Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo

In Parkinson's disease, brain dopamine neurons degenerate most prominently in the substantia nigra. Neurotrophic factors promote survival, differentiation and maintenance of neurons in developing and adult vertebrate nervous system. The most potent neurotrophic factor for dopamine neurons described so far is the glial-cell-line-derived neurotrophic factor (GDNF). Here we have identified a conserved dopamine neurotrophic factor (CDNF) as a trophic factor for dopamine neurons. CDNF, together with its previously described vertebrate and invertebrate homologue the mesencephalic-astrocyte-derived neurotrophic factor, is a secreted protein with eight conserved cysteine residues, predicting a unique protein fold and defining a new, evolutionarily conserved protein family. CDNF (Armetl1) is expressed in several tissues of mouse and human, including the mouse embryonic and postnatal brain. In vivo, CDNF prevented the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in a rat experimental model of Parkinson's disease. A single injection of CDNF before 6-OHDA delivery into the striatum significantly reduced amphetamine-induced ipsilateral turning behaviour and almost completely rescued dopaminergic tyrosine-hydroxylase-positive cells in the substantia nigra. When administered four weeks after 6-OHDA, intrastriatal injection of CDNF was able to restore the dopaminergic function and prevent the degeneration of dopaminergic neurons in substantia nigra. Thus, CDNF was at least as efficient as GDNF in both experimental settings. Our results suggest that CDNF might be beneficial for the treatment of Parkinson's disease.     

Inducing dopaminergic differentiation of expanded rat mesencephalic neural stem cells by ascorbic acid in vitro

Ascorbic acid (AA) induced differentiation of neural stem cells (NSCs) into dopaminergic (DAergic) neurons is reported.NSCs derived from rat mesencephalon were maintained and expanded in a defined medium containing mitogens of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).Compared with the control, ascorbic acid treatment led to more DAergic neuronal differentiation as indicated by the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT), which are specific markers of dopamine neurons.AA induction also enhanced expression of Nurr1 and Shh.PD98059, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, could block AA-induced Nurr1, TH and DAT mRNA expression.The results might suggest a new strategy to provide enough dopaminergic cells for the therapy of Parkinson's disease (PD), and Nurr1 and ERK signaling pathway might participate in the AA-induced DAergic differentiation.     

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease

Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.     

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.     

Evidence for neuromelanin involvement in MPTP-induced neurotoxicity

Exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) reproduces certain clinical, pathological, and neurochemical features of Parkinson's disease. MPTP is metabolized by monoamine oxidase Type B to 1-methyl-4-phenylpyridine (MPP+), which is selectively accumulated by high-affinity uptake mechanisms into dopaminergic neurons. Lyden et al. described low-affinity binding of MPTP to synthetic and retinal melanin. We showed that MPP+ binds to neuromelanin with high affinity, suggesting that in MPTP neurotoxicity, MPP+ enters nigral neurons by the dopamine uptake system and binds to neuromelanin, which serves as a depot, continuously releasing MPP+ until it destroys the cells. This model predicts that agents which compete with MPP+ binding to neuromelanin should partially protect the dopamine neurons from MPTP-induced toxicity. The most potent identified competitor for MPP+ binding to melanin is the antimalarial drug chloroquine, which has a high affinity for melanins. In the present study, chloroquine, administered to monkeys in conventional anti-malarial doses before MPTP, protects them from MPTP-induced parkinsonian motor abnormalities, dopamine depletion in the striatum, and neuropathological changes in the substantia nigra.     

Salsolinol合成酶的克隆和表达

 Salsolinol 合成酶是一种催化多巴胺和乙醛生成Salsolinol 的酶,与帕金森病发病机制密切相关。研究发现Salsolinol 合成酶与泛素的氨基酸序列高度相似,只有4 个氨基酸位点有差异。本研究以泛素基因为模板,采用聚合酶链式反应技术对4 个位点进行定点突变,将突变基因片段克隆到载体pET30a-GST 上,构建pET30a-GST-Sal synthase 重组载体,转化BL21 后,IPTG 诱导重组菌表达融合蛋白,经亲和层析柱纯化。结果表明,实现目的位点的定点突变,获得Sal 合成酶基因,成功构建了GST-Sal synthase 原核表达质粒,在大肠杆菌中表达纯化后得到较高纯度的GST-Sal synthase 融合蛋白。     

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1

Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.     

