SR proteins and the nonsense-mediated decay mechanism are involved in human GLB1 gene alternative splicing

<p>Abstract</p> <p>Background</p> <p>The human <it>GLB1 </it>gene is known to give rise to two alternatively spliced mRNAs, which encode two different proteins: lysosomal β-galactosidase (β-gal) and elastin-binding protein (EBP). The β-gal transcript includes the 16 exons of the <it>GLB1 </it>gene. In the EBP transcript, exons 3, 4 and 6 are skipped, while exon 5 has a different reading frame. However, little is known on how this alternative splicing is regulated.</p> <p>Findings</p> <p>Cycloheximide treatment of HeLa cells and human fibroblasts revealed the presence of new transcripts that are otherwise degraded by nonsense-mediated decay (NMD). A minigene carrying the exons involved in the alternative splicing of <it>GLB1 </it>was constructed. Improving the acceptor-site scores of exons 3 or 4 increased the relative inclusion of these exons, but did not stop them being skipped in some transcripts. Overexpression of different SR proteins altered the relative proportion of the different transcripts produced by the minigene, indicating a possible mechanism for the regulation of the alternative splicing of <it>GLB1</it>. Finally, a comparison of this gene among different species was performed.</p> <p>Conclusion</p> <p>In the processing of the <it>GLB1 </it>RNA several transcripts are generated, but only those with a correct reading frame are not degraded. The differential inclusion/exclusion of exons could be partially explained by the relatively weak splice sites in the exons involved. Different SR proteins have an effect on the process of skipping of these exons, at least in the minigene conditions, indicating a possible mechanism for the regulation of the alternative splicing of the <it>GLB1 </it>gene.</p

Sanfilippo syndrome: molecular basis, disease models and therapeutic approaches

Sanfilippo syndrome or mucopolysaccharidosis III is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. The exact molecular and cellular mechanisms by which impaired degradation and storage lead to cellular dysfunction and neuronal degeneration are still not fully understood. Here, we compile the knowledge on this issue and review all available animal and cellular models that can be used to contribute to increase our understanding of Sanfilippo syndrome disease mechanisms. Moreover, we provide an update in advances regarding the different and most successful therapeutic approaches that are currently under study to treat Sanfilippo syndrome patients and discuss the potential of new tools such as induced pluripotent stem cells to be used for disease modeling and therapy development

EXTL2 and EXTL3 inhibition with siRNAs as a promising substrate reduction therapy for Sanfilippo C syndrome

Sanfilippo syndrome is a rare lysosomal storage disorder caused by an impaired degradation of heparan sulfate (HS). It presents severe and progressive neurodegeneration and currently there is no effective treatment. Substrate reduction therapy (SRT) may be a useful option for neurological disorders of this kind, and several approaches have been tested to date. Here we use different siRNAs targeting EXTL2 and EXTL3 genes, which are important for HS synthesis, as SRT in Sanfilippo C patients' fibroblasts in order to decrease glycosaminoglycan (GAG) storage inside the lysosomes. The results show a high inhibition of the EXTL gene mRNAs (around 90%), a decrease in GAG synthesis after three days (30-60%) and a decrease in GAG storage after 14 days (up to 24%). Moreover, immunocytochemistry analyses showed a clear reversion of the phenotype after treatment. The in vitro inhibition of HS synthesis genes using siRNAs shown here is a first step in the development of a future therapeutic option for Sanfilippo C syndrome

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

Generation of two NAGLU-mutated homozygous cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo B syndrome

Mutations in the NAGLU gene cause Sanfilippo B syndrome (mucopolysaccharidosis IIIB), a rare lysosomal storage disorder whose main symptom is a severe and progressive neurodegeneration for which no treatment is still available. Here, we generated two homozygous NAGLU-mutated cell lines using CRISPR/Cas9 editing in a healthy human induced pluripotent stem cell (hiPSC) line. These novel cell lines express pluripotency specific markers and maintain their capability to differentiate into all three germ layers in vitro while exhibit a normal karyotype. These mutated lines in combination with the isogenic control line will be useful to model in vitro Sanfilippo B syndrome

Mapping the genetic and clinical characteristics of Gaucher disease in the Iberian Peninsula

<p>Abstract</p> <p>Background</p> <p>Gaucher disease (GD) is due to deficiency of the glucocerebrosidase enzyme. It is panethnic, but its presentation reveals ethnicity-specific characteristics.</p> <p>Methods</p> <p>We evaluated the distribution, and clinical and genetic characteristics of GD patients in the Iberian Peninsula (IP). We analysed geographical distribution, demographic, genetic and clinical data, age at diagnosis, type, and years of therapy in 436 GD patients from the IP.</p> <p>Results</p> <p>The prevalence of GD was 1/149,000 inhabitants; 88.3% were type 1, 6.7% type 2, and 5.0% type 3. The mean age at diagnosis in type 1 was 28.7 years. A total of 72.7% were classified as having mild forms, 25.5% moderate, and 1.7% severe. Anemia and thrombocytopenia were present in 56% and 55%, respectively. Bone disease and hepatomegaly were reported in 62% and 68%, respectively, and were more likely in asplenic than in non-splenectomized patients. Sixty-nine mutant alleles were identified, and five mutations accounted for 75% of the <it>GBA </it>alleles. Several patients described in our series had interesting phenotypes. A total of 58.7% of patients had received enzyme replacement therapy and 12.6% were treated with miglustat.</p> <p>Conclusions</p> <p>A broad spectrum of <it>GBA </it>mutations is present in the IP, with 98.2% of type 1 GD being mild and 23.0% never treated. These data highlight genetic and phenotypic heterogeneities among geographic populations.</p

Pharmacological inhibition of soluble epoxide hydrolase protects cognitive impairment in a Niemann-Pick mice model

Niemann-Pick type C (NPC) disease is a childhood autosomal recessive inherited rare neurodegenerative disease characterized by accumulation of cholesterol and glycosphingolipids and where the autophagy-lysosome system and inflammatory processes are implicated in the pathogenesis of the disease. We follow a novel approach to deal with NPC disease, by modulating key features of the disease such as inflammation and autophagy, through inhibition of soluble epoxide hydrolase (sEH)

Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations

Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides. In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome. Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding. We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications

Neuronal and astrocytic differentiation from Sanfilippo C syndrome iPSCs for disease modeling and drug development

Sanfilippo syndrome type C (mucopolysaccharidosis IIIC) is an early-onset neurodegenerative lysosomal storage disorder, which is currently untreatable. The vast majority of studies focusing on disease mechanisms of Sanfilippo syndrome were performed on non-neural cells or mouse models, which present obvious limitations. Induced pluripotent stem cells (iPSCs) are an efficient way to model human diseases in vitro. Recently developed transcription factor-based differentiation protocols allow fast and efficient conversion of iPSCs into the cell type of interest. By applying these protocols, we have generated newneuronal and astrocyticmodels of Sanfilippo syndrome using our previously established disease iPSC lines. Moreover, our neuronal model exhibits disease-specific molecular phenotypes, such as increase in lysosomes and heparan sulfate. Lastly, we tested an experimental, siRNA-based treatment previously shown to be successful in patients' fibroblasts and demonstrated its lack of efficacy in neurons. Our findings highlight the need to use relevant human cellular models to test therapeutic interventions and shows the applicability of our neuronal and astrocyticmodels of Sanfilippo syndrome for future studies on disease mechanisms and drug development

Activity and high-order effective connectivity alterations in Sanfilippo C patient-specific neuronal networks

Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration