CDNF and MANF in an experimental model of Parkinson's disease in rats

Parkinson s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra (SN). Current therapies of PD do not stop the progression of the disease and the efficacy of these treatments wanes over time. Neurotrophic factors are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. Neurotrophic factors are attractive candidates for neuroprotective or even neurorestorative treatment of PD. Thus, searching for and characterizing trophic factors are highly important approaches to degenerative diseases. CDNF (cerebral dopamine neurotrophic factor) and MANF (mesencephalic astrocyte-derived neurotrophic factor) are secreted proteins that constitute a novel, evolutionarily conserved neurotrophic factor family expressed in vertebrates and invertebrates. The present study investigated the neuroprotective and restorative effects of human CDNF and MANF in rats with unilateral partial lesion of dopamine neurons by 6-hydroxydopamine (6-OHDA) using both behavioral (amphetamine-induced rotation) and immunohistochemical analyses. We also investigated the distribution and transportation profiles of intrastriatally injected CDNF and MANF in rats. Intrastriatal CDNF and MANF protected nigrostriatal dopaminergic neurons when administered six hours before or four weeks after the neurotoxin 6-OHDA. More importantly, the function of the lesioned nigrostriatal dopaminergic system was partially restored even when the neurotrophic factors were administered four weeks after 6-OHDA. A 14-day continuous infusion of CDNF but not of MANF restored the function of the midbrain neural circuits controlling movement when initiated two weeks after unilateral injection of 6-OHDA. Continuous infusion of CDNF also protected dopaminergic TH-positive cell bodies from toxin-induced degeneration in the substantia nigra pars compacta (SNpc) and fibers in the striatum. When injected into the striatum, CDNF and GDNF had similar transportation profiles from the striatum to the SNpc; thus CDNF may act via the same nerve tracts as GDNF. Intrastriatal MANF was transported to cortical areas which may reflect a mechanism of neurorestorative action that is different from that of CDNF and GDNF. CDNF and MANF were also shown to distribute more readily than GDNF. In conclusion, CDNF and MANF are potential therapeutic proteins for the treatment of PD.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.

Role of GDNF and its Cross-Talk with Other Growth Factors in the Dopaminergic System

Parkinson´s Disease (PD) is a neurodegenerative movement disorder resulting from loss of dopaminergic (DA) neurons in substantia nigra (SN). Possible causative treatment strategies for PD include neurotrophic factors, which protect and in some cases restore the function of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors have been to date the most promising candidates for treatment of PD, demonstrating both neuroprotective and neurorestorative properties. We have investigated the role of GDNF in the rodent dopaminergic system and its possible crosstalk with other growth factors. We characterized the GDNF-induced gene expression changes by DNA microarray analysis in different neuronal systems, including in vitro cultured Neuro2A cells treated with GDNF, as well as midbrains from GDNF heterozygous (Hz) knockout mice. These microarray experiments, resulted in the identification of GDNF-induced genes, which were also confirmed by other methods. Further analysis of the dopaminergic system of GDNF Hz mice demonstrated about 40% reduction in GDNF levels, revealed increased intracellular dopamine concentrations and FosB/DeltaFosB expression in striatal areas. These animals did not show any significant changes in behavioural analysis of acute and repeated cocaine administration on locomotor activity, nor did they exhibit any changes in dopamine output following treatment with acute cocaine. We further analysed the significance of GDNF receptor RET signalling in dopaminergic system of MEN2B knock-in animals with constitutively active Ret. The MEN2B animals showed a robust increase in extracellular dopamine and its metabolite levels in striatum, increased tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels by immunohistochemical staining and Western blotting, as well as increased Th mRNA levels in SN. MEN2B mice had increased number of DA neurons in SN by about 25% and they also exhibited increased sensitivity to the stimulatory effects of cocaine. We also developed a semi-throughput in vitro micro-island assay for the quantification of neuronal survival and TH levels by computer-assisted methodology from limited amounts of tissue. This assay can be applied for the initial screening for dopaminotrophic molecules, as well as chemical drug library screening. It is applicable to any neuronal system for the screening of neurotrophic molecules. Since our microarray experiments revealed possible GDNF-VEGF-C crosstalk we further concentrated on studying the neurotrophic effects of VEGF-C. We showed that VEGF-C acts as a neurotrophic molecule for the DA neurons both in vitro and in vivo, however without additive effect when used together with GDNF. The neuroprotective effect for VEGF-C in vivo in rat 6-OHDA model of PD was demonstrated. The possible signalling mechanisms of VEGF-C in the nervous system were investigated - infusion of VEGF-C to rat brain induced ERK activation, however no direct activation of RET signalling in vitro was found. VEGF-C treatment of rat striatum lead to up-regulation of VEGFR-1-3, indicating that VEGF-C can regulate the expression level of its own receptor. VEGF-C dopaminotrophic activity in vivo was further supported by increased vascular tissue in the neuroprotection experiments.

