GDF10 is a signal for axonal sprouting and functional recovery after stroke

Journal name:
Nature Neuroscience
Volume:
18,
Pages:
1737–1745
Year published:
DOI:
doi:10.1038/nn.4146
Received
Accepted
Published online

Abstract

Stroke produces a limited process of neural repair. Axonal sprouting in cortex adjacent to the infarct is part of this recovery process, but the signal that initiates axonal sprouting is not known. Growth and differentiation factor 10 (GDF10) is induced in peri-infarct neurons in mice, non-human primates and humans. GDF10 promotes axonal outgrowth in vitro in mouse, rat and human neurons through TGFβRI and TGFβRII signaling. Using pharmacogenetic gain- and loss-of-function studies, we found that GDF10 produced axonal sprouting and enhanced functional recovery after stroke; knocking down GDF10 blocked axonal sprouting and reduced recovery. RNA sequencing from peri-infarct cortical neurons revealed that GDF10 downregulated PTEN, upregulated PI3 kinase signaling and induced specific axonal guidance molecules. Using unsupervised genome-wide association analysis of the GDF10 transcriptome, we found that it was not related to neurodevelopment, but may partially overlap with other CNS injury patterns. Thus, GDF10 is a stroke-induced signal for axonal sprouting and functional recovery.

At a glance

Figures

  1. GDF10 expression in peri-infarct cortex after stroke in mice, macaques and humans.
    Figure 1: GDF10 expression in peri-infarct cortex after stroke in mice, macaques and humans.

    Top two rows, immunohistochemical staining in peri-infarct cortex in mice 7 d after stroke (n = 5). GDF10 staining (red) was apparent in peri-infarct tissue, overlapping with NeuN staining (green). Arrows in bottom right panel indicate representative NeuN+, GDF10+ cells. Schematic at right shows location of stroke as red shaded area; box is the position of photomicrographs. The panels below the mouse schematic are higher magnification of GDF10 (red) colocalizing to neurons whose dendrites are MAP2+ (white), denoted by arrows. Middle two rows, immunohistochemical staining in peri-infarct cortex in non-human primate (n = 2 stroke, n = 3 control) 2 d after stroke. Data are presented as above. Arrows in bottom right indicate double-labeled NeuN+, GDF10+ neurons after stroke. Bottom two rows, immunohistochemical staining in human control (n = 4) and stroke (n = 7). Arrows indicate neurons labeled with both GDF10 and either NeuN or TuJ1 after stroke. In this human case, the stroke is chronic, or greater than 3 months after the event. Scale bar in the bottom right panel represents 50 μm and applies to all photomicrographs. Scale bar in images beneath mouse schematics represents 20 μm.

  2. GDF10 enhances axonal outgrowth in primary neurons in vitro.
    Figure 2: GDF10 enhances axonal outgrowth in primary neurons in vitro.

    (ac) Axonal outgrowth in P4 mouse primary cortical neurons. Axon length was measured after 3 d in culture. Cyto C = cytochrome C, a protein control for the addition of growth factor, used in the in vivo studies (Fig. 4a). In c, wells were plated with CSPG before cell growth. In all graphs, boxes extend from the 25th–75th percentiles and the lines in the boxes are the mean. The whiskers show the minimum and maximum values. (d) P4 cortical neurons stained with SMI-312 after 2 additional days culture in medium alone or medium + GDF10 (500 ng ml−1). Scale bar represents 20 μm. (e) Rat adult RGCs cultured in the presence of GDF10, forskolin or mannitol. n = 7 in culture medium only; n = 8 for the other three groups. Two independent cultures per condition and, in each culture, four wells repeating the condition. *P < 0.05, **P < 0.01 compared with medium only; #P < 0.05, ##P < 0.01 compared with scrambled + GDF10; SSP < 0.05 compared with scrambled siRNA. All conditions were tested in quadruplicate, in two separate experiments. a, F(6, 105) = 7.220; b, F(6, 105) = 8.384; c, F(6, 105) = 22.44; e, medium versus GDF10: t = 2.852, df = 13; fosk/mann versus fosk/mann GDF10: t = 2.371, df = 14. All observations were normalized to the number of NeuN+ cells in each sample (Supplementary Fig. 13). Statistical testing was repeated-measures ANOVA followed by Tukey-Kramer's post hoc test (ac) or one-tail unpaired t test (e).

  3. GDF10 enhances axonal outgrowth in human neurons via TGF[beta] signaling.
    Figure 3: GDF10 enhances axonal outgrowth in human neurons via TGFβ signaling.

    (a,b) P4 mouse cortical neuron culture with TGFβRI and TGFβRII and Smad blockade. SB431542 is a TGFβRI antagonist, added at initial plating. In all graphs, boxes extend from the 25th–75th percentiles and the lines in the boxes are the mean. The whiskers show the minimum and maximum values. (c,d) Human iPS-neurons cultured in the presence of GDF10, SB431542, or TGFβRII, Smad2 and Smad3 siRNA. Each condition consisted of 2–4 observations in 2–3 independent experiments. (e,f) iPS-NPCs in culture with GDF10 for 2 d, stained with SMI-312 for axons. Scale bar represents 20 μm. (g) TGFβ1 and Smad2 enhanced axonal outgrowth of P4 primary cortical neurons. Axon length with treatment of TGFβ1 at ascending concentrations is shown. n = 3 for each experiment. (h) Axonal outgrowth with transfection of Smad2 expression plasmid. Data are presented as in g. *P < 0.05, **P < 0.01, ***P < 0.005 compared with medium only; P < 0.05 compared with medium + GDF10; #P < 0.05 compared with scrambled + GDF10. All conditions were tested in quadruplicate in two separate experiments. a, F(5, 186) = 10.28; b, F(2, 93) = 6.138; c, F(4, 155) = 10.23; d, F(4, 155) = 11.49; g, F(2, 93) = 4.435; h, t test, two-tailed t = 3.073, df = 62. All observations were normalized to the number of NeuN+ cells in each sample (Supplementary Fig. 13). Statistical testing was repeated-measures ANOVA followed by Tukey-Kramer's post hoc test (ad,g) or one-tail unpaired t test (h).

