Development Of Dopaminergic Neurons Research Articles

Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

Long-term exposure to 6-PPD quinone at environmentally relevant concentrations causes neurotoxicity by affecting dopaminergic, serotonergic, glutamatergic, and GABAergic neuronal systems in Caenorhabditis elegans

6-PPD quinone (6-PPDQ), an emerging environmental pollutant, is converted based on 6-PPD via ozonation. However, a systematic evaluation on possible neurotoxicity of long-term and low-dose 6-PPDQ exposure and the underlying mechanism remain unknown. In the present work, 0.1–10 μg/L 6-PPDQ was added to treat Caenorhabditis elegans for 4.5 days, with locomotion behavior, neuronal development, sensory perception behavior, neurotransmitter content, and levels of neurotransmission-related genes being the endpoints. 6-PPDQ exposure at 0.1–10 μg/L significantly reduced locomotion behavior, and that at 1–10 μg/L decreased sensory perception behavior in nematodes. Moreover, 6-PPDQ exposure at 10 μg/L notably induced damage to the development of dopaminergic, glutamatergic, serotonergic, and GABAergic neurons. Importantly, nematodes with chronic 6-PPDQ exposure at 10 μg/L were confirmed to suffer obviously decreased dopamine, serotonin, glutamate, dopamine, and GABA contents and altered neurotransmission-related gene expression. Meanwhile, the potential binding sites of 6-PPDQ and neurotransmitter synthesis-related proteins were further shown by molecular docking method. Lastly, Pearson's correlation analysis showed that locomotion behavior and sensory perception behavior were positively correlated with the dopaminergic, serotonergic, glutamatergic, and GABAergic neurotransmission. Consequently, 6-PPDQ exposure disturbed neurotransmitter transmission, while such changed molecular foundation for neurotransmitter transmission was related to 6-PPDQ toxicity induction. The present work sheds new lights on the mechanisms of 6-PPDQ and its possible neurotoxicity to organisms at environmentally relevant concentrations.

Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.

Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.

Inhibition of 7α,26-dihydroxycholesterol biosynthesis promotes midbrain dopaminergic neuron development

Dysregulated cholesterol metabolism has been linked to neurodegeneration. We previously found that free, non-esterified, 7α,(25R)26-dihydroxycholesterol (7α,26-diHC), was significantly elevated in the cerebrospinal fluid of patients with Parkinson's disease (PD). In this study we investigated the role of 7α,26-diHC in midbrain dopamine (mDA) neuron development and survival. We report that 7α,26-diHC induces apoptosis and reduces the number of mDA neurons in hESC-derived cultures and in mouse progenitor cultures. Voriconazole, an oxysterol 7α-hydroxylase (CYP7B1) inhibitor, increases the number of mDA neurons and prevents the loss of mDA neurons induced by 7α,26-diHC. These effects are specific since neither 7α,26-diHC nor voriconazole alter the number of Islet1+ oculomotor neurons. Furthermore, our results suggest that elevated 24(S),25-epoxycholesterol, which has been shown to promote mDA neurogenesis, may be partially responsible for the effect of voriconazole on mDA neurons. These findings suggest that voriconazole, and/or other azole CYP7B1 inhibitors may have implications in PD therapy development.

Neurog1 and Olig2 integrate patterning and neurogenesis signals in development of zebrafish dopaminergic and glutamatergic dual transmitter neurons

Dopaminergic neurons develop in distinct neural domains by integrating local patterning and neurogenesis signals. While the proneural proteins Neurog1 and Olig2 have been previously linked to development of dopaminergic neurons, their dependence on local prepatterning and specific contributions to dopaminergic neurogenesis are not well understood. Here, we show that both transcription factors are differentially required for the development of defined dopaminergic glutamatergic subpopulations in the zebrafish posterior tuberculum, which are homologous to A11 dopaminergic neurons in mammals. Both Olig2 and Neurog1 are expressed in otpa expressing progenitor cells and appear to act upstream of Otpa during dopaminergic neurogenesis. Our epistasis analysis confirmed that Neurog1 acts downstream of Notch signaling, while Olig2 acts downstream of Shh, but upstream and/or in parallel to Notch signaling during neurogenesis of A11-type dopaminergic clusters. Furthermore, we identified Olig2 to be an upstream regulator of neurog1 in dopaminergic neurogenesis. This regulation occurs through Olig2-dependent repression of the proneural repressor and Notch target gene her2. Our study reveals how Neurog1 and Olig2 integrate local patterning signals, including Shh, with Notch neurogenic selection signaling, to specify the progenitor population and initiate neurogenesis and differentiation of A11-type dopaminergic neurons.

