PKAN and control astrocytes were prepared as detailed in Santambrogio et al. (2022). In brief, hiPS clones for each control and PKAN patient were obtained and characterized as previously reported (Orellana et al., 2016). hiPS clones from control and PKAN patients were then differentiated into pure population of Neuornal Precursor Cells (NPCs) as previously described (Orellana et al., 2016). NPCs were plated onto Matrigel-coated dishes and maintained in DMEM-F12 supplemented with 1% Pen/Strep (Sigma Aldrich, St. Louis, United States), N2 (1:100, Life Technologies, Carlsbad, United States), B27 (1:200, Life Technologies, Carlsbad, United States), 2 mM L-glutamine (Sigma Aldrich, St. Louis, United States), and hFGF (20 ng/ml, Tebu-Bio Italy, Magenta, Italy). As the NPCs culture reached 60% confluence, 20% heat-inactivated (56°C, 30 min) FBS was added to the culture media and the culture produced mature astrocytes. The culture was maintained for 30 days before checking maturation (Supplementary Figure 1).

Immunoblotting was performed after the separation of soluble proteins by SDS-PAGE as described in Orellana et al. (2016). The signal was revealed using an ECL kit (GE Healthcare, Chicago, United States) and collected by ChemiDoc MP Imaging System (BIORAD, Hercules, United States) (Abs: mouse TfR1, Zymed Laboratories, San Francisco, United States Cat #13-6800, 1:1,500; mouse β-actin, Sigma Aldrich, St. Louis, United States Cat #A5441, 1:600). Cells grown on coverslips were fixed in 4% paraformaldehyde immunofluorescence was done as in Santambrogio et al. (2020). Specific primary antibodies used were: rabbit anti-GFAP (Glial Fibrillary Acidic Protein Agilent Technologies Italia, MIlan, Italia Cat# GA524, RRID:AB_2811722, 1:250 dilution) and mouse anti-EAAT2 (Excitatory Amino Acid Transporter 2, Santa Cruz Biotechnology, Dallas, United States Cat# sc-365634, RRID:AB_10844832). Specific Donkey secondary antibodies Alexa-488 and Alexa-546 conjugated were used (#IS20015, #IS20305, 1:800 dilution, Immunological Sciences, Rome, Italy). Images were acquired by fluorescence microscope Zeiss Axio Observer.Z1 equipped with Hamamatsu EM-CCD 9100-02 camera and Volocity acquisition software.

Lentiviral vectors encoding SynaptoZip (SZ) were prepared and used as described (Ferro et al., 2017) to infect 35 days old astrocytes. Pulse and chase experiments with fluorescent transferrin and Synbond (SB) were performed on 50 to 60 days old astrocytes.

Astrocytes were differentiated and maintained for 50–60 days in 20% heat-inactivated FBS, single doses of CoA 25 μM, 3 days (Sigma Aldrich, St. Louis, United States, Cat #C4780) and 4-phenylbutyric acid (4-PBA) 0.5 μM, 24 h (Sigma Aldrich, St. Louis, United States, Cat #P21005) were directly added in the culture medium. In these conditions (heat-inactivation 56°C, 30 min), the serum enzymes responsible for the quick degradation of CoA are inactivated, as demonstrated in Srinivasan et al. (2015). Cells were switched in serum-free media only for the short timeframes of pulse and chase or live-tracking experiments.

After addition of CoA and 4-PBA, cells were maintained in 5% CO2, 37°C for the treatment times, and then culture media was changed based on the programmed experiment (see above).

Statistical tests to predetermine the sample size in the experiments were not used. Statistical analysis and graphs generation were run in GraphPad Prism 9 (GraphPad Prism, RRID:SCR_002798, San DIego, United States), statistical tests used were Student’s t-test, Mann–Whitney test, KS test, and One-Way ANOVA as detailed in the text, Figure legends, and Tables. Figures and schemes were arranged by Serif Affinity Designer 1.10 software (Serif Europe, West Bridgford, United Kingdom). All experiments were replicated at least three times and normalized as detailed in the text, Figure legends, and Tables. A p-value < 0.05 was considered statistically significant (*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001).

