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SDF-1 induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells

Robyn S. Klein^1,*,, Joshua B. Rubin^2,*, Hilary D. Gibson¹, Elliot N. DeHaan¹, Xavier Alvarez-Hernandez⁴, Rosalind A. Segal^2,3 and Andrew D. Luster¹

¹ Center for Immunology and Inflammatory Diseases, Division Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
² Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
³ Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
⁴ Department of Pathology, New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772, USA
^* These two authors contributed equally

View larger version (32K): [in a new window]   Fig. 1. Expression of CXCR4 and SDF-1 mRNA in postnatal mouse cerebella. (A) Northern blot analysis was performed on 20 µg total RNA isolated from pooled cerebella of litters taken at P3, P5, P7, P9 and P12. CXCR4 and SDF-1 mRNA were expressed at all ages. (B) Phosphoimager analysis was used to quantify the CXCR4/GAPDH and SDF-1/GAPDH mRNA signals for each sample. CXCR4 expression declined during the second postnatal week to approximately 30% of its P3 value. SDF-1 expression remained more constant over this time period.

View larger version (99K): [in a new window]   Fig. 2. Localization of CXCR4 and SDF-1 mRNA and protein in postnatal cerebellar cortex and distribution of CXCR4 on granule cells in vitro. (A) CXCR4 mRNA expression was detected in both the EGL and IGL of P4 and P8 (not shown) cerebella. (B) SDF-1 mRNA expression was primarily limited to the pia mater overlying the cerebella of P4 and P8 (not shown) animals. A small amount of staining is also present throughout the cerebellum. (C) Control sections hybridized with sense RNA probes did not demonstrate expression of CXCR4 or SDF-1 (not shown). Bar represents 50 µm. (D) Immunostaining for CXCR4 using Cy3-conjugated secondary antibodies (red) demonstrates expression in the EGL and IGL of P4 and P8 (not shown) cerebella, consistent with localization of CXCR4 mRNA. Nuclei are localized with DAPI (blue). (E) Immunostaining for SDF-1 using Cy3-conjugated secondary antibodies (red) demonstrates high levels of expression in cells composing the pia mater of P4 and P8 (not shown) cerebella, consistent with localization of SDF-1 mRNA. There is also some staining of cells throughout the cerebellum, as observed for SDF-1 mRNA expression. The location of this staining suggests it may be in glial elements. Nuclei are localized with DAPI (blue). Bar represents 50 µm. (F) Confocal imaging of CXCR4-positive neurons detected with FITC-conjugated secondary antibodies indicates that granule cells express CXCR4 in vitro with punctate staining evident over the cell bodies and along the axonal process. (G) Cultured neurons stained with non-immune serum and detected with FITC-conjugated (green) secondary antibodies demonstrate no background (green) staining of cell bodies or processes; only nuclear staining with topro-3 is detected (blue). Bar represents 10 µm.

View larger version (13K): [in a new window]   Fig. 3. SDF-1 induces chemotactic responses in granule cells. (A) Increasing concentrations of SDF-1 () or MIP-1 () were added to the bottom wells of a modified Boyden chemotaxis chamber. The chemotactic response of purified granule cells was determined by counting migrating cells per high-powered field (Cells/HPF ±s.e.m) in five fields from replicate wells. Data shown is combined from 5 separate experiments using cells derived from P8-P10 mice where each data point was performed in triplicate. Chemotactic responses of granule cells to SDF-1 are significantly above background (0 concentration) and significantly greater than chemotactic responses to MIP-1 at every concentration (P<0.05). Chemotactic responses to MIP-1 are not significantly greater than background (negative control). (B) Granule cells derived from P8 mice were pretreated with 200 ng/ml pertussis toxin for 1 hour, 10 µg/ml polyclonal antibodies to CXCR4 for 15 minutes, or medium alone before being placed in a modified Boyden chemotaxis chamber. For some wells, 1000 ng/ml SDF-1 was added to both the top wells with cells and the bottom wells to test for SDF-1 mediated random migration (chemokinesis). The chemotactic response of purified granule cells was determined by counting migrating cells per high-powered field (Cells/HPF) in five fields from replicate wells and results are expressed ± s.e.m. All cells were examined for chemotactic responses to 1000 ng/ml SDF-1, which was deemed to produce maximal chemotactic responses in dose response experiments (see A). Data shown is combined from 2 separate experiments using cells derived from P8 mice where each data point was performed in triplicate. (*P<0.002; #P<0.005). (C) The chemotactic response of purified granule cells derived from P3-4 vs. P7-8 rats to 0 and 1000 ng/ml SDF-1 was determined by counting migrating cells per high-powered field (Cells/HPF ±s.e.m.) in five fields from replicate wells. Data shown is combined from 2 separate experiments where each data point was performed in triplicate. (**P<0.05).

