Reactive astrocytes fail to de-differentiate

Mature astrocytes have been shown to regain neural stem cell potential under controlled conditions in vitro. However, one of the major questions remains, namely why they do not de-differentiate in vivo, refilling the pool of lost neurons e.g., in a neurodegenerative disease, after a stroke, or other injuries. FGF2 is produced by e.g., astrocytes, neurons, or endothelial cells after stab wound injury (Logan et al. 1992), and thus astrocytes could be triggered to proliferate and de-differentiate. Nevertheless, there are many more factors produced and released by astrocytes or other cells, e.g., microglia, during disease or in-jury, which might inhibit the de-differentiation. To simulate an activation of microglia, which occurs in almost every pathological insult in the brain, inflammatory cytokines that are released by reactive microglia and other cells, have been tested during the de-differ-entiation of mAGES with FGF2.

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Fig. 20. Block of the generation of NSC2 from mAGES by IFNγ signaling

(A) The mAGES were exposed to 0 (ctrl, w/o factors) or 20 ng/ml FGF2 for 8 days. FGF-exposed cells were co-treated with various combinations of inflammatory cytokines (10 ng/ml TNFα, 10 ng/ml IL1β, 20 ng/ml IFNγ) or a complete cytokine mix (CCM). At the end of the exposure, proliferation was measured by quantification of additional resazurin reduction. Data are means ± SEM. ***, p <.0001; *, p <.01; ns, not significant. (B) The experiment was performed as in (A), and EdU incorporation was measured by high throughput imaging. (C)The mAGES were exposed to 0 (ctrl, w/o factors) or 20 ng/ml FGF2 for 8 days (data points for systems calibration). FGF2-exposed cells were co-treated with 20 ng/ml IFNγ and various concentrations of ruxolitinib (Rux). EdU was quantified as in (B). Data are means±SEM. (D) The experi-ment was performed as in (C), with CCM instead of IFNγ. (E) The mAGES were pre-incubated for 30 min with ruxolitinib, and exposed to 20 ng/ml FGF2 and/or 20 ng/ml IFNγ for 20 min. Phospho-STAT1 (pSTAT1) was analyzed by Western blot analysis, with actin as loading control. (F) The experiment was performed as in (E), but with CCM instead of IFNγ. The experiment was repeated once with a similar result.

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The mAGES were exposed to 10 ng/ml IL1β, 10 ng/ml TNFα, 20 ng/ml IFNγ, or combi-nations thereof during de-differentiation with FGF2. Resazurin reduction already re-vealed that FGF2 failed to induce the proliferation of cells in the presence of specific cytokine combinations (Fig. 20A). While TNFα and IL1β alone did not reduce prolifera-tion, a combination of both cytokines significantly decreased it. Interestingly, IFNγ alone strongly inhibited the proliferation of mAGES. The same result could be found, when measuring EdU incorporation. TNFα and IL1β in combination significantly inhibited EdU incorporation induced by FGF2 in mAGES (Fig. 20B). IFNγ alone strongly de-creased EdU incorporation, and the effect was even enhanced with the complete cytokine mix (CCM). Nestin upregulation and GFAP downregulation in mAGES induced by FGF2 was significantly, but not strongly inhibited by the presence of cytokines (Supplemental Fig. 23). IFNα or IFNβ did not show any effect on the de-differentiation of mAGES (data not shown).

As IFNγ alone was sufficient to inhibit the de-differentiation of mAGES, it was used for further studies on the mechanisms of inhibition. Since IFNγ induced a strong induction of iNOS (inducible nitric oxide synthase) in astrocytes, which is even enhanced with CCM (data not shown), we assumed NO-production being responsible for the inhibition.

Several inhibitors of NOS enzymes were tested (L-NNA, AMT, 7-NINA) in mAGES exposed to FGF2, but none of them inhibited proliferation induction as measured by resazurin reduction or EdU incorporation (data not shown). Moreover, the addition of an NO-donor during FGF2 exposure did not show any effect at all (data not shown). Thus, a mechanism different from NO-production must be responsible for the inhibition of de-differentiation.

IFNγ stimulation of cells involves the activation of the JAK/STAT pathway. We therefore tested ruxolitinib (Rux), an inhibitor of JAK1/2. Western blot analysis revealed that 1 µM of the inhibitor was sufficient to completely block phosphorylation of STAT1, which was induced by the presence of IFNγ (Fig. 20E). When mAGES were exposed to FGF2 and IFNγ in combination, ruxolitinib blocked the proliferation inhibition caused by IFNγ, as measured by resazurin reduction (Supplemental Fig. 24A) as well as EdU incorporation (Fig. 20C). The same effect was seen, when mAGES were de-differentiated in the pres-ence of CCM. Ruxolitinib (1 µM) completely blocked STAT1 phosphorylation (Fig.

20F), and proliferation inhibition in mAGES by CCM was recovered by ruxolitinib (Fig.

20D, Supplemental Fig. 24B). Thus, ruxolitinib restored de-differentiation of mAGES

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even in the presence of cytokines. A direct effect of either IFNγ or ruxolitinib on FGF2 signaling (i.e., phosphorylation of ERK) could not be observed (Supplemental Fig. 24D).

Moreover, IFNγ induced phosphorylation at different sites of STAT1. While short term exposure to IFNγ (30 min) only induced a phosphorylation of the tyrosine residue Y701, long-term exposure (24 h) lead to the phosphorylation of the serine residue S727. The phosphorylation at both sites were inhibited by ruxolitinib (Supplemental Fig. 24C).

Hence, the presence of cytokines disturbed the de-differentiation of mAGES, involving an activation of the JAK/STAT pathway.

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4 D ISCUSSION

4.1 The generation of astrocytes from stem cells

The study of metabolic and functional features of astrocytes, in their resting state or in different defined activation scenarios, faces a number of challenges: 1) measurements in vivo require a distinction of astrocytes from surrounding cells for the analytical endpoints chosen; 2) ex vivo studies, using e.g., FACS-purified adult astrocytes, suffer from a com-promised viability of the obtained cells, and from undefined activation states, when put in culture; 3) in vitro studies mainly rely on studies of mixed populations, prepared from relatively immature cells. They may contain precursor cells, reactive astrocytes and other cell types, such as microglia. Additional approaches would thus be desirable to further explore astrocyte biology. In our study, we present such an alternative strategy that allows generation of a pure population of non-proliferative, non-activated astrocytes.

4.1.1 Homogeneity of mAGES in contrast to primary astrocyte cultures