Downregulation of Sphingosine 1-Phosphate Receptor 1 Promotes the Switch from Tangential to Radial Migration in the OB

Abstract

Neuroblast migration is a highly orchestrated process that ensures the proper integration of newborn neurons into complex neuronal circuits. In the postnatal rodent brain, neuroblasts migrate long distances from the subependymal zone of the lateral ventricles to the olfactory bulb (OB) within the rostral migratory stream (RMS). They first migrate tangentially in close contact to each other and later radially as single cells until they reach their final destination in the OB. Sphingosine 1-phosphate (S1P) is a bioactive lipid that interacts with cell-surface receptors to exert different cellular responses. Although well studied in other systems and a target for the treatment of multiple sclerosis, little is known about S1P in the postnatal brain. Here, we report that the S1P receptor 1 (S1P1) is expressed in neuroblasts migrating in the RMS. Using in vivo and in vitro gain- and loss-of-function approaches in both wild-type and transgenic mice, we found that the activation of S1P1 by its natural ligand S1P, acting as a paracrine signal, contributes to maintain neuroblasts attached to each other while they migrate in chains within the RMS. Once in the OB, neuroblasts cease to express S1P1, which results in cell detachment and initiation of radial migration, likely via downregulation of NCAM1 and β1 integrin. Our results reveal a novel physiological function for S1P1 in the postnatal brain, directing the path followed by newborn neurons in the neurogenic niche.

Significance statement: The function of each neuron is highly determined by the position it occupies within a neuronal circuit. Frequently, newborn neurons must travel long distances from their birthplace to their predetermined final location and, to do so, they use different modes of migration. In this study, we identify the sphingosine 1-phosphate (S1P) receptor 1 (S1P1) as one of the key players that govern the switch from tangential to radial migration of postnatally generated neuroblasts in the olfactory bulb. Of interest is the evidence that the ligand, S1P, is provided by nearby astrocytes. Finally, we also propose adhesion molecules that act downstream of S1P1 and initiate the transition from tangential chain migration to individual radial migration outside of the stream.

Keywords: cell adhesion; migration; olfactory bulb; retrovirus; sphingosine; time lapse.

Figure 1.

S1P1 is expressed in RMS neuroblasts. A, In situ hybridization of S1P1 in sagittal mouse brain sections at the indicated ages showing strong expression of the receptor in the RMS (images obtained from the Allen Brain Atlas). B, C, Double immunostainings showing S1P1 expression (red) in DCX-positive neuroblasts (green) along the RMS before entering the OB (B) and inside the OB (C). The rectangles delineate the region depicted at a higher magnification below. D, Overview of the OB costained with the mature neuronal marker NeuN (green) and S1P1 (red). The insets in the upper corner indicate the brain area shown in the picture. Note the decline in S1P1 expression in radially migrating neuroblasts inside the OB. Scale bars, 50 μm.

Figure 2.

S1P1 expression levels regulate the fraction of neuroblasts outside the RMS. A, Scheme of the control and knock-down retroviral constructs used to infect SEZ-derived newborn cells by stereotactical brain injections. B, Western blot assays with total protein extracts from HEK cells cotransfected with S1P1 and either a control plasmid expressing scrambled shRNA or shRNA against S1P1 (“a” and “b” are different sequences targeting S1P1). Top graph shows the quantification of the blot shown below. C, Experimental scheme. Mice were injected in the SEZ with a retroviral mixture and the OB was imaged 6 d later. D, Confocal image of a sagittal brain section containing the OB from a mouse injected with the viral mixture in the SEZ and killed 6 d later. Enlargements show infected cells (red, control; green, S1P1 knock-down) outside the RMS^OB (top) and migrating within the RMS^OB (bottom). E, Quantification of control and knock-down infected cells within and outside the RMS^OB plotted as ratios between knock-down/control cells. A higher proportion of knock-down cells are found outside the RMS^OB compared with control cells. n = 8 mice for shRNAa (2317 and 5945 control and knock-down cells, respectively) and n = 7 mice for shRNAb (941 and 3858 control and knock-down cells, respectively). **p = 0.0006, *p = 0.012, t test. F, Experimental scheme. P4 mice were injected in the SEZ with a mixture of control and S1P1 knock-down virus and, 4 d later, the RMS at the level indicated with the red square was analyzed. The confocal picture on the right shows infected neuroblasts migrating within the RMS and outside the RMS in the corpus callosum toward the cortex (arrows indicate S1P1 KD cells outside the RMS). G, Quantification of control and knock-down infected cells within and outside the RMS plotted as ratios between knock-down/control cells. A higher proportion of knock-down cells exited the RMS in direction to the cortex. n = 4 mice (254 and 635 control and knock-down cells, respectively). CC, Corpus callosum. *p = 0.0286, Mann–Whitney test. H, Scheme of the control and overexpressing retroviral constructs used to infect SEZ-derived newborn cells. I, Example of HEK cells infected with control (green) or S1P1 overexpression (red) viruses immunostained with anti-S1P1 antibodies (white), confirming the correct expression and membrane localization of the recombinant protein. Blue nuclei were stained with DAPI. J, Confocal image of a sagittal brain section from a mouse injected with the viral mixture in the SEZ and killed 6 d later. Enlargements show infected cells (green, control; red, S1P1 overexpression) outside (top) and within the RMS^OB (bottom). K, Quantification of control and S1P1-overexpressing cells within and outside the RMS^OB plotted as ratios between overexpression/control cells. A higher proportion of S1P1-overexpressing cells remained in the RMS^OB compared with control cells. n = 12 mice (11583 and 3063 control and overexpressing cells, respectively). ***p = 2.4E-5, t test. Scale bars, 50 μm.

