Mesencephalic Dopamine Neurons Research Articles

Heterogeneity of mesencephalic dopaminergic neurons: From molecular classifications, electrophysiological properties to functional connectivity.

The mesencephalic dopamine (DA) system is composed of neuronal subtypes that are molecularly and functionally distinct, are responsible for specific behaviors, and are closely associated with numerous brain disorders. Existing research has made significant advances in identifying the heterogeneity of mesencephalic DA neurons, which is necessary for understanding their diverse physiological functions and disease susceptibility. Moreover, there is a conflict regarding the electrophysiological properties of the distinct subsets of midbrain DA neurons. This review aimed to elucidate recent developments in the heterogeneity of midbrain DA neurons, including subpopulation categorization, electrophysiological characteristics, and functional connectivity to provide new strategies for accurately identifying distinct subtypes of midbrain DA neurons and investigating the underlying mechanisms of these neurons in various diseases.

The State of the Dopaminergic and Glutamatergic Systems in the Valproic Acid Mouse Model of Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a progressive neurodevelopmental disorder mainly characterized by deficits in social communication and stereotyped behaviors and interests. Here, we aimed to investigate the state of several key players in the dopamine and glutamate neurotransmission systems in the valproic acid (VPA) animal model that was administered to E12.5 pregnant females as a single dose (450 mg/kg). We report no alterations in the number of mesencephalic dopamine neurons or in protein levels of tyrosine hydroxylase in either the striatum or the nucleus accumbens. In females prenatally exposed to VPA, levels of dopamine were slightly decreased while the ratio of DOPAC/dopamine was increased in the dorsal striatum, suggesting increased turn-over of dopamine tone. In turn, levels of D1 and D2 dopamine receptor mRNAs were increased in the nucleus accumbens of VPA mice suggesting upregulation of the corresponding receptors. We also report decreased protein levels of striatal parvalbumin and increased levels of p-mTOR in the cerebellum and the motor cortex of VPA mice. mRNA levels of mGluR1, mGluR4, and mGluR5 and the glutamate receptor subunits NR1, NR2A, and NR2B were not altered by VPA, nor were protein levels of NR1, NR2A, and NR2B and those of BDNF and TrkB. These findings are of interest as clinical trials aiming at the dopamine and glutamate systems are being considered.

Genomic, transcriptomic, and metabolomic profiles of hiPSC-derived dopamine neurons from clinically discordant brothers with identical PRKN deletions

We previously reported on two brothers who carry identical compound heterozygous PRKN mutations yet present with significantly different Parkinson’s Disease (PD) clinical phenotypes. Juvenile cases demonstrate that PD is not necessarily an aging-associated disease. Indeed, evidence for a developmental component to PD pathogenesis is accumulating. Thus, we hypothesized that the presence of additional genetic modifiers, including genetic loci relevant to mesencephalic dopamine neuron development, could potentially contribute to the different clinical manifestations of the two brothers. We differentiated human-induced pluripotent stem cells (hiPSCs) derived from the two brothers into mesencephalic neural precursor cells and early postmitotic dopaminergic neurons and performed wholeexome sequencing and transcriptomic and metabolomic analyses. No significant differences in the expression of canonical dopamine neuron differentiation markers were observed. Yet our transcriptomic analysis revealed a significant downregulation of the expression of three neurodevelopmentally relevant cell adhesion molecules, CNTN6, CNTN4 and CHL1, in the cultures of the more severely affected brother. In addition, several HLA genes, known to play a role in neurodevelopment, were differentially regulated. The expression of EN2, a transcription factor crucial for mesencephalic dopamine neuron development, was also differentially regulated. We further identified differences in cellular processes relevant to dopamine metabolism. Lastly, wholeexome sequencing, transcriptomics and metabolomics data all revealed differences in glutathione (GSH) homeostasis, the dysregulation of which has been previously associated with PD. In summary, we identified genetic differences which could potentially, at least partially, contribute to the discordant clinical PD presentation of the two brothers.

Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery

Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.