  4. GDF10 promotes axonal connections in peri-infarct cortex after stroke.
    Figure 4: GDF10 promotes axonal connections in peri-infarct cortex after stroke.

    (a) Quantitative cortical mapping of connections in layers II/III of the flattened mouse cortical hemisphere ipsilateral to the forelimb motor cortex in stroke with protein control (Cyto C) (blue, n = 8), GDF10 + stroke (red, n = 8) and areas of dense overlap of these two conditions (dark blue). x and y axes are distances in millimeters from the center of the BDA tracer injection (empty circle). P value is Hotellings T2. (b) Polar plot of connections of forelimb motor cortex projections relative to the tracer injection in forelimb motor cortex as the origin. Filled polygons represent the 70th percentile of the distances of all BDA-labeled connections from the injection site in each segment of the graph. Weighted polar vectors represent the median vector multiplied by the median of the normal distribution of the number of points in a given segment of the graph. P value is Watson's nonparametric two-sample U2 test. Inset shows schematic lateral view of mouse brain. The horizontal line shows the position in which neuronal label was quantified c. (c) Projections from forelimb motor cortex after stroke with GDF10 delivery (red) and protein control (Cyto C) (cyan) taken from counts along the line in (a). *P < 0.05, **P < 0.01. Inset shows schematic mouse brain with the location of the BDA injection (black dot) and the linear quantification construct (line). In c and f, boxes extend from the 25th–75th percentiles and the lines in the boxes are the mean. The whiskers show the minimum and maximum values. (d) Quantitative cortical mapping of GDF10 knockdown in stroke. Data are presented as in a. (e) Polar plots of GDF10 siRNA and scrambled siRNA after stroke. Data are presented as in b. (f) Linear quantification of neuronal connections in treatment groups of GDF10 siRNA+Stroke and scrambled siRNA+Stroke. Data are presented as in c. c, F(1, 10) = 12.03; f, F(1, 10) = 20.24; b, U2 = 647.176, df = 90939, df2 = 180911; e, U2 = 78.616, df = 38554, df2 = 5906. The circle in a and d indicates the center of the stroke site.

  5. Astrocyte, endothelial and inflammatory responses in peri-infarct cortex with GDF10 after stroke.
    Figure 5: Astrocyte, endothelial and inflammatory responses in peri-infarct cortex with GDF10 after stroke.

    All data were obtained 28 d after stroke. (a) GFAP immunoreactivity was increased in all stroke conditions compared with control (*P < 0.05, **P < 0.01 versus control; ^P = 0.05 versus stroke only; #P < 0.05 versus stroke + protein control) and was significantly decreased in GDF10 siRNA + stroke compared with the scrambled siRNA (P < 0.05 versus scrambled siRNA + stroke). In a, c and e, boxes extend from the 25th–75th percentiles and the lines in the boxes are the mean. The whiskers show the minimum and maximum values. (b) Photomicrographs of GFAP immunostaining in stroke and stroke + GDF10. (c) PECAM/CD31 immunoreactivity for endothelial cells in control and gain and loss of function in GDF10 after stroke. Data are presented as in a. GDF10 induced an increase and GDF10 siRNA reduced a decrease in PECAM-immunoreactive vessels in peri-infarct cortex after stroke. (d) PECAM staining in peri-infarct cortex in stroke and stroke + GDF10. (e,f) IBA-1 immunoreactivity for microglia/macrophages in peri-infarct cortex. Stroke increased the microglial staining in peri-infarct cortex. GDF10 knockdown significantly reduced the staining of microglia/macrophages compared with scrambled siRNA+stroke. However, there was a significant difference in IBA-1–immunoreactive signal between groups of GDF10 + stroke and cyto C + stroke. Scale bar represents 50 μm. See Supplementary Table 10 for sample size and ANOVA statistics.

