Parkin Maintains Robust Pacemaking in Human Induced Pluripotent Stem Cell-Derived A9 Dopaminergic Neurons.

Background: The degeneration of nigral (A9) dopaminergic (DA) neurons results in cardinal motor symptoms that define Parkinson's disease (PD). Loss-of-function mutations in parkin are linked to a rare form of early-onset PD that is inherited recessively.

Objective: We generated isogenic human A9 DA neurons with or without parkin mutations to establish the causal relationship between parkin mutations and the dysfunction of human A9 DA neurons.

Generation of human A9 dopaminergic pacemakers from induced pluripotent stem cells.

The degeneration of nigral (A9) dopaminergic (DA) neurons causes motor symptoms in Parkinson's disease (PD). We use small-molecule compounds to direct the differentiation of human induced pluripotent stem cells (iPSCs) to A9 DA neurons that share many important properties with their in vivo counterparts. The method generates a large percentage of TH neurons that express appropriate A9 markers, such as GIRK2 and ALDH1A1, but mostly not the A10 marker CALBINDIN.

Molecular Features of Parkinson's Disease in Patient-Derived Midbrain Dopaminergic Neurons.

Background: Despite intense efforts to develop an objective diagnostic test for Parkinson's disease, there is still no consensus on biomarkers that can accurately diagnose the disease.

Objective: Identification of biomarkers for idiopathic Parkinson's disease (PD) may enable accurate diagnosis of the disease. We tried to find molecular and cellular differences in dopaminergic (DA) neurons derived from healthy subjects and idiopathic PD patients with or without rest tremor at onset.

Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells.

Direct conversion of adult human retinal pigmented epithelium cells to neurons with photoreceptor properties.

Degeneration of photoreceptors is a major cause of blindness. Identifying new methods for the generation of photoreceptors offers valuable options for a cell replacement therapy of blindness. Here, we show that primary adult human retinal pigmented epithelium (hRPE) cells were directly converted to postmitotic neurons with various properties of photoreceptors by the neurogenic transcription factor ASCL1 and microRNA124.

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm.

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells.

Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos.

It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts.

Attenuation of PRRX2 and HEY2 enables efficient conversion of adult human skin fibroblasts to neurons.

The direct conversion of accessible cells such as human fibroblasts to inaccessible cells, particularly neurons, opens up many opportunities for using the human model system to study diseases and discover therapies. Previous studies have indicated that the neuronal conversion of adult human skin fibroblasts is much harder than that for human lung fibroblasts, which are used in many experiments. Here we formally report this differential plasticity of human skin versus lung fibroblasts in their transdifferentiation to induced neurons.

Modeling Parkinson's Disease Using Patient-specific Induced Pluripotent Stem Cells.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is characterized by the degeneration of nigral dopaminergic (DA) neurons. While over 90% of cases are idiopathic, without a clear etiology, mutations in many genes have been linked to rare, familial forms of PD.

Dopamine Induces Oscillatory Activities in Human Midbrain Neurons with Parkin Mutations.

Locomotor symptoms in Parkinson's disease (PD) are accompanied by widespread oscillatory neuronal activities in basal ganglia. Here, we show that activation of dopamine D1-class receptors elicits a large rhythmic bursting of spontaneous excitatory postsynaptic currents (sEPSCs) in midbrain neurons differentiated from induced pluripotent stem cells (iPSCs) of PD patients with parkin mutations, but not normal subjects. Overexpression of wild-type parkin, but not its PD-causing mutant, abolishes the oscillatory activities in patient neurons.

Induced dopaminergic neurons: A new promise for Parkinson's disease.

Motor symptoms that define Parkinson's disease (PD) are caused by the selective loss of nigral dopaminergic (DA) neurons. Cell replacement therapy for PD has been focused on midbrain DA neurons derived from human fetal mesencephalic tissue, human embryonic stem cells (hESC) or human induced pluripotent stem cells (iPSC). Recent development in the direct conversion of human fibroblasts to induced dopaminergic (iDA) neurons offers new opportunities for transplantation study and disease modeling in PD.

Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons.

The direct conversion of fibroblasts to induced dopaminergic (iDA) neurons and other cell types demonstrates the plasticity of cell fate. The low efficiency of these relatively fast conversions suggests that kinetic barriers exist to safeguard cell-type identity. Here we show that suppression of p53, in conjunction with cell cycle arrest at G1 and appropriate extracellular environment, markedly increase the efficiency in the transdifferentiation of human fibroblasts to iDA neurons by Ascl1, Nurr1, Lmx1a and miR124.

Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.

