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Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination

Abstract

Enhancing repair of myelin is an important but still elusive therapeutic goal in many neurological disorders1. In multiple sclerosis, an inflammatory demyelinating disease, endogenous remyelination does occur but is frequently insufficient to restore function. Both parenchymal oligodendrocyte progenitor cells and endogenous adult neural stem cells resident within the subventricular zone are known sources of remyelinating cells2. Here we characterize the contribution to remyelination of a subset of adult neural stem cells, identified by their expression of Gli1, a transcriptional effector of the sonic hedgehog pathway. We show that these cells are recruited from the subventricular zone to populate demyelinated lesions in the forebrain but never enter healthy, white matter tracts. Unexpectedly, recruitment of this pool of neural stem cells, and their differentiation into oligodendrocytes, is significantly enhanced by genetic or pharmacological inhibition of Gli1. Importantly, complete inhibition of canonical hedgehog signalling was ineffective, indicating that the role of Gli1 both in augmenting hedgehog signalling and in retarding myelination is specialized. Indeed, inhibition of Gli1 improves the functional outcome in a relapsing/remitting model of experimental autoimmune encephalomyelitis and is neuroprotective. Thus, endogenous neural stem cells can be mobilized for the repair of demyelinated lesions by inhibiting Gli1, identifying a new therapeutic avenue for the treatment of demyelinating disorders.

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Figure 1: Gli1-expressing cells are recruited to, and generate myelinating oligodendrocytes at sites of demyelination.
Figure 2: Loss of Gli1 enhances oligodendrogliogenesis during remyelination.
Figure 3: Pharmacological inhibition of Gli1 promotes NSC recruitment and differentiation during remyelination.
Figure 4: GANT61 improves functional outcomes and is neuroprotective in a RR-EAE model.

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Acknowledgements

We thank A. Joyner for providing mouse lines and for advice during the course of this project, M. Bhat, M. Wegner, and M. Rasband for providing antibodies, G. Zanazzi for providing human brain tissue, A. Liang for assistance with immunoelectron microscopy, and G. Multani for technical assistance during initial studies. This research was supported by grants to J.L.S. from the New York State Department of Health Stem Cell Board and the National Multiple Sclerosis Society. J.S. was a recipient of a postdoctoral fellowship from the National Multiple Sclerosis Society.

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Contributions

J.S. performed the experiments, analysed the data and co-wrote the paper with J.L.S. All authors contributed to the design of individual experiments, reviewed individual results and assisted with portions of manuscript preparation.

Corresponding authors

Correspondence to Jayshree Samanta or James L. Salzer.

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Competing interests

A patent on the method of targeting Gli1 as a strategy to promote remyelination has been awarded, with J. L. Salzer, J. Samanta and G. Fishell listed as co-inventors.

Extended data figures and tables

Extended Data Figure 1 Gli1-expressing cells generate oligodendrocytes after demyelination.

Additional markers used to analyse fate-mapped Shh-responsive NSCs are shown. a, Two weeks after removal from cuprizone diet, GFP-labelled cells in the CC of Gli1CE/+ mice co-expressed the oligodendrocyte progenitor markers PDGFR-α, NG2 and Sox10, the mature oligodendrocyte marker CC1, and the astrocytic markers GFAP and S100β. Scale bar, 10 µm. b, Four weeks after removal from cuprizone diet, GFP-labelled processes co-localized with myelin proteins MBP and MOG but not with peripheral myelin protein P0. GFP-labelled processes also overlaid the axonal, paranodal marker Caspr. N = 5 mice per group. Scale bar, 10 µm.

Extended Data Figure 2 Gli1-expressing neural stem cells in the SVZ egress and generate labelled cells in the CC.

a, Expression of Gli1 in the forebrain was confirmed in Gli1nLacZ/+ mice by immunofluorescence for LacZ. Labelled cells were observed in the SVZ, cortex and basal forebrain but not in the CC of mice. The right panel shows the magnified images for the corresponding boxes in the left panel. N = 5 mice. Scale bar, 50 µm. b, Double staining for PDGFR-α and LacZ in the Gli1nLacZ/+ forebrain does not show any co-labelled cells, indicating that Gli1 is not expressed by OPCs. N = 5 mice. Scale bar, 50 µm. c, The ventral SVZ lining the body of the lateral ventricle from a human brain specimen shows co-localization of Gli1 with GFAP+ cells (yellow arrow) as well as a Gli1+ cell not expressing GFAP (arrowhead). N = 1 brain. Scale bar, 50 µm. df, Time-course analysis of the SVZ and CC of Gli1CE/+ mice stereotactically injected with saline (control, left panels) or LPC (right panels) to induce demyelination. N = 3 mice per group. No labelled cells were seen within the CC after saline injections; areas of ingress into the LPC-injected CC are boxed. At 1 day post-lesion (d.p.l.) (d), GFP-labelled cells diverted towards the CC; at 2 d.p.l. (e), a few labelled cells were seen within the CC; at 6 d.p.l. (f), many GFP+ cells had accumulated at the site of LPC injection (arrowhead). Scale bar, 50 µm.

