-
Transcription Factor-Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells.Methods in Molecular Biology (Clifton,... 2023The ability to differentiate pluripotent stem cells and to generate specific cell types is a long-standing goal of regenerative medicine. This can be accomplished by...
The ability to differentiate pluripotent stem cells and to generate specific cell types is a long-standing goal of regenerative medicine. This can be accomplished by recreating the developmental trajectories using sequential activation of the corresponding signaling pathways, or more recently-by direct programming of cell identities using lineage-specific transcription factors. Notably, to be functional in cell replacement therapies, generation of complex cell types, such as specialized neuronal sub-types of the brain, requires precise induction of molecular profiles and regional specification of the cells. However, the induction of the correct cellular identity and marker gene expression can be hampered by technical challenges, one of which is the robust co-expression of multiple transcription factors that is often required for correct cell identity specification. Here, we describe in detail a method for co-expression of seven transcription factors required for efficient induction of dopaminergic neurons with midbrain characteristics from human embryonic and induced pluripotent stem cells.
Topics: Humans; Transcription Factors; Dopaminergic Neurons; Neural Stem Cells; Pluripotent Stem Cells; Neurogenesis; Induced Pluripotent Stem Cells; Cell Differentiation
PubMed: 37300765
DOI: 10.1007/978-1-0716-3287-1_4 -
Frontiers in Immunology 2023Tourette syndrome (TS) is associated with immunological dysfunction. The DA system is closely related to TS development, or behavioral stereotypes. Previous evidence...
BACKGROUND
Tourette syndrome (TS) is associated with immunological dysfunction. The DA system is closely related to TS development, or behavioral stereotypes. Previous evidence suggested that hyper-M1-polarized microglia may exist in the brains of TS individuals. However, the role of microglia in TS and their interaction with dopaminergic neurons is unclear. In this study, we applied iminodipropionitrile (IDPN) to establish a TS model and focused on the inflammatory injury in the striatal microglia-dopaminergic-neuron crosstalk.
METHODS
Male Sprague-Dawley rats were intraperitoneally injected with IDPN for seven consecutive days. Stereotypic behavior was observed to verify the TS model. Striatal microglia activation was evaluated based on different markers and expressions of inflammatory factors. The striatal dopaminergic neurons were purified and co-cultured with different microglia groups, and dopamine-associated markers were assessed.
RESULTS
First, there was pathological damage to striatal dopaminergic neurons in TS rats, as indicated by decreased expression of TH, DAT, and PITX3. Next, the TS group showed a trend of increased Iba-1 positive cells and elevated levels of inflammatory factors TNF-α and IL-6, as well as an enhanced M1-polarization marker (iNOS) and an attenuated M2-polarization marker (Arg-1). Finally, in the co-culture experiment, IL-4-treated microglia could upregulate the expression of TH, DAT, and PITX3 in striatal dopaminergic neurons LPS-treated microglia. Similarly, the TS group (microglia from TS rats) caused a decreased expression of TH, DAT, and PITX3 compared with the Sham group (microglia from control rats) in the dopaminergic neurons.
CONCLUSION
In the striatum of TS rats, microglia activation is M1 hyperpolarized, which transmits inflammatory injury to striatal dopaminergic neurons and disrupts normal dopamine signaling.
Topics: Rats; Male; Animals; Microglia; Dopaminergic Neurons; Rats, Sprague-Dawley; Dopamine; Tourette Syndrome
PubMed: 37187752
DOI: 10.3389/fimmu.2023.1178113 -
Journal of Neurochemistry Feb 2021Glial cell line-derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those...
Glial cell line-derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those results are still being debated. Glial cell line-derived neurotrophic factor (gdnf) is highly conserved in zebrafish and plays a role in enteric nervous system function. However, little is known about gdnf function in the teleost brain. Here, we employed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to impede gdnf function in the maintenance of dopaminergic neuron development. Genotyping of gdnf crispants revealed successful deletions of the coding region with various mutant band sizes and down-regulation of gdnf transcripts at 1, 3 and 7 day(s) post fertilization. Notably, ~20% reduction in ventral diencephalic dopaminergic neuron numbers in clusters 8 and 13 was observed in the gdnf-deficient crispants. In addition, gdnf depletion caused a modest reduction in dopaminergic neurogenesis as determined by 5-ethynyl-2'-deoxyuridine pulse chase assay. These deleterious effects could be partly attributed to deregulation of dopaminergic neuron fate specification-related transcription factors (otp,lmx1b,shha,and ngn1) in both crispants and established homozygous mutants with whole mount in-situ hybridization (WISH) on gdnf mutants showing reduced otpb and lmx1b.1 expression in the ventral diencephalon. Interestingly, locomotor function of crispants was only impacted at 7 dpf, but not earlier. Lastly, as expected, gdnf deficiency heightened crispants vulnerability to 1-methyl-4-phenylpyridinium toxic insult. Our results suggest conservation of teleost gdnf brain function with mammals and revealed the interactions between gdnf and transcription factors in dopaminergic neuron differentiation.
