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) qPCR analysis of neuronal markers (FOXG1 and NEUN) 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.

Altered mitochondrial parameters in OCRL-deficient iPSC-derived neurons

(a) qPCR analysis of mitochondrial DNA (mtDNA) levels, assessed using CO2 and D-loop regions, in 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 bars as indicated. (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.

Altered neuronal and astrocytic marker expression in the Lowe syndrome mouse model

(a) qPCR analysis of progenitor-associated marker (Pax6) and neuronal markers (NeuN) 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 INPP5B/OCRL double knockout (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.

Altered mitochondrial parameters in the Lowe syndrome mouse brain.

(a) qPCR analysis of mitochondrial DNA (mtDNA), assessed using mito1 and COX1 in brain tissue from 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.

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 (KO) 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.

Altered ciliary parameters and Sonic Hedgehog signaling in OCRL-deficient models

(a) qPCR analysis of Shh pathway genes (GLI1, PTCH1, SHH) in iPSC-derived iN cells. (b) Immunofluorescence staining of brain sections for 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 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.

OCRL deficiency disrupts neuronal development through mitochondrial dysfunction, oxidative stress, and impaired 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.