Involvement of DA D3 Receptors in Structural Neuroplasticity of Selected Limbic Brain Circuits: Possible Role in Treatment-Resistant Depression.

Structural neuroplasticity in the adult brain is a process involving quantitative changes of the number and size of neurons and of their dendritic arborization, axon branching, spines, and synapses. These changes can occur in specific neural circuits as adaptive response to environmental challenges, exposure to stressors, tissue damage or degeneration. Converging studies point to evidence of structural plasticity in circuits operated by glutamate, GABA, dopamine, and serotonin neurotransmitters, in concert with neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF) or Insulin Growth Factor 1 (IGF1) and a series of modulators that include circulating hormones. Intriguingly, most of these endogenous agents trigger the activation of the PI3K/Akt/mTOR and ERK1/2 intracellular pathways that, in turn, lead to the production of growth-related structural changes, enhancing protein synthesis, metabolic enzyme functions, mitogenesis for energy, and new lipid-bilayer membrane apposition. The dopamine (DA) D3 receptor has been shown to play a specific role by inducing structural plasticity of the DAergic neurons of the nigrostriatal and mesocorticolimbic circuit, where they are expressed in rodents and humans, via activation of the mTORC1 and ERK1/2 pathways. These effects are BDNF-dependent and require the recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to allow the structural changes. Since in mood disorders, depression and anhedonia have been proposed to be associated with impaired neuroplasticity and reduced DAergic tone in brain circuits connecting prefrontal cortex, ventral striatum, amygdala, and ventral mesencephalon, activation of D3 receptors could provide a therapeutic benefit. Sustained improvements of mood and anhedonia were observed in subjects with an unsatisfactory response to serotonin uptake inhibitors (SSRI) when treated with D3-preferential D2/D3 agonists such as pramipexole and ropinirole. The recent evidence that downstream mTOR pathway activation in human mesencephalic DA neurons is also produced by ketamine, probably the most effective antidepressant currently used in subjects with treatment-resistant depression, further supports the rationale for a D3 receptor activation in mood disorders.

Mesencephalic dopamine neurons are essential for modafinil-induced arousal.

Modafinil is a potent eugeroic (wakefulness-promoting) drug that is prescribed to treat narcolepsy and has a low incidence of abuse. Although previous studies have shown that modafinil-induced arousal depends on the dopamine receptors and transporters, the specific part/s of the dopamine transmitter system underlying this mechanism remained unclear. Here, we investigated the role of mesencephalic dopamine neurons in modafinil-evoked arousal. A dopamine indicator (dLight1.1) was employed to detect dopamine changes in the nucleus accumbens (NAc) and dorsal striatum. We specifically lesioned mesencephalic dopamine neurons via diphtheria toxin (DTA) in the dopamine transporter (DAT)-Cre mice. Then, the sleep-wake states were recorded to evaluate the effect of modafinil on arousal. Finally, the extent of DTA-induced lesions was determined by immunohistochemistry. Modafinil promptly increased dopamine levels in the NAc and dorsal striatum in a dose-dependent manner. Lesioning of dopamine neurons in the substantia nigra pars compacta (SNc) or ventral tegmental area (VTA) had no significant effects on physiological sleep-wake cycles. Modafinil at 90 mg·kg-1 increased continuous wakefulness for 355 min in control mice. However, these effects were slightly decreased by 6.7% in the SNc-lesioned mice and were prominently diminished by 32.8% in VTA-lesioned mice. Furthermore, the modafinil-induced arousal was completely blocked in the SNc-VTA-lesioned mice, whereas lesions of the dorsal raphé nucleus had no effect. Taken together, our findings indicate that mesencephalic dopamine neurons are essential for modafinil-induced arousal.

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

T-Type Ca2+ Enhancer SAK3 Activates CaMKII and Proteasome Activities in Lewy Body Dementia Mice Model.