MPTP activates ASK1-p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model

Activation of apoptosis signal regulating kinase 1 (ASK1)-p38 MAPK death signaling cascade is irn plicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream 1)38 MAPK in a time dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thiorcdoxin (Trxl), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase ji 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein ovariation netvvork analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mecliated TNF signaling through intrathecal administration of TNFneutralizing antibody prevented Trxl oxidation and downstream ASK1-p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1-p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states. (C) 2015 Published by Elsevier Inc.

The Role of the Subthalamic Nucleus in L-DOPA Induced Dyskinesia in 6-Hydroxydopamine Lesioned Rats

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The locus coeruleus Is Directly Implicated in L-DOPA-Induced Dyskinesia in Parkinsonian Rats: An Electrophysiological and Behavioural Study

Despite being the most effective treatment for Parkinson's disease, L-DOPA causes a development of dyskinetic movements in the majority of treated patients. L-DOPA-induced dyskinesia is attributed to a dysregulated dopamine transmission within the basal ganglia, but serotonergic and noradrenergic systems are believed to play an important modulatory role. In this study, we have addressed the role of the locus coeruleus nucleus (LC) in a rat model of L-DOPA-induced dyskinesia. Single-unit extracellular recordings in vivo and behavioural and immunohistochemical approaches were applied in rats rendered dyskinetic by the destruction of the nigrostriatal dopamine neurons followed by chronic treatment with L-DOPA. The results showed that L-DOPA treatment reversed the change induced by 6-hydroxydopamine lesions on LC neuronal activity. The severity of the abnormal involuntary movements induced by L-DOPA correlated with the basal firing parameters of LC neuronal activity. Systemic administration of the LC-selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not modify axial, limb, and orolingual dyskinesia, whereas chemical destruction of the LC with ibotenic acid significantly increased the abnormal involuntary movement scores. These results are the first to demonstrate altered LC neuronal activity in 6-OHDA lesioned rats treated with L-DOPA, and indicate that an intact noradrenergic system may limit the severity of this movement disorder.

Relação entre a doença de Parkinson e o gene LRRK2: um estudo na população brasileira

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente depois da Doença de Alzheimer, afetando aproximadamente 1% da população com idade superior a 65 anos. Clinicamente, esta doença caracteriza-se pela presença de tremor em repouso, bradicinesia, rigidez muscular e instabilidade postural, os quais podem ser controlados com a administração do levodopa. As características patológicas da DP incluem a despigmentação da substância nigra devido à perda dos neurônios dopaminérgicos e a presença de inclusões proteicas denominadas corpos de Lewy nos neurônios sobreviventes. As vias moleculares envolvidas com esta patologia ainda são obscuras, porém a DP é uma doença complexa, resultante da interação entre fatores ambientais e causas genéticas. Mutações no gene leucine-rich repeat kinase 2 (LRRK2; OMIM 609007) constituem a forma mais comum de DP. Este gene codifica uma proteína, membro da família de proteínas ROCO, que possui, entre outros domínios, dois domínios funcionais GTPase (ROC) e quinase (MAPKKK). Neste estudo, os principais domínios do gene LRRK2 foram analisados em 204 pacientes brasileiros com DP por meio de sequenciamento dos produtos da PCR. Através da análise de 14 exons correspondentes aos domínios ROC, COR e MAPKKK foram identificadas 31 variantes. As alterações novas, p.C1770R e p.C2139S, possuem um potencial papel na etiologia da DP. Três alterações exônicas (p.R1398R, p.T1410M e p.Y2189C) e nove intrônicas (c.4317+16C>T, c.5317+59A>C, c.5509+20A>C, c.5509+52T>C, c.5509+122A>G, c.5657-46C>T, c.6382-36G>A, c.6382-37C>T e c.6576+44T>C) são potencialmente não patogênicas. Ao todo, dezessete variantes exônicas e intrônicas constituem polimorfismos já relatados na literatura (p.R1398H, p.K1423K, p.R1514Q, p.P1542S, c.4828-31T>C, p.G1624G, p.K1637K, p.M1646T, p.S1647T, c.5015+32A>G, c.5170+23T>A, c.5317+32C>T, p.G1819G, c.5948+48C>T, p.N2081D, p.E2108E e c.6381+30A>G). A frequência total de alterações potencialmente patogênicas ou patogênicas detectadas em nossa amostra foi de 3,4% (incluindo a mutação p.G2019S, anteriormente descrita em 2 artigos publicados por nosso grupo: Pimentel et al., 2008; Abdalla-Carvalho et al., 2010), sendo a frequência de mutações nos casos familiares (11,1%) cerca de seis vezes maior do que a encontrada nos casos isolados da DP (1,8%). Os resultados alcançados neste estudo revelam que mutações no gene LRRK2 desempenham um papel significativo como fator genético para o desenvolvimento da DP em pacientes brasileiros.

