Mesencephalic Dopaminergic Neurons Research Articles

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules.

BackgroundThe mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.ResultsBy taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.ConclusionsOur study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.

Potential of Cellular and Animal Models Based on a Prion-Like Propagation of α-Synuclein for Assessing Antiparkinson Agents.

The pathological hallmark of Parkinson's disease (PD) is the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies (LBs). LBs are intracellular inclusions typically found in these neurons and in noradrenergic neurons of the locus coeruleus in patients with PD. However, LBs can be found more widely in neurons of the olfactory bulb, cerebral cortex, and spinal cord. Additionally, LBs appear in neurons of the cardiac, cutaneous, and intestinal autonomic nervous systems. LBs are composed of fibrillar aggregates of α-synuclein (α-syn). The widespread distribution of LBs indicates that α-syn aggregation occurs in neurons in various areas, supporting the concept that PD is not only a simple movement disorder but also a complex one with nonmotor impairments. However, it is unclear how α-syn pathology spreads in the nervous system. Postmortem analyses of patients with PD who received transplants of fetal mesencephalic dopaminergic neurons revealed LB formation in surviving grafts, providing a crucial clue regarding the host-to-graft disease propagation. Recent experiments demonstrated that fibrillar α-syn is transferred from neurons to neurons in cellular and animal models, suggesting that fibrillar α-syn is repeatedly generated in cells by triggering the continuous conversion of normal soluble species into fibrillar ones. These findings suggest a "prion-like" mechanism for α-syn propagation in the pathogenesis of PD. This review summarizes the experimental findings on the prion-like propagation of α-syn and discusses the potential of cellular and animal models for testing the protective effects of chemical agents against neurodegeneration in PD.

Products of the Parkinson's disease-related glyoxalase DJ-1, D-lactate and glycolate, support mitochondrial membrane potential and neuronal survival.

Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes linked with the onset of Parkinson's disease, DJ-1/PARK7, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease.

Epigenetic reprogramming of cortical neurons through alteration of dopaminergic circuits.

Alterations of the dopaminergic system are associated with the cognitive and functional dysfunctions that characterize complex neuropsychiatric disorders. We modeled a dysfunctional dopaminergic system using mice with targeted ablation of dopamine (DA) D2 autoreceptors in mesencephalic dopaminergic neurons. Loss of D2 autoreceptors abolishes D2-mediated control of DA synthesis and release. Here we show that this mutation leads to a profound alteration of the genomic landscape of neurons receiving dopaminergic afferents at distal sites, specifically in the prefrontal cortex. Indeed, we observed a remarkable down-regulation of gene expression in this area of ∼ 2,000 genes, which involves a widespread increase in the histone repressive mark H3K9me2/3. This reprogramming process is coupled to psychotic-like behaviors in the mutant mice. Importantly, chronic treatment with a DA agonist can revert the genomic phenotype. Thus, cortical neurons undergo a profound epigenetic reprogramming in response to dysfunctional D2 autoreceptor signaling leading to altered DA levels, a process that may underlie a number of neuropsychiatric disorders.

Suppression of glia maturation factor expression prevents 1-methyl-4-phenylpyridinium (MPP+)-induced loss of mesencephalic dopaminergic neurons

Inflammation mediated by glial activation appears to play a critical role in the pathogenesis of Parkinson disease (PD). Glia maturation factor (GMF), a proinflammatory protein predominantly localized in the central nervous system was isolated, sequenced and cloned in our laboratory. We have previously demonstrated immunomodulatory and proinflammatory functions of GMF, but its involvement in 1-methyl-4-phenylpyridinium (MPP+), active metabolite of classical parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inducing loss of dopaminergic (DA) neurons has not been studied. Here we show that altered expression of GMF has direct consequences on the production of reactive oxygen species (ROS) and nuclear factor-kappa B (NF-κB)- mediated production of inflammatory mediators by MPP+. We examined MPP+-induced DA neuronal loss in primary cultures of mouse mesencephalic neurons/glia obtained from GMF-deficient (GMF knockout (GMF-KO)) and GMF-containing wild-type (Wt) mice. We demonstrate that deficiency of GMF in GMF-KO neurons/glia led to decreased production of ROS and downregulation of NF-κB-mediated production of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) as compared to Wt neurons/glia. Additionally, overexpression of GMF induced DA neurodegeneration, whereas GMF downregulation by GMF-specific shRNA protected DA neurons from MPP-induced toxicity. Subsequently, GMF deficiency ameliorates antioxidant balance, as evidenced by the decreased level of lipid peroxidation, less ROS production along with increased level of glutathione; and attenuated the DA neuronal loss via the downregulation of NF-κB-mediated inflammatory responses. In conclusion, our overall data indicate that GMF modulates oxidative stress and release of deleterious agents by MPP+ leading to loss of DA neurons. Our study provides new insights into the potential role of GMF and identifies targets for therapeutic interventions in neurodegenerative diseases.