Lewy bodies are pathological characteristics of Lewy body dementia (LBD) and are composed of α-synuclein (α-Syn), which is mostly degraded via the ubiquitin–proteasome system. More importantly, 26S proteasomal activity decreases in the brain of LBD patients. We recently introduced a T-type calcium channel enhancer SAK3 (ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)- 2H-spiro[cyclopentane-1,3-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate) for Alzheimer’s disease therapeutics. SAK3 enhanced the proteasome activity via CaMKII activation in amyloid precursor protein knock-in mice, promoting the degradation of amyloid-β plaques to improve cognition. At this point, we addressed whether SAK3 promotes the degradation of misfolded α-Syn and the aggregates in α-Syn preformed fibril (PFF)-injected mice. The mice were injected with α-Syn PFF in the dorsal striatum, and SAK3 (0.5 or 1.0 mg/kg) was administered orally for three months, either immediately or during the last month after injection. SAK3 significantly inhibited the accumulation of fibrilized phosphorylated-α-Syn in the substantia nigra. Accordingly, SAK3 significantly recovered mesencephalic dopamine neurons from cell death. Decreased α-Syn accumulation was closely associated with increased proteasome activity. Elevated CaMKII/Rpt-6 signaling possibly mediates the enhanced proteasome activity after SAK3 administration in the cortex and hippocampus. CaMKII/Rpt-6 activation also accounted for improved memory and cognition in α-Syn PFF-injected mice. These findings indicate that CaMKII/Rpt-6-dependent proteasomal activation by SAK3 recovers from α-Syn pathology in LBD.

Quantitative Immunohistochemistry to Measure Regional Expression of Nurr1 in the Brain and the Effect of the Nurr1 Heterozygous Genotype.

The transcription factor Nurr1 is a member of the steroid hormone nuclear receptor superfamily. Ablation of Nurr1 expression arrests mesencephalic dopamine neuron differentiation while attenuation of Nurr1 in the subiculum and hippocampus impairs learning and memory. Additionally, reduced Nurr1 expression has been reported in patients with Parkinson’s disease and Alzheimer’s disease. In order to better understand the overall function of Nurr1 in the brain, quantitative immunohistochemistry was used to measure cellular Nurr1 protein expression, across Nurr1 immunoreactive neuronal populations. Additionally, neuronal Nurr1 expression levels were compared between different brain regions in wild-type mice (+/+) and Nurr1 heterozygous mice (+/−). Regional Nurr1 protein was also investigated at various time points after a seizure induced by pentylenetetrazol (PTZ). Nurr1 protein is expressed in various regions throughout the brain, however, a wide range of Nurr1 expression levels were observed among various neuronal populations. Neurons in the parietal and temporal cortex (secondary somatosensory, insular, auditory, and temporal association cortex) had the highest relative Nurr1 expression (100%) followed closely by the claustrum/dorsal endopiriform cortex (85%) and then subiculum (76%). Lower Nurr1 protein levels were found in neurons in the substantia nigra pars compacta and ventral tegmental area (39%) followed by CA1 (25%) and CA3 (19%) of the hippocampus. Additionally, in the parietal and temporal cortex, two distinct populations of high and medium Nurr1 expressing neurons were observed. Comparisons between +/− and +/+ mice revealed Nurr1 protein was reduced in +/− mice by 27% in the parietal/temporal cortex, 49% in the claustrum/dorsal endopiriform cortex, 25% in the subiculum, 33% in substantia nigra pars compacta, 22% in ventral tegmental area, and 21% in CA1 region of the hippocampus. Based on these data, regional mechanisms appear to exist which can compensate for a loss of a Nurr1 allele. Following a single PTZ-induced seizure, Nurr1 protein in the dentate gyrus peaked around 2 h and returned to baseline by 8 h. Since altered Nurr1 expression has been implicated in neurologic disorders and Nurr1 agonists have showed protective effects, understanding regional protein expression of Nurr1, therefore, is necessary to understand how changes in Nurr1 expression can alter brain function.

NMDA receptor membrane dynamics tunes the firing pattern of midbrain dopaminergic neurons.

