Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus

Abstract

Congenital hydrocephalus (CH), occurring in approximately 1/1,000 live births, represents an important clinical challenge due to the limited knowledge of underlying molecular mechanisms. The discovery of novel CH genes is thus essential to shed light on the intricate processes responsible for ventricular dilatation in CH. Here, we identify FLVCR1 (feline leukemia virus subgroup C receptor 1) as a gene responsible for a severe form of CH in humans and mice. Mechanistically, our data reveal that the full-length isoform encoded by the FLVCR1 gene, FLVCR1a, interacts with the IP3R3-VDAC complex located on mitochondria-associated membranes (MAMs) that controls mitochondrial calcium handling. Loss of Flvcr1a in mouse neural progenitor cells (NPCs) affects mitochondrial calcium levels and energy metabolism, leading to defective cortical neurogenesis and brain ventricle enlargement. These data point to defective NPCs calcium handling and metabolic activity as one of the pathogenetic mechanisms driving CH.

Graphical abstract

Figure 1

FLVCR1, responsible for CH in humans, is highly expressed by NPCs during development (A) Sonographic examinations of 32 + 4 weeks of pregnancy showing extreme microcephaly with anechoic skull and no evidence of cerebral tissue in a human fetus carrying the c.160delC, p.Arg54GlyfsTer59 mutation in the FLVCR1 gene. (B) Family tree of the affected fetus. (C) FLVCR1a expression data from developing human brain extracted from the dataset GSE25219 of the Human BrainSpan Atlas. This dataset consists of RNA sequencing and exon microarrays obtained at sequential developmental stages of the human brain. Heatmap indicates low (blue) and high (red) expression values. Gray color: no available data. PCW, post-conception weeks; A1C, auditory cortex; AMY, amygdala; CBC, cerebellar cortex; DFC, dorsolateral prefrontal cortex; HIP, hippocampus; IPC, posterior inferior parietal cortex; ITC, inferior temporal cortex; M1C, primary motor cortex; MD, mediodorsal nucleus of the thalamus; MFC, medial pre-frontal cortex; OFC, orbital prefrontal cortex; S1C, primary somatosensory cortex; STC, superior temporal cortex; STR, striatum; V1C, primary visual cortex; VFC, ventrolateral prefrontal cortex. (D) FLVCR1a staining (red) in 11 post-conception weeks (PCW) human brain sections shows expression in RGCs (PAX6+ and SOX2+ cells) and IPs (TBR2+ cells). Scale bar: 500 μm. Magnified images of the insets on the left are shown on the right. Scale bar: 100 μm. (E) Western blot analysis of FLVCR1a expression in the brains isolated from E12.5 to E18.5 embryos, 2 days (P2) and 3-month-old Flvcr1-myc mice. An anti-Myc-Tag antibody was used to detect endogenous FLVCR1a. A representative image is shown. (F) Flvcr1a mRNA levels in “FlashTag”-labeled cell populations in the developing mouse cortex. The different isolation time points correspond to specific populations: 1 h = RGCs; 10 h = IPs; 24 h = newborn neurons; 4 days = neurons. Data represent mean ± SEM. See also Figure S1 and S2.

Figure 2

Loss of Flvcr1a in murine NPCs impairs neurogenesis and causes CH (A) Micro-CT analyses performed on Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/+;NesCRE+ and Flvcr1a^fl/fl embryos at E18.5. Quantification of the area of the brain, subarachnoid space, ventricles, spinal cord, spinal canal, and the cortical thickness with ImageJ software. Data represent mean ± SEM. (n = 3; one-way ANOVA, ∗ = p < 0.05; ∗∗ = p < 0.005; ∗∗∗ = p < 0.001.) (B) Immunostaining of PAX6+ and TUJ1+ cells of E14.5 Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/+;NesCRE+ cortexes. Each dot represents the mean of 3 images quantified from each animal. N = 3. Scale bar, 100 μm. (C) Relative quantification of PAX6+ cells and TUJ1 thickness layer. Each dot represents the mean of 3 images quantified from each animal. Data represent mean ± SEM. n = 3. (D) EdU staining of E14.5 Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/+;NesCRE+ cortexes. DAPI (blue) was used as a nuclear marker. Scale bar, 100 μm. (E) Relative quantification of EdU+ cells. Each dot represents the mean of three images quantified from each animal. Data represent mean ± SEM. n = 3. (F) EdU and PAX6 co-staining of E14.5 Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/+;NesCRE+ cortexes. DAPI (blue) was used as a nuclear marker. Scale bar, 100 μm. (G) Relative quantification of EdU+/Pax6+ cells. Each dot represents the mean of three images quantified from each animal. Data represent mean ± SEM. n = 3. (H) EdU and TBR2 co-staining of E14.5 Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/+;NesCRE+ cortex. DAPI (blue) was used as a nuclear marker. Scale bar, 100 μm. (I) Relative quantification of EdU+/TBR2+ cells. Each dot represents the mean of 3 images quantified from each animal. Data represent mean ± SEM. n = 3. (J) Neurospheres isolated from Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/fl cortexes and quantification of their diameter (t test; ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001; ∗∗∗∗ = p < 0.0001). (K) Proliferation rate of Flvcr1a^fl/fl NPCs overexpressing an empty vector and Flvcr1a^fl/fl;NesCRE+ NPCs overexpressing an empty vector, FLVCR1a-myc, or FLVCR1a∗c.160del-myc. Each time point represents the mean ± SEM of 5 different biological replicates (two-way ANOVA, ∗∗∗ = p < 0.001). See also Figures S3, S4, S5, S6.

Figure 3

FLVCR1a interacts with the IP3R3-VDAC complex (A) STRING analysis of the top 50 FLVCR1a interactors. (B) Gene Ontology term analysis for subcellular compartments of the top 50 FLVCR1a interactors. (C) Subcellular fractioning of HeLa cells. H, homogenate; ER, endoplasmic reticulum; MAMs, mitochondria-associated membranes; Mito, pure mitochondria. (D) PLA was performed in HeLa cells using the FLVCR1a and VDAC1 antibodies (FLVCR1a-ATP5i pair was used as a negative control), or the FLVCR1a and IP3R3 antibodies (FLVCR1a-PDI pair was used as a negative control), or the FLVCR1a and GRP75 antibodies (FLVCR1a-Laminin pair was used as a negative control). (non parametric Mann-Whitney U test; ∗∗∗∗ = p < 0.0001.) (E) Immunoprecipitation assay to detect the interaction between IP3R3-GFP and FLVCR1a-Myc. The protein complex was immune-precipitated using anti-GFP antibody, and the eluted proteins were detected by immunoblotting using either an anti Myc-Tag or an anti-GFP antibody. The vector expressing GFP alone was used as a negative control. (F) Immunoprecipitation assay to detect the interaction between FLVCR1a-Myc and IP3R3-GFP. The protein complex was immune-precipitated using anti-Myc-Tag antibody, and the eluted proteins were detected by immunoblotting using either an anti-GFP or an anti Myc-Tag antibody. (G) Immunoprecipitation assay to detect the interaction between FLVCR1a-Myc and VDAC-HA. The protein complex was immune-precipitated by an anti-Myc-Tag antibody, and the eluted proteins were detected by immunoblotting using either an anti-HA or an anti Myc-Tag antibody. (H) Immunoprecipitation of endogenous FLVCR1a from HEK293 cells followed by immunoblotting of the eluted proteins by an anti-VDAC antibody. (I) Immunoprecipitation of endogenous VDAC from E18.5 brains collected from the Flvcr1-myc embryos followed by immunoblotting using an anti-myc-Tag antibody. See also Figures S7 and S8.