PLCγ1 in dopamine neurons critically regulates striatal dopamine release via VMAT2 and synapsin III

Dopamine neurons are essential for voluntary movement, reward learning, and motivation, and their dysfunction is closely linked to various psychological and neurodegenerative diseases. Hence, understanding the detailed signaling mechanisms that functionally modulate dopamine neurons is crucial for the development of better therapeutic strategies against dopamine-related disorders. Phospholipase Cγ1 (PLCγ1) is a key enzyme in intracellular signaling that regulates diverse neuronal functions in the brain. It was proposed that PLCγ1 is implicated in the development of dopaminergic neurons, while the physiological function of PLCγ1 remains to be determined. In this study, we investigated the physiological role of PLCγ1, one of the key effector enzymes in intracellular signaling, in regulating dopaminergic function in vivo. We found that cell type-specific deletion of PLCγ1 does not adversely affect the development and cellular morphology of midbrain dopamine neurons but does facilitate dopamine release from dopaminergic axon terminals in the striatum. The enhancement of dopamine release was accompanied by increased colocalization of vesicular monoamine transporter 2 (VMAT2) at dopaminergic axon terminals. Notably, dopamine neuron-specific knockout of PLCγ1 also led to heightened expression and colocalization of synapsin III, which controls the trafficking of synaptic vesicles. Furthermore, the knockdown of VMAT2 and synapsin III in dopamine neurons resulted in a significant attenuation of dopamine release, while this attenuation was less severe in PLCγ1 cKO mice. Our findings suggest that PLCγ1 in dopamine neurons could critically modulate dopamine release at axon terminals by directly or indirectly interacting with synaptic machinery, including VMAT2 and synapsin III.

Perinatal exposure to potential endocrine disrupting chemicals and autism spectrum disorder: From Norwegian birth cohort to zebrafish studies

BackgroundThe etiology of autism spectrum disorder (ASD) is multifactorial, involving genetic and environmental contributors such as endocrine-disrupting chemicals (EDCs). ObjectiveTo evaluate the association between perinatal exposure to 27 potential EDCs and ASD among Norwegian children, and to further examine the neurodevelopmental toxicity of associated chemicals using zebrafish embryos and larvae. Method1,199 mothers enrolled in the prospective birth-cohort (HUMIS, 2002–2009) study. Breastmilk levels of 27 chemicals were measured: polychlorinated biphenyls, organochlorine pesticides, polybrominated diphenyl ethers, and perfluoroalkyl substances as a proxy for perinatal exposure. We employed multivariable logistic regression to determine association, utilized elastic net logistic regression as variable selection method, and conducted an in vivo study with zebrafish larvae to confirm the neurodevelopmental effect. ResultsA total of 20 children had specialist confirmed diagnosis of autism among 1,199 mother–child pairs in this study. β-Hexachlorocyclohexane (β-HCH) was the only chemical associated with ASD, after adjusting for 26 other chemicals. Mothers with the highest levels of β-HCH in their milk had a significant increased risk of having a child with ASD (OR = 1.82, 95 % CI: 1.20, 2.77 for an interquartile range increase in ln-transformed β-HCH concentration). The median concentration of β-HCH in breast milk was 4.37 ng/g lipid (interquartile range: 2.92–6.47), and the estimated daily intake (EDI) for Norwegian children through breastfeeding was 0.03 µg/kg of body weight. The neurodevelopmental and social behavioral effects of β-HCH were established in zebrafish embryos and larvae across various concentrations, with further analysis suggesting that perturbation of dopaminergic neuron development may underlie the neurotoxicity associated with β-HCH. ConclusionsPrenatal exposure to β-HCH was associated with an increased risk of specialist-confirmed diagnoses of ASD among Norwegian children, and the EDI surpasses the established threshold. Zebrafish experiments confirm β-HCH neurotoxicity, suggesting dopaminergic neuron disruption as a potential underlying mechanism.

