Mesencephalic Dopaminergic Neurons Research Articles

Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease.

ABSTRACTParkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.

Role of the Peripheral Nervous System in PD Pathology, Diagnosis, and Treatment.

Studies on Parkinson disease (PD) have mostly focused on the central nervous system—specifically, on the loss of mesencephalic dopaminergic neurons and associated motor dysfunction. However, the peripheral nervous system (PNS) is gaining prominence in PD research, with increasing clinical attention being paid to non-motor symptoms. Researchers found abnormal deposition of α-synuclein and neuroinflammation in the PNS. Attempts have been made to use these pathological changes during the clinical diagnosis of PD. Animal studies demonstrated that combined transplantation of autologous peripheral nerves and cells with tyrosine hydroxylase activity can reduce dopaminergic neuronal damage, and similar effects were observed in some clinical trials. In this review, we will systematically explain PNS performance in PD pathology and its clinical diagnostic research, describe PNS experimental results [especially Schwann cell (SC) transplantation in the treatment of PD animal models] and the results of clinical trials, and discuss future directions. The mechanism by which SCs produce such a therapeutic effect and the safety of transplantation therapy are briefly described.

A Propagated Skeleton Approach to High Throughput Screening of Neurite Outgrowth for In Vitro Parkinson's Disease Modelling.

Neuronal models of neurodegenerative diseases such as Parkinson’s Disease (PD) are extensively studied in pathological and therapeutical research with neurite outgrowth being a core feature. Screening of neurite outgrowth enables characterization of various stimuli and therapeutic effects after lesion. In this study, we describe an autonomous computational assay for a high throughput skeletonization approach allowing for quantification of neurite outgrowth in large data sets from fluorescence microscopic imaging. Development and validation of the assay was conducted with differentiated SH-SY5Y cells and primary mesencephalic dopaminergic neurons (MDN) treated with the neurotoxic lesioning compound Rotenone. Results of manual annotation using NeuronJ and automated data were shown to correlate strongly (-value for SH-SY5Y cells and -value for MDN). Pooled linear regressions of results from SH-SY5Y cell image data could be integrated into an equation formula (; for normalized results) with y depicting automated and x depicting manual data. This automated neurite length algorithm constitutes a valuable tool for modelling of neurite outgrowth that can be easily applied to evaluate therapeutic compounds with high throughput approaches.

Neuroprotective effect of Na+ /H+ exchangers isoform-1 inactivation against 6-hydroxydopamine-induced mitochondrial dysfunction and neuronal apoptosis in Parkinson's disease models.

Parkinson's disease (PD) is a disabling neurodegenerative disease mainly caused by degeneration of mesencephalic dopaminergic neurons in the substantia nigra pars compacta (SNpc). The neuroprotective role of Na+ /H+ exchangers isoform-1 (NHE1) inactivation in cerebral ischemic damage has been elucidated. The current study aimed to investigate the impacts of NHE1 in PD. In this study, 6-hydroxydopamine (6-OHDA)-induced PD rat models were established to attempt to illuminate the role and underlying mechanisms of NHE1 in SNpc neurons of PD. Meanwhile, nerve growth factor-stimulated PC12 cells followed by 6-OHDA treatment was used to mimic PD in vitro. Results showed that the protein levels of NHE1 were significantly increased in the SNpc neurons of rats and differentiated PC12 cells after 6-OHDA treatment. Inactivation of NHE1 with chemical inhibitor HOE642 suppressed SNpc neuronal loss and NHE1 expression in PD rats. The overlays of tyrosine hydroxylase and NHE1 displayed that NHE1 expression was not colocalized but closely associated with TH. Besides, treatment with HOE642 relieved the dyskinesia, mitochondrial dysfunction, and neuronal apoptosis. Further in vitro evidence confirmed that inhibition of NHE1 by genetic-knockdown prevented mitochondrial dysfunction and apoptosis. Our study represents the first experimental evidence of a potential role for NHE1 in the pathogenesis of PD.

