A novel purification method for CNS projection neurons leads to the identification of brain vascular cells as a source of trophic support for corticospinal motor neurons

Abstract

One of the difficulties in studying cellular interactions in the CNS is the lack of effective methods to purify specific neuronal populations of interest. We report the development of a novel purification scheme, cholera toxin beta (CTB) immunopanning, in which a particular CNS neuron population is selectively labeled via retrograde axonal transport of the cell-surface epitope CTB, and then purified via immobilization with anti-CTB antibody. We have demonstrated the usefulness and versatility of this method by purifying both retinal ganglion cells and corticospinal motor neurons (CSMNs). Genomic expression analyses of purified CSMNs revealed that they express significant levels of many receptors for growth factors produced by brain endothelial cells; three of these factors, CXCL12, pleiotrophin, and IGF2 significantly enhanced purified CSMN survival, similar to previously characterized CSMN trophic factors BDNF and IGF1. In addition, endothelial cell conditioned medium significantly promoted CSMN neurite outgrowth. These findings demonstrate a useful method for the purification of several different types of CNS projection neurons, which in principle should work in many mammalian species, and provide evidence that endothelial-derived factors may represent an overlooked source of trophic support for neurons in the brain.

Figure 1.

CTB panning can be used to purify RGCs. A, Retrogradely transported CTB is cleaved from the surfaces of labeled cells during enzymatic dissociation, but internal stores of CTB bound to Retrobeads IX replace lost CTB during the postdissociation recovery step and can then be selectively bound by immunopanning. N, Nucleus; A, axon. B, CTB conjugated to fluorescent tracer Retrobeads injected into the superior collicular brachium is retrogradely transported to the retina. C, Fluorescent tracer is localized to the RGC layer in the retina 48 h after the injection. Scale bar, 100 μm. RGC, RGC layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PE, pigmented epithelium. D, Phase-contrast image of CTB-panned RGCs in culture. Scale bar, 100 μm. E, Fluorescent image of the same field shows that purified RGCs are fluorescently labeled with Retrobeads (arrows). F, Retrogradely labeled RGCs that adhere to the CTB panning plate (CTB panned) and RGCs purified from unlabeled retinas by Thy1.1 antibody (T11D7 panned) respond similarly to BDNF, CNTF, and full growth medium (BDNF, CNTF, insulin, and forskolin). Survival of RGCs purified from P6 rats assayed after 3 DIV (±SEM) is shown (n = 2; >150 cells/replicate condition scored).

Figure 2.

CSMNs can be purified by CTB panning. A, CTB conjugated to fluorescent tracer Retrobeads is injected into the pyramidal decussation of the spinal cord and retrogradely transported to cortical layer V. B, C, Successfully retrogradely labeled dissected cortices (B) can easily be discerned from unsuccessful injections that fail to label layer V (C) using an inverted fluorescent microscope. D, E, Immunohistochemistry shows that cells labeled with green fluorescent Retrobeads are layer V CSMNs. All retrogradely labeled (green) cells also label with CTIP2 (red), a nuclear marker for CSMNs (D). Blue DAPI costain shows that CTIP2 is specific for layer V cells (E). F, Immunocytochemistry of cultured purified cells shows that they are CSMNs. All purified cells labeled by neuronal marker MAP2 (green) coexpress CTIP2 (red). Scale bars: (in B) B, C, 1 mm; D (for D, E), F, 100 μm.

Figure 3.

Purified CSMNs express CSMN and neuronal genes and not other neural genes. A, B, Comparison of expression levels of stereotypical neuronal, astrocyte, oligodendrocyte/oligodendrocyte precursor cell, and vascular cell (A) and CSMN-enriched (B) genes in purified CSMNs (black) and whole dissociated brain (gray) from P3 rats. Probe sets and expression values (arbitrary units) are shown in supplemental Table S2 (available at www.jneurosci.org as supplemental material). In cases where multiple probe sets are present for a single gene, the probe set with the highest expression was chosen. To fit the graph, the expression values for Syt1 and Snap25 were scaled down threefold for both samples.

Figure 4.

Survival of CTB-immunopurified CSMNs in vitro. A, Percentage of purified P3 CSMNs surviving after 5 DIV in the various media listed. Neg, Basal serum-free medium (see Materials and Methods); CPT-cAMP, CPT-cAMP added to basal medium; all remaining single/multiple factors were added in the presence of CPT-cAMP; All, CXCL12 plus IGF2 plus PTN plus BDNF with or without IGF1 (no significant differences noted with/without IGF1 when the other four factors were present); ECM, fresh 10×ECM added at plating and day 3 feeding. All data presented were normalized to negative controls (Neg or CPT-cAMP) (±SEM). *p < 0.001, multiple pairwise ANOVA post hoc Student–Newman–Keuls test; **p < 0.0001, two-tailed Student's t test; all statistical differences were found with control CPT-cAMP, no significant differences were detected between the various factor/ECM conditions. Neg, n = 20; CPT-cAMP, n = 41; BDNF, n = 33; IGF1, n = 21; IGF2, n = 33; PTN, n = 26; CXCL12, n = 15; All, n = 19; ECM, n = 11; ECM+All, n = 13; for each “n” value, 50–300 cells per well were counted. B1, B2, Insulin+BDNF supports short-term but not long-term CSMN survival. Two independently performed assays of purified P3 CSMN survival at various DIV in media containing CPT-cAMP and insulin, with or without BDNF are shown. All data are ±SEM (n = 3). C, Survival of purified P7 CSMNs 1–3 DIV in media containing CPT-cAMP and insulin, with or without BDNF.

Figure 5.

Morphology of CTB-immunopurified CSMNs in vitro. A–F, Representative images of purified P3 CSMNs cultured for 5 DIV in basal serum-free medium (A), basal media containing CPT-cAMP alone (B), CPT-cAMP plus BDNF (C), IGF2 (D), BDNF plus CXCL12 plus IGF2 plus PTN (E), or 10× ECM (F). All live cells were visualized by incubation in calcein AM. Scale bar, 100 μm. G, H, Histograms showing percentages of purified P3 CSMNs alive at 5 DIV in indicated media, visualized by calcein AM incubation, demonstrating <50, 50–400, 400–800, or >800 μm total process outgrowth (G), or 0–1, 2–9, 10–50, or >50 total process branch points (H). In each condition, >20 randomly selected live cells were analyzed.