Our results and analyses suggest that

Our results and analyses suggest that the disorder notably disrupts the topology of the network. In the context of the patients, these alterations could significantly affect the normal operation of the brain. Such an aspect is important in the framework of studies that highlight the relation between altered network topology upon disease and the degradation of brain’s operability and cognitive tasks (Bassett and Bullmore, 2009; van den Heuvel and Sporns, 2013). The spontaneous activity of SFC-derived neurons was recovered after transduction, in the SNM stage, with lentiviruses carrying the WT HGSNAT cDNA, and subsequent differentiation. The lack of recovery at 3 weeks and the partial recovery at 6 weeks contrast with the total recovery at 9 weeks of differentiation. We believe these differences are due to the fact that LV transduction was around 60% efficient, which could initially slow down the development of the network compared with the WT case. At later stages, we hypothesize that the large fraction of healthy neurons suffices to foster broad circuit connectivity and, ultimately, high neuronal activity. We note that the high expression levels of the transduced cells do not seem to alter their individual activity. Indeed, the WT-derived cultures overexpressing the HGSNAT cDNA did not show any significant alteration in these properties. However, our results indicate that the transduced cells do play a role in maintaining or restoring broad network activity and connectivity. Thus, we conclude that neuronal network activity and development could be reestablished with a therapeutic approach that rescues only a fraction (though sufficiently high) of the total neurons. However, GAG storage or lysosome appearance by TEM showed only partial reversion after 9 weeks of differentiation. Whether longer times are needed for complete reversion of these features, or whether this is not at all possible, will require further investigation. The availability of the human cellular model described here provides an excellent tool to investigate this and other issues. In summary, the cellular model introduced here reproduces the major features of the Sanfilippo type C syndrome, especially specific neuronal traits. We have demonstrated that most of the phenotypic features of this neuronal model can be reversed after gene complementation, using lentiviruses overexpressing the cDNA of the HGSNAT gene. Moreover, our neuronal model could be used as a tool to test different possible therapeutic strategies. This is particularly relevant because no cellular model was available for Sanfilippo type C syndrome, and a mouse model has only very recently been developed (Martins et al., 2015). Our findings prove the usefulness of iPSC-derived neuronal models to detect early functional phenotypes that can shed light onto the molecular and cellular processes that lead to the myd88 signaling dysfunction in these patients, as well as providing valuable readouts for screening of potential therapeutic compounds that prevent, rather than revert, the onset of neurodegeneration. Moreover, the neuronal activity and effective connectivity analyses could be applicable to other neurodegenerative diseases in which iPSC-based models are available, such as Parkinson’s or Alzheimer’s disease, autism, and others. Further studies are needed to establish whether this technique would be able to detect differences in the neuronal activity or the network structure before the onset of disease. Such an approach could foster the development of in vivo analyses for early diagnosis of patients affected by neurological diseases, as well as their monitoring during potential treatments.
Experimental Procedures A complete description of experimental procedures can be found in Supplemental Experimental Procedures.
Acknowledgments
Introduction Bone is a highly specialized tissue under constant renewal that takes place through coordinated balanced destruction and reconstruction, respectively, played by osteoclasts and osteoblasts, known as bone remodeling (Teitelbaum and Ross, 2003). Osteoclasts are large fused multinucleated cells deriving from myeloid precursors belonging to the hematopoietic lineage. Defects in their resorbing activity lead to autosomal recessive osteopetrosis (ARO), a rare life-threatening bone disease that causes increased bone density, decreased bone strength with risk of multiple fractures, and progressive narrowing of the medullary cavity. In turn, these defects lead to anemia, thrombocytopenia, and compensatory extramedullary hematopoiesis and in some cases may associate to immune dysfunction (Sobacchi et al., 2013). The most frequently mutated gene is the T cell immune regulator 1 (TCIRG1), encoding the a3 subunit of the vacuolar-type proton transporting ATPase pump (Frattini et al., 2000), which mediates the acidification of the bone/osteoclast interface. The spontaneous mutant oc/oc mouse bears a deletion in the Tcirg1 gene and well mimics the clinical features of human osteopetrosis (Scimeca et al., 2000). To date, the only available treatment for ARO is the hematopoietic stem cell (HSC) transplantation (A.S. Schulz et al., 2013, abstract, 39th Annual Meeting of the European Group for Blood and Marrow Transplantation). However, several patients do not have access to grafts from HLA-matched siblings, a treatment that ensures the best 5-year disease-free survival rate (62% versus 40% with alternative donor transplantation including HLA-matched unrelated) (Orchard et al., 2015). To overcome the problem of donor availability, generation of autologous corrected HSCs followed by transplantation might represent a valid therapeutic option. HSC gene therapy is being utilized with efficacy in a number of genetic diseases (Aiuti et al., 2009, 2013; Biffi et al., 2013; Cavazzana-Calvo et al., 2010; Hacein-Bey-Abina et al., 2010). However, the use of integrating viral vectors to deliver wild-type (WT) cDNA does not allow to restore the integrity of the locus. For this reason, HSCs generated from iPSCs (Robinton and Daley, 2012; Takahashi and Yamanaka, 2006) might represent an alternative source of autologous donor cells, since iPSCs can be easily expanded in large quantities, manipulated to perform gene targeting, and then differentiated into corrected autologous HSCs. Initial experiments on gene targeting in human CD34+ cells by delivering artificial endonucleases and donor DNA template have recently been performed with success, providing a new promising technology (Genovese et al., 2014). Nevertheless, the relative low efficiency by which gene targeting occurs in long-term repopulating HSCs and progenitors may limit application of this technology to diseases in which there is a strong in vivo selective advantage of the corrected cells. Generation of iPSCs from patients who do not have access to an HLA-identical sibling donor could allow their site-specific genetic correction by homologous recombination, followed by differentiation toward hematopoietic progenitors and autologous transplantation.