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C O M P L E T E T I T L E : Stem cell modelling of neuroferritinopathy reveals iron as a determinant of senescence and ferroptosis during neural agin.
S O U R C E : Stem Cell Reports
Highlights
• NF is a unique pathophysiological model for analyzing cellular iron dysregulation
• NF fibroblast/iPSC-derived NPCs and neurons show a phenotype of iron mobilization
• Free iron is sufficient per se to cause both cell senescence and ferroptosis
• Iron has a primary role in neuronal aging and degeneration
Summary
Neuroferritinopathy (NF) is a movement disorder caused by alterations in the L-ferritin gene that generate cytosolic free iron. NF is a unique pathophysiological model for determining the direct consequences of cell iron dysregulation. We established lines of induced pluripotent stem cells from fibroblasts from two NF patients and one isogenic control obtained by CRISPR/Cas9 technology. NF fibroblasts, neural progenitors, and neurons exhibited the presence of increased cytosolic iron, which was also detectable as: ferritin aggregates, alterations in the iron parameters, oxidative damage, and the onset of a senescence phenotype, particularly severe in the neurons. In this spontaneous senescence model, NF cells had impaired survival and died by ferroptosis. Thus, non-ferritin-bound iron is sufficient per se to cause both cell senescence and ferroptotic cell death in human fibroblasts and neurons. These results provide strong evidence supporting the primary role of iron in neuronal aging and degeneration.
Introduction
Neuroferritinopathy (NF) (OMIM, no. 606159) is a rare dominantly inherited late-onset monogenic disorder with 100% penetrance caused by mutations in the L-ferritin gene (FTL1) (Curtis et al., 2001). NF belongs to a group of monogenic extrapyramidal disorders called neurodegeneration with brain iron accumulation (NBIA) and is characterized by the focal accumulation of iron in the basal ganglia (Levi and Finazzi, 2014). The main common symptoms of NBIA include movement disorders, spasticity, and cognitive impairment; the age of onset varies among these diseases (Di Meo and Tiranti, 2018), averaging around 40 years. At present, 11 different causative mutations have been described worldwide, all of which are located in the last part of exon 4 in the FTL1 sequence (Levi and Rovida, 2015). These mutations affect both length and sequence of the C terminus peptide, disturbing the amino acid contacts involved in the shaping of the hydrophobic channels along the 4-fold axis of the molecule (Levi and Rovida, 2015). In humans, cytosolic ferritin is a heteropolymeric protein with a spherical shape obtained by the assembly of 24 structurally similar subunits of two different types, namely H and L, and encoded by two genes, FTH1 and FTL1, with iron stored in the internal cavity as ferrihydrite (rosio and Levi, 201). The overexpression of disease-associated gene variants in vitro (Cozzi et al., 2010) and in vivo (Maccarinelli et al., 2015, Vidal et al., 2008) revealed that the NF causative mutations act in a negative-dominant manner to impair the iron-storage function of ferritin, resulting in increased level of intracellular free iron (Cozzi et al., 2010, Luscieti et al., 2010). Emerging evidence supports the key role of iron in aging (ecca et al., 200) and neurodegeneration processes (Rouault, 2013), mainly because iron accumulates in the brain during aging (Ward et al., 2014) and its surplus renders cells more susceptible to oxidative stress (Koskenkorva-Frank et al., 2013). Thus, NF cellular models represent valuable tools for investigations of the controversial role of this metal in the cellular processes occurring during aging and neurodegeneration. However, the precise role of iron in the development of these two cellular processes is not completely elucidated, and its role in the neuronal compartment is particularly obscure due to the lack of faithful experimental models recapitulating spontaneous occurrence of these alterations.
Cellular senescence is normally induced in vitro by several stressful events (radiation, oxidants, and oncogenes) and in vivo by ablation of anti-senescent genes, such as p66 (Berry et al., 2008) and nuclear receptor co-activator 4 (NCOA4) (Bellelli et al., 2014). Ferroptosis is prevalently studied in cancer cell lines (Dixon et al., 2012), where it is revealed only after ferroptosis-inducing reagents (Xu et al., 2019).
The scarcity of human primary neuronal models to study the action of iron in aging and neurodegeneration stimulated us to develop a model characterized by the presence of excess free iron. We applied cellular reprogramming techniques (Orellana et al., 2016) to fibroblasts, obtaining induced pluripotent stem cell (iPSC)-derived neuronal precursor cells (NPCs) and neurons derived from two patients affected by NF, one isogenic control and three healthy subjects. A significant increase of cytosolic free iron content, alteration of iron homeostasis, DNA/protein/lipid oxidative damage, a clear senescence phenotype, and spontaneous death by ferroptosis were observed in NF fibroblasts, iPSC-derived NPCs, and neurons compared with controls.
These results, when interpreted in view of the pathogenetic mechanism of NF, confirm the detrimental effect of free iron in neuronal cells. In fact, in conditions such as NF in which iron is not safely removed from cytosol due to alterations of ferritin structure/function, it triggers a cascade of damaging events leading to senescence and ferroptosis, thereby accelerating the aging process.
