Caveolin-1 Autonomously Regulates Hippocampal Neurogenesis Via Mitochondrial Dynamics

Abstract

The mechanisms underlying adult hippocampal neurogenesis (AHN) are not fully understood. AHN plays instrumental roles in learning and memory. Understanding the signals that regulate AHN has implications for brain function and therapy. Here we show that Caveolin-1 (Cav-1), a protein that is highly enriched in endothelial cells and the principal component of caveolae, autonomously regulates AHN. Conditional deletion of Cav-1 in adult neural progenitor cells (nestin +) led to increased neurogenesis and enhanced performance of mice in contextual discrimination. Proteomic analysis revealed that Cav-1 plays a role in mitochondrial pathways in neural progenitor cells. Importantly, Cav-1 was localized to the mitochondria in neural progenitor cells and modulated mitochondrial fission-fusion, a critical process in neurogenesis. These results suggest that Cav-1 is a novel regulator of AHN and underscore the impact of AHN on cognition.

Keywords: Adult Hippocampal Neurogenesis; Caveolin-1; Mitochondria Dynamics; Neural Stem Cells; Neuronal Differentiation.

Figure 1.. Characterization of Cav-1 expression in adult hippocampal NSPC.

(A) Expression of Cav-1 in WT hippocampal NSPC shown by confocal imaging of Cav-1 (green), Nestin (red), and DAPI (blue). Scale bar, 20 μm. (B-C) Correlation plot of Cav-1 fluorescence intensity per cell by nestin signal intensity per cell (B) and nestin area per cell (C). Each dot represents the fluorescence intensity of an individual cell analyzed from WT mice (n=26 cells). (D) Schematic of NestinCre^ERT2/+;Cav-1^fl/fl mice, where mice were injected at 4-5 weeks of age with either corn oil (Corn) or tamoxifen (TAM) for 5 consecutive days to generate control mice (iNSC Cav-1 WT) and Cav-1 knockout mice (iNSC Cav-1 KO), respectively. (E) RT-qPCR quantification of Cav-1 transcript expression from NSPC isolated from the iNSC Cav-1 KO and iNSC Cav-1 WT mice. (F-G) Cav-1 immunoblot and quantification normalized to β-actin of protein lysate from NSPC isolated from the iNSC Cav-1 KO and iNSC Cav-1 WT mice. (H) Immunocytochemistry of Cav-1 and confocal imaging of Cav-1 (green) and DAPI (blue) in hippocampal NSPC isolated from iNSC Cav-1 WT and iNSC Cav-1 KO mice shown. Scale bar, 20 μm. (I) Ultrastructural electron micrographs of caveolae and clathrin coated vesicles in hippocampal NSPC isolated from iNSC Cav-1 WT and iNSC Cav-1 KO mice. Solid green arrowhead indicate caveolae. Open green arrowhead indicate clathrin coated vesicle. Scale bar, 200 nm. Data represented as mean ± SEM. Data analyzed by Spearman Correlation Analysis (B-C) **p < 0.01 and unpaired two-tailed Student’s t-test (D-G). **p < 0.01, ****p < 0.0001.

Figure 2.. iNSC Cav-1 KO mice have reduced levels of proliferating NSC in the dentate gyrus.

(A) Strategy to quantify NSC populations in dentate gyrus (DG) of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 3 and 6 months of age. (B) Representative confocal images of GFAP (white), Nestin (red), MCM2 (green) and DAPI (blue) markers in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 3 months of age. Yellow arrowheads indicate GFAP⁺Nestin⁺MCM2⁺ cells. Scale bar, 25 μm. (C-E) Quantification of total NSC (GFAP⁺Nestin⁺), quiescent NSC (GFAP⁺Nestin⁺MCM2⁻) and proliferating NSC (GFAP⁺Nestin⁺MCM2⁺) in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 3 months of age. n=5 mice per group. (F) Representative confocal images of GFAP (white), Nestin (red), MCM2 (green) and DAPI (blue) positive cells in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 3 months of age. Yellow arrowheads indicate GFAP⁺Nestin⁺MCM2⁺ cells. Scale bar, 25 μm. (G-I) Quantification of total NSC (GFAP⁺Nestin⁺), quiescent NSC (GFAP⁺Nestin⁺MCM2⁻) and proliferating NSC (GFAP⁺Nestin⁺MCM2⁺) in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 6 months of age. n=5 mice per group. (J-M) Clonogenic proliferation assay in NSPC isolated from iNSC Cav-1 WT and iNSC Cav-1 KO mice. The number of clones (neurospheres) (L), average clone diameter (M) and number of cells after dissociation of clones (N) on day 5 of the assay were quantified per well. Scale bar, 100 μm. (N-P) EdU uptake assay in NSPC isolated from iNSC Cav-1 WT and iNSC Cav-1 KO mice. (O) Quantification of the percentage of EdU+ cells to the total DAPI between NSPC isolated from iNSC Cav-1 WT and iNSC Cav-1 KO mice. (P) EdU fluorescence intensity per DAPI nuclei between NSPC isolated from iNSC Cav-1 WT and iNSC Cav-1 KO mice. Scale bar, 20 μm. Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. ns p>0.05, *p< 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.