Extended Data Figure 3 Neural stem cells generate oligodendrocytes in various white matter tracts in Gli1-null brains upon demyelination.

a, b, Fate-mapped Gli1-null cells migrated into the anterior commissure (AC) (a) and striatum (b) after cuprizone-mediated demyelination (inset shows the area of the forebrain) in Gli1Null (Gli1CE/nLacZ) mice. Scale bar, 50 µm. c, Two weeks after removal from cuprizone diet, GFP-labelled cells are present throughout the CC of Gli1Null (Gli1CE/nLacZ) mice. N = 5 mice per group. Scale bar, 100 µm.

Extended Data Figure 4 Myelination starts earlier in developing Gli1 null mice.

a, Gli1nLacZ/nLacZ (Gli1Null) mice (right panel) show increased MBP levels in the forebrain at postnatal day 9 (P9) compared with Gli1nLacZ/+ (Gli1Het) mice. b, Quantification of the extent of MBP expression at P9 in the CC of Gli1 nulls (47.12 ± 10.44%) versus heterozygotes (29.71 ± 1.77%) corroborates that myelination is accelerated. N = 5 mice per genotype. c, Analysis of healthy adult forebrain shows the intensity of Black-Gold myelin stain in Gli1 heterozygotes and nulls was comparable. d, Quantification of the sizes of the CC shows the CC in Gli1Null (Gli1nLacZ/nLacZ) was slightly larger on average than that in Gli1Het (Gli1nLacZ/+) mice, although the difference was not statistically significant. N = 5 mice per genotype. e, Quantification of G ratios from electron micrographs of healthy Gli1Het (Gli1nLacZ/+) and Gli1Null (Gli1nLacZ/nLacZ) mice revealed no difference in the thickness of myelin sheaths in the CC. N = 3 mice per genotype. Scale bar, 50 µm. Data are mean ± s.e.m., Student’s t-test.

Extended Data Figure 5 Effects of gain of smoothened function in Gli1-het versus Gli1-null cells during remyelination.

Gli1Het (Gli1CE/+), Gli1Het;SmoM2 (Gli1CE/+;SmoM2) and Gli1Null;SmoM2 (Gli1CE/nLacZ;SmoM2) mice were injected with tamoxifen, fed either a regular or a cuprizone-supplemented diet for 6 weeks and analysed by immunofluorescence 2 weeks after removal of cuprizone. a, GFP+ cells are only seen in the CC of mice on cuprizone diet (right) and not in the control mice (left). b, Quantification of the GFP+ cells in the CC shows significantly higher numbers of cells in Gli1Null;SmoM2 mice compared with Gli1Het and Gli1Het;SmoM2 mice. c, Quantification of the proportion of GFP-labelled co-expressing glial markers in the CC of cuprizone-treated Gli1Het, Gli1Het;SmoM2 and Gli1Null;SmoM2 mice shows an increase in percentage of GFP-labelled OPCs (PDGFR-α+) in Gli1Het;SmoM2 mice and mature oligodendrocytes (CC1+) in Gli1Null;SmoM2 mice. N = 3 mice per group for each genotype. Data are mean ± s.e.m., Student’s t-test.

Extended Data Figure 6 Proliferation of NSCs and expression of Shh in Gli1-null mice.