  6. GDF10 improves behavioral recovery after stroke.
    Figure 6: GDF10 improves behavioral recovery after stroke.

    (a) Cylinder test of forelimb symmetry in exploratory rearing (n = 7 all conditions in behavioral testing). The y axis shows bilaterally symmetric rearing as 0.0 and percent of left (unaffected) forelimb rearing as negative values. Left, stroke caused a significant increase in the number of rears with the left forelimb (**P < 0.01). GDF10 treatment produced a significant recovery compared with stroke + vehicle (#P < 0.05) and stroke + cyto C (^P < 0.05). Right, stroke + GDF10 siRNA impaired the normal recovery seen in stroke + vehicle (#P < 0.01) and in stroke + scrambled siRNA (P < 0.05). (b) Gridwalking test of forelimb function in gait. The y axis is the number of footfaults of the forelimb contralateral to the stroke (right forelimb). Left, stroke + GDF10 produced a significant recovery in forelimb function compared with stroke + cyto C (^P < 0.05). Right, stroke + GDF10 siRNA reduced the normal process of motor recovery after stroke (*P < 0.05, **P < 0.01, ***P < 0.005 compared with stroke only) and impaired the forelimb function compared with stroke + scrambled siRNA (P < 0.05). (c) Pasta handling task after stroke. The y axis is the percentage of handling time using right forepaw relative to both paws. Delivery of GDF10 resulted in a significant recovery in forepaw use compared to delivery of protein control cyto C (^P < 0.05). Injection of GDF10 siRNA complex significantly reduced right forepaw function compared with injection of the scrambled siRNA (P < 0.05). *P < 0.05, **P < 0.01 in forepaw use compared with normal control. (d) Timeline of behavioral studies and schematics of GDF10 delivery and behavioral testing procedures. a, F(1.958, 11.75) = 22.07; b, F(1.869, 11.21) = 10.70; c, F(2.101, 12.61) = 9.382. Error bars represent s.e.m. Statistics are multiple comparisons ANOVA followed by Tukey-Kramer's post hoc test.

  7. GDF stroke transcriptome.
    Figure 7: GDF stroke transcriptome.

    Genes differentially regulated at false discovery rate (FDR) < 0.1 analyzed for relationship across stroke conditions, developmental state and molecular pathway. (a) Schematic of experimental approach of neuron isolation and deep sequencing (n = 3 sample for each condition of two pooled brains per sample). (b) Differences in gene expression among conditions. Red represents genes upregulated and green represents genes downregulated greater than 1.2-fold. (c) Heat map and unsupervised clustering of transcriptomes. Red represents genes upregulated and green represents genes downregulated. Differentially expressed genes were identifed using the Bioconductor package EdgeR, which were then considered and ranked based on adjusted P values (FDR) of <0.1. For hierarchical cluster analysis, the distances between clusters were computed using the complete linkage clustering method (R hclust function).

  8. GDF10 canonical signaling pathways and genome-wide associations.
    Figure 8: GDF10 canonical signaling pathways and genome-wide associations.

    (a) Top canonical pathways significantly regulated in stroke + GDF10 versus stroke. The y axis is the inverse log of the P value corrected for multiple comparisons in Benjamini-Hochberg (B-H) test. Significance was set to a B-H P < 0.05 = −log(B-H P) of 1.3. Red represents net upregulation of this gene in this pathway and green represents net downregulation. Gray represents mixed up or downregulation in pathway genes such that there was no net trend. (b) Genome-wide associations of stroke + GDF10 transcriptome to learning and memory, neurodevelopmental and CNS injury transcriptomes. Statistical testing was Fisher's exact P value, Benjamini-Hochberg correction for multiple comparisons (a) and principle component analysis of 180 transcriptomes (Supplementary Fig. 12).

  9. TGF[beta] superfamily
    Supplementary Fig. 1: TGFβ superfamily

    (a) TGFβ superfamily members are classified into TGFβ, Activin/inhibin/Nodal group and BMP groups. In the canonical signaling pathway, shown here, TGFβ family members signal through type I and type II receptors for each member to receptor-associated SMAD proteins (R-SMAD), SMAD2 and SMAD3. Activin/inhibin/Nodal proteins signal through distinct type I and II receptors: ACVR1B or ALK-4 acts as a transducer of Activin/inhibin/Nodal signals. These Activin ligands bind to either ACVR2A or ACVR2B and then bind ACVR1B and then activate (phosphorylate) SMAD2/SMAD3. BMP signals are transduced through their distinct type I and type II receptors for each member to R-SMAD proteins SMAD1, SMAD5 and SMAD8. Phosphorylated R-SMADs associate with SMAD4 and are then translocated to the nucleus to activate transcription of target genes. GDF family members signal through both Activin and BMP pathways, except for GDF10, which signals through TGFβ receptors. This summary is simplified and, for example, does not represent the full complexity of signaling that can come through heterotetrameric binding between TGFβ, Activin/inhibin/Nodal and BMP families. (b) The genetic relationship of GDF/BMP proteins as established by sequence homology. Figures modified from12,14.

  10. GDF10 is upregulated in peri-infarct cortex.
    Supplementary Fig. 2: GDF10 is upregulated in peri-infarct cortex.

    (a) GDF10 protein expression in the ipsilateral hemisphere after stroke. This low magnification photomicrograph of the ipsilesional hemisphere shows axonal loss, as indicated by loss of MAP2, in the stroke core. GDF10 is induced in the cortex bordering the stroke core, as seen by the increased intensity of staining (arrows). The stroke core contains an abundance of microglia and macrophages, labeled by CD11b. (b) GDF10 expression in the peri-infarct cortex. Image is taken from the region of interest indicated by box in panel (a) GDF10 protein expression is upregulated compared to contralateral control in panel 1(c). (b) Higher magnification view of peri-infarct cortex shows that GDF10 is present in MAP2+ neuronal dendrites (arrows) in addition to neuronal cell bodies (Fig. 1) and not present in CD11b+ microglia/macrophages. (c) GDF10 expression in the non-injured cortex from the contralateral hemisphere. GDF10 has a sparse low level of endogenous expression. This indicates that GDF10 induction is specific to cortex immediately adjacent to the site of stroke injury. In the absence of stroke, CD11b is not induced in the control cortex. (d) GDF10 expression in the peri-infarct colocalizes to neurons labeled with MAP2 and excludes microglia and macrophages. GDF10 immunoreactivity is present in MAP2+ neuronal somas and in dendrites (arrows). GDF10 does not colocalize with CD11b+ microglia/ macrophages. (e) Secreted GDF10 is also found in the extracellular space in the peri-infarct cortex near surviving processes (MAP2).

