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. 2014 Jan 28:3:28.
doi: 10.12688/f1000research.3-28.v2. eCollection 2014.

Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury

Laura Genis  1 , David Dávila  1 , Silvia Fernandez  1 , Andrea Pozo-Rodrigálvarez  2 , Ricardo Martínez-Murillo  2 , Ignacio Torres-Aleman  1
Affiliations

Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury

Laura Genis et al. F1000Res. .

Abstract

Oxidative stress is a proposed mechanism in brain aging, making the study of its regulatory processes an important aspect of current neurobiological research. In this regard, the role of the aging regulator insulin-like growth factor I (IGF-I) in brain responses to oxidative stress remains elusive as both beneficial and detrimental actions have been ascribed to this growth factor. Because astrocytes protect neurons against oxidative injury, we explored whether IGF-I participates in astrocyte neuroprotection and found that blockade of the IGF-I receptor in astrocytes abrogated their rescuing effect on neurons. We found that IGF-I directly protects astrocytes against oxidative stress (H 2O 2). Indeed, in astrocytes but not in neurons, IGF-I decreases the pro-oxidant protein thioredoxin-interacting protein 1 and normalizes the levels of reactive oxygen species. Furthermore, IGF-I cooperates with trophic signals produced by astrocytes in response to H 2O 2 such as stem cell factor (SCF) to protect neurons against oxidative insult. After stroke, a condition associated with brain aging where oxidative injury affects peri-infarcted regions, a simultaneous increase in SCF and IGF-I expression was found in the cortex, suggesting that a similar cooperative response takes place in vivo. Cell-specific modulation by IGF-I of brain responses to oxidative stress may contribute in clarifying the role of IGF-I in brain aging.

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Conflict of interest statement

Competing interests: No competing interests were disclosed.

