Dissecting the transcriptome of Parkin (PRKN)-linked Parkinson’s disease using 2D and 3D hiPSC-neuronal models

The human LRRK2-R1441G mutation drives age-dependent oxidative stress and mitochondrial dysfunction in dopaminergic neurons.

BACKGROUND Mitochondrial dysfunction and oxidative stress are central to the pathogenesis of Parkinson's disease (PD), particularly affecting substantia nigra pars compacta (SNc) dopamine (DA) neurons. Here, we investigate how the R1441G mutation in leucine-rich repeat kinase 2 (LRRK2), a key genetic contributor to familial and sporadic PD, impacts mitochondrial function in midbrain DA neurons. METHODS We employed a BAC transgenic mouse model overexpressing human LRRK2-R1441G (BAC-hR1441G) and crossed it with TH-mito-roGFP mice to enable mitochondria-targeted redox imaging specifically in DA neurons. Acute midbrain slices from 3-, 6-, and 10-month-old mice were imaged using two-photon microscopy to assess mitochondrial oxidative stress. In parallel, mitochondrial respiratory function, membrane potential flickering events, and expression of uncoupling proteins (UCP4/UCP5) were analyzed. Spatial transcriptomic profiling was performed using the GeoMx® Digital Spatial Profiler to uncover associated molecular alterations. RESULTS We observed a progressive increase in mitochondrial oxidative stress in SNc DA neurons of BAC-hR1441G mice at 3, 6, and 10 months of age. This was accompanied by reduced respiratory complex activity, attenuated mitochondrial membrane potential flickering, and diminished expression of UCP4 and UCP5. Spatial transcriptomic analysis revealed dysregulation of genes linked to mitochondrial uncoupling, calcium signaling, and redox regulation in BAC-hR1441G SNc DA neurons. CONCLUSIONS These findings reveal an age-dependent progression of mitochondrial dysfunction in BAC-hR1441G SNc DA neurons. Dysregulation of calcium channels and uncoupling proteins emerges as a key mechanism contributing to bioenergetic failure, suggesting potential therapeutic targets to mitigate PD progression.

Mitochondrial Metabolism and Calcium Handling in Parkinson’s Disease hiPSC-derived Astrocytes

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide, and mutations in the LRRK2 and PRKN genes are among the most common familial causes of the disease. In neurodegenerative diseases such as PD, disturbances in Ca²⁺ homeostasis and cellular bioenergetics impair the function of neurons and glial cells, contributing to disease progression. These changes are not limited to neurons; mitochondrial dysfunction and disrupted Ca2+ homeostasis in astrocytes are increasingly recognized as key contributors to PD, impairing bioenergetics, redox balance, neuroinflammatory responses, and metabolic support essential for dopaminergic neuron survival. In this study, we investigated mitochondrial calcium homeostasis, mitochondrial oxidative phosphorylation, morphology and distribution in human induced pluripotent stem cell (hiPSC)-derived astrocytes with mutations in the PD genes LRRK2 (G2019S) and PRKN (c.155delA; Ex3-4del) and wild-type controls. Intracellular calcium dynamics were assessed using Fura-2 AM. Compared with control astrocytes, LRRK2-related PD patient-derived mutant astrocytes exhibited lower intracellular calcium levels, and slower calcium extrusion following stimulation with ATP. Mitochondrial morphology was analyzed using MitoTracker Deep Red, revealing increased mitochondrial fragmentation and redistribution of mitochondria toward the cell periphery in both PD mutant cell types. Because oxidative phosphorylation is tightly regulated by mitochondrial morphology and calcium homeostasis, we next assessed oxygen consumption rates using a continuous metabolic monitoring system (Resipher) and quantified the expression of genes (RT-qPCR) and proteins (capillary electrophoresis-based western detection) involved in mitochondrial calcium transport and bioenergetics. These analyses showed that PRKN mutant astrocytes exhibit a more oxidative bioenergetic phenotype than LRRK2 mutant astrocytes, while both mutant lines displayed altered phosphorylation of mitochondrial morphology regulator DRP1 as well as decreased levels of respiratory complexes relative to control astrocytes. In summary, this study identifies astrocyte-specific mitochondrial dysfunctions and calcium dysregulation as key features of LRRK2- and PRKN-related pathology, providing new insights into how glial metabolic alterations contribute to neurodegeneration in PD.

