Figures and data
Injury induces ROS and cell stress signaling reflected by changes in the transcriptome and chromatin landscape of gliogenic-NEPs.
(A) Schematic summarizing the experimental plan.
(B-C) UMAP projections of 11,878 cells (6,978 nonIR and 4,900 IR) showing cluster annotations (B), treatment (black: nonIR, red: IR) and the age of the samples (red: P1, green: P2, blue: P3, purple: P5) (C).
(D) Dot plot showing the expression levels of key marker genes used for cluster annotation (gliogenic-NEPs: Hopx, Gdf10, Slc1a3, neurogenic-NEPs: Ascl1, Ptf1a, immature neurons: Pax2, GCPs: Atoh1, postmitotic neurons: Tubb3, microglia: Cx3cr1, oligodendrocyte progenitors: Olig2, oligodendrocytes: Gpr17, Ependymal cells: Foxj1).
(E) Feature plots showing Hopx (gliogenic-NEPs), Ascl1 (neurogenic-NEPs) and Atoh1 (GCPs) expression highlighting the three main populations of interest. Clusters containing Hopx-NEPs (clusters 2, 3, 6, 10), Ascl1-NEPs (clusters 5, 8, 11), or GCPs (clusters 1, 4, 7, 12, 14) were subsetted from the original data set and were divided according to age (P2 or P3+P5) for the downstream differential expression analyses.
(F) Volcano plot showing differentially expressed genes in the P2 gliogenic-NEPs (red: adjusted p-value≤0.05, log2fold-change>|1|).
(G) Violin plots showing some of the top differentially expressed genes in P2 gliogenic-NEPs and how their expression changes over time with respect to their expression in control cells.
(H) Top GO terms associated with differentially expressed genes in P2 gliogenic-NEPs that were either upregulated in nonIR (top panel) or IR (bottom panel) cells (adjusted p-value≤0.05, Table S3).
(I) Heatmap showing differentially open chromatin regions in P2 nonIR and IR NEPs, identified by bulk ATAC-seq (1168 differentially open regions, adjusted p-value<0.05, Table S4).
(J) Tracks highlighting the injury-induced open chromatin regions around Cdkn1a and Phlda3, the top differentially expressed genes identified in (F).
(K) Gene network analysis of ATAC-seq data revealed an active transcriptional network involved in regulating cell death and apoptosis. Genes colored in red (Ppara, Egln3, Foxo3, Jun and Nos1ap) have been implicated as upregulated with increased ROS levels or involved in ROS signaling.
Cerebellar injury at P1 results in increased superoxide production, a reduction in mitochondria and increased cell death in the EGL that peaks 24h after injury.
(A) Examples of flow cytometry analysis of mitochondrial ROS at P2 from nonIR and IR cerebella using MitoSOX dye. Gating determined the top 90% MitoSOX signal (MitoSOX high cells).
(B, C) Quantification of MitoSOX high (B) and MitoTtracker high (C) expression in nonIR and IR cerebella at P2.
(D, E) Quantification of TUNEL+ cell density in the EGL at P2 (D) and P3 (E) in lobules 3-5 of nonIR and IR mice.
(F, G) Quantification of TUNEL+ cell density outside the EGL at P2 (F) and P3 (G) in lobules 3-5 of nonIR and IR mice.
EGL, External granular layer; SSC, side scatter; P, postnatal day; nonIR, non-irradiated; IR, irradiated. All statistical significance was determined using an unpaired t-test and data are represented as mean ± SEM.
Cerebellar injury at P1 induces transient microglial recruitment to the EGL and prolonged astroglial microenvironment changes in the cerebellum.
(A-H) Immunohistochemical (IHC) staining of medial sagittal cerebellar sections for GFAP (green) in lobules 4/5 of nonIR and IR cerebellum at the stages indicated. Nuclei were counterstained with Hoechst. (B’) and (F’) show high-power images of white dashed line boxes in (B) and (F), respectively.
