Ventral Midbrain Research Articles

Verification of a genetic locus for methamphetamine intake and the impact of morphine.

A quantitative trait locus (QTL) on proximal chromosome (Chr) 10 accounts for > 50% of the genetic variance in methamphetamine (MA) intake in mice selectively bred for high (MAHDR) and low (MALDR) voluntary MA drinking. The µ-opioid receptor (MOP-r) gene, Oprm1, resides at the proximal end of Chr 10, and buprenorphine reduces MA intake in MAHDR mice. However, this drug has only partial agonist effects at MOP-r. We investigated the impact of a full MOP-r agonist, morphine, on MA intake and saccharin intake, measured MOP-r density and affinity in several brain regions of the MA drinking lines and their C57BL/6J (B6) and DBA/2J (D2) progenitor strains, and measured MA intake in two congenic strains of mice to verify the QTL and reduce the QTL interval. Morphine reduced MA intake in the MAHDR line, but also reduced saccharin and total fluid intake. MOP-r density was lower in the medial prefrontal cortex of MAHDR, compared to MALDR, mice, but not in the nucleus accumbens or ventral midbrain; there were no MOP-r affinity differences. No significant differences in MOP-r density or affinity were found between the progenitor strains. Finally, Chr 10 congenic results were consistent with previous data suggesting that Oprm1 is not a quantitative trait gene, but is impacted by the gene network underlying MA intake. Stimulation of opioid pathways by a full agonist can reduce MA intake, but may also non-specifically affect consummatory behavior; thus, a partial agonist may be a better pharmacotherapeutic.

Parkinson's Disease-Associated LRRK2 Hyperactive Kinase Mutant Disrupts Synaptic Vesicle Trafficking in Ventral Midbrain Neurons.

Parkinson's disease (PD) is characterized pathologically by the selective loss of substantia nigra (SN) dopaminergic (DAergic) neurons. Recent evidence has suggested a role of LRRK2, linked to the most frequent familial PD, in regulating synaptic vesicle (SV) trafficking. However, the mechanism whereby LRRK2 mutants contribute to nigral vulnerability remains unclear. Here we show that the most common PD mutation LRRK2 G2019S impairs SV endocytosis in ventral midbrain (MB) neurons, including DA neurons, and the slowed endocytosis can be rescued by inhibition of LRRK2 kinase activity. A similar endocytic defect, however, was not observed in LRRK2 mutant neurons from the neocortex (hereafter, cortical neurons) or the hippocampus, suggesting a brain region-specific vulnerability to the G2019S mutation. Additionally, we found MB-specific impairment of SV endocytosis in neurons carrying heterozygous deletion of SYNJ1 (PARK20), a gene that is associated with recessive Parkinsonism. Combining SYNJ1+/- and LRRK2 G2019S does not exacerbate SV endocytosis but impairs sustained exocytosis in MB neurons and alters specific motor functions of 1-year-old male mice. Interestingly, we show that LRRK2 directly phosphorylates synaptojanin1 in vitro, resulting in the disruption of endophilin-synaptojanin1 interaction required for SV endocytosis. Our work suggests a merge of LRRK2 and SYNJ1 pathogenic pathways in deregulating SV trafficking in MB neurons as an underlying molecular mechanism of early PD pathogenesis.SIGNIFICANCE STATEMENT Understanding midbrain dopaminergic (DAergic) neuron-selective vulnerability in PD is essential for the development of targeted therapeutics. We report, for the first time, a nerve terminal impairment in SV trafficking selectively in MB neurons but not cortical neurons caused by two PARK genes: LRRK2 (PARK8) and SYNJ1 (PARK20). We demonstrate that the enhanced kinase activity resulting from the most frequent G2019S mutation in LRRK2 is the key to this impairment. We provide evidence suggesting that LRRK2 G2019S and SYNJ1 loss of function share a similar pathogenic pathway in deregulating DAergic neuron SV endocytosis and that they play additive roles in facilitating each other's pathogenic functions in PD.

Vitamin C-Induced Epigenetic Modifications in Donor NSCs Establish Midbrain Marker Expressions Critical for Cell-Based Therapy in Parkinson's Disease.

