An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke

Abstract

Stroke is an age-related disease. Recovery after stroke is associated with axonal sprouting in cortex adjacent to the infarct. The molecular program that induces a mature cortical neuron to sprout a new connection after stroke is not known. We selectively isolated neurons that sprout a new connection in cortex after stroke and compared their whole-genome expression profile to that of adjacent, non-sprouting neurons. This 'sprouting transcriptome' identified a neuronal growth program that consists of growth factor, cell adhesion, axonal guidance and cytoskeletal modifying molecules that differed by age and time point. Gain and loss of function in three distinct functional classes showed new roles for these proteins in epigenetic regulation of axonal sprouting, growth factor-dependent survival of neurons and, in the aged mouse, paradoxical upregulation of myelin and ephrin receptors in sprouting neurons. This neuronal growth program may provide new therapeutic targets and suggest mechanisms for age-related differences in functional recovery.

Figure 1

Experimental approach and laser capture microdissection of sprouting neurons after stroke. (a) Stroke was produced by permanently occluding two anterior branches of the distal middle cerebral artery over the parietal cortex and transiently occluding bilateral jugular veins. Two different fluorescent conjugates of the tracer cholera toxin B subunit (CTB) were sequentially injected into the same site of forelimb sensorimotor cortex, the first at the time of stroke (Alexa 488–CTB) and the second at 2/3 the volume either 7 or 21 d after stroke (Alexa 647–CTB). Neurons that project to this forelimb sensorimotor site at the time of stroke are labeled with Alexa 488–CTB; neurons that establish a projection to this site only after stroke are labeled solely with Alexa 647–CTB. (b) Injection sites from three separate cases. Only mice in which the second tracer injection was entirely within the first were used for further analysis. (c) Young adult sprouting neurons are shown in the top row and sprouting neurons from an aged brain are shown in the bottom row. The left column shows neurons labeled by the first tracer injection; the middle column neurons labeled by the second tracer injection; the right column neurons seen after laser capture of a tracer 2-only neuron. Neurons with double label (non-sprouting neurons; arrowheads) or labeled by CTB-Alexa 647( tracer-2 only, sprouting neurons; arrows) were separately laser-captured in the peri-infarct cortex. Scale bars, 50 μm (c) and 10 μm (b).

Figure 2

Cellular pattern of ATRX, IGF1 and Lingo1 expression in the brain after stroke. (a) Nissl-stained photomicrograph of mouse barrel field stroke model. Boxes indicate region of contralateral cortex (left column) and peri-infarct cortex in young adult (middle column) and aged adult (right column). (b–j) Panels for ATRX (b–d), IGF1 (e–g) and Lingo1 (h–j) show immunoreactivity in each condition. Insets: Colocalized staining of ATRX (red) and NeuN (green) is seen as yellow (d). IGF1 staining (red) localizes to astrocytes and not neurons (green) in young adult peri-infarct cortex (f). In aged adult peri-infarct cortex, IGF1 (red) co-localizes to NeuN positive neurons (green) (g). Lingo1 staining (red) localizes to NeuN positive neurons (green) in aged peri-infarct cortex (j). Scale bars, 1 mm (a), 50 μm (b–d) and 20 μm (insets).

Figure 3

Quantitative connectional mapping. (a) Timeline of experimental design for all in vivo axonal tracer experiments. Mice received a sham surgery or stroke, followed 1 week later by siRNA or drug delivery. Three weeks later biotinylated dextran amine (BDA) was microinjected into forelimb motor cortex. (b,c) The cortex was then removed, flattened, tangentially cut (b) and stained for cytochrome oxidase (c) and BDA in the same sections. (d,e)The location of BDA-labeled axons was digitally mapped in x, y coordinates relative to the injection site. (d) The quantitative connectional map of forelimb sensorimotor connections in sham-operated animals (n = 5). (e) The quantitative connectional map of forelimb sensorimotor cortex connections after stroke (n = 7). (f) Cytochrome oxidase staining in layer IV identifies the mouse somatosensory body map after stroke. (g) Each connectional map was registered to the somatosensory body map from the same brain to produce a group connectional and functional map of cortex. Axonal sprouting was identified when a pattern of cortical connections was precisely mapped and statistically different across treatment conditions. Scale bars, 1 mm.

