RTP801/REDD1 regulates the timing of cortical neurogenesis and neuron migration

Abstract

The generation, differentiation, and migration of newborn neurons are critical features of normal brain development that are subject to both extracellular and intracellular regulation. However, the means of such control are only partially understood. Here, we show that expression of RTP801/REDD1, an inhibitor of mTOR (mammalian target of rapamycin) activation, is regulated during neuronal differentiation and that RTP801 functions to influence the timing of both neurogenesis and neuron migration. RTP801 levels are high in embryonic cortical neuroprogenitors, diminished in newborn neurons, and low in mature neurons. Knockdown of RTP801 in vitro and in vivo accelerates cell cycle exit by neuroprogenitors and their differentiation into neurons. It also disrupts migration of rat newborn neurons to the cortical plate and results in the ectopic localization of mature neurons. On the other hand, RTP801 overexpression delays neuronal differentiation. These findings suggest that endogenous RTP801 plays an essential role in temporal control of cortical development and in cortical patterning.

Figure 1.

RTP801 is rapidly and transiently induced with NGF exposure and regulates neurite outgrowth by PC12 cells via TSC2 and mTOR signaling. A, PC12 cells were exposed to NGF for the indicated times, and cell extracts were analyzed by Western immunoblotting for RTP801 and ERK1 [loading control (Ctr)]. B, PC12 cells were transfected with pSIREN-ShLuciferase, pSIREN-ShRTP801, pCMS eGFP, or pCMS-eGFP RTP801 and exposed to NGF 2 d later. After 3 d, cultures were fixed and stained with red fluorescent protein (DsRed) or eGFP antibodies to identify transfected cells. Fluorescence photomicrographs of cultures are shown on the left. Scale bar, 10 μm. Graphs on the right indicate the proportions of transfected cells with neurites in each condition. Values are means ± SEM of at least three independent experiments. Student's t test: *p < 0.01 vs pSIREN ShLuciferase; **p < 0.01 vs pCMSeGFP. C, PC12 cells were transfected with pSIREN-ShLuciferase or pSIREN-ShTSC2 and exposed to NGF for 3 and 6 d. Cultures were then fixed and stained with DsRed antibody to identify transfected cells. Fluorescence photomicrographs of cultures are shown on the left. Scale bar, 10 μm. Graphs below indicate the proportions of transfected cells with processes in each condition. Values are means ± SEM of at least three independent experiments. Student's t test: **p < 0.01 vs pSIREN ShLuciferase; ***p < 0.001 vs pSIREN ShLuciferase. D, PC12 cells were transfected with control (ShG) or RTP801-4 shRNA. At 48 h posttransfection, cultures were exposed to NGF with or without 1 μm rapamycin for 3 d. The graph shows the proportion of transfected cells bearing processes in each condition. *p < 0.05 vs Ctl ShG, ^$p < 0.05 vs Ctl ShG, ⁺⁺⁺p < 0.001 vs Ctl ShRTP801 4.

Figure 2.

Temporal expression of various differentiation markers and of RTP801 in plated neurosphere cultures. A, Time course of neural stem cell differentiation after plating on an adherent substrate. After disaggregation and plating, murine hippocampal neurosphere cultures were stained at the indicated times with specific antibodies to detect undifferentiated neuroprogenitor/neural stem cells (nestin), neurons (β-III tubulin), astrocytes (GFAP), and oligodendrocytes (O1). Proliferating cells were also visualized by incubating with and immunostaining for bromodeoxyuridine (BrdU). B, RTP801 is transiently induced in plated neurosphere cultures. Extracts from floating neurospheres (NS), disaggregated neurospheres (dNS) (as a control for mechanical stress), and neurospheres that were disaggregated and plated for the indicated times were analyzed by Western immunoblotting for RTP801 expression. Membrane was reprobed against Erk1/2 as a loading control. C, RTP801 is present at high levels in neuroprogenitor cells and newborn neurons, but not in astroglia or mature neurons in plated neurosphere cultures. Plated neurospheres cultures were subjected to fluorescence immunostaining for RTP801 and the indicated markers 1 and 8 d after disaggregation and plating. Hoechst dye 33242 was used to stain nuclei. Scale bars: top, 10 μm; bottom, 20 μm.

Figure 3.