Astrocytes were differentiated from previously obtained hiPSC and characterized as previously described (Santambrogio et al., 2022; Supplementary Figure 1). These hiPS-clones (Orellana et al., 2016) originated from three neonatal normal subjects, here called controls, and two PKAN patients, one carrying a c.[569_570insA] homozygous mutation leading to the expression of a protein with a premature stop codon and one a c.[1259delG] homozygous mutation leading to the expression of a protein with a frameshift, respectively (see Table 1 for details) (Santambrogio et al., 2015; Orellana et al., 2016). Both mutations result in the complete absence of the PANK2 enzyme (Santambrogio et al., 2015). Experiments were run on cells after 50–60 days from the beginning of differentiation from NPCs. At this early stage (young), PKAN astrocytes do not show detectable iron accumulation. Moreover, no evidence of significant overexpression of TfR1, compared to controls, emerged by Western blot analyses (Figure 1C and Supplementary Figure 2A). Thus, the analysis of young astrocytes could allow dissecting the early mechanisms that lead to iron burden in cells older than 70 days (Santambrogio et al., 2022). The post-endocytic fate of TfR1 has been well-characterized, and it can be used to identify different classes of endosomes based on its enrichment after different incubation time points (Lin et al., 2004; Drecourt et al., 2018) as resumed in Figure 1A. We performed pulse and chase experiments in which astrocytes, from controls and PKAN patients, were live-incubated with fluorescent Tf (Alexa-546-Tf or Alexa-647-Tf) at given time points to selectively label different endosomal compartments. As shown in Figures 1B,D and Supplementary Figure 2B, at 5 and 15 min of incubation, there is a significant increase of Tf uptake in both PKAN young astrocytes as compared to controls, thus suggesting the hypothesis of a potential alteration of the TfR1 recycling pathway. Since no alteration in the expression levels of TfR1 was seen (Figure 1C and Supplementary Figure 2A), this increased incorporation of Tf, in young astrocytes, could represent a potential mechanism for the iron overload and subsequent accumulation previously seen in older cells (Santambrogio et al., 2022).

