The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis

Abstract

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.

Figures

Figure 1

G-CSF has stable neuroprotective activity in focal cerebral ischemia and passes the intact BBB. (A) G-CSF has efficacy in the transient MCAO stroke model when given 2 hours after onset of ischemia, as shown by reduction in infarct volume (dose: 60 μg/kg i.v.; vehicle, n = 7; G-CSF, n = 10; **P < 0.01 by 2-sided t test). (B) G-CSF reduces infarct volume in the rat cortical combined CCA/distal MCA occlusion model when given 1 hour after onset of ischemia (dose: 50 μg/kg i.v.; n = 5 each; *P < 0.05). (C) Behavioral measurements in the cortical combined CCA/distal MCA occlusion model. G-CSF–treated animals have a better composite neurological deficit score (NDS) (*P < 0.05). (D) Comparison of the brain/serum ratios of i.v. injected iodinated G-CSF and albumin at 1, 4, and 24 hours following injection. Albumin does not pass the BBB. Radiolabeled proteins (G-CSF and BSA) were injected via the tail vein of healthy female Sprague-Dawley rats. The relative amount of radiolabeled G-CSF and BSA in serum and brain was measured, and the ratio of brain/serum was plotted against the time. The brain/serum ratio of G-CSF was significantly greater than that of albumin, which indicated passage of the intact BBB in non-ischemic animals.

Figure 2

The G-CSF receptor (A–D) and its ligand (E–I) are expressed by neurons in a variety of brain regions in the rat. Among other areas, expression of the receptor was detected in pyramidal cells in cortical layer V (A); Purkinje cells in the cerebellum (B); and cerebellar nuclei (C). Importantly, this neuronal staining pattern could also be detected in the human brain (frontal cortex, D). G-CSF is expressed by neurons in many areas of the CNS. Immunohistochemistry identifies G-CSF–positive cells in the CA3 region of the hippocampus (E) and the subgranular zone and hilus of the dentate gyrus (E, arrows), the entorhinal cortex (F), the olfactory bulb (G), and cerebellar nuclei (H). Expression was also seen in cells in the SVZ (I).

Figure 3

G-CSF is specifically expressed by neurons in the rat CNS. (A–C) Double-immunofluorescence staining with the astrocytic marker GFAP revealed absence of G-CSF expression in astrocytes in the hilus of the dentate gyrus (A) or cortex (Cx; B). In contrast, there was perfect colocalization of G-CSF with cells expressing the neuronal marker NeuN (C). (D–I) In situ hybridization confirmed the neuronal expression of G-CSF and demonstrated an expression pattern that paralleled results obtained by immunohistochemistry. For example, G-CSF mRNA was detected in pyramidal neurons in the cortex (D; original magnification, ×40), in the hippocampus CA3 field (F; original magnification, ×40), and in specific cells located in or near the subgranular zone in the dentate gyrus (DG) (H; original magnification, ×40). Sense probes did not yield any specific staining in corresponding sections (E, G, and I; original magnification, ×40). (J) Using amplified mRNA from laser-excised neurons or astrocytes from the mouse cortex (100 cells each), a G-CSF–specific PCR signal could only be obtained in the neuronal pool but not from astrocytes after 50 amplification cycles. As a control, GFAP was amplified only from the astrocytic population, whereas the ubiquitous housekeeping gene cyclophilin was amplified from both cell pools. A brain cDNA library served as positive control (Pos.) for all PCR reactions. PCR reactions using water as input served as negative control (Neg.). M, size marker.

Figure 4

The G-CSF receptor and its ligand colocalize in neurons in the cortex. Double-immunofluorescence detected G-CSF receptor (G-CSFR) (A) and G-CSF itself (B) in identical layer V neurons in the frontal cortex. Note the relatively stronger presence of the receptor in dendritic processes (C).