Figure 4

ATRX function in post-stroke axonal sprouting. (a) Knockdown of ATRX by siRNA reduced axonal outgrowth in vitro compared to scrambled siRNA treatment. ATRX overexpression induced axonal sprouting compared to expression of GFP alone. (b) Axon numbers in normal control or peri-infarct cortex layer 2/3 with Atrx siRNA or scrambled siRNA. (c) Sham, non stroke forelimb motor cortex connections (blue, n = 10) compared to those from Atrx siRNA siRNA group (red, n = 12). The blue label indicates the position of axons projecting from motor cortex summed from the entire sample of mice in the sham, non-stroke condition. The red label indicates that position of axons from the entire sample of mice treated with Atrx siRNA. The dark blue is the areas of dense overlap of the two projection systems. There was no significant difference in the pattern of cortical connections between normal (non-stroke, sham operated (d) Quantitative connectional map of forelimb sensorimotor connections in stroke + scrambled siRNA (blue, n = 11) and stroke + ATRX siRNA (red, n = 12), registered to the body map of underlying cortex with stroke. Dark blue shows area of dense overlap of connections in the two conditions. There is a significant increase in motor cortex projections between stroke + scrambled siRNA and stroke + ATRX siRNA. (e) Polar plots of axonal label from studies in (d). Shaded regions represent 70th percentile of the distances of labeled axons from the injection site; weighted polar vectors represent the normalized distribution of the number of points in a given segment of the graph. There was a significant difference in distribution of cortical projections between stroke + Atrx siRNA (red) and stroke + scrambled siRNA (blue) and between stroke + scrambled siRNA (blue) and non-stroke vehicle (yellow), and no significant change between stroke + Atrx siRNA (red) and non-stroke vehicle (yellow) (n = 10, P > 0.05). Scale bars, 1 mm. Error bars, s.d.; @, * and ** indicate a significant difference compared to saline normal control, GFP control and scrambled siRNA stroke, respectively.

Figure 5

IGF1 maintains neuronal viability after stroke. (a) There was no significant difference in the pattern of cortical connections between stroke + saline (n = 5) and stroke + IGF1 (n = 7). (b) There was no significant change in the polar distribution of connections with IGF1 administration compared to stroke + saline (P > 0.05). (c) There was a significant loss of cortical connections between stroke + saline (n = 5) and stroke + JB1 (n = 8). (d) There was a significant decrease in polar distribution of connections with IGF1 blockade (P < 0.001). (e) Photomicrographs of neurons in peri-infarct cortex, showing no change in number across IGF1 treatment conditions. (f) (f) Effects of IGF1 and JB1 on neuronal number in layer 5 in normal control or peri-infarct cortex. IGF1 or JB1 were delivered beginning 7 days after stroke. Stroke (n = 6) induces neuronal cell death in peri-infarct cortex (p < 0.01). This neuronal death is not altered by delivery of IGF1 (n = 5, p > 0.05) or vehicle (n = 5, p > 0.05) into peri-infarct cortex. However, IGF1 blockade with JB1 + hydrogel significantly (n = 5, p < 0.05) increases cell death in peri-infarct cortex when compared to the saline + hydrogel (n = 5) –treated animals post-stroke. (g) Photomicrographs of neurons in peri-infarct cortex, showing decrease in number of neurons with JB1-induced IGF1 signaling blockade. Scale bars, 500 μm (c,g) and 1 mm (a,b,d,e). Note that the location of BDA tracer injection was more lateral in ATRX studies (Fig. 4) than in IGF1/JB1 and Lingo1-Fc/NgR1/NgR2 studies, the latter two of which use the same tracer coordinates; this produces slightly different patterns of intracortical connections. Error bars, s.d, *** p < 0.001 and ** p < 0.01 indicate a significant difference compared to normal control; # < 0.05 indicates a significant difference compared to stroke saline + hydrogel.

Figure 6

Lingo1/NgR1 restricts cortical axonal sprouting after stroke. Quantitative connectional maps of forelimb motor connections with NgR1 signaling blockade after hydrogel delivery of Lingo1-Fc + stroke (n = 6) versus hydrogel control-Fc + stroke (n = 7). (a) Lingo1-Fc release into peri-infarct cortex induced a significantly different pattern of forelimb motor cortex connections. (b) Registration of motor cortical connections with the underlying cytochrome oxidase body map shows that Lingo1-Fc blocked the connections that form lateral and rostral to mouse somatosensory cortex, in secondary somatosensory cortex and motor cortex. (c) Polar analysis of forelimb sensorimotor connections from same maps. Lingo1-Fc induced a significant increase in motor cortex connections in the sites seen in the body map registration motor cortex, SI and SII (P < 0.001). (d). Quantitative connectional map of forelimb sensorimotor connections in NgR1 knockout mice with stroke (n = 5) versus control mouse stroke (n = 5). There was a significant increase in the pattern of cortical connections in NgR1 knockout. (e) Registering the underlying mouse body map to the motor cortical projections indicates that new projections could be seen in the NgR1 knockout in lateral SI, SII and to a lesser extent in motor cortex. (f) Polar analysis of forelimb motor connections in stroke + NgR1 knockout versus control. Significantly different connections were seen in motor and lateral somatosensory regions (P < 0.005). Scale bars, 1 mm.