a, b, At the start of demyelination (3 weeks of cuprizone diet), Gli1Null (Gli1nLacZ/nLacZ) brains have a higher proportion of proliferating nLacZ+ neural stem cells indicated by the percentage of EdU-incorporating cells co-expressing nLacZ in the SVZ compared with Gli1Het (Gli1nLacZ/+) brains, namely 31 ± 8.9% versus 8.22 ± 5.33%, respectively. N = 3 mice per group for each genotype. Scale bar, 50 µm. c, The numbers of fate-mapped Gli1+ neural stem cells in the SVZ were quantified as the proportion of GFP+ cells co-expressing GFAP in Gli1Het (Gli1CE/+) and Gli1Null (Gli1CE/nLacZ) mice at 2 weeks of recovery from cuprizone diet. The percentage of GFAP+GFP+ cells in the SVZ of mice receiving cuprizone diet was comparable to those on a control diet, suggesting that the stem cell pool is not depleted during remyelination. N = 3 mice per group for each genotype. Data are mean ± s.e.m., Student’s t-test. d, Fate-mapping of Shh expressing cells using an mGFP reporter labels neurons in the basal forebrain (left) with their neurites reaching the ventral SVZ (right) in Gli1Het and Gli1Null brains. e, Immunostaining of Gli1Null mice shows Shh in the SVZ and CC after demyelination is mostly co-localized to GFAP-expressing cells. Thus, Shh is produced by neurons of the basal forebrain and binds to a responsive set of astrocytes and neural stem cells. f, Quantification of the proportional area of the CC expressing Shh does not show any significant difference between Gli1Het and Gli1Null mice either on control or cuprizone diet. N = 3 mice per group for each genotype. Data are mean ± s.e.m., Student’s t-test. Scale bar, 50 µm.

Extended Data Figure 7 GANT61 reduces Gli1 levels but does not deplete neural stem cells in the SVZ.

a, Relative expression of Gli1 and Gli2 mRNA in the forebrain of Gli1CE/+ mice was examined by qPCR after administration of GANT61 (50 mg/kg/day) for 4 weeks. GANT61 decreases the mRNA levels of Gli1 significantly without changing Gli2 levels. N = 3 mice per group. Data are mean ± s.e.m., Student’s t-test. b, c, The numbers of fate-mapped Gli1+ neural stem cells in the SVZ were analysed by immunofluorescence as the proportion of GFP+ cells co-expressing GFAP in Gli1CE/+ mice treated with vehicle or GANT61 at 2 weeks of recovery from cuprizone diet (b). The percentage of GFAP+GFP+ cells in the SVZ of mice treated with GANT61 was comparable to those treated with vehicle, suggesting that the stem cell pool is not depleted by GANT61 (c). N = 3 mice per group. Data are mean ± s.e.m., Student’s t-test.

Extended Data Figure 8 Pharmacological inhibition of Gli1 does not affect OPC recruitment or differentiation during remyelination.

a, NG2CE/+ mice were treated with two doses of intraperitoneal tamoxifen to sparsely label OPCs and analysed at 2 weeks of recovery from cuprizone. Scale bar, 50 µm. b, Numbers ( 40 GFP+ cells per field) and c, proportions of GFP-labelled OPCs (PDGFR-α) and mature oligodendrocytes (CC1) were similar in the GANT61- versus the vehicle-treated mice in the CC, indicating GANT61 does not alter OPC remyelination. Scale bar, 50 µm. N = 3 mice per group. Data are mean ± s.e.m., Student’s t-test.

Extended Data Figure 9 Gli1 is not expressed by immune cells of spleen, thymus and liver of healthy mice.

a–d, Cells from the spleen, liver and thymus of tamoxifen-treated Gli1Het (Gli1CE/+) and Gli1Null (Gli1CE/nLacZ) mice were analysed by flow cytometry for GFP expression. Wild-type mice did not express GFP and were used as controls. Representative flow cytometry scatter plots showing absence of GFP expression in CD45+/CD3+/CD4+/CD19+ T and B cells (a), CD45+/CD3/CD19/B220+/MPDCA+ plasmacytoid dendritic cells (b), CD45+/CD3/CD19/B220/CD11b+ macrophages, monocytes, dendritic and natural killer (NK) cells (c) and CD45+/CD3/CD19/B220/CD11b cells (d) in Gli1CE/+ and Gli1CE/nLacZ mice. N = 3 mice per genotype.

Extended Data Figure 10 Effects of GANT61 on spinal cord axons in the PLP-induced EAE model.

a, Scatter plot of G ratios with respect to axonal diameters (n = 500 axons in three mice per group, exponential trend line). b, Analysis of electron microscopy images showing the relative proportion of axons binned by their diameters in the four groups. c, Analysis of electron microscopy images, indicating the G ratios of axons relative to their diameters in the four groups (N = 500 axons in three mice per group; data are mean ± s.e.m., Student’s t-test). d, Immunofluorescence image of a spinal cord section from Gli1nLacZ/+ mice shows that LacZ is not expressed by NG2+ OPCs. The inset shows expression of LacZ in the germinal zone around the central canal. N = 3 mice. Scale bar, 50 µm.

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Samanta, J., Grund, E., Silva, H. et al. Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination. Nature 526, 448–452 (2015). https://doi.org/10.1038/nature14957

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