Iron induces two distinct Ca2+ signalling cascades in astrocytes

Guan, Wenzheng; Xia, Maosheng; Ji, Ming; Chen, Beina; Li, Shuai; Zhang, Manman; Liang, Shanshan; Chen, Binjie; Gong, Wenliang; Dong, Chengyi; Wen, Gehua; Zhan, Xiaoni; Zhang, Dianjun; Li, Xinyu; Zhou, Yuefei; Guan, Dawei; Verkhratsky, Alexei; Li, Baoman

doi:10.1038/s42003-021-02060-x

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Published: 05 May 2021

Iron induces two distinct Ca²⁺ signalling cascades in astrocytes

Wenzheng Guan^1,2^na1,
Maosheng Xia^1,3^na1,
Ming Ji^1,4,
Beina Chen^1,4,
Shuai Li^1,4,
Manman Zhang^1,4,
Shanshan Liang^1,4,
Binjie Chen^1,4,
Wenliang Gong^1,4,
Chengyi Dong³,
Gehua Wen^1,4,
Xiaoni Zhan^1,4,
Dianjun Zhang^1,4,
Xinyu Li^1,4,
Yuefei Zhou³,
Dawei Guan ORCID: orcid.org/0000-0002-9439-6557⁵,
Alexei Verkhratsky ORCID: orcid.org/0000-0003-2592-9898^4,6,7,8 &
…
Baoman Li ORCID: orcid.org/0000-0002-3959-9570^1,4

Communications Biology volume 4, Article number: 525 (2021) Cite this article

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Abstract

Iron is the fundamental element for numerous physiological functions. Plasmalemmal divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of ferrous (Fe²⁺), whereas transferrin receptors (TFR) carry transferrin (TF)-bound ferric (Fe³⁺). In this study we performed detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca²⁺]_i) in astrocytes. Administration of Fe²⁺ or Fe³⁺ in μM concentrations evoked [Ca²⁺]_i in astrocytes in vitro and in vivo. Iron ions trigger increase in [Ca²⁺]_i through two distinct molecular cascades. Uptake of Fe²⁺ by DMT1 inhibits astroglial Na⁺-K⁺-ATPase, which leads to elevation in cytoplasmic Na⁺ concentration, thus reversing Na⁺/Ca²⁺ exchanger and thereby generating Ca²⁺ influx. Uptake of Fe³⁺ by TF-TFR stimulates phospholipase C to produce inositol 1,4,5-trisphosphate (InsP₃), thus triggering InsP₃ receptor-mediated Ca²⁺ release from endoplasmic reticulum. In summary, these findings reveal the mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload.

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Introduction

Iron contributes to numerous cellular and biochemical processes and acts as a co-factor in various molecular cascades in the nervous tissue including the synthesis and metabolism of several brain-specific enzymes and neurotransmitters^1,2. In biological systems iron is present in either reduced ferrous (Fe²⁺) or oxidized ferric (Fe³⁺) state. The brain has the second (after liver) highest quantity of iron in the human body with total non-haem iron in the brain reaching about 60 mg³. The non-haem iron concentration in the serum ranges between 9 and 30 μM, whereas the iron concentration in cerebrospinal fluid (CSF) is much smaller being around 0.3–0.75 μM^4,5. Transport of iron across the blood–brain barrier (BBB) is mediated either by transferrin receptor (TFR)-mediated endocytosis of Fe³⁺-bound to transferrin (holo-TF), or, for non-TF-bound iron, by vesicular and non-vesicular pathways⁶. Membrane transport of Fe²⁺ is also mediated by divalent metal ion transporter 1 (DMT1/SLC11A2) which underlies Fe²⁺ uptake through the plasma membrane or from endosomes⁶. Under physiological conditions, the intracellular cytosolic ionised iron levels fluctuate around 0.5–1.5 μM⁷.

In the brain, up to three-fourths of total iron is accumulated within neuroglial cells⁸. Astrocytes in particular are fundamental elements of ionostatic control over CNS environment⁹. Ionised Fe²⁺ enters astrocytes through DMT1/SLC11A2 transporters which are particularly concentrated in the endfeet of cerebral and hippocampal astrocytes¹⁰. In physiological condition, DMT1 is widely distributed in the nervous system, being expressed in neurones^11,12,13, astrocytes^13,14,15 and oligodendrocytes^14,16 in vitro and in the brain tissue. In astrocytes, DMT1 mediates non‐transferrin‐bound iron (NTBI) transport, thus contributing to the brain iron homoeostasis in development and adulthood^17,18,19. Conditional deletion of DMT1 form oligodendrocyte precursors substantially inhibited both myelination in development and remyelination in pathology¹⁶. Evidence for the expression of TFR in astroglial cells remains controversial^6,20,21, while iron overload may influence the expression or distribution of TFR in astrocytic compartments^21,22. Cellular uptake of Fe³⁺ requires internalization of TF–TFR complex²³. An adaptor protein Disabled-2 (Dab2) plays an essential role in cell signalling, migration and development²⁴. In mice the Dab2 has two isoforms of 96 and 67 kDa (p96 and p67²⁴). In human K562 cells, Dab2 regulates internalization of TFR and uptake of TF²⁵. Dab2 is also widely distributed in immune cells and in neuroglia²⁴, although the functional link between Dab-2 and TFR in astrocytes has not been demonstrated.