Figure 3.

Proliferation and apoptosis of neuroblasts is unaltered after S1P1 knock-down. A, Experimental scheme. A mixture of control and S1P1-knock-down retroviruses was injected in the SEZ, followed by 2 intraperitoneal BrdU injections at 3 d.p.i. (with an interval of 6 h), and mice were killed at 15 d.p.i. B, Examples of virally infected neurons in the OB positive for the proliferative marker BrdU (white arrows). C, Quantification of control and S1P1-knock-down cells positive for BrdU shows no difference in proliferation between groups (n = 8, 1151 and 1323 control and knock-down cells, respectively). D, Experimental scheme. A mixture of control and S1P1-knock-down retroviruses was injected in the SEZ and the mice were killed at 3 or 6 d.p.i. E, Examples of virally infected neuroblasts in the RMS positive for the apoptotic marker Active Casp-3 (white arrows). F, Quantification of control and S1P1-knock-down cells positive for Active Casp-3 in the RMS^OB at 3 d.p.i. (left) and 6 d.p.i. (right) shows no difference in apoptosis between groups (n = 4239 and 1175 control and knock-down cells, respectively, p = 0.7 for 3 d.p.i., t test; n = 5231 and 773 control and knock-down cells, respectively, p = 0.29 for 6 d.p.i., t test. Scale bars, 50 μm.

Figure 4.

S1P1 knock-down triggers radial migration in the OB. A, Experimental scheme. A mixture of control and S1P1-knock-down retroviruses was injected in the SEZ and the mice were killed between 5 and 9 d.p.i. to perform time-lapse imaging in OB sections. B, Initial (time = 0) and final image (time = 351 min) from one video exemplifying different types of migration of labeled neuroblasts: tangential, detaching, and radial. Yellow and blue arrowheads indicate the starting and the final location, respectively. White arrows show the trajectory of the cells. C, Example of a S1P1-knock-down neuroblast migrating out of the RMS^OB. D, Quantification of the distance covered per hour for control and S1P1-knock-down neuroblasts while they migrate tangentially within the RMS^OB (n = 104 and 135 control and knock-down cells, respectively), detaching from the RMS^OB (n = 36 and 73 control and knock-down cells, respectively), or radially outside of the RMS^OB (n = 17 and 25 control and knock-down cells, respectively) shows no difference between cell types. E, The proportion of S1P1-knock-down cells (over control neuroblasts) shows a significant increase when comparing cells migrating tangentially and radially (n = 757 and 1053 control and knock-down cells, respectively, from 9 independent videos). ***p = 0.0009, t test. F, Experimental scheme. A mixture of control and S1P1-knock-down retroviruses was injected in the SEZ and mice were killed between 5 and 7 d.p.i. to perform time-lapse imaging in the RMS (area indicated with the square). G, Initial (time = 0) and final image (time = 235 min) from one video showing the trajectory of labeled neuroblasts. H, Quantification of the distance covered per hour for control and S1P1-knock-down neuroblasts while they migrate within the RMS (n = 80 and 56 control and knock-down cells, respectively) shows no difference between cell types. Scale bars, 50 μm.

Figure 5.

S1P1 regulates neuroblast cell adhesion. A, Experimental scheme. SEZ explants were plated on top of astrocytes and incubated with control medium, an agonist of S1P1 (CYM5442, 0.5 μm), or an antagonist of S1P1 (W146, 1 μm; top scheme in orange) or with either a control AAV-virus or an S1P1-overexpressing AAV-virus and plated in Matrigel (bottom scheme in red). Two to 4 d later, the explants were fixed and stained with a neuroblast marker. B, Representative pictures of fixed explants stained with anti-tuj-1 and treated with control medium, CYM5442, or W146. Bottom images are an enlargement of the area indicated in the top. C, D, Quantification of neuroblasts migrating out of the explants as single cells in control or treatment conditions. Activating S1P1 resulted in enhanced cell–cell adhesion, whereas blocking the receptor induced neuroblast detachment (n = 3148 and 3773 cells per group from 13–14 explants and 4870 and 3896 cells per group from 13–14 explants for C and D, respectively). *p = 0.01, **p = 0.0004, t test. E, Representative images from fixed explants incubated with control AAV-Tomato or AAV-S1P1-IRES-Tomato virus stained with anti-tuj-1 antibodies (white) and DAPI (blue). F, Quantification of cells migrating out of the explant as single cells for each group normalized to the control value (n = 4214 and 2707 cells per group from 19 to 20 explants). ***p = 1.E-9, t test. Scale bars, 50 μm.

