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

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

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

Genetic and molecular analysis or Sanfilippo C syndrome. Generation of a neuronal model using human induced pluripotent stem (iPS) cells and therapeutic strategies

[cat] La síndrome de Sanfilippo és una malaltia monogènica hereditària que presenta una severa i progressiva neurodegeneració que s’inicia durant els primers anys de vida dels pacients. Està causada per mutacions en el gen HGSNAT, identificat l’any 2006 en el cromosoma 8, el qual codifica per l’enzim acetil-CoA α-glucosaminida N-acetiltransferasa, una proteïna de membrana lisosomal. La seva funció és acetilar les glucosamines terminals de les cadenes de heparà sulfat que estan sent degradades. Quan la proteïna està mutada, es promou l’acumulació de cadenes d’heparà sulfat parcialment degradades en els lisosomes, les quals augmenten en nombre i mida, provocant la seva disfunció. Per aquesta raó, la síndrome de Sanfilippo es classifica com a malaltia d’acumulació lisosòmica, en concret com a una mucopolisacaridosi, degut a la naturalesa del substrat acumulat. L’heparà sulfat és un dels glicosaminoglicans, anteriorment coneguts com a mucopolisacàrids, que es troba en la matriu extracel·lular formant part dels proteoglicans. Aquestes molècules participen en diferents i importants funcions cel·lulars com ara la migració i l’adhesió. La desregulació de la seva homeòstasi provoca una disfunció de múltiples processos cel·lulars. Aquesta tesi contribueix de manera important a l’estudi molecular de la malaltia. S’ha portat a terme un anàlisi mutacional i la conseqüent caracterització de les mutacions identificades per tal d’aprofundir en el coneixement de la malaltia. Per altra banda, s’han provat diferents possibles aproximacions terapèutiques com un primer pas en l’obtenció d’una teràpia exitosa per a aquesta devastadora malaltia neurodegenerativa per la qual encara no hi ha un tractament efectiu. Finalment, s’ha generat un model neuronal utilitzant cèl·lules mare pluripotents induïdes. Aquest model serà d’utilitat per estudiar i entendre els processos moleculars i cel·lulars que contribueixen al desenvolupament de la malaltia a nivell de la neurona i representarà una ajuda molt valuosa en la cerca de tractaments efectius.[eng] Sanfilippo C syndrome is a lysosomal storage disorder that presents an autosomal recessive inheritance pattern and is caused by mutations in the HGSNAT gene, identified in 2006 in the chromosome 8. This gene codes for a lysosomal transmembrane protein, acetyl-CoA α-glucosaminide N-acetyltransferase, which acetylates the terminal glucosamine in the heparan sulfate chain during its degradation, a crucial step previous to the action of the next enzyme of the pathway. Heparan sulfate is a glycosaminoglycan localized in the extracellular matrix being part of proteoglycans and participate in several and important cellular processes. The HGSNAT protein dysfunction promotes the storage of partially degraded heparan sulfate chains inside the lysosomes, causing an alteration in many different cellular processes and affecting especially neurons. This fact promotes the progressive and severe neurodegeneration that appears during childhood as the main phenotypic feature in patients. This thesis represents an important study on the molecular basis of Sanfilippo C syndrome. Firstly, a mutational analysis has been performed, identifying the mutations causing the disease in 15 patients from different origins. A total of 13 different mutations have been found, seven of which were not previously described. The pathogenicity of four missense mutations identified has been proved by measuring the enzyme activity after in vitro expression of the proteins. Also, the pathogenicity of five mutations affecting different conserved splice sites has been demonstrated since they were shown to alter the splicing process. It has been established that two prevalent mutations in Spanish patients accounts for almost the 70% of the total and, using a haplotype analysis, a single origin for each of them has been suggested. Secondly, some therapeutic approaches have been tested, as a first step in the pursuit of an effective therapy that to date does not exist for this disease. The use of modified U1 snRNAs that present a higher complementarity to the mutated splice site sequences than the wild type U1 snRNA has been proved to partially restore the normal splicing process for one of the splicing mutations analyzed. In the case of missense mutations or mutations that result in the loss of some amino acids, this work suggests the possibility to use glucosamine as a chaperone to prevent the incorrect folding of the protein and to facilitate the trafficking process of the protein from the endoplasmic reticulum to the Golgi apparatus. Finally, the use of siRNAs to inhibit important genes in the heparan sulfate synthetic pathway, specifically the EXTL genes, has been suggested as a possible substrate reduction therapy, with the best results obtained on the inhibiton of EXTL2 expression. Finally, during this thesis, a neuronal model for Sanfilippo C syndrome has been obtained. This represents an important progress in the study of this disease since to date, neither cellular nor animal model exists. To achieve this goal, fibroblasts from two different Sanfilippo C patients have been reprogrammed to produce induced pluripotent cells that later have been differentiated to neurons. It has been demonstrated that these neurons present the typical phenotypic features of the disease such as the lack of enzyme activity, the heparan sulfate storage, the increased size and number of lysosomes, an alteration in the autophagy process and an increase in the number of apoptotic cells. Using specific experiments to study the neuronal activity in these cultures, a progressive decrease in the patients’ neurons activity and problems in the maintenance of the developed neuronal networks has been detected. This model will be a good platform to study profoundly the molecular, cellular and brain basis of the disease and to develop and test different therapeutic approaches for Sanfilippo C syndrome in the cellular type most affected in patients

Genetic and molecular analysis or Sanfilippo C syndrome. Generation of a neuronal model using human induced pluripotent stem (iPS) cells and therapeutic strategies

