Ependymal cells-CSF flow regulates stress-induced depression

Abstract

Major depressive disorder (MDD) is a severe, common mood disorder. While reduced cerebrospinal fluid (CSF) flow adversely affects brain metabolism and fluid balance in the aging population and during development, only indirect evidence links aberrant CSF circulation with many diseases including neurological, neurodegenerative, and psychiatric disorders, such as anxiety and depression. Here we show a very high concentration of p11 as a key molecular determinant for depression in ependymal cells, which is significantly decreased in patients with MDD, and in two mouse models of depression induced by chronic stress, such as restraint and social isolation. The loss of p11 in ependymal cells causes disoriented ependymal planar cell polarity (PCP), reduced CSF flow, and depression-like and anxiety-like behaviors. p11 intrinsically controls PCP core genes, which mediates CSF flow. Viral expression of p11 in ependymal cells specifically rescues the pathophysiological and behavioral deficits caused by loss of p11. Taken together, our results identify a new role and a key molecular determinant for ependymal cell-driven CSF flow in mood disorders and suggest a novel strategy for development of treatments for stress-associated neurological, neurodegenerative, and psychiatric disorders.

Fig. 1. Enrichment of p11 in ependymal cells from mice and humans.

a Immunofluorescence image illustrating p11-positive cells (EGFP⁺) in the lateral ventricle (LV) and the choroid plexus (ChP) from p11-promoter-driven EGFP (p11-EGFP) mice. Scale bar, 100 μm. b Representative image obtained by immuno-electron microscopy using p11 antibody (p11 IEM) illustrating expression of p11 (dots) in ependymal cells in the ventricular–subventricular zone (V–SVZ) of lateral ventricle. VL, ventricle lumen; EL, ependymal cell layer; BP, brain parenchyma; EP, ependymal cells; CSF, cerebrospinal fluid; Nu, nucleus; Mt, mitochondria; Mv, microvilli. Scale bar, 2 μm. c Human p11 (green) expression in ependymal cells in the V–SVZ of lateral ventricle. Scale bar, 10 μm. d Co-localization of ependymal p11 (green, mouse p11-EGFP⁺, up; and human p11, down) with S100β (blue), as ependymal cell marker in the V–SVZ of lateral ventricle from mice and humans. Scale bars, 10 μm, up; and 5 μm, down. e Co-expression of p11 fluorescence in situ hybridization (yellow; mouse p11-FISH, up; and human p11-FISH, down) with FoxJ1 (red) and S100β (blue), ependymal cell markers in the V–SVZ of the lateral ventricle. Scale bar, 30 μm. f Immunofluorescence image of EGFP⁺ cells from ependymal cell bacTRAP (Ep; Dcdc2a-L10a/EGFP) mice. Scale bar, 500 μm. g, h TRAP and RNAseq analysis of cell type-specific translated mRNA expression in ependymal cell bacTRAP (Ep; Dcdc2a-L10a/EGFP) mice, striatal dopamine D1 receptor-expressing neuron-bacTRAP (Drd1; Drd1-L10a/EGFP) mice, and cholinergic neuron-bacTRAP (Chat; ChAT-L10a/EGFP) mice, visualized by Integrative Genomics Viewer (IGV 2.3). Expression of ependymal cell markers (FoxJ1, S100β, Dcdc2a, and Tppp3, g), p11 and p11 binding partners (AnxA2, Smarca3 and Ahnak1, h) in those cells from the bacTRAP mouse lines.

Fig. 2. Chronic stress decreases p11 expression in ependymal cells and reduces CSF flow.

a Immunofluorescence images illustrating p11-EGFP-positive ependymal cells in the V–SVZ of lateral ventricle from control (CON) and chronic restraint stressed (RST) mice with or without antidepressants. Imi; imipramine, Flu; fluoxetine, Esci; escitalopram. Scale bar, 5 μm. Quantification of p11 expression (EGFP⁺) in ependymal cells from those mice (n = 8 for CON, n = 5 for each group of RST, RST + Imi, RST + Flu, RST + Esci). b Expression of p11 mRNA in LV ependymal cells TRAP and qPCR measurements from CON, RST and RST + Flu ependymal cell bacTRAP mice (n = 10 for CON, n = 12 for each group of RST and RST + Flu). c Ependymal p11 expression in the V–SVZ of lateral ventricle and quantification from patients with major depressive disorder (MDD) and unaffected control (CON) from postmortem brain sections (n = 15 for each group). CSF, cerebrospinal fluid; EP, ependymal cells; BP, brain parenchyma. Scale bar, 10 μm. See Supplementary Table S1 for the detailed sample information. d Magnetic resonance imaging (MRI) images represent CSF flow in the cerebral aqueduct (Aq) from control and stressed mice. Scale bar, 850 μm. Quantification of CSF flow from the control and stressed mice (n = 7 for each group). ^#P < 0.05, compared to CON; *P < 0.05 and **P < 0.01, compared to RST, ANOVA test. *P < 0.05 and **P < 0.01, Student’s t-test. Data are mean ± s.e.m.