We investigated the activity and behavior of the constitutive endosomal trafficking, independently from receptor-mediated iron endocytosis, by using SynaptoZip-Synbond (SZ-SB) system (Figure 2A; Ferro et al., 2017). Thanks to this tool, it is possible to study the cumulative activity and the intracellular fate of single vesicles by analyzing the fluorescent Synbond (SB) peptide uptake (Ferro et al., 2017). This couple works as an activity marker generating a long-lasting fluorescent signal that can be acquired both live and retrospectively. The cumulative fluorescent signal results from the specific SynaptoZip-Synbond bound and subsequent entrapping of Synbond into an exocytosed vesicle. Synbond vesicle enriching relates to the number of exo-endocytic events and the average number of Synbond molecules that bind to available SynaptoZip at each fusion throughout the chosen experimental timeframe. In Figure 2B, we showed some representative young astrocytes expressing SZ (green) and their SB uptake (red and correspondent LUT transformation in right panels). No significant difference in viability and SZ expression was observed in PKAN astrocytes compared to controls (data not shown). To first investigate SB uptake, astrocytes expressing SZ were live incubated with SB and retrospectively analyzed by confocal microscopy. Single vesicles expressing SZ were segmented based on size and fluorescence, as described in the “Materials and Methods” section, and fluorescence for each SB-enriched endosome was analyzed; only endosomes with a SB fluorescence higher than the established threshold (see “Materials and Methods” section for details) were considered as active and included in the analysis (Figure 2C 15 min uptake and Supplementary Figures 3A,B 5 min uptake). PKAN astrocytes showed a significant increase of SB vesicular fluorescence compared to controls, indicating a general higher level of activity of constitutive trafficking of PKAN endosomes (Figure 2C 15 min SB incubation and Supplementary Figures 3A,B 5 min SB incubation). An estimate of endosomes “recruitment,” which is the fraction of active vesicles over time, was also obtained in astrocytes live pulsed with 5 nM SB for 5 and 15 min (Figure 2D and Supplementary Figure 5). Assuming this, with the SZ-SB pair, we can tag the entire endosomal compartment and detect the cumulative constitutive activity at the end of different epochs (5 and 15 min in this case), and in these conditions, an increase in cumulative activity is predictable as SB incubation time is augmented. However, while we observed a consistent increase in endosome recruitment in control astrocytes, this trend did not emerge in PKAN astrocytes. At first glance, these results appear to be in contrast with the evidence that PKAN endosomal compartment is more active at 15 min, but this increased activity is not due to an increment in vesicles recruitment. Therefore, we hypothesized that the same vesicle could be entrapped in a portion of aberrant plasma membrane, thus resulting in a different retention time affecting the normal endocytosis cycle. In this way, the vesicle trip toward its normal intracellular fate is severly impaired. To clarify this point, we followed SB uptake in living cells for 15 min to define SB-binding dynamics from PKAN and control astrocytes (Figure 2E and Supplementary Figure 3E). PKAN astrocytes showed faster SB binding dynamics compared to controls and apparently tend to saturate their available sites over the investigated time range (15 min). On the contrary, controls in the same timeframes still seem to follow a linear binding behavior (Figure 2E and Table 2). To corroborate this finding, we exploited two different SB peptides, conjugated with different fluorophores (Alexa-647, Synbond-647, Alexa-568, Synbond-568), to label endosomal compartment activity in two sequential epochs. The protocol is detailed in the “Materials and Methods” section and graphically resumed in Figure 2F. Since the SZ-SB bound is practically irreversible, it is possible to tag the same vesicle if it re-exposes to the plasma membrane in two different (Figure 2G) (Ferro et al., 2017). In this way, vesicles that are recycled back for a new exo-endocytosis cycle will be stained by both SB-tagged peptides (Synbond-647 and Synbond-568). Given the 1:1 SynaptoZip:Synbond binding ratio and the irreversibility of this bound, a similar amount of uptaken SB, and relative fluorescence, is expected to occur during the second epoch in the following cases: (i) a comparable number of exocytosis events of the same vesicle, (ii) a comparable exposure time of the same fusing vesicle to SB present in the extracellular environment, and (iii) a comparable availability of free binding sites for SB left from the first epoch. Vesicular fluorescence signals and their cumulative probability distributions were calculated from SB fluorescence in the two sequential epochs and the data obtained are presented in Figures 2H,I, respectively. As expected, considering known constitutive endosomal dynamics, a physiological reduction in SB uptake during the second epoch emerged in all cases (Supplementary Figure 3C,D), but a significant decrease of SB uptake during the second epoch emerged in PKAN astrocytes, if compared to the reduction seen in controls (Figure 2H). Cumulative functions of controls are almost completely overlapped, suggesting a similar ability, of the same endosome, to bind SB in the first and second epoch. On the contrary, PKAN endosomes cumulative functions are indicative of a significantly lower SB incorporation during the second epoch respect to the first, as predicted by the left shift of the curve seen in the second epoch (Figure 2I). PKAN endosomes reduced binding ability in the second epoch can be due to the higher amount of uptaken SB during the first epoch and the subsequent lower availability of SB binding sites during the second epoch respect to controls, as also suggested by binding dynamics (Figure 2E). This condition is also reinforced by the altered endosomal recruitment shown in Figure 2D and consequent higher exposure times or exocytosis events, for the same endosome, during the 1st epoch. Taken together, these data support the hypothesis of an impaired intracellular fate of endosomes in PKAN astrocytes, which lead to compromised exo-endocytosis cycles before being free to follow their natural routes.