Rastreamento de mutações no gene GBA como fator de risco ao desenvolvimento da doença de Parkinson na população brasileira

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente, depois da doença de Alzheimer, com uma incidência de aproximadamente 3,3% na população brasileira acima dos 60 anos. Ela é caracterizada por uma perda dos neurônios dopaminérgicos da parte compacta da substância negra e pela presença de inclusões protéicas intracelulares denominadas corpúsculos de Lewy nos neurônios sobreviventes. A DP tem uma etiologia complexa que envolve interações genes-ambiente e múltiplos genes de susceptibilidade. Nesse contexto, mutações de perda de função no gene da glicocerebrosidase (GBA) têm sido bem validadas como importantes fatores de risco para a DP. Esse gene está localizado na região 1q21 e compreende 11 exons que codificam a enzima lisossômica glicocerebrosidase. O principal objetivo deste estudo foi investigar se alterações no gene GBA constituem um fator de predisposição para o desenvolvimento da DP na população brasileira. Para isso, um grupo de 126 pacientes brasileiros, não-aparentados, com DP (24 casos familiares e 102 isolados; idade média 66,4 11,4) foram analisados para mutações no GBA através do seqüenciamento completo de todos os exons e alguns íntrons. Sete alterações e um alelo recombinante, anteriormente encontrados em pacientes com a DP analisados em outros estudos, foram detectados (K(-)27R, IVS2+1G>A, N370S, L444P, T369M, A456P, E326K e RecNciI), assim como, uma variante nunca antes identificada associada à DP (G325G) e uma nova mutação (W378C), num total de 18 pacientes (14,3%). Além disso, foram encontradas três alterações intrônicas (c.454+47G>A, c.589-86A>G e c.1225-34C>A), que constam do banco de SNPs, entretanto, não foram associadas a nenhuma doença. Dentre todas as variantes identificadas, três são comprovadamente patogênicas (IVS2+1G>A, L444P e N370S) e foram encontradas em 5,5% da amostra, não sendo detectadas na amostra controle, indicando uma freqüência significativamente alta dessas mutações em pacientes com DP quando comparadas aos controles (P=0,0033). Esses resultados reforçam a associação entre o gene GBA e a DP na população brasileira, além de apoiar a hipótese de que alterações nesse gene representam um importante fator de susceptibilidade ao desenvolvimento da DP

Interaction between the 5-HT system and the basal ganglia: functional implication and therapeutic perspective in Parkinson's disease

The neurotransmitter serotonin (5-HT) has a multifaceted function in the modulation of information processing through the activation of multiple receptor families, including G-protein-coupled receptor subtypes (5-HT1, 5-HT2, 5-HT4-7) and ligand-gated ion channels (5-HT3). The largest population of serotonergic neurons is located in the midbrain, specifically in the raphe nuclei. Although the medial and dorsal raphe nucleus (DRN) share common projecting areas, in the basal ganglia (BG) nuclei serotonergic innervations come mainly from the DRN. The BG are a highly organized network of subcortical nuclei composed of the striatum (caudate and putamen), subthalamic nucleus (STN), internal and external globus pallidus (or entopeduncular nucleus in rodents, GPi/EP and GPe) and substantia nigra (pars compacta, SNc, and pars reticulata, SNr). The BG are part of the cortico-BG-thalamic circuits, which play a role in many functions like motor control, emotion, and cognition and are critically involved in diseases such as Parkinson's disease (PD). This review provides an overview of serotonergic modulation of the BG at the functional level and a discussion of how this interaction may be relevant to treating PD and the motor complications induced by chronic treatment with L-DOPA.

