Dopaminergic Substantia Nigra Research Articles

Guanabenz promotes neuronal survival via enhancement of ATF4 and parkin expression in models of Parkinson disease

Reduced function of parkin appears to be a central pathogenic event in Parkinson disease (PD). Increasing parkin levels enhances survival in models of PD-related neuronal death and is a promising therapeutic objective. Previously, we demonstrated that the transcription factor ATF4 promotes survival in response to PD-mimetic stressors by maintaining parkin levels. ATF4 translation is up-regulated by phosphorylation of the translation initiation factor eIF2α. The small molecule guanabenz enhances eIF2α phosphorylation by blocking the function of GADD34, a regulatory protein that promotes eIF2α dephosphorylation. We tested the hypothesis that guanabenz, by inhibiting GADD34 and consequently increasing eIF2α phosphorylation and elevating ATF4, would improve survival in models of PD by up-regulating parkin.We found that GADD34 is strongly induced by 6-OHDA, and that GADD34 localization is dramatically altered in dopaminergic substantia nigra neurons in PD cases. We further demonstrated that guanabenz attenuates 6-hydroxydopamine (6-OHDA) induced cell death of differentiated PC12 cells and primary ventral midbrain dopaminergic neurons in culture, and of dopaminergic neurons in the substantia nigra of mice. In culture models, guanabenz also increases eIF2α phosphorylation and ATF4 and parkin levels in response to 6-OHDA. Furthermore, if either ATF4 or parkin is silenced, then the protective effect of guanabenz is lost. We also found similar results in a distinct model of neuronal death: primary cultures of cortical neurons treated with the topoisomerase I inhibitor camptothecin, in which guanabenz limited camptothecin-induced neuronal death in an ATF4- and parkin-dependent manner.In summary, our data suggest that guanabenz and other GADD34 inhibitors could be used as therapeutic agents to boost parkin levels and thereby slow neurodegeneration in PD and other neurodegenerative conditions.

Vagus nerve stimulation improves locomotion and neuronal populations in a model of Parkinson's disease

BackgroundParkinson's disease (PD) is a progressive, neurodegenerative disorder with no disease-modifying therapies, and symptomatic treatments are often limited by debilitating side effects. In PD, locus coeruleus noradrenergic (LC-NE) neurons degenerate prior to substantia nigra dopaminergic (SN-DA) neurons. Vagus nerve stimulation (VNS) activates LC neurons, and decreases pro-inflammatory markers, allowing improvement of LC targets, making it a potential PD therapeutic. ObjectiveTo assess therapeutic potential of VNS in a PD model. MethodsTo mimic the progression of PD degeneration, rats received a systemic injection of noradrenergic neurotoxin DSP-4, followed one week later by bilateral intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine. At this time, a subset of rats also had vagus cuffs implanted. After eleven days, rats received a precise VNS regimen twice a day for ten days, and locomotion was measured during each afternoon session. Immediately following final stimulation, rats were euthanized, and left dorsal striatum, bilateral SN and LC were sectioned for immunohistochemical detection of monoaminergic neurons (tyrosine hydroxylase, TH), α-synuclein, astrocytes (GFAP) and microglia (Iba-1). ResultsVNS significantly increased locomotion of lesioned rats. VNS also resulted in increased expression of TH in striatum, SN, and LC; decreased SN α-synuclein expression; and decreased expression of glial markers in the SN and LC of lesioned rats. Additionally, saline-treated rats after VNS, had higher LC TH and lower SN Iba-1. ConclusionsOur findings of increased locomotion, beneficial effects on LC-NE and SN-DA neurons, decreased α-synuclein density in SN TH-positive neurons, and neuroinflammation suggest VNS has potential as a novel PD therapeutic.