  11. GDF10 protein levels and siRNA knockdown in vivo, P4 neuron outgrowth, and siRNA protein knockdown
    Supplementary Fig. 3: GDF10 protein levels and siRNA knockdown in vivo, P4 neuron outgrowth, and siRNA protein knockdown

    (a) GDF10 siRNA, vehicle or scrambled siRNA was delivered into the stroke cavity and peri-infarct tissue processed for Western blot. X axis shows days after stroke and siRNA delivery. * = p< 0.05, ** = p< 0.01 compared to the scrambled siRNA; # = p<0.05 compared to Stroke only. Lower panel shows representative Western blot images from (a). N = 3 for each experiment. (b) Both myelin and CSPGs inhibit the outgrowth or P4 cortical neurons. *** = p<0.001, multiple comparisons ANOVA, Tukey-Kramer post-hoc. (c) Western blot of GDF10 protein level with two-day treatment of siRNA in P4 neurons. Numbers in the columns indicate the distinct siRNA construct. Lower panel shows a representative blot from each experiment. Each experiment represents 4 samples and 2 technical replicates. (d) (b) Western blot results for knockdown of TGFβRI, II and Smads1, 2, 3, 5. Same conventions as in (a). ** = p<0.01 compared to the scrambled siRNA.

  12. Primary cortical neurons plated in vitro express GDF10
    Supplementary Fig. 4: Primary cortical neurons plated in vitro express GDF10

    (a) Primary cortical neurons stained with GDF10 and MAP2 antibody demonstrates neuronal expression of GDF10 in vitro. This substantiates the in vitro GDF10 knockdown experiments using siRNAs to study axonal outgrowth, without a need for addition of GDF10 to the system to mimic induction. In contrast to the low endogenous expression of GDF10 in uninjured cortical neurons in vivo, GDF10 expression in vitro is likely induced by mechanical stress of dissection and plating of primary neurons. (b) High magnification of neuronal soma expressing GDF10. GDF10 immunoreactivity is also evident along neuronal processes (arrows).

  13. pSmad2/3 quantification in peri-infarct cortex after TGF[beta] antagonism in vivo
    Supplementary Fig. 5: pSmad2/3 quantification in peri-infarct cortex after TGFβ antagonism in vivo

    Stroked animals were treated with two different TGFβ antagonists, SB431542 and Losartan at 10mg/kg and 100mg/kg, respectively, based on published i.p. doses16,17 for 5 days after stroke (n=3 mice per group). pSmad puncta of 0.45μm were quantified at 100x imaging fields in two sections per animal, and mean puncta values were statistically compared. As seen with in vitro axon outgrowth studies in primary cortical neurons from mouse (Fig. 2) and human iPSC-derived neurons (Fig. 3), in vivo administration of TGFβ antagonists, SB431542 (p=0.0056) and Losartan (p=0.0244), each significantly decreases pSmad2/3 signaling within 300µm of the infarct core. *=p<0.05.

  14. Quantitative connectional mapping and axonal connections in peri-infarct cortex
    Supplementary Fig. 6: Quantitative connectional mapping and axonal connections in peri-infarct cortex

    (a) Left panel. Quantitative connectional map of neurons back-labeled from a retrograde tracer injection (cholera toxin b subunit) into cervical level 5 spinal cord (n = 5). This labels all neurons in motor, somatosensory and premotor cortex that send projections to the spinal cord. The location of all neurons is plotted in tangential sections and the x y coordinates are made relative to bregma49. Middle panel. Axonal label plotted from BDA injections into forelimb motor cortex in stroke+GDF10 and Cyto C+stroke (protein control). This is the same experiment as in Fig 4a. Right panel: alignment of corticospinal projections with GDF10/stroke map, to show location of areas of axonal sprouting relative to motor, premotor and somatosensory areas. Note that a substantial region of new connections formed in Stroke+GDF10 is in cortex rostral to the corticospinal populations of neurons. (b) The effect of stroke on motor cortex connections. Left panel: quantitative connectional map of connections from tracer injections into forelimb motor cortex in stroke only (red label) and in control, non-stroke (light blue label) mice. The cortical areas with dense overlap of connections in stroke and control are in dark blue. Note the presence of connections only in the stroke condition in posterior cortical areas that localize to motor cortex and second somatosensory cortex in registering to the corticospinal map and to primary somatosensory cortex by cytochrome oxidase stain (not shown). P value is significant (Hotellings T2 test). Middle panel: Polar plot of connections in stroke (red) and control (blue). Conventions as in Fig. 4b. Right panel: quantification of neuronal connections in linear array through cortical hemisphere.

  15. Infarct volume, BDA volume and injection locations.
    Supplementary Fig. 7: Infarct volume, BDA volume and injection locations.

    (a) Left Y axis: BDA injection volume, right Y axis infarct volume. Columns are for in vivo axonal tracing studies. There are no significant differences among groups. (b) Location of BDA injections for each group relative to the midline of the cortical hemisphere and to the rostral pole of the brain. There are no significant differences among groups. Error bars are SEM.

  16. Quantitative connectional mapping and axonal connections in GDF10 controls compared with normal control.
    Supplementary Fig. 8: Quantitative connectional mapping and axonal connections in GDF10 controls compared with normal control.