NMDA receptors (NMDARs) expressed by dopamine neurons of the ventral tegmental area (VTA) play a central role in glutamate synapse plasticity, neuronal firing and adaptative behaviours. The NMDAR surface dynamics shapes synaptic adaptation in hippocampal networks, as well as associative memory. We investigated the basic properties and role of the NMDAR surface dynamics on cultured mesencephalic and VTA dopamine neurons in rodents. Using a combination of single molecule imaging and electrophysiological recordings, we demonstrate that NMDARs are highly diffusive at the surface of mesencephalic dopamine neurons. Unexpectedly, the NMDAR membrane dynamics per se regulates the firing pattern of VTA dopaminergic neurons, probably through a functional interplay between NMDARs receptors and small-conductance calcium-dependent potassium (SK) channels. Midbrain dopaminergic (DA) neurons play a central role in major physiological brain functions, and their dysfunctions have been associated with neuropsychiatric diseases. The activity of midbrain DA neurons is controlled by ion channels and neurotransmitter receptors, such as the glutamate NMDA receptor (NMDAR) and small-conductance calcium-dependent potassium (SK) channels. However, the cellular mechanisms through which these channels tune the firing pattern of midbrain DA neurons remain unclear. Here, we investigated whether the surface dynamics and distribution of NMDARs tunes the firing pattern of midbrain DA neurons. Using a combination of single molecule imaging and electrophysiological recordings, we report that NMDARs are highly diffusive at the surface of cultured midbrain DA neurons from rodents and humans. Reducing acutely the NMDAR membrane dynamics, which leaves the ionotropic function of the receptor intact, robustly altered the firing pattern of midbrain DA neurons without altering synaptic glutamatergic transmission. The reduction of NMDAR surface dynamics reduced apamin (SK channel blocker)-induced firing change and the distribution of SK3 channels in DA neurons. Together, these data show that the surface dynamics of NMDAR, and not solely its ionotropic function, tune the firing pattern of midbrain DA neurons partly through a functional interplay with SK channel function.

HOTAIR drives autophagy in midbrain dopaminergic neurons in the substantia nigra compacta in a mouse model of Parkinson's disease by elevating NPTX2 via miR-221-3p binding.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive cell loss, largely confined to mesencephalic dopamine neurons of the substantia nigra. This study investigated the functional relevance of the HOX transcript antisense intergenic RNA (HOTAIR)/microRNA-221-3 (miR-221-3p)/neuronal pentraxin II (NPTX2) axis in the process of dopaminergic neuron autophagy using PD mouse models. The PD mouse models were established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP), while PD cell model was constructed by pretreatment with 1-methyl-4-phenylpyridinium (MPP+). The expression of HOTAIR was then examined using RT-qPCR. In addition, the interactions between HOTAIR, miR-221-3p, and NPTX2 were detected through RIP and dual-luciferase reporter gene assays. CCK-8 assay was performed to measure cell viability, and the expression of autophagy-related genes was determined using Western blot analysis. HOTAIR was found to be significantly expressed in the substantia nigra compact tissues and MN9D cells following PD modeling. HOTAIR could bind to miR-221-3p and elevate the NPTX2 expression, which resulted in diminished cell viability and enhanced autophagy of dopaminergic neurons both in vitro and in vivo. In summary, down-regulation of HOTAIR could potentially inhibit the autophagy of dopaminergic neurons in the substantia nigra compacta in a mouse model of PD, thus saving the demise of dopaminergic neurons.

Role of Glial Cell Line-Derived Neurotrophic Factor in the Maintenance of Adult Mesencephalic Catecholaminergic Neurons.

