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  • In the present study enzyme

    2018-10-24

    In the present study, enzyme activity was dispensable for reprogramming and iPSC maintenance. This is in contrast with Fanconi anemia (Raya et al., 2009; Navarro et al., 2014), and also with Sanfilippo type B (Lemonnier et al., 2011) and Pompe disease (Higuchi et al., 2014), in all of which gene complementation was Atglistatin needed to achieve reprogramming. We hypothesize that the dispensability of enzyme activity for iPSC generation and maintenance might be related to overall low lysosomal activity in these cells. Thus, all the iPSCs generated in the present study (from either controls or SFC patients) showed relatively small numbers of lysosomes in comparison with fibroblasts, as judged by immunostaining with LAMP2 (Figure S4). Moreover, enzyme activity was very low in control iPSCs compared with fibroblasts (Figures 3A and 3B), as were the activities of other lysosomal enzymes (β-hexosaminidase and β-glucocerebrosidase; data not shown), in agreement with previous results (Tiscornia et al., 2013). Taken together, all these data suggest that iPSCs present a small number of lysosomes in comparison with other cell types. To reduce the variability associated with neural differentiation of iPSCs (Falk et al., 2012), we established iPSC-derived SNMs consisting of neural progenitor Atglistatin that can be expanded and subsequently differentiated to neurons and glia (Cho et al., 2008). Mature human neurons that exhibit the principal features of the disease have been successfully generated after culturing iPSC-derived neural precursors cells for 3 to 9 weeks in neuronal differentiation medium. iPSC differentiation, including mature and functional neurons as the main cellular type, was proved through the expression of mature neuronal markers as well as synaptic proteins. Importantly, neuronal cultures derived from SFC patients showed lack of enzyme activity and an accumulation of GAGs and alteration of lysosomes. Apart from the lack of enzyme activity, which is always lacking in SFC cultures, other alterations appeared to be progressive. GAGs accumulated over time, but in our experiments, these differences did not reach statistical significance versus controls until 9 weeks (around 50% respect to control), which could indicate a lower rate of synthesis and storage in this cell type compared to fibroblasts. Lysosomal alterations displayed through TEM/LAMP1 and immunostaining with LAMP2 were first noticed at 3 to 6 weeks and became dramatic at 9 weeks (almost doubled in size). This timeline of appearance of alterations is in concordance with the progressive nature of the disease and highlights current difficulties in predicting the extent of neurological decline, because the lack of enzyme activity is not predictive, and the analysis of GAG storage and lysosome size and number requires invasive techniques. The fact that our SFC-iPSC-derived neural cultures developed alterations that recapitulated those seen in patients prompted us to investigate whether we could detect early functional alterations predating known pathological signs of the disease. For this purpose, we used calcium imaging to analyze neuronal function in patients’ cells. An important decrease in spontaneous activity of SFC neurons compared with WT controls was already detected at 3 weeks of differentiation, particularly for the most severe case (SFC6). The different behavior in SFC6 and SFC7 could be due to the particular features of their mutations. In this regard, we have recently showed that the c.372-2G > A mutation, borne by SFC7, gives rise to a protein lacking 4 amino acids that has some residual activity (Matos et al., 2014). These data suggest that the decline of neuronal activity correlates with the severity of the neurological phenotype observed in patients. Moreover, we combined a direct quantification of neuronal activity with advanced functional connectivity analyses framed in the context of transfer entropy (TE) (Stetter et al., 2012). The latter provided evidences for broad changes in network structure, unraveling extensive disconnection of neurons, the emergence of a subset of highly connected cells, and the evolution of the network toward a disassortative structure. Although the presence of unconnected neurons is a clear indicator of a dysfunctional network, the existence of assortative or disassortative traits is not. Indeed, several naturally occurring networks may fall into one kind or another (Honey et al., 2007; Pan et al., 2010). Theoretical studies (Schmeltzer et al., 2014) and experiments in vitro (Teller et al., 2014) have shown that assortative networks are resilient to attack since the highly connected nodes shape a core that preserves the functionality of network. In contrast, disassortative networks are highly vulnerable, since the targeted loss of the few highly connected nodes may cause network-wide failure (Newman, 2002). These ideas, translated to our study, suggest that the affected, disassortative cultures could completely collapse upon such targeted damage, making them highly vulnerable networks.