WDR45 variants cause ferrous iron loss due to impaired ferritinophagy associated with nuclear receptor coactivator 4 and WD repeat domain phosphoinositide interacting protein 4 reduction

Abstract

Static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration is a neurodegenerative disorder with brain iron accumulation caused by the variants of WDR45, a core autophagy-related gene that encodes WD repeat domain phosphoinositide interacting protein 4. However, the pathophysiology of the disease, particularly the function of WDR45/WD repeat domain phosphoinositide interacting protein 4 in iron metabolism, is largely unknown. As no other variants of core autophagy-related genes show abnormalities in iron metabolism, the relation between autophagy and iron metabolism remains to be elucidated. Since iron deposition in the brain is the hallmark of static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration, iron chelation therapy has been attempted, but it was found to worsen the symptoms; thus, the establishment of a curative treatment is essential. Here, we evaluated autophagy and iron metabolism in patient-derived cells. The expression of ferritin and ferric iron increased and that of ferrous iron decreased in the patient cells with WDR45 variants. In addition, the expression of nuclear receptor coactivator 4 was markedly reduced in patient-derived cells. Furthermore, divalent metal transporter 1, which takes in ferrous iron, was upregulated, while ferroportin, which exports ferrous iron, was downregulated in patient-derived cells. The transfer of WDR45 via an adeno-associated virus vector restored WD repeat domain phosphoinositide interacting protein 4 and nuclear receptor coactivator 4 expression, reduced ferritin levels, and improved other phenotypes observed in patient-derived cells. As nuclear receptor coactivator 4 mediates the ferritin-specific autophagy, i.e. ferritinophagy, its deficiency impaired ferritinophagy, leading to the accumulation of ferric iron-containing ferritin and insufficiency of ferrous iron. Because ferrous iron is required for various essential biochemical reactions, the changes in divalent metal transporter 1 and ferroportin levels may indicate a compensatory response for maintaining the intracellular levels of ferrous iron. Our study revealed that the pathophysiology of static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration involves ferrous iron insufficiency via impaired ferritinophagy through nuclear receptor coactivator 4 expression reduction. Our findings could aid in developing a treatment strategy involving WDR45 manipulation, which may have clinical applications.

Keywords: NCOA4; SENDA/BPAN; WDR45/WIPI4; ferittinophagy; iron metabolism.

Graphical abstract

Figure 1

Fibroblast WDR45/WIPI4 expression under conditions involving the availability of nutrients and lysosomal inhibitory conditions. (A) An illustration of WDR45, which comprises 12 exons (rectangles). The UTRs and coding regions are shown in white and black, respectively. Three variants were confirmed as de novo (patients 1, 3 and 4); the other could not be confirmed as the parental samples were unavailable (patient 2). (B) WDR45 mRNA expression ratio evaluated via qRT-PCR using TaqMan. The expression ratio was normalized to that of the endogenous hGAPDH. (C) WIPI4 protein expression was confirmed using immunoblotting. Actin was used as the loading control. A comparison of the quantitative expression ratio to that of the control is shown. A one-way ANOVA with Tukey’s HSD test was used for analysis (n = 4, control versus Patients 1–4; ****P < 0.0001) (Supplementary material for uncropped blots). (D) The protein expression of WIPI4 was analyzed using immunoblotting under nutrient condition and starvation conditions with or without Baf A1 treatment. Actin was used as the loading control. The quantitative expression ratio compared to that of the control is shown. One-way ANOVA with Wilcoxon test was used for analysis (n = 3, *P = 0.0049). All data are represented as the mean ± SEM of a minimum of three independent experiments (Supplementary material for uncropped blots).

