Induced pluripotent stem cell-based modeling of mutant LRRK2-associated Parkinson's disease

Abstract

Recent advances in cell reprogramming have enabled assessment of disease-related cellular traits in patient-derived somatic cells, thus providing a versatile platform for disease modeling and drug development. Given the limited access to vital human brain cells, this technology is especially relevant for neurodegenerative disorders such as Parkinson's disease (PD) as a tool to decipher underlying pathomechanisms. Importantly, recent progress in genome-editing technologies has provided an ability to analyze isogenic induced pluripotent stem cell (iPSC) pairs that differ only in a single genetic change, thus allowing a thorough assessment of the molecular and cellular phenotypes that result from monogenetic risk factors. In this review, we summarize the current state of iPSC-based modeling of PD with a focus on leucine-rich repeat kinase 2 (LRRK2), one of the most prominent monogenetic risk factors for PD linked to both familial and idiopathic forms. The LRRK2 protein is a primarily cytosolic multi-domain protein contributing to regulation of several pathways including autophagy, mitochondrial function, vesicle transport, nuclear architecture and cell morphology. We summarize iPSC-based studies that contributed to improving our understanding of the function of LRRK2 and its variants in the context of PD etiopathology. These data, along with results obtained in our own studies, underscore the multifaceted role of LRRK2 in regulating cellular homeostasis on several levels, including proteostasis, mitochondrial dynamics and regulation of the cytoskeleton. Finally, we expound advantages and limitations of reprogramming technologies for disease modeling and drug development and provide an outlook on future challenges and expectations offered by this exciting technology.

Keywords: LRRK2; Parkinson's disease; disease modeling; iPSC; mitophagy.

Figure 1

Reprogramming approaches in combination with genome editing facilitate disease modeling and drug discovery. Somatic cells (skin fibroblasts, blood cells) from healthy or diseased donors are reprogrammed into induced pluripotent stem cells (iPSCs) via various approaches, including ectopic expression of reprogramming factors, combinations of transcription factors and microRNAs or application of small molecules. The genome of resulting iPSCs can be edited to insert disease‐specific mutations into cells of healthy donors or to repair mutants in patient‐derived cells. Both approaches result in isogenic pairs of iPSCs which differ only by the respective disease variant, thereby eliminating phenotypic variability due to interindividual genetic differences. Optimized differentiation routines are next applied to generate the desired cell type(s) for in vitro disease modeling and drug discovery

Figure 2

Leucine‐rich repeat kinase 2 protein structure and functions. (a) LRRK2 protein architecture and disease‐linked variants. Colored boxes, domains comprising the catalytic core; white boxes, protein–protein interaction domains, color‐coding of disease‐associated variants: red, Parkinson's disease; blue, Crohn disease; gray, leprosy. (b) Pleiotropic functions of LRRK2. The LRRK2 protein is contributing to the homeostasis and regulation of several cellular pathways and compartments including autophagy, mitochondrial function, vesicle transport and nuclear architecture. CMA, chaperone‐mediated autophagy; Ca²⁺, Calcium²⁺; ER, endoplasmatic reticulum; GA, Golgi apparatus; ΔΨ, mitochondrial membrane potential. See main text for details

Figure 3

An isogenic iPSC‐based model of LRRK2^G2019S‐linked PD reveals alterations in mitochondrial morphology and impaired mitophagy in mesDA progenitors. (a) Schematic overview of the experimental setup. Isogenic iPSCs expressing the mito‐RFP‐EGFP reporter were subjected to differentiation into floor plate‐derived mesDA neurons. Mitochondrial morphology and mitophagy were analyzed at day 14 (d14). LDN, LDN193189; SB, SB431542; B, BDNF; D, DAPT; Db, dbcAMP; G, GDNF; L, LAAP; T, TGFβ. (b) Illustration of the mitophagy reporter mito‐RFP‐EGFP, which is imported into the mitochondrial matrix upon translation and indicates mitophagy by a pH‐dependent color shift from yellow (RFP+EGFP) to red (RFP only) upon lysosomal delivery. (c–e) Quantification and exemplary images of mitochondrial morphologies in isogenic G2019S mesDA progenitors at d14. LRRK2^G2019S cultures display reduced levels of filamentous mitochondria (c) and increased levels of intermediate and punctate mitochondria (d). (e) At d14, levels of active mitophagy are reduced in G2019S cultures. For each condition, 5–6 wells were quantified per experiment (n = 3). The percentages of cells containing mitochondria of a specific category per well are depicted as dot plots. Lines indicate the median. For details on the methodology, see Supporting Information

Figure 4

LRRK2^G2019S mesDA neuronal cultures exhibit decreased neurite length which can be rescued by LRRK2 kinase inhibitor treatment. (a) Schematic overview of the experimental set up. Control (Ctrl) and isogenic G2019S (GS) mesDA neuronal cultures were either untreated (left), treated with DMSO or treated with 1 μM LRRK2‐IN1 (IN‐1) and subjected to a neurite outgrowth assay. LDN, LDN193189; SB, SB431542. (b) Quantification of neurite length demonstrates that decreased neurite lengths in LRRK2^G2019S neurons can be rescued by treatment with IN‐1. For each condition, 2–3 wells were analyzed in three independent experimental sets (n = 3). A total of 4982 neurites were quantified. Each dot represents the neurite length of one individual neurite (expressed as percentage from the mean of the respective healthy control). Red lines indicate the median. (c) Exemplary images of isogenic control and LRRK2^G2019S TH ⁺ neurons under different experimental conditions. For details on the methodology, see Supporting Information