Results
Development and Characterization of NF Fibroblasts and iPSC-Derived Neuronal Models
Fibroblasts were obtained from skin biopsies of two NF affected patients: one with heterozygous FTL1 469_484dup (Storti et al., 2013), and the other with heterozygous FTL1 351delG_InsTTT (hereafter referred to as NF1 and NF2, respectively) (Figure S1). Control fibroblasts from three healthy adult subjects were purchased from ATTC (hereafter referred to as Ctr1, Ctr2, and Ctr3). To develop a neuronal model we established multiple iPSC lines by reprogramming fibroblasts from all subjects as previously described (Orellana et al., 2016). Isogenic control cells were obtained by CRISPR/Cas9 technology on clone no. 7 of NF1-iPSC. We used one clone from each healthy subject (Ctr1 no. 203, Ctr2 no. 37, and Ctr3 no. 151), and three from each patient and isogenic control (NF1 no. 1, no. 7, and no. 8; NF2 no. 8, no. 11, and no. 12; and R-NF1 no. 38, no. 40, and no. 41). Characterization of the obtained clones of iPSCs, embryoid bodies (EBs), derived NPCs, and neurons are shown and described in Supplemental Information (Figures S2 and S3). Electrophysiological recordings in iPSC-derived neurons are reported in Figure S4. All clones were subjected to the same reported analysis, an example of which is provided in each panel.
NF Fibroblast/iPSC-Derived NPCs and Neurons Showed Cellular Iron Mobilization and Ferritin/Iron Aggregates
NF mutations caused structural modification in the ferritin E-helix, which is involved in the formation of the hydrophobic pores of the molecule (Figure S5A) (Cozzi et al., 2010, Levi and Rovida, 2015). In-silico-generated structural models of the ferritins containing the mutated subunits (Figures S5B, S5C, S1B, and S1C) showed the 4-fold channel dramatically altered, with a larger pore that likely impairs the retention of iron into the cavity. In the NF2 variant (Figure S1C), this effect is directly related to the premature stop site caused by the mutation, resulting in a shortened sequence and a smaller subunit lacking the E-helix and a part of the D-helix. In the NF1 variant (Figure S1B), this effect can be attributed to conformational changes that expose the E-helix to proteolytic cleavage, as confirmed experimentally by proteinase K digestion (Figure S1D).
To investigate the ferritin functionality, we treated the fibroblasts with 2 μCi 55Fe-ammonium citrate (FeAC) for 18 h. The incorporation of iron into ferritin was reduced to approximately 20% in the NF1 and NF2 fibroblasts (Figure S5D) and to 50% in the iPSC-derived neurons (Figure S5H) compared with controls, probably leaving a high amount of intracellular free iron. To confirm this effect, we explored the activity of the IRE/IRP machinery, which post-transcriptionally controls the expression of the major iron proteins (ferritin, transferrin receptor 1, and ferroportin) as a function of cytosolic iron concentration (Muckenthaler et al., 2008).
ELISA assay, specific for cytosolic ferritin-L (FtL) and ferritin-H (FtH), performed in fibroblasts, revealed similar contents of FtL and of FtH among the three controls; in patients the concentration of FtL was found to be similar to controls, while FtH was 2-fold higher (Table S1). Immunoblotting revealed that the expression of transferrin receptor 1 was decreased ∼2.5-fold in the NF fibroblasts and NPCs (Figures S5E and S5F) and was undetectable in neurons (Figure S5I); accordingly, ferroportin appeared unchanged in fibroblasts (not shown) and NPCs (Figure S5G), and showed a tendency to increase in neurons (Figure S5L). These data suggest that the reduced efficiency of iron incorporation of mutant ferritin results in an increased free cellular iron availability.
To stress this phenotype, the NF fibroblasts were treated with 0.1 mM ferric ammonium citrate (FAC) for 14 days and subjected to immunostaining using an antibody specific for H-ferritin. The higher number of ferritin-positive granules observed in the NF1 and NF2 fibroblasts in basal conditions (Figure 1A , upper panel) was further increased after treatment with iron (Figure 1A, lower panel). Analysis at ultrastructural level by electron microscopy (EM) and electron spectroscopy imaging (ESI) in patient cells showed that the granules were localized in the cytosol, mainly associated with lipid droplets and surrounded by bilayer membranous formations (Figure 1B), similarly to the ones detected in the NF disease mouse model (Maccarinelli et al., 2015). ESI defined iron as a component of these aggregates (Figure 1B), the number of which was ∼3-fold greater in NF than in control cells.
Figure 1Representative Images of NF Fibroblasts and iPSC-Derived Neurons Showed Formation of Ferritin/Iron Aggregates
(A) Control and variant fibroblasts untreated (UT) or treated (Fe) with 100 μM FeAC for 14 days and stained with an anti-human H-ferritin antibody (Hoechst staining to detect the nuclei). The arrows show the ferritin aggregates. Scale bars, 20 μm.