SummaryCultured neural stem/precursor cells (NSCs) are regarded as a potential systematic cell source to treat Parkinson's disease (PD). However, the therapeutic potential of these cultured NSCs is lost during culturing. Here, we show that treatment of vitamin C (VC) enhances generation of authentic midbrain-type dopamine (mDA) neurons with improved survival and functions from ventral midbrain (VM)-derived NSCs. VC acted by upregulating a series of mDA neuron-specific developmental and phenotype genes via removal of DNA methylation and repressive histone code (H3K9m3, H3K27m3) at associated gene promoter regions. Notably, the epigenetic changes induced by transient VC treatment were sustained long after VC withdrawal. Accordingly, transplantation of VC-treated NSCs resulted in improved behavioral restoration, along with enriched DA neuron engraftment, which faithfully expressed midbrain-specific markers in PD model rats. These results indicate that VC treatment to donor NSCs could be a simple, efficient, and safe therapeutic strategy for PD in the future.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

A role of BAG3 in regulating SNCA/α-synuclein clearance via selective macroautophagy

Many studies reveal that BAG3 plays a critical role in the regulation of protein degradation via macroautophagy. However, it remains unknown whether BAG3 affects the quality control of α-synuclein (SNCA), a Parkinson's disease–related protein. In this study, we demonstrated the increases of BAG3 expression in the ventral midbrain of SNCAA53T transgenic mice and also in MG132-treated PC12 cells overexpressing wild-type SNCA (SNCAWT-PC12). Moreover, we showed that BAG3 overexpression was sufficient to enhance the autophagy activity while knockdown of Bag3 reduced it in SNCAWT-PC12 cells. Immunoprecipitation revealed that BAG3 interacted with heat shock protein 70 and sequestosome 1. The immunostaining also showed the perinuclear accumulation and colocalization of BAG3 with these 2 proteins, as well as with LC3 dots in tyrosine hydroxylase–positive neurons in the midbrain of SNCAA53T mice. BAG3 overexpression was able to modulate SNCA degradation via macroautophagy which was prevented by Atg5 knockdown. Taken together, these results indicate that BAG3 plays a relevant role in regulating SNCA clearance via macroautophagy, and the heat shock protein 70-BAG3-sequestosome 1 complex may be involved in this process.

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.

Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation.

Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. We show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts. By specifying how to perform quality control (QC), troubleshooting and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate reproducibility of experiments and enable shipping of cells between centers, we present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.

Homophilic binding of the neural cell adhesion molecule CHL1 regulates development of ventral midbrain dopaminergic pathways

Abnormal development of ventral midbrain (VM) dopaminergic (DA) pathways, essential for motor and cognitive function, may underpin a number of neurological disorders and thereby highlight the importance of understanding the birth and connectivity of the associated neurons. While a number of regulators of VM DA neurogenesis are known, processes involved in later developmental events, including terminal differentiation and axon morphogenesis, are less well understood. Recent transcriptional analysis studies of the developing VM identified genes expressed during these stages, including the cell adhesion molecule with homology to L1 (Chl1). Here, we map the temporal and spatial expression of CHL1 and assess functional roles of substrate-bound and soluble-forms of the protein during VM DA development. Results showed early CHL1 in the VM, corresponding with roles in DA progenitor migration and differentiation. Subsequently, we demonstrated roles for CHL1 in both axonal extension and repulsion, selectively of DA neurons, suggestive of a role in guidance towards forebrain targets and away from hindbrain nuclei. In part, CHL1 mediates these roles through homophilic CHL1-CHL1 interactions. Collectively, these findings enhance our knowledge of VM DA pathways development, and may provide new insights into understanding DA developmental conditions such as autism spectrum disorders.

Neuroendoscopy to Achieve Superior Glioma Resection Outcomes.

Neuroendoscopy to Achieve Superior Glioma Resection Outcomes.

