreover, the defects observed in these zebrafish models resemble the most common GD phenotypes, like hepatosplenomegaly, anemia and skeletal deformity, making them good models to study the molecular mechanisms of the bone phenotype. By analyzing the main molecular markers involved in bone development, as col10a1, runx2b and osx, I could point out that bone defects observed in these models are determined by an alteration in the osteoblasts differentiation process. Also, using zebrafish transgenic lines in which fluorescent proteins such as GFP are expressed under the control of specific promoters for major molecular signaling pathways, allowed me to identify alterations of Wnt and BMP due to deficiency of the enzyme β-glucocerebrosidase. In this work, characterization of a novel animal model for the study of Gaucher disease, highlighted that dysfunction of the lysosomal enzyme β-glucocerebrosidase can lead to alteration of major molecular signaling involved in the embryonic development, such as Wnt and BMP. Both these pathways have an important role in the formation and maintenance of osteoblasts lineage and early defects in these signal during embryogenesis could lead to defect in the differentiation program of mesenchymal stem cells progenitors. The results showed in this doctoral thesis, highlight for the first time the early involvement of two pathways, the Wnt and BMP signaling, behind the bone pathogenesis of Gaucher disease.