Differential Neuroprotective Effects of 5 '-Deoxy-5 '-Methylthioadenosine

Background: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. Methods: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. Results: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A(2A) adenosine receptor antagonist did produce significant neuroprotection in this brain region. Conclusion: MTA may potentially offer therapeutic neuroprotection.

Efeitos da hipóxia-isquemia perinatal sobre o comportamento motor, distribuição da Tirosina Hidroxilase na substância negra e da NADPH diaforase no hipocampo durante o desenvolvimento em ratos

A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento.

Increased antiparkinson efficacy of the combined administration of VEGF- and GDNF-loaded nanospheres in a partial lesion model of Parkinson's disease

Current research efforts are focused on the application of growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), as neuroregenerative approaches that will prevent the neurodegenerative process in Parkinson's disease. Continuing a previous work published by our research group, and with the aim to overcome different limitations related to growth factor administration, VEGF and GDNF were encapsulated in poly(lactic-co-glycolic acid) nanospheres (NS). This strategy facilitates the combined administration of the VEGF and GDNF into the brain of 6-hydroxydopamine (6-OHDA) partially lesioned rats, resulting in a continuous and simultaneous drug release. The NS particle size was about 200 nm and the simultaneous addition of VEGF NS and GDNF NS resulted in significant protection of the PC-12 cell line against 6-OHDA in vitro. Once the poly(lactic-co-glycolic acid) NS were implanted into the striatum of 6-OHDA partially lesioned rats, the amphetamine rotation behavior test was carried out over 10 weeks, in order to check for in vivo efficacy. The results showed that VEGF NS and GDNF NS significantly decreased the number of amphetamine-induced rotations at the end of the study. In addition, tyrosine hydroxylase immunohistochemical analysis in the striatum and the external substantia nigra confirmed a significant enhancement of neurons in the VEGF NS and GDNF NS treatment group. The synergistic effect of VEGF NS and GDNF NS allows for a reduction of the dose by half, and may be a valuable neurogenerative/neuroreparative approach for treating Parkinson's disease.

Depression of Serotonin Synaptic Transmission by the Dopamine Precursor L-DOPA

Imbalance between the dopamine and serotonin (5-HT) neurotransmitter systems has been implicated in the comorbidity of Parkinson's disease (PD) and psychiatric disorders. L-DOPA, the leading treatment of PD, facilitates the production and release of dopamine. This study assessed the action of L-DOPA on monoamine synaptic transmission in mouse brain slices. Application of L-DOPA augmented the D2-receptor-mediated inhibitory postsynaptic current (IPSC) in dopamine neurons of the substantia nigra. This augmentation was largely due to dopamine release from 5-HT terminals. Selective optogenetic stimulation of 5-HT terminals evoked dopamine release, producing D2-receptor-mediated IPSCs following treatment with L-DOPA. In the dorsal raphe, L-DOPA produced a long-lasting depression of the 5-HT1A-receptor-mediated IPSC in 5-HT neurons. When D2 receptors were expressed in the dorsal raphe, application of L-DOPA resulted in a D2-receptor-mediated IPSC. Thus, treatment with L-DOPA caused ectopic dopamine release from 5-HT terminals and a loss of 5-HT-mediated synaptic transmission.

M-type channels selectively control bursting in rat dopaminergic neurons

Midbrain dopaminergic neurons in the substantia nigra, pars compacta and ventral tegmental area are critically important in many physiological functions. These neurons exhibit firing patterns that include tonic slow pacemaking, irregular firing and bursting, and the amount of dopamine that is present in the synaptic cleft is much increased during bursting. The mechanisms responsible for the switch between these spiking patterns remain unclear. Using both in-vivo recordings combined with microiontophoretic or intraperitoneal drug applications and in-vitro experiments, we have found that M-type channels, which are present in midbrain dopaminergic cells, modulate the firing during bursting without affecting the background low-frequency pacemaker firing. Thus, a selective blocker of these channels, 10,10-bis(4-pyridinylmethyl)-9(10H)- anthracenone dihydrochloride, specifically potentiated burst firing. Computer modeling of the dopamine neuron confirmed the possibility of a differential influence of M-type channels on excitability during various firing patterns. Therefore, these channels may provide a novel target for the treatment of dopamine-related diseases, including Parkinson's disease and drug addiction. Moreover, our results demonstrate that the influence of M-type channels on the excitability of these slow pacemaker neurons is conditional upon their firing pattern. © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.