Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons

Abstract

Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson’s disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

Mitochondrial Phenotypes in Parkinson’s Diseases—A Focus on Human iPSC-Derived Dopaminergic Neurons

“…Conversely, Parkin patient-derived NPCs showed a subtle, but significant elevation in mitochondrial fragmentation that dose-dependently increased in severity with copper exposure, a known environmental risk factor for PD [ 100 ]. A recent study using Parkin KO iPSC-derived neurons elegantly showed that smaller and less functional mitochondria were present in dopaminergic, but absent in non-dopaminergic neurons using a tyrosine hydroxylase reporter cassette and correlative light electron microscopy [ 101 ]. This suggests that dopaminergic neurons possess intrinsic physiological differences, making them particularly vulnerable compared to other types of neurons.…”

Genetic Dissection of Temperament Personality Traits in Italian Isolates

“…The PRKN gene encodes the Parkin RBR E3 ubiquitin protein ligase, a protein involved in the pathway of protein ubiquitination. In particular it has been studied in the mitochondrial mechanisms of quality control and associated with the regulation of mitochondrial apoptosis and neurodegenerative diseases [ 31 ]. PRKN mutations are known to be involved with early onset familial and autosomal recessive juvenile Parkinson’s disease [ 32 , 33 ], likely due to the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting from the accumulation of damaged mitochondria [ 31 ].…”

“…In particular it has been studied in the mitochondrial mechanisms of quality control and associated with the regulation of mitochondrial apoptosis and neurodegenerative diseases [ 31 ]. PRKN mutations are known to be involved with early onset familial and autosomal recessive juvenile Parkinson’s disease [ 32 , 33 ], likely due to the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting from the accumulation of damaged mitochondria [ 31 ]. Interestingly, dopamine plays a central role in the Reward system, which regulates the effects of decision-making and induces approach behavior.…”

From the Structural and (Dys)function of ATP Synthase to Deficiency in Age-Related Diseases

“…iPSC-derived mutant PARK2 neurons exhibited increased oxidative stress and aberrant activation of the NRF2 pathway with impaired mitochondrial function and α-synuclein accumulation [106]. Similarly, iPSC-derived mutant PARK2 dopaminergic neurons, carrying a GFP expression cassette at the TH locus, showed small and less functional neurons with a decline in mitochondrial membrane potential, suggesting a mitochondria-dependent mechanism involved in dopaminergic neuron susceptibility to PD [107]. A potential link between α-synuclein and impairments in mitochondrial dynamics and bioenergetics is suggested by the direct interaction with the OXPHOS components as demonstrated in proteomic studies [108,109].…”