Fig. 3. p11 regulates ependymal cell planar cell polarity and CSF flow.

a Scatter plot displaying ependymal cell translational profiling in the lateral ventricle (LV) from WT (Dcdcd2a-L10a/EGFP) and p11 KO (p11 KO x Dcdc2a-L10a/EGFP) bacTRAP mice (n = 12, WT; n = 8, p11 KO). See Supplementary Table S2 for numerical data. b Rank-based gene set enrichment of selected planar cell polarity (PCP)-related processes on p11-regulated LV ependymal cell gene differential expression, presented as z-score (positive z-scores indicate upregulation of processes in WT compared to p11 KO). See Supplementary Fig. S7 and Supplementary Tables S3 and S4 for numerical data. c The LV ependymal PCP core genes functional interaction network with p11; only functional interactions with larger than three fold over random are shown. See Supplementary Table S5 for additional data compendium used to build the network. d Expression of PCP gene mRNA in the ependymal cell-specific TRAP profiling from WT and p11 KO bacTRAP mice (n = 10, WT; n = 16, p11 KO). e Scanning electron microscopy (SEM) images illustrating ependymal cell PCP orientation in the V–SVZ of lateral ventricle from WT and p11 KO mice. Quantification of multidirectional cilia orientation in LV ependymal cells from WT and p11 KO mice (n = 20 cells from 3 mice in each group). BB, basal body (arrowhead); BF, basal foot (arrow). Scale bar, 2 μm. f Transmission electron microscopy (TEM) images illustrating bosal body and basal foot orientation in ependymal cells in the V–SVZ of lateral ventricle from WT and p11 KO mice. g MRI images represent CSF flow and quantification of CSF flow from p11 KO mice and WT littermates (n = 7 for each group). Scale bar, 850 μm. **P < 0.01, ***P < 0.001, Student’s t-test. Data are mean ± s.e.m.

Fig. 4. Overexpression of p11 in ependymal cells from depressive mice ameliorates disrupted PCP and CSF flow, as well as depression-like behaviors.

a SEM images and quantification of multidirectional cilia orientation in ependymal cells in the V–SVZ of lateral ventricle from conditional p11 knockout (p11 cKO; Tppp3-Cre x p11^f/f) mice with Cre-dependent viral overexpression of AAV1-DIO-p11 (AAV_p11) or AAV1-DIO-eYFP (AAV_eYFP) into intracerebroventricularly (n = 11 for each group). Scale bar, 2 μm. b MRI images and quantification of CSF flow from p11 cKO with the expression of AAV_p11 or AAV_eYFP in ependymal cells (n = 7, p11 cKO+AAV_eYFP; n = 8, p11 cKO+AAV_p11). Scale bar, 850 μm. c–f Depression- and anxiety-like behaviors in WT and p11 cKO mice with viral overexpression of AAV_p11 in ependymal cells, as measured by immobility time in tail suspension test (TST, c), forced swim test (FST, d), the latency to feed in novelty suppressed feeding test (NSF, e), and the total travel distance in locomotor activity test (f) (n = 10, WT + AAV_eYFP and WT + AAV_p11; n = 14, p11 cKO+AAV_eYFP and p11 cKO+AAV_p11). g Quantification of CSF flow from constitutive p11 KO crossed to Tppp3-Cre [p11 KO (Tppp3); p11 KO x Tppp3-Cre] mice with viral overexpression of AAV_p11 or AAV_eYFP into intracerebroventricularly. h–f Depression-like behaviors in p11 KO (Tppp3) mice with viral overexpression of AAV_p11 or AAV_eYFP in ependymal cells, as measured by TST (h), FST(i), NSF(j), and locomotor activity (k) (g–k, n = 8, p11 KO (Tppp3)+AAV_eYFP; n = 9, p11 KO (Tppp3)+AAV_p11). l Quantification of CSF flow from chronic stressed Tppp3-Cre [RST(Tppp3)] mice with viral overexpression of AAV_p11 or AAV_eYFP into intracerebroventricularly. (n = 5, RST(Tppp3)+AAV_eYFP; n = 8, RST(Tppp3)+AAV_p11). m–p Depression-like behaviors in WT and p11 KO (Tppp3) mice with viral overexpression of AAV_p11 in ependymal cells, as measured by TST (m), FST(n), NSF (o), and locomotor activity (p) (n = 8, RST(Tppp3)+AAV_eYFP; n = 7, RST(Tppp3)+AAV_p11). *P < 0.05 and ***P < 0.001, Student’s t-test. *P < 0.05 and **P < 0.01, ANOVA test. Data are mean ± s.e.m.