(I, J) Quantification of IBA1+ cell density in the WM at P2 (I) and P3 (J) in lobules 3-5 of nonIR and IR mice. (K, L) IHC staining of medial sagittal cerebellar sections for IBA1 and TUNEL in lobule 3 of nonIR and IR cerebellum at P2. Nuclei were counterstained with Hoechst. White matter (WM) and external granular layer (EGL) are delineated by white dotted lines and dashed lines, respectively. High-power image in (L) of the area indicated by the white dashed line represents an IBA1+ cell present in the EGL. White arrowheads indicate additional IBA1+ cells in the EGL.
(M, N) Quantification of IBA1+ cell density in the EGL at P2 (M) and P3 (N) in lobules 3-5 of nonIR and IR mice. EGL, External granular layer; WM, White matter; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Scale bar: A and E 100µm, B, C, D, E, F, G and H: 250µm, B’ and F’: 50µm, I and J: 50µm. All statistical significance was determined using an unpaired t-test and data are represented as mean ± SEM.
Reduction of ROS impairs adaptive reprogramming and cerebellar repair.
(A, B) IHC staining of medial sagittal cerebellar sections for human catalase in control (A) AND mCAT/+ mice (B) at P7. Nuclei were counterstained with Hoechst (blue). Similar staining was seen in four mCAT/+ mice.
(C) Quantification of MitoSOX high expression at P2 in control and mCAT/+ cerebella, with and without irradiation at P1 (Two-way ANOVA, F(1,34)=6.768, p=0.0136).
(D) Quantification of MitoTracker high expression at P2 in control and mCAT/+ cerebella, with and without irradiation at P1 (Two-way ANOVA, F(1,31)=25.06, p<0.0001).
(E) Quantification of TUNEL+ cell density in the EGL at P2 in control and mCAT/+ cerebella, with and without irradiation at P1 (Two-way ANOVA, F(1,14)=87.56, p<0.0001).
(F) Quantification of IBA1+ cell density in the EGL at P2 in control and mCAT/+ cerebella, with and without irradiation at P1 (Two-way ANOVA, F(1,14)=15.58, p=0.0015).
(G-J) Hematoxylin and eosin staining on mid-sagittal sections of P30 control and mCAT/+ cerebellum with or without irradiation.
(K) Quantification of P30 cerebellar mid-sagittal section area in controls and mCAT/+ nonIR and IR mice (Two-way ANOVA, F(1,20)=11.82, p=0.0026).
(L) Graph showing the average area of mid-sagittal cerebellar sections at P3, P5, P8, P12 and P30 in control and mCAT/+ non-irradiated and irradiated mice. Detailed statistics are shown in Supplementary Figure 4.
EGL, External granular layer; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Scale bar: A and B: 100µm, F-I: 1mm. Significant Tukey’s post hoc multiple comparison tests are shown in the figures and data are represented as mean ± SEM.
Reduced ROS impairs expansion of BgL-NEPs and their recruitment to the EGL after injury.
(A, B) Quantification of EGL thickness at P5 (Two-way ANOVA, F(1,21)=36.64, p<0.0001) (A) and P8 (Two-way ANOVA, F(1,12)=11.34, p=0.0056)(B) in lobules 3-5 of Nes-Cfp/+ control and Nes-Cfp/+; mCAT/+ mutant mice with and without irradiation at P1
(C-F) IHC staining of medial sagittal cerebellar sections showing expression of CFP (green) in the lobules 4/5 of Nes-Cfp/+ control and Nes-Cfp/+; mCAT/+ mutant mice at P5. Nuclei were counterstained with Hoechst (blue). (D’) and (F’) show a high-power images of the yellow boxed area in the single channel CFP. EGL is delineated by the dashed white lines. (G, H) Quantification of CFP+ cell density in the EGL (Two-way ANOVA, F(1,19)=5.192, p=0.0359) (G) and BgL (Two-way ANOVA, F(1,17)=6.191, p=0.0223) (H) at P5 in Nes-Cfp/+ control or Nes-Cfp/+; mCAT/+ mutant non-irradiated and irradiated mice.