Interleukin-6 regulates iron-related proteins through c-Jun N-terminal kinase activation in BV2 microglial cell lines.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and subsequent DA depletion in the striatum. Microglia activation and nigral iron accumulation play important roles in the pathogenesis of PD. Activated microglia show increased iron deposits. However, the relationship between microglia activation and iron accumulation remains unclear. In the present study, we aimed to determine how iron levels affect interleukin-6 (IL-6) synthesis, and the effect of IL-6 on cellular iron metabolism in BV2 microglial cells.IL-6 mRNA was up-regulated after FAC treatment for 12 h in BV2 cells. Iron regulatory protein 1 (IRP1) and divalent metal transporter 1 (DMT1) were up-regulated and iron exporter ferroportin 1 (FPN1) was down-regulated in BV2 cells after 24 h of IL-6 treatment. Phosphorylated JNK increased significantly compared to the control after BV2 cells were treated with IL-6 for 1 h. Pretreatment with SP600125 attenuated the up-regulation of IRP1 and DMT1 and down-regulation of FPN1 (compared to IL-6-treated group). These results suggest that iron load could increase IL-6 mRNA expression in BV2 cells. Further, IL-6 likely up-regulates IRP1 and DMT1 expression and down-regulates FPN1 expression in BV2 microglial cells through JNK signaling pathways.

Lower Affinity of Isradipine for L-Type Ca2+ Channels during Substantia Nigra Dopamine Neuron-Like Activity: Implications for Neuroprotection in Parkinson's Disease.

Ca2+-influx through L-type Ca2+-channels (LTCCs) is associated with activity-related stressful oscillations of Ca2+ levels within dopaminergic (DA) neurons in the substantia nigra (SN), which may contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by in vivo pretreatment with therapeutically relevant isradipine plasma levels, or Cav1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Cav1.2 α1-subunit, long and short Cav1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Cav1.3 variants conducted Ca2+ current (ICa) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated ICa during (Cav1.2 plus Cav1.3) and after (Cav1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Cav1.3 variants during SN DA-like pacemaking compared with Cav1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca2+ oscillations. Our data predict that Cav1.2 and Cav1.3 splice variants contribute differentially to Ca2+ load in SN DA neurons, with prominent Cav1.3-mediated ICa between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Cav1.2.SIGNIFICANCE STATEMENT The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCCs), in particular Cav1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Cav1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca2+ oscillations of SN DA neurons.

Proximity of Substantia Nigra Microstimulation to Putative GABAergic Neurons Predicts Modulation of Human Reinforcement Learning.

Neuronal firing in the substantia nigra (SN) immediately following reward is thought to play a crucial role in human reinforcement learning. As in Ramayya et al. (2014a) we applied microstimulation in the SN of patients undergoing deep brain stimulation (DBS) for the treatment of Parkinson's disease as they engaged in a two-alternative reinforcement learning task. We obtained microelectrode recordings to assess the proximity of the electrode tip to putative dopaminergic and GABAergic SN neurons and applied stimulation to assess the functional importance of these neuronal populations for learning. We found that the proximity of SN microstimulation to putative GABAergic neurons predicted the degree of stimulation-related changes in learning. These results extend previous work by supporting a specific role for SN GABA firing in reinforcement learning. Stimulation near these neurons appears to dampen the reinforcing effect of rewarding stimuli.

Reward Dependent Invigoration Relates to Theta Oscillations and Is Predicted by Dopaminergic Midbrain Integrity in Healthy Elderly.

Motivation can have invigorating effects on behavior via dopaminergic neuromodulation. While this relationship has mainly been established in theoretical models and studies in younger subjects, the impact of structural declines of the dopaminergic system during healthy aging remains unclear. To investigate this issue, we used electroencephalography (EEG) in healthy young and elderly humans in a reward-learning paradigm. Specifically, scene images were initially encoded by combining them with cues predicting monetary reward (high vs. low reward). Subsequently, recognition memory for the scenes was tested. As a main finding, we can show that response times (RTs) during encoding were faster for high reward predicting images in the young but not elderly participants. This pattern was resembled in power changes in the theta-band (4–7 Hz). Importantly, analyses of structural MRI data revealed that individual reward-related differences in the elderlies’ response time could be predicted by the structural integrity of the dopaminergic substantia nigra (SN; as measured by magnetization transfer (MT)). These findings suggest a close relationship between reward-based invigoration, theta oscillations and age-dependent changes of the dopaminergic system.

