Environmental impact on direct neuronal reprogramming in vivo in the adult brain

Abstract

Direct reprogramming of non-neuronal cells to generate new neurons is a promising approach to repair damaged brains. Impact of the in vivo environment on neuronal reprogramming, however, is poorly understood. Here we show that regional differences and injury conditions have significant influence on the efficacy of reprogramming and subsequent survival of the newly generated neurons in the adult rodent brain. A combination of local exposure to growth factors and retrovirus-mediated overexpression of the neurogenic transcription factor Neurogenin2 can induce new neurons from non-neuronal cells in the adult neocortex and striatum where neuronal turnover is otherwise very limited. These two regions respond to growth factors and Neurogenin2 differently and instruct new neurons to exhibit distinct molecular phenotypes. Moreover, ischaemic insult differentially affects differentiation of new neurons in these regions. These results demonstrate strong environmental impact on direct neuronal reprogramming in vivo.

Figure 1

Focal labeling of striatal and neocortical cells with GFP retroviruses in the adult rat brain. (a-c) Schematic diagrams illustrating the location of virus injection sites in parasagittal (a) and coronal (b,c) views. Boxes indicate the areas shown in d-k. (d-k) Distributions of GFP⁺ cells (dashed circles and arrowheads) in the striatum (d-h) and neocortex (i-k) at DAI-3. Sections were counter-stained with methylgreen. (l-w) Co-expression of neuronal markers and GFP in virus-infected cells (arrows) in the striatum (l-s) and neocortex (t-w). Dashed lines in l, o, q, and r indicate the border of the matrix (M) and patch (P) compartments of the striatum. Arrows and arrowheads indicate GFP⁺ cells co-expressing and non-expressing, respectively, the marker shown above each panel. The lower panels m, p, s, u, w show the co-localization of relevant markers in single cells in orthogonal views of confocal z-stack images in I, o, r, t, and v, respectively. Scale bar: d, e, f, I, 1 mm; e, g, h, j, k, 100 μm; l, n, o, q, r, t, v, 50 μm; m, p, s, u, w, 20 μm. Abbreviations: Cx, neocortex; CC, corpus callosum; DG, dentate gyrus; LV, lateral ventricle; RMS, rostral migratory stream; Str, striatum; SVZ, subventricular zone.

Figure 2

Differential impacts of GFs and Neurog2 in neuronal induction in the adult rat striatum and neocortex. The percentages of GFP⁺ cells expressing Dcx and NeuN in the striatum (a) and neocortex under various conditions are shown (mean ± s.d. of 3-4 animals). GFP⁺/Dcx⁺ cells were examined at DAI-3 and DAI-7 in the neocortex and striatum, respectively. *, p < 0.01 compared with control viruses in Student’s t test; ^$, p < 0.01 compared with treatment with GFs or Neurog2 viruses alone in Student’s t test.

Figure 3

Combinatorial actions of GFs and Neurog2. (a-c) The numbers of total GFP⁺ (a), GFP⁺/Dcx⁺ (b), and GFP⁺/NeuN⁺ (c) cells detected at different time points after GF/virus infection (mean ± s.d., n = 3-4 animals). The number of GFP⁺ cells in GF-untreated animals is shown in Supplementary Table S1. *, p < 0.01 compared with control viruses in Student’s t test. (d, e) The estimated numbers of GFP⁺/Dcx⁺ (left) and GFP⁺/NeuN⁺ (right) cells in the striatum (d) and neocortex (e) under various conditions (mean ± s.d., n = 3-4 animals). The numbers in parentheses show the percentage of GFP⁺/NeuN⁺ cells at DAI-14 compared with GFP⁺/Dcx⁺ cells at earlier time pints. *, p < 0.01 compared with control viruses in Student’s t test; ^$, p < 0.01 compared with GFs or Neurog2 alone in Student’s t test. nd, not detected.

Figure 4

Region-specific differentiation of GFP-labeled neurons in the striatum and neocortex. (a-g) Co-labeling of various neuronal markers and BrdU in GFP⁺ cells (arrows) in the striatum. Time points after infection, types of manipulations used, and markers stained are shown above individual panels. In c-e, BrdU was administered twice each day for three days between DAI-0 and DAI-2. Dashed lines in a, b, c, and f indicate the border of the matrix (M) and patch (P) compartments of the striatum. Lower panels in a-d and g show orthogonal views of confocal z-stack images of the cells indicated by arrows. Note that the overlap of green, red, and blue colors in c, d, and e is indicated as white color. (h-y) Region-specific phenotypes of GFP-labeled neurons in the striatum (h-p) and neocortex (q-y) at DAI-28. Arrows indicate GFP⁺/NeuN⁺ neurons expressing relevant markers, whereas arrowheads indicate marker-negative GFP⁺ and/or NeuN⁺ cells. Images in n-p were obtained from control virus-infected animals, whereas all others were from Neurog2 virus-infected animals. (z-b2) Retrograde labeling of GFP⁺/NeuN⁺ cells in the striatum by FG. FG was injected into the globus pallidus ipsilateral to the virus injection site at DAI-84, and animals were analyzed at DAI-91. z shows the distribution of FG fluorescence (white dots) in the striatum (the virus injection site in a dashed circle). a2 shows GFP⁺/NeuN⁺ cells co-labeled with FG detected in the area boxed in z. b2 shows confocal images (an orthogonal view in lower panels) of a neuron boxed in a2. Scale bar: a-g, 50 μm; h-y, 25 μm; z, 1 mm; a2, 50 μm; b2 and lower panels of a, b, c, d, g, and b2, 20 μm.