EGL, External granular layer; BgL, Bergmann glia Layer; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Scale bar: D-F: 100µm. Significant Tukey’s post hoc multiple comparison tests are shown in the figures and data are represented as mean ± SEM.
Microglia promote recruitment of NEPs to the EGL during cerebellar adaptive reprogramming after injury.
(A-D) IHC staining of medial sagittal cerebellar sections for IBA1 (red) in control and mCAT/+ mice at P5. Nuclei were counterstained with Hoechst (blue).
(E, F) Quantification of IBA1+ cell density in the external granular layer (E) and white matter (Two-way ANOVA, F(1,13)=24.74, p=0.0003) (F) at P5 on midsagittal sections of lobules 3-5 in the cerebellum of control and mCAT/+ animals, with or without irradiation.
(G-J) IHC staining of medial sagittal cerebellar sections at P5 for CFP (green) in lobules 4/5 of Nes-Cfp/+ mice treated with PLX5622 or control DMSO with or without irradiation. Nuclei were counterstained with Hoechst (blue). (H’) and (J’) show a high-power image of area indicated by yellow boxes. EGL is delineated by the white dashed lines.
(K) Quantification of IBA1+ cell density in the white matter at P5 on mid-sagittal sections in lobules 3-5 of Nes-Cfp/+ mice treated with PLX5622 or control DMSO, with or without irradiation (Two-way ANOVA, F(1,12)=42.40, p<0.001).
(L) Quantification of EGL thickness at P5 in lobules 3-5 of Nes-Cfp/+ mice treated with PLX5622 or control DMSO with or without irradiation (Two-way ANOVA, F(1,12)=109.5, p<0.001).
(M) Quantification of CFP+ cells density in the EGL at P5 on mid-sagittal sections in lobules 3-5 of Nes-Cfp/+ mice treated with PLX5622 or control DMSO with or without irradiation (Two-way ANOVA, F(1,12)=10.62, p=0.0068).
(N) Measurement of cerebellar mid-sagittal section area at P30 in controls or mice treated with PLX, with or without irradiation at P1 (Two-way ANOVA, F(1,12)=13.29, p=0.0034).
EGL, External granular layer; WM, White matter; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Scale bar: A-D and G-J: 250 µm. Significant Tukey’s post hoc multiple comparison tests are shown in the figures and data are represented as mean ± SEM.
scRNA-seq quality metrics and number of cells sequenced in each condition and biological replicate.
(A) Violin plots showing the number of features, RNA and percent mitochondrial RNA count across the biological replicates of the scRNA-seq data set after filtering the bad quality cells (cells were filtered out where number of detected genes was ≤ 1500, the number of detected transcripts was ≥ 40,000 and mitochondrial gene percentage ≥ 5%).
(B) Number of cells from each replicate and condition used for downstream analyses after filtering.
(C) UMAPs showing the distribution of cells across different clusters based on the samples.
Injury induces distinct transcriptional changes in NEP subtypes and GCPs during adaptive reprograming.
(A-E) Volcano plot showing differentially expressed genes in the P3+5 gliogenic-NEPs (A), P2 and P3+5 neurogenic NEPs (B, C) and P2 or P3+5 GCPs (D, E) (red: adjusted p-value≤0.05, log2fold-change=1, Table S2).
(F-J) Top GO terms associated with differentially expressed genes in the P3+5 gliogenic-NEPs (F), P2 or P3+5 neurogenic NEPs (G, H) and P2 or P3+5 GCPs (I, J) that were either upregulated in nonIR (top panel) or IR (bottom panel) (p-value≤0.05).
Irradiation of cerebella at P1 results in increased superoxide production and cell death and recruitment of microglia to the EGL that peaks at 24h.