Figure 5

G-CSF and its receptor are induced by cerebral ischemia. (A–C) Quantitative PCR demonstrates induction of mRNA following cerebral ischemia. (A) In the MCAO model, G-CSF mRNA is induced more than 100-fold in the ipsilateral and contralateral forebrain hemisphere at 2 hours following ischemia. At 6 hours, induction levels dropped, and overexpression became more specific to the ipsilateral hemisphere. At 20 hours, induction was no longer detectable (data not shown). (B) Moderate induction of the G-CSF receptor mRNA in forebrain hemispheres was seen 6 hours following MCAO. (C) Receptor induction was also detected 6 hours after ischemia in another ischemic model, cortical photothrombotic ischemia in biopsy material from the periinfarct cortex. The substantially higher induction reflects the strong induction in the infarct penumbral zone. (D–O) Immunohistochemical detection of receptor and ligand in the corresponding ischemia models. (D–I) Staining for G-CSF receptor (D–F) and ligand (G–I) in the MCAO model, 6 hours after ischemia: ipsilateral cortex (D and G) and corresponding areas of the contralateral hemisphere (E and H) and the cortex of a sham-operated rat (F and I). (J–O) Staining for G-CSF receptor (J–L) and G-CSF itself (M–O) in the photothrombotic model: ipsilateral cortex (J and M) and corresponding areas of the contralateral hemisphere (K and N) and the cortex of a sham-operated rat (L and O). The infarct border zone is shown in the upper-right quadrant in D, G, J, and M and is particularly clear in the photothrombotic model. Note the strong dendritic staining for the G-CSFR. Original magnification ×20.

Figure 6

G-CSF receptor is neuronally induced upon human stroke. Immunohistochemical detection of G-CSF receptor in the ipsilateral (A) and contralateral (B) cortex of a human brain obtained at autopsy 3 days after onset of ischemic stroke as well as the frontal cortex of an age-matched neuropathologically normal control brain (C). Scale bars: 100 μm. Note increased staining of neurons in the ipsilateral cortex compared with the contralateral cortex and control brain (insets).

Figure 7

G-CSF counteracts programmed cell death in rat cortical neurons (A–D) and human neuroblastoma cells (SHSY-5Y) (E) in vitro. (A) The G-CSF receptor is present on primary cortical neurons in culture as shown by immunocytochemistry. (B) G-CSF of both human (h) and mouse (m) origin counteracts camptothecin-induced programmed cell death in primary neurons as determined by caspase-3/7 activity. (C) Preincubation of primary neurons with an antibody against the G-CSF receptor abolishes the antiapoptotic activity of G-CSF (not significant). (D) The NO donor NOR3 [(±)E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (150 μM) induces apoptosis in primary neurons as evidenced by PARP and caspase-3 cleavage (immunoblots, first and second lanes), which is reduced by G-CSF treatment (third lane). (E) Also in the human neuroblastoma cell line SHSY-5Y, human or mouse G-CSF reduces caspase activation by the NO donor NOR3. Bar graphs show relative caspase activity levels after normalization to control values. Rel. units, relative units.

Figure 8

Signal transduction events evoked by G-CSF treatment of rat cortical neurons. G-CSF activates STAT3, ERK, and PI3K/Akt pathways in primary cortical neurons. Western blots for phosphorylated proteins were stripped and reprobed with antibodies nonselective for phosphorylation. (A) STAT3 was rapidly but moderately phosphorylated (pSTAT3) 5 minutes after addition of G-CSF to the medium. Neuronal expression of the G-CSF receptor was also confirmed by Western blot analysis. Addition of the JAK2 inhibitor AG490 inhibited hyperphosphorylation of STAT3 5 minutes after addition of G-CSF. (B) Quantification of phosphorylation ratios from Western blots illustrates the moderate and transient but reproducible activation of STAT3 by G-CSF (data from 3 independent experiments). (C) G-CSF leads to increase of protein levels of the antiapoptotic STAT3 target Bcl-XL in primary cortical neurons over 8 hours. (D) Induction of ERK1/2 (double band) and ERK5 by G-CSF. While ERK1/2 activation appears to be very transient (upper rows), ERK5 is induced for at least 60 minutes following G-CSF exposure (lower rows). (E) Stable induction of Akt phosphorylation upon addition of G-CSF to the medium (50 ng/ml) shown by immunoblotting with a Ser437 phosphorylation–specific antibody. In accordance with the known Akt activation pathway, the PDK kinase upstream of Akt was phosphorylated, and Akt phosphorylation could be blocked by preincubation of the neurons with the PI3K inhibitor LY294002. (F and G) Inhibition of PI3K by LY294002 diminishes G-CSF–mediated protection from apoptosis (measured by luminometric caspase-3/7 activity assay) in rat cortical neurons treated with staurosporine (F) or in the human neuroblastoma cell line SHSY-5Y, where cell death was elicited by camptothecin (G). Bars indicate mean relative protection (%) against the cell death stimulus ± SEM; values were normalized to the appropriate controls (n = 16 each).