Disruption of dopamine metabolism by exposure to 6-PPD quinone in Caenorhabditis elegans

Caenorhabditis elegans is a useful model for examining metabolic processes and related mechanisms. We here examined the effect of exposure to N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6-PPDQ) on dopamine metabolism and underling molecular basis in nematodes. The dopamine content was reduced by 6-PPDQ (1 and 10 μg/L). Meanwhile, dopamine related behaviors (basal slowing response and area restricted searching) were changed by 6-PPDQ (1 and 10 μg/L). Exposure to 6-PPDQ (1 and 10 μg/L) decreased expressions of genes (cat-2 and bas-1) encoding enzymes governing dopamine synthesis and cat-1 encoding dopamine transporter. Development of dopaminergic neurons was also affected by 10 μg/L 6-PPDQ as reflected by decrease in fluorescence intensity, neuronal loss, and defect in dendrite development. Exposure to 6-PPDQ (1 and 10 μg/L) altered expressions of ast-1 and rcat-1 encoding upregulators of cat-2 and bas-1. The dopamine content and expressions of cat-2 and bas-1 were inhibited by RNAi of ast-1 and increased by RNAi of rcat-1 in 6-PPDQ exposed nematodes. Using endpoints of locomotion behavior and brood size, in 6-PPDQ exposed nematodes, the susceptibility to toxicity was caused by RNAi of ast-1, cat-2, bas-1, and cat-1, and the resistance to toxicity was induced by RNAi of rcat-1. Therefore, 6-PPDQ exposure disrupted dopamine metabolism and the altered molecular basis for dopamine metabolism was associated with 6-PPDQ toxicity induction. Moreover, the defects in dopamine related behaviors and toxicity on locomotion and reproduction could be rescued by treatment with 0.1 mM dopamine.

Effects of melatonin on dopaminergic neuron development via IP3-mediated mitochondrial Ca2+ regulation in autism spectrum disorder

Melatonin entrainment of suprachiasmatic nucleus-regulating circadian rhythms is mediated by MT1 and MT2 receptors. Melatonin also has neuroprotective and mitochondrial activating effects, suggesting it may affect neurodevelopment. We studied melatonin's pharmacological effects on autism spectrum disorder (ASD) neuropathology. Deciduous tooth-derived stem cells from children with ASD were used to model neurodevelopmental defects and differentiated into dopaminergic neurons (ASD-DNs) with or without melatonin. Without melatonin, ASD-DNs had reduced neurite outgrowth, mitochondrial dysfunction, lower mitochondrial Ca2+ levels, and Ca2+ accumulation in the endoplasmic reticulum (ER) compared to control DNs from typically developing children-derived stem cells. Melatonin enhanced IP3-dependent Ca2+ release from ER to mitochondria, improving mitochondrial function and neurite outgrowth in ASD-DNs. Luzindole, an MT1/MT2 antagonist, blocked these effects. Thus, melatonin supplementation may improve dopaminergic system development in ASD by modulating mitochondrial Ca2+ homeostasis via MT1/MT2 receptors.

Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases. They also represent a potential source of transplanted cells for therapeutic applications. In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development. Emerging evidence and impressive advances in human induced pluripotent stem cells, with tuned neural induction and differentiation protocols, makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible. Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol, we obtained multiple subtypes of neurons, including 20% tyrosine hydroxylase-positive dopaminergic neurons. To obtain more dopaminergic neurons, we next added sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) on day 8 of induction. This increased the proportion of dopaminergic neurons, up to 75% tyrosine hydroxylase-positive neurons, with 15% tyrosine hydroxylase and forkhead box protein A2 (FOXA2) co-expressing neurons. We further optimized the induction protocol by applying the small molecule inhibitor, CHIR99021 (CHIR).This helped facilitate the generation of midbrain dopaminergic neurons, and we obtained 31-74% midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining. Thus, we have established three induction protocols for dopaminergic neurons. Based on tyrosine hydroxylase and FOXA2 immunostaining analysis, the CHIR, SHH, and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons, which could be an ideal resource for tackling midbrain-related diseases.

Mechanisms of NURR1 Regulation: Consequences for Its Biological Activity and Involvement in Pathology.

