Defective neuronal differentiation in Lowe syndrome is associated with mitochondrial dysfunction and impaired cilia-related Sonic Hedgehog signaling
- Department of Ophthalmology, Stanford University School of Medicine, United States
- Palo Alto Veterans Administration, United States
- Maternal Children Health Research Institute at Stanford, Stanford University School of Medicine, United States
- BioX, Stanford University School of Medicine, United States
Figures
Figure 1
Increased astrocyte production during neuronal differentiation in OCRL-deficient and Lowe syndrome iPSCs.
(a and b) Immunofluorescence analysis of OCRL expression and pluripotency markers in iPSCs. Cells were stained for OCRL (red) and pluripotency markers NANOG and OCT4 (green), with nuclei counterstained with DAPI (blue). Scale bars are as indicated. (c) Schematic representation of Ngn2-mediated direct conversion of iPSCs into induced neurons (iNs) using lentiviral vectors (adapted from Zhang et al., 2013). (d) and (e) Immunofluorescence analysis of iPSC-derived iNs following neuronal induction. Cells were stained for GFAP (red), with Ngn2-EGFP marking transduced cells. Nuclei were counterstained with DAPI (blue). Scale bars as indicated. (f) Quantitative real-time PCR (qPCR) analysis of neuronal markers (FOXG1 and RBFOX3) in iN cells derived from control and OCRL-deficient iPSCs. (g) qPCR analysis of GFAP expression in iN cells. Gene expression values were normalized to GAPDH. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined using Student’s t-test. Changes in gene expression reflect relative marker levels and do not directly quantify cell-type proportions.
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Figure 1—source data 1
Numerical values corresponding to Figure 1f.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig1-data1-v1.xlsx
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Figure 1—source data 2
Numerical values corresponding to Figure 1g.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig1-data2-v1.xlsx
Figure 2
Altered mitochondrial parameters in OCRL-deficient iPSC-derived neurons.
(a) Quantitative real-time PCR (qPCR) analysis of mitochondrial DNA (mtDNA) levels, assessed using CO2 and D-loop regions, in induced neuron (iN) cells derived from control and OCRL-deficient iPSCs. (b) Immunofluorescence staining for 8-oxo-dG (red), a marker of oxidative DNA damage, in iN cells. Ngn2-EGFP (green) marks induced neurons. Nuclei are counterstained with DAPI (blue). Scale bar shown in the top-left image applies to all images in panel b. (c) Quantification of the percentage of 8-oxo-dG-positive cells. More than 100 cells were analyzed per independent experiment. (d) Mitochondrial respiration was assessed by oxygen consumption rate (OCR) using Seahorse extracellular flux analysis. Gene expression values were normalized to GAPDH. Data represent mean ± SEM from three independent experiments. Statistical significance was determined using Student’s t-test.
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Figure 2—source data 1
Numerical values corresponding to Figure 2a.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig2-data1-v1.xlsx
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Figure 2—source data 2
Numerical values corresponding to Figure 2c.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig2-data2-v1.xlsx
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Figure 2—source data 3
Numerical values corresponding to Figure 2d.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig2-data3-v1.xlsx
Figure 3
Altered neuronal and astrocytic marker expression in the Lowe syndrome mouse model.
(a) Quantitative real-time PCR (qPCR) analysis of progenitor-associated marker (Pax6) and neuronal markers (Rbfox3) in brain tissue. (b) qPCR analysis of astrocytic marker GFAP in brain tissue. (c) Quantification of NeuN and GFAP signal intensity in brain sections. More than 100 cells were analyzed per independent experiment. (d) Representative images of brains from wild-type (WT) and Ocrl−/− Inpp5b−/− INPP5B+/+ (IOB) mice. Immunofluorescence staining of brain sections for NeuN (red) and GFAP (green). Nuclei are counterstained with DAPI (blue). Scale bars as indicated. Gene expression values were normalized to GAPDH. Data represent mean ± SEM. Statistical significance was determined using Student’s t-test.
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Figure 3—source data 1
PDF file containing genotyping processing for IOB mice.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig3-data1-v1.zip
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Figure 3—source data 2
Original files of gels used for the genotyping.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig3-data2-v1.zip
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Figure 3—source data 3
Numerical values corresponding to Figure 3a.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig3-data3-v1.xlsx
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Figure 3—source data 4
Numerical values corresponding to Figure 3.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig3-data4-v1.xlsx
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Figure 3—source data 5
The file contains the numerical values corresponding to Figure 3c.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig3-data5-v1.xlsx
Figure 4
Altered mitochondrial parameters in the Lowe syndrome mouse brain.
(a) Quantitative real-time PCR (qPCR) analysis of mitochondrial DNA (mtDNA), assessed using Mto1 and Mt-Co1 in brain tissue from wild-type (WT) and IOB mice. (b) Quantification of 8-oxo-dG-positive signal in brain sections. More than 100 cells were analyzed per independent experiment. (c) Immunofluorescence staining for 8-oxo-dG (red) in brain sections. Nuclei are counterstained with DAPI (blue). Scale bars as indicated. Gene expression values were normalized to GAPDH. Data represent mean ± SEM. Statistical significance was determined using Student’s t-test.
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Figure 4—source data 1
Numerical values corresponding to Figure 4a.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig4-data1-v1.xlsx
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Figure 4—source data 2
Numerical values corresponding to Figure 4b.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig4-data2-v1.xlsx
Figure 5
OCRL loss is associated with mitochondrial dysfunction, oxidative stress, and reduced survival in zebrafish.
(a) Representative brightfield images of zebrafish larvae at 2 and 5 days post-fertilization (dpf), including wild-type (WT), control gRNA-injected, and ocrl knockout groups. (b) Kaplan-Meier survival analysis of zebrafish larvae. OCRL-deficient larvae exhibit reduced survival compared to control gRNA-injected larvae. (c) Quantification of phenotype penetrance over time (1–5 dpf), presented as (i) percentage of affected larvae among living animals and (ii) percentage of affected larvae relative to total injected embryos. (d) Mitochondrial reactive oxygen species (ROS) assessed by MitoSOX staining. Representative images and quantification of MitoSOX-positive area fraction (%) in cranial and ocular regions are shown. (e) Mitochondrial content assessed by TOM20 immunostaining. Representative images and quantification of TOM20-positive area fraction (%) are shown. (f) Mitochondrial membrane potential (ΔΨm) assessed by MitoTracker CMXRos staining. Representative images and quantification of MitoTracker CMXRos intensity (a.u.) and positive area fraction (%) in cranial and ocular regions are shown. Data are presented as mean ± SEM from n=10–15 larvae per group. Statistical significance was determined using Student’s t-test unless otherwise indicated. Imaging and quantification were performed under identical conditions across all groups.
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Figure 5—source data 1
PDF file containing the genotyping analysis of ocrlKO zebrafish.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data1-v1.zip
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Figure 5—source data 2
Original raw uncropped and unedited gel files used for genotyping.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data2-v1.zip
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Figure 5—source data 3
Numerical values corresponding to Figure 5b.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data3-v1.xlsx
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Figure 5—source data 4
Numerical values corresponding to Figure 5c.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data4-v1.xlsx
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Figure 5—source data 5
Numerical values corresponding to Figure 5d.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data5-v1.xlsx
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Figure 5—source data 6
Numerical values corresponding to Figure 5e.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data6-v1.xlsx
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Figure 5—source data 7
Numerical values corresponding to Figure 5f.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig5-data7-v1.xlsx
Figure 6
Altered ciliary parameters and Sonic Hedgehog signaling in OCRL-deficient models.
