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

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.Le patologie da accumulo lisosomiale sono malattie metaboliche rare a carattere ereditario determinate da carenze di specifici enzimi o trasportatori lisosomiali, che hanno complessivamente un’incidenza di ~1/7000 nuovi nati nella popolazione mondiale. Al giorno d’oggi, almeno 50 disordini genetici sono causati da difetti in enzimi lisosomiali, che determinano l’incompleta degradazione e/o il riciclaggio di molecole a livello intracellulare con conseguente accumulo all’interno del lisosoma dei substrati enzimatici. Nonostante la presenza di proteine lisosomiali in quasi tutti i tessuti ed organi del corpo, l’accumulo del materiale non digerito è generalmente limitato solo a quelle cellule, tessuti od organi nel quale il ricambio del substrato enzimatico è molto elevato. Questa caratteristica determina differenti fenotipi per le varie patologie da accumulo lisosomiale, in quanto diversi organi o cellule possono essere coinvolti. Tra queste patologie, la malattia di Gaucher è la più frequente con un’incidenza di 1 su 200.000 nati vivi nella popolazione mondiale. La frequenza di questa patologia, aumenta drasticamente a 1 su 850 all’interno della popolazione degli ebrei Ashkenazi (Europa dell’Est). Questa malattia è causata da mutazioni a carico del gene che codifica l’enzima lisosomiale β-glucocerebrosidase (GBA). Tali mutazioni determinano l’incorretto ripiegamento della proteina enzimatica che, di conseguenza, non è in grado di degradare il suo substrato, la glucosilceramide, che si accumula nel lisosoma. Una delle caratteristiche di questa patologia è la presenza delle così dette “cellule di Gaucher”, ovvero macrofagi ad elevato contenuto di substrato non degradato, in differenti tessuti. Insieme alla presenza di questi macrofagi alterati, pazienti affetti dalla malattia di Gaucher presentano ingrossamento di fegato e milza (epatosplenomegalia), anemia, trombocitopenia e gravi disfunzioni a carico del sistema scheletrico quali osteonecrosi, riduzione della densità ossea, dolori cronici e frequenti fratture a carico delle ossa lunghe. Si possono distinguere tre sottocategorie di pazienti affetti da GD, generalmente classificati sulla base della presenza e gravità dei difetti a carico del sistema nervoso centrale (SNC). I pazienti affetti da GD di tipo I sono i più frequenti, hanno un’insorgenza della patologia in età tardiva ma non presentano coinvolgimento del SNC. I pazienti affetti da GD di tipo II, invece, manifestano i primi sintomi della malattia fin nei primi anni di vita e spesso i gravi difetti a carico del sistema nervoso possono portare alla morte del paziente. La terza categoria di pazienti, GD tipo III, manifestano i sintomi durante l’età infantile e i difetti neurologici sono meno gravi rispetto a quelli dei pazienti di tipo II. Al giorno d’oggi, questa classificazione basata sulla presenza di difetti neurologici è poco credibile a causa della presenza di fenotipi diversificati all’interno della stessa sottocategoria di pazienti. Il concetto di uno spettro continuo di fenotipi che variano dal meno grave (GD tipo I) al più severo (GD tipo II e III) è più appropriato per descrivere questa patologia. La terapia maggiormente utilizzata per il trattamento della sintomatologia di questa malattia è la terapia enzimatica sostitutiva (ERT), che consiste nella somministrazione di un enzima ricombinante in grado si sopperire alla mancanza della β-glucocerebrosidasi. Nonostante sia ben tollerata dalla maggioranza dei pazienti e sia in grado di far regredire l’ingrossamento di fegato e milza, l’anemia e la trombocitopenia, tale terapia ha effetti davvero limitati sui difetti scheletrici e neurologici. Nel corso degli anni, diversi modelli murini sono stati sviluppati per cercare di comprendere quali siano i meccanismi patogenetici della malattia che inducono questo ampio spettro di fenotipi. Sfortunatamente, la maggior parte di questi modelli animali non sono vitali o non manifestano tutti i difetti della malattia. Lo scopo del mio progetto di dottorato è stato quello di generare un nuovo modello animale per comprendere i meccanismi patogenetici a monte dei difetti ossei della malattia di Gaucher. A tal fine, mi sono avvalsa dell’uso dello zebrafish per la sua facilità di manipolazione e la trasparenza delle uova che permettono di seguire lo sviluppo embrionale fin dalle prime fasi. Utilizzando la tecnica del morfolino e avvalendomi di un modello genetico mutante stabile in zebrafish, ho potuto studiare quale fosse l’effetto della mancanza dell’enzima Gba1 fin dalle prime fasi dello sviluppo embrionale. Questi modelli, inoltre, manifestano insieme ai principali difetti di questa patologia, come l’ingrossamento di milza e fegato e l’anemia, anche i difetti a carico del sistema scheletrico, rendendoli dei buoni modelli per studiare i meccanismi molecolari a monte del fenotipo osseo. Analizzando i principali marcatori molecolari coinvolti nello sviluppo osseo, come col10a1, runx2b e osx, ho potuto evidenziare che i difetti ossei osservati in questi modelli sono determinati da un difetto nel processo di differenziamento degli osteoblasti. Inoltre, l’utilizzo di linee transgeniche di zebrafish nelle quali proteine fluorescenti, come la GFP, sono espresse sotto il controllo di promotori specifici per le principali vie di segnale molecolari, mi ha permesso di individuare alterazioni a carico delle vie di segnale Wnt e BMP in conseguenza alla carenza dell’enzima β-glucocerebrosidasi. Con questo lavoro di dottorato, la caratterizzazione di un nuovo modello animale per lo studio della malattia di Gaucher ha permesso di evidenziare che, disfunzioni a carico di un enzima lisosomiale come la β-glucocerebrosidasi, può determinare alterazioni in segnali molecolari molto importanti per lo sviluppo embrionale, quali il Wnt ed il BMP. Entrambe queste vie molecolari svolgono ruoli importanti nel processo di formazione e mantenimento degli osteoblasti e alterazioni precoci di questi segnali durante l’embriogenesi possono determinare difetti nel processo di differenziamento cellulare da progenitori mesenchimali staminali. I risultati ottenuti durante questo lavoro di dottorato, hanno evidenziato per la prima volta il precoce coinvolgimento di due vie di segnale molecolari, il Wnt e il BMP, nella patogenesi ossea della malattia di Gaucher