NURR1 (Nuclear receptor-related 1 protein or NR4A2) is a nuclear protein receptor transcription factor with an essential role in the development, regulation, and maintenance of dopaminergic neurons and mediates the response to stressful stimuli during the perinatal period in mammalian brain development. The dysregulation of NURR1 activity may play a role in various diseases, including the onset and progression of neurodegenerative diseases, and several other pathologies. NURR1 is regulated by multiple mechanisms, among which phosphorylation by kinases or SUMOylation are the best characterized. Both post-translational modifications can regulate the activity of NURR1, affecting its stability and transcriptional activity. Other non-post-translational regulatory mechanisms include changes in its subcellular distribution or interaction with other protein partners by heterodimerization, also affecting its transcription activity. Here, we summarize the currently known regulatory mechanisms of NURR1 and provide a brief overview of its participation in pathological alterations.

Identification of signaling pathways modifying human dopaminergic neuron development using a pluripotent stem cell-based high-throughput screening automated system: purinergic pathways as a proof-of-principle.

Introduction: Alteration in the development, maturation, and projection of dopaminergic neurons has been proposed to be associated with several neurological and psychiatric disorders. Therefore, understanding the signals modulating the genesis of human dopaminergic neurons is crucial to elucidate disease etiology and develop effective countermeasures. Methods: In this study, we developed a screening model using human pluripotent stem cells to identify the modulators of dopaminergic neuron genesis. We set up a differentiation protocol to obtained floorplate midbrain progenitors competent to produce dopaminergic neurons and seeded them in a 384-well screening plate in a fully automated manner. Results and Discussion: These progenitors were treated with a collection of small molecules to identify the compounds increasing dopaminergic neuron production. As a proof-of-principle, we screened a library of compounds targeting purine- and adenosine-dependent pathways and identified an adenosine receptor 3 agonist as a candidate molecule to increase dopaminergic neuron production under physiological conditions and in cells invalidated for the HPRT1 gene. This screening model can provide important insights into the etiology of various diseases affecting the dopaminergic circuit development and plasticity and be used to identify therapeutic molecules for these diseases.

Genetic analysis of transcription factors in dopaminergic neuronal development in Parkinson's disease.

Genetic variants of dopaminergic transcription factor-encoding genes are suggested to be Parkinson's disease (PD) risk factors; however, no comprehensive analyses of these genes in patients with PD have been undertaken. Therefore, we aimed to genetically analyze 16 dopaminergic transcription factor genes in Chinese patients with PD. Whole-exome sequencing (WES) was performed using a Chinese cohort comprising 1917 unrelated patients with familial or sporadic early-onset PD and 1652 controls. Additionally, whole-genome sequencing (WGS) was performed using another Chinese cohort comprising 1962 unrelated patients with sporadic late-onset PD and 1279 controls. We detected 308 rare and 208 rare protein-altering variants in the WES and WGS cohorts, respectively. Gene-based association analyses of rare variants suggested that MSX1 is enriched in sporadic late-onset PD. However, the significance did not pass the Bonferroni correction. Meanwhile, 72 and 1730 common variants were found in the WES and WGS cohorts, respectively. Unfortunately, single-variant logistic association analyses did not identify significant associations between common variants and PD. Variants of 16 typical dopaminergic transcription factors might not be major genetic risk factors for PD in Chinese patients. However, we highlight the complexity of PD and the need for extensive research elucidating its etiology.

Extracellular matrix protein anosmin-1 overexpression alters dopaminergic phenotype in the CNS and the PNS with no pathogenic consequences in a MPTP model of Parkinson’s disease

The development and survival of dopaminergic neurons are influenced by the fibroblast growth factor (FGF) pathway. Anosmin-1 (A1) is an extracellular matrix protein that acts as a major regulator of this signaling pathway, controlling FGF diffusion, and receptor interaction and shuttling. In particular, previous work showed that A1 overexpression results in more dopaminergic neurons in the olfactory bulb. Prompted by those intriguing results, in this study, we investigated the effects of A1 overexpression on different populations of catecholaminergic neurons in the central (CNS) and the peripheral nervous systems (PNS). We found that A1 overexpression increases the number of dopaminergic substantia nigra pars compacta (SNpc) neurons and alters the striosome/matrix organization of the striatum. Interestingly, these numerical and morphological changes in the nigrostriatal pathway of A1-mice did not confer an altered susceptibility to experimental MPTP-parkinsonism with respect to wild-type controls. Moreover, the study of the effects of A1 overexpression was extended to different dopaminergic tissues associated with the PNS, detecting a significant reduction in the number of dopaminergic chemosensitive carotid body glomus cells in A1-mice. Overall, our work shows that A1 regulates the development and survival of dopaminergic neurons in different nuclei of the mammalian nervous system.