Carnosine Exerts Neuroprotective Effect Against 6-Hydroxydopamine Toxicity in Hemiparkinsonian Rat

Parkinson's disease (PD) is the second most common disorder of the central nervous system due to the degeneration of mesencephalic dopaminergic neurons. Current treatments for PD have a symptomatic relief strategy with no prevention of disease progression. Due to the neuroprotective and antiapoptotic potential of the natural dipeptide carnosine, this study was conducted to assess its beneficial effect in 6-hydroxydopamine (6-OHDA)-induced model of PD in rat. Unilateral intrastriatal 6-OHDA-lesioned rats received i.p. carnosine at a dose of 250mg/kg twice at an interval of 24h, which started presurgery. Apomorphine caused contralateral rotations, a significant reduction in the number of Nissl-stained neurons on the left side of the substantia nigra, and increased apoptosis was observed with enhanced oxidative stress burden in 6-OHDA-lesioned rats. Carnosine pretreatment significantly reduced rotations, attenuated apoptosis, and restored malondialdehyde and nitrite content and catalase activity with no significant effect on reduced glutathione (GSH). These results indicate that prelesion administration of carnosine could exert neuroprotection against 6-OHDA toxicity, and this may be of benefit in patients with early PD.

MPTP로 유도된 Parkinson's disease 동물 모델에서 항염증효과를 통한 측백엽의 도파민신경보호 효과

Objectives : The aim of this study was to investigate the protective effect of extract of Thuja orientalis leaves (TOFE) against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity by inhibition of inflammation in in vitro and in vivo models of Parkinson's disease (PD). Methods : We evaluated the effect of TOFE against lipopolysaccharide (LPS)/1-methyl-4-phenylpyridinium (<TEX>$MPP^+$</TEX>) toxicity using nitric oxide (NO) assay, inducible NO synthase and cyclooxygenase 2 western blot, tyrosine hydroxylase and microglia activation immunohistochemistry (IHC) in BV2 cell, primary rat mesencephalic neurons, or C57BL/6 mice. We also evaluated the effect of TOFE in mice PD model induced by MPTP. C57BL/6 mice were treated with TOFE 50 mg/kg for 5 days and were injected intraperitoneally with four administrations of MPTP on the last day. We conducted behavioral tests and IHC analysis to see how TOFE affect MPTP-induced neuronal loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and striatum (ST) of mice. To assess the anti-inflammation effects, we carried out glial fibrillary acidic protein and macrophage-1 antigen integrin alpha M in IHC in SNpc and ST of mice. Results : In an in vitro system, TOFE decreasesd NO generations in BV2 cells. TOFE protected dopaminergic cells against LPS or <TEX>$MPP^+$</TEX>-induced toxicity in primary mesencephalic dopaminergic neurons. In vivo system, TOFE at 50 mg/kg treated group showed improved motor deteriorations than the MPTP only treated group and TOFE significantly protected striatal dopaminergic damage from MPTP-induced neurotoxicity in mice. Moreover, TOFE inhibited activation of astrocyte and microglia in SNpc and ST of the mice. Conclusions : We concluded that TOFE showed anti-parkinsonian effect by protection of dopaminergic neurons against MPTP toxicity through anti-inflammatory actions.

Potential for cell therapy in Parkinson's disease using genetically programmed human embryonic stem cell-derived neural progenitor cells.

Neural transplantation is a promising strategy for restoring dopaminergic dysfunction and modifying disease progression in Parkinson's disease (PD). Human embryonic stem cells (hESCs) are a potential resource in this regard because of their ability to provide a virtually limitless supply of homogenous dopaminergic progenitors and neurons of appropriate lineage. The recent advances in developing robust cell culture protocols for directed differentiation of hESCs to near pure populations of ventral mesencephalic (A9-type) dopaminergic neurons has heightened the prospects for PD cell therapy. Here, we focus our review on current state-of-the-art techniques for harnessing hESC-based strategies toward development of a stem cell therapeutic for PD. Importantly, we also briefly describe a novel genetic-programming approach that may address many of the key challenges that remain in the field and that may hasten clinical translation.

Inhibition of prothrombin kringle-2-induced inflammation by minocycline protects dopaminergic neurons in the substantia nigra in vivo

Prothrombin kringle-2 (pKr-2), a domain of prothrombin, can cause the degeneration of mesencephalic dopaminergic neurons through microglial activation. However, the chemical products that inhibit pKr-2-induced inflammatory activities in the brain are still not well known. The present study investigated whether minocycline, a semisynthetic tetracycline derivative, could inhibit pKr-2-induced microglial activation and prevent the loss of nigral dopaminergic (DA) neurons in vivo. To address this question, rats were administered a unilateral injection of pKr-2 in the substantia nigra in the presence or absence of minocycline. Our results show that pKr-2 induces the production of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and inducible nitric oxide synthase from the activated microglia. In parallel, 7 days after pKr-2 injection, tyrosine hydroxylase immunocytochemical analysis and western blot analysis showed a significant loss of nigral DA neurons. This neurotoxicity was antagonized by minocycline and the observed neuroprotective effects were associated with the ability of minocycline to suppress the expression of tumor necrosis factor-α, interleukin-1β, and nitric oxide synthase. These results suggest that minocycline may be promising as a potential therapeutic agent for the prevention of DA neuronal degeneration associated with pKr-2-induced microglial activation.

Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways.

The lifelong maintenance of mesencephalic dopaminergic neurons by Nurr1 and engrailed

Specific vulnerability and degeneration of the dopaminergic neurons in the substantia nigra pars compacta of the midbrain is the pathological hallmark of Parkinson’s disease. A number of transcription factors regulate the birth and development of this set of neurons and some remain constitutively expressed throughout life. These maintenance transcription factors are closely associated with essential neurophysiological functions and are required ultimately for the long-term survival of the midbrain dopaminergic neurons. The current review describes the role of two such factors, Nurr1 and engrailed, in differentiation, maturation, and in normal physiological functions including acquisition of neurotransmitter identity. The review will also elucidate the relationship of these factors with life, vulnerability, degeneration and death of mesencephalic dopaminergic neurons in the context of Parkinson’s disease.

Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: Effects on locomotor behavior and seizure susceptibility

The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1Cre/+; tOtx2ov/+), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1Cre/+; tOtx2ov/+ adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1Cre/+; tOtx2ov/+ adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1Cre/+; tOtx2ov/+ mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1Cre/+; Otx2floxv/flox mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1Cre/+; tOtx2ov/+ and control mice. No difference was observed between mutant and control mice, but En1Cre/+; tOtx2ov/+ mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1Cre/+; tOtx2ov/+ mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor activity and seizure-induced gene expression. Our results strengthen the notion that Otx2 mutant mouse models are a powerful genetic tool to unravel the molecular and behavioral consequences of altered development of the DAergic system.

Dopamine D2 Receptor-mediated Epidermal Growth Factor Receptor Transactivation through a Disintegrin and Metalloprotease Regulates Dopaminergic Neuron Development via Extracellular Signal-related Kinase Activation

Dopamine D2 receptor (D2R)-mediated extracellular signal-regulated kinase (ERK) activation plays an important role in the development of dopaminergic mesencephalic neurons. Here, we demonstrate that D2R induces the shedding of heparin-binding epidermal growth factor (EGF) through the activation of a disintegrin and metalloprotease (ADAM) 10 or 17, leading to EGF receptor transactivation, downstream ERK activation, and ultimately an increase in the number of dopaminergic neurons and their neurite length in primary mesencephalic cultures from wild-type mice. These outcomes, however, were not observed in cultures from D2R knock-out mice. Our findings show that D2R-mediated ERK activation regulates mesencephalic dopaminergic neuron development via EGF receptor transactivation through ADAM10/17.

향부자(香附子)의 염증 억제 작용을 통한 항파킨슨 효과

ABSTRACT Objectives : The aim of this study was to investigate the neuroprotective ef fects and mechanisms of Cyperi Rhizoma extracts (CRE) using in vitro and in vivo models of Parkinson's disease (PD).Methods : We evaluated the neuroprotective effect of CRE against 1-methy l-4-phenylpyridinium (MPP+) toxicity using tyrosine hydroxylase immunohistochemistry (IHC) in primar y rat mesencephalic dopaminergic neurons. In addition, the effect of CRE was evaluated in mice PD model indu ced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). For evaluations, C57bl/6 mice were orally treated with CRE 50 mg/kg for 5 days and were injected intraperitoneally with MPTP (20 mg/kg) at 2 h intervals on the last day. To identify the CRE affects on MPTP-induced neuronal loss of dopaminergic neurons in substanti a nigra pars compacta (SNpc) and striatum of mice, the behavioral tests and IHC analysis were carried out. Also, we conducted nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) assay in dopaminergic neurons and IHC using glial markers in SNpc of mice to assess the anti-inflammation effects.Results : In primary mesencephalic culture system, CRE protected dopaminergic cells against 10 µM MPP+-induced toxicity at 0.2 and 1.0 µg/mL. In the behavior tests, CRE treated group showed improved motor deteriorations than those in the MPTP only treated group. CRE significantly protected striatal dopaminergic damage from MPTP-induced neurotoxicity in mice. Moreover, CRE inhibited pro ductions of NO and TNF-α in dopaminergic culture system and activation of astrocyte and microglia in SNpc of the mice.Conclusion : We concluded that CRE shows anti-parkinsonian effect by protect ing dopaminergic neurons against MPP+/MPTP toxicities through anti-inflammatory actions.Key words : Cyperi Rhizoma, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Parkinson’s disease, Neuroinflammation

ApoER2 and VLDLr Are Required for Mediating Reelin Signalling Pathway for Normal Migration and Positioning of Mesencephalic Dopaminergic Neurons

The migration of mesencephalic dopaminergic (mDA) neurons from the subventricular zone to their final positions in the substantia nigra compacta (SNc), ventral tegmental area (VTA), and retrorubral field (RRF) is controlled by signalling from neurotrophic factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM). Reelin and the cytoplasmic adaptor protein Disabled-1 (Dab1) have been shown to play important roles in the migration and positioning of mDA neurons. Mice lacking Reelin and Dab1 both display phenotypes characterised by the failure of nigral mDA neurons to migrate properly. ApoER2 and VLDLr are receptors for Reelin signalling and are therefore part of the same signal transduction pathway as Dab1. Here we describe the roles of ApoER2 and VLDLr in the proper migration and positioning of mDA neurons in mice. Our results demonstrate that VLDLr- and ApoER2-mutant mice have both a reduction in and abnormal positioning of mDA neurons. This phenotype was more pronounced in VLDLr-mutant mice. Moreover, we provide evidence that ApoER2/VLDLr double-knockout mice show a phenotype comparable with the phenotypes observed for Reelin- and Dab1- mutant mice. Taken together, our results demonstrate that the Reelin receptors ApoER2 and VLDLr play essential roles in Reelin-mediated migration and positioning of mDA neurons.

Ghrelin amplifies the nicotine-induced dopamine release in the rat striatum

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward mechanisms and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Nicotine activates the mesencephalic dopaminergic neurons via nicotinic acetylcholine receptors (nAchR). Ghrelin stimulates the dopaminergic neurons via growth hormone secretagogue receptors (GHS-R1A) in the ventral tegmental area and the substantia nigra pars compacta resulting in the release of dopamine in the ventral and dorsal striatum, respectively. In the present study an in vitro superfusion of rat striatal slices was performed, in order to investigate the direct action of ghrelin on the striatal dopamine release and the interaction of ghrelin with nicotine through this neurotransmitter release. Ghrelin increased significantly the dopamine release from the rat striatum following electrical stimulation. This stimulatory effect was reversed by both the selective nAchR antagonist mecamylamine and the selective GHS-R1A antagonist GHRP-6. Nicotine also increased significantly the dopamine release under the same conditions. This stimulatory effect was antagonized by mecamylamine, but not by GHRP-6. Ghrelin further stimulated the nicotine-induced dopamine release and this effect was abolished by mecamylamine and was partially inhibited by GHRP-6. The present results demonstrate that ghrelin stimulates directly the dopamine release and amplifies the nicotine-induced dopamine release in the rat striatum. We presume that striatal cholinergic interneurons also express GHS-R1A, through which ghrelin can amplify the nicotine-induced dopamine release in the striatum. This study provides further evidence of the impact of ghrelin on the mesolimbic and nigrostriatal dopaminergic pathways. It also suggests that ghrelin signaling may serve as a novel pharmacological target for treatment of addictive and neurodegenerative disorders.

Calcium Entry and -Synuclein Inclusions Elevate Dendritic Mitochondrial Oxidant Stress in Dopaminergic Neurons

The core motor symptoms of Parkinson's disease (PD) are attributable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed a major factor in PD pathogenesis. Previous work has shown that activity-dependent calcium entry through L-type channels elevates perinuclear mitochondrial oxidant stress in SNc dopaminergic neurons, providing a potential basis for their selective vulnerability. What is less clear is whether this physiological stress is present in dendrites and if Lewy bodies, the major neuropathological lesion found in PD brains, exacerbate it. To pursue these questions, mesencephalic dopaminergic neurons derived from C57BL/6 transgenic mice were studied in primary cultures, allowing for visualization of soma and dendrites simultaneously. Many of the key features of in vivo adult dopaminergic neurons were recapitulated in vitro. Activity-dependent calcium entry through L-type channels increased mitochondrial oxidant stress in dendrites. This stress progressively increased with distance from the soma. Examination of SNc dopaminergic neurons ex vivo in brain slices verified this pattern. Moreover, the formation of intracellular α-synuclein Lewy-body-like aggregates increased mitochondrial oxidant stress in perinuclear and dendritic compartments. This stress appeared to be extramitochondrial in origin, because scavengers of cytosolic reactive oxygen species or inhibition of NADPH oxidase attenuated it. These results show that physiological and proteostatic stress can be additive in the soma and dendrites of vulnerable dopaminergic neurons, providing new insight into the factors underlying PD pathogenesis.

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Hemoglobin is present as a canonical α2β2 tetramer in dopaminergic neurons

Hemoglobin is the oxygen carrier in blood erythrocytes. Oxygen coordination is mediated by α2β2 tetrameric structure via binding of the ligand to the heme iron atom. This structure is essential for hemoglobin function in the blood. In the last few years, expression of hemoglobin has been found in atypical sites, including the brain. Transcripts for α and β chains of hemoglobin as well as hemoglobin immunoreactivity have been shown in mesencephalic A9 dopaminergic neurons, whose selective degeneration leads to Parkinson's disease. To gain further insights into the roles of hemoglobin in the brain, we examined its quaternary structure in dopaminergic neurons in vitro and in vivo. Our results indicate that (i) in mouse dopaminergic cell line stably over-expressing α and β chains, hemoglobin exists as an α2β2 tetramer; (ii) similarly to the over-expressed protein, endogenous hemoglobin forms a tetramer of 64kDa; (iii) hemoglobin also forms high molecular weight insoluble aggregates; and (iv) endogenous hemoglobin retains its tetrameric structure in mouse mesencephalon in vivo. In conclusion, these results suggest that neuronal hemoglobin may be endowed with some of the biochemical activities and biological function associated to its role in erythroid cells. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

The 6-hydroxydopamine-induced lesion of A8 and A9 mesencephalic dopaminergic neurons decreases the harmaline-induced glutamate release in the cerebellum

The 6-hydroxydopamine-induced lesion of A8 and A9 mesencephalic dopaminergic neurons decreases the harmaline-induced glutamate release in the cerebellum