Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy

Abstract

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Keywords: CME; DNAJC6; auxilin; gene therapy; neurodevelopmental; parkinsonism.

Figure 1

Schematic representation of CME. Clathrin-mediated endocytosis with (1) nucleation of clathrin-coated vesicles and clathrin coat assembly; (2) uncoating and release of cargo; (3) endolysosomal recycling of synaptic vesicles (SV); and (4) synaptic vesicle exocytosis. CME = clathrin-mediated endocytosis. Created with BioRender.com.

Figure 2

Patient-derived neurons show auxilin deficiency and disturbance of CME and synaptic vesicle homeostasis. (A) Immunoblot analysis for auxilin protein in control, patient and CRISPR Patient P1-derived neurons at Day 65 of differentiation. Quantification of protein abundance relative to the loading control (β-actin) (auxilin n = 5, 4, 4, 3, 3, 3). (B) Representative images of FM1–43 dye uptake assay for control, patient and CRISPR Patient P1-derived neurons at Day 65 of differentiation. Arrows in the high magnification images (bottom row) show representative synaptic boutons. Scale bar = 20 μm. (C) Quantification of FM1–43 mean fluorescence intensity in patients relative to the control and Patient 1 versus CRISPR Patient P1, respectively. (D) Electron microscopy analysis of presynaptic terminals in control, patient and CRISPR Patient P1-derived neuronal cultures at Day 65 of differentiation. Scale bar = 500 nm. Dotted circles indicate area with synaptic vesicles. (E) Quantification of synaptic vesicle (SV) density in the presynaptic terminal on electron microscopy images. CME = clathrin-mediated endocytosis; ns = not significant.

Figure 3

Patient-derived neurons show alterations in ventral midbrain patterning without neurodegeneration. (A) Representative immunofluorescence images for LMX1A and FOXA2 in control, patient and CRISPR Patient P1-derived neural progenitors at Day 11 of differentiation. Scale bar = 100 μm. (B) Quantification of total number of FOXA2-positive cells and LMX1A/FOXA2 double-positive cells at Day 11 (n = 3 for all). (C) Representative immunofluorescence images for MAP2 and TH in control, patient and CRISPR Patient P1-derived midbrain dopaminergic (mDA) neurons at Day 65 of differentiation. Scale bar = 150 μm. (D) Quantification of total number of MAP2 and TH positive cells, and TH/MAP2 double-positive cells at Day 65 of differentiation (n = 3 for all). (E) Representative immunofluorescence images for cCASP3 in control, patient and CRISPR Patient P1-derived mDA neurons at Day 65 of differentiation. Scale bar = 150 μm. (F) Quantification of total numbers of cCASP3-positive cells and cCASP3/TH double-positive cells at Day 65 of differentiation (n = 3 for all). ns = not significant.

Figure 4

Patient-derived neurons show defects in neuronal maturation. (A) Representative immunofluorescence images for NeuN and TH in control, patient and CRISPR Patient P1-derived midbrain dopaminergic (mDA) neurons. Scale bar = 150 μm. (B) Quantification of NeuN positive and TH/NeuN double-positive cells in control, patient and CRISPR Patient P1-derived mDA neurons at Day 65 of differentiation (n = 3 for all). (C) Representative immunofluorescence images for control, patient and CRISPR Patient P1-derived mDA neuron branching. Scale bar = 20 μm. (D) Quantification of average primary neurite branching in control, patient-and CRISPR Patient P1-derived mDA neurons at Day 65 of differentiation (n = 4 for all). ns = not significant.

Figure 5

Bulk RNA sequencing analysis highlights developmental and synaptic vesicle defects in patient-derived neurons. (A) Heat map showing hierarchical clustering of protein-coding differentially expressed genes (DEGs) in all patients compared to Control 1 (n = 3). (B) Gene Ontology (GO) terms enrichment for biological process of total (black), under-expressed (blue) and over-expressed (red) protein-coding DEGs in patients compared to control. The top 10 or five categories are shown. (C) Heat map showing hierarchical clustering of protein-coding DEGs in Patient 1 compared to CRISPR Patient P1 (n = 3). (D) GO terms enrichment for biological process of total (black), under-expressed (blue) and over-expressed (red) protein-coding DEGs in Patient 1 compared to CRISPR Patient P1. The top 10 or five categories are shown. (E) GO terms enrichment for cellular component of total (black) and under-expressed (blue) protein-coding DEGs in Patient 1 compared to CRISPR Patient P1.

Figure 6

Synaptic location enrichment analysis reveals structural abnormalities in patient-derived neurons. (A) Pie charts showing synaptic location mapping of under-expressed protein-coding differentially expressed genes (DEGs) in Patient 1 compared to CRISPR Patient P1. Left: general synapse; right: presynapse zoom-in. (B) Pie charts showing synaptic location mapping of under-expressed protein-coding DEGs in all patients compared to Control 1. Left: general synapse; right: presynapse zoom-in. (C) Pie charts showing synaptic function mapping of under-expressed protein-coding DEGs in Patient 1 compared to CRISPR Patient P1. (D) Pie charts showing synaptic function mapping of under-expressed protein-coding DEGs in all patients compared to Control 1.

Figure 7

Lentiviral DNAJC6 gene transfer restores auxilin and improves CME. (A) Left: Representative immunoblot images for auxilin and loading control (β-actin) in patient-derived midbrain dopaminergic (mDA) neurons transfected with LV GFP (lentivirus encoding green fluorescent protein) and LV DNAJC6-GFP (lentivirus encoding DNAJC6-green fluorescent protein) at Day 65 of maturation. Right: Quantification relative to the loading control (β-actin) (n = 3). (B) Representative immunofluorescence images for FM1–43 dye uptake assay in patient-derived mDA neurons transfected with LV GFP and LV DNAJC6-GFP at Day 65 of maturation. Scale bar = 20 μm. (C) Quantification of FM1–43 mean fluorescence intensity in patient-derived mDA neurons transfected with LV GFP and LV DNAJC6-GFP.