Figure 3.. Cav-1 regulates differentiation of hippocampal NSPCs.

(A) Confocal images of DCX and NeuN immunostaining in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 6 months of age. Scale bar, 25 μm. (B-D) Quantification of NPCs and neuroblasts (DCX⁺NeuN⁻) and immature neurons (DCX+NeuN+) and total DCX expressing cells in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice. n=4 mice per group. (E) Schematic representation of the protocol used for neural differentiation of primary hippocampal NSPCs. (F-H) Quantification of Cav-1, Nestin, and MAP2 transcript expression by RT-qPCR between iNSC Cav-1 WT and iNSC Cav-1 KO NSPCs undergoing differentiation for 5 days. Expression level normalized to d0 of iNSC Cav-1 WT NSPC (n=3 replicates). (I) Immunoblot of Cav-1, Sox2 and β-III-Tubulin in NSPC isolated from iNSC Cav-1 KO and iNSC Cav-1 WT mice undergoing differentiation for 7 days. (J-L) Quantification of Cav-1, Sox2 and β-III-Tubulin normalized to GAPDH expression and then normalized to d0 iNSC Cav-1 WT NSPC (n=3 replicates). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test (B-D) and two-way ANOVA with Tukey multiple comparisons correction (F-G and J-L). ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 4.. Deletion of Cav-1 in NSC causes premature differentiation of newborn neurons in the dentate gyrus.

(A) 5-bromo-2'-deoxyuridine (BrdU) pulse strategy to quantify changes in NSC fate and differentiation in DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 6 months of age. Mice were injected daily for 12 consecutive days and sacrificed 24 hr after the last injection. (B-C) Confocal images of BrdU immunostaining and quantification total number of BrdU⁺ cells in the DG of 6-month-old iNSC Cav-1 KO and iNSC Cav-1 WT mice. Scale bar, 25 μm. n=4 mice per group. (D) Representative confocal images of BrdU (red), Sox2 (white), GFAP (green) and DAPI (blue) markers in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 6 months of age. Solid yellow arrowheads indicate BrdU⁺GFAP⁺Sox2⁺ cells. Outlined yellow arrowheads indicate BrdU⁺GFAP⁻Sox2⁻ cells. Scale bar, 25 μm. (E-G) Quantification of total BrdU⁺ NSCs (GFAP⁺Sox2⁺), BrdU⁺ NPCs (GFAP⁺Sox2⁻) and percentage of BrdU⁺ cell phenotypes normalized to total number of BrdU ⁺ cells in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice. n=4 mice per group. (H) Representative confocal images of BrdU (white), DCX (Green), NeuN (red) and DAPI (blue) markers in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice at 6 months of age. Outlined yellow arrowheads indicate BrdU⁺DCX⁺NeuN⁺ cells. Scale bar, 25 μm. (I-K) Quantification of BrdU⁺ DCX expressing NPCs and neuroblasts (DCX⁺NeuN⁻), immature neurons (DCX+NeuN+), and percentage of BrdU⁺ immature neurons (DCX+NeuN+) normalized to total number of BrdU + cells in the DG of iNSC Cav-1 WT and iNSC Cav-1 KO mice. n=4 mice per group. Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test, except (G) which was analyzed by two-way ANOVA with Tukey multiple comparisons correction. ns p > 0.05, *p < 0.05, and **p < 0.01.

Figure 5.. iNSC Cav-1 KO mice display enhanced contextual discrimination learning and memory.

(A) Schematic of contextual fear discrimination paradigm between Context A and B. See Materials and methods for details. (B-C) Quantification of percent freeze (30 min post-shock) and discrimination index in context A and B on Day 2 between the NSC Cav-1 WT and iNSC Cav-1 KO mice. n=12 per genotype. (D-E) Quantification of percent freeze (24 hr post-shock) and discrimination index in context A and B on Day 3 between the NSC Cav-1 WT and iNSC Cav-1 KO mice. n=12 per genotype. (F) Schematic of contextual fear generalization paradigm between Context A and C. See Materials and methods for details. (G-H) Quantification of percent freeze (30 min post-shock) and discrimination index in context A and C on Day 2 between the NSC Cav-1 WT and iNSC Cav-1 KO mice. n=12 iNSC Cav-1 WT and n=8 iNSC Cav-1 KO. (H-I) Quantification of percent freeze (24 hr post-shock) and discrimination index in context A and C on Day 3 between the NSC Cav-1 WT and iNSC Cav-1 KO mice. n=12 iNSC Cav-1 WT and n=8 iNSC Cav-1 KO. Data represented as mean ± SEM. Data analyzed in by two-way ANOVA with Tukey’s multiple comparisons correction expect for data in C,E,H,J which was analyzed by unpaired two-tailed Student’s t-test. ns p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 6.. Expression of mitochondria and metabolism related proteins are altered in hippocampal NSPCs lacking Cav-1.

