An unconventional role for mirna: let-7 activates Toll-like receptor 7 and causes neurodegeneration

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1 An unconventional role for mirna: let-7 activates Toll-like receptor 7 and causes neurodegeneration Sabrina M. Lehmann, Christina Krüger, Boyoun Park, Katja Derkow, Karen Rosenberger, Jan Baumgart, Thorsten Trimbuch, Gina Eom, Michael Hinz, David Kaul, Piet Habbel, Roland Kälin, Eleonora Franzoni, Agnieszka Rybak, Duong Nguyen, Rüdiger Veh, Olaf Ninnemann, Oliver Peters, Robert Nitsch, Frank L. Heppner, Douglas Golenbock, Eckart Schott, Hidde L. Ploegh, F. Gregory Wulczyn, Seija Lehnardt Supplementary Figure 1. let-7a, let-7c, let-7g, and mir-599 induce the release of TNF-α from macrophages through TLR7. Immortalized wild-type (WT) or TLR7 / bone marrowderived macrophages (BMDMs) were incubated for 12 h with various doses of let-7a (a), let-7c (c), let-7g (e), or mir-599 (g) or with 5 µg/ml of let-7a (b), let-7c (d), let-7g (f), or mir-599 (h) for various durations, as indicated. 10 µg/ml mutant oligoribonucleotide were used as negative control; 100 ng/ml LPS were used as positive control. Subsequently, TNF-α amounts in the culture supernatants were determined by ELISA. Results are presented as mean ± SD. One representative experiment of at least 3 independent experiments is shown. n.d., not detected. 1

2 Supplementary Figure 2. Extracellularly delivered let-7a, let-7c, let-7g, and mir-599 induce neuronal cell death through TLR7. Neurons from C57Bl/6J (WT) and TLR7 / mice were incubated with let-7a (a), let-7c (c), let-7g (e), or mir-599 (g) at various doses for 4 d or with 5 µg/ml of let-7a (b), let-7c (d), let-7g (f), or mir-599 (h) for various durations, as indicated. 10 µg/ml mutant oligoribonucleotide were used. Quantity of NeuN-positive cells was expressed as relative neuronal viability. Results are presented as mean ± SD. p= (a), p= (b), p= (c), p= (d), p= (e), p= (f), p= (g), p= (h) over all WT groups, and not significant over all TLR7 / groups (Kruskal-Wallis test). p*=0.045, p**=0.0161, p***=0.0103, p#=0.0064, p##=0.005, p###= (Mann-Whitney U test) for the comparison of indicated groups. One representative experiment of 3 independent experiments is shown. 2

3 Supplementary Figure 3. Purified neuronal cultures do not contain significant numbers of glial cells. (a) Purified cortical neurons from C57Bl/6J mice were immunostained with NeuN antibody and with IB4 or GFAP antibody to mark neurons, microglia, and astrocytes, respectively. Scale bar, 50 µm. (b) Quantification of neurons (NeuN + ), microglia (IB4 + ), astrocytes (GFAP + ), and Oligodendrocytes (O4 + ) in purified neuronal cultures at DIV3 and DIV7, as indicated. Results are presented as mean ± SD. 3

4 Supplementary Figure 4. Stimulation of neurons with let-7b does not activate NF-κB. (a) Purified neurons were incubated with 5 µg/ml let-7b for various time periods,, as indicated or with 5 µg/ml mutant oligoribonucleotide for 2 h. 100 ng/ml TNF-α served as positive control. (b) Purified microglia were incubated with 5 µg/ml let-7b or with 5 µg/ml mutant oligoribonucleotide for 2 h. 1 µg/ml LPS served as positive control. Subsequently, cell lysates were assayed for NF-κB activation by EMSA. Oct-1 binding to H2B served as loading control. One representative experiment of at least 3 independent experiments is shown. 4

5 Supplementary Figure 5. Astrocytes do not affect neuronal cell death induced by let-7b in vitro. (a) Astrocytes from C57Bl/6J mice were incubated for 12 h with various doses of let-7b, as indicated. 10 µg/ml mutant oligoribonucleotide were used as negative control. The following doses of other compounds were used: LPS 100 ng/ml, imiquimod 10 µg/ml. Subsequently, the amount of TNF-α in the culture supernatants was determined by ELISA. (b) Neurons with or without astrocytes from C57Bl/6J mice were incubated with various doses of let-7b, as indicated, 10 µg/ml mutant oligoribonucleotide, or PBS (control) for 4 d. Relative neuronal viability was assessed by quantification of NeuN-positive cells. Results are presented as mean ± SD. One representative experiment of 3 independent experiments is shown. n.d., not detected. 5

6 Supplementary Figure 6. let-7-induced neurotoxic effects do not require phosphorothioate modification of the oligoribonucleotide. (a) Cortical neurons from C57Bl/6J mice were incubated with 5 µg/ml biotinylated let-7b or PBS (control) for 12 h and subsequently stained with Avidin-Alexa 488 and DAPI. Scale bar, 10 µm. (b) Neurons were incubated with the indicated doses of non-phosphorothioated, native let-7b oligoribonucleotide or with let-7b modified by phosphorothioate linkages for 4 d. Subsequently, surviving NeuN-positive cells were quantified, and expressed as relative neuronal viability compared to control cells. Results are presented as mean ± SD. p< over all groups (Kruskal-Wallis test). p-values for relevant groups as determined using the Mann-Whitney U test are shown. One representative experiment of at least 3 independent experiments is shown. 6

