Roberta Gioia

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: A new target for osteogenesis imperfecta pharmacological therapy

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well‐characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony‐forming unit‐adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony‐forming unit‐osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder. STEM CELLS2012;30:1465–1476

TREX1 C-terminal frameshift mutations in the systemic variant of retinal vasculopathy with cerebral leukodystrophy

AbstractRetinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset disorder caused by C-terminal heterozygous frameshift (fs) mutations in the human 3′–5′ DNA exonuclease TREX1. Hereditary systemic angiopathy (HSA) is considered a variant of RVCL with systemic involvement of unknown genetic cause, described in a unique family so far. Here we describe the second case of RVCL with systemic involvement, characterized by cerebral calcifications and pseudotumoral lesions, retinopathy, osteonecrosis, renal and hepatic failure. The genetic screening of TREX1 in this patient revealed the novel heterozygous T270fs mutation on the C-terminal region. On the same gene, we found the V235fs mutation, formerly shown in RVCL, in one patient previously reported with HSA. These mutations lead to important alterations of the C-terminal of the protein, with the loss of the transmembrane helix (T270fs) and the insertion of a premature stop codon, resulting in a truncated protein (V235fs). Functional analysis of T270fs-mutated fibroblasts showed a prevalent localization of the protein in the cytosol, rather than in the perinuclear region. RVCL with systemic involvement is an extremely rare condition, whose diagnosis is complex due to multiorgan manifestations, unusual radiological and histopathological findings, not easily attributable to a single disease. It should be suspected in young adults with systemic microangiopathy involving retina, liver, kidney, bones and brain. Here we confirm the causative role played by TREX1 autosomal dominant fs mutations disrupting the C-terminal of the protein, providing a model for the study of stroke in young adults.

Characterization of stress response in human retinal epithelial cells.

The pathogenesis of age‐related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE‐19) cells to 5‐(N,N‐hexamethylene)amiloride (HMA), an inhibitor of Na+/H+ exchangers. We observed that ARPE‐19 cells treated with HMA are unable to activate ‘classical’ apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli.

Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin.

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases.

Altered cytoskeletal organization characterized lethal but not surviving Brtl+/− mice: insight on phenotypic variability in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-β signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment.

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

Differential response to intracellular stress in the skin from osteogenesis imperfecta Brtl mice with lethal and non lethal phenotype: A proteomic approach☆

Phenotypic variability in the presence of an identical molecular defect is a recurrent feature in heritable disorders and it was also reported in osteogenesis imperfecta (OI). OI is a prototype for skeletal dysplasias mainly caused by mutations in the two genes coding for type I collagen. No definitive cure is available for this disorder, but the understanding of molecular basis in OI phenotypic modulation will have a pivotal role in identifying possible targets to develop novel drug therapy. We used a functional proteomic approach to address the study of phenotypic variability using the skin of the OI murine model Brtl. Brtl mice reproduce the molecular defect, dominant transmission and phenotypic variability of human OI patients. In the presence of a Gly349Cys substitution in α1(I)-collagen Brtl mice can have a lethal or a moderately severe outcome. Differential expression of chaperones, proteasomal subunits, metabolic enzymes, and proteins related to cellular fate demonstrated that a different ability to adapt to cellular stress distinguished mutant from wild-type mice and mutant lethal from surviving mutant animals. Interestingly, class discovery analysis identified clusters of differentially expressed proteins associated with a specific outcome, and functional analysis contributed to a deeper investigation into biochemical and cellular pathways affected by the disease. This article is part of a Special Issue entitled: Translational Proteomics.

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta.

Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta

Abstract Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.