Figure 2

Autophagic activity in patient fibroblasts was reduced to ∼50% of that in the healthy control. (A)The levels of LC3-I and II under starvation conditions with or without Baf A1 treatment were evaluated for the indicated intervals using fibroblasts. LC3-II was rapidly degraded in control fibroblasts; however, its degradation was suppressed in patient fibroblasts under starvation conditions. Moreover, LC3-II accumulation was increased in control fibroblasts; however, it was suppressed in patient fibroblasts under starvation conditions with Baf A1 treatment. Actin was used as the loading control. Data are representative of a minimum of three independent experiments (Supplementary material for uncropped blots). (B, C) Quantitative expression ratio under starvation conditions with or without Baf A1 treatment. N, nutrient condition; S, starvation condition; SB, starvation condition with Baf A1 (D) Autophagic activities were calculated as follows: LC3-II of starvation + Baf A1 (Fig. 2c) minus LC3-II of starvation (Fig. 2b) for the same time. Autophagic activities in patient fibroblasts were significantly decreased compared to those in the control fibroblasts at all indicated times. Data are represented as mean ± SEM of three or more experiments. One-way ANOVA with Wilcoxon test was used for analysis. (n = 3, 2 h; **P = 0.0029, 4 h; **P = 0.0029, 6 h; **P = 0.0029) (E) Fibroblast stable single clones expressing the GFP-LC3-RFP probe were incubated in starvation conditions with or without Baf A1 treatment over time. The fluorescence of GFP and RFP was measured using a multimode plate reader. GFP/RFP ratios were plotted over time under starvation conditions with or without BafA1 treatment. (F) The sum of the GFP-LC3 degradation and accumulation ratios in each fibroblast were plotted for the indicated times. Autophagy activities were significantly decreased compared with those observed in control fibroblasts at all indicated times. One-way ANOVA with Wilcoxon test was used for statistical analysis. (n = 6, 2 h; **P = 0.0010, 4 h; ***P = 0.0005, and 6 h; ***P = 0.0001) Data are represented as the mean ± SEM of a minimum of three independent experiments.

Figure 3

Microscopic imaging of the intracellular ferrous and ferric iron. (A) Fibroblasts were incubated with 1 μM FerroOrange for 30 min. RFP fluorescence was attributed to the intracellular ferrous iron content. The nuclei were stained blue using Hoechst staining. Ferrous iron contents were lower in patient fibroblasts than in the control fibroblasts. Scale bar, 200 μm. Statistical analyses are shown in Supplementary Fig. 7. (B) FerroOrange signals normalized by Hoechst 33342 measured using a multimode plate reader under starvation conditions were plotted for all fibroblasts over time. Ferrous iron content increased rapidly and then decreased in the control fibroblast. However, the levels of ferrous iron only decreased in patient fibroblasts (n = 4). (C) Fibroblasts were subjected to Berlin blue staining for the detection of ferric iron. Ferric iron was more densely stained in patient fibroblasts than in the control fibroblasts. Scale bar, 50 μm. (D) Fibroblasts were stained via Berlin blue staining under conditions involving the availability of nutrients and starvation conditions with or without Baf A1 treatment for 8 h. Notably, the enhanced staining in Baf A1-treated fibroblasts relative to that under other conditions was observed only in the control fibroblasts. Berlin blue staining intensity decreased upon starvation and increased under starvation conditions with Baf A1 treatment in the control fibroblasts. However, the signals corresponding to starvation conditions with or without Baf A1 treatment showed little change in the patient fibroblasts. Scale bar, 500 μm. Statistical analyses are shown in Supplementary Fig. 7.

Figure 4

Immunoblotting and immunofluorescence analyses of ferritin heavy and light chains. (A) Ferritin heavy and light chains were significantly accumulated in patient fibroblasts, as examined with immunoblotting using whole cell lysates prepared from fibroblasts. Actin was used as the loading control. One-way ANOVA with Wilcoxon test was used for analysis. (n = 3, FTH; **P = 0.0095, FTL; **P = 0.0095) (Supplementary material for uncropped blots). (B) Immunofluorescence indicated that ferritin accumulated robustly in patient fibroblasts. Nuclei were stained with DAPI. Scale bar, 20 μm. (C) Immunoblotting of ferritin heavy and light chains under starvation conditions with or without Baf A1 treatment demonstrated that the ferritin heavy and light chains were degraded under starvation over time, and the degradation was inhibited under starvation conditions with Baf A1 treatment in the control fibroblasts. However, the degradation of ferritin heavy and light chains was minimal in all patient fibroblasts under both conditions. Actin was used as the loading control (Supplementary material for uncropped blots). (D) The degradation rates of the ferritin heavy and light chains detected by immunoblotting were plotted over time. The degradation rates rapidly decreased under starvation conditions and were stable under starvation conditions with Baf A1 treatment in the control fibroblasts. However, the degradation rates were nearly stable under both conditions in patient fibroblasts (n = 3). Data are represented as mean ± SEM of values from three experiments.