Inhibiting purinergic P2X7 receptors with the antagonist brilliant blue G is neuroprotective in an intranigral lipopolysaccharide animal model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder, which is characterized by the selective and progressive death of dopaminergic (DA) neurons in the substantia nigra. Increasing evidence suggests that inflammation is important in the degeneration of DA neurons. The purinergic receptor subtype P2X7 receptor (P2X7R) is key in the activation and proliferation of microglia. The present study aimed to examine whether inhibiting purinergic P2X7 receptors is neuroprotective in a rat model of PD, specifically via inhibiting p38 mitogen-activated protein kinase (MAPK). In an intranigral lipopolysaccharide (LPS) rat model of PD, immunohistochemical analysis revealed enhanced expression of P2X7R was observed in microglia. The administration of the P2X7R antagonist, brilliant blue G (BBG), reduced activation of the microglia and the loss of nigral DA neurons. In addition, immunohistochemistry and western blot analysis revealed the phosphorylation level of p38 MAPK increased in the microglia of the LPS-injected rats, which was inhibited by BBG treatment. The p38 MAPK inhibitor, SB203580, reduced microglial activation and the loss of DA neurons. Thus, these findings suggested that inhibition of P2X7R by BBG attenuated microglial activation and the loss of substantia nigra DA neurons via p38 MAPK in the rat LPS model of PD.

Defective mitochondrial DNA homeostasis in the substantia nigra in Parkinson disease.

Increased somatic mitochondrial DNA (mtDNA) mutagenesis causes premature aging in mice, and mtDNA damage accumulates in the human brain with aging and neurodegenerative disorders such as Parkinson disease (PD). Here, we study the complete spectrum of mtDNA changes, including deletions, copy-number variation and point mutations, in single neurons from the dopaminergic substantia nigra and other brain areas of individuals with Parkinson disease and neurologically healthy controls. We show that in dopaminergic substantia nigra neurons of healthy individuals, mtDNA copy number increases with age, maintaining the pool of wild-type mtDNA population in spite of accumulating deletions. This upregulation fails to occur in individuals with Parkinson disease, however, resulting in depletion of the wild-type mtDNA population. By contrast, neuronal mtDNA point mutational load is not increased in Parkinson disease. Our findings suggest that dysregulation of mtDNA homeostasis is a key process in the pathogenesis of neuronal loss in Parkinson disease.

Cannabinoid Type 2 (CB2) Receptors Activation Protects against Oxidative Stress and Neuroinflammation Associated Dopaminergic Neurodegeneration in Rotenone Model of Parkinson's Disease.

The cannabinoid type two receptors (CB2), an important component of the endocannabinoid system, have recently emerged as neuromodulators and therapeutic targets for neurodegenerative diseases including Parkinson's disease (PD). The downregulation of CB2 receptors has been reported in the brains of PD patients. Therefore, both the activation and the upregulation of the CB2 receptors are believed to protect against the neurodegenerative changes in PD. In the present study, we investigated the CB2 receptor-mediated neuroprotective effect of β-caryophyllene (BCP), a naturally occurring CB2 receptor agonist, in, a clinically relevant, rotenone (ROT)-induced animal model of PD. ROT (2.5 mg/kg BW) was injected intraperitoneally (i.p.) once daily for 4 weeks to induce PD in male Wistar rats. ROT injections induced a significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and DA striatal fibers, following activation of glial cells (astrocytes and microglia). ROT also caused oxidative injury evidenced by the loss of antioxidant enzymes and increased nitrite levels, and induction of proinflammatory cytokines: IL-1β, IL-6 and TNF-α, as well as inflammatory mediators: NF-κB, COX-2, and iNOS. However, treatment with BCP attenuated induction of proinflammatory cytokines and inflammatory mediators in ROT-challenged rats. BCP supplementation also prevented depletion of glutathione concomitant to reduced lipid peroxidation and augmentation of antioxidant enzymes: SOD and catalase. The results were further supported by tyrosine hydroxylase immunohistochemistry, which illustrated the rescue of the DA neurons and fibers subsequent to reduced activation of glial cells. Interestingly, BCP supplementation demonstrated the potent therapeutic effects against ROT-induced neurodegeneration, which was evidenced by BCP-mediated CB2 receptor activation and the fact that, prior administration of the CB2 receptor antagonist AM630 diminished the beneficial effects of BCP. The present study suggests that BCP has the potential therapeutic efficacy to elicit significant neuroprotection by its anti-inflammatory and antioxidant activities mediated by activation of the CB2 receptors.