Pitx3 and En1 determine the size and molecular programming of the dopaminergic neuronal pool.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral midbrain. These neurons form the substantia nigra (SNc) and the ventral tegmental area (VTA). Two transcription factors that play important roles in the process of terminal differentiation and subset-specification of mdDA neurons, are paired-like homeodomain transcription factor 3 (Pitx3), and homeobox transcription factor Engrailed 1 (En1). We previously investigated the single Pitx3KO and En1KO and observed important changes in the survival of mdDA neurons of the SNc and VTA as well as altered expression of pivotal rostral- and caudal-markers, Ahd2 and Cck, respectively. To refine our understanding of the regional-specific relationships between En1 and Pitx3 and their (combined) role in the programming mdDA neurons on the rostral-to-caudal axis, we created double En1tm1Alj/tm1Alj;Pitx3gfp/gfp (En1KO;Pitx3GFP/GFP) animals. Here we report, that in absence of En1 and Pitx3, only a limited number of mdDA neurons are present at E14.5. These mdDA neurons have a rudimentary dopaminergic cell fate, as they express Nurr1, Pbx3 and Otx2 but have lost their rostral or caudal subset identity. Furthermore, we report that the expression of Cck depends on En1 expression, while (in contrast) both Pitx3 and En1 are involved in the initiation of Ahd2 expression. Thus we reveal in this manuscript that regulated levels of Pitx3 and En1 control the size and rostral/caudal-identity of the mdDA neuronal population.

Neuronal firing activity in the basal ganglia after striatal transplantation of dopamine neurons in hemiparkinsonian rats

The loss of nigral dopaminergic neurons and the resulting dopamine (DA) depletion in the striatum (STR) lead to altered neuronal activity and enhanced beta activity in various regions of the basal ganglia (BG) motor loop in patients with Parkinson’s disease and in rodents in the 6-hydroxydopamine (6-OHDA)-lesioned rat model. Intrastriatal DA graft implantation has been shown to re-innervate the host brain and restore DA input.Here, DA cell grafts were implanted into the STR of 6-OHDA-lesioned rats and the effect on neuronal activity under urethane anesthesia (1.4g/kg, injected intraperitoneally) was tested in the entopeduncular nucleus (EPN, the equivalent to the human globus pallidus internus), the output nucleus of the BG, and the globus pallidus (GP, the equivalent to the human globus pallidus externus), a key region in the indirect pathway.In animals, which were transplanted with cells derived from the ventral mesencephalon of embryonic day 12 rat embryos into the STR, the rotational behavior induced by DA agonists in 6-OHDA-lesioned rats was significantly improved. This was accompanied by alleviated EPN firing rate and reinstated patterns of neuronal activity in the GP and EPN. Analysis of oscillatory activity revealed enhanced beta activity in both regions, which was reduced after grafting.In summary these data indicate restoration of BG motor loop toward normal activity by DA graft integration.

Critical role of TRPC1 in thyroid hormone-dependent dopaminergic neuron development

Thyroid hormones play a crucial role in midbrain dopaminergic (DA) neuron development. However, the underlying molecular mechanisms remain largely unknown. In this study, we revealed that thyroid hormone treatment evokes significant calcium entry through canonical transient receptor potential (TRPC) channels in ventral midbrain neural stem cells and this calcium signaling is essential for thyroid hormone-dependent DA neuronal differentiation. We also found that TRPC1 is the dominant TRPC channel expressed in ventral midbrain neural stem cells which responds to thyroid hormone. In addition, thyroid hormone increases TRPC1 expression through its receptor alpha 1 during DA neuron differentiation, and, importantly, produces calcium signals by activating TRPC1 channels. In vivo and in vitro gene silencing experiments indicate that TRPC1-mediated calcium signaling is required for thyroid hormone-dependent DA neuronal differentiation. Finally, we confirmed that the activation of OTX2, a determinant of DA neuron development and the expression of which is induced by thyroid hormone, is dependent on TRPC1-mediated calcium signaling. These data revealed the molecular mechanisms of how thyroid hormone regulates DA neuron development from ventral midbrain neural stem cells, particularly endowing a novel physiological relevance to TRPC1 channels.

Issue Cover (August 2017)

Front cover: Detection of mouse brain Kir2.3 by immunohistochemistry. Frank et al in this issue show genetic rules specify Kir2.3 assembly into anatomically restricted subfamilies of ~1.5 MDa PSD95-containing postsynaptic supercomplexes, including NMDAR-Kir2.3 ion channel-channel supercomplexes in the ventral midbrain. Read the full article ‘Hierarchical organization and genetically separable subfamilies of PSD95 postsynaptic supercomplexes’ by R. A. W. Frank, F. Zhu, N. H. Komiyama and S. G. N. Grant (J. Neurochem. 2017, vol. 142 (4), pp. 504–511) on doi: 10.1111/jnc.14056 Read the Editorial Highlight for this article on doi: 10.1111/jnc.14084

Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, invitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes invivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Altered nicotine reward-associated behavior following α4 nAChR subunit deletion in ventral midbrain.

