Stem Cell Reports Ar ticle Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks Isaac Canals, 1,2,3 Jordi Soriano, 4 Javier G. Orlandi, 4 Roger Torrent, 3 Yvonne Richaud-Patin, 5,6 Senda Jime ´nez-Delgado, 5,6 Simone Merlin, 7 Antonia Follenzi, 7 Antonella Consiglio, 3,8 Lluı ¨sa Vilageliu, 1,2,3 Daniel Grinberg, 1,2,3, * and Angel Raya 5,6,9, * 1 Departament de Gene `tica, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain 2 Centro de Investigacio ´ n Biome ´dica en Red de Enfermedades Raras, 28029 Madrid, Spain 3 Institut de Biomedicina de la Universitat de Barcelona, 08028 Barcelona, Spain 4 Departament d’Estructura i Constituents de la Mate `ria, Universitat de Barcelona, 08028 Barcelona, Spain 5 Centre de Medicina Regenerativa de Barcelona and Control of Stem Cell Potency Group, Institut de Bioenginyeria de Catalunya, 08028 Barcelona, Spain 6 Centro de Investigacio ´ n Biome ´dica en Red en Bioingenierı ´a, Biomaterials y Nanomedicina, 28029 Madrid, Spain 7 Health Sciences Department, Universita’ del Piemonte Orientale, 28100 Novara, Italy 8 Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy 9 Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats, 08010 Barcelona, Spain *Correspondence: dgrinberg@ub.edu (D.G.), araya@cmrb.eu (A.R.) http://dx.doi.org/10.1016/j.stemcr.2015.08.016 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). SUMMARY 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 connec- tivity. 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 read- outs to screen compounds, which can prevent the onset of neurodegeneration. INTRODUCTION Sanfilippo syndrome, also known as mucopolysaccharido- sis type III (MPS III), is a lysosomal storage disorder (LSD) with an autosomal recessive inheritance pattern. Four different subtypes have been described (type A, OMIM 252900; type B, OMIM 252920; type C, OMIM 252930; and type D, OMIM 252940), which share clinical character- istics, including severe early onset CNS degeneration that typically results in death within the second or third decade of life (Valstar et al., 2008). Each subtype is caused by mu- tations in a different gene encoding for enzymes involved in the degradation pathway of the glycosaminoglycan (GAG) heparan sulfate (Neufeld and Muenzer, 2001). The lack of activity of any of these enzymes leads to the accu- mulation of partially degraded heparan sulfate chains within the lysosomes. Subtype C (MPS IIIC) is caused by mutations in the HGSNAT gene, encoding acetyl-CoA a-glucosaminide N-acetyltransferase (EC 2.3.1.78), a lyso- somal membrane enzyme. The prevalence of MPS IIIC ranges between 0.07 and 0.42 per 100,000 births, depend- ing on the population (Poupetova ´ et al., 2010). The HGSNAT gene was identified by two independent groups in 2006 (Fan et al., 2006; H rebı ´ cek et al., 2006), and 64 different mutations have been identified since then (Human Gene Mutation Database Professional 2014.3). A mouse model has been very recently developed (Martins et al., 2015), but a cellular model for Sanfilippo type C has yet to be developed. The ability to reprogram somatic cells back to a pluripo- tent state (Takahashi and Yamanaka, 2006; Takahashi et al., 2007) has created new opportunities for generating in vitro models of disease-relevant cells differentiated from patient-specific induced pluripotent stem cell (iPSC) lines (recently reviewed by Cherry and Daley, 2013; Inoue et al., 2014; Trounson et al., 2012). This approach has been shown to be particularly useful in the case of congenital or early-onset monogenic diseases. In particular, iPSC-based models of various LSD have been established, including Gaucher disease (Mazzulli et al., 2011; Panicker et al., 2012; Park et al., 2008; Scho ¨ndorf et al., 2014; Tiscornia et al., 2013), Hurler syndrome (Tolar et al., 2011), Pompe disease (Higuchi et al., 2014; Huang et al., 2011), Sanfilippo B syndrome (Lemonnier et al., 2011), and Niemann-Pick type C1 (Maetzel et al., 2014; Trilck et al., 2013). In all these cases, disease-relevant cell types derived from patient-spe- cific iPSCs not only displayed morphologic, biochemical, and/or functional hallmarks of the disease but also have Stem Cell Reports j Vol. 5 j 1–12 j October 13, 2015 j ª2015 The Authors 1 Please cite this article in press as: Canals et al., Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.08.016