Figures

Figure 1.
Figure 1.. IGF-I signalling participates in astrocyte neuroprotection against oxidative injury.
A) Neurons are protected from oxidative stress in the presence of astrocytes whereas when cultured alone they rapidly die. Viability of GFP neurons was measured as the number of green (GFP +) cells two days after H 2O 2 treatment in the presence or absence of wild type astrocytes (F=41.85; ***p<0.001 vs. neurons alone. B) Both astrocytes and neurons secrete IGF-I, although astrocytes produce much higher levels (*p<0.05 vs neurons). H 2O 2 lowers IGF-I secretion. C) In the presence of a dominant negative IGF-IR (IGF-IR DN) signalling by IGF-I was markedly reduced. Astrocytes were transfected with IGF-IR DN or mock transfected, and the ratio pAkt/Akt (histograms) was measured as an index of IGF-I signalling. Representative blots and quantitative histograms are shown (2 way ANOVA, IGF-I and IGF-IR DN interaction: p<0.05, F=6.99; IGF-I p<0.01, F=13.46; IGF-IR DN p<0.05, F=7.06; Post-hoc: **p<0.01 vs control (mock-transfected) and *p<0.05 vs. IGF-I+IGF-IR DN). D) Blockade of IGF-IR function with IGF-IR DN compromises neuroprotection by astrocytes. GFP neurons were seeded on top of wild type astrocytes transfected with an IGF-IR DN construct or mock-transfected (control) and exposed to 100 µM H 2O 2. Viability of GFP neurons was measured after 5 days (2 way ANOVA, H 2O 2 and IGF-IR interaction: p<0.05, F=10.77; H 2O 2 p<0.01, F=68.92; IGF-IR DN p<0.05 F=17.86; post-hoc: ***p<0.001, *p<0.05 vs. Control; ##p<0.01 vs mock). Experiments were done at least 3 times in this and following figures. Bars are SEM in all figures.
Figure 2.
Figure 2.. Endogenously produced IGF-I protects neurons against oxidative injury.
A) The IGF-IR inhibitor PPP blocks IGF-I signalling in astrocytes. Astrocytes were treated with 120 nM PPP 1h before adding IGF-I while pAkt levels were measured 10 minutes after adding IGF-I. Ratios are shown in histograms (2 way ANOVA, IGF-I and PPP interaction: p<0.01, F=33.07; IGF-I p<0.01, F=27.38; PPP p<0.001, F=112.3; post-hoc: **p<0.01 vs. IGF-I alone). Representative blot is shown. B) Blockade of IGF-IR signalling with PPP in neurons cultured alone does not affect H 2O 2 toxicity after 3–4 days of exposure (2 way ANOVA, H 2O 2 and PPP interaction: F=0.069; H 2O 2 p<0.01, F=12.43; PPP F=3.66; post-hoc: **p<0.01 vs. respective controls). Note that PPP alone does not affect neuronal survival. C) Viability of cerebellar neurons co-cultured with forebrain astrocytes decreased significantly when treated with PPP for six days. PPP treatment in the presence of H 2O 2 decreased neuronal viability even further (2 way ANOVA, H 2O 2 and PPP interaction: F=0.097; H 2O 2 p<0.05, F=9.65; PPP p<0.01, F=31.33; post-hoc: *p<0.05 vs untreated control and #p<0.05 vs H 2O 2). D) Viability of forebrain neurons co-cultured with forebrain astrocytes decreased significantly when treated with PPP for five days. PPP treatment in the presence of H 2O 2 decreased neuronal viability even further (2 way ANOVA, H 2O 2 and PPP interaction: p<0.001, F=23.74; H 2O 2 p<0.001, F=321.6; PPP p<0.001, F=151.3, post-hoc: ***p<0.01 vs. untreated control and ### p<0.01 vs. H 2O 2). E) When co-cultured with wild type astrocytes, neuronal survival after five days of exposure to 100 µM H 2O 2 was moderately increased in the presence of 100 nM IGF-I (2 way ANOVA, H 2O 2 and IGF-I interaction: F=0.542; IGF-I p<0.05, F=7.28; H 2O 2 p<0.001, F=25.9; post-hoc: *p<0.05 vs. control or H 2O 2). I+H: IGF-I + H 2O 2.
Figure 3.
Figure 3.. IGF-I protects astrocytes against oxidative stress.
A) Whereas IGF-I increases neuronal survival under control conditions, it does not protect neurons from H 2O 2 induced death. This confirms previous observations . Neuronal mortality was measured by counting PI + cells 6h after treatment. H 2O 2 induces neuronal death in a dose-dependent manner irrespective of the presence of IGF-I (2 way ANOVA, H 2O 2 and IGF-I interaction: p<0.001, F=10.3; IGF-I p<0.05, F=9.98; H 2O 2 p<0.001, F=128.7; post-hoc: ***p<0.001 vs. no H 2O 2¸, # p<0.05 vs control). B) IGF-I treatment protects astrocytes from H 2O 2 induced death. Astrocyte demise was measured by counting PI + cells 24 h after H 2O 2 (100 µM). H 2O 2 exerts a dose-dependent effect that is reduced by IGF-I (2 way ANOVA, H 2O 2 and IGF-I interaction: p<0.01, F=5.36; IGF-I p<0.001, F=30.29; H 2O 2 p<0.001, F=60.42; post-hoc: *p<0.05 vs control). C) IGF-I blocks FOXO activity induced by H 2O 2 (100 µM). FOXO activity was measured with a luciferase reporter in astrocytes treated with IGF-I, H 2O 2 or both for 24 h (2 way ANOVA, H 2O 2 and IGF-I interaction: p<0.001, F=25.98; IGF-I p<0.001, F=49.58; H 2O 2 p<0.01, F=10.47; post-hoc: ***p<0.001 vs no treatment). D) Protection by IGF-I against cell death induced by H 2O 2 requires blockade of FOXO activity. Astrocyte viability was measured by counting GFP + astrocytes after co-transfection of GFP and a FOXO wild type (wt) or an Akt-insensitive mutant of FOXO (M-FOXO; 2 way ANOVA, M-FOXO and IGF-I interaction: p<0.01, F=59.99; IGF-I p<0.05, F=13.31; M-FOXO p<0.01, F=21.84; post-hoc: *p<0.05 vs no IGF-I). E) IGF-I increases phosphorylation of Akt (pAkt) in the presence of H 2O 2 in a dose-dependent fashion. Representative blots are shown. Lower histograms indicate quantification of pAkt/Akt ratio in the presence of IGF-I as shown in the right blot. pAkt levels were measured after 15 min. (*p<0.05 and ***p<0.001 vs. no H 2O 2).
Figure 4.
Figure 4.. IGF-I reduces oxidative stress in astrocytes.