The glial cell line-derived neurotrophic factor has a potent neuroprotective action on mesencephalic dopamine neurons, which are progressively lost in Parkinson's disease. Intrastriatal administration of this factor is a promising therapy for Parkinson's disease. Glial cell line-derived neurotrophic factor is naturally produced in restricted cerebral regions, such as the striatum, septum, and thalamus; however, its effects in the adult brain remain under debate. We sought to clarify the physiologic role of endogenous glial cell line-derived neurotrophic factor in the survival of catecholaminergic neurons of the substantia nigra pars compacta and the locus coeruleus in adult mice. We used 2 new Cre recombinase-based mouse models to delete a floxed-glial cell line-derived neurotrophic factor gene. The first model had Cre expression in the parvalbumin expressing interneurons, as these cells represent the major source of striatal glial cell line-derived neurotrophic factor. The second model was an estrogen receptor 2-based inducible Cre triggered by tamoxifen at 2 months of age. We found that the floxed-glial cell line-derived neurotrophic factor gene was resilient to ablation by Cre-induced recombination and that parvalbumin-driven Cre was particularly inefficient to do so. The inducible-Cre model allowed an average 70% to 80% reduction in glial cell line-derived neurotrophic factor messenger ribonucleic acid and protein in striatum and septum with moderate significant loss of catecholamine neurons in the nigrostriatal pathway and, more markedly, in the locus coeruleus. This was accompanied with mild locomotor decline. Our data support qualitatively the view that brain glial cell line-derived neurotrophic factor is needed for the maintenance of adult central catecholaminergic neurons. © 2020 International Parkinson and Movement Disorder Society.

Existence of working memory in teleosts: Establishment of the delayed matching-to-sample task in adult zebrafish

Operant conditioning is a powerful tool to study animal perception and cognition. Compared to mammals and birds, there are very few behavioral studies using operant conditioning paradigm in teleosts. Here we aim to establish matching-to-sample task (MTS) in adult zebrafish, using visual cues (colors) as discriminative stimuli. Unlike simple one-to-one color-reward association learning, MTS requires ability for context integration. In this study, zebrafish learned to perform the simultaneous-matching-to-sample (SMTS) within 15 sessions. After the SMTS training, working memory was tested by inserting a delay period (delayed matching-to-sample; DMTS). Zebrafish could perform the DMTS with a delay of at least 3–4 seconds. They could also learn to perform the DMTS even with a delay period from the beginning of the training session. These results strongly suggest that adult zebrafish possess working memory. However, our study also indicates limitations of zebrafish in cognitive flexibility or attention: they could perform SMTS/DMTS only in a certain set-up. The presence of working memory without the mesencephalic dopamine neurons indicates the convergent evolution of this function in amniotes and teleosts.

In‐Vitro Estradiol Treatment Increases the Expression of Metabotropic Glutamate Receptors in Embryonic Dopamine Neurons

Sex differences in the manifestation, prevalence, and progression of dopamine‐related diseases have been documented, suggesting a role of gonadal hormones (Bodzean et al., 2014; Kokras and Dalla, 2014). Although dopamine neurons are sensitive to estradiol (E2), main estrogen metabolite in women, it is uncertain how estradiol affects the function and survival of dopamine neurons. Eight metabotropic glutamate receptor subtypes (mGluR1‐8) have been identified and classified in three different groups according to the receptor signal transduction, pharmacology and amino acid sequence homology. mGluR subtypes are important modulators of cell proliferation (Plenz and Kitaj 1998), neuronal excitability and neurotransmitter release (Meltzer et al. 1997; Mercuri et al. 1993). The objective of our research was to study the effects of E2 on the expression of group I (mGluR1 and 5) and group II (mGluR2 and 3) mGluRs. We hypothesized that E2 increases the protein expression of group I mGluRs without affecting the expression of group II mGluRs. To test our hypothesis cultures of embryonic mesencephalic dopamine neurons were treated with either cyclodextrin or E2 at different concentrations (10nM and 10μM). Immunocytochemistry experiments showed the expression of estrogen receptors alpha (ERα), mGluR1, and mGluR2/3 subtypes in our experimental cellular model. Western bot analyses demonstrated that E2 treatment upregulates the expression of mGluR1, a group I mGluR subtype, but not group II mGluRs subtypes. Estradiol's effect was concentration dependent producing mGluR1 upregulation at 10μM but not at 10nM (F(2,9)=4.03, p=0.05). Changes in mGluRs protein expression by differences in estradiol levels may underlay variations in cells excitability and function, inducing sex differences in the neurophysiology of dopamine neurons.Support or Funding InformationThis research was supported by the Deanship of Academic Affairs of the University of Puerto Rico, Cayey Campus (FIDI‐UPR CAYEY 2017–2018) and in part by Puerto Rico Center for Environmental Neuroscience (NSF grant HRD 1137725). We also thank Barreto‐Estrada J and Perez‐Acevedo N for her support, collaboration and mentoring during this project.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Region-Specific Neuroprotective Features of Astrocytes against Oxidative Stress Induced by 6-Hydroxydopamine.