Activating transcription factor-4 promotes neuronal death induced by Parkinson's disease neurotoxins and α-synuclein aggregates.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra resulting in severe and progressive motor impairments. However, the mechanisms underlying this neuronal loss remain largely unknown. Oxidative stress and ER stress have been implicated in PD and these factors are known to activate the integrated stress response (ISR). Activating transcription factor 4 (ATF4), a key mediator of the ISR, and has been reported to induce the expression of genes involved in cellular homeostasis. However, during prolonged activation ATF4 can also induce the expression of pro-death target genes. Therefore, in the present study, we investigated the role of ATF4 in neuronal cell death in models of PD. We demonstrate that PD neurotoxins (MPP+ and 6-OHDA) and α-synuclein aggregation induced by pre-formed human alpha-synuclein fibrils (PFFs) cause sustained upregulation of ATF4 expression in mouse cortical and mesencephalic dopaminergic neurons. Furthermore, we demonstrate that PD neurotoxins induce the expression of the pro-apoptotic factors Chop, Trb3, and Puma in dopaminergic neurons in an ATF4-dependent manner. Importantly, we have determined that PD neurotoxin and α-synuclein PFF induced neuronal death is attenuated in ATF4-deficient dopaminergic neurons. Furthermore, ectopic expression of ATF4 but not transcriptionally defective ATF4ΔRK restores sensitivity of ATF4-deficient neurons to PD neurotoxins. Finally, we demonstrate that the eIF2α kinase inhibitor C16 suppresses MPP+ and 6-OHDA induced ATF4 activation and protects against PD neurotoxin induced dopaminergic neuronal death. Taken together these results indicate that ATF4 promotes dopaminergic cell death induced by PD neurotoxins and pathogenic α-synuclein aggregates and highlight the ISR factor ATF4 as a potential therapeutic target in PD.

Human Amniotic Epithelial Cells Alleviate a Mouse Model of Parkinson's Disease Mainly by Neuroprotective, Anti-Oxidative and Anti-Inflammatory Factors.

Human amniotic epithelial cells (hAECs) have been reported to have neuroprotective roles in Parkinson's disease (PD) animal models. However, the molecular mechanism is not fully understood. The present study was designed to explore the possible mechanism by which hAECs ameliorate PD symptoms and the important paracrine factors produced by hAECs that attribute to the recovery of dopaminergic neurons. Thus, we performed in vivo and in vitro experiments with hAECs in PD models or lesioned dopaminergic neurons, respectively. First, hAECs were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and motor deficits were significantly attenuated. Second, the grafts prevented the loss of nigral dopaminergic neurons and promoted the outgrowth of neurites and striatal axon fibers in PD mice. In addition, decreased microglial activation, inflammatory factor levels and MPTP-induced excessive reactive oxygen species (ROS) levels were also observed in hAEC-treated PD mice. In vitro, we found that the conditioned medium (CM) from hAECs promoted the survival of mesencephalic dopaminergic neurons stimulated with 1-methyl-4-phenylpyridine (MPP+) and induced neurite outgrowth. Next, analysis of hAEC-CM with an antibody array of 507 soluble target proteins revealed that the levels of many neurotrophic factors, growth factors, neuronal cell adhesion molecule (NrCAM) and anti-inflammatory factors were evidently high. In addition, antibody neutralization experiments showed that many of these factors contributed to the survival and growth of dopaminergic neurons and neurite outgrowth. More importantly, we found that the anti-inflammatory factor interleukin-1 receptor antagonist (IL-1ra) also augmented the survival of dopaminergic neurons, demonstrating for the first time an anti-oxidative and anti-inflammatory role of hAECs in PD mice, which represents a novel molecular mechanism of hAECs in the treatment of PD. The molecular mechanism of hAECs recovering lesioned dopaminergic neurons and attenuating PD symptoms. First, hAECs secret manyneurotrophic factors, growth factors, and neuronal cell adhesion molecule (NrCAM) which promote the growth of the damaged dopaminergic neurons andtheir neurites. Second, hAECs produce many anti-inflammatory factors and other factors contributing to reducing the activation of microglia andsuppressing the neuroinflammation. Third, hAECs reduce the excessive ROS levels by upregulating some anti-oxidative signals.