Selective brain penetrable Nurr1 transactivator for treating Parkinson's disease.

Parkinson's disease (PD) is one of the most common movement disorders, and currently there is no effective treatment that can slow disease progression. Preserving and enhancing DA neuron survival is increasingly regarded as the most promising therapeutic strategy for treating PD. IRX4204 is a second generation retinoid X receptor (RXR) agonist that has no cross reactivity with retinoic acid receptors, farnesoid X receptor, liver X receptors or peroxisome proliferator-activated receptor PPARγ. We found that IRX4204 promotes the survival and maintenance of nigral dopaminergic (DA) neurons in a dose-dependent manner in primary mesencephalic cultures. Brain bioavailability studies demonstrate that IRX4204 can cross the blood brain barrier and reach the brain at nM concentration. Oral administration of IRX4204 can activate nuclear receptor Nurr1 downstream signaling in the substantia nigra (SN) andattenuate neurochemical and motor deficits in a rat model of PD. Our study suggests that IRX4204 represents a novel, potent and selective pharmacological means to activate cellular RXR-Nurr1 signaling and promote SN DA neuron survival in PD prevention and/or treatment.

Copper and iron changes in Parkinson's disease brain; implications for aetiology and potential for novel treatments

Event Abstract Back to Event Copper and iron changes in Parkinson's disease brain; implications for aetiology and potential for novel treatments Katherine Davies1, Benjamin Trist2, Veronica Cottam2, Dominic J. Hare3, Sylvain Bohic4, Asuncion Carmona5, Richard Ortega5 and Kay L. Double2* 1 Neuroscience Research Australia, Australia 2 The University of Sydney, School of Medical Sciences, Sydney Medical School, Australia 3 Florey Institute of Neuroscience and Mental Health, University of Technology Sydney, and The University of Melbourne , Australia 4 Universite Grenoble Alpes, Inserm, France 5 and Centre National de la Recherche Scientifique, University of Bordeaux, France We have used sensitive, high resolution biophysical imaging methods to investigate biometals changes at the single cell level in the Parkinson’s disease brain. Consistent with a large body of bulk tissue reports, we identified increased intraneuronal Fe in the degenerating substantia nigra in this disorder, a change not present in non-degenerating brain regions. We have suggested that Fe and dopamine form a potent biochemical redox couple, thus the accumulation of Fe within the dopaminergic substantia nigra in the Parkinson’s disease brain may contribute to the selective vulnerability of this brain region. We also identified significantly decreased Cu levels in these same neurons, associated with altered cellular Cu transport pathways. Decreased intraneuronal Cu was also present in the substantia nigra in incidental Lewy body disease prior to the onset of neuronal loss, suggesting this change occurs early in the disease process. The decrease in Cu in these vulnerable neurons is associated with alterations in cuproproteins. Regional changes in cuproproteins in the Parkinson’s disease brain may contribute to the accumulation of intraneuronal Fe in this disorder, but also to mechanisms of degeneration linked to dysfunction of the cuproprotein superoxide dismutase 1. We suggest that normalisation of brain Fe and Cu levels in Parkinson’s disease brain represents a tractable target for neuroprotective therapy. Keywords: Copper, Iron, Substantia Nigra, Parkinson, neurodegeneration Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 3: Metals in the Aetiology and Treatment of Neurodegenerative Diseases Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Davies K, Trist B, Cottam V, Hare DJ, Bohic S, Carmona A, Ortega R and Double KL (2016). Copper and iron changes in Parkinson's disease brain; implications for aetiology and potential for novel treatments. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Kay L Double, The University of Sydney, School of Medical Sciences, Sydney Medical School, Sydney, NSW, Australia, kay.double@sydney.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Katherine Davies Benjamin Trist Veronica Cottam Dominic J Hare Sylvain Bohic Asuncion Carmona Richard Ortega Kay L Double Google Katherine Davies Benjamin Trist Veronica Cottam Dominic J Hare Sylvain Bohic Asuncion Carmona Richard Ortega Kay L Double Google Scholar Katherine Davies Benjamin Trist Veronica Cottam Dominic J Hare Sylvain Bohic Asuncion Carmona Richard Ortega Kay L Double PubMed Katherine Davies Benjamin Trist Veronica Cottam Dominic J Hare Sylvain Bohic Asuncion Carmona Richard Ortega Kay L Double Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of siRNA-induced silencing of cellular prion protein on tyrosine hydroxylase expression in the substantia nigra of a rat model of Parkinson's disease.