In previous studies, we found regional differences in the induction of antioxidative molecules in astrocytes against oxidative stress, postulating that region-specific features of astrocytes lead region-specific vulnerability of neurons. We examined region-specific astrocytic features against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) as an oxidative stress using co-culture of mesencephalic neurons and mesencephalic or striatal astrocytes in the present study. The 6-OHDA-induced reduction of mesencephalic dopamine neurons was inhibited by co-culturing with astrocytes. The co-culture of midbrain neurons with striatal astrocytes was more resistant to 6-OHDA than that with mesencephalic astrocytes. Furthermore, glia conditioned medium from 6-OHDA-treated striatal astrocytes showed a greater protective effect on the 6-OHDA-induced neurotoxicity and oxidative stress than that from mesencephalic astrocytes. The cDNA microarray analysis showed that the number of altered genes in both mesencephalic and striatal astrocytes was fewer than that changed in either astrocyte. The 6-OHDA treatment, apparently up-regulated expressions of Nrf2 and some anti-oxidative or Nrf2-regulating phase II, III detoxifying molecules related to glutathione synthesis and export in the striatal astrocytes but not mesencephalic astrocytes. There is a profound regional difference of gene expression in astrocytes induced by 6-OHDA. These results suggest that protective features of astrocytes against oxidative stress are more prominent in striatal astrocytes, possibly by secreting humoral factors in striatal astrocytes.

2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside attenuates MPP+/MPTP-induced neurotoxicity in vitro and in vivo by restoring the BDNF-TrkB and FGF2-Akt signaling axis and inhibition of apoptosis.

The major bioactive ingredient THSG of Polygonum multiflorum is well established for its anti-oxidation, anti-aging and anti-inflammation properties. Increasing evidence supports the capacity of THSG to ameliorate the biochemistry of neurotrophins and their downstream signaling axis in mouse models to attenuate neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In this study, the neuroprotective effects of THSG were studied in vitro and in vivo. In cultured mesencephalic dopamine neurons and SH-SY5Y cell line, it was found that THSG protected the integrity of the cell body and neurite branching from MPP+-induced toxicity by restoring the expression of FGF2 and BDNF and their downstream signaling pathways to inhibit apoptosis and promote cell survival. The inhibition of Akt signaling by LY294002 or TrkB activity by K252a eliminated the neuroprotective effects of THSG. In the MPTP-induced mouse models of Parkinson's disease, THSG ameliorated the animal behaviors against MPTP-induced neurotoxicity, which was demonstrated by the pole test and the tail suspension test. Biochemical and immunohistochemical analysis verified the THSG-mediated restoration of the FGF2-Akt and BDNF-TrkB signaling axis in the substantia nigra and corpus striatum and the recovery of dopaminergic neurons. These results establish the neuroprotective effects of THSG in vitro and in vivo and unravel the underlying mechanism against toxin-induced neural atrophy, providing a new avenue for the use and pharmacological research of edible medicine for anti-neurodegenerative diseases.

The Healing Effect of Human Milk Fat Globule-EGF Factor 8 Protein (MFG-E8) in A Rat Model of Parkinson's Disease.