Figure 5

NCOA4 protein expression was markedly reduced in all patient fibroblasts. Protein levels of molecules involved in iron influx/outflux, and lysosomes were altered in patient fibroblasts. (A) Immunoblot analysis demonstrated that the NCOA4 expression was markedly reduced under nutrient-availability conditions in all patient fibroblasts. Asterisks indicate non-specific immunoreactive bands. Actin was used as the loading control (Supplementary material for uncropped blots). (B) The NCOA4 mRNA was detected following qRT-PCR using the TaqMan probe. The expression ratio was normalized to that of endogenous hGAPDH. (C) FPN protein expression decreased and DMT1 protein expression increased in patient fibroblasts. No differences were observed in the expression of TfR protein between control and patient fibroblasts (Supplementary material for uncropped blots). (D) The levels of lysosomal proteins, membrane protein LAMP2, and major lysosomal enzymes CTSD and TPP1 were increased notably in all patient fibroblasts (Supplementary material for uncropped blots). Actin was used as the loading control. Quantitative expression ratios compared to those observed in the control patient are shown. Data are represented as mean ± SEM of a minimum of three experiments. A one-way ANOVA with Wilcoxon test was used for analysis. P-values are shown in Supplementary Table 6. n.s., not significant.

Figure 6

WDR45 gene transfer by AAV vector restored autophagic activity and related phenotypes of patients. (A) Illustration of the vector constructs. The expression cassette consisted of CMV promoter, human WDR45 complementary DNA, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and simian virus (SV) 40 poly A signal inserted between the ITR of AAV3. The capsids were tyrosine-mutant AAV9. The vectors were prepared at a titre of 2.3× 10¹¹ vg/μlL. (B) The WDR45 mRNA expression ratio was determined by qRT-PCR using the TaqMan probe. The expression ratio was normalized to that of endogenous hGAPDH. (C) WIPI4 protein expression after WDR45 gene transfer using AAV-WDR45 was confirmed via immunoblotting. Actin was used as the loading control (Supplementary material for uncropped blots). (D) NCOA4 protein expression was completely restored by the WDR45 gene transfer. Moreover, FTH, FTL, FPN, DMT1, LMP2, CTSD, and TTP1 expression levels were restored compared to those observed in control fibroblast. Actin was used as the loading control. The quantitative expression ratio compared to the control is shown. A one-way ANOVA with Tukey’s HSD test was used for analysis. P-values are shown in Supplementary Table 7. n.s., not significant (Supplementary material for uncropped blots). (E) The autophagic activity was measured using patient 1 and 4 fibroblasts expressing the GFP-LC3-RFP probe. The autophagic activities were increased approximately twice and improved compared to those observed in the control fibroblasts following WDR45 gene transfer using AAV-WDR45. A one-way ANOVA with Wilcoxon’s test was used for analysis (Patient 1; *P = 0.037, Patient 4; *P = 0.012). All data are represented as mean ± SEM of values obtained from a minimum of three experiments.

Figure 7

Iron metabolism in healthy and SENDA/BPAN patient cells. (HeaIthy) Iron metabolism in healthy cells. Ferrous iron enters cells from the extracellular space via DMT1, while ferric iron combined with transferrin enters via the transferrin receptor by endocytosis. Ferric iron is reduced to ferrous iron by STEAP3, and ferrous iron is then released into the cytoplasm. Intracellular ferrous iron is used for essential biochemical reactions. Excess ferrous iron is exported by FPN or oxidized in the ferritin heavy chain, changed to a non-toxic iron form, ferric iron and stored in the ferritin light chain. Depending on the demand of intracellular ferrous iron, ferritin is degraded via ferritinophagy and ferric iron included in ferritin is released as ferrous iron into the cytoplasm. (Patient) Impaired iron metabolism in patient cells. NCOA4 expression is decreased by deprivation of WIPI4. NCOA4-mediated ferritinophagy is impaired and leads to the accumulation of ferritin. The decreased supply of ferrous iron from ferritinophagy impacts the essential biochemical reactions. The expression of DMT1 increases and that of FPN decreases to improve the supply and reduce the export of ferrous iron.