The most significant pathological feature of Parkinson's disease (PD) is the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra. Currently, available treatments for PD cannot prevent the loss of DA neurons. Tyrosine hydroxylase (TH) expressed in substantia nigra neurons catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the rate-limiting step of DA biosynthesis. Major reasons for PD occurrence include decreased TH activity in the substantia nigra and secondary DA suppression. Decreased TH activity and the resulting suppression of DA synthesis (or neurotransmission) in the substantia nigra are key factors underlying the development of PD. Cellular prion protein (PRP) is a membrane glycoprotein expressed in the central nervous system. Although the sequence of PRP is highly conserved, its physiological function is unclear. The purpose of this study was to investigate the effect of PRP-targeted small interfering RNA (siRNA) on TH expression in a rat model of PD. Thirty male Wistar rats were injected with 6-hydroxydopamine (6-OHDA) to generate a model of PD. The rats then received injections of PRP-siRNA or nonsense siRNA in the lateral ventricles. Substantia nigra samples were collected for quantification of PRP and TH expression using real-time polymerase chain reaction and western blotting. PRP-siRNA decreased PRP expression in the substantia nigra. TH expression was decreased in PD model rats but was increased after PRP silencing. We conclude that PRP-siRNA may increase TH expression in vivo and may therefore exert protective effects on neurons in a model of PD.

Aquaporin-4 deficiency diminishes the differential degeneration of midbrain dopaminergic neurons in experimental Parkinson’s disease

Parkinson’s disease (PD) is primarily due to the progressive, selective and irreversible loss of dopaminergic (DA) neurons in the substantia nigra (SN). Interestingly, DA neurons in the ventral and lateral SN are much more susceptible than adjacent dopamine neurons in the ventral tegmental area (VTA) not only in human PD but in many PD model systems. However, the molecular causes of regional vulnerability in PD remain unknown. In our previous studies, we established acute PD animal models by administration of MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine), and found that AQP4 knockout mice were significantly more prone to MPTP-induced neurotoxicity. Here, we further observe that AQP4 deficiency resulted in the same susceptible to MPTP between SN DA neuron and VTA neurons both in acute and chronic PD model. Moreover, we show that AQP4 deficiency increased the numbers of reactive astrocytes and microglias not only in the SN and but also in the VTA under basal and MPTP-induced situations. Meanwhile, AQP4 deficiency disrupted the balance of the pro-inflammatory cytokine/neurotrophin in midbrain. Taken together, these results demonstrate that glial AQP4 is involved in the susceptibility differences of DA neurons between SN and VTA, although the precise mechanism of AQP4 remains to be explored. Moreover, these findings also suggest that these susceptibility differences are not only due to intrinsic neuronal factors, but also attribute to differences in astrocytes of these regions.

Differential Regulation of Action Potential Shape and Burst-Frequency Firing by BK and Kv2 Channels in Substantia Nigra Dopaminergic Neurons.