    (a-c) Cortical connections of forelimb motor cortex in normal control animals only compared to stroke+protein control, cytochrome C delivery from the infarct core via biopolymer hydrogel. (n = 8 for each group). Figure conventions are as in Supplementary Figure 8 and Figure 4. There is a significant difference in the cortical connectional map (a), polar plots (b) and linear measurement of neuronal connections (c) between normal control forelimb motor cortex and stroke+scrambled siRNA. This indicates that the normal mode axonal sprouting that is seen in stroke compared to control brains is also seen in the control conditions for GDF10 delivery. (d-f) Cortical connections of forelimb motor area in normal control compared to scrambled siRNA delivered into infarct core. siRNA GDF10 knocks down GDF10 protein levels (Supplementary Fig 5a) and blocks post-stroke axonal sprouting (Fig. 4d-f). To verify that the siRNA is not having effects outside of GDF10 knock down, scrambled sequence siRNA was tested. Figure conventions are as in Supplementary Fig. 8 and Fig. 4. There is a significant difference in cortical connection map (a), polar plots (b) or linear measurement of neuronal connections (c) between control and scrambled siRNA (n = 7-8 for each group). * = p<0.05. Note that Cyto C+stroke and scrambled siRNA+stroke produce very similar maps of forelimb motor cortex connections, and are comparable to stroke only (Supplementary Fig. 6b).

  17. Quantitative connectional mapping and axonal connections in GDF10 controls compared with stroke only.
    Supplementary Fig. 9: Quantitative connectional mapping and axonal connections in GDF10 controls compared with stroke only.

    (a-c) Cortical connections of forelimb motor area in stroke only compared to scrambled siRNA delivered into infarct core. siRNA GDF10 knocks down GDF10 protein levels (Supplementary Fig 3a) and blocks post-stroke axonal sprouting (Fig. 4d-f). To verify that the siRNA is not having effects outside of GDF10 knock down, scrambled sequence siRNA was tested. Figure conventions are as in Supplementary Fig. 8 and Fig. 4. There is no significant difference in cortical connection map (a), polar plots (b) or linear measurement of neuronal connections (c) between stroke and scrambled siRNA. (d-f) Cortical connections of forelimb motor cortex in stroke only compared to stroke+protein control, cytochrome C delivery from the infarct core via biopolymer hydrogel (n = 8 each group). Figure conventions are as in Supplementary Fig 8 and Figure 4. There is no significant difference in cortical connection map (a), polar plots (b) or linear measurement of neuronal connections (c) between stroke and Cyto C. Cohorts (n=8) of mice treated with GDF10 (red) or control protein (Cytochrome C) (light blue).

  18. Synaptic protein localization in peri-infarct cortex axons that have undergone axonal sprouting after GDF-10 treatment.
    Supplementary Fig. 10: Synaptic protein localization in peri-infarct cortex axons that have undergone axonal sprouting after GDF-10 treatment.

    (a) Synaptic protein analysis was performed in the same peri-infarct tissues from animals used for BDA axonal sprouting maps in Fig. 4(a) and (b). Presynaptic VGLUT2 and postsynaptic Homer1 antibodies were used for identification of synaptic contacts. Marker colocalization analyses were performed on Imaris Imaging software to uniquely identify the synaptic connections formed by GDF10-induced sprouting cortical neurons after stroke (mapped in Fig. 4). (b) Video through a 10.5μm thick section of peri-infarct cortex taken at 100x. BDA surface is shown in light blue. VGLUT2 presynaptic marker is shown by green spots, and Homer1 postsynaptic marker in red spots.

  19. Comparison of forelimb motor system connections in CytoC + stroke versus GDF10 siRNA + stroke.
    Supplementary Fig. 11: Comparison of forelimb motor system connections in CytoC + stroke versus GDF10 siRNA + stroke.

    Conventions as in Supplementary Figures 10 and 11.

  20. Pipeline for incorporating microarray and RNA-seq data sets for neurodevelopmental and CNS injury experiments.
    Supplementary Fig. 12: Pipeline for incorporating microarray and RNA-seq data sets for neurodevelopmental and CNS injury experiments.

    Raw data from different platforms are processed to have gene symbols which were subsequently used to merge the datasets. Merged datasets were normalized, then batch effect was adjusted. In one case, the datasets have very few common genes on different array platforms and these datasets were combined (unionized) instead of taking intersections.

  21. Neuronal numbers in in vitro axonal outgrowth assays.
    Supplementary Fig. 13: Neuronal numbers in in vitro axonal outgrowth assays.

    The numbers of neurons used for each in vitro experiment to generate the data on axonal outgrowth effect of GDF10 and other experimental manipulations in Figures 2 and 3. There is no significant difference in neuronal sampling number across experiments.

Videos

  1. Synaptic proteins identified in axons that have undergone axonal sprouting in peri-infarct cortex after GDF10 treatment.
    Video 1: Synaptic proteins identified in axons that have undergone axonal sprouting in peri-infarct cortex after GDF10 treatment.
    This movie shows the process of identifying pre- and post-synaptic connections that are present in axons that have undergone axonal sprouting with GDF10 after stroke. The movie is derived from a confocal image stack taken from a BDA-labelled area in premotor cortex that has axons only when axonal sprouting is induced with GDF10 delivery. The BDA-labelled axons are seen at the outset of the movie (white axonal label). This is then filamented (blue label). The tissue has been stained for Homer (post-synaptic, green) and VGlut2 (a subset of excitatory pre-synaptic terminals, red) and filtered so that only those VGlut2 positive pre-synaptic terminals that co-localize with BDA-filled axons are present. Further, the Homer positive post-synaptic boutons are filtered so that only those that co-localize with the VGlut2 that is within BDA-labelled axons are displayed. The movie that zooms and rotates to show the 3D distribution of this network of axonal connections.