Loss of lysosomal iduronate sulfatase function perturbs FGF signaling and the expression of downstream key osteogenic factors.

Mucopolisaccaridosis II (MPSII), also called Hunter syndrome, is a rare X-linked lysosomal storage disease caused by defects in the activity of the lysosomal enzyme iduronate-2-sulfatase (IDS). IDS is an ubiquitously expressed hydrolase that catalyses the removal of O-linked sulfates from glycosaminoglycans heparan and dermatan sulfate. The disease is multi-systemic and patients exhibit a wide spectrum of clinical features with a distinct early childhood-onset and chronic progressive course. Clinical manifestations include severe skeletal abnormalities, a marked cardiomyopathy and cardiac valve alterations. The current hypothesis suggests that loss of IDS function is tightly associated toa progressive accumulation of partially degraded glycosaminoglycans, which in turn is responsible for the multi-organ failure. However, the cascade of pathogenetic mechanisms is not well understood yet. We recently demonstrated that iduronate-2-sulfatase activity is critical during early vertebrate development (Moro et al.; 2010). We, therefore, hypothesize that major skeletal defects may occur during early development, before the onset of GAG accumulation. To explore this hypothesis we generated a fish model for Hunter syndrome in order to assess early molecular defects occurring as a consequence of IDS loss of function. To shed light on bone impairments we have analyzed several key markers on fish morphants at different developmental stages. Moreover, we carried out IDS loss of function analysis in transgenic reporter lines for major developmental signaling pathways. We found a significant decrease of FGF signaling in morphants, and altered expression of key transcription factors during osteogenesis. Therefore, our preliminary results support the hypothesis that IDS deficiency causes an early dysregulation of the FGF signaling pathway which may be associated with impaired expression of genes involved in bone development

Glucocerebrosidase deficiency in zebrafish leads to primary osteogenic defects.

Ostopenia and other skeletal complications have a considerable influence on the morbidity of patients affected by Gaucher disease (GD). Despite the development of novel therapeutic approaches, bone response to current enzymatic replacement is slow and bone manifestations may worsen or persist in affected patients. The pathogenetic mechanisms responsible for bone alterations are currently unknown. Aim: This study was aimed to analyze bone defects occurring in a fish model with a morpholino-induced glucocerebrosidase (GBA) deficiency. Moreover, key molecular pathways affected by GBA loss of function were investigated. Methods: We used a set of transgenic biosensor fish to identify the involvement of targeted cell signaling pathways as a consequence of GBA deficiency. Confocal live imaging on cartilage and bone specific trangenics and transcriptomic analysis were additionaly used to perform a detailed spatiotemporal characterization of key molecular genes affected by GBA loss of function. Results. Our study suggests that well-defined osteogenic defects occur during early life stages in fish larvae lacking correct glucocerebrosidase functional activity. Moreover, when testing a set of biosensor reporter fish, we found a specific impairment of signaling pathways, which are good candidates in bone formation. Discussion. An early onset of cell signaling alterations detected in our fish model supports a view that GBA loss of function leads to premature primary bone defects , which significantly compromise bone remodeling in later stages. This study emphasizes the use of an early therapeutic intervention in GD affected children and suggests potential novel key targets for therapy of the skeletal disorders in GD

Perturbations in cell signaling elicit early cardiac defects in mucopolysaccharidosis type II

Morphogens release and activity can be negatively affected by an impaired glycosaminoglycans (GAGs) turnover and proteoglycans assembly in the extracellular matrix, leading to altered tissue morphogenesis. In this work, we show that loss of Iduronate-2-sulfatase (IDS) activity, affecting GAGs catabolism and responsible for a life-threatening valvulopathy in mucopolysaccharidosis type II (MPSII), triggers early Sonic Hedgehog (Shh) and Wnt/\u3b2-catenin signaling defects, leading to aberrant heart development and atrioventricular valve formation in a zebrafish model. In addition, we consistently found impaired Shh signaling activity and cardiac electrophysiological abnormalities in IDS knockout mice at postnatal stages before any evident massive GAGs accumulation. These results suggest that IDS activity substantially affect cardiac morphogenesis through impaired Shh signaling and document an unexplored role of the enzyme in the fine-tuning of cell signaling pathways

Generation and application of signaling pathway reporter lines in zebrafish

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach