ABSTRACT
Iron is the fundamental element for numerous physiological functions. Reduced ferrous (Fe2+) and oxidized ferric (Fe3+) are the two ionized iron states in the living organisms. In the cell membrane, divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of Fe2+, whereas transferrin receptors (TFR) carry transferrin (TF)-bound Fe3+. In this study we performed, for the first time, detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Using qPCR and immunocytochemistry we identified DMT1 and TFR in astrocytes in primary cultures, in acutely isolated astrocytes and in brain tissue preparations; in situ both DMT1 and TFR are concentrated in astroglial perivascular endfeet. Administration of Fe2+ or Fe3+ in low μM concentrations evoked Ca2+ signals in astrocytes in vitro and in vivo. Iron ions triggered increase in [Ca2+]i by acting through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibited astroglial Na+-K+-ATPase (NKA), which led to an elevation in cytoplasmic Na+ concentration (as measured by SBFI probe), thus reversing Na+/Ca2+ exchanger (NCX) thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulated phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus trigering InsP3 receptor-mediated Ca2+ release from the endoplasmic reticulum. Iron-induced Ca2+ signals promote astroglial release of arachidonic acid and prostaglandin E2 cytokines by activating cytosolic phospholipase A2 (cPLA2) and NF-κB signalling cascade. In summary, these findings reveal new mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload, in response to which astrocytes actively accumulate excessive iron and activate neuroprotective pathways.
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-heme iron in the brain reaching about 60 mg 3. The non-heme iron concentration in the serum ranges between 9-30 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 means of transferrin receptor (TFR)-mediated internalisation of Fe3+-bound to transferrin (holo-TF), or, for non-TF-bound iron, by vesicular and non-vesicular pathways 6. 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 endosomes 6. Under physiological conditions, the intracellular cytosolic ionized iron levels fluctuate around 0.5-1.5 μM 7.
In the brain, up to three-fourths of total iron is accumulated within neuroglial cells 8. Astrocytes in particular are fundamental elements of ionostatic control over CNS environment 9. Ionized Fe2+ enters astrocytes through DMT1/SLC11A2 transporters which are particularly concentrated in endfeet of cerebral and hippocampal astrocytes 10. The evidence for the expression of TFR in astroglial cells remains controversial 6, 11, 12, while iron overload may influence the expression or distribution of TFR in astrocytic compartments 12, 13. Cellular uptake of Fe3+ requires internalization of TF-TFR complex 14. An adaptor protein Disabled-2 (Dab2) plays an essential role in cell signalling, migration and development 15. In mice the Dab2 has two isoforms of 96 and 67 kDa (p96 and p67 15). In human K562 cells, Dab2 regulates internalization of TFR and uptake of TF 16. Dab2 is also widely distributed in immune cells and in neuroglia 15, 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 17, 18. 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+ sugnals may also be generated by plasmalemmal Ca2+ entry through Ca2+-permeable channels or by sodium-calcium exchanger (NCX) operating in the reverse mode 17, 19. 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 20–22; as well as by Na+ extrusion through the sodium-potassium pump (NKA). Both Na+ and Ca2+ signalling systems are closely coordinated, with NKA and NCX accomplishing this coordination at the molecular level 23. Astrocytes specifically express α2-subunit containing NKA which is fundamental for astroglial K+ buffering 24. Astrocytes express all three isoforms of NCX - NCX1/SLC8A1, NCX2/ SLC8A2 and NCX3/SLC8A3, with some evidence indicating higher expression of NCX1 25. 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 26, 27. The NCX are also known to localise at caveolae with caveolin-3 (Cav-3), the latter isoform being predominantly expressed in astrocytes 28.
The excess of iron may cause abnormal [Ca2+]i signalling and the dysregulation of the downstream kinase cascades. Iron can increase the protein expression of P65 subunit of nuclear factor κB (NF-κB) 29, which was claimed to contribute to neuroinflammation and neuroprotection through activation of cytosolic phospholipase A2 (cPLA2) in astrocytes 30. The cPLA2 is a Ca2+-dependent phospholipase, which can hydrolyze the fatty acid at sn-2 position of glycerophospholipids to produce arachidonic acid (AA), which is an astroglia-specific process 31. In our previous studies we demonstrated that the Ca2+-stimulated phosphorylation of cPLA2 contributes to the fast secretion of AA and prostaglandin E2 (PGE2) from spinal cord astrocytes 32, 33.
In the present paper we performed an in depth analysis of the action of ferrous and ferric (Fe2+ and Fe3+) on astroglial Ca2+ and Na+ dynamics. We found that Fe2+ (via DMT1) and Fe3+-TF (via TFR) evoke [Ca2+]i transients in astrocytes in culture and in vivo. Effects of Fe2+ on [Ca2+]i were mediated mainly by the reversed NCX, whereas Fe3+ triggered Ca2+ release from the endoplasmic reticulum by stimulations of InsP3R. Finally, we quantified iron-induced downstream activity of cPLA2 and NF-κB as well as cPLA2-regulted up-regulation of arachidonic acid (AA) and prostaglandin E2 (PGE2).
RESULTS
Fe2+/Fe3+ trigger [Ca2+]i increase in cortical astrocytes in vitro and in vivo
We analysed Ca2+ dynamics in astrocytes in primary cultures and in vivo in the GFAP-eGFP transgenic mice for cell identification (Fig. 1). In the primary cultured astrocytes, administration of both 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 rise (Fig 1A). Both ions induced [Ca2+]i responses in a concentration dependent manner with apparent EC50 of 0.635 μM for Fe2+ and 0.711 μM for Fe3+ (Fig. 1C).
When imaging cortical astrocytes in vivo (the cells were identified by specific eGFP fluorescence) we found that addition of either Fe2+ or Fe3+ to the superfusing solution 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 florescent signal to 308.50% ± 13.01% (n = 10, p < 0.0001) of the basal value.
DMT1 and TFR mediate Fe2+ and Fe3+ uptake
As mentioned above, Fe2+ uptake is mediated by plasmalemmal transporter DMT1, whereas Fe3+ is 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 showed preferential localisation at privascular endfeet. Meanwhile, expression of specific DMT1 and TFR mRNA was also detected in the freshly isolated and FACS-sorted astrocytes and neurones, as well as and in the cerebral tissues (Fig. 2C).
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 protein 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). 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). In un-stimulated astrocytes in culture the DMT1 fluorescence was the highest around the nucleus, suggesting its preferred intracellular localisation. After treatment of the cultures with Fe2+ for 5 minutes we observed redistribution of DMT1 from the nuclear region to the plasma membrane (Fig. 2F).
Sources of iron-induced [Ca2+]i mobilisation
The main sources of [Ca2+]i, increase in astrocytes are (i) Ca2+ release from the ER following 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 highlighted the role for plasmalemmal Ca2+ influx in Ca2+ signalling triggered by Fe2+ and ER Ca2+ release for Ca2+ signals triggered by Fe3+.
Incubation of astrocytes with an 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 selective agonist KB-R7943 at 10 μM completely eliminated [Ca2+]i response to Fe2+, without affecting Fe3+-induced [Ca2+]i transients (Fig. 3D). Thus, two forms of iron, the ferrous and 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 34, 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 Ca2+-induced Ca2+ release ER channels) 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 usually triggered following an increase in the [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 19, 21. The activity of NKA was acutely 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 further to 71.80 ± 7.81% (n = 10, p < 0.0001) (Fig. 4A).
Inhibition of NKA in astrocytes results in a substantial elevation in [Na+]i. When monitoring the [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). When the cells were pre-treated with ouabain for 30 min, application of Fe2+ had no effect on [Na+]i (n = 10, data not shown), whereas acute application of ouabain and Fe2+ lead to an increase in [Na+]i (Fig 4B). These changes in [Na+]i were paralleled by [Ca2+]i dynamics. Exposure of astrocytes to Fe2+, ouabain 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 was decreased to 7.41 ± 4.32% (n = 6, p < 0.0001) of the control (Fig. 4E). An in vitro knock-down of Cav3 significantly reduced the man 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); whereas in control astrocytes treated with negative 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 to only 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 knock down Cav3, Fe2+ only elevated the level of NCX1 and NKA 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).
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 affects 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 resting InsP3 level of 24.40 ± 6.35 ng/ml in control group. 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, expressions of 96KD and 67KD 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 to Fe3+ in Dab2-deficient astrocytes 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).
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).
Iron increases phosphorylation of cPLA2 and stimulates secretion of arachidonic acid and prostaglandin E2
To characterise downstream cascades induced by iron ions we measured phosphorylation of cPLA2 and NF-κB. As compared to the control group, Fe2+ and Fe3+ increased the ratio of p-cPLA2 and cPLA2 respectively to 210.00 ± 19.38% (n = 10, p < 0.0001) and 177.42 ± 15.05% (n = 10, p < 0.0001) (Fig. 7A, B). Similarly, the ratio of the phosphorylated P65 and P65 was increased by Fe2+ and Fe3+ to 164.58 ± 10.66 (n = 10, p < 0.0001) and 141.20 ± 11.85% (n = 10, p < 0.0001) of the control values (Fig. 7A, C). The pre-treatments with Ca2+ chelator BAPTA-AM abolished an increase in the phosphorylation of cPLA2 following exposure to Fe2+ or Fe3+ to 100.65 ± 19.93% (n = 10, p = 0.9429) and 83.87 ± 17.86% (n = 10, p = 0.0716) of the control. Likewise, BAPTA-AM suppressed phosphorylation of P65 stimulated by Fe2+ or Fe3+ to 90.28 ± 13.80% (n = 10, p = 0.0939) and 96.99 ± 19.26% (n = 10, p = 0.6700) of the controls (Fig. 7A, C). Treatment with NCX inhibitor KB-R7943 and InsP3 receptor blocker Xe-C also abolished the phosphorylation of cPLA2 and P65 (Fig. 7A-C). The KB-R7943 decreased the phosphorylation of cPLA2 induced by Fe2+ to 98.46 ± 20.20% (n = 10, p = 0.8534) of control group, while Xe-C suppressed the Fe3+-stimulated phosphorylation of cPLA2 to 103.75 ± 23.32% (n = 10, p = 0.7065) of the control value. These two blockers had the same effect on phosphorylation of P65: Xe-C decreased the Fe2+-induced phosphorylation of P65 to 99.51 ± 16.82% (n = 10, p = 0.9418) of the control, whereas Xe-C decreased phosphorylation of P65 stimulated Fe3+ to 102.86 ± 17.71% (n = 10, p = 0.7048) of the control (Fig. 7B, C).
We also measured secretion of AA and PGE2 using ELISA. As compared with control group, Fe2+ and Fe3+ increased extracellular level of AA to 180.90 ± 7.43% (n = 10, p < 0.0001) and 164.60 ± 4.35% (n = 10, p < 0.0001) of the control. Pre-treatment with BAPTA-AM (that eliminates any increase in [Ca2+]i) completely inhibited Fe2+ and Fe3+ -induced rise in AA secretion (to 100.10 ± 5.43% (n = 10, p = 0.9651) and 102.00 ± 3.74% (n = 10, p = 0.3014) of the controls (Fig. 7D). Similarly, inhibitors of NCX and InsP3 receptors eliminated effects of Fe2+ and Fe3+. Incubation with KB-R7943 decreased the level of AA in the presence of Fe2+ to 101.70 ± 5.06% (n = 10, p = 0.5803) of the control, while XeC suppressed the level of AA induced by Fe3+ to 100.40 ± 5.70% (n = 10, p = 0.8951) of the control (Fig. 7D). Treatment with Fe2+ and Fe3+ also stimulated secretion of PGE2; the levels of the latter increased, respectively, to 174.12 ± 12.39% (n = 10, p < 0.0001) and 152.21 ± 6.24% (n = 10, p < 0.0001) of the control values. Again, pre-treatment with BAPTA-AM completely inhibited stimulatory effects of iron on secretion of PGE2 (levels of PGE2 in the presence of Fe2+ and Fe3+ were 98.23 ± 11.19% (n = 10, p = 0.7538) and 99.33 ± 8.44% (n = 10, p = 0.8968) of the controls (Fig. 7E). Likewise, KB-R7943 suppressed Fe2+-induced, whereas XeC inhibited Fe3+-indueced stimulation of PGE2 release (with PGE2 values of 99.79 ± 8.48% (n = 10, p = 0.9550) 97.42 ± 11.38% (n = 10, p = 0.5991) of the controls (Fig. 7E). Finally, Fe2+ and Fe3+-induced phosphorylation of P65 was blocked by the specific inhibitor of cPLA2, AACOCF3 (Fig. 7F). After the pre-treatment with 2 μM AACOCF3, the level of p-P65 in the presence of Fe2+ was 98.77 ± 10.48% (n = 10, p = 0.7795) and the level of p-P65 in the presence of Fe3+ was 98.77 ± 7.81% (n = 10, p = 0.7451).
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 0.635 μM for Fe2+ and 0.711 μM for Fe3+. We further 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+.
Iron transport in astrocytes is mediated by DMT1 and TFR
Glial cells, and astrocytes in particular, store up to 75% of ionised iron in the CNS 35, being arguably active players in brain protection against iron overloads 36. Transmembrane transport of iron in astroglial cells is has not been studied in details. There is a general agreement of primary role of plasmalemmal divalent metal transporter 1, DMT1/SLC11A2, which selectively transports Fe2+; the DMT1 was identified in astrocytes in culture and there are limited data indicating its presence in astroglial endfeet in situ 37–39. The Fe2+ was also suggested to enter reactive astrocytes by diffusion through transient receptor potential “canonical” (TRPC) channels 36. Expression of TF-Fe3+-transporting TFR has been noted in astrocytes in culture (ref 12, 40); it is, however, generally believed that astrocytes in vivo are not in a possession of TFR and hence can not accumulate Fe3+ 41–43. This conclusion, however, has been made on the basis of rather limited investigations 40, 44; and expression of TFR-specific mRNA was detected in astroglial transriptome 45. 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). Subsequently we detected astroglial TFR expression at mRNA level in the transcriptase of astutely 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 the cortical tissue TFR labelling was concentrated in perivascular astrocytic endfeet (Fig. 2B).
Mechanisms of iron induced Ca2+ signalling
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 neurones 46. 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 when administered to the cortices of alive animals studied with transcranial confocal microscopy. Both ions acted in the low μM range of concentrations, however the kinetics of [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 recovered to the baseline within ~ 200 – 300 s in the presence of Fe3+ (Fig. 1B, 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 eliminated Ca2+ signal (Fig. 2). The Fe2+-induced [Ca2+]i changes originate from plasmalemmal Ca2+ entry, because removal of Ca2+ from the extracellular medium inhibited [Ca2+]i response. Finally, Fe2+-induced [Ca2+]i signals require NCX, as pharmacological inhibition of the latter effectively suppressed [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 which generates Ca2+ influx into the cell in exchange for Na+. This scenario requires increase in astroglial [Na+]i, which readily reverses the NCX 19, 47. An increase in [Na+]i is likely to follow an inhibition of NKA, which represents the major Na+ efflux mechanism in astroglial cells 21. Activity of NKA indeed was suppressed by Fe2+, and probing astrocytes with Na+-sensitive indicator SBFI revealed Fe2+-induced [Na+]i elevation (Fig. 4). These effects of Fe2+ were eliminated by NKA inhibitor ouabain thus demonstrating the central role of NKA in Fe2+-induced Ca2+ signalling (Figs. 4, 8).
The mechanism of Fe3+-induced Ca2+ signalling is associated with intracellular Ca2+ release. The Fe3+-induced [Ca2+]i responses were 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 eliminated 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 homeostasis, inflammation and receptors internalization 48. For example, Dab2 influences signalling pathways of fibrinogen and its receptors (integrin αIIbβ3) by regulating the complex internalization thus modulating the platelet aggregation 48, 49. In addition, Dab2 facilitates the binding of the internalized integrin αIIbβ3 and phosphatidylinositol 4,5-bisphosphate (PIP2) in the activated platelet 48, 50. Ablation of Dab2 in astrocytes with specific siRNA interrupts signalling chain and blocks Fe3+-dependent Ca2+ signalling (Figs. 6, 8).
Role of caveolae in iron-induced Ca2+ signalling
Caveoale are specific plasmalemmal structures that form functional microdomains involved in various signalling, endocytotic and transporting events 51. Caveolae and its main structural and regulatory proteins Caveolin-1,2,3 are present in astrocytes (with predominant expression of Cav3); astroglial caveolae contribute to signal transduction, formation of signalling protein complexes and are involved in action of various neuroactive substances and drugs 52, 53. Caveolae are known to form functional Ca2+ signalling units, establish links between Ca2+ channels and various transports and may be a substrate for plasmalemmal/ER functional domains operational in astrocytes 54. 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. 8) and moreover exposure to iron increases formation of such units.
Do astrocytes protect the brain against iron overload?
Iron homeostasis is of fundamental importance for cells, tissues and organisms, as iron contributes to a wide range of vital biological pathways 55. The brain contains high concentrations of bound and free iron, which participates in multiple processes from energy production to synaptic transmission 35. Iron overload and failures in iron homoeostatic cascades triggers neurotoxicity and is implicated in brain diseases 42. Genetic mutation of iron regulatory proteins (the key elements of iron homoeostasis) results in iron deposition in the brain with subsequent neurodegeneration characteristic for aceruloplasminemia 56 and neuroferritinopathy 57. Similarly, iron accumulation has been characterised in neurodegenerative diseases including Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis and Huntington disease to name but a few 42. Specific class of neurodegeneration with brain iron accumulation (NBIA) has been also categorised in recent years 58.
Based on our data we propose that astrocytes mount the defence against 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 stimulate NF-κB cascade and increase secretion of neuroprotective molecules such as arachidonic acid and prostaglandin E2. Iron-induced Ca2+ signalling is activated by 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 59, 60. Astrogliosis plays important, if not defining, role in the evolution of many neurological diseases 61. Our previous experiments have shown that formation of brain deposits of iron up-regulates astroglial expression of TFR and instigates reactive astrogliosis 62. 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 presents a novel phenomenon that iron ions (Fe2+ and Fe3+) directly induce intracellular Ca2+ signalling and stimulate astroglial protective mechanisms against iron overload in broad pathological contexts.
MATERIALS and METHODS
Materials
The culture medium including DMEM and foetal 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, p-cPLA2 antibody, cPLA2 antibody, p-NF-κB P65 antibody, NF-κB P65 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, AACOCF3, BAPTA-AM, ferrous sulfate heptahydrate (FeSO4) and ferric ammonium citrate were purchase from Sigma-Aldrich (St. Louis, MO, USA). Ryanodine and KB-R7943 was 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).
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◻; light/dark cycle of 12/12h), 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 as described previously 63, 64. 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 ◻ in a humidified atmosphere of CO2/air (5:95%). The cultures are highly enriched in astrocytes, the purity is >95% as judged by GFAP staining 65.
Iron Treatments
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 hour at 37 ◻ 66, 67. 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 ◻ and used immediately, the same volume of serum-free culture medium was used as the control for Fe2+ group.
RNA Interfering
As described previously 65, 68, 69, cultured astrocytes were incubated in DMEM without serum for 12 hours before transfection. 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 as previously described 70, 71. In brief, primary cultured astrocytes were collected and homogenised in SET (0.315 M sucrose, 20 mM TrisCl, 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 subseqeunet western blotting to check the conjunction level between NCXs and DMT1, as described previously 68. After homogenization, protein content was determined by the Bradford method 72 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).
Western Blotting
As described previously 64, 73, for quantifying expressions of DMT1, TFR, NCX1-3, Cav-3 or Dab2, and for detection the phosphorylation of cPLA2 and P65, 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 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™ FC 32-bit software.
Monitoring of [Ca2+]i
For [Ca2+]i monitoring and imaging in cultured astrocytes, experiments were run as previously described 74, 75. After the pre-treatment with or without inhibitors or siRNA duplex chains, the primarily cultured astrocytes were loaded with 5 μM fluo-4-AM, molecular probes (Thermo Fisher Scientific (Waltham, MA USA)), for 30 min. Fluo-4 signals were visualised by fluorescent microscopy (Olympus IX71, Japan). The fluorescence intensity was normalised to the baseline intensity before stimulation.
Two-photon in vivo Ca2+ imaging
As described previously 75, 76, adult FVB/N-Tg(GFAP-eGFP)14Mes/J transgenic mice (10 to 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 cortical cells were loaded with Ca2+ indicator Rhod-2 AM (50 μM, 1 hour). 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 540nm/40nm for eGFP and 850nm/70nm for rhod-2 signals. Time-lapse images of astrocytic Ca2+ signalling were recorded every five second using FluoView with a custom-built two-photon laser-scanning setup (Nikon AR1, Japan).
Intracellular Na+ measurements
For monitoring intracellular ionized Na+ ([Na+]i) in cultured astrocytes, the measurements were performed as described in 47. Primary cultured astrocytes were loaded with 10 μM of Na+-sensitive indicator SBFI-AM for 30 min in serum-free medium, with subsequent 1 hour 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).
Immunofluorescence
The brain tissue was fixed by immersion in 4% paraformaldehyde and cut into 100 μm slices (see also 64, 73). The cultured cells were fixed with 100% methanol at −20 °C. Brain slices or cells were permeabilised by incubation for 1 hour 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 markers 4’, 6’-diamidino-2-phenylindole (DAPI) at 1:1000 dilution. The incubation with the primary antibodies were 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 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 control group. To assay the InsP3 concentration 77, the supernatant was collected and the concentration of InsP3 assayed using a commercial ELISA kit (E-EL-0059c; Elabscience Biotechnology, Wuhan, China), the sensitivity is 10 pg/mL. PGE2 concentrations were measured using a specific ELISA kit (SEKM-0173, Solarbio Life Sciences, Shanghai, China), the assay was performed as the manufacturer’s protocols 32, 33.
PGE2 production was evaluated from a standard curve of PGE2, and the released level was finally calibrated by the protein content. The sensitivity of the assay allowed detection is 3 pg/ml. When necessary, the samples were diluted in the assay buffer. The level of AA was determined by using a commercial ELISA kit (E-EL-0051c; Elabscience Biotechnology, Wuhan, China), the sensitivity is 1 ng/mL. The results were normalized by control group and presented as the percentage 32, 33.
Astrocytes were incubated at 37 °C in 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 determine the NKA activity, a commercial ELISA kit (abx255202; Abbexa, Cambridge, UK) was used and operated as prescribed; the sensitivity is 0.19 ng/mL, the optical density (OD) was measured at 450 nm and the OD value was normalized to the control group. To assay the InsP3 concentration 77, the cultured cells were placed into lysate and centrifuged at 10,000×g at 4°C for 10 min, the supernatant was collected and the concentration of IP3 assayed using a commercial ELISA kit (E-EL-0059c; Elabscience Biotechnology, Wuhan, China). Preparation of the reference standard and sample diluent, adding working solution, substrate reagent and stop solution in sequence. Determine the OD value with a micro-plate reader at 450 nm, the sensitivity is 10 pg/mL. PGE2 concentrations were measured using a specific ELISA kit (SEKM-0173, Solarbio Life Sciences, Shanghai, China), the assay was performed as the manufacturer’s protocols 32, 33. PGE2 production was evaluated, the amounts were calculated from a standard curve of PGE2, and the released level was finally calibrated by the protein content. The sensitivity of the assay allowed detection is 3 pg/ml. When necessary, the samples were diluted in the assay buffer. And the level of AA was assayed by using a commercial ELISA kit (E-EL-0051c; Elabscience Biotechnology, Wuhan, China), the sensitivity is 1 ng/mL. The results were normalized by control group and presented as the percentage 32, 33.
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 neurones expressing fluorescent marker YFP (Thy1-YFP mice) were used; we also extracted the cerebral hemispheres tissues from wild type mice. As previously described 76, 78, the cells from transgenic mice were used for specific sorting of astrocytes or neurones with FACS. 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, as per previous description 73, 76. 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.
Statistical Analysis
For statistical analysis we used one-way analysis of variance (ANOVA) followed by a Tukey 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). All statistical data in the text are presented as the mean ± SD, the value of significance was set at p < 0.05.
Author Contributions
A.V., M.X. and B.L. designed and supervised the study; M.X., W.G., S.L., G.W., N.X., BN.C., BJ.C., M.J. and W.G. collected the data in vitro and analysed the relevant data; SS.L., Z.L., C.D., D.Z. and X.L. performed the experiments in vivo and analysed the data; B.L. and A.V. wrote the manuscript.
Conflict of interest
The authors have no conflicts of interest to disclose.
Acknowledgments
This study was supported by Grant No. 81871852 to BL from the National Natural Science Foundation of China, Grant No. XLYC1807137 to BL from LiaoNing Revitalization Talents Program, and Grant No. 20151098 to BL from the Scientific Research Foundation for Returned Scholars of Education Ministry of China. Grant No. 20170541030 to MX from the Natural Science Foundation of Liaoning Province.
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