View larger version (18K): [in a new window]   Fig. 4. SDF-1 augments SHH-induced proliferation in a pertussis toxin sensitive manner. Primary cultures of neonatal mouse cerebella derived from P6 mice were treated with SHH (0.7 µg/ml) or not in the presence or absence of forskolin (10 µM), SDF-1 (1000 ng/ml) or SDF-1 and pertussis toxin (25 ng/ml) for 20 hours. SHH induced a 2.5-fold increase in proliferation as compared with untreated cultures (*P<0.001). SDF-1 enhanced SHH-induced proliferation by 50% as compared with SHH treatment alone (#P<0.001) but had no effect on its own. The SDF-1 effect on SHH-induced proliferation was blocked by pertussis toxin (##P<0.001). SHH-induced proliferation was blocked by forskolin (**P<0.001). Average values are from 3-7 experiments performed in triplicate and presented, ± s.e.m.

View larger version (34K): [in a new window]   Fig. 5. Neuronal calcium flux responses to SDF-1. PBMC preparations or replicate granule cell cultures derived from P8 mice were loaded with 5 µM fura-2 and analyzed using a cuvette- (leukocytes) or microscope- (neurons) based calcium flux apparatus. (A) Leukocyte responses to SDF-1 are not altered by pretreatment with 20 mM KCl. (B) Granule cell neurons display calcium flux responses to 100 ng/ml of SDF-1 only after predepolarization with 20 mM KCl (tracings 1 vs. 2). (C) Although all cells produce calcium flux responses during predepolarization, pretreatment with polyclonal antibodies to CXCR4 (tracing 1) or pertussis toxin (tracing 2) abolishes neuronal responses to SDF-1. (D) Pretreatment of granule cell neurons with glutamate (100 µM) also produces maximal responses to 100 ng/ml SDF-1. Leukocyte populations of 5x106 cells/ml were analysed and groups of 10-20 neurons were visualized and analyzed. Data are presented as the relative ratio of fluorescence at emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of 2 separate experiments with leukocyte preparations and 4 separate experiments with neuronal preparations.

View larger version (23K): [in a new window]   Fig. 6. Proposed model for SDF-1 effects on granule cell position and proliferation during cerebellar development. Depicted is a granule cell in the EGL of the neonatal cerebellum where its position enables it to respond to two separate factors: SHH and SDF-1. SHH, produced by Purkinje cells, is known to stimulate granule cell proliferation by releasing the inhibition of smoothened (SMO) that is exerted by the SHH receptor, patched (PTC). SHH effects can be blocked by PKA or forskolin. We propose that pial derived SDF-1, through the activation of Gi, reduces cAMP and PKA activity and thereby enhances SHH-induced proliferation. In addition, SDF-1 also promotes the localization of granule cells to the EGL through a chemotactic effect that is directed towards the pia. SDF-1 thus contributes to granule cell proliferation both directly, by augmenting SHH effects, and indirectly by maintaining granule cell position within the proliferative environment of the EGL. EGL, external granule cell layer; ML, molecular layer; IGL, internal granule cell layer; SDF-1, stromal derived factor-1;-SHH, Sonic hedgehog; PTC, patched; SMO, smoothened; PKA, protein kinase A.