Knockdown of RTP801 in plated neurosphere cultures accelerates cell cycle exit and neuronal differentiation of neuroprogenitor cells. Floating neurospheres were transfected with pSiren-shRTP801 or with control vector pSiren-shLuciferase and 2 d later were disaggregated and plated on an adherent substrate. At 1, 3, and 7 d after plating, cultures were fixed and immunostained for expression of nestin, Ki67 antigen, β-III tubulin, GFAP, and DsRed. Immunostained cultures were scored under fluorescence microscopy for proportions of transfected cells (DsRed⁺) expressing the various markers. A, Fluorescence photomicrographs illustrating that RTP801 knockdown diminishes nestin expression in neurosphere cultures 1 d after plating. Arrows show locations of transfected cells. Scale bar, 20 μm. B–E, Quantification of marker expression by control and RTP801 knockdown cells. RTP801 knockdown accelerates loss of nestin (B) and Ki67 antigen (C) expression at 1 and 3 d postplating and, at 7 d, increases the proportions of cells expressing the neuronal marker β-III tubulin (D) and the glial marker GFAP (E). F, Transfection efficiency for the two vectors determined at 1 and 3 d after plating. Values are expressed as means ± SEM of at least three independent experiments done in triplicate. Student's t test: ***p < 0.001 vs nestin/ShLuc 24 h, *p < 0.05 vs nestin/ShLuc 3 d; ***p < 0.001 vs Ki67/ShLuc 24 h, *p < 0.05 vs Ki67/ShLuc 3 d; ***p < 0.001 all vs β-III tubulin/ShLuc; ***p < 0.001 vs GFAP/ShLuc.

Figure 4.

Expression of RTP801 in embryonic mouse brain. Sections (12 μm, sagittal) of fixed E14.5 and E16.5 mouse brains were immunostained for RTP801 (green), nestin (red) or Ki67 antigen (also in red), all as described in Materials and Methods. A–C, RTP801 is highly expressed in the E14.5 VZ along with nestin and shows progressively reduced expression outside of the VZ. V, Ventricle; PS, pial surface. D–I, Higher-power view of RTP801 and nestin expression in the VZ (D–F) and CP (G–I). J–L, RTP801 is expressed in Ki67⁺ cells in the E14.5 periventricular region. M–O, RTP801 is coexpressed with nestin in the E16.5 ventricular zone. V, Ventricle. P–R, Low expression of RTP801 and absence of nestin expression near the pial surface (PS). Immunostaining conditions and image acquisition were identical in D–I and M–R. Scale bars: A–C, 50 μm; D–R, 20 μm. S–U, RTP801 is coexpressed with β-III tubulin in the periventricular zone of the developing cerebral cortex. Sagittal sections (12 mm thick) of E16.5 murine embryo cerebral cortex were fixed and stained with RTP801 and β-III tubulin antiserum. Arrows indicate examples of costained cells. Scale bar, 20 μm. V, Ventricle. CP indicates direction of the cortical plate.

Figure 5.

Knockdown of RTP801 in developing rat brain impairs neuron migration to the cortical plate and induces precocious neuronal differentiation. E17 rat brains were electroporated with the indicated constructs, harvested at E20 and processed as in Materials and Methods for immunostaining. A, RTP801 knockdown affects migration of electroporated cells in the developing cerebral cortex. Panels show immunofluorescence micrographs (stained for eGFP) of brains electroporated with control or RTP801 shRNAs. The approximate positions of the various zones of the developing cortex are shown on the left. Scale bar, 50 μm. B, RTP801 knockdown impairs cell migration into the CP. Graph shows proportion (normalized as percentage) of total electroporated cells that are present in each zone. Values are means ± SEM of counts from at least three different embryos per condition. Student's t test:**p < 0.01 vs ShG at SVZ, ⁺⁺p < 0.01 vs ShG at CP. C, Knockdown of RTP801 affects the morphology of cells in the IZ. E17 rat brains were electroporated with ShG (control shRNA) or ShRTP801-4, harvested at E20, and processed as in Materials and Methods for eGFP immunostaining. Arrows indicate examples of cells electroporated with shRTP801-4 exhibiting a multipolar morphology. Such cells were absent in the IZ of brains electroporated with the control construct. Scale bar, 20 μm. D, Knockdown of RTP801 impairs neuron migration and causes ectopic localization of NeuN⁺ neurons in the developing cerebral cortex. Sections were immunostained for NeuN (red) and eGFP (green). Fluorescent micrographs show that double-stained cells are absent from the CP and ectopically present in the IZ of brains electroporated with RTP801 shRNAs. Scale bars: top, 20 μm; bottom, 10 μm. E, Quantification of the effect of RTP801 knockdown on neuron migration. Graph shows proportion (normalized as percentages) of eGFP⁺ cells in each zone that are also NeuN⁺. Values are means ± SEM of counts from at least three different embryos per condition. Student's t test: ***p < 0.001 vs ShG at the SVZ; *p < 0.05 and **p < 0.01 vs ShG at the IZ.