Sanfilippo C syndrome is a lysosomal storage disorder that presents an autosomal recessive inheritance pattern and is caused by mutations in the HGSNAT gene, identified in 2006 in the chromosome 8. This gene codes for a lysosomal transmembrane protein, acetyl-CoA α-glucosaminide N-acetyltransferase, which acetylates the terminal glucosamine in the heparan sulfate chain during its degradation, a crucial step previous to the action of the next enzyme of the pathway. Heparan sulfate is a glycosaminoglycan localized in the extracellular matrix being part of proteoglycans and participate in several and important cellular processes. The HGSNAT protein dysfunction promotes the storage of partially degraded heparan sulfate chains inside the lysosomes, causing an alteration in many different cellular processes and affecting especially neurons. This fact promotes the progressive and severe neurodegeneration that appears during childhood as the main phenotypic feature in patients. This thesis represents an important study on the molecular basis of Sanfilippo C syndrome. Firstly, a mutational analysis has been performed, identifying the mutations causing the disease in 15 patients from different origins. A total of 13 different mutations have been found, seven of which were not previously described. The pathogenicity of four missense mutations identified has been proved by measuring the enzyme activity after in vitro expression of the proteins. Also, the pathogenicity of five mutations affecting different conserved splice sites has been demonstrated since they were shown to alter the splicing process. It has been established that two prevalent mutations in Spanish patients accounts for almost the 70% of the total and, using a haplotype analysis, a single origin for each of them has been suggested. Secondly, some therapeutic approaches have been tested, as a first step in the pursuit of an effective therapy that to date does not exist for this disease. The use of modified U1 snRNAs that present a higher complementarity to the mutated splice site sequences than the wild type U1 snRNA has been proved to partially restore the normal splicing process for one of the splicing mutations analyzed. In the case of missense mutations or mutations that result in the loss of some amino acids, this work suggests the possibility to use glucosamine as a chaperone to prevent the incorrect folding of the protein and to facilitate the trafficking process of the protein from the endoplasmic reticulum to the Golgi apparatus. Finally, the use of siRNAs to inhibit important genes in the heparan sulfate synthetic pathway, specifically the EXTL genes, has been suggested as a possible substrate reduction therapy, with the best results obtained on the inhibiton of EXTL2 expression. Finally, during this thesis, a neuronal model for Sanfilippo C syndrome has been obtained. This represents an important progress in the study of this disease since to date, neither cellular nor animal model exists. To achieve this goal, fibroblasts from two different Sanfilippo C patients have been reprogrammed to produce induced pluripotent cells that later have been differentiated to neurons. It has been demonstrated that these neurons present the typical phenotypic features of the disease such as the lack of enzyme activity, the heparan sulfate storage, the increased size and number of lysosomes, an alteration in the autophagy process and an increase in the number of apoptotic cells. Using specific experiments to study the neuronal activity in these cultures, a progressive decrease in the patients’ neurons activity and problems in the maintenance of the developed neuronal networks has been detected. This model will be a good platform to study profoundly the molecular, cellular and brain basis of the disease and to develop and test different therapeutic approaches for Sanfilippo C syndrome in the cellular type most affected in patients.La síndrome de Sanfilippo és una malaltia monogènica hereditària que presenta una severa i progressiva neurodegeneració que s’inicia durant els primers anys de vida dels pacients. Està causada per mutacions en el gen HGSNAT, identificat l’any 2006 en el cromosoma 8, el qual codifica per l’enzim acetil-CoA α-glucosaminida N-acetiltransferasa, una proteïna de membrana lisosomal. La seva funció és acetilar les glucosamines terminals de les cadenes de heparà sulfat que estan sent degradades. Quan la proteïna està mutada, es promou l’acumulació de cadenes d’heparà sulfat parcialment degradades en els lisosomes, les quals augmenten en nombre i mida, provocant la seva disfunció. Per aquesta raó, la síndrome de Sanfilippo es classifica com a malaltia d’acumulació lisosòmica, en concret com a una mucopolisacaridosi, degut a la naturalesa del substrat acumulat. L’heparà sulfat és un dels glicosaminoglicans, anteriorment coneguts com a mucopolisacàrids, que es troba en la matriu extracel·lular formant part dels proteoglicans. Aquestes molècules participen en diferents i importants funcions cel·lulars com ara la migració i l’adhesió. La desregulació de la seva homeòstasi provoca una disfunció de múltiples processos cel·lulars. Aquesta tesi contribueix de manera important a l’estudi molecular de la malaltia. S’ha portat a terme un anàlisi mutacional i la conseqüent caracterització de les mutacions identificades per tal d’aprofundir en el coneixement de la malaltia. Per altra banda, s’han provat diferents possibles aproximacions terapèutiques com un primer pas en l’obtenció d’una teràpia exitosa per a aquesta devastadora malaltia neurodegenerativa per la qual encara no hi ha un tractament efectiu. Finalment, s’ha generat un model neuronal utilitzant cèl·lules mare pluripotents induïdes. Aquest model serà d’utilitat per estudiar i entendre els processos moleculars i cel·lulars que contribueixen al desenvolupament de la malaltia a nivell de la neurona i representarà una ajuda molt valuosa en la cerca de tractaments efectius

Generation of two compound heterozygous HGSNAT-mutated lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo C syndrome

Sanfilippo C syndrome (Mucopolysaccharidosis IIIC) is a rare lysosomal storage disorder caused by mutations in the HGSNAT gene. It is characterized by a progressive and severe neurodegeneration, for which there is no treatment available. Here, we report the generation of two HGSNAT-mutated cell lines from a healthy human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 editing. These novel cell lines have a normal karyotype, express pluripotency specific markers and have the capability to differentiate into all three germ layers in vitro. These hiPSC lines will be useful for the generation of in vitro models of Sanfilippo C syndrome

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