In Vitro Protective Action of Monomeric and Fibrillar α-Synuclein on Neuronal Cells Exposed to the Dopaminergic Toxins Salsolinol and DOPAL

The aggregation of α-synuclein (aSyn) is believed to be mechanistically linked to the degeneration of dopamine (DA)-producing neurons in Parkinson's disease (PD). In this respect, one crucial question that yet remains unsolved is whether aSyn aggregation is associated with either a gain- or loss-of-function of the protein in neuronal cells. Herein, we investigated the effect of monomeric versus fibrillar aSyn on mesencephalic dopaminergic neurons in primary cultures challenged with the neurotoxic catechols: salsolinol (SALSO; 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) and 3,4-dihydroxyphenylacetaldehyde (DOPAL). aSyn monomer protected cells against either SALSO- or DOPAL-induced toxicity via inhibition of caspase-3-mediated apoptosis. While fibrillar aSyn failed in attenuating SALSO neurotoxicity, it increased the viability of DOPAL-treated cells, which was apparently not associated with the inhibition of caspase-3 cleavage. The fact that DOPAL-derived aSyn adducts exhibit lower toxicity compared with DOPAL itself raises the question of whether the generation of these adducts could be part of or a collateral effect of aSyn-mediated protection in neurons exposed to DOPAL. Overall, our work provides important evidence on the impact of the fibrillation of aSyn on its protective role in neuronal cells exposed to the toxic catechols SALSO and DOPAL.

The Neuroprotective Effect of N-Docosahexaenoyldopamine on Degenerating Dopaminergic Neurons of the Mesencephalon

This study was aimed at evaluating the neuroprotective effect of N-docosahexaenoyldopamine (DHA-DA), an endogenous bioactive lipid of the neurolipin family, on mesencephalic dopaminergic neurons of mouse fetuses in a cell culture under exposure to 1-methyl-4-phenylpyridinium+ (MPP+), a specific neurotoxin for catecholaminergic neurons. The state of neurons in the experiment (MPP+) and in the control (0.9% NaCl) was estimated according to three parameters: the number of surviving dopaminergic neurons, the length of the neurites, and the total content of dopamine in the neurons. DHA-DA was shown to have a dose-dependent neuroprotective effect on degenerating dopaminergic neurons under the influence of MPP+. DHA-DA at concentrations of 0.5 μM or lower does not have a neuroprotective effect. However, at higher concentrations it has a neuroprotective effect, preventing the death of dopaminergic neurons and the degradation of neuritis. At a concentration of 1 μM, DHA-DA protects the neuron cell bodies from degradation to a higher extent than their processes, whereas at a concentration 2 μM it protects and/or stimulates to a greater extent the growth of processes of the surviving neurons. In addition, DHA-DA reduces significantly the loss of dopamine in neurons under exposure to MPP+. Thus, it is shown that DHA-DA has a dose-dependent neuroprotective effect on degenerating dopaminergic neurons.

Combined 1-Deoxynojirimycin and Ibuprofen Treatment Decreases Microglial Activation, Phagocytosis and Dopaminergic Degeneration in MPTP-Treated Mice.