7 Supplementary Figure 7. Prior inhibition of let-7 in neurons undergoing apoptosis reduces neurotoxic properties of the supernatant. Neurons were transfected with 100 nm let-7 family inhibitor (let-7 FI) or non-specific inhibitor (neg. co). After 24 h, cells were forced to undergo apoptosis, and recovered supernatants were added to cortical neurons from wild-type (WT) or TLR7 / mice. After a further 4 d neurons were immunostained with NeuN and Neurofilament antibodies. Scale bar, 50 µm. One representative experiment of at least 3 independent experiments is shown. 7

8 Supplementary Figure 8. Direct inhibition of let-7 in supernatants from apoptotic and necrotic neurons leads to reduced neurotoxicity. Supernatants derived from apoptotic (a) and necrotic (b) neurons were supplemented with 100 nm let-7 family inhibitor (let-7 FI) or nonspecific inhibitor (neg. co) before application to cortical neurons derived from wild-type (WT) or TLR7 / mice. After 4 d, neurons were immunostained with NeuN antibody, and relative neuronal viability was assessed. Results are presented as mean ± SD. p< over all groups (Kruskal-Wallis test). Statistical comparison of indicated groups was performed using the Mann- Whitney U test. One representative experiment of at least 3 independent experiments is shown. 8

9 Supplementary Figure 9. Validation of LNA-based mirna inhibitor specificity. (a) A mirna sensor assay based on egfp plasmids containing binding sites for let-7, mir-124 or mir-128 in the 3 UTR was performed using HEK293 cells co-transfected with the indicated sensor plasmid and the synthetic mirna, as indicated. Each transfection mix contained either the negative control inhibitor (neg. co, 10 nm or 100 nm, as indicated) or the let-7 family inhibitor (let-7 FI, 10 nm or 100 nm, as indicated). egfp mean fluorescence intensity was determined for three independent experiments and expressed relative to each sensor with the scrambled mirna and negative control inhibitor (100%). egfp expression of each sensor was reduced 5-20-fold upon co-transfection of the corresponding mirna. let-7b and let-7e activity was blocked by let-7 FI. Values >100% reflect inhibition of endogenous let-7 activity by let-7 FI. mir-124 and mir-128 were unaffected by let-7 FI. (b) Neurons from C57Bl/6J mice were incubated with 5 µg/ml let-7b, mir-599, or mut. oligo with 100 nm let-7 FI or negative control inhibitor (neg. co). After 4 d, neurons were immunostained with NeuN antibody. Relative neuronal viability was assessed. Whereas let-7b-induced neurotoxicity was abolished by let-7 FI but not by neg. co, mir-599-induced neurodegeneration was not affected by let-7 FI. Similarly, imiquimod-induced neurodegeneration was not affected by let-7 FI. Results are presented as 9

10 mean ± SD. p= over all groups (Kruskal-Wallis test). p-values for relevant groups as determined using the Mann-Whitney U test are shown. n.s., not significant. One representative experiment of 3 independent experiments is shown. 10

11 Supplementary Figure 10. Intrathecal administration of let-7b causes neuronal cell death that involves activation of IRAK µg let-7b, 10 µg mutant oligoribonucleotide, or water (control) were injected intrathecally into C57Bl/6J (WT, let-7b n=10; mut. oligo n=8; water n=4) or TLR7-deficient (TLR7 /, let-7b n=10; mut. oligo n=9; water n=4) mice. After 3 d, brain sections were immunostained with NeuN antibody (a), stained with TUNEL and DAPI (b), or stained with NeuN antibody and an antibody against phosphorylated IRAK-4 (e). 10 µg let-7b or 10 µg mutant oligoribonucleotide were injected intrathecally into C57Bl/6J (WT, let-7b n=4; mut. oligo n=4) or TLR7-deficient (TLR7 /, let-7b n=3; mut. oligo n=4) mice. After 2 weeks, brain sections were immunostained with NeuN antibody, and NeuN-positive cortical cells were quantified (c). (d) Quantification of striatal neurons (NeuN+) from animals analyzed in (a). Results are presented as mean ± SD. p= (c) and p= (d) over all groups (KruskalWallis test). p-values for relevant groups as determined using the Mann-Whitney U test are shown. Scale bar, 50 µm. 11

12 Supplementary Figure 11. Brain sections of mice intrathecally injected with let-7b do not display signs of glial activation. 10 µg let-7b, 10 µg mutant oligoribonucleotide, or water (control) were injected intrathecally into C57Bl/6J mice (WT, let-7b n=10; mut. oligo n=8; water n=4). After 3 d, brain sections were immunostained for (a) Iba1, CD11b, or MHCII and DAPI. (b) Sections of mice intrathecally injected with let-7b or mutant oligoribonucleotide were immunostained with GFAP to mark astrocytes and with DAPI. As a positive staining image of strongly activated microglia and astrocytes, brain sections of stroked mice (focal cerebral ischemia, FCI for 72 h) were immunostained with the respective markers in parallel. Scale bar 50 µm for all images except cut-outs in (a) with scale bar 10 µm. 12

13 Supplementary Figure 12. Glial cells in TLR7 / mice electroporated in utero with TLR7/GFP do not express TLR7. TLR7 / embryos were electroporated in utero with an enhanced green-fluorescent protein (GFP) plasmid containing tlr7. At day 19 after birth, the cerebral cortex was analyzed by immunostaining with GFAP and Iba1 antibodies as well as by DAPI staining. One representative image of the immunostainings is shown. Scale bar, 50 µm. 13

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