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Author information

  1. These authors contributed equally to this work.

    • Songlin Li &
    • Esther H Nie

Affiliations

  1. Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    • Songlin Li,
    • Esther H Nie &
    • S Thomas Carmichael
  2. Neuroscience Interdepartmental Graduate Program, University of California Los Angeles, Los Angeles, California, USA

    • Esther H Nie
  3. Laboratories for Neuroscience Research in Neurosurgery, Children's Hospital, Boston, Massachusetts, USA

    • Yuqin Yin &
    • Larry I Benowitz
  4. Department of Pathology and Laboratory Medicine (Neuropathology), David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    • Spencer Tung &
    • Harry V Vinters
  5. Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, USA

    • F Rena Bahjat &
    • Mary P Stenzel-Poore
  6. Program in Neurogenetics, Department of Neurology and Department of Psychiatry, Semel Institute for Neuroscience and Human Behavior, Los Angeles, California, USA.

    • Riki Kawaguchi &
    • Giovanni Coppola

Contributions

S.T.C., S.L. and E.H.N. conceived the project. S.T.C., S.L. and E.H.N. designed the experiments. S.L. performed most of the experiments. E.H.N. performed immunohistochemical characterization of GDF10 expression, FACS, RNA isolation, synapse analyses and in vivo TGFβ blockade experiments. Y.Y. and L.I.B. performed rat neuronal experiments. S.T. and H.V.V. performed human tissue preparation. F.R.B. and M.P.S.-P. performed primate stroke experiments. R.K. and G.C. performed RNA-seq and bioinformatics experiments. S.T.C., E.H.N. and S.L. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: TGFβ superfamily (114 KB)

    (a) TGFβ superfamily members are classified into TGFβ, Activin/inhibin/Nodal group and BMP groups. In the canonical signaling pathway, shown here, TGFβ family members signal through type I and type II receptors for each member to receptor-associated SMAD proteins (R-SMAD), SMAD2 and SMAD3. Activin/inhibin/Nodal proteins signal through distinct type I and II receptors: ACVR1B or ALK-4 acts as a transducer of Activin/inhibin/Nodal signals. These Activin ligands bind to either ACVR2A or ACVR2B and then bind ACVR1B and then activate (phosphorylate) SMAD2/SMAD3. BMP signals are transduced through their distinct type I and type II receptors for each member to R-SMAD proteins SMAD1, SMAD5 and SMAD8. Phosphorylated R-SMADs associate with SMAD4 and are then translocated to the nucleus to activate transcription of target genes. GDF family members signal through both Activin and BMP pathways, except for GDF10, which signals through TGFβ receptors. This summary is simplified and, for example, does not represent the full complexity of signaling that can come through heterotetrameric binding between TGFβ, Activin/inhibin/Nodal and BMP families. (b) The genetic relationship of GDF/BMP proteins as established by sequence homology. Figures modified from12,14.

  2. Supplementary Figure 2: GDF10 is upregulated in peri-infarct cortex. (842 KB)

    (a) GDF10 protein expression in the ipsilateral hemisphere after stroke. This low magnification photomicrograph of the ipsilesional hemisphere shows axonal loss, as indicated by loss of MAP2, in the stroke core. GDF10 is induced in the cortex bordering the stroke core, as seen by the increased intensity of staining (arrows). The stroke core contains an abundance of microglia and macrophages, labeled by CD11b. (b) GDF10 expression in the peri-infarct cortex. Image is taken from the region of interest indicated by box in panel (a) GDF10 protein expression is upregulated compared to contralateral control in panel 1(c). (b) Higher magnification view of peri-infarct cortex shows that GDF10 is present in MAP2+ neuronal dendrites (arrows) in addition to neuronal cell bodies (Fig. 1) and not present in CD11b+ microglia/macrophages. (c) GDF10 expression in the non-injured cortex from the contralateral hemisphere. GDF10 has a sparse low level of endogenous expression. This indicates that GDF10 induction is specific to cortex immediately adjacent to the site of stroke injury. In the absence of stroke, CD11b is not induced in the control cortex. (d) GDF10 expression in the peri-infarct colocalizes to neurons labeled with MAP2 and excludes microglia and macrophages. GDF10 immunoreactivity is present in MAP2+ neuronal somas and in dendrites (arrows). GDF10 does not colocalize with CD11b+ microglia/ macrophages. (e) Secreted GDF10 is also found in the extracellular space in the peri-infarct cortex near surviving processes (MAP2).

  3. Supplementary Figure 3: GDF10 protein levels and siRNA knockdown in vivo, P4 neuron outgrowth, and siRNA protein knockdown (89 KB)

    (a) GDF10 siRNA, vehicle or scrambled siRNA was delivered into the stroke cavity and peri-infarct tissue processed for Western blot. X axis shows days after stroke and siRNA delivery. * = p< 0.05, ** = p< 0.01 compared to the scrambled siRNA; # = p<0.05 compared to Stroke only. Lower panel shows representative Western blot images from (a). N = 3 for each experiment. (b) Both myelin and CSPGs inhibit the outgrowth or P4 cortical neurons. *** = p<0.001, multiple comparisons ANOVA, Tukey-Kramer post-hoc. (c) Western blot of GDF10 protein level with two-day treatment of siRNA in P4 neurons. Numbers in the columns indicate the distinct siRNA construct. Lower panel shows a representative blot from each experiment. Each experiment represents 4 samples and 2 technical replicates. (d) (b) Western blot results for knockdown of TGFβRI, II and Smads1, 2, 3, 5. Same conventions as in (a). ** = p<0.01 compared to the scrambled siRNA.