Figure 5

Neurogenesis in the ischemic brain. (a-n) BrdU- and GFP-labeled neurons in the adult rat striatum (a-f) and neocortex (g-n). Circles and boxes in a, d, g, and k indicate the location of virus-infected cells and the areas shown in fluorescence images, respectively. Dashed lines in b and e indicate the ventricular wall. b and e show BrdU-labeled neurons near the LV, whereas c and f show GFP-labeled neurons detected around virus-infected regions in the striatum. h and j show GFP⁺/NeuN⁺ and GFP⁺/GABA⁺ cells in the neocortex that received control viruses, whereas l and n show GFP⁺/NeuN⁺ and GFP⁺/NeuN⁺/Glu⁺ cells detected in Neurog2 virus-infected animals. I and m show orthogonal views of z stack confocal images of neurons indicated by arrows in h and l, respectively. (o, p) Estimated numbers of GFP⁺/Dcx⁺ cells at DAI-3 (neocortex) and DAI-7 (striatum) (o), and GFP⁺/NeuN⁺ cells at DAI-14 (both regions) (p) under various manipulation conditions (mean ± s.d., n = 3-4 animals). The data regarding non-ischemic animals are adopted from Fig. 3d and 3e. The numbers in parentheses show the percentage of GFP⁺/NeuN⁺ cells at DAI-14 compared with GFP⁺/Dcx⁺ cells at earlier time pints. *, p < 0.01 compared with control viruses in Student’s t test; ^$, p < 0.01 compared with non-ischemic animals treated with GFs and Neurog2 in Student’s t test. nd, not detected. Scale bar: a, d, g, k, 2 mm; b, c, e, f, h, l, 50 μm; and insets in b, c, e, f, i, j, m, n, 20 μm.

Figure 6

Neurosphere-forming cells induced by stab wound and GFs in the adult rat neocortex and striatum. (a, b) GFP⁺ Neurospheres formed in vitro by cells exposed to GFs and GFP viruses in vivo. (c-h) Phase-contrast images (c-e) and staining for BrdU (f-h, red) of growing secondary neurospheres. Cell nuclei were stained with Hoechst 33258 (blue). (i) Frequencies of neurosphere-forming cells 14 days after tissue isolation (primary spheres, indicated as 1st) and 14 days after the first passage (secondary spheres, 2^nd) (mean ± s.d., n = 10). *, p < 0.01 in Student’s t test. (j-m) Differentiation of a secondary neurosphere derived from the striatum. The cells (phase-contrast image in j) were stained for TuJ1 (k, red), GFAP (l, blue), and O4 (m, green). (n) Percentages of TuJ1⁺, GFAP⁺, and O4⁺ cells among total cells in culture of secondary neurospheres (mean ± s.d., n = 4). *, p < 0.01 compared to SVZ-derived cells in Student’s t test. Scale bar: a, b, d-i, and k, 50 μm.

Figure 7

Differential gene expression profiles of neurosphere-forming cells derived from distinct regions of the adult mouse brain. (a) Venn diagram showing the number of genes commonly and differentially expressed in neurospheres derived from the SVZ, neocortex and striatum. Among 3,003 genes that were selected as those that gave more than a 5.0-fold higher hybridization signal with one or more of the adult neurosphere samples compared with the whole brain of adult mice. (b) Heat-map view of cluster analysis of 716 probe sets (637 genes) that showed more than a 5-fold difference in the expression level between cortical and SVZ cells (three stripes represent 3 independent cultures). Neurospheres from the dorsal and ventral forebrains (dFB and vFB) of E14.5 embryos were used for comparison. (c-i) Quantitative RT-PCR analyses of the expression of transcription factor mRNAs. The levels are normalized using GAPDH as internal control, and data are expressed as values relative to the SVZ-derived cells (designated as 1.0) (mean ± s.d., n = 3). Abbreviation: dFB and vFB, dorsal and ventral embryonic forebrain culture, respectively.