To better understand the fate of an endocytosed endosome, cells expressing SZ were live-pulsed 5 min with SB to tag only active early endosomes which undergone at least one exocytosis cycle, then SB was washed, and fluorescence live-recorded at 20 fps. Feature point tracking of endosomes was then run on deconvoluted videos by the Mosaic/Particle Tracker 2D/3D ImageJ plugin (Sbalzarini and Koumoutsakos, 2005). From the mean square displacement (MSD) of the tracked particle, it is possible to dissect different characteristics of its motion such as diffusion coefficient (DC) and Moment Scaling Spectra slope (MSSs) (Ferrari et al., 2001; Sbalzarini and Koumoutsakos, 2005; Briane et al., 2018). Endosomes trafficking inside the cell follows biophysical laws and the aforementioned MSSs value is a good index to understand the type of motion and can be used to classify the behavior of endosomes (MSSs = 0 stationary object, MSSs < 0.5 confined motion, MSSs = 0.5 diffusive motion, 0.5 < MSSs < 1 directed motion). From a biological point of view, confined motion can represent an object that is moving in a closed region or in a wider but crowded area. The diffusive motion is the typical Brownian motion and can represent a free-floating endosome in the cytosol; instead, the directed motion represents an active motion that involves microtubular structures (Sbalzarini and Koumoutsakos, 2005; Briane et al., 2018). A representative tracked SB-enriched endosome is shown in Figure 3A (Supplementary Movies 1, 2), while in Figure 3B are shown exemplar trajectories from tracked endosomes with a directed (left) and diffusive (right) behavior. A generalized impairment of constitutive endosomal trafficking arose in PKAN astrocytes; in particular, a lower percentage of diffusive trajectories (Figure 3C middle panel), a higher percentage of directed trajectories (Figure 3C, lower panel), a lower diffusion coefficient (Figure 3D), and a lower average distance are covered by endosomes (Figure 3E) in PKAN astrocytes as compared to controls. The overall data are summarized in Table 3. These alterations agree with the hypothesis of an altered membrane exo-endocytic fusion process and, given the lower DC found, with a potential struggle for the endosome in moving along its physiological intracellular pathways. Likewise, the differences in directed motion and the lower average distance covered by motile endosomes in PKAN astrocytes are suggestive of a possible alteration in motor-driven motility along tubular pathways.

We then tested potential effects of acute treatments with CoA (72 h) and 4-PBA (24 h) on endosomal behavior. Direct treatments with CoA have been demonstrated to be efficient in rescuing aberrant PKAN behavior in hiPS-derived neurons and astrocytes (Orellana et al., 2016; Santambrogio et al., 2022). 4-PBA is an orphan drug with several properties, such as inhibition of pyruvate dehydrogenase kinase (PDK) that leads to an increase of activated pyruvate dehydrogenase complex (PDHC). PDHC catalyzes the oxidative decarboxylation of pyruvate to produce acetyl-CoA, which depends on mitochondrial CoA. It is plausible to hypothesize that the activated PDHC boosts, among others, acetyl-CoA levels, and lipogenic pathways in conditions of PDH deficiency (Ferriero and Brunetti-Pierri, 2013; Ferriero et al., 2013). Obtained data showed a partial rescue of endosomal trafficking and, for some features, 4-PBA was effective where CoA did not produce significant effects (Figure 4E). The failure in endosomal recruitment seen in PKAN astrocytes (Figure 2D) is partially restored by CoA and 4-PBA treatments. Indeed, an expected increase in endosome recruitment in PKAN astrocytes emerges only in treated conditions (Figures 4B,C). In control astrocytes treated with 4-PBA, a slight dampening of the endosomal recruitment trend also emerged, but this trend/effect is not statistically significant compared to UT condition (Figure 4A). Treatments also had an effect on the SB uptake in both PKAN patients, in particular, 4-PBA was able to bring back single vesicle SB uptake to the basal levels seen in controls (Figure 4D). CoA showed a similar ability on both PKAN patients, lowering the excess of SB uptake seen in UT conditions, restoring the endosomal activity to control values only in PKAN[GlyValfs*30] but not PKAN[Tyr190*] astrocytes, whose activity persisted as significantly higher than in controls. These data suggest that 4-PBA is more efficient in rescuing this feature, even in unhealthier conditions, such as the one found in PKAN[Tyr190*] (Figure 4D). CoA and 4-PBA also help in a partial rescue of some diffusional features of the endosomal compartment in treated astrocytes from both PKAN patients that showed a significant increase in DC compared to UT condition (Figure 4F). However, only 4-PBA proved to be efficient in restoring the percentage of diffusive trajectories toward values comparable to UT controls (Figure 4E and Table 3), while a similar, although not significant trend also emerged for directed trajectories (Table 3). The amelioration of global endosomal features carried out by CoA and 4-PBA treatments led to the restoration of correct Tf trafficking, significantly decreasing cellular Tf uptake in PKAN patients, and bringing back Tf uptake to levels comparable to UT controls in both PKAN patients (Figure 4G).