The Influence of Prenatal Exposure to Methamphetamine on the Development of Dopaminergic Neurons in the Ventral Midbrain.

Methamphetamine, a highly addictive central nervous system (CNS) stimulant, is used worldwide as an anorexiant and attention enhancer. Methamphetamine use during pregnancy, even at therapeutic doses, may harm fetal development. Here, we examined whether exposure to methamphetamine affects the morphogenesis and diversity of ventral midbrain dopaminergic neurons (VMDNs). The effects of methamphetamine on morphogenesis, viability, the release of mediator chemicals (such as ATP), and the expression of genes involved in neurogenesis were evaluated using VMDNs isolated from the embryos of timed-mated mice on embryonic day 12.5. We demonstrated that methamphetamine (10 µM; equivalent to its therapeutic dose) did not affect the viability and morphogenesis of VMDNs, but it reduced the ATP release negligibly. It significantly downregulated Lmx1a, En1, Pitx3, Th, Chl1, Dat, and Drd1 but did not affect Nurr1 or Bdnf expression. Our results illustrate that methamphetamine could impair VMDN differentiation by altering the expression of important neurogenesis-related genes. Overall, this study suggests that methamphetamine use may impair VMDNs in the fetus if taken during pregnancy. Therefore, it is essential to exercise strict caution for its use in expectant mothers.

Genes critical for development and differentiation of dopaminergic neurons are downregulated in Parkinson's disease.

We performed transcriptome analysis using RNA sequencing on substantia nigra pars compacta (SNpc) from mice after acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment and fromParkinson's disease (PD) patients. Acute and chronic exposure to MPTP resulted in decreased expression of genes involved in sodium channel regulation. However, upregulation of pro-inflammatory pathways was seen after single dose but not after chronic MPTP treatment. Dopamine biosynthesis and synaptic vesicle recycling pathways were downregulated in PD patients and after chronic MPTP treatment in mice. Genes essential for midbrain development and determination of dopaminergic phenotype such as, LMX1B, FOXA1, RSPO2, KLHL1, EBF3, PITX3, RGS4, ALDH1A1, RET, FOXA2, EN1, DLK1, GFRA1, LMX1A, NR4A2, GAP43, SNCA, PBX1, and GRB10 were downregulated in human PD and overexpression of GFP tagged LMX1B rescued MPP+ induced death in SH-SY5Y neurons. Downregulation of gene ensemble involved in development and differentiation of dopaminergic neurons indicate their potential involvement in pathogenesis and progression of human PD.

Isoliquiritigenin induces neurodevelopmental-toxicity and anxiety-like behavior in zebrafish larvae

Isoliquiritigenin, a flavonoid compound, exhibits a variety of pharmacological properties, including anti-inflammatory, anti-oxidative, anti-microbial, anti-viral, and anti-tumor effects. In the past few years, the consumption of isoliquiritigenin-containing dietary supplements has increased due to their health benefits. Although the neuroprotective effects of isoliquiritigenin have been well-investigated, these studies were performed in cells and adult animals. The potential effects of isoliquiritigenin on the development, especially the neurodevelopment, of certain populations, such as zebrafish larvae, have not been investigated. In this study, zebrafish larvae were employed as a model to investigate the effects of isoliquiritigenin on development and neurodevelopment. Zebrafish embryos treated with high concentrations of isoliquiritigenin (10 and 15 μM) exhibited high rates of mortality, hatching, and malformation, indicating that isoliquiritigenin can affect zebrafish development. In addition, isoliquiritigenin impeded the development of central nervous system regions and the length of dopaminergic neurons located in midbrains and thalami of transgenic zebrafish larvae. The locomotor ability of zebrafish larvae exposed to high concentrations of isoliquiritigenin was negatively affected. The total distance and the average velocity significantly decreased, and anxiety-related behaviors were observed under light–dark challenge. Furthermore, the levels of gap43, tuba1b, mbp, hcrt, vmat2, and pomc, which mediate neurodevelopment, neurotoxicity, and anxiety were significantly decreased in zebrafish larvae exposed to isoliquiritigenin. These results indicate that isoliquiritigenin can disrupt the development of dopaminergic neurons and the function of the central nervous system in zebrafish, causing anxiety-like symptoms.