(A) Schematic of proteomic workflow for hippocampal NSPCs isolated from iNSC Cav-1 KO and iNSC Cav-1 WT mice. See Materials and methods for details. (B) Volcano plot of proteins identified in iNSC Cav-1 KO vs iNSC Cav-1 WT cells. Significantly upregulated proteins in red and significantly downregulated proteins in blue. Data represented as Log₂FoldChange (FC) of mean abundance of proteins normalized to iNSC Cav-1 WT (n=3 iNSC Cav-1 KO and n=3 iNSC Cav-1 WT). DEP determined by a one-way ANOVA with a p-value of < 0.05. (C) Bar chart showing the top 10 Ingenuity Pathway Analysis (IPA) altered pathways in the iNSC Cav-1 KO compared to iNSC Cav-1 WT hippocampal NSPCs (ANOVA, p < 0.05). (D) Cytoscape cluster mapping of altered GO pathways in hippocampal NSPCs isolated from iNSC Cav-1 KO and iNSC Cav-1 WT mice. Analysis based on Log₂FC ratio of DEPs with a significant cut off value of p < 0.05. (E) Weighted Venn diagram depicting overlap of DEP with genes encoding proteins localizing to mitochondria based on Mitocarta 3.0 curation. (F) Volcano plot and (G) Table of Mitocarta 3.0 proteins identified in iNSC Cav-1 KO vs iNSC Cav-1 WT cells. Metabolism related protein in orange; Protein Import, Sorting & Homeostasis in green; Signaling and Molecule Transport in blue, Mitochondria Central Dogma in purple; and Mitochondria Dynamics and Surveillance in pink.

Figure 7.. Cav-1 regulates mitochondrial morphology and dynamics in hippocampal NSPC.

(A) Representative confocal image of Caveolin-1 (Cav-1, Green), Mitochondrial import receptor subunit TOM20 homolog (Tom-20, green) and DAPI (blue) immunostaining in WT hippocampal NSPCs. Scale bar 10 μm. Mander’s Correlation Coefficient of co-localization of Cav-1 with Tom-20 determined with ImageJ. (B) Immunoblot of Cav-1, Dynamin-related protein 1 (Drp-1) and Tom-20 in total cell lysate, cytosol fraction and mitochondrial enriched fraction of WT hippocampal NSPCs. Data represented as mean ± SEM. (C) Immunoblot of Drp-1, mitofusion-2 (Mfn-2) and Tom-20 in total cell lysate (TC), cytosol fraction (Cyto) and mitochondrial enriched fraction (Mito) of iNSC Cav-1 WT and iNSC Cav-1 KO hippocampal NSPCs. (D) Normalization of iNSC Cav-1 KO Drp-1 and Mfn-2 in cytosol enriched fraction to iNSC Cav-1 WT expression. (E) Normalization of iNSC Cav-1 KO Drp-1 and Mfn-2 expression in mitochondrial enriched fraction to iNSC Cav-1 WT expression. Data representative of n=1 T-75 flask of NSPC per genotype. (F) Representative transmission electron microscopy (TEM) images of NSPCs isolated from iNSC Cav-1 KO and iNSC Cav-1 WT mice. Mitochondria are shaded in green; scale bar = 1 μm with zoomed image scale bar = 500 nm. (G) Representative live-cell images of TMRM staining in NSPCs isolated from iNSC Cav-1 KO and iNSC Cav-1 WT mice. NSPCs were incubated with 50 nM TMRM for 30 min followed by confocal microcopy visualization. Mitochondria were skeletonized in ImageJ. Scale bar, 10 μm. (H-J) Quantification of mitochondria area per cell, mitochondria perimeter per cell, and circularity per cell in iNSC Cav-1 WT and iNSC Cav-1 KO NSCPs. N=25 cells per group. (K) Quantification of skeletonized mitochondria branch length per mitochondria in iNSC Cav-1 WT and iNSC Cav-1 KO NSCPs. N=25 cells per group. (L) Quantification of TMRM fluorescence in iNSC Cav-1 WT and iNSC Cav-1 KO NSPC. N=25 cells per group. (M) Representative images of mitochondria trafficking per minute using Trackmate in iNSC Cav-1 WT and iNSC Cav-1 KO NSPCs. Scale bar 10 μm. (N) Quantification of mitochondria velocity. n=10 cells per genotype with n=948 particles (mitochondria) for iNSC Cav-1 WT and n=1259 particles (mitochondria) for iNSC Cav-1 KO analyzed. (O) Quantification of mitochondria total distanced traveled. n=10 cells per genotype with n=948 particles (mitochondria) for iNSC Cav-1 WT and n=1259 particles (mitochondria) for iNSC Cav-1 KO analyzed. Data represented as mean ± SEM. Data analyzed in by unpaired two-tailed Student’s t-test except in (N,O) was analyzed by two-way ANOVA with Tukey’s multiple comparisons correction. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.