The lack of robust Parkinson's disease (PD) phenotype in parkin knockout rodents and the identification of defective dopaminergic (DA) neurotransmission in midbrain DA neurons derived from induced pluripotent stem cells (iPSC) of PD patients with parkin mutations demonstrate the utility of patient-specific iPSCs as an effective system to model the unique vulnerabilities of midbrain DA neurons in PD. Significant efforts have been directed at developing efficient genomic engineering technologies in human iPSCs to study diseases such as PD. In the present study, we converted patient-specific iPSCs from the primed state to a naivetropic state by DOX-induced expression of transgenes (Oct4, Sox2, Klf4, c-Myc, and Nanog) and the use of 2iL (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021, and human LIF).

Parkin mutations reduce the complexity of neuronal processes in iPSC-derived human neurons.

Parkinson's disease (PD) is characterized by the degeneration of nigral dopaminergic (DA) neurons and non-DA neurons in many parts of the brain. Mutations of parkin, an E3 ubiquitin ligase that strongly binds to microtubules, are the most frequent cause of recessively inherited PD. The lack of robust PD phenotype in parkin knockout mice suggests a unique vulnerability of human neurons to parkin mutations.

Nucleolar molecular signature of pluripotent stem cells.

Induced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells to the pluripotent state. Identification and quantitative characterization of changes in the molecular organization of the cell during the process of cellular reprogramming is valuable for stem cell research and advancement of its therapeutic applications. Here we employ quantitative Raman microspectroscopy and biomolecular component analysis (BCA) for a comparative analysis of the molecular composition of nucleoli in skin fibroblasts and iPSC derived from them.

Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria.

Redefining Parkinson's disease research using induced pluripotent stem cells.

Parkinson's disease (PD) is a movement disorder associated with the degeneration of nigral dopaminergic (DA) neurons. One of the greatest obstacles for PD research is the lack of patient-specific nigral DA neurons for mechanistic studies and drug discovery. The advent of induced pluripotent stem cells (iPSCs) has overcome this seemingly intractable problem and changed PD research in many profound ways.

Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells.

Parkinson's disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Here we generate induced pluripotent stem cells from normal subjects and PD patients with parkin mutations.

Isoform specific regulation of divalent metal (ion) transporter (DMT1) by proteasomal degradation.

Divalent metal ion transporter (DMT1) is the major transporter for iron entrance into mammalian cells and iron exit from endosomes during the transferrin cycle. Four major mRNA isoforms correspond to four protein isoforms, differing at 5'/3' and N-/C-termini, respectively. Isoforms are designated 1A versus 1B reflecting where transcription starts or +iron responsive element (+IRE) versus -IRE reflecting the presence/absence of an IRE in the 3' end of the mRNA.

Parkin degrades estrogen-related receptors to limit the expression of monoamine oxidases.

Parkin, whose mutations cause Parkinson disease (PD), controls oxidative stress by limiting the expression of monoamine oxidases (MAO)--mitochondrial enzymes responsible for the oxidative de-amination of dopamine. Here, we show that parkin performed this function by increasing the ubiquitination and degradation of estrogen-related receptors (ERR), orphan nuclear receptors that play critical roles in the transcription regulation of many nuclear-encoded mitochondrial proteins. All three ERRs (α, β and γ) increased the transcription of MAOs A and B; the effects were abolished by parkin, but not by its PD-linked mutants.

Protein kinase C inhibits autophagy and phosphorylates LC3.

During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation.

Parkin protects dopaminergic neurons against microtubule-depolymerizing toxins by attenuating microtubule-associated protein kinase activation.

Mitogen-activated protein kinases, originally known as microtubule-associated protein (MAP) kinases, are activated in response to a variety of stimuli. Here we report that microtubule-depolymerizing agents such as colchicine or nocodazole induced strong activation of MAP kinases including JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose mutations are linked to Parkinson disease (PD).

Parkin suppresses the expression of monoamine oxidases.

Mutations of parkin are linked to early onset Parkinson disease. Here we show that stable transfection of parkin in the human dopaminergic neuroblastoma cell line SH-SY5Y markedly reduced the activities of both monoamine oxidase (MAO) A and B. The amount of 3,4-dihydroxyphenylacetic acid, which is produced during dopamine oxidation by MAO, was greatly reduced by parkin overexpression.

Selective vulnerability of dopaminergic neurons to microtubule depolymerization.

Parkinson disease (PD) is characterized by the specific degeneration of dopaminergic (DA) neurons in substantia nigra and has been linked to a variety of environmental and genetic factors. Rotenone, an environmental PD toxin, exhibited much greater toxicity to DA neurons in midbrain neuronal cultures than to non-DA neurons. The effect was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole.

Parkin increases dopamine uptake by enhancing the cell surface expression of dopamine transporter.

Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to Parkinson's disease (PD). Although a variety of parkin substrates have been identified, none of these is selectively expressed in dopaminergic neurons, whose degeneration plays a critical role in PD. Here we show that parkin significantly increased dopamine uptake in the human dopaminergic neuroblastoma cell line SH-SY5Y.