Bone development and remodeling in metabolic disorders

There are many metabolic disorders that present with bone phenotypes. In some cases, the pathological bone symptoms are the main features of the disease whereas in others they are a secondary characteristic. In general, the generation of the bone problems in these disorders is not well understood and the therapeutic options for them are scarce. Bone development occurs in the early stages of embryonic development where the bone formation, or osteogenesis, takes place. This osteogenesis can be produced through the direct transformation of the pre-existing mesenchymal cells into bone tissue (intramembranous ossification) or by the replacement of the cartilage by bone (endochondral ossification). In contrast, bone remodeling takes place during the bone's growth, after the bone development, and continues throughout the whole life. The remodeling involves the removal of mineralized bone by osteoclasts followed by the formation of bone matrix by the osteoblasts, which subsequently becomes mineralized. In some metabolic diseases, bone pathological features are associated with bone development problems but in others they are associated with bone remodeling. Here, we describe three examples of impaired bone development or remodeling in metabolic diseases, including work by others and the results from our research. In particular, we will focus on hereditary multiple exostosis (or osteochondromatosis), Gaucher disease, and the susceptibility to atypical femoral fracture in patients treated with bisphosphonates for several years

Generació d'un model cel·lular osteoblàstic i aproximacions terapèutiques per a la malaltia de Gaucher