Inflammation is a predominant aspect of neurodegenerative diseases and experimental studies performed in animal models of Parkinson's disease (PD) suggesting that a sustained neuroinflammation exacerbates the nigrostriatal degeneration pathway. The central role of microglia in neuroinflammation has been studied as a target for potential neuroprotective drugs for PD, for example nonsteroidal anti-inflammatory drugs (NSAIDs) and matrix metalloproteinases (MMP) inhibitors that regulates microglial activation and migration. The aim of this study was to investigate the neuroprotective response of the iminosugar 1-deoxynojirimycin (1-DNJ) and compare its effect with a combined treatment with ibuprofen. MPTP-treated mice were orally dosed with ibuprofen and/or 1-DNJ 1. Open-field test was used to evaluate behavioral changes. Immunohistochemistry for dopaminergic neurons marker (TH+) and microglia markers (Iba-1+; CD68+) were used to investigate neuronal integrity and microglial activation in the substantia nigra pars compacta (SNpc). The pro-inflammatory cytokines TNF-α and IL-6 were analysed by qPCR. Treatments with either 1-DNJ or Ibuprofen alone did not reduce the damage induced by MPTP intoxication. However, combined treatment with 1-DNJ and ibuprofen prevents loss of mesencephalic dopaminergic neurons, decreases the number of CD68+/ Iba-1+ cells, the microglia/neurons interactions, and the pro-inflammatory cytokines, and improves behavioral changes when compared with MPTP-treated animals. In conclusion, these data demonstrate that the combined treatment with a MMPs inhibitor (1-DNJ) plus an anti-inflammatory drug (ibuprofen) has neuroprotective effects open for future therapeutic interventions. Graphical Abstract MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a protoxicant that, after crossing the Blood Brain Barrier, is metabolized by astrocytic MAO-B to MPDP+, a pyridinium intermediate, which undergoes further two-electron oxidation to yield the toxic metabolite MPP+ (methyl-phenyltetrahydropyridinium) that is then selectively transported into nigral neurons via the mesencephalic dopamine transporter. In this study, we demonstrated that MPTP induced death of dopaminergic neurons, microgliosis, increase of gliapses, motor impairment and neuroinflammation in mice, which were inhibited by combined 1-deoxynojirimycin and ibuprofen treatment.

Toward Generating Subtype-Specific Mesencephalic Dopaminergic Neurons in vitro.

Mesencephalic dopaminergic (mDA) neurons derived from pluripotent stem cells (PSCs) have proven to be pivotal for disease modeling studies and as a source of transplantable tissue for regenerative therapies in Parkinson’s disease (PD). Current differentiation protocols can generate standardized and reproducible cell products of dopaminergic neurons that elicit the characteristic transcriptional profile of ventral midbrain. Nonetheless, dopamine neurons residing in the mesencephalon comprise distinct groups of cells within diffusely defined anatomical boundaries and with distinct functional, electrophysiological, and molecular properties. Here we review recent single cell sequencing studies that are shedding light on the neuronal heterogeneity within the mesencephalon and discuss how resolving the complex molecular profile of distinct sub-populations within this region could help refine patterning and quality control assessment of PSC-derived mDA neurons to subtype-specificity in vitro. In turn, such advances would have important impact in improving cell replacement therapy, disease mechanistic studies and drug screening in PD.

Astrocyte-Derived Exosomal microRNA miR-200a-3p Prevents MPP+-Induced Apoptotic Cell Death Through Down-Regulation of MKK4.

Astrocytes release exosomes that regulate neuronal cell function. 1-methyl-4-phenylpyridinium (MPP+) is a well-known neurotoxin used to induce cell death in in vitro Parkinson's disease models, and microRNA (miRNA) transferred by released exosomes can regulate its mechanisms. Here, we demonstrated that exosomes released from normal astrocytes (ADEXs), but not exosomes derived from MPP+-stimulated astrocytes (MPP+-ADEXs), significantly attenuate MPP+-induced cell death in SH-SY5Y cells and primary mesencephalic dopaminergic neuron cultures, and reduce expression of mitogen-activated protein kinase kinase 4 (MKK4), an important upstream kinase in the c-Jun N-terminal kinase cell death pathway. Similar neuroprotective results were obtained from primary hippocampal neuron cultures, an in vitro glutamate excitotoxicity model. Through small-RNA sequencing of exosomal miRNA, we identified miR-200a-3p as the most down-regulated miRNA expressed in MPP+-ADEXs. miRNA target analysis and reporter assay confirmed that miR-200a-3p targets MKK4 through binding to two independent sites on the 3'-UTR of Map2k4/MKK4 mRNA. Treatment with miR-200a-3p mimic suppressed both MKK4 mRNA and protein expressions, and attenuated cell death in MPP+-treated SH-SY5Y cells and glutamate-treated hippocampal neuron cultures. Our results suggest that normal astrocytes release miR-200a-3p which exhibits a neuroprotective effect through down-regulation of MKK4.