Topics: Animals; Animals, Genetically Modified; Cell Differentiation; Diencephalon; Dopaminergic Neurons; Glial Cell Line-Derived Neurotrophic Factor; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 32583440
DOI: 10.1111/jnc.15108 -
ELife Jun 2022New findings cast doubt on whether suppressing the RNA-binding protein PTBP1 can force astrocytes to become dopaminergic neurons.
New findings cast doubt on whether suppressing the RNA-binding protein PTBP1 can force astrocytes to become dopaminergic neurons.
Topics: Astrocytes; Cells, Cultured; Dopaminergic Neurons; Gatekeeping
PubMed: 35723428
DOI: 10.7554/eLife.80232 -
Phytomedicine : International Journal... Jul 2022Parkinson's disease (PD) is an age-related neurodegenerative disorder without effective treatments. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been...
BACKGROUND
Parkinson's disease (PD) is an age-related neurodegenerative disorder without effective treatments. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been suggested to be capable of protecting against PD by inhibiting endoplasmic reticulum (ER) stress-mediated neuronal apoptosis.
PURPOSE
This study was aimed to evaluate the antiparkinsonian effect of dendrobine and reveal its underlying mechanisms from the perspective of MANF-mediated ER stress suppression.
METHODS
Behavioral assessments of PD mice as well as LDH/CCK-8 assay in SH-SY5Y cells and primary midbrain neurons were carried out to detect the antiparkinsonian effect of dendrobine. Immunofluorescence, western blot, flow cytometry and shRNA-mediated MANF knockdown were used to determine the apoptosis of dopaminergic neurons and the expressions of ER stress-related proteins for investigating the underlying mechanism of dendrobine.
RESULTS
Dendrobine significantly ameliorated the motor performance of PD mice and attenuated the injuries of dopaminergic neurons. Dendrobine could also relieve neuronal apoptosis, up-regulate MANF expression and inhibit ER stress, which were largely abolished by shRNA-mediated MANF knockdown in PD model.
CONCLUSION
Dendrobine might protect against PD by inhibiting dopaminergic neuron apoptosis, which was achieved by facilitating MANF-mediated ER stress suppression. Our study suggested that dendrobine could act as a MANF up-regulator to protect against PD, and provided a potential candidate for exploring etiological agents of PD.
Topics: Alkaloids; Animals; Antiparkinson Agents; Apoptosis; Dopamine; Dopaminergic Neurons; Endoplasmic Reticulum Stress; Humans; Mice; Nerve Growth Factors; Neuroblastoma; Parkinson Disease; RNA, Small Interfering
PubMed: 35636177
DOI: 10.1016/j.phymed.2022.154193 -
Journal of Neural Transmission (Vienna,... Apr 2019Neurodegeneration of the nigrostriatal dopaminergic system and concurrent dopamine (DA) deficiency in the basal ganglia represent core features of Parkinson's disease... (Review)
Review
Neurodegeneration of the nigrostriatal dopaminergic system and concurrent dopamine (DA) deficiency in the basal ganglia represent core features of Parkinson's disease (PD). Despite the central role of DA in the pathogenesis of PD, dopaminergic systems outside of the midbrain have not been systematically investigated for Lewy body pathology or neurodegeneration. Dopaminergic neurons show a surprisingly rich neurobiological diversity, suggesting that there is not one general type of dopaminergic neuron, but rather a spectrum of different dopaminergic phenotypes. This heterogeneity on the cellular level could account for the observed differences in susceptibility of the dopaminergic systems to the PD disease process. In this review, we will summarize the long history from the first description of PD to the rationally derived DA replacement therapy, describe the basal neuroanatomical and neuropathological features of the different dopaminergic systems in health and PD, explore how neuroimaging techniques broadened our view of the dysfunctional dopaminergic systems in PD and discuss how dopaminergic replacement therapy ameliorates the classical motor symptoms but simultaneously induces a new set of hyperdopaminergic symptoms.
Topics: Animals; Brain; Dopaminergic Neurons; Humans; Parkinson Disease
PubMed: 30643975
DOI: 10.1007/s00702-019-01970-9 -
Brain, Behavior, and Immunity May 2021Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children. The orexigenic hormone ghrelin is important in...
Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children. The orexigenic hormone ghrelin is important in neuroprotection and neurodevelopment, which may play an important role in psychopathogenesis of ADHD. This study aimed to systematically investigate the genomic and pharmacological manipulations of ghrelin functioning in ADHD-like symptoms in zebrafish models and validated the effects of ghrelin polymorphisms in human subjects with ADHD. We firstly generated ghrelin zebrafish mutant, which displayed hyperactive, attention deficit-like and impulsive-like behaviors, as well as endophenotypes, mimicking human ADHD. Ghrelin zebrafish exhibited downregulated expression levels of wnt1, wnt3a, wnt5a that are critical for dopaminergic neuron development to possibly regulate their number and spatial organization. Pharmacological blockade of wnt signaling with XAV939 induced a reduced moving activity and less dopaminergic neurons; whereas, wnt agonist SB415286 rescued hyperactivity and dopaminergic neuron loss in ghrelin zebrafish. In addition, we further identified and validated a SNP, rs696217, on orexigenic hormone preproghrelin/ghrelin (T408T, Met72Met) to be associated with a higher risk of ADHD in a case-controlled association study with 248 subjects with ADHD and 208 subjects of healthy controls. Together, our results reveal a novel endogenous role for orexigenic hormone ghrelin in ADHD, which provides insights into genetic regulation and drug screens for the identification of novel treatments of ADHD.