  4. Supplementary Figure 4: Primary cortical neurons plated in vitro express GDF10 (225 KB)

    (a) Primary cortical neurons stained with GDF10 and MAP2 antibody demonstrates neuronal expression of GDF10 in vitro. This substantiates the in vitro GDF10 knockdown experiments using siRNAs to study axonal outgrowth, without a need for addition of GDF10 to the system to mimic induction. In contrast to the low endogenous expression of GDF10 in uninjured cortical neurons in vivo, GDF10 expression in vitro is likely induced by mechanical stress of dissection and plating of primary neurons. (b) High magnification of neuronal soma expressing GDF10. GDF10 immunoreactivity is also evident along neuronal processes (arrows).

  5. Supplementary Figure 5: pSmad2/3 quantification in peri-infarct cortex after TGFβ antagonism in vivo (79 KB)

    Stroked animals were treated with two different TGFβ antagonists, SB431542 and Losartan at 10mg/kg and 100mg/kg, respectively, based on published i.p. doses16,17 for 5 days after stroke (n=3 mice per group). pSmad puncta of 0.45μm were quantified at 100x imaging fields in two sections per animal, and mean puncta values were statistically compared. As seen with in vitro axon outgrowth studies in primary cortical neurons from mouse (Fig. 2) and human iPSC-derived neurons (Fig. 3), in vivo administration of TGFβ antagonists, SB431542 (p=0.0056) and Losartan (p=0.0244), each significantly decreases pSmad2/3 signaling within 300µm of the infarct core. *=p<0.05.

  6. Supplementary Figure 6: Quantitative connectional mapping and axonal connections in peri-infarct cortex (81 KB)

    (a) Left panel. Quantitative connectional map of neurons back-labeled from a retrograde tracer injection (cholera toxin b subunit) into cervical level 5 spinal cord (n = 5). This labels all neurons in motor, somatosensory and premotor cortex that send projections to the spinal cord. The location of all neurons is plotted in tangential sections and the x y coordinates are made relative to bregma49. Middle panel. Axonal label plotted from BDA injections into forelimb motor cortex in stroke+GDF10 and Cyto C+stroke (protein control). This is the same experiment as in Fig 4a. Right panel: alignment of corticospinal projections with GDF10/stroke map, to show location of areas of axonal sprouting relative to motor, premotor and somatosensory areas. Note that a substantial region of new connections formed in Stroke+GDF10 is in cortex rostral to the corticospinal populations of neurons. (b) The effect of stroke on motor cortex connections. Left panel: quantitative connectional map of connections from tracer injections into forelimb motor cortex in stroke only (red label) and in control, non-stroke (light blue label) mice. The cortical areas with dense overlap of connections in stroke and control are in dark blue. Note the presence of connections only in the stroke condition in posterior cortical areas that localize to motor cortex and second somatosensory cortex in registering to the corticospinal map and to primary somatosensory cortex by cytochrome oxidase stain (not shown). P value is significant (Hotellings T2 test). Middle panel: Polar plot of connections in stroke (red) and control (blue). Conventions as in Fig. 4b. Right panel: quantification of neuronal connections in linear array through cortical hemisphere.

  7. Supplementary Figure 7: Infarct volume, BDA volume and injection locations. (163 KB)

    (a) Left Y axis: BDA injection volume, right Y axis infarct volume. Columns are for in vivo axonal tracing studies. There are no significant differences among groups. (b) Location of BDA injections for each group relative to the midline of the cortical hemisphere and to the rostral pole of the brain. There are no significant differences among groups. Error bars are SEM.

  8. Supplementary Figure 8: Quantitative connectional mapping and axonal connections in GDF10 controls compared with normal control. (75 KB)

    (a-c) Cortical connections of forelimb motor cortex in normal control animals only compared to stroke+protein control, cytochrome C delivery from the infarct core via biopolymer hydrogel. (n = 8 for each group). Figure conventions are as in Supplementary Figure 8 and Figure 4. There is a significant difference in the cortical connectional map (a), polar plots (b) and linear measurement of neuronal connections (c) between normal control forelimb motor cortex and stroke+scrambled siRNA. This indicates that the normal mode axonal sprouting that is seen in stroke compared to control brains is also seen in the control conditions for GDF10 delivery. (d-f) Cortical connections of forelimb motor area in normal control compared to scrambled siRNA delivered into infarct core. siRNA GDF10 knocks down GDF10 protein levels (Supplementary Fig 5a) and blocks post-stroke axonal sprouting (Fig. 4d-f). To verify that the siRNA is not having effects outside of GDF10 knock down, scrambled sequence siRNA was tested. Figure conventions are as in Supplementary Fig. 8 and Fig. 4. There is a significant difference in cortical connection map (a), polar plots (b) or linear measurement of neuronal connections (c) between control and scrambled siRNA (n = 7-8 for each group). * = p<0.05. Note that Cyto C+stroke and scrambled siRNA+stroke produce very similar maps of forelimb motor cortex connections, and are comparable to stroke only (Supplementary Fig. 6b).