Effect of Wenshen-Yanggan Decoction on Movement Disorder and Substantia Nigra Dopaminergic Neurons in Mice with Chronic Parkinson's Disease.

This study aimed to explore the protective effects of Wenshen-Yanggan decoction on dopaminergic (DA) neuron injury in a rotenone-induced mouse model with chronic Parkinson's disease (PD) and explore its mechanism of action. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure the content of six main components in the Wenshen-Yanggan decoction. The chronic PD mouse model was established by treating 10-month-old healthy wild C57BL/6 male mice with rotenone 30 mg/kg/day for 28 days in succession. The pole test and rotarod test were applied to detect the rescue effect of Wenshen-Yanggan decoction in high, medium, and low dosages, respectively, on PD-like behaviors in mice with chronic PD. The protective effect of Wenshen-Yanggan decoction on the mesencephalic nigrostriatal DA neuron injury was determined employing tyrosine hydroxylase (TH) immunofluorescence staining. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the inflammatory cytokines in serum, including TNF-α (tumor necrosis factor-alpha), IFN-γ (interferon gamma), NF-κB (nuclear factor kappa-B), and IL-1β (interleukin-1 beta). Western blotting was performed to quantify the expression of phosphorylated c-Jun N-terminal kinase (p-JNK), cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), and NF-κB in the brain. Our results showed that the Wenshen-Yanggan decoction in high, medium, and low dosages reduced the turning time of mice (P < 0.01, P < 0.01, and P < 0.05). The high and medium dosages shortened the total climbing time of PD mice in the pole test (P < 0.01 and P < 0.05). Meanwhile, the high, medium, and low dosages increased the rod-standing time of PD mice in the rotarod test (P < 0.01, P < 0.05, and P < 0.05). Besides, the decoction reversed the decrease in TH-positive neurons induced by rotenone, upregulated TH protein expression, and downregulated the α-syn expression in the PD model. Moreover, the decoction in high dosage significantly inhibited the expression of p-JNK, cleaved caspase-3, and NF-κB in the midbrain of PD mice (P < 0.05, P < 0.05, and P < 0.01), upregulated the expression of Bcl-2 (P < 0.05), and decreased the content of TNF-α, IFN-γ, NF-κB, and IL-1β in the serum (P < 0.001, P < 0.001, P < 0.001, and P < 0.001). Taken together, the Wenshen-Yanggan decoction could protect mice from rotenone-induced chronic PD, which might be related to the reduction of the DA neuron apoptosis via suppressing the inflammatory reaction and the neuronal apoptosis pathway.

Fatty Acid-Binding Protein 3 is Critical for α-Synuclein Uptake and MPP+-Induced Mitochondrial Dysfunction in Cultured Dopaminergic Neurons.

α-Synuclein is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinson′s disease and other synucleinopathies. Fatty acids partially regulate α-Synuclein accumulation, and mesencephalic dopaminergic neurons highly express fatty acid-binding protein 3 (FABP3). We previously demonstrated that FABP3 knockout mice show decreased α-Synuclein oligomerization and neuronal degeneration of tyrosine hydroxylase (TH)-positive neurons in vivo. In this study, we newly investigated the importance of FABP3 in α-Synuclein uptake, 1-methyl-4-phenylpyridinium (MPP+)-induced axodendritic retraction, and mitochondrial dysfunction. To disclose the issues, we employed cultured mesencephalic neurons derived from wild type or FABP3−/− C57BL6 mice and performed immunocytochemical analysis. We demonstrated that TH+ neurons from FABP3+/+ mice take up α-Synuclein monomers while FABP3−/− TH+ neurons do not. The formation of filamentous α-Synuclein inclusions following treatment with MPP+ was observed only in FABP3+/+, and not in FABP3−/− neurons. Notably, detailed morphological analysis revealed that FABP−/− neurons did not exhibit MPP+-induced axodendritic retraction. Moreover, FABP3 was also critical for MPP+-induced reduction of mitochondrial activity and the production of reactive oxygen species. These data indicate that FABP3 is critical for α-Synuclein uptake in dopaminergic neurons, thereby preventing synucleinopathies, including Parkinson′s disease.