Figure 4

FLVCR1a regulates ER-mitochondria membrane tethering and ER-mitochondria calcium uptake (A) Representative immunoblotting showing FLVCR1a protein levels upon FLVCR1a downregulation using short hairpin RNA (shRNA). A scramble shRNA was used as a control. (B) Quantification of Mander’s coefficients (M1 and M2) on confocal images of scramble or FLVCR1a shRNA HeLa cells expressing mt-DsRed and sec61-GFP. Superplot quantification showing each analyzed cell (small dot) and the mean of the three independent experiments (big dots). The three independent experiments are depicted with different colors. Data represent mean ± SEM. n = 3. (C) Representative images of scramble or FLVCR1a shRNA HeLa cells expressing mt-DsRed (MITO, red) and sec61-GFP (ER, green). White arrows indicate signal colocalization (ER-mitochondria contact sites).Scale bar: 5 μm. (D) DAPI staining (blue) and PLA (red dots) on scramble or FLVCR1a shRNA HeLa cells performed with the IP3R3-VDAC antibody pair. (E) IP3R3-VDAC PLA dot count collected from 5 different 96-wells for each condition. The total dot count was normalized on the number of nuclei in each well. (Non-parametric Mann-Whitney U test; ∗∗∗∗ = p < 0.0001.) (F) Immunoblotting showing VDAC and IP3R3 protein expression levels in HeLa cells. A representative image is shown. n = 3. (G) Mitochondrial calcium uptake measured as Ca²⁺ responses to agonist stimulation (100 μM histamine) in HeLa cells expressing a mitochondrial aequorin-based probe. Representative calcium traces are shown. (H) Quantification of peak mitochondrial calcium amplitude in HeLa cells upon agonist stimulation. Each dot represents the mean of 5 different wells from five independent experiments. Data represent mean ± SEM. n = 5. (I) Mitochondrial calcium elevation following agonist stimulation (histamine) in HeLa cells using a fluorescence resonance energy transfer (FRET)-based probe. The calcium increase is calculated based on FRET efficiency. Each dot represents the FRET efficiency of each cell group analyzed arising from 2 independent replicates. n= 2. (J) Immunoblotting showing FLVCR1a protein levels upon stable overexpression of FLVCR1a or an empty vector. A representative image is shown. (K) Quantification of Mander’s coefficients (M1 and M2) on confocal images of over empty vector of over FLVCR1a-myc HeLa cells expressing mt-DsRed and sec61-GFP. Superplot quantification showing each analyzed cell (small dot) and the mean of the three independent experiments (big dots). Three independent experiments are depicted with different colors. Data represent mean ± SEM. n = 3. (L) Mitochondrial calcium uptake measured as calcium responses to agonist stimulation (100 μM histamine) in HeLa cells expressing a mitochondrial aequorin-based probe. Representative calcium traces are shown. (M) Quantification of peak mitochondrial calcium amplitude in HeLa cells upon agonist stimulation. Each dot represents the mean of 5 different wells from 3 independent experiments. Data represent mean ± SEM. n = 3 (paired t test ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001; ∗∗∗∗ = p < 0.0001). See also Figure S9.