Cloning and expression of human and rat D1 dopamine receptors

The importance of the dopaminergic system in brain function has been emphasized by its association with neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. On the basis of their biochemical and pharmacological characteristics, dopamine receptors are classified into D1 and D2 subtypes. As the most abundant dopamine receptor in the central nervous system, D1 receptors seem to mediate some behavioural responses, modulate activity of D2 dopamine receptors, and regulate neuron growth and differentiation. The D dopamine receptor has been cloned by low-stringency screening. We report here the cloning of human and rat D1 dopamine receptors by applying an approach based on the polymerase chain reaction. The cloned human D1 dopamine receptor has been characterized on the basis of four criteria: the deduced amino-acid sequence, which reveals that it is a G protein-coupled receptor; the tissue distribution of its messenger RNA, which is compatible with that of the D1 dopamine receptor; its pharmacological profile when transfected into COS-7 cells; and its ability to stimulate the accumulation of cyclic AMP in human 293 cells.     

<Emphasis Type="Italic">In vitro</Emphasis> induced dopaminergic differentiation of expanded rat mesencephalic neural stem cell

Recent progress in stem cell biology and recognition of the unique biological properties of stem cell have made it possible to treat the neurodegenerative diseases includ- ing Parkinson抯 disease (PD) by the approach of cell-re- placement and nutritional support with NSCs. Tissue re-construction based on the stem cells endowed us some unpredicted application and market opportunities. Studies in a variety of systems have highlighted perfect prospects for the application of stem cells in the t…     

BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra

Brain-derived neurotrophic factor (BDNF), present in minute amounts in the adult central nervous system, is a member of the nerve growth factor (NGF) family, which includes neurotrophin-3 (NT-3). NGF, BDNF and NT-3 all support survival of subpopulations of neural crest-derived sensory neurons; most sympathetic neurons are responsive to NGF, but not to BDNF; NT-3 and BDNF, but not NGF, promote survival of sensory neurons of the nodose ganglion. BDNF, but not NGF, supports the survival of cultured retinal ganglion cells but both NGF and BDNF promote the survival of septal cholinergic neurons in vitro. However, knowledge of their precise physiological role in development and maintenance of the nervous system neurons is still limited. The BDNF gene is expressed in many regions of the adult CNS, including the striatum. A protein partially purified from bovine striatum, a target of nigral dopaminergic neurons, with characteristics apparently similar to those of BDNF, can enhance the survival of dopaminergic neurons in mesencephalic cultures. BDNF seems to be a trophic factor for mesencephalic dopaminergic neurons, increasing their survival, including that of neuronal cells which degenerate in Parkinson's disease. Here we report the effects of BDNF on the survival of dopaminergic neurons of the developing substantia nigra.     

SH-SY5Y与U87细胞共培养条件性培养基可降低N-甲基-salsolinol介导THP-1细胞的凋亡

 神经免疫在帕金森病(PD)的致病机理中发挥重要的作用,PD 患者的外周血淋巴细胞的数量发生了变化,提示外周免疫系统在PD 的发生发展中发挥一定的作用。但是外周单核细胞(PBMC)在其中发挥的具体作用尚不清楚。外源性神经毒素(MPTP)类似物,内源性神经毒素(NMSal)可能是导致PD 发生的一种因素。研究采用NMSal 损伤的SH-SY5Y与U87 细胞共培养的条件性培养基培养外周单核细胞THP-1,探讨NMSal 损伤的多巴胺能神经元细胞对外周单核细胞的影响。结果表明,该条件性培养基可以降低NMSal 毒性诱导的THP-1 细胞的凋亡、氧化应激水平(MDA 和H₂O₂)、线粒体的损伤和凋亡相关蛋白FADD、Bax 和caspase3 的表达和活化水平。PD 病人中损伤的多巴胺能神经元与星形胶质细胞的相互作用可能会影响PBMC,进而影响PD 病情的进展。     