Gastrointestinal non-motor dysfunction in Parkinson's disease model rats with 6-hydroxydopamine.

Parkinson's disease (PD) is a neurodegenerative disease with a progressive loss of mesencephalic dopaminergic neurons of the substantia nigra (SN). To further evaluate its pathophysiology, accurate animal models are needed. The current study aims to verify the impact of a 6-hydroxydopamine (6-OHDA) bilateral microinjection into the SN on gastrointestinal symptoms in rats and confirm that the 6-OHDA rat model is an appropriate tool to investigate the mechanisms of Parkinsonian GI disorders. Immunohistochemistry, digital X-ray imaging, short-circuit current, FITC-dextran permeability and ultra-performance liquid chromatography tandem mass spectrometry were used in this study. The results indicated that the dopaminergic neurons in SN and fibres in the striatum were markedly reduced in 6-OHDA rats. The 6-OHDA rats manifested reductions in occupancy in a rotarod test and increases in daily food debris but no difference in body mass or daily consumption. Compared with control rats, faecal pellets and their contents were significantly decreased, whereas gastric emptying and intestinal transport were delayed in 6-OHDA rats. The increased in vivo FITC-dextran permeability and decreased intestinal transepithelial resistance in the model suggest attenuated barrier function in the digestive tract in the PD model. Moreover, inflammatory factors in the plasma showed that pro-inflammatory factors IL-1? and IL-8 were significantly increased in 6-OHDA rats. Collectively, these findings indicate that the model is an interesting experimental tool to investigate the mechanisms involved in the progression of gastrointestinal dysfunction in PD.

Dopaminergic modulation of striatal function and Parkinson's disease.

The striatum is richly innervated by mesencephalic dopaminergic neurons that modulate a diverse array of cellular and synaptic functions that control goal-directed actions and habits. The loss of this innervation has long been thought to be the principal cause of the cardinal motor symptoms of Parkinson's disease (PD). Moreover, chronic, pharmacological overstimulation of striatal dopamine (DA) receptors is generally viewed as the trigger for levodopa-induced dyskinesia (LID) in late-stage PD patients. Here, we discuss recent advances in our understanding of the relationship between the striatum and DA, particularly as it relates to PD and LID. First, it has become clear that chronic perturbations of DA levels in PD and LID bring about cell type-specific, homeostatic changes in spiny projection neurons (SPNs) that tend to normalize striatal activity. Second, perturbations in DA signaling also bring about non-homeostatic aberrations in synaptic plasticity that contribute to disease symptoms. Third, it has become evident that striatal interneurons are major determinants of network activity and behavior in PD and LID. Finally, recent work examining the activity of SPNs in freely moving animals has revealed that the pathophysiology induced by altered DA signaling is not limited to imbalance in the average spiking in direct and indirect pathways, but involves more nuanced disruptions of neuronal ensemble activity.

Lethal Factor Domain-Mediated Delivery of Nurr1 Transcription Factor Enhances Tyrosine Hydroxylase Activity and Protects from Neurotoxin-Induced Degeneration of Dopaminergic Cells