(a) Quantitative real-time PCR (qPCR) analysis of SHH pathway genes (GLI1, PTCH1, SHH) in iPSC-derived induced neuron (iN) cells. (b) Immunofluorescence staining of brain sections for Sonic Hedgehog (SHH) (red) and the ciliary marker ARL13B (green). Nuclei are counterstained with DAPI (blue). Scale bars as indicated. (c) Quantification of the ciliated cells and cilia length in brain sections. More than 100 cells were analyzed per independent experiment. (d) qPCR analysis of Hedgehog pathway genes (Gli1, Gli2, Gli3, Ptch1) in brain tissue from wild-type (WT) and IOB mice. (e) Western blot analysis of SHH and GLI1 protein levels in brain tissue. β-actin was used as a loading control. Gene expression values were normalized to GAPDH. Data represent mean ± SEM. Statistical significance was determined using Student’s t-test.
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Figure 6—source data 1
Original western blots for Figure 6e indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig6-data1-v1.zip
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6e.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig6-data2-v1.zip
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Figure 6—source data 3
Numerical values corresponding to Figure 6a.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig6-data3-v1.xlsx
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Figure 6—source data 4
Numerical values corresponding to Figure 6c.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig6-data4-v1.xlsx
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Figure 6—source data 5
Numerical values corresponding to Figure 6d.
- https://cdn.elifesciences.org/articles/104055/elife-104055-fig6-data5-v1.xlsx
Figure 7
OCRL deficiency disrupts neuronal development through mitochondrial dysfunction, oxidative stress, and impaired Sonic Hedgehog (SHH)-cilia signaling.
Schematic representation of the integrated findings across experimental systems. OCRL deficiency leads to mitochondrial dysfunction, characterized by reduced mitochondrial DNA, decreased oxidative phosphorylation, reduced mitochondrial content, and increased oxidative stress. Elevated oxidative stress is associated with two parallel processes: (i) altered balance between neuronal and astrocytic cell states and (ii) reduced Shh signaling, accompanied by changes in ciliary parameters, including decreased proportion of ciliated cells and increased cilia length. These combined alterations are associated with impaired neuronal development in Lowe syndrome.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | LS100 iPSC | Herbert M. Lachman laboratory (Barnes et al., 2018) | Lowe syndrome patient-derived | |
| Cell line (Homo sapiens) | LS200 iPSC | Herbert M. Lachman laboratory (Barnes et al., 2018) | Unaffected sibling | |
| Cell line (Homo sapiens) | 690 Ctrl iPSC | Barnes et al., 2018 | Control iPSC | Healthy unrelated donor |
| Cell line (Homo sapiens) | 690 OCRL KO iPSC | Barnes et al., 2018 | OCRL knockout | CRISPR-generated derivative of 690 Ctrl |
| Strain, strain background (Mus musculus) | IOB mouse | Robert L. Nussbaum laboratory | - | Ocrl−/− Inpp5b−/− INPP5B+/+; 2-month-old mice |
| Strain, strain background (Mus musculus) | C57BL/6 | The Jackson Laboratory | 000664 | Wild-type (WT) control |
| Strain, strain background (Danio rerio) | WT zebrafish | Zebrafish International Resource Center (ZIRC) | AB | Used for CRISPR/Cas9 generation of ocrl-deficient larvae; mitochondrial assays performed at 2 dpf |
| Sequence-based reagent | ocrl crRNA | IDT | Custom. gRNA1: 5’-TCTAACAAGGACAGGAGTCTTGG-3’ gRNA2: 5’-TCTGCGAGGTGAACGAACACCGG-3’ | CRISPR RNA targeting zebrafish ocrl. |