(A, B) Quantification of high MitoSOX expression in nonIR and IR cerebella at P3 (A) and P5 (B).
(C) Examples of flow cytometry analysis of mitochondria at P2 from nonIR and IR cerebella using MitoTracker dye. Gating determined the top 90% MitoTracker signal (MitoTracker high cells).
(D, E) Quantification of MitoTracker high expression in nonIR and IR cerebella at P3 (D) and P5 (E).
(F) Quantification of IBA1+ cell density in the WM at P5 in lobules 3-5 of nonIR and IR mice.
(G) Quantification of IBA1+ cell density in the EGL at P5 in lobules 3-5 of nonIR and IR mice.
EGL, External granular layer; WM, White matter; P, postnatal day; nonIR, non-irradiated; IR, irradiated. All statistical significance was determined using an unpaired t-test and data are represented as mean ± SEM.
Reduction of ROS impairs adaptive reprogramming and cerebellar repair.
(A, B) Quantification of high MitoSOX expression at P3 (A) and P5 (B) in control and mCAT/+ cerebella, with and without irradiation at P1.
(C, D) Quantification of MitoTracker high expression at P3 (C) and P5 (D) in control and mCAT/+ cerebella, with and without irradiation at P1.
(E-G) Quantification of TUNEL+ cell density outside EGL at P2 (Two-way ANOVA, F(1,10)=14.20, p=0.0037) (E), at P3 (G), and in the EGL at P3 (F) in lobules 3-5 of nonIR and IR mice.
(H-J) Quantification of IBA1+ cell density in WM at P2 (H), at P3 (J), and in the EGL at P3 (I) in lobules 3-5 of nonIR and IR mice.
(K-N) Quantification of cerebellar midsagittal section area in controls and mCAT/+ nonIR and IR mice at P3 (K), P5 (Two-way ANOVA, F(1,15)=28.52, p<0.001) (L), P8 (Two-way ANOVA, F(1,12)=21.21, p=0.0006) (M) and P12 (Two-way ANOVA, F(1,14)=9.682, p=0.0077) (N).
EGL, External granular layer; WM, White matter; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Significant Tukey’s post hoc multiple comparison tests are shown in the figures and data are represented as mean ± SEM.
Reduced ROS impairs expansion of BgL-NEPs and their migration to the EGL after injury.
(A) Quantification of CFP+ cell density in the EGL at P3 in Nes-Cfp/+ control or Nes-Cfp/+ mCAT mutant nonIR and IR mice. (Two-way ANOVA, F(1,14)=33.77, p<0.0001).
(B) Quantification of CFP+ cell normalized on BgL length at P5 in Nes-Cfp/+ control or Nes-Cfp/+ mCAT mutant nonIR and IR mice.
(C, D) Quantification of SOX2+ NEP cell density on BgL length at P8 (C) and P12 (Two-way ANOVA, F(1,12)=12.50, p=0.0033) (D) in control or mCAT mutant nonIR and IR mice.
EGL, External granular layer; BgL, Bergmann glia Layer; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Significant Tukey’s post hoc multiple comparison tests are shown in the figures and data are represented as mean ± SEM.
Microglia promote recruitment of NEPs to the EGL during cerebellar adaptive reprogramming after injury.
(A) Quantification of IBA1+ cell density in the WM at P8 in control or mCAT mutant nonIR and IR mice.
(B-E) Immunostaining of medial sagittal cerebellar sections at P5 showing expression of IBA1 (red) in mice treated with PLX5622 or control DMSO, with or without irradiation.
(F) Quantification of SOX2+ NEP cell density in the BgL at P5 in control or PLX treated nonIR and IR mice. BgL, Bergmann glia Layer; WM, White Matter; P, postnatal day; nonIR, non-irradiated; IR, irradiated. Scale bar: 500 µm. Data are represented as mean ± SEM.