The orphan transcription factor nuclear receptor-related 1 protein (Nurr1, also known as NR4A2) plays a key role in embryonic development and maintenance of mesencephalic dopaminergic neurons in the substantia nigra. Nurr1 deficiency is associated with Parkinson’s disease where dopaminergic neurons degenerate suggesting that counter-regulation of Nurr1 activity may have therapeutic effects. Here, we bacterially expressed and isolated a human Nurr1 fusion protein containing a N-terminal cell delivery domain derived from detoxified anthrax lethal factor followed by wild type ubiquitin with deubiquitinating enzyme recognition site for intracellular cleavage. Addition of the Nurr1 fusion protein to dopaminergic SH-SY5Y cells generated a cleaved, cytosolic Nurr1-containing fragment which was associated with increased levels of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Promoter-activity assays confirmed that exposure of cells to full-length Nurr1 fusion protein activated not only its cognate human tyrosine hydroxylase promoter but also the corresponding mouse sequence, although at a reduced efficiency. Using 6-hydroxydopamine as a dopaminergic cell specific neurotoxin, we demonstrate that full-length Nurr1 fusion protein promotes a concentration-dependent protection from this toxic insult. Altogether, the enhancement of tyrosine hydroxylase in naïve dopaminergic cells and the protective effects in a cellular model of Parkinson’s disease suggest that full-length Nurr1 fusion protein may contribute to the development of a novel concept of protein-based therapy.

Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression.

LINE-1 mobile genetic elements have shaped the mammalian genome during evolution. A minority of them have escaped fossilization which, when activated, can threaten genome integrity. We report that LINE-1 are expressed in substantia nigra ventral midbrain dopaminergic neurons, a class of neurons that degenerate in Parkinson's disease. In Engrailed-1 heterozygotes, these neurons show a progressive degeneration that starts at 6weeks of age, coinciding with an increase in LINE-1 expression. Similarly, DNA damage and cell death, induced by an acute oxidative stress applied to embryonic midbrain neurons in culture or to adult midbrain dopaminergic neurons invivo, are accompanied by enhanced LINE-1 expression. Reduction of LINE-1 activity through (i) direct transcriptional repression by Engrailed, (ii) a siRNA directed against LINE-1, (iii) the nucleoside analogue reverse transcriptase inhibitor stavudine, and (iv) viral Piwil1 expression, protects against oxidative stress invitro and invivo We thus propose that LINE-1 overexpression triggers oxidative stress-induced DNA strand breaks and that an Engrailed adult function is to protect mesencephalic dopaminergic neurons through the repression of LINE-1 expression.

Sequential Application of Discrete Topographical Patterns Enhances Derivation of Functional Mesencephalic Dopaminergic Neurons from Human Induced Pluripotent Stem Cells

Parkinson’s Disease is a progressive neurodegenerative disorder attributed to death of mesencephalic dopaminergic (DA) neurons. Pluripotent stem cells have great potential in the study for this late-onset disease, but acquirement of cells that are robust in quantity and quality is still technically demanding. Biophysical cues have been shown to direct stem cell fate, but the effect of different topographies in the lineage commitment and subsequent maturation stages of cells have been less examined. Using human induced pluripotent stem cells (iPSCs), we applied topographical patterns sequentially during differentiation stages and examined their ability to influence derivation yield and functionality of regionalized subtype-specific DA neurons. Gratings showed higher yield of DA neurons and may be beneficial for initial lineage commitment. Cells derived on pillars in the terminal differentiation stage have increased neuronal complexity, and were more capable of firing repetitive action potentials, showing that pillars yielded better network formation and functionality. Our topography platform can be applied to patient-derived iPSCs as well, and that cells harbouring LRRK2 mutation were more functionally mature when optimal topographies were applied sequentially. This will hopefully accelerate development of robust cell models that will provide novel insights into discovering new therapeutic approaches for Parkinson’s Disease.

The role of p27kip1 in the development, differentiation and maturation of mesencephalic dopaminergic neurons.

The role of p27kip1 in the development, differentiation and maturation of mesencephalic dopaminergic neurons.

Central and Peripheral Nervous System Progenitors Derived from Human Pluripotent Stem Cells Reveal a Unique Temporal and Cell-Type Specific Expression of PMCAs.

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca2+ regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca2+ changes, which can ultimately correspond to changes in specific stem cell transcription states.