Challenges in the clinical advancement of cell therapies for Parkinson's disease.

Cell therapies as potential treatments for Parkinson's disease first gained traction in the 1980s, owing to the clinical success of trials that used transplants of foetal midbrain dopaminergic tissue. However, the poor standardization of the tissue for grafting, and constraints on its availability and ethical use, have hindered this treatment strategy. Recent advances in stem-cell technologies and in the understanding of the development of dopaminergic neurons have enabled preclinical advancements of promising stem-cell therapies. To move these therapies to the clinic, appropriate levels of safety screening, as well as optimization of the cell products and the scalability of their manufacturing, will be required. In this Review, we discuss how challenges pertaining to cell sources, functional and safety testing, manufacturing and storage, and clinical-trial design are being addressed to advance the translational and clinical development of cell therapies for Parkinson's disease.

The role of crm-1 in ionizing radiation-induced nervous system dysfunction in Caenorhabditis elegans.

Ionizing radiation can cause changes in nervous system function. However, the underlying mechanism remains unclear. In this study, Caenorhabditis elegans (C. elegans) was irradiated with 75 Gy of 60Co whole-body γ radiation. Behavioral indicators (head thrashes, touch avoidance, and foraging), and the development of dopaminergic neurons related to behavioral function, were evaluated to assess the effects of ionizing radiation on nervous system function in C. elegans. Various behaviors were impaired after whole-body irradiation and degeneration of dopamine neurons was observed. This suggests that 75 Gy of γ radiation is sufficient to induce nervous system dysfunction. The genes nhr-76 and crm-1, which are reported to be related to nervous system function in human and mouse, were screened by transcriptome sequencing and bioinformatics analysis after irradiation or sham irradiation. The expression levels of these two genes were increased after radiation. Next, RNAi technology was used to inhibit the expression of crm-1, a gene whose homologs are associated with motor neuron development in other species. Downregulation of crm-1 expression effectively alleviated the deleterious effects of ionizing radiation on head thrashes and touch avoidance. It was also found that the expression level of crm-1 was regulated by the nuclear receptor gene nhr-76. The results of this study suggest that knocking down the expression level of nhr-76 can reduce the expression level of crm-1, while down-regulating the expression level of crm-1 can alleviate behavioral disorders induced by ionizing radiation. Therefore, inhibition of crm-1 may be of interest as a potential therapeutic target for ionizing radiation-induced neurological dysfunction.

Inorganic arsenic alters the development of dopaminergic neurons but not serotonergic neurons and induces motor neuron development via Sonic hedgehog pathway in zebrafish

The mechanism of inorganic arsenic-induced neurotoxicity at the cellular level is not known. In zebrafish, teratological effects of inorganic arsenic have been shown at various concentrations. Here, we used similar concentrations of inorganic arsenic to evaluate the effects on specific neuron types. Exposure of zebrafish embryos at 5 h post fertilization (hpf) to sodium arsenite induced developmental toxicity (reduced body length) in 72 hpf larvae, beginning at a concentration of 300 mg/L concentration. Mortality or overt morphological deformity was detected at 500 mg/L sodium arsenite. While 200 mg/L sodium arsenite induced development of tyrosine hydroxylase-positive (dopaminergic) neurons, there was no significant effect on the development of 5-hydroxytryptamine (serotonergic) neurons. Sodium arsenite reduced acetylcholinesterase activity. In the hb9-GFP transgenic larvae, both 200 and 400 mg/L sodium arsenite produced supernumerary motor neurons in the spinal cord. Inhibition of the Sonic hedgehog (Shh) pathway that is essential for motor neuron development, by Gant61, prevented sodium arsenite-induced supernumerary motor neuron development. Inductively coupled plasma mass spectrometry (ICP-MS) revealed that with 200 mg/L and 400 mg/L sodium arsenite treatment, each larva had an average of 387.8 pg and 847.5 pg arsenic, respectively. The data show for the first time that inorganic arsenic alters the development of dopaminergic and motor neurons in the zebrafish larvae and the latter occurs through the Shh pathway. These results may help understand why arsenic-exposed populations suffer from psychiatric disorders and motor neuron disease and Shh may, potentially, serve as a plasma biomarker of arsenic toxicity.