We searched for drugs that alleviate the reduction of dopaminergic neurons caused by the administration of lipopolysaccharide (LPS) to the substantia nigra of the rat brain. Human milk fat globule-EGF factor 8 protein (MFG-E8) is similar to MFG-E8-S, a short isoform, of the mouse MFG-E8. However, the function of MFG-E8-S was not clear. Rats with LPS-induced Parkinson’s disease were prepared and the effects of human MFG-E8 were examined. MFG-E8 improved the significant reduction in mesencephalic dopamine neurons induced by the administration of LPS. LPS was administered to human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons to induce inflammation and the effect of MFG-E8 was examined. MFG-E8 showed no toxicity toward neurons. We reanalyzed the results using public microarray data. MFG-E8 mRNA was found to be expressed in all parts of the body, particularly by adipose-derived stem cells (ADSCs). Furthermore, we investigated the culture supernatant of ADSCs using the liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis method and successfully identified the peptide of the MFG-E8 F5/8 type C domain. The results suggested that MFG-E8-S may have a preventive effect against Parkinson’s disease.

Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat.

Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional‐motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits. It has previously been proposed that dopaminergic neurons in the ventral mesencephalon may play a role in this circuit interaction. Therefore, we studied in rats convergence of basal ganglia circuits by depositing an anterograde neuroanatomical tracer into the ventral striatum together with a retrograde fluorescent tracer ipsilaterally in the dorsolateral striatum. In the mesencephalon, using confocal microscopy, we looked for possible appositions of anterogradely labeled fibers and retrogradely labeled neurons, “enhancing” the latter via intracellular injection of Lucifer Yellow. Tyrosine hydroxylase (TH) immunofluorescence served to identify dopaminergic neurons. In neurophysiological experiments, we combined orthodromic stimulation in the medial ventral striatum with recording from ventral mesencephalic neurons characterized by antidromic stimulation from the dorsal striatum. We observed terminal fields of anterogradely labeled fibers that overlap populations of retrogradely labeled nigrostriatal cell bodies in the substantia nigra pars compacta and lateral ventral tegmental area (VTA), with numerous close appositions between boutons of anterogradely labeled fibers and nigrostriatal, TH‐immunopositive neurons. Neurophysiological stimulation in the medial ventral striatum caused inhibition of dopaminergic nigrostriatal neurons projecting to the ventrolateral striatal territory. Responding nigrostriatal neurons were located in the medial substantia nigra and adjacent VTA. Our results strongly suggest a functional link between ventromedial, emotional‐motivational striatum, and the sensorimotor dorsal striatum via dopaminergic nigrostriatal neurons.

Long 3'UTR of Nurr1 mRNAs is targeted by miRNAs in mesencephalic dopamine neurons.

The development of mesencephalic dopamine neurons and their survival later in life requires the continuous presence of the transcription factor Nurr1 (NR4A2). Nurr1 belongs to the nuclear receptors superfamily. However, it is an orphan member that does not require a ligand to regulate the transcription of its target genes. Therefore, controlling the expression of Nurr1 is an important manner to control its function. Several reports have shown that microRNAs (miRNAs) regulate Nurr1 expression. However, Nurr1 has several splicing variants, posing the question what variants are subjected to miRNA regulation. In this work, we identified a long 3'UTR variant of rat Nurr1 mRNA. We used bioinformatics analysis to identify miRNAs with the potential to regulate Nurr1 expression. Reporter assays performed with the luciferase gene fused to the short (658 bp) or long (1,339 bp) 3'UTR of rat Nurr1 mRNAs, showed that miR-93, miR-204 and miR-302d selectively regulate the mRNA with the longest 3'UTR. We found that the longest variant of Nurr1 mRNA expresses in the rat mesencephalon as assessed by PCR. The transfection of rat mesencephalic neurons with mixed miR-93, miR-204 and miR-302d resulted in a significant reduction of Nurr1 protein levels. In conclusion, Nurr1 mRNA variant with the longest 3'UTR undergoes a specific regulation by miRNAs. It is discussed the importance of fine-tuning Nurr1 protein levels in mesencephalic dopamine neurons.

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

SummaryDevelopment of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.