Topics: Animals; Attention Deficit Disorder with Hyperactivity; Child; Dopaminergic Neurons; Ghrelin; Humans; Impulsive Behavior; Zebrafish
PubMed: 33412253
DOI: 10.1016/j.bbi.2020.12.029 -
Methods in Molecular Biology (Clifton,... 2021Dopaminergic (DA) neurons regulate various physiological functions, including motor function, emotion, learning, sleep, and arousal. Degeneration of DA neurons in the...
Dopaminergic (DA) neurons regulate various physiological functions, including motor function, emotion, learning, sleep, and arousal. Degeneration of DA neurons in the substantia nigra of the midbrain causes motor disturbance in Parkinson's disease (PD). Studies on familial PD have revealed that a subset of PD genes encode proteins that regulate mitochondrial function and synaptic dynamics. Drosophila is a powerful model of PD, whereby genetic interactions of PD genes with well-conserved cellular signaling can be evaluated. Morphological changes in mitochondria, along with dysfunction and degeneration of DA neurons, have been reported in many studies using Drosophila PD models. In this chapter, we will describe imaging methods to visualize mitochondria in DA neurons and to evaluate spontaneous neural activity of DA neurons in the Drosophila brain.
Topics: Animals; Disease Models, Animal; Dopamine; Dopaminergic Neurons; Drosophila; Mesencephalon; Mitochondria; Parkinson Disease; Substantia Nigra
PubMed: 34043204
DOI: 10.1007/978-1-0716-1495-2_18 -
International Journal of Molecular... Apr 2018Oxidative stress is increasingly recognized as a central event contributing to the degeneration of dopaminergic neurons in the pathogenesis of Parkinson's disease (PD).... (Review)
Review
Oxidative stress is increasingly recognized as a central event contributing to the degeneration of dopaminergic neurons in the pathogenesis of Parkinson's disease (PD). Although reactive oxygen species (ROS) production is implicated as a causative factor in PD, the cellular and molecular mechanisms linking oxidative stress with dopaminergic neuron death are complex and not well characterized. The primary insults cause the greatest production of ROS, which contributes to oxidative damage by attacking all macromolecules, including lipids, proteins and nucleic acids, leading to defects in their physiological function. Consequently, the defects in these macromolecules result in mitochondrial dysfunction and neuroinflammation, which subsequently enhance the production of ROS and ultimately neuronal damage. The interaction between these various mechanisms forms a positive feedback loop that drives the progressive loss of dopaminergic neurons in PD, and oxidative stress‑mediated neuron damage appears to serve a central role in the neurodegenerative process. Thus, understanding the cellular and molecular mechanisms by which oxidative stress contributes to the loss of dopaminergic neurons may provide a promising therapeutic approach in PD treatment.
Topics: Animals; Cell Death; Dopaminergic Neurons; Humans; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Parkinson Disease; Reactive Oxygen Species
PubMed: 29393357
DOI: 10.3892/ijmm.2018.3406 -
Neuroscience Letters Feb 2022Our previous investigation showed Wnt signal pathway was significantly activated during DA neuron differentiation of epiblast-derived stem cells. In this study, we next...
Our previous investigation showed Wnt signal pathway was significantly activated during DA neuron differentiation of epiblast-derived stem cells. In this study, we next attempt to examine the therapeutic potential of the purified exosomes derived bone marrow mesenchymal stem cells (BMSCs) by administrating exosomes into the rat striatum of parkinson's disease (PD) animal model. Results revealed that the protein levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-alpha (TNF-α), and reactive oxygen species (ROS) in the substantia nigra of PD rats were down regulated after injection of BMSC induced-Exosomes into the striatum of PD model compared to BMSC quiescent-Exosomes. In addition, the expression of ionized calcium binding adaptor molecule 1 (Iba1) mRNA was significantly decreased, while the expression of tyrosine hydroxylase (TH) mRNA was increased after injection of BMSC induced-Exosomes. Injection of BMSC induced-Exosomes into the striatum rescued the rotation behavior and climbing speed in the PD rats. More importantly, Wnt5a was found to be enriched in BMSC induced Exosomes, which could be effectively transferred to the substantia nigra of PD rats. In conclusion, these findings demonstrated that exosomes isolated during dopaminergic neuron differentiation could rescue the pathogenic features of Parkinson's disease by reshaping the inflammatory microenvironment in the substantia nigra and repairing the injury to DA nerves.
Topics: Animals; Calcium-Binding Proteins; Cells, Cultured; Dopaminergic Neurons; Exosomes; Interleukin-6; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Microfilament Proteins; Neurogenesis; Parkinson Disease; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Substantia Nigra; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase
PubMed: 34954117
DOI: 10.1016/j.neulet.2021.136414