[cat] La malaltia de Gaucher (GD) forma part d'un conjunt de malalties que es coneixen amb el nom de "malalties d'acumulació lisosómica". La GD n'és la més prevalent i en la majoria de casos és causada per mutacions en el gen GBA1, que codifica l'enzim glucocerebrosidasa (GBA1) la qual hidrolitza, principalment, l'esfingolipid glucosilceramida (GlcCer) en glucosa i ceramida. En la GD aquest enzim presenta una activitat deficient i la GlcCer s'acumula en els lisosomes dels macròfags que formen les anomenades "cèl. lules Gaucher". La GD és autosòmica recessiva i compren 3 subtipus clínics, en funció de la presència i el grau de les manifestacions neurològiques: la de tipus 1 (GD1; no neuronopàtica), la de tipus 2 (GD2; neuronopàtica aguda), i la de tipus 3 (GD3; neuronopàtica subaguda). La GD1 es caracteritza per presentar problemes viscerals, hematològics i ossis. La patofisiologia de les manifestacions òssies d'aquesta és poc coneguda però tot apunta a que les cèl. lules de Gaucher, el sistema immunològic i les cèl• lules òssies hi juguen un paper important. De totes les aproximacions terapèutiques pel tractament de la malaltia, cal destacar que la teràpia de substitució enzimàtica (ERT) és la més utilitzada, però en els últims 10 anys la teràpia amb xaperones farmacològiques ha esdevingut una opció atractiva per millorar el plegament i transport d'aquells enzims mutats que ho requereixin. El treball que es presenta en aquesta tesi doctoral ha permès identificar algunes xaperones que presenten una bona capacitat d'augmentar l'activitat enzimàtica d'algunes glucocerebrosidases mutades. A més, s'ha elaborat un model mesenquimal i osteoblàstic de la GD1 en el que s'està avaluant la implicació de la deficient activitat GBA1 en la diferenciació osteoblàstica i, indirectament, en l'activació de la formació dels osteoclasts.[eng] Gaucher disease (GD) is the most prevalent lysosomal storage disorder and in most cases is caused by mutations in the GBA1 gene, which encodes the enzyme glucocerebrosidase (GBA1). This protein mainly hydrolyzes the sphingolipid glucosylceramide (GlcCer) into glucose and ceramide. Gaucher patients have a deficient GBA1 enzyme activity which causes that GlcCer accumulates in lysosomes of macrophages forming the so-called "Gaucher cells". GD is an autosomal recessive disorder and can be classified in three clinical subtypes, depending on the presence and degree of neurological manifestations: type 1 (GD1, no neuronopathic), type 2 (GD2; acute neuronopathic) and type 3 (GD3; subacute neuronopathic). The GD1 is characterized by visceral, haematological and bone manifestations. The pathological mechanisms of bone alterations in GD are still poorly understood. But it seems that Gaucher cells, immunitary system and bone cells play an important role. Of the current therapeutic approaches for treatment of the disease, enzyme replacement therapy (ERT) is the most commonly used. However, in the last 10 years pharmacological chaperones therapy has become an attractive option to improve the folding and transport of some mutated enzymes. The aim of this work has been the identification and characterization of compounds with positive effect on the activity of some mutant glucocerebrosidases to act as chaperones. Also, a model of mesenchymal and osteoblastic GD1 has been generated. In this model is being assessed the involvement of deficient GBA1 activity in the osteoblast differentiation process and, indirectly, in the capacity of osteoclasts formation.[spa] La enfermedad de Gaucher (GD) forma parte de un conjunto de enfermedades que reciben el nombre de "enfermedades de acumulo lisosómico". La GD es la más prevalente y en la mayoría de los casos es debida a mutaciones en el gen GBA1 que codifica la enzima glucocerebrosidasa (GBA1) la cual hidroliza, principalmente el esfingolípido glucosilceramida (GlcCer) en glucosa y ceramida. En la GD ésta enzima presenta una actividad deficiente y la GlcCer se acumula en los lisosomas de los macrófagos formando las células Gaucher. La GD es una patología autosómica recesiva e incluye 3 subtipos clínicos, en función de la presencia y el grado de las manifestaciones neurológicas: tipo 1 (GD1; no neurológica), tipo 2 (GD2; neurológica aguda), y tipo 3 (GD3; neurológica subaguda). La GD1 se caracteriza por presentar problemas viscerales, hematológicos y óseos. La patofisiología de las manifestaciones óseas de la GD1 es poco conocida pero parece ser que las células Gaucher, el sistema inmunológico y las células óseas juegan un papel importante. De todas las aproximaciones terapéuticas para el tratamiento de la GD, la terapia de substitución enzimática (ERT) es la más habitual, pero en los últimos 10 años la terapia con chaperones farmacológicas ha resultado ser una buena opción para mejorar el plegamiento y el transporte de aquellos enzimas mutados que lo requieren. El trabajo que se presenta en ésta tesis doctoral ha permitido identificar algunos productos que presentan una buena capacidad de aumentar la actividad de algunas glucerebrosidasas mutadas y actuar por lo tanto como chaperonas farmacológicas. Además, se ha elaborado un modelo mesenquimal y osteoblástico de la GD1 en el que se está evaluando la implicación de la deficiente actividad de la enzima GBA1 en la diferenciación osteoblástica y, indirectamente, en la activación de la formación de los osteoclastos

Induced pluripotent stem cell models of lysosomal storage disorders

Induced pluripotent stem cells (iPSCs) have provided new opportunities to explore the cell biology and pathophysiology of human diseases, and the lysosomal storage disorder research community has been quick to adopt this technology. Patient-derived iPSC models have been generated for a number of lysosomal storage disorders, including Gaucher disease, Pompe disease, Fabry disease, metachromatic leukodystrophy, the neuronal ceroid lipofuscinoses, Niemann-Pick types A and C1, and several of the mucopolysaccharidoses. Here, we review the strategies employed for reprogramming and differentiation, as well as insights into disease etiology gleaned from the currently available models. Examples are provided to illustrate how iPSC-derived models can be employed to develop new therapeutic strategies for these disorders. We also discuss how models of these rare diseases could contribute to an enhanced understanding of more common neurodegenerative disorders such as Parkinson’s disease, and discuss key challenges and opportunities in this area of research

Pseudopeptidic fluorescent on-off pH sensor based on pyrene excimer emission: Imaging of acidic cellular organelles

