Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. To function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated different aspects involved in ESCs’ response to iron accumulation following stable knockdown of the ferritin heavy chain (FTH1) gene, which encodes for a major iron storage protein with ferroxidase activity. Experimental findings highlight unexpected and, to a certain extent, paradoxical results. If on one hand FTH1 silencing does not correlate with increased ROS production nor with changes in the redox status, strengthening the concept that hESCs are extremely resistant and, to a certain extent, even refractory to intracellular iron imbalance, on the other, the differentiation potential of hESCs seems to be affected and apoptosis is observed. Interestingly, we found that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP), which crosstalk in driving hESCs antioxidant cascade events. These findings shed new light on how hESCs perform under oxidative stress, dissecting the molecular mechanisms through which Nrf2, in combination with PPP, counteracts oxidative injury triggered by FTH1 knockdown.

Mutations in SCN1A gene, encoding the voltage-gated sodium channel (VGSC) NaV1.1, are widely recognized as a leading cause of genetic febrile seizures (FS), due to the decrease in the Na+ current density, mainly affecting the inhibitory neuronal transmission. Here, we generated induced pluripotent stem cells (iPSCs)-derived neurons (idNs) from a patient belonging to a genetically well-characterized Italian family, carrying the c.434T>C mutation in SCN1A gene (hereafter SCN1AM145T). A side-by-side comparison of diseased and healthy idNs revealed an overall maturation delay of SCN1AM145T cells. Membranes isolated from both diseased and control idNs were injected into Xenopus oocytes and both GABA and AMPA currents were successfully recorded. Patch-clamp measurements on idNs revealed depolarized action potential for SCN1AM145T, suggesting a reduced excitability. Expression analyses of VGSCs and chloride co-transporters NKCC1 and KCC2 showed a cellular “dysmaturity” of mutated idNs, strengthened by the high expression of SCN3A, a more fetal-like VGSC isoform, and a high NKCC1/KCC2 ratio, in mutated cells. Overall, we provide strong evidence for an intrinsic cellular immaturity, underscoring the role of mutant NaV1.1 in the development of FS. Furthermore, our data are strengthening previous findings obtained using transfected cells and recordings on human slices, demonstrating that diseased idNs represent a powerful tool for personalized therapy and ex vivo drug screening for human epileptic disorders.

Unverricht-Lundborg disease (ULD), also known as progressive myoclonic epilepsy 1 (EPM1), is a rare autosomal recessive neurodegenerative disorder characterized by a complex symptomatology that includes action- and stimulus-sensitive myoclonus and tonic-clonic seizures. The main cause of the onset and development of ULD is a repeat expansion of a dodecamer sequence localized in the promoter region of the gene encoding cystatin B (CSTB), an inhibitor of lysosomal proteases. Although this is the predominant mutation found in most patients, the physio-pathological mechanisms underlying the disease complexity remain largely unknown. In this work, we used patient-specific iPSCs and their neuronal derivatives to gain insight into the molecular and genetic machinery responsible for the disease in two Italian siblings affected by different phenotypes of ULD. Specifically, fragment length analysis on amplified CSTB promoters found homozygous status for dodecamer expansion in both patients and showed that the number of dodecamer repeats is the same in both. Furthermore, the luciferase reporter assay showed that the CSTB promoter activity was similarly reduced in both lines compared to the control. This information allowed us to draw important conclusions: (1) the phenotypic differences of the patients do not seem to be strictly dependent on the genetic mutation around the CSTB gene, and (2) that some other molecular mechanisms, not yet clearly identified, might be taken into account. In line with the inhibitory role of cystatin B on cathepsins, molecular investigations performed on iPSCs-derived neurons showed an increased expression of lysosomal cathepsins (B, D, and L) and a reduced expression of CSTB protein. Intriguingly, the increase in cathepsin expression does not appear to be correlated with the residual amount of CSTB, suggesting that other mechanisms, in addition to the regulation of cathepsins, could be involved in the pathological complexity of the disease.

Background: Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. As such, to function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated and characterized different aspects involved in ESCs response to iron accumulation following stable knockdown of ferritin heavy chain (FTH1) gene, encoding for a major iron storage protein with ferroxidase activity. Methods: stable FTH1 knockdown of H9-hES cell line was achieved with the use of shRNA lentiviral particles. Upon FTH1 silencing, we speculated whether hESCs still retained their pluripotency capability were first monitored for their capability the pluripotent status,FTH1 stable knock-down ESCs were obtained using lentiviral vector plasmids. The effect of FTH1 silencing on hESCs pluripotency was evaluated through alkaline phosphatase (AP) staining, immunofluorescence and embryoid bodies (EBs) formation assay. Western blotting and qRT-PCR analysis were performed to assess the involvement of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and pentose phosphate pathway (PPP) in the antioxidant response. ROS levels and mitochondrial functionality were explored by flow cytometry. Seahorse Analyzer was used to evaluate metabolic and bioenergetic profiles. Results: Our findings clearly show that FTH1 silencing in hESCs does not correlate with increased ROS production nor with redox status strengthening the concept that hESCs are extremely resistant and, to certain extent, even refractory to the pattern of results produced in other cell lines. Collectively, our results demonstrate that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP) which crosstalk in driving hESCs antioxidant cascade events able to antagonize the effects of FTH1 silencing.Conclusion: to our knowledge, this is the first evidence of a crosstalk between FTH1 silencing and Nrf2 pathway activation in hESCs, casting a new light on how human ESCs perform under oxidative stress conditions. Our findings go beyond previous reports, showing how the Nrf2 pathway, in combination with PPP activation, regulates the molecular signature underlying ESCs defence mechanisms against oxidative stress mediated by FTH1 downregulation.