Nicotinic acetylcholine receptors containing α4 subunits (α4β2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 μg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.

Functional determination of the differentiation potential of ventral mesencephalic neural precursor cells during dopaminergic neurogenesis

The ventral mesencephalic neural precursor cells (vmNPCs) that give rise to dopaminergic (DA) neurons have been identified by the expression of distinct genes (e.g., Lmx1a, Foxa2, Msx1/2). However, the commitment of these NPCs to the mesencephalic DA neuronal fate has not been functionally determined. Evaluation of the plasticity of vmNPCs suggests that their commitment occurs after E10.5. Here we show that E9.5 vmNPCs implanted in an ectopic area of E10.5 mesencephalic explants, retained their specification marker Lmx1a and efficiently differentiated into neurons but did not express the gene encoding tyrosine hydroxylase (Th), the limiting enzyme for dopamine synthesis. A proportion of E10.5-E11.5 implanted vmNPCs behaved as committed, deriving into Th+ neurons in ectopic sites. Interestingly, implanted cells from E12.5 embryos were unable to give rise to a significant number of Th+ neurons. Concomitantly, differentiation assays in culture and in mesencephalic explants treated with Fgf2+LIF detected vmNPCs with astrogenic potential since E11.5. Despite this, a full suspension of E12.5 vmNPCs give rise to DA neurons in a similar proportion as those of E10.5 when they were transplanted into adult brain, but astrocytes were only detected with the former population. These data suggest that the subventricular postmitotic progenitors present in E12.5 ventral mesencephalon are unable to implant in embryonic explants and are the source of DA neurons in the transplanted adult brain. Based on our findings we propose that during DA differentiation committed vmNPCs emerge at E10.5 and they exhaust their neurogenic capacity with the rise of NPCs with astrogenic potential.

A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway

BackgroundAutosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson’s disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson’s disease patients.MethodsCo-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis’ early development.ResultsOur proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo.ConclusionsOur study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/β-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson’s disease.

Translation of WNT developmental programs into stem cell replacement strategies for the treatment of Parkinson's disease.

This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Cell replacement therapy is the remedial solution for treating Parkinson's disease.

The selective degeneration of dopaminergic (DA) neurons in Parkinson's disease (PD) has made an idol target for cell replacement therapies and other emerging surgical treatments. Certainly, by transplantation method, the therapeutic regimens such as human fetal ventral midbrain (hfVM) cells, human embryonic stem cells (hESCs), human neural stem/precursor/ progenitor cells (hNSCs/hNPCs), human mesenchymal stem cells (hMSCs), human induced neural stem cells (hiNSCs), and human induced pluripotent stem cells (hiPSCs) have been used into DA deficient striatum. In recent decades, surgical methods such as deep brain stimulation (DBS) and gene therapies have been used with the aim of treating PD. Though the technology has improved and many treating options arise, the permanent source for curing PD has not been identified yet. In this review, we examine how stem cell therapies have made advancement as a therapeutic source for PD when compared with surgical treatments.

TCDD induces UbcH7 expression and synphilin-1 protein degradation in the mouse ventral midbrain.

UbcH7 is an ubiquitin-conjugating enzyme that interacts with parkin, an E3 ligase. The UbcH7-parkin complex promotes the ubiquitination and degradation of several proteins via the 26S proteasome. Cellular accumulation of the UbcH7-parkin targets alpha-synuclein and synphilin-1 has been associated with Parkinson disease. In mouse liver, 2,3,7,8-tetrachlorodibenzo-p-dioxin, an aryl hydrocarbon receptor ligand, induces UbcH7 expression. Therefore, the aim of the present study was to determine whether 2,3,7,8-tetrachlorodibenzo-p-dioxin induces Ubch7 mRNA and UbcH7 protein expression in the mouse brain, to characterize the molecular mechanism, and the effect on synphilin-1 half-life. We found that 2,3,7,8-tetrachlorodibenzo-p-dioxin promotes the aryl hydrocarbon receptor binding to Ubch7 gene promoter as well as its transactivation, resulting in an induction of UbcH7 levels in the olfactory bulb, ventral midbrain, hippocampus, striatum, cerebral cortex, brain stem, and medulla oblongata. In parallel, 2,3,7,8-tetrachlorodibenzo-p-dioxin promoted synphilin-1 degradation in an aryl hydrocarbon receptor-dependent way.