A pseudopeptidic compound comprising a central C2 symmetric bis(aminoamide) moiety derived from the amino acid serine and two pyrene groups at the ends, Et(SerPyr)2, has been efficiently prepared. In aqueous solution, it displays fluorescence bands typical of both monomer and excimer pyrene emission. In particular, the emission at 490 nm, due to the excimer, is strongly pH-dependent, with a linear 4.7-fold increase from pH 6.0 to pH 4.2. This feature makes Et(SerPyr)2 suitable for intracellular pH sensing in the acidic range. A fluorescence microscopy study revealed that Et(SerPyr)2 allows the visualization of acidic organelles in human lung adenocarcinoma A549 cells, as well as their pH changes over time. Other advantages of this probe include its easy and modular synthesis, good photostability, high quantum yield, fast optical response, large Stokes shift (ca. 145 nm) and low cytotoxicity.We acknowledge financial support from the Ministry of Economy and Competitiveness, Spain (CTQ2012-38543-C03, CTQ2014-54743-R, CTQ2015-68429-R and CTQ2015-70117-R projects), Generalitat de Catalunya (AGAUR, 2014 SGR 231), Generalitat Valenciana (PROMETEO/2012/020) and UJI (PI-1B2013-38). We thank Mr. Pedro Rayo for excellent technical assistance

Gaucher disease: Biochemical and molecular findings in 141 patients diagnosed in Greece

Gaucher disease (GD) is characterized by a marked phenotypic and genetic diversity. It is caused by the functional deficiency of the lysosomal enzyme β-glucocerebrosidase (GCase), which in most instances results from mutations in the GBA1 gene and over 500 different disease causing mutations have been described. We present the biochemical and molecular findings in 141 GD cases (14 were siblings) with the three types of the disorder diagnosed in Greece over the last 35 years. 111/141 (78%) GD patients were of Greek origin. The remaining patients were Albanian (24/141; 17%), Syrian (2/141; 1.4%), Egyptian (2/141; 1.4%), Italian (1/141; 0.7%) and Polish (1/141; 0.7%). Mutation analysis identified 28 different mutations and 37 different genotypes. Seven of the mutations were not previously reported (T231I, D283N, N462Y, LI75P, F81L, Y135S and T482K). The most frequent mutations were N370S, D409H;H255Q and L444P. Mutation D409H;H255Q was only identified in Greek and Albanian patients. Sixteen mutations, including the novel ones, were identified only in one allele. Although the N370S mutation was identified only in type 1 patients, not all of type 1 patients carried this mutation. Our results highlight the heterogeneity of Gaucher disease and support the Balkan origin of the double mutant allele D409H;H255Q

Investigation of original multivalent iminosugars as pharmacological chaperones for the treatment of Gaucher disease

Multivalent iminosugars conjugated with a morpholine moiety and/or designed as prodrugs have been prepared and evaluated as new classes of pharmacological chaperones for the treatment of Gaucher disease. This study further confirms the interest of the prodrug concept and shows that the addition of a lysosome-targeting morpholine unit into iminosugar cluster structures has no significant impact on the chaperone activity on Gaucher cell

Glucocerebrosidase enhancers for selected Gaucher disease genotypes by modification of α-1-C-substituted imino-D-xylitols (DIXs) by click chemistry

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9-DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α-1-C-propargyl-1,5-dideoxy-1,5-imino-D-xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β-glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX-28 (α-1-C-[(1-(3-trimethylsilyl)propyl)-1H-1,2,3-triazol-4-yl)methyl]-1,5-dideoxy-1,5-imino-D-xylitol), bearing the 3-trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM. Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non-competitive inhibitors, with the exception of the mixed-type inhibitor DIX-2

Expression profiling of microRNAs in human bone tissue from postmenopausal women

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.This work was supported by the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF; RD12/0043/0022) and Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES; CB16/10/00245), and the Grants PI10/01537, PI13/00116, and PI13/00444 from FIS (Carlos III Health Institute, Science and Innovation Ministry); SAF2016-75948-R, from Ministerio de Economia y Competitividad, and 2014SGR-932 from Generalitat de Catalunya. FEDER funds also supported this study