Figure 6.

Knockdown of RTP801 in developing rat cerebral cortex causes premature cell cycle exit of neuroprogenitor cells while RTP801 overexpression results in ectopic localization of mitotically active cells. A–C, E17 rat brains were electroporated with the indicated constructs, harvested at E20 and processed as in Materials and Methods for immunostaining. A, Knockdown of RTP801 accelerates cell cycle exit in the VZ. Sections of electroporated brains were immunostained for eGFP and phospho-histone 3 (P-H3), an M-phase marker. Graph shows proportions (normalized as percentages) of eGFP⁺ cells in each zone that were also P-H3⁺. Values are mean ± SEM of data from at least three different embryos per condition. Student's t test: *p < 0.05 vs ShG at VZ. B, RTP801 knockdown accelerates loss of cells from the VZ with the progenitor marker BLBP. Left panel shows fluorescence photomicrographs of the SVZ (coronal sections) in brains electroporated with constructs expressing control and RTP801 shRNA and immunostained for eGFP and BLBP. Scale bar, 20 μm. Arrows indicate examples of electroporated cells that are negative for BLBP staining. Right panel shows quantification of proportions (normalized as percentages) of eGFP⁺ cells in each zone that were also BLBP⁺. Values are mean ± SEM of data from at least three different embryos per condition. Student's t test: **p < 0.01 vs ShG at VZ. C, Knockdown of RTP801 accelerates neuronal differentiation. Graph shows proportion (normalized to percentage) of eGFP⁺ cells per electroporated brain that were also NeuN⁺. Values are mean ± SEM of data from at least three different embryos per condition. Student's t test: **p < 0.01 vs ShG. D, RTP801 overexpression causes ectopic localization of mitotic cells. Rat embryos were electroporated at E17 with either empty vector pCMSeGFP or with pCMSeGFP-RTP801. At E18, coronal sections were evaluated by immunohistochemistry for expression of phospho-histone 3 (red) and eGFP (green). Fluorescence micrographs show examples (depicted by arrows) of electroporated cells in the VZ/SVZ that are positive for phospho-histone 3. Asterisks mark autofluorescent blood and/or endothelial cells. Scale bar, 20 μm. E, Distribution of electroporated cells among the various zones for brains electroporated with either control or RTP801 constructs. Values are mean ± SEM of data from at least three different embryos per condition. F, Proportions (normalized to percentages) of eGFP⁺ cells in each zone that were also P-H3. Values are mean ± SEM of data from at least three different embryos per condition. Student's t test: *p < 0.05 vs ShG at SVZ and **p < 0.01 vs ShG at the IZ + CP.

Figure 7.

RTP801 regulates phosphorylation of the mTOR signaling target ribosomal protein S6 in the developing rat cerebral cortex. E17 rat brains were electroporated with the indicated constructs, harvested at E20 and processed as in Materials and Methods for immunostaining. A, Fluorescence micrographs of coronal brain sections comparing phospho-S6 staining (red) in cells electroporated (eGFP, green) with either control or RTP801 vectors (top) or with control or RT801 shRNA vectors (bottom). Arrows show examples of electroporated cells positive for phospho-S6. Asterisks show examples of electroporated cells with no detectable staining for phospho-S6. Scale bar, 20 μm. Ventricle (V) is to the right. B, RTP801 overexpression decreases expression of phospho-S6. Brain sections were scored for percentage of electroporated cells in VZ and SVZ that were positive for phospho-S6. C, RTP801 knockdown increases expression of phospho-S6. Brain sections were scored for percentage of electroporated cells in VZ and SVZ that were positive for phospho-S6. Values in B and C are mean ± SEM of data from at least two different embryos per condition. Statistics are based on Student's t test. B, *p < 0.05 vs pCMSeGFP at the VZ; C, *p < 0.05 and **p < 0.01 vs ShG at the VZ; ⁺p < 0.05 vs ShG at the SVZ.