Abstract
ABSTRACT Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) – the predominant neuronal sub-type afflicted in PD – have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells. Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4ʹ,6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco’s modified eagle’s medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.
Highlights
Parkinson disease (PD) is a progressive neurodegenerative disorder that causes motor, physical and cognitive impairments, leading to a reduction in quality of life and eventually, an inability to live independently
It is clear that there are substantial differences in midbrain dopaminergic neuron (mDAN) differentiation protocols being applied, and varying standards for assessing mDAN identity/contribution in culture, highlighting a need for greater consistency. mDANs can be induced from iPSCs by overexpression of LMX1A
Neurite morphology defects were reported in LRRK2G2019 S mutant cells, corresponding with evidence of failed autophagic flux [59]
Summary
Parkinson disease (PD) is a progressive neurodegenerative disorder that causes motor, physical and cognitive impairments, leading to a reduction in quality of life and eventually, an inability to live independently. PD affects around 1% of the population aged over 65 and around 4% aged over 85. Classic PD symptoms (e.g., tremors at rest, postural instability, stiffness, dyskinesia) result from falling levels of the catecholaminergic neurotransmitter, dopamine, caused by the degeneration of midbrain dopamineproducing neurons (mDANs) [2]. There is a need to better understand how PD pathophysiology affects these neurons. To achieve this goal, relevant and amenable cell-based assay systems are required for functional tests in vitro
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