Astrocytes possess a special form of intracellular ionic excitability, mediated by temporal and spatial fluctuations in the intracellular ion concentration^26,27. Astroglial Ca²⁺ signalling is mediated by Ca²⁺ release from the endoplasmic reticulum (ER) following activation of inositol-1,4,5-trisphosphate receptor (InsP₃R), or intracellular Ca²⁺-gated Ca²⁺ channels known as ryanodine receptors (RyR). Astroglial Ca²⁺ signals may also be generated by plasmalemmal Ca²⁺ entry through Ca²⁺-permeable channels or by sodium-calcium exchanger (NCX) operating in the reverse mode^26,28. Astroglial Na⁺ signalling is shaped by plasmalemmal Na⁺ entry through cationic channels and numerous Na⁺-dependent transporters, of which the major role belongs to Na⁺-dependent glutamate transporters^29,30,31; Na⁺ extrusion is mediated by the sodium-potassium pump (NKA). Both Na⁺ and Ca²⁺ signalling systems are closely coordinated, with NKA and NCX accomplishing this coordination at the molecular level³². Astrocytes specifically express α2-subunit containing NKA which is fundamental for astroglial K⁺ buffering³³. Astrocytes express all three isoforms of NCX - NCX1/SLC8A1, NCX2/SLC8A2 and NCX3/SLC8A3, with some evidence indicating higher expression of NCX1³⁴. The NKA, the NCX and glutamate transporters are known to be preferentially concentrated in the perisynaptic astroglial membranes indicating intimate relationship between these ion-transporting molecules^35,36. The NCX is also known to localise at caveolae rich in caveolin-3 (Cav-3), the latter isoform being predominantly expressed in astrocytes³⁷.

In the present paper we performed an in depth analysis of the action of ferrous and ferric (Fe²⁺ and Fe³⁺) on astrocytic Ca²⁺ and Na⁺ dynamics. We found that Fe²⁺ (through DMT1) and Fe³⁺-TF (through TFR) evoke [Ca²⁺]_i transients in astrocytes in culture and in vivo. Effects of Fe²⁺ on [Ca²⁺]_i are mediated mainly by the reversed NCX, whereas Fe³⁺ triggers Ca²⁺ release from the endoplasmic reticulum by stimulations of InsP₃R. In conclusion, we discovered that iron ions trigger astrocytic Ca²⁺ signalling by acting through two distinct molecular cascades.

Results

Fe²⁺/Fe³⁺ trigger [Ca²⁺]_i increase in cortical astrocytes in vitro and in vivo

Exposure of cultured astrocytes to Fe²⁺ leads to a gradual increase in cytoplasmic Fe²⁺ concentration as revealed by quenching of Fura-2 (Supplementary Figure 1). Washout of Fe²⁺ leads to a full recovery of Fura-2 fluorescence; these data indicate the existence of plasmalemmal Fe²⁺ transport system as well as absence of non-specific damaging effect of ionised iron on the cellular membrane. We analysed effects of Fe²⁺ and Fe³⁺ Ca²⁺ dynamics in astrocytes in primary cultures and in vivo in GFAP-eGFP transgenic mice (Fig. 1). In the primary cultured astrocytes, administration of either FeSO₄ (Fe²⁺) or ferric ammonium citrate-TF (Fe³⁺) increased [Ca²⁺]_i in concentration-dependent manner, albeit with different kinetics. In the presence of Fe²⁺ an increase in [Ca²⁺]_i demonstrated prominent plateau, whereas Fe³⁺ triggered transient, relatively rapidly decaying [Ca²⁺]_i response (Fig. 1a).

**Fig. 1: Iron ions, Fe²⁺ and Fe³⁺, evoke astrocytic intracellular Ca²⁺ signals in vitro and in vivo.**

When imaging cortical astrocytes in vivo (the cells were identified by specific eGFP fluorescence) we found that addition of either Fe²⁺ or Fe³⁺ for 30 s induced transient [Ca²⁺]_i increase (Fig. 1b). Administration of Fe²⁺ increased fluorescent intensity of Rhod-2 to 456.30% ± 18.46% (n = 10, p < 0.0001) whereas Fe³⁺ increased the peak of fluorescent signal to 308.50% ± 13.01% (n = 10, p < 0.0001) of the basal value. In experiments in vitro we characterised concentration-dependence of [Ca²⁺]_i responses: the EC₅₀ was around 0.4–0.6 μM for both Fe²⁺ and Fe³⁺ (Fig. 1c).

DMT1 and TFR mediate Fe²⁺ and Fe³⁺ uptake

As mentioned above, Fe²⁺ uptake could be mediated by plasmalemmal transporter DMT1, whereas Fe³⁺ is mainly accumulated in TF-bound form by TFRs (Fig. 2a). Immunostaining of cortical tissue preparations and primary cultured astrocytes demonstrated co-localisation of DMT1 and TFR with astroglial GFAP-positive profiles (Fig. 2b). In the cortical tissue both DMT1 and TFR are preferentially localised at perivascular endfeet. Meanwhile, expression of specific DMT1 and TFR mRNA was also detected in the freshly isolated and sorted astrocytes and neurones, as well as and in the cerebral tissue (Fig. 2c).

**Fig. 2: Astrocytic expression of DMT1 and TFR.**

To reveal the contribution of DMT1 and TFR to Fe²⁺/Fe³⁺-induced [Ca²⁺]_i dynamics, we inhibited expression of DMT1 or TFR using siRNA duplex chains. The representative western blots demonstrating the efficacy of knockdown are shown in Fig. 2d. When compared to the control group, the DMT1 siRNA reduced expression of DMT1 to 9.52% ± 2.58% (n = 6, p < 0.0001), whereas treatment with TFR siRNA decreased TFR levels to 7.84% ± 2.10% (n = 6, p < 0.0001) from the control values. Administration of Fe²⁺ to DMT1-deficient astrocytes failed to induce any changes in [Ca²⁺]_i. At the same time Fe²⁺ induced robust [Ca²⁺]_i elevation in astrocytes treated with siRNA(−) (Fig. 2e). Similarly, Fe³⁺ did not produce [Ca²⁺]_i, transients in astrocytes treated with TFR siRNA duplex chains, whereas in cells exposed to negative control siRNA Fe³⁺ evoked [Ca²⁺]_i elevations (Fig. 2e). At the same time Fe³⁺-induced [Ca²⁺]_i transients were fully preserved in DMT1-deficient astrocytes (Supplementary Figure 2), hence questioning the role of DMT1 in release of Fe³⁺ from endosomes.

In unstimulated astrocytes in culture the DMT1 fluorescence was the highest around the nucleus, suggesting its preferred intracellular localisation. After treatment of cultures with Fe²⁺ for 5 min we observed redistribution of DMT1 from the nuclear region to the plasma membrane (Fig. 2f). The levels of DMT1 were measured in the extracted proteins of the nucleus and cytoplasm (Fig. 2g). After treatment with Fe²⁺ for 5 min, the level of DMT1 in the nuclei decreased to 36.41% ± 4.33% of control group (n = 10, p < 0.0001), whereas the level of cytoplasmic DMT1 increased to 117.37% ± 5.79% of control group (n = 10, p < 0.0001) (Fig. 2g).

Sources of iron-induced [Ca²⁺]_i mobilisation

The main sources for [Ca²⁺]_i increase in astrocytes are (i) Ca²⁺ release from the ER following the opening of InsP₃Rs or RyRs, or (ii) plasmalemmal Ca²⁺ influx through either Ca²⁺ permeable channels (such as L-type Ca²⁺ channels or TRP channels) or NCX operating in the reverse mode (Fig. 3a) or (iii) combination of some or all of these pathways. To dissect Ca²⁺ sources we first determined the influence of extracellular Ca²⁺ on iron-evoked [Ca²⁺]_i transients. Removal of Ca²⁺ from the extracellular milieu completely abolished Fe²⁺-induced [Ca²⁺]_i elevations but left Fe³⁺-evoked [Ca²⁺]_i transients largely intact (Fig. 3b). This highlights the role for plasmalemmal Ca²⁺ influx in Ca²⁺ signalling triggered by Fe²⁺ and ER Ca²⁺ release for Ca²⁺ signals triggered by Fe³⁺.

**Fig. 3: Ca²⁺ sources for Fe²⁺ or Fe³⁺-induced [Ca²⁺]_i transients.**

Incubation of astrocytes with inhibitor of L-type voltage-gated Ca²⁺ channel nifedipine (10 μM) did not affect [Ca²⁺]_i responses to Fe²⁺ or to Fe³⁺ (Fig. 3c). In contrast, inhibition of NCX with 10 μM of selective agonist KB-R7943 completely eliminated [Ca²⁺]_i responses to Fe²⁺, without affecting Fe³⁺-induced [Ca²⁺]_i transients (Fig. 3d). Thus, two forms of iron, the ferrous and the ferric, mobilise intracellular Ca²⁺ through distinct pathways: Fe²⁺ stimulates Ca²⁺ influx by NCX, whereas Fe³⁺ triggers intracellular Ca²⁺ release. This suggestion was further corroborated by pharmacological inhibition of InsP₃ receptors with potent antagonist Xestospongin C (XeC)³⁸, Exposure of cultured astrocytes to 10 μM of XeC effectively suppressed [Ca²⁺]_i response to Fe³⁺, without much affecting Fe²⁺-induced [Ca²⁺]_i transient (Fig. 3e). Finally, treatment with 10 μM ryanodine (which at this concentration inhibits RyRs) somewhat decreased the plateau phase of Fe²⁺-induced [Ca²⁺]_i transient without modifying [Ca²⁺]_i response to Fe³⁺ (Fig. 3f).

DMT1 transports Fe²⁺, which inhibits NKA, increases [Na⁺]_i and reverses NCX

Experiments described above have demonstrated that Fe²⁺, after being transported into the cell by DMT1, leads to a reversal of the NCX, which results in Ca²⁺ influx. The NCX reversal in astrocytes is triggered by an increase in [Na⁺]_i. Such an increase may originate either from the activation of plasmalemmal Na⁺ entry or from inhibition of the NKA, which maintains basal [Na⁺]_i^28,30. The activity of NKA was suppressed by exposure to 10 μM Fe²⁺ to 82.40 ± 5.74% (n = 10, p < 0.0001) of the control. Exposure to 100 nM of the specific NKA inhibitor, ouabain, reduced NKA activity to 72.30 ± 5.91% (n = 10, p < 0.0001) of the control (Fig. 4a). When 10 μM Fe²⁺ and 100 nM ouabain were added together, the NKA activity was reduced to 71.80 ± 7.81% (n = 10, p < 0.0001) (Fig. 4a).

**Fig. 4: Fe²⁺ reverses NCXs by inhibiting NKA.**

Inhibition of NKA in astrocytes results in a substantial elevation in [Na⁺]_i. When monitoring [Na⁺]_i in cultured astrocytes with Na⁺-sensitive probe SBFI we found that both Fe²⁺ (10 μM) and ouabain (100 nM) triggered rapid and substantial elevation of [Na⁺]_i (Fig. 4b). These changes in [Na⁺]_i were paralleled by [Ca²⁺]_i dynamics. Exposure of astrocytes to Fe²⁺, or ouabaine or mixture of Fe²⁺ and ouabain caused [Ca²⁺]_i elevation (Fig. 4c). When Fe²⁺ was applied in the presence of ouabain it failed to change [Ca²⁺]_i (Fig. 4d); at the same time application of Fe³⁺ in the presence of ouabain still triggered additional [Ca²⁺]_i elevation (Fig. 4d).

Fe²⁺-induced Ca²⁺ mobilisation is associated with caveolae

Treatment of cultured astrocytes with interfering Cav3 siRNA duplex chains decreased the level of Cav3 to 7.41 ± 4.32% (n = 6, p < 0.0001) of the control (Fig. 4e). An in vitro knock-down of Cav3 significantly reduced amplitudes of Fe²⁺-induced [Ca²⁺]_i increase; maximal increase in Fluo-4 F/F₀ after Cav3 knockdown was 242.00 ± 16.44% (n = 10, p < 0.0001). In control astrocytes treated with siRNA(−) the amplitude of Fe²⁺-induced [Ca²⁺]_i increase reached 363.34 ± 11.62% (n = 10, p < 0.0001, Fig. 4f). To further analyse the effects of Cav3 on Fe²⁺-induced [Ca²⁺]_i dynamics the levels of relevant proteins were measured in the extracted caveolae (Fig. 5a). As shown in Fig. 5B, exposure to 10 μM Fe²⁺ for 5 min significantly increased the level of DMT1 to 293.24 ± 24.89% (n = 10, p < 0.0001) of the control values. After pre-treatment with Cav3 siRNA duplex chains, Fe²⁺ increased the level of DMT1 only to 195.77 ± 20.19% (n = 10, p < 0.0001) of control group (Fig. 5b). The levels of NCX1 and NKA were similarly affected by Fe²⁺ and the knocking down of Cav3. Exposure to Fe²⁺ increased the level of NCX1 and NKA to 248.71 ± 19.58% (n = 10, p < 0.0001) and 263.66 ± 25.93% (n = 10, p < 0.0001) of the controls, after knocking down Cav3, Fe²⁺ elevated the level of NCX1 and NKA only to 172.96 ± 11.76% (n = 10, p < 0.0001) and 200.86 ± 18.14% (n = 10, p < 0.0001) of control values (Fig. 5b). Of note, Fe²⁺ did not affect the levels of NCX2 and NCX3 (Fig. 5b).

**Fig. 5: Caveolae integrate DMT1, NKA and NCX.**

Fe³⁺ triggers Ca²⁺ release through stimulation of PLC and increase in InsP₃ production

As shown in Fig. 3e, inhibition of InsP₃ receptors with XeC suppressed Fe³⁺-induced [Ca²⁺]_i mobilisation. We therefore analysed effects of Fe³⁺ on the InsP₃ signalling cascade in cultured astrocytes. Incubation of astrocytes with Fe³⁺ increased the level of InsP₃ in cell lysates to 74.10 ± 8.14 ng/ml (n = 10, p < 0.0001), from the control InsP₃ level of 24.40 ± 6.35 ng/ml. In cells treated with TF alone the InsP₃ level was 28.00 ± 12.62 ng/ml (n = 10, p = 0.4309) (Fig. 6a). Subsequently we analysed the links between scaffolding/signalling protein Dab2 and Fe³⁺-induced Ca²⁺ signalling. We suppressed expression of two isoforms of Dab2 by siRNA duplex chains (Fig. 6b). After RNA interfering, expression of 96 kD and 67 kD Dab2 isoforms decreased, respectively, to 5.81 ± 3.56% (n = 6, p < 0.0001) and to 11.88 ± 6.51% (n = 6, p < 0.0001) of the control values (Fig. 6b). The knock-down of Dab2 rendered Fe³⁺ ineffective: exposure of Dab2-deficient astrocytes to Fe³⁺ did not affect InsP₃ production (control: 31.60 ± 12.51 ng/ml (n = 10); Dab2 knockdown: 30.60 ± 10.42 ng/ml (n = 10, p = 0.1253; Fig. 6c).

**Fig. 6: Mechanisms of Fe³⁺ induced Ca²⁺ signalling.**

When Dab2-deficient astrocytes were challenged with Fe³⁺, no [Ca²⁺]_i increase was recorded (Fig. 6d). Similarly, after inhibition of the PLC with U-73122, application of Fe³⁺ did not change [Ca²⁺]_i (Fig. 6d). Hence, we may surmise that uptake of Fe³⁺ through TFR requires Dab2 protein; after entering the cytosol Fe³⁺ activates the PLC, which produces InsP₃ that triggers InsP₃-induced Ca²⁺ release from the ER (Fig. 6e).

Discussion

In this paper, we describe previously unknown effects of iron ions on cellular [Ca²⁺]_i in astrocytes. Administration of either Fe²⁺ or Fe³⁺ triggered a concentration-dependent increase in [Ca²⁺]_i with EC₅₀ of about 0.4–0.6 μM. We performed an in depth analysis of the mechanisms underlying iron transport and iron induced Ca²⁺ signalling. We also demonstrated that, contrary to the previous beliefs, astrocytes express functional TFR in vitro and in vivo thus allowing accumulation of Fe³⁺.

In the brain, majority of cells express a full complement of proteins responsible for iron homoeostasis, including TFR and DMT1 for iron uptake, heavy and light chains ferritin for iron sequestration, cytosolic iron exporter ferroportin 1 (FPN1), and iron regulatory protein 1 and 2 (IRP1and IRP2) for regulating intracellular iron homoeostasis^39,40. Glial cells, and astrocytes in particular, store up to 75% of ionised iron in the CNS⁴¹, and protect the brain against iron overloads⁴². Transmembrane transport of iron in astrocytes has been identified, but was not studied in details. There is a general agreement on the primary role of plasmalemmal divalent metal transporter 1, DMT1/SLC11A2, which selectively transports Fe²⁺; the DMT1 was detected in astrocytes in culture and there is evidence indicating its presence in astroglial endfeet in situ^43,44,45. In addition, Fe²⁺ was suggested to enter reactive astrocytes by diffusion through transient receptor potential canonical (TRPC) channels⁴². Expression of TF-Fe³⁺-transporting TFR has been noted in astrocytes in culture^21,46; it is, nonetheless, generally believed that astrocytes in vivo are not in a possession of TFR and hence cannot accumulate Fe³⁺ ^47,48,49. This conclusion, however, has been made on the basis of rather limited investigations^14,46; while expression of TFR-specific mRNA was detected in astrocyte transriptome⁵⁰. In our study we confirmed expression of DMT1, at mRNA and protein levels as well as by immunostaining, in acutely isolated astrocytes, in astroglial primary culture and in situ in cortical tissue; the DMT1 was particularly enriched in the endfeet (Fig. 2b–d). We also detected astroglial expression of TFR at mRNA level in the transcriptome of acutely isolated and FACS-sorted astrocytes (Fig. 2c). We further confirmed expression of TFR in astrocytes at a protein level and in immunohistochemical analysis of astrocytes in culture and in cortical preparations (Fig. 2b–d). In the cortical tissue TFR labelling was concentrated in perivascular astrocytic endfeet (Fig. 2b–d).

Not much is known about the links between ionised iron and Ca²⁺ signalling in the cellular elements of the CNS. In the literature, we found only a single example of Fe³⁺-induced [Ca²⁺]_i transient in cultured hippocampal neurones⁵¹. To the best of our knowledge here we present the first recordings of Fe²⁺/Fe³⁺ induced Ca²⁺ signals in astrocytes. Both ions evoked [Ca²⁺]_i elevation in primary cultured astrocytes and in EGFP-labelled astrocytes in the cortices of alive animals studied with transcranial confocal microscopy. Both ions acted in the low μM range of concentrations, however the kinetics of respective [Ca²⁺]_i transients are different. Exposure of cultured astrocytes to Fe²⁺ triggered rapid [Ca²⁺]_i increase with long-lasting plateau; the [Ca²⁺]_i barely declined in the presence of Fe²⁺. In contrast, Fe³⁺-induced transient elevation of [Ca²⁺]_i recovers to the baseline within ~ 200–300 s in the presence of Fe³⁺ (Figs. 1b and 2e). These distinct kinetics reflect different signalling cascades activated by iron ions.

The Fe²⁺-induced [Ca²⁺]_i responses require DMT1: in vitro knockdown of DMT1 expression with silencing mRNA completely eliminates Ca²⁺ signal (Fig. 2). The Fe²⁺-induced [Ca²⁺]_i changes originate from plasmalemmal Ca²⁺ entry, because removal of Ca²⁺ from the extracellular medium inhibits [Ca²⁺]_i response. Finally, Fe²⁺-induced [Ca²⁺]_i signals require NCX, as pharmacological inhibition of the latter effectively suppresses [Ca²⁺]_i elevation (Fig. 3). These data indicate that Fe²⁺, after being accumulated in the astrocyte, switches the NCX into the reverse mode of operation thus generating Ca²⁺ influx into the cell in exchange for Na⁺. This scenario requires increase in astroglial [Na⁺]_i, which readily reverses the NCX^28,52. An increase in [Na⁺]_i is likely to result from the inhibition of NKA, which represents the major Na⁺ efflux mechanism in astrocytes³⁰. Activity of NKA was effectively suppressed by Fe²⁺, and probing astrocytes with Na⁺-sensitive indicator SBFI revealed Fe²⁺-induced [Na⁺]_i elevation (Fig. 4). In the presence of NKA inhibitor ouabain Fe²⁺-induced [Ca²⁺]_i responses were eliminated thus corroborating the central role of NKA and NCX in Fe²⁺-induced Ca²⁺ signalling (Figs. 4, 7). Of note, exposure to Fe²⁺ initiated rapid redistribution of DMT1 from nucleus into cytoplasm (Fig. 2g) and arguably to the plasmalemma thus increasing astrocyte capacity for iron uptake.

**Fig. 7: Mechanisms of Fe²⁺ and Fe³⁺ -induced intracellular Ca²⁺ signalling.**

The mechanism of Fe³⁺-induced Ca²⁺ signalling is associated with intracellular Ca²⁺ release. The Fe³⁺-induced [Ca²⁺]_i responses are preserved in Ca²⁺-free extracellular solution while being blocked by XeC (inhibitor of InsP₃ receptor) and by U-73122 (inhibitor of PLC) thus revealing the central role for InsP₃-medaited ER Ca²⁺ release. Initiation of this signalling cascade requires transmembrane transport of Fe³⁺: in vitro knockdown of TFR eliminates Fe³⁺-evoked [Ca²⁺]_i dynamics. The internalisation of TF-Fe³⁺-TFR complex also requires functional Dab2 protein. This protein is a multi-modular scaffold protein with signalling roles in ion homoeostasis, inflammation and receptors internalization⁵³. Ablation of Dab2 in astrocytes with specific siRNA interrupts signalling chain and blocks Fe³⁺-dependent Ca²⁺ signalling (Figs. 6 and 7).

Import of TF-Fe³⁺-TFR complex involves endocytosis; acidic (pH ~5.6) environment of the endosome achieved through the action of ATP-dependent H⁺ pumps⁵⁴, reduces the affinity of TF to Fe³⁺ and the latter is released⁵⁵. Subsequently, an endosomal reductase reduces Fe³⁺ to Fe²⁺, and Fe²⁺ can be transported into the cytosol by DMT1 or by ZIP8 or ZIP14 Zn transporters⁵⁶. Our data question the DMT1 pathway because siRNA knockout of DMT1 did not affect Fe³⁺-induced Ca²⁺ signalling; the detailed mechanism of Fe³⁺ transport in astrocytes remains to be fully characterised.

Caveolae are specific plasmalemmal structures that form functional microdomains involved in various signalling, endocytotic and transporting events⁵⁷. Caveolae and their main structural and regulatory proteins Caveolin-1,2,3 are present in astrocytes (with predominant expression of Cav3); astrocytic caveolae contribute to signal transduction, formation of signalling protein complexes and are involved in action of various neuroactive substances and drugs^58,59. Caveolae are known to form functional Ca²⁺ signalling units, establish links between Ca²⁺ channels and various transports and may contribute to formation of plasmalemmal/ER functional domains operational in astrocytes⁶⁰. We found that down-regulation of expression of Cav3 in cultured astrocytes substantially reduced the amplitude of Fe²⁺-evoked [Ca²⁺]_i responses. We suggest therefore that Cav3 and caveolae integrate DMT1, NKA and NCX into a single Ca²⁺ signalling unit (Fig. 7); moreover exposure to iron potentiates formation of such units.

Iron homoeostasis is of fundamental importance for cells, tissues and organisms, as iron contributes to a wide range of vital biological pathways⁶¹. The brain contains high concentrations of bound and free iron, which participates in multiple processes from energy production to synaptic transmission⁴¹. Iron overload and failures in iron homoeostatic triggers neurotoxicity and is implicated in brain diseases⁴⁸. Genetic mutations of iron regulatory proteins (the key elements of iron homoeostasis) result in iron deposition in the brain with subsequent neurodegeneration characteristic for aceruloplasminemia⁶² and neuroferritinopathy⁶³. In aceruloplasminemia patients, iron overload has been observed in astrocytes, particularly in the basal ganglia⁶⁴. Ceruloplasmin oxidises Fe²⁺ to Fe³⁺ in order to generate the oxidised form of iron that can bind to extracellular transferrin; in astrocytes ceruloplasmin is also required for iron export⁶⁴. Thus, in aceruloplasminemia, iron entering the CNS as ferrous might escape oxidation; hence cells exposed to the excess of Fe²⁺ may readily become iron overloaded⁶⁵. Similarly, abnormal iron accumulation has been characterised in neurodegenerative diseases including Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis and Huntington disease to name but a few⁴⁸. Specific class of neurodegeneration with brain iron accumulation (NBIA) has been also categorised in recent years⁶⁶.

Based on our data we propose that astrocytes mount the defence against acute iron overload. This defence includes iron accumulation through both DMT1 and TFR, redistribution of DMT1 from intracellular locations to plasmalemma and generation of Ca²⁺ signals, which control astrocytic reactivity. Iron-induced Ca²⁺ signalling is activated at low pathological iron concentrations (>1 μM; while physiological iron concentration in the CSF ranges between 0.3 and 0.75 μM). Importantly, two distinct signalling cascades (DMT1 Fe²⁺ transport, inhibition of NKA and reversal of NCX versus Fe³⁺-TF-TFR transport, activation of PLC and generation of InsP₃-induced Ca²⁺ release) distinguish between ferric and ferrous. These distinct pathways may define very different outputs: it is known for example that activation of astroglial InsP₃ receptors type II is linked to initiation of reactive astrogliosis^67,68. In our recent report, exposure to iron was found to stimulate the over-expression of DMT1 in astrocytes and microglia, but not in neurones, which may result in the neuroprotection by glial uptake of excessive iron¹³. Astrogliosis plays important, if not defining role in evolution of many neurological diseases⁶⁹. Our previous experiments have shown that formation of brain deposits of iron up-regulates astroglial expression of TFR and instigates reactive astrogliosis⁷⁰. What characterises the iron-induced reactive phenotype and what is the role of astroglial reactivity in managing excessive iron in the brain remains to be found. In conclusion, our study presented a phenomenon that iron ions (Fe²⁺ and Fe³⁺) directly induce intracellular Ca²⁺ signalling and stimulate astroglial protective mechanisms against iron overload in broad pathological contexts.

Methods

Materials

The culture medium including DMEM and fetal bovine serum were purchased from Gibco Life Technology Invitrogen (Grand Island, NY, USA). Oligo-fectamine, MEMI, fluo-4 AM, sodium-binding benzofuran isophthalate (SBFI) AM, G-agarose bead, TFR antibody, β-actin antibody, GFAP antibody, DMT1 antibody and DMT1 siRNA duplex chains were from Thermo Fisher Scientific (Waltham, MA USA); siRNA duplex chains of TFR, NCX1-3, Cav-3 and Dab2, NCX2 antibody, Na⁺/K⁺-ATPase alpha1/2 antibody and the secondary antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NCX1 antibody, NCX3 antibody and native mouse apo-transferrin (apo-TF; i.e., iron-free) were from Abcam (Cambridge, MA, USA). Donkey serum, xestospongin C (Xe-C), nifedipine, ferrous sulfate heptahydrate (FeSO₄), sulforhodamine 101 (SR101) and ferric ammonium citrate were purchase from Sigma-Aldrich (St. Louis, MO, USA). Ryanodine and KB-R7943 were purchased from Calbiochem (La Jolla, CA, USA). Secondary antibody staining with donkey anti-mouse or anti-rabbit Cy-2/3 were from Jackson Immuno-Research (West Grove, PA, USA). Primary antibody of histone H3 was purchased from EarthOx (Millbrae, CA, USA).

Animals

The C57BL/6 mice, FVB/N-Tg(GFAP-eGFP)14Mes/J and B6.Cg-Tg(Thy1-YFP)HJrs/J transgenic mice were all purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The animals were raised in standard housing conditions (22 ± 1 °C; light/dark cycle of 12/12 h), with water and food available ad libitum. All experiments were performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023) and its 1978 revision, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University, No. [2019]059.

Primary culture of astrocytes

Astrocytes were cultured from newborn mice^71,72. In brief, the cerebral hemispheres were isolated, dissociated and filtered. Isolated astrocytes were grown in Dulbecco’s Minimum Essential Medium (DMEM) with 7.5 mM glucose supplemented with 10% foetal bovine serum. Astrocytes were incubated at 37 °C in a humidified atmosphere of CO₂/air (5:95%). The cultures are highly enriched in astrocytes, the purity is >95% as judged by GFAP staining⁷³.

Iron treatment

For preparing Fe³⁺-TF solution, ferric ammonium citrate and mouse apo-TF were incubated at a 2:1 ratio in serum-free culture medium for 1 h at 37 °C^74,75. The same concentration of apo-TF in the same volume of culture medium but without Fe³⁺ was used for control treatments. For the Fe²⁺ solution, FeSO₄ was freshly dissociated in serum-free culture medium at 37 °C and used immediately, the same volume of serum-free culture medium was used as the control for Fe²⁺ group.

RNA interfering

The cultured astrocytes were incubated in DMEM without serum for 12 hours before transfection^73,76,77. A transfection solution containing 2 μl oligo-fectamine (Promega, Madison, WI, USA), 40 μl MEMI, and 2.5 μl siRNA (DMT1, TFR, NCX1-3, Cav-3 or Dab2) was added to the culture medium in every well for 8 h. In the siRNA-negative control cultures, transfection solution without siRNA was added. Thereafter, DMEM with three times serum was added to the cultures. These siRNA duplex chains were purchased from Santa Cruz Biotechnology (CA, USA).

Preparation of membrane caveolae

Cell homogenization and the caveolae preparation from astrocytes was made^78,79. In brief, primary cultured astrocytes were collected and homogenised in SET (0.315 M sucrose, 20 mM Tris-HCl, and 1 mM EDTA, pH 7.4), and centrifuged for 1 h at 1,000 × g. The pellets were re-solubilised in SET and layered on Percoll (30% in SET) followed centrifugation at 1,000 × g. The pellets were re-homogenised and re-layered to three sucrose density gradient solution (80%, 30% and 5%) with ultra-centrifugation at 175,000 × g. Finally, the purified caveolae were collected and re-suspended in SET.

Co-immunoprecipitation

We used technologies of co-immunoprecipitation and subsequent western blotting to check the conjunction level between NCXs and DMT1⁷⁶. After homogenization, protein content was determined by the Bradford method⁸⁰ using bovine serum albumin as the standard. For immunoprecipitation of NCX1-3, whole cell lysates (500 μg) were incubated with 20 μg of anti-NCX1, anti-NCX2 or anti-NCX3 antibody for overnight at 4 °C. Then, 200 μl of washed protein G-agarose bead slurry was added, and the mixture was incubated for another 2 hours at 4 °C. The agarose beads were washed three times with cold phosphate buffer solution (PBS) and collected by pulsed centrifugation (5 s in a microcentrifuge at 14,000 × g), the supernatant was drained off, and the beads were boiled for 5 min. Thereafter, the supernatant was collected by pulsed centrifugation, and the entire immunoprecipitates were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Cytoplasm and nucleus protein extraction

The subcellular fraction was analyzed using the protein extraction Kit (P0028, Beyotime, Shanghai, China)⁸¹, according to the manufacturer’s protocol. For further western blotting assays, cytoplasmic and nucleic protein extractions were kept under −20 °C.

Western blotting

For quantifying expressions of DMT1, TFR, NCX1-3, Cav-3 or Dab2, the samples containing 100 μg of protein were added to slab gels. After transferring to PVDF membranes, the samples were blocked by 10% skimmed milk powder for 1 h, and membranes were incubated overnight with the primary antibodies, specific to either DMT1 at a 1:300 dilution, TFR at a 1:200 dilution, NCX1 at a 1:100 dilution, NCX2 at a 1:200 dilution, NCX3 at a 1:150 dilution, Cav-3 at a 1:200 dilution, Dab2 at a 1:100 dilution, Histone H3 at 1:500 dilution or β-actin at a 1:1000 dilution. After washing, specific binding was detected by horseradish peroxidase-conjugated secondary antibodies. Images were analysed with an Electrophoresis Gel Imaging Analysis System (MF-ChemiBIS 3.2, DNR Bio-Imaging Systems, Israel). Band density was measured with Window AlphaEase^TM FC 32-bit software^72,82.

Monitoring of [Ca²⁺]_i

For [Ca²⁺]_i monitoring and imaging in cultured astrocytes^83,84, after the pre-treatment with or without inhibitors or siRNA duplex chains, the primarily cultured astrocytes were loaded with 5 μM fluo-4-AM, (Thermo Fisher Scientific (Waltham, MA USA)), for 30 min. Fluo-4 signals were visualised by fluorescent microscopy (Olympus IX71, Japan). The readings from all fluo-4 positive cells in one measured field of view in each culture were included in the statistics, the fluorescence intensity of fluo-4 was normalised to the baseline intensity before stimulation. The measurements were repeated in 10 different cultures. Cultured astrocytes were also stained with specific marker sulforhodamine 101 (SR101) at 1:2000 dilution.

Two-photon in vivo Ca²⁺ imaging

Adult FVB/N-Tg(GFAP-eGFP)14Mes/J transgenic mice (10–12 weeks old) were anesthetized with ketamine (80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Body temperature was monitored using a rectal probe, and the mice were maintained at 37 °C by a heating blanket. A custom made metal plate was glued to the skull with dental acrylic cement and a cranial window was prepared over the right hemisphere at 2.5 mm lateral and 2 mm posterior to bregma. The somato-sensory cortical cells were loaded with Ca²⁺ indicator Rhod-2 AM (50 μM, 1 h). The transcranial window was superfused with artificial CSF. After a stable baseline recording was obtained, Fe²⁺ (100 μM) or Fe³⁺-TF (100 μM) was added for 1 min. Bandpass filters (Chroma) were 540 nm/40 nm for eGFP and 850 nm/70 nm for rhod-2 signals. Time-lapse images of astrocytic Ca²⁺ signalling were recorded every 5 s using FluoView with a custom-built two-photon laser-scanning setup (Nikon AR1, Japan)^84,85.

Intracellular Na⁺ measurements

For monitoring intracellular ionised Na⁺ ([Na⁺]_i) in cultured astrocytes, primary cultured astrocytes were loaded with 10 μM of Na⁺-sensitive indicator SBFI-AM for 30 min in serum-free medium, with subsequent 1 h of washout. SBFI was alternatively excited at 340 nm and 380 nm, and the emission was monitored at 500 nm. The SBFI signals were measured by fluorescent microscopy (Olympus IX71, Japan) and expressed as a ratio (R = F₃₄₀/F₃₈₀)⁵².

Immunofluorescence

The brain tissue was fixed by immersion in 4% paraformaldehyde and cut into 100 μm slices^72,82. The cultured cells were fixed with 100% methanol at −20 °C. Brain slices or cells were permeabilised by incubation for 1 h with donkey serum. Primary antibodies against DMT1 or TFR were used at a 1:100 dilution, against glial fibrillary acidic protein (GFAP) was used at 1:200 dilution. And nuclei were stained with marker 4′, 6′-diamidino-2-phenylindole (DAPI) at 1:1000 dilution. The primary antibodies were incubated overnight at 4 °C and then donkey anti-mouse or anti-rabbit Cy-2/3 conjugated secondary antibody for 2 h at room temperature. Images were captured using a confocal scanning microscope (DMi8, Leica, Wetzlar, Germany).

ELISA assays

Astrocytes were incubated at 37 °C in a fresh serum-free culture medium; after the treatment with Fe²⁺/Fe³⁺ or inhibitors, the astrocytes were collected and centrifuged at 10,000 × g for 10 min to remove floating cells and/or cell debris at 4 °C. To assay the NKA activity, a commercial ELISA kit (abx255202; Abbexa, Cambridge, UK) was used and operated as the protocols, the sensitivity is 0.19 ng/mL, the optical density (OD) was measured at 450 nm and the OD value was normalized by the control group. To assay the InsP₃ concentration⁸⁶, the supernatant was collected and the concentration of InsP₃ assayed using a commercial ELISA kit (E-EL-0059c; Elabscience Biotechnology, Wuhan, China).

Sorting neural cells through fluorescence activated cell sorter (FACS) and quantitative PCR (qPCR)

To measure the mRNA for TFR and DMT1, astrocytes expressing fluorescent marker GFP (GFAP-GFP mice) and neurons expressing fluorescent marker YFP (Thy1-YFP mice) were used; we also extracted the cerebral hemispheres tissues from wild type mice. The cells from transgenic mice were used for specific sorting of astrocytes or neurons with FACS^85,87. The RNA of the sorted cells and cerebral tissue was extracted by Trizol. Total RNA was reverse transcribed and PCR amplification was performed in a Robo-cycler thermocycler^81,84. The relative quantity of transcripts was assessed using five-folds serial dilutions of RT product (200 ng). RNA quantity was normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and values are expressed as the ratio TFR/GAPDH or DMT1/GAPDH.

Statistics and reproducibility

For statistical analysis, we used one-way analysis of variance (ANOVA) followed by a Tukey’s or Dunnett’s post hoc multiple comparison test for unequal replications using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA) and SPSS 24 software (International Business Machines Corp., NY, USA). One-way ANOVA for comparisons including more than two groups; unpaired two-tailed t-test for two-group comparisons. All statistical data in the text are presented as the mean ± SD, the value of significance was set at p < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Source data underlying the main and supplementary figures are available in Supplementary Data. The data that support the findings of this study are available from the corresponding author Baoman Li upon reasonable request.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China, B.L. [Grant number 81871852]; LiaoNing Revitalization Talents Program, B.L. [Grant number XLYC1807137], the Scientific Research Foundation for Returned Scholars of Education Ministry of China, B.L. [grant number 20151098], LiaoNing Thousands Talents Program, B.L. [Grant number 202078], “ChunHui” Program of China Education Ministry, B.L. [grant number 2020703], and the National Natural Science Foundation of China D.G. [Grant number 81671862] and [Grant number 81871529].

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These authors contributed equally: Wenzheng Guan, Maosheng Xia.

Authors and Affiliations

Practical Teaching Centre, School of Forensic Medicine, China Medical University, Shenyang, PR China
Wenzheng Guan, Maosheng Xia, Ming Ji, Beina Chen, Shuai Li, Manman Zhang, Shanshan Liang, Binjie Chen, Wenliang Gong, Gehua Wen, Xiaoni Zhan, Dianjun Zhang, Xinyu Li & Baoman Li
The First Department of Reproduction, Shengjing Hospital, China Medical University, Shenyang, China
Wenzheng Guan
Department of Orthopaedics, The First Hospital, China Medical University, Shenyang, PR China
Maosheng Xia, Chengyi Dong & Yuefei Zhou
Department of Forensic Analytical Toxicology, School of Forensic Medicine, China Medical University, Shenyang, China
Ming Ji, Beina Chen, Shuai Li, Manman Zhang, Shanshan Liang, Binjie Chen, Wenliang Gong, Gehua Wen, Xiaoni Zhan, Dianjun Zhang, Xinyu Li, Alexei Verkhratsky & Baoman Li
Department of Forensic Pathology, School of Forensic Medicine, China Medical University, Shenyang, China
Dawei Guan
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
Alexei Verkhratsky
Achucarro Center for Neuroscience, IKERBASQUE, Bilbao, Spain
Alexei Verkhratsky
Sechenov First Moscow State Medical University, Moscow, Russia
Alexei Verkhratsky

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Wenzheng Guan
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Maosheng Xia
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Ming Ji
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Beina Chen
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Shuai Li
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Manman Zhang
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Shanshan Liang
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Binjie Chen
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Wenliang Gong
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Chengyi Dong
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Gehua Wen
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Xiaoni Zhan
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Dianjun Zhang
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Xinyu Li
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Yuefei Zhou
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Dawei Guan
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Alexei Verkhratsky
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Baoman Li
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Contributions

A.V., D.G., M.X. and B.L. designed and supervised the study; M.X., WZ.G., S.L., G.W., BN.C., BJ.C., M.J. and WL.G. collected the data in vitro and analysed the relevant data; WZ.G., SS.L., C.D., X.Z., Y.Z., M.Z., D.Z. and X.L. performed the experiments in vivo and analysed the data; B.L. and A.V. wrote the manuscript. BN.C. and M.Z. collected the images of immunofluorescence, Figs. 1a, b and 2b, f.

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Correspondence to Dawei Guan, Alexei Verkhratsky or Baoman Li.

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Guan, W., Xia, M., Ji, M. et al. Iron induces two distinct Ca²⁺ signalling cascades in astrocytes. Commun Biol 4, 525 (2021). https://doi.org/10.1038/s42003-021-02060-x

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Received: 18 September 2020
Accepted: 30 March 2021
Published: 05 May 2021
DOI: https://doi.org/10.1038/s42003-021-02060-x

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