A) H 2O 2 increases the number of astrocytes expressing mitochondrial O 2 -. This increase is prevented when cells are pre-treated with IGF-I. Mitochondrial O 2 - levels were detected with MitoSOX by flow cytometry. Astrocytes were treated overnight with IGF-I and for 1 hour more with 200 µM H 2O 2 (2 way ANOVA, H 2O 2 and IGF-I interaction: F=1.27; IGF-I p<0.05, F=8.18; H 2O 2 p<0.01, F=16.18; post-hoc: **p<0.01 H 2O 2 vs control, *p<0.05 H 2O 2 vs IGF-I + H 2O 2). B) IGF-I lowers ROS levels after treatment of astrocytes with H 2O 2 (100 µM). Left: representative photomicrographs of astrocytes stained with carboxy-H 2DCFDA to detect ROS and DAPI to stain cell nuclei. The increase in fluorescent cells elicited by H 2O 2 was markedly diminished by IGF-I. Right histograms: fluorimetric quantification of ROS levels with carboxy-H 2DCFDA confirmed the rescuing action of IGF-I on astrocytes exposed to H 2O 2. (2 way ANOVA, H 2O 2 and IGF-I interaction: p<0.05, F=7.38; IGF-I p<0.05, F=5.89; H 2O 2 p<0.05, F=8.49; post-hoc: **p<0.01 H 2O 2 vs control, IGF-I, or IGF-I + H 2O 2).
Figure 5.
Figure 5.. SOD responses to oxidative stress in astrocytes.
A) Cu/ZnSOD levels in astrocytes are modulated by IGF-I and H 2O 2. B) MnSOD levels are enhanced by H 2O 2 but not by IGF-I (*p<0.05 and **p<0.01 vs control).
Figure 6.
Figure 6.. Both H 2O 2 and IGF-I reduce TXNIP1 in astrocytes.
A) Levels of the pro-oxidant protein TXNIP1 are reduced by IGF-I and H 2O 2. Inhibition is greater when both are added together (F=156.6; ***p<0.001 vs. control and ###p<0.001 (vs. IGF-I) and #p<0.05 (vs. H 2O 2). Levels of actin in each sample were measured to normalize TXNIP1 levels. B) Western blot: transfection of astrocytes with shRNA TXNIP1 results in reduced TXNIP1 levels as compared to astrocytes transfected with scrambled shRNA (SCR). Left panel: TXNIP1 shRNA silencing makes astrocytes less sensitive to H 2O 2 toxicity. Astrocyte viability was measured by FDA in the presence of 200µM H 2O 2 (2 way ANOVA, TXNIP1 and H 2O 2 interaction: F=2.94; TXNIP1 shRNA, F=2.94; H 2O 2, p<0.001, F=35.5; post-hoc: **p<0.01 vs control). Right panel: However, neuronal viability is not increased by reduced TXNIP1 in astrocytes as neurons die in the same proportion after H 2O 2 challenge. Viability of neurons was determined after co-culture for three days with astrocytes transfected with TXNIP1 shRNA (2 way ANOVA, TXNIP1 and H 2O 2 interaction: F=0.93; TXNIP1 shRNA, F=0.0097; H 2O 2; p<0.05, F=10.95). C) In neurons, only H 2O 2 decreases TXNIP1 levels, whereas IGF-I does not (***p<0.001 vs control). D) Reduction of TXNIP1 by IGF-I and H 2O 2 in astrocytes depends on Ca 2+ as in the presence of the calcium chelator BAPTA-AM, the decrease is abrogated. (F=7.226; *p<0.05 and ***p<0.001 vs. control). C=control, I=IGF-I, H=H 2O 2, H+I=H 2O 2 + IGF-I.
Figure 7.
Figure 7.. IGF-I cooperates with SCF to promote neuronal survival.
A1) Upper panel: H 2O 2 stimulates SCF mRNA levels in astrocytes after 16 h of exposure whereas IGF-I partially counteracts this increase (F=38.67; *p<0.05 vs. control and IGF-I, #p<0.05 vs. H 2O 2). A2) Lower panel: H 2O 2 stimulates SCF secretion. SCF levels in supernatants from astrocyte cultures treated or not with H 2O 2 and/or IGF-I for 24 h. A representative western blot is shown (*p<0.05 vs control). B) SCF and IGF-I cooperate to protect neurons from oxidative stress. Neurons were pre-treated with SCF, IGF-I or both 48 h before adding H 2O 2 (50 µM) and viability was assessed after overnight treatment (F=12.09, ***p<0.0001 vs H 2O 2), H: H 2O 2; I: IGF-I. C) When H 2O 2 is present, Erk phosphorylation is significantly increased only when both SCF and IGF-I are added to the cultures but not with either alone. Neurons were treated with 100 nM IGF-I, 20 ng/ml SCF and 50 µM H 2O 2 for 5 minutes and pErk levels were measured by western blot and normalized for total Erk. (*p<0.05 and **p<0.01 vs. control without H 2O 2 and #p<0.05 vs. H 2O 2). D) SCF and IGF-I mRNA levels increased 16 hours after middle cerebral artery occlusion (MCAO) in the contralateral side (CONTRA) in the case of SCF (F=31.53; ***p<0.001 vs. intact control mice) and in both sides in the case of IGF-I (F=7.853; *p<0.05 and **p<0.01 vs. control). E) SCF protein levels increase after MCAO in both sides of the cortex (F=12.38; *p<0.05 and ***p<0.001 vs. control). A representative blot is shown. Six, five and four animals were used per group, respectively. Levels of actin in each sample were measured to normalize for total protein levels.
Figure 8.
Figure 8.. Schematic representation of IGF-I neuroprotection through astrocytes.
Left: under basal conditions IGF-I exerts potent neuroprotective actions directly onto neurons, as extensively documented previously (also shown in Figure 3A), and probably also through astrocytes. In the presence of H 2O 2 (right side) the actions of IGF-I on neurons and astrocytes can be summarized in 5 points: 1) IGF-I loses its ability to directly protect neurons, 2) IGF-I secretion by astrocytes is diminished, 3) IGF-I reinforces astrocyte defences against oxidative stress by down-regulating pro-oxidant mechanisms such as TXNIP1. 4) IGF-I cooperates with SCF secreted by astrocytes to promote neuronal survival. 5) However, the precise mechanism(s) downstream of astrocyte IGF-I receptors underlying enhanced astrocyte neuroprotection remains to be determined. Cytotoxic effects are depicted in red while cytoprotective actions are indicated in blue trace.
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Grants and funding

This work was funded by grants of the Spanish Ministry of Science (SAF2010-17036) and Centro Investigacion Biomedica en red Enfermedades Neurodegenerativas (CIBERNED) to IT-A.
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