| Sequence-based reagent | Alt-R CRISPR-Cas9 tracrRNA | IDT | 1072532 | Used for CRISPR/Cas9 RNP assembly |
| Peptide, recombinant protein | Alt-R Cas9 Nuclease V3 | IDT | 1081058 | Used for CRISPR/Cas9 RNP assembly |
| Other | Matrigel | Corning | 354277 | Substrate for iPSC culture |
| Other | mTeSR1 Plus | STEMCELL Technologies | 85850 | iPSC maintenance medium |
| Other | ReLeSR | STEMCELL Technologies | 05872 | Used for iPSC passaging |
| Other | Accutase | STEMCELL Technologies | 07920 | Used for iPSC dissociation |
| Chemical compound, drug | MitoTracker Red CMXRos | Thermo Fisher | M7512 | 100 nM; 20 min incubation |
| Chemical compound, drug | MitoSOX Red | Thermo Fisher | M36008 | 2 µM; 20 min incubation |
| Antibody | anti-NANOG (rabbit polyclonal) | CST | 3580 S; RRID:AB_2150399 | IF 1:250 |
| Antibody | Anti-OCT4A, clone C30A3 (rabbit monoclonal) | CST | 2840 S; RRID:AB_2167691 | IF 1:250 |
| Antibody | Anti-GFAP(chicken polyclonal) | Abcam | ab4674; RRID:AB_304558 | IF 1:250 |
| Antibody | Anti-OCRL/INPP5B, clone N166A/26 (mouse monoclonal) | UC Davis/NIH NeuroMab Facility | N166A/26; RRID:AB_2877449 | IF 1:250 |
| Antibody | Anti-8-oxo-dG (mouse monoclonal) | R&D Systems | 4354-MC-050; RRID:AB_1857195 | IF 1:250 |
| Antibody | Anti-SHH, clone EP1190Y (rabbit monoclonal) | Abcam | ab53281 | IF 1:200; WB 1:500 |
| Antibody | Anti-GLI1 (rabbit monoclonal) | Abcam | ab217326; | WB 1:500 |
| Antibody | Anti-ARL13B, clone N295B/66 (mouse monoclonal) | Antibodies Inc. | 75–287 | IF 1:500 |
| Antibody | Anti-NeuN, clone A60 (mouse monoclonal) | Sigma | MAB377; RRID:AB_2298772 | IF 1:200 |
| Antibody | Anti-TOM20 (mouse monoclonal) | Abcam | ab56783; RRID:AB_945896 | IF 1:200 |
| Antibody | Anti-β-actin (mouse monoclonal) | Proteintech | 66009–1; RRID:AB_2687938 | WB 1:5000 |
| Antibody | Alexa Fluor 488-, 555-, or 647-conjugated goat secondary antibodies (goat polyclonal) | Thermo Fisher | A-21235; RRID:AB_2535804 A-32723 A-21428; RRID:AB_2535849 | IF 1:500 |
| Antibody | HRP-conjugated anti-mouse secondary antibody | Invitrogen | 31430; RRID:AB_228307 | WB 1:10000 |
| Antibody | HRP-conjugated anti-rabbit secondary antibody | Invitrogen | 31460; RRID:AB_228341 | WB 1:10000 |
| Commercial assay, kit | HiScript III RT SuperMix for qPCR + gDNA wiper | Vazyme | R323-01 | Reverse transcription for qPCR |
| Commercial assay, kit | FastSYBR Mixture (2 x) | CWBio | CW0955L | qPCR; primers used at 0.5 µM |
| Commercial assay, kit | BCA Protein Assay | Thermo Fisher | 23227 | Protein concentration measurement |
| Other | RIPA lysis buffer | Millipore | 20–188 | Protein extraction |
| Software, algorithm | Fiji/ImageJ | NIH | RRID:SCR_002285 | Image quantification and manual cilia-length measurements |
| Software, algorithm | GraphPad Prism | GraphPad Software | Version 8; RRID:SCR_002798 | Statistical analysis |
| Software, algorithm | ZEN | Carl Zeiss | RRID:SCR_013672 | Confocal image acquisition and processing |
Additional files
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MDAR checklist
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Source data 1
Primer sequences used for qPCR analysis.
- https://cdn.elifesciences.org/articles/104055/elife-104055-data1-v1.xlsx
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Source data 2
Numeric data for tables and figures.
- https://cdn.elifesciences.org/articles/104055/elife-104055-data2-v1.xlsx
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Defective neuronal differentiation in Lowe syndrome is associated with mitochondrial dysfunction and impaired cilia-related Sonic Hedgehog signaling
eLife 13:RP104055.
https://doi.org/10.7554/eLife.104055.3