  9. Supplementary Figure 9: Quantitative connectional mapping and axonal connections in GDF10 controls compared with stroke only. (76 KB)

    (a-c) Cortical connections of forelimb motor area in stroke only compared to scrambled siRNA delivered into infarct core. siRNA GDF10 knocks down GDF10 protein levels (Supplementary Fig 3a) and blocks post-stroke axonal sprouting (Fig. 4d-f). To verify that the siRNA is not having effects outside of GDF10 knock down, scrambled sequence siRNA was tested. Figure conventions are as in Supplementary Fig. 8 and Fig. 4. There is no significant difference in cortical connection map (a), polar plots (b) or linear measurement of neuronal connections (c) between stroke and scrambled siRNA. (d-f) Cortical connections of forelimb motor cortex in stroke only compared to stroke+protein control, cytochrome C delivery from the infarct core via biopolymer hydrogel (n = 8 each group). Figure conventions are as in Supplementary Fig 8 and Figure 4. There is no significant difference in cortical connection map (a), polar plots (b) or linear measurement of neuronal connections (c) between stroke and Cyto C. Cohorts (n=8) of mice treated with GDF10 (red) or control protein (Cytochrome C) (light blue).

  10. Supplementary Figure 10: Synaptic protein localization in peri-infarct cortex axons that have undergone axonal sprouting after GDF-10 treatment. (213 KB)

    (a) Synaptic protein analysis was performed in the same peri-infarct tissues from animals used for BDA axonal sprouting maps in Fig. 4(a) and (b). Presynaptic VGLUT2 and postsynaptic Homer1 antibodies were used for identification of synaptic contacts. Marker colocalization analyses were performed on Imaris Imaging software to uniquely identify the synaptic connections formed by GDF10-induced sprouting cortical neurons after stroke (mapped in Fig. 4). (b) Video through a 10.5μm thick section of peri-infarct cortex taken at 100x. BDA surface is shown in light blue. VGLUT2 presynaptic marker is shown by green spots, and Homer1 postsynaptic marker in red spots.

  11. Supplementary Figure 11: Comparison of forelimb motor system connections in CytoC + stroke versus GDF10 siRNA + stroke. (39 KB)

    Conventions as in Supplementary Figures 10 and 11.

  12. Supplementary Figure 12: Pipeline for incorporating microarray and RNA-seq data sets for neurodevelopmental and CNS injury experiments. (54 KB)

    Raw data from different platforms are processed to have gene symbols which were subsequently used to merge the datasets. Merged datasets were normalized, then batch effect was adjusted. In one case, the datasets have very few common genes on different array platforms and these datasets were combined (unionized) instead of taking intersections.

  13. Supplementary Figure 13: Neuronal numbers in in vitro axonal outgrowth assays. (124 KB)

    The numbers of neurons used for each in vitro experiment to generate the data on axonal outgrowth effect of GDF10 and other experimental manipulations in Figures 2 and 3. There is no significant difference in neuronal sampling number across experiments.

Video

  1. Video 1: Synaptic proteins identified in axons that have undergone axonal sprouting in peri-infarct cortex after GDF10 treatment. (93.59 MB, Download)
    This movie shows the process of identifying pre- and post-synaptic connections that are present in axons that have undergone axonal sprouting with GDF10 after stroke. The movie is derived from a confocal image stack taken from a BDA-labelled area in premotor cortex that has axons only when axonal sprouting is induced with GDF10 delivery. The BDA-labelled axons are seen at the outset of the movie (white axonal label). This is then filamented (blue label). The tissue has been stained for Homer (post-synaptic, green) and VGlut2 (a subset of excitatory pre-synaptic terminals, red) and filtered so that only those VGlut2 positive pre-synaptic terminals that co-localize with BDA-filled axons are present. Further, the Homer positive post-synaptic boutons are filtered so that only those that co-localize with the VGlut2 that is within BDA-labelled axons are displayed. The movie that zooms and rotates to show the 3D distribution of this network of axonal connections.

PDF files

  1. Supplementary Text and Figures (1,892 KB)

    Supplementary Figures 1–13

  2. Supplementary Methods Checklist (496 KB)

Excel files

  1. Supplementary Table 1 (30 KB)

    Canonical Pathways Mult Comp Stroke+GDF10 vs Stroke

  2. Supplementary Table 2 (8 KB)

    PI3K Pathway Mult Comp Stroke+GDF10 vs Stroke

  3. Supplementary Table 3 (8 KB)

    PTEN Pathway Mult Comp Stroke+GDF10 vs Stroke

  4. Supplementary Table 4 (11 KB)

    PI3K Pathway Mult Comp Stroke+GDF10 vs Stroke

  5. Supplementary Table 5 (11 KB)

    PTEN Pathway Mult Comp Stroke+GDF10 vs P4

  6. Supplementary Table 6 (9 KB)

    Axonal Guidance Molecules Differentially Regulated in Stroke+GDF10 vs Stroke

  7. Supplementary Table 7 (48 KB)

    Genes Differentially Regulated in Stroke+GDF10 vs Stroke at FDR<0.1

  8. Supplementary Table 8 (11 KB)

    Sources for Genome Wide Assocation Analysis

  9. Supplementary Table 9 (9 KB)

    Primary Antibodies

  10. Supplementary Table 10 (10 KB)

    Multiple Comparsions ANOVA and Tukey-Kramer Test for Differences Between Means in Figure 5

  11. Supplementary Table 11 (11 KB)

    Human cases

Additional data