Figure 5

Alteration of MAM structure and function in PCARP/HSAN fibroblasts (A) Representative western blot depicting FLVCR1a abundance in fibroblasts derived from patients with PCARP/HSAN (P1 and P3) and healthy subjects (C1 and C3). Vinculin was used as a loading control. (B) PLA (red dots) performed in control and patient-derived fibroblasts with the IP3R3-VDAC antibody pair. DAPI (blue) was used to stain the nuclei. (C)IP3R3-VDAC PLA dot count was collected from five 96-wells for each condition. The total dot count was normalized on the total number of nuclei (Non-parametric Mann-Whitney U test; ∗∗∗∗ = p < 0.0001.). (D) Immunoblotting of VDAC and IP3R3 proteins in control and patient-derived fibroblasts. Vinculin was used as loading control. Relative quantification of protein abundance. Each dot represents normalized protein abundance of 2 technical replicates of 3 independent experiments. Data represent mean ± SEM. n = 3. (E) Mitochondrial calcium uptake measured as Ca²⁺ responses to agonist stimulation (100 μM histamine) in control and patient-derived fibroblasts. Representative calcium traces are shown. (F) Quantification of peak mitochondrial calcium amplitude in control and patient-derived fibroblasts upon agonist stimulation. Each dot represents the mean of 5 different wells from 3 independent experiments. Data represent mean ± SEM. n = 3 (t test; ∗∗ = p < 0.01). (G) Immunoblotting showing MCU overexpression in patients and control fibroblasts. Vinculin was used as a loading control. (H) Quantification of mitochondrial ATP levels in control and patient-derived fibroblasts, under basal conditions or upon MCU overexpression. Results are shown as nmoles ATP/mg of mitochondrial proteins. Data represent means ± SEM, n = 2. (I–L) Activity of the ETC complex I–IV in patient-derived fibroblasts under basal conditions or upon MCU overexpression. Results are shown as nmoles of NAD+/min/mg of mitochondrial protein for complex I, nmoles reduced cytochrome c/min/mg of mitochondrial protein for complex II and III, and nmoles oxidized cytochrome c/min/mg of mitochondrial protein. Data represent means ± SEM, n = 2 (two-way ANOVA; ∗∗∗∗ = p < 0.0001). See also Figure S10.

Figure 6

Loss of Flvcr1a in murine NPCs impairs mitochondrial calcium handling and energetic metabolism (A) RNA sequencing data showing altered expression of genes involved in calcium transport and homeostasis in Flvcr1a^fl/fl;NesCRE+ compared to Flvcr1a^fl/+;NesCRE+ E14.5 brains. (B) Transmission electron microscopy images from Flvcr1a^fl/fl;NesCRE+ compared to Flvcr1a^fl/fl NPCs. Pink arrows show ER-mitochondria contacts. Quantification of contact lengths and number is shown. Data represent mean ± SD. (t test; ∗∗ = p < 0.01). (C) Mitochondrial calcium uptake measured as Ca²⁺ responses to agonist stimulation (500 μM carbachol) in Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/fl NPCs. The mito-GEM-GECO1 probe was used. Representative Ca²⁺ traces are shown. (D) Quantification of peak mitochondrial Ca²⁺ amplitude upon agonist stimulation. Each dot represents the peak amplitude of single cells from 3 independent experiments. Data represent mean ± SEM. n = 3 (t test; ∗∗ = p < 0.01). (E) Mitochondrial calcium uptake measured as Ca²⁺ responses to agonist stimulation (500 μM carbachol) in Flvcr1a^fl/fl NPCs overexpressing an empty vector and Flvcr1a^fl/fl;NesCRE+ NPCs overexpressing an empty vector, FLVCR1a-myc, or FLVCR1a∗c.160del protein. The mito-GEM-GECO1 probe was used. Representative Ca²⁺ traces are shown. (F) Quantification of peak mitochondrial Ca²⁺ amplitude upon agonist stimulation. Each dot represents the peak amplitude of a single cell from two independent experiments. Data represent mean ± SEM. n = 2 (one-way ANOVA; ∗∗ = p < 0.01, ∗∗∗∗ = p < 0.0001). (G) Activity of the pyruvate, isocitrate, and α-ketoglutarate dehydrogenases in NPCs isolated from Flvcr1a^fl/fl;NesCRE+, Flvcr1a^fl/+;NesCRE+, and Flvcr1a^fl/fl embryos. Results are expressed as nmoles NADH/min/mg of mitochondrial protein. Each dot represents a single replicate of 3 different replicates of 2 independent experiments. Data represent mean ± SEM. n = 2. (H) TCA cycle flux is expressed as pmol CO₂/h/mg of protein. Data represent mean ± SEM. n = 2. (I) Overall ETC activity in NPCs isolated from Flvcr1a^fl/fl;NesCRE+, Flvcr1a^fl/+;NesCRE+, and Flvcr1a^fl/fl embryos. Results are shown as nmoles reduced cytochrome c/min/mg of mitochondrial protein. Each dot represents a single replicate of 3 different replicates of 2 independent experiments. Data represent mean ± SEM. n = 2. (J) Activity of the different mitochondrial ETC complexes in NPCs isolated from Flvcr1a^fl/fl;NesCRE+, and Flvcr1a^fl/+;NesCRE+ and Flvcr1a^fl/fl embryos. Results are shown as nmoles of NAD+/min/mg of mitochondrial protein for complex I, nmoles reduced cytochrome c/min/mg of mitochondrial protein for complex II and III, and nmoles oxidized cytochrome c/min/mg of mitochondrial protein. Each dot represents a single replicate of 3 different replicates of 2 independent experiments. Data represent mean ± SEM. n = 2. (K) Quantification of mitochondrial ATP levels in NPCs isolated from Flvcr1a^fl/fl;NesCRE+ and Flvcr1a^fl/+;NesCRE+ and Flvcr1a^fl/fl embryos. Results are shown as nmoles ATP/mg of mitochondrial proteins. Each dot represents a single replicate of 3 different replicates of 2 independent experiments. Data represent mean ± SEM. n = 2 (one-Way ANOVA; ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001; ∗∗∗∗ = p < 0.0001). See also Figure S11.