(B) Ultrastructural analysis of fibroblasts examined under an electron microscope (EM). The arrows indicate the aggregates. Cells untreated (UT) or treated (Fe) as described in (A) were subjected to an ESI analysis. The images showing the ultrastructural organization observed at 250 eV with a superimposed iron map represented by pseudo-colors. The iron granules were counted and are represented as a ratio to the total number in the counted fields (means ± SD of three independent experiments). The data were analyzed by unpaired, two-tailed t test, ∗∗∗p < 0.001.
© Immunofluorescence of β-III-tubulin (Tuj1) and H-ferritin (FtH) in neurons obtained from a control subject and patient NF1 (Hoechst staining for the nuclei), and co-treatment with the iron chelator (5 μM DFO) and antioxidant (100 μM NAC). Digital higher-magnification of images (dashed rectangles) is shown. Scale bars, 20 μm.
(D) Control and NF1 Perls staining under basal conditions and after treatment with 5 μM DFO and 100 μM NAC. The arrows show the presence of iron aggregates. Scale bars, 20 μm.
A similar phenotype was present in the NF iPSC-derived neurons. Immunofluorescence analysis on NF iPSC-derived neurons showed increased FtH, visualized as granular formations localized to the cytosolic part of the soma and along the axon (Figure 1C). These formations were also detectable by Perls staining (a specific dye for ferric iron deposits), suggesting the presence of a large amount of iron (Figure 1D). A 3-week treatment with the iron chelator deferoxamine (DFO) (5 μM) and the antioxidant n-acetyl-cysteine (NAC) (100 μM) reverted this phenotype (Figures 1C and 1D).
Cellular Iron Mobilization-Induced Oxidative Damage and Cellular Death in the NF Fibroblast/iPSC-Derived NPCs and Neurons
The free redox-active form of iron promotes the formation of free oxygen/nitrogen radicals, and thus increased cellular iron availability may affect cell resistance to oxidative stress. We searched for any sign of an altered cellular oxidative status under basal conditions and compared NF fibroblasts, iPSC-derived NPCs, and neurons with controls. For each cell type, we set up appropriate methods to reveal different types of oxidative damage products. The ROS levels were assayed by the specific fluorescent probe 2′,7′-dichlorofluorescein (DCF). Carbonylated proteins were assessed using an Oxy-Blot analysis, while lipid peroxidation was assessed using an Oxy-MDA assay (Cozzi et al., 2010) and C11-Bodipy (581/591) staining. A low but significant increase in the ROS levels was revealed in the NF fibroblasts (Figure 2A ), which was corroborated by an enhancement of oxidized proteins in NF fibroblasts, iPSC-derived NPCs, and neurons (Figures 2B, 2D, and 2F). Lipid peroxidation was significantly increased only in the NF1, but not NF2, fibroblasts (Figure 2C); however, the phenotype was more severe in the NPCs and neurons, and the cells from both patients showed a statistically significant difference from the controls (Figures 2E and 2G). Furthermore, the reduced form of glutathione was significantly decreased in the NF iPSC-derived neurons (Figure 2H).
Figure 2Cellular Iron Mobilization-Induced Oxidative Damage in NF Fibroblast/iPSC-Derived NPCs and Neurons
(A) Fibroblasts were incubated with the ROS-sensitive 2′,7′-dichlorofluorescein. The results are presented as the mean of three independent experiments in octuplicate.
(B and C) Quantification by densitometry of Oxyblot (B) and lipid-malondialdehyde (MDA) peroxidation © of fibroblasts.
(D and E) Quantification of Oxyblot (D) and lipid-MDA peroxidation (E) of NPCs.
(F) Oxyblot of neurons.
(G) BODIPY of neurons.
(H) Glutathione (GSH)-sensitive ThiolTracker Violet fluorescence signal detected on Tuj1-positive human neurons.
The data are presented as the means ± SD (B–F) or ± SEM (G and H) of at least three independent experiments. All the data were analyzed by unpaired, two-tailed t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
To investigate whether this altered oxidative status was sufficient to promote cellular death, we performed DAPI and trypan blue staining and LDH determination. No significant differences were detected between controls and NF fibroblasts (not shown), indicating that the NF fibroblasts did not die by apoptosis or necrosis. However, the MTT assay revealed a significantly decreased number of viable cells in the NF fibroblasts (NF1 ∼27%, NF2 ∼22%), which was more consistent in the iPSC-derived NPCs (NF1 ∼46%, NF2 ∼52%) and neurons (NF1 ∼52%, NF2 ∼51%) compared with the controls. To confirm the specific involvement of free iron in eliciting cell death, neurons were grown for 3 weeks in the presence of the iron chelator deferiprone (5 μM) (Pinto et al., 2018). The addition of deferiprone prevented the death of the NF-derived neurons (∼90% NF1 and ∼94% NF2 viable cells), demonstrating that iron is the leading cause of neuronal death.
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Edited by Engadin, 10 October 2019 - 07:51 PM.