Figure 6.

SK1/2 double knock-down in astrocytes reduces neuroblast cell adhesion. A, Confocal images of RMS and OB tissue sections showing the expression of SK1 (top, red) and SK2 (bottom, red) in astrocytes immunostained with GFAP (green). B, Top, Scheme of the plasmid constructs that were transfected to HEK cells. Bottom, Examples of cells cotransfected with the indicated plasmids and immunostained with anti-SK1 antibodies. C, Western blots with protein extracts from HEK cells cotransfected with SK2-IRES-Tomato and a plasmid expressing shRNA scrambled (first and second lanes), shRNA against SK2 (third and fourth lanes), shRNA against SK1 (fifth and sixth lanes), or shRNA against SK2 and SK1 (seventh and eighth lanes). Because SK2 and Tomato sequences are encoded in the same transcript, an shRNA targeting SK2 will also silence Tomato. D, Experimental scheme. Primary astrocytic cultures were infected with AAV control virus (shRNA scrambled + Tomato) or AAV double SK1/2 knock-down virus (+ Tomato). Six days later, SEZ explants were plated on top of the infected astrocytes and fixed after 3 d in vitro. E, Examples of explants plated on top of control (left) or double SK1/2 knock-down infected astrocytes (right), stained with a neuroblast marker (DCX in green) and anti-tomato (red). Bottom images are an enlargement of the area indicated in the top images. F, Quantification of the neuroblasts migrating out of the explant as single cells (shown as percentage) on top of noninfected astrocytes (black bars) or on top of infected astrocytes with either control or double SK1/2 knock-down virus (gray bars). Neuroblasts in contact with SK1/2 knock-down astrocytes tend to detach from each other more frequently than cells migrating on top of control astrocytes (n = 699, 3665, 2743, and 1317 neuroblasts per group, in the order shown in the graph). ***p < 0.0001, Kruskal–Wallis test. Scale bars, 50 μm.

Figure 7.

S1P1 genetic deletion in the RMS results in enhanced neuroblast dispersion in the OB. A, Experimental scheme. S1P1^lx/lx (control) and S1P1^lx/lx MashCRE^ERT2 mice were treated with tamoxifen twice per day for 5 d to delete S1P1 expression in the RMS. For one set of experiments, total RNA was extracted from the RMS and the OB and used as template for cDNA quantification. For another set of experiments, BrdU was injected at day 2, the mice were perfused, and brains were analyzed at day 6. B, Quantitative real-time PCR using RMS samples revealed similar expression levels of DCX (normalized to cyclophilin values), but reduced S1P1 expression (normalized to DCX values) in S1P1^loxP/loxP MashCRE^ERT2 compared with control mice (n = 4–8 mice per group). **p = 0.0044, Mann–Whitney test. C, Examples of BrdU stainings in sections containing the OB showing the width of the RMS^OB as indicated by the yellow bars. D, Quantification of the bulb size and the size of RMS^OB in control and S1P1^loxP/loxP MashCRE^ERT2 mice (n = 13 mice per group). **p = 0.001, t test.

Figure 8.

S1P1 deletion in the RMS downregulates the cell adhesion molecule NCAM1 and β1 integrin. A, NCAM1 and β1 integrin (Itgb1) expression levels (normalized to DCX values) were quantified in the RMS and OB of S1P1^lx/lx and S1P1 ^lx/lx MashCRE-ERT2 mice. In control mice, NCAM1 and Itgb1 expression levels were reduced in the OB compared with the RMS, whereas knock-out mice showed reduced levels of NCAM1 and Itgb1 both in the RMS and the OB (one-way ANOVA followed by Bonferroni's multiple-comparisons test). ***p < 0.0001; **p < 0.01. B, Immunostaining with anti-DCX (red) and anti-PSA-NCAM (green) antibodies in OB tissue sections showing a high expression of PSA-NCAM in neuroblasts migrating in chains (yellow arrowheads) and a low expression in neuroblasts migrating radially as single cells (white arrows). C, Experimental scheme. Mice were injected in the SEZ with a mixture of control and S1P1 knock-down virus and, 7 d later, cells migrating in the RMS at the level indicated with the square were analyzed by immunostainings with anti-PSA-NCAM antibodies. D, Example of a control-infected cell (red) positive for PSA-NCAM (white) is indicated by the orange arrowheads and examples of S1P1 knock-down infected cells (green) negative for PSA-NCAM (white) are pointed out by light blue arrowheads. Scale bars, 50 μm.

Figure 9.

Summary. A, Neuroblasts (green cells) migrating in chains in the RMS express high levels of S1P1, PSA-NCAM, and β1 integrin. S1P is secreted by the surrounding astrocytes (blue cells) and activates S1P1, resulting in strong cell adhesion. B, When neuroblasts reach the OB, they stop expressing S1P1, which leads to a downregulation of NCAM1 and β1 integrin and subsequent cell detachment from migrating chains. C, Neuroblasts lacking S1P1 and exhibiting low levels of PSA-NCAM and β1 integrin are able to initiate single-cell radial migration to eventually reach their final position in the OB.