This work shows that BK calcium-activated potassium channels and Kv2 voltage-activated potassium channels both regulate action potentials in dopamine neurons of the substantia nigra pars compacta. Although both channel types participate in action potential repolarization about equally, they have contrasting and partially opposite effects in regulating neuronal firing at frequencies typical of bursting. Our analysis shows that this results from their different kinetic properties, with fast-activating BK channels serving to short-circuit activation of Kv2 channels, which tend to slow firing by producing a deep afterhyperpolarization. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell.

Efficient Expression of Igf-1 from Lentiviral Vectors Protects In Vitro but Does Not Mediate Behavioral Recovery of a Parkinsonian Lesion in Rats.

Gene therapy approaches delivering neurotrophic factors have offered promising results in both preclinical and clinical trials of Parkinson's disease (PD). However, failure of glial cell line-derived neurotrophic factor in phase 2 clinical trials has sparked a search for other trophic factors that may retain efficacy in the clinic. Direct protein injections of one such factor, insulin-like growth factor (IGF)-1, in a rodent model of PD has demonstrated impressive protection of dopaminergic neurons against 6-hydroxydopamine (6-OHDA) toxicity. However, protein infusion is associated with surgical risks, pump failure, and significant costs. We therefore used lentiviral vectors to deliver Igf-1, with a particular focus on the novel integration-deficient lentiviral vectors (IDLVs). A neuron-specific promoter, from the human synapsin 1 gene, excellent for gene expression from IDLVs, was additionally used to enhance Igf-1 expression. An investigation of neurotrophic effects on primary rat neuronal cultures demonstrated that neurons transduced with IDLV-Igf-1 vectors had complete protection on withdrawal of exogenous trophic support. Striatal transduction of such vectors into 6-OHDA-lesioned rats, however, provided neither protection of dopaminergic substantia nigra neurons nor improvement of animal behavior.

No parkinsonism in SCA2 and SCA3 despite severe neurodegeneration of the dopaminergic substantia nigra.

See Klockgether (doi:10.1093/awv253) for a scientific commentary on this article.The spinocerebellar ataxias types 2 (SCA2) and 3 (SCA3) are autosomal dominantly inherited cerebellar ataxias which are caused by CAG trinucleotide repeat expansions in the coding regions of the disease-specific genes. Although previous post-mortem studies repeatedly revealed a consistent neurodegeneration of the dopaminergic substantia nigra in patients with SCA2 and with SCA3, parkinsonian motor features evolve only rarely. As the pathophysiological mechanism how SCA2 and SCA3 patients do not exhibit parkinsonism is still enigmatic, we performed a positron emission tomography and a post-mortem study of two independent cohorts of SCA2 and SCA3 patients with and without parkinsonian features. Positron emission tomography revealed a significant reduction of dopamine transporter levels in the striatum as well as largely unaffected postsynaptic striatal D2 receptors. In spite of this remarkable pathology in the motor mesostriatal pathway, only 4 of 19 SCA2 and SCA3 patients suffered from parkinsonism. The post-mortem investigation revealed, in addition to an extensive neuronal loss in the dopaminergic substantia nigra of all patients with spinocerebellar ataxia, a consistent affection of the thalamic ventral anterior and ventral lateral nuclei, the pallidum and the cholinergic pedunculopontine nucleus. With the exception of a single patient with SCA3 who suffered from parkinsonian motor features during his lifetime, the subthalamic nucleus underwent severe neuronal loss, which was clearly more severe in its motor territory than in its limbic or associative territories. Our observation that lesions of the motor territory of the subthalamic nucleus were consistently associated with the prevention of parkinsonism in our SCA2 and SCA3 patients matches the clinical experience that selective targeting of the motor territory of the subthalamic nucleus by focal lesions or deep brain stimulation can ameliorate parkinsonian motor features and is likely to counteract the manifestation of parkinsonism in SCA2 and SCA3 despite a severe neurodegeneration of the dopaminergic substantia nigra.

"Provocation test" as a novel approach to early diagnosis of Parkinson's disease.

Parkinson's disease (PD) is one of the most severe socially important neurodegenerative disorders. The loss of dopaminergic neurons of the nigrostriatal sys� tem is considered to be a key aspect of the pathogene� sis of PD. The first clinical symptoms of the disease including motor dysfunction develop many years after the onset of the pathological process, which is believed to be a consequence of the brain plasticity (or com� pensatory mechanisms). However, when 50-70% of dopaminergic neurons in the substantia nigra (SN) are degenerated and the striatal dopamine (DA) is reduced by 80% those compensatory mechanisms lose the ability to mask motor impairments (1-5). Conse� quently, preclinical diagnosis of the masked patholog� ical process is of great importance because it may become the basis for preventive neuroprotective ther� apy. The therapeutic approach in this case can be aimed at slowingdown the process of neuronal loss and thus extending the preclinical stages of the disease or, in other words, the period of patient's comfortable state. Currently, the methods of preclinical diagnosis of PD including positron emission tomography and singlephoton emission computed tomography are very costly and technically challenging, which results in their limited use in mass health screening. The purpose of this study was to experimentally develop a "provocation test" that would allow to diag� nose PD at its preclinical stage. This approach is based on the temporary and reversible "impairment" of dopaminergic nigrostriatal system by pharmacological inhibition of DA synthesis to the threshold level, where the first motor symptoms can be observed. Moreover, it is necessary to use the minimal dose of the DA inhibitor that would cause motor abnormali� ties in an experimental model of presymptomatic PD, but would not affect the behavior of normal animals in the control. Experiments were performed on male C57BL/6JSto mice (2.5� to 3�monthold animals with a body weight of 22-26 g) using the model of PD at a preclinical stage that was developed in our laboratory (6). Accord� ing to this model, the mice (n = 42) were subcutane� ously injected with the neurotoxin 1�methyl�4�phe� nyl�1,2,3,6�tetrahydropyridine (MPTP) at a single dose of 12 mg/kg in 0.9% NaCl. The injection was repeated twice at a 2�h interval. Animals in the control group (n = 42) were administered with 0.9% NaCl in a similar way. The model is characterized by the absence of any motor function impairment, the loss of 26% in dopaminergic neurons SN 59% of axon terminals in the striatum, and a 60% reduction of the striatal level of DA. The locomotor and exploratory activities of the animals were assessed 15 days after MPTP or saline administration during the 6�min period in the "open� field" test using an OptoVarimex�3 system (Columbus Instruments International, United States). Based on the data obtained, all animals were grouped into two subgroups with equal average and variance values; the subgroups were designated as NaCl 1 and NaCl 2 , MPTP 1 and MPTP 2 , respectively.

Effects of inflammation on hippocampus and substantia nigra responses to novelty in healthy human participants.

Humans are naturally inquisitive. This tendency is adaptive, aiding identification of potentially valuable novel outcomes. The dopaminergic substantia nigra (SN) is implicated in the drive to explore novel stimuli and situations. However, infection and inflammation inhibit the motivation to seek out novelty. This likely serves to limit exposure to uncertain, potentially detrimental outcomes when metabolic resources are limited. Nevertheless, the neural mechanisms through which inflammation constrains novelty seeking are poorly understood. We therefore scanned 16 healthy participants (6 male, mean 27.2±7.3 years), using fMRI, once following experimental inflammation (intramuscular (i.m.) typhoid vaccination) and once after placebo (i.m. saline), with the aim of characterizing effects of inflammation on neural processing of novel and familiar place, and face stimuli. We specifically tested the effects of inflammation on the hypothesized roles of SN and hippocampus in novelty processing. Typhoid vaccination evoked a nearly threefold increase in circulating pro-inflammatory cytokine (interleukin-6) levels 3 h after injection, indicating induction of mild systemic inflammation. Enhanced hippocampal responses to novel (compared with familiar) stimuli were observed following both vaccine and placebo, consistent with intact central novelty detection. However, the normal bilateral reactivity of SN to stimulus novelty was significantly attenuated following inflammation. Correspondingly, inflammation also markedly impaired novelty-related functional coupling between the SN and hippocampus. These data extend previous findings of SN sensitivity to mild inflammation associated with changes in psychomotor responding, and suggest that inflammation-induced blunting of SN responses to hippocampal novelty signals may represent a plausible mechanism through which inflammation impairs motivational responses to novelty.

Nifedipine and nimodipine protect dopaminergic substantia nigra neurons against axotomy-induced cell death in rat vibrosections via modulating inflammatory responses

Neurodegeneration of cholinergic and dopaminergic neurons is a major hallmark in Alzheimer׳s or Parkinson׳s disease, respectively. A dysregulation in calcium homeostasis may be part of this process and counteracting calcium influx may have neuroprotective properties in both diseases. Therefore, we investigated the putative neuroprotective or neurotoxic activity of L-type calcium channel (LTCC) inhibitors on cholinergic and dopaminergic neurons in a rat organotypic vibrosection model. Sagittal or coronal vibrosections (200μm thick) of postnatal day 10 rats were cultured on 0.4μm semipermeable membranes for 2 weeks with 10ng/ml nerve growth factor (NGF) and/or glial-cell line derived neurotrophic factor (GDNF) to maintain survival of cholinergic or dopaminergic neurons, respectively. Thereafter, sections were incubated with 0.1, 1 or 10μM isradipine, nicardipine or verapamil for 2 weeks to explore cytotoxicity. Alternatively, in order to explore neuroprotective activity, vibrosections were incubated without growth factors but with isradipine or verapamil or with nicardipine, nimodipine or nifedipine from the beginning for 4 weeks. Our data show that all LTCC inhibitors exhibited no neurotoxic effect on cholinergic and dopaminergic neurons. Further, LTCC inhibitors did not have any neuroprotective activity on cholinergic neurons. However, nimodipine and nifedipine significantly enhanced the survival of dopaminergic substantia nigra (SN) but not ventral tegmental area (VTA) neurons, while nicardipine, isradipine and verapamil had no effect. Nifedipine (and more potently GDNF) reduced inflammatory cytokines (macrophage inflammatory protein-2, tumor necrosis factor-α), but did not influence oxidative stress or caspase-3 activity and did not interfere with iron-mediated overload. Our data show that nifedipine and nimodipine are very potent to enhance the survival of axotomized SN neurons, possibly influencing inflammatory processes.

Evidence for topographic guidance of dopaminergic axons by differential Netrin-1 expression in the striatum.

There are two main subgroups of midbrain dopaminergic (DA) neurons: the more medially located ventral tegmental area (VTA) DA neurons, which have axons that innervate the ventral-lateral (VL) striatum, and the more laterally located substantia nigra (SN) DA neurons, which preferentially degenerate in Parkinson's disease (PD) and have axons that project to the dorsal-medial (DM) striatum. DA axonal projections in the striatum are not discretely localized and they arborize widely, however they do not stray from one zone to the other so that VTA axons remain in the VL zone and SN axons in the DM zone. Here we provide evidence that Netrin-1 acts in a novel fashion to topographically pattern midbrain DA axons into these two striatal zones by means of a gradient of Netrin-1 in the striatum and by differential attraction of the axons to Netrin-1. Midbrain DA neurons are attracted to the striatum in culture and this attraction is blocked by an anti-DCC (Netrin receptor) antibody. Mechanistically, outgrowth of both VTA and SN DA axons is stimulated by Netrin-1, but the two populations of DA axons respond optimally to overlapping but distinct concentrations of Netrin-1, with SN axons preferring lower concentrations and VTA axons preferring higher concentrations. In vivo this differential preference is closely mirrored by differences in Netrin-1 expression in their respective striatal target fields. In vivo in mice lacking Netrin-1, DA axons that reach the striatum fail to segregate into two terminal zones and to fully innervate the striatum. Our results reveal novel actions for Netrin-1 and provide evidence for a mechanism through which DA axons can selectively innervate one of two terminal zones in the striatum but have free reign to arborize widely within a terminal zone.