Characterizing the Roles of Dysferlin Deficiency in Function, Polarization, and Secretome of Human iPSC-Derived Macrophages 2251399

Limb-girdle muscular dystrophy R2/2B (LGMDR2/2B) is an untreatable and progressive late-onset skeletal muscle disease caused by the loss of a membrane-repair protein dysferlin. Even before disease symptom onset, LGMDR2 muscles are infiltrated by pro-inflammatory macrophages (MP), implicating immune cells in disease pathogenesis. While MPs express dysferlin, defining the cell-autonomous roles of dysferlin in MP function has been challenging in vivo due to complex multicellular interactions and altered microenvironment in LGMDR2 muscle. To address this, we generated human induced pluripotent stem cell (hiPSC)-derived macrophages (iMPs) from three healthy and three LGMDR2 donors to delineate cell-autonomous roles of dysferlin in macrophage: 1) polarization, 2) transcriptional profile, 3) secretome, and 4) phagocytotic and endocytic function. Despite exhibiting comparable polarization under well-characterized pro- and anti-inflammatory cues, RNAseq analyses revealed downregulation of Gene Ontology terms related to cytokine secretion, phagocytosis, and receptor-mediated endocytosis in LGMDR2 iMPs. Proteomic analysis of iMP conditioned media revealed significant differences in 72 secreted proteins, including numerous chemokines, cytokines, and growth factors, suggesting an altered secretory phenotype. Functional assays found no significant differences in the phagocytosis of E. coli bioparticles or fluorescent myotube debris. However, receptor-mediated endocytosis of AcLDL was significantly lower in both M0 and M2 LGMDR2 vs. healthy iMPs. Pharmacological screens identified clathrin-dependent endocytosis as the primary pathway for AcLDL uptake in both genotypes, with altered clathrin trafficking and reduced scavenger receptor expression likely underlying LGMDR2 endocytic deficits. Overall, dysferlin loss in iMPs results in cell-autonomously altered transcriptome, secretome, and endocytic function, which may contribute to LGMDR2 muscle pathology and disease progression. Jain Foundation grant, NIH grant 1R01AR082979-01, National Science Foundation Graduate Research Fellowship Immune Mechanisms of Human Disease (HUM)

Neurogenesis defects in iPSC-derived midbrain organoids of early-onset Parkinson's disease with 22q11.2 deletion syndrome

Sporadic Parkinson's disease (PD) is typically a late-onset disorder caused by a combination of genetics, environment, and aging, manifesting when the loss of midbrain dopaminergic neurons exceeds a critical threshold, usually after the age of 50. Conversely, early-onset PD, as observed in cases linked to parkin (PRKN) gene mutations, suggests mechanisms involving either accelerated postnatal neuron loss or an insufficient number of neurons at birth. Patients with the 22q11.2 deletion syndrome (DS) have a significantly higher prevalence of early-onset PD. The absence of known genes associated with hereditary PD in the deleted region suggests the involvement of novel, non-traditional risk factors. This could potentially implicate the neurodevelopmental origin of dopaminergic neurons arising from the floor plate. To investigate this hypothesis, we generated midbrain organoids from induced pluripotent stem cells derived from a patient with 22q11.2 DS. The organoids recapitulated key aspects of in vivo neurogenesis, revealing enhanced differentiation of dopaminergic neurons in 22q11.2DS- and PRKN-derived organoids compared to controls on days 28 and 56 of culture. These findings suggest that, in early-onset PD patients with 22q11.2 DS or PRKN mutation, enhanced neurogenesis could result in reduced number of dopaminergic neurons during early development. The organoids of early-onset PD demonstrated that progenitors undergo enhanced differentiation at an early stage. This suggests that the atypical developmental process could reduce progenitors before there are enough mature dopaminergic neurons. This in turn indicates that the onset of PD may occur as early as the embryonic stage.

Modeling Parkinson’s pathology in human iPSC dopaminergic neurons uncovers key mechanisms of Lewy body formation and heterogeneity

The aggregation of alpha-synuclein (aSyn) into intraneuronal inclusions of heterogeneous morphology, known as Lewy bodies (LBs), is a defining hallmark of Parkinson’s disease (PD); yet, our understanding of the mechanisms underpinning their formation and heterogeneity remains incomplete. Here, we present a human isogenic induced pluripotent stem cell–derived dopaminergic neuron (iDA) model that faithfully recapitulates the diverse biochemical, morphological, and ultrastructural features of LB neuropathology. The iDA model accurately reproduces the temporal relationships between neuritic and cell-body aSyn pathology and recapitulates the proteome, posttranslational modifications, and morphological diversity of aSyn aggregates found in human PD tissue. Moreover, our work provides critical insight into how different pathways to aSyn fibrillization and the complex interaction between aSyn fibrils and membranous organelles shape the morphological diversity of LB-like inclusions. This model represents a versatile platform to investigate the mechanisms of pathology formation, maturation, and neuronal dysfunction and to develop diagnostics and therapeutics that account for the diversity of aSyn pathology in PD and related synucleinopathies.

Restoring Parkin Function: An AAV Gene Therapy Approach for Early-Onset Parkinson’s Disease