Figure 9

The G-CSF receptor is present on adult neural stem cells (NSCs) and drives neural progenitor differentiation in vitro. (A) Results of PCR analysis for the G-CSF receptor on neural stem cells demonstrate presence of G-CSF receptor mRNA in neurospheres in culture. (B) Double-fluorescence immunocytochemistry on neural stem cells plated onto coated 96-well plates. Almost all cells were positive for the stem cell marker nestin and for the G-CSF receptor (original magnification, ×20). (C–E) G-CSF drives neuronal phenotype induction in adult neural stem cells in vitro. (C) As 1 approach, we assayed β-III-tubulin promoter activity by a luciferase reporter assay. Treatment with G-CSF in increasing concentrations for 48 hours increased promoter activity. As a positive control, neural stem cells were treated with the standard differentiation protocol involving withdrawal of EGF and bFGF and addition of FCS (+FCS/–EGF/–bFGF). (D) G-CSF treatment induces a concentration-dependent upregulation of the neuronal markers NSE and β-III-tubulin 4 days after G-CSF treatment, as indicated by quantitative PCR. The glial markers PLP and GFAP are moderately induced in response to increasing G-CSF concentrations. Error bars indicate standard deviations calculated from measurements done with serial dilutions of the cDNA samples (1:3, 1:9, 1:27, and 1:81). (E) FACS analysis for MAP2-positive cells served to confirm the data presented above on an individual cell basis. Cells were treated for 4 days prior to analysis. Left: Examples of FACS runs of vehicle-treated and G-CSF–treated (100 ng/ml) neural stem cells. Right: The bar graph summarizes the results of several experiments (vehicle, n = 4; G-CSF, n = 7; P < 0.005).

Figure 10

G-CSF treatment improves long-term functional outcome after cortical ischemia. (A–D) G-CSF significantly improved motor recovery as measured by rotarod performance (A) and NSS, which included the results of the beam balance test (B), compared with those in nontreated, ischemic control animals. Sensory-motor function as measured by adhesive tape removal was significantly better in G-CSF–treated, ischemic animals compared with ischemic controls when the contralateral forepaw was tested (C) and borderline significant in the ipsilateral forepaw (D). Bar graphs represent an analysis of area under the curve (AUC) for each rat over time in an experimental group. *P < 0.05; **P < 0.01; #P < 0.001.

Figure 11

G-CSF induces neural progenitor cells and their migration in subcortical areas. (A–I) G-CSF induced substantially more neural progenitor cells and immature neurons (DCX in red) in subcortical regions adjacent to the ischemic lesion (NeuN in green, BrdU in blue). (A) Control: unlesioned hemisphere of G-CSF–treated ischemic animals. (B) Lesioned hemisphere with G–CSF treatment. (C) Lesioned hemisphere with sham treatment. Note in B that G-CSF induced a stream of DCX-positive cells migrating toward the ischemic lesion (upper right). (D–I) Details from the boxed areas in A–C. Note the density of DCX expression in F. Images represent cumulative confocal image Z-stacks throughout the whole slice thickness.

Figure 12

G-CSF increases neurogenesis in the dentate gyrus. (A) Example of BrdU/NeuN-double-positive cells within the basal layer of the dentate gyrus (scale bar: 40 &____m). The arrow in A indicates the enlarged double-stained cell in B (scale bar: 10 &____m). (C) DCX in red. (D) BrdU in green. (E) NeuN in blue. (F) G-CSF increased the number of newly generated neurons (BrdU+/NeuN+) on the side of the ischemic lesion (red bars, ipsilateral + vehicle vs. ipsilateral + G-CSF; **P < 0.01). Contralateral to the lesion, there was a trend toward an increase in newly generated neurons compared with vehicle-treated ischemic animals that was not statistically significant (blue bars, contralateral + vehicle vs. contralateral + G-CSF). However, G-CSF increased neurogenesis in sham-operated, nonischemic animals (green bars, sham + vehicle vs. sham + G-CSF; *P < 0.05). (G) The total number of BrdU+ cells in the dentate gyrus was not significantly further increased by G-CSF treatment in the ischemic animals (red and blue bars), which implies a true induction of neuronal differentiation by G-CSF in the postischemic brain. In contrast, sham-lesioned animals showed an elevation of the total number of BrdU+ cells after G-CSF treatment (green bars; *P < 0.05).