Figure 7

Mitochondrial calcium restoration improves Flvcr1a^fl/fl-NesCRE+ NPC proliferation and metabolism (A) Immunoblotting showing MCU overexpression in Flvcr1a^fl/fl;NesCRE+, Flvcr1a^fl/+;NesCRE+, and Flvcr1a^fl/fl NPCs. Vinculin was used as a loading control. (B) Representative images of Flvcr1a^fl/fl NPCs overexpressing an empty vector and Flvcr1a^fl/fl;NesCRE+ NPCs overexpressing an empty vector or MCU. NPC proliferation was measured for 48 h post-transfection using Incucyte SX5 Live-Cell Analysis. (C) Proliferation rate of Flvcr1a^fl/fl NPCs overexpressing an empty vector and Flvcr1a^fl/fl;NesCRE+ NPCs overexpressing an empty vector or MCU. Each time point represents the mean ± SEM of 5 different biological replicates (two-way ANOVA; ∗∗∗∗ = p < 0.0001). (D) Activity of the pyruvate, isocitrate, and α-ketoglutarate dehydrogenases in Flvcr1a^fl/fl;NesCRE+, Flvcr1a^fl/+;NesCRE+ and Flvcr1a^fl/fl NPCs overexpressing an empty vector or MCU. Results are expressed as nmoles NADH/min/mg of mitochondrial protein. Data represent means ± SEM, n = 2. (E) Activity of the ETC complexes in Flvcr1a^fl/fl;NesCRE+, Flvcr1a^fl/+;NesCRE+, and Flvcr1a^fl/fl NPCs overexpressing an empty vector or MCU. Results are shown as nmoles of NAD+/min/mg of mitochondrial protein for complex I, nmoles reduced cytochrome c/min/mg of mitochondrial protein for complex II and III, and nmoles oxidized cytochrome c/min/mg of mitochondrial protein. Data represent means ± SEM, n = 2. (F) Quantification of mitochondrial ATP levels in Flvcr1a^fl/fl;NesCRE+, Flvcr1a^fl/+;NesCRE+, and Flvcr1a^fl/fl NPCs overexpressing an empty vector or MCU. Results are shown as nmoles ATP/mg of mitochondrial proteins. Data represent means ± SEM, n = 2 (two-way ANOVA; ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001; ∗∗∗∗ = p < 0.0001).