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

Abstract

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.