A Drosophila model of Parkinson's disease

Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder. Mutations in the alpha-synuclein gene are linked to familial Parkinson's disease and alpha-synuclein accumulates in Lewy bodies and Lewy neurites. Here we express normal and mutant forms of alpha-synuclein in Drosophila and produce adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein and locomotor dysfunction. Our Drosophila model thus recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease.     

BDNF controls dopamine D3 receptor expression and triggers behavioural sensitization

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, is a polypeptidic factor initially regarded to be responsible for neuron proliferation, differentiation and survival, through its uptake at nerve terminals and retrograde transport to the cell body. A more diverse role for BDNF has emerged progressively from observations showing that it is also transported anterogradely, is released on neuron depolarization, and triggers rapid intracellular signals and action potentials in central neurons. Here we report that BDNF elicits long-term neuronal adaptations by controlling the responsiveness of its target neurons to the important neurotransmitter, dopamine. Using lesions and gene-targeted mice lacking BDNF, we show that BDNF from dopamine neurons is responsible for inducing normal expression of the dopamine D3 receptor in nucleus accumbens both during development and in adulthood. BDNF from corticostriatal neurons also induces behavioural sensitization, by triggering overexpression of the D3 receptor in striatum of hemiparkinsonian rats. Our results suggest that BDNF may be an important determinant of pathophysiological conditions such as drug addiction, schizophrenia or Parkinson's disease, in which D3 receptor expression is abnormal.     

Expression of dopaminergic phenotypes in the mouse olfactory bulb induced by the calcitonin gene-related peptide

In the olfactory bulb, tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is expressed after birth when the axons of olfactory epithelial neurons have made synapses in the bulb. It has been suggested that expression of TH is regulated trans-synaptically because on deafferentation of the bulb there is a marked decrease in the contents of TH, dopamine and 3,4-dihydroxyphenylacetic acid, which, however, return to normal levels after regeneration of the primary afferents. To date the molecular signalling involved in this trans-synaptic induction has not yet been characterized; I have therefore studied the expression of dopaminergic properties (presence of TH and dopamine uptake) in dissociated cell cultures from embryonic mouse olfactory bulb. I report that the number of dopaminergic cells increases fivefold when olfactory bulb neurons are co-cultured with olfactory epithelial neurons and that soluble factors, rather than cell interactions, mediate this effect. The dopaminergic-inducing factor is the calcitonin gene-related peptide (CGRP) which is present in chemosensory neurons of the olfactory epithelium and when added at nanomolar concentrations to olfactory bulb cultures mimics the effect of olfactory epithelial neurons. Significantly the induction of dopaminergic phenotypes brought about by olfactory epithelial neurons is abolished by an antiserum to CGRP. These observations show that CGRP is involved in the differentiation of dopaminergic olfactory bulb neurons.     

Modelling schizophrenia using human induced pluripotent stem cells

Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85%. Although post-mortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (Supplementary Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cyclic AMP and WNT signalling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood. We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder.     

Facilitation of a dendritic calcium conductance by 5-hydroxytryptamine in the substantia nigra

Within the substantia nigra, the dendrites of dopaminergic neurons that project to the striatum appear to play an active and nonclassical role in the physiology of the neuron in that they release transmitter and protein, but little is known of the factors controlling release of substances from these dendrites. In this study, we show that 5-hydroxytryptamine, which is contained in afferent fibres to the substantia nigra, is present in terminals making direct synaptic contact with dopaminergic neurons and also that it has a site-dependent, receptor-mediated, facilitatory effect on a specific dendritic calcium-dependent potential in nigrostriatal neurons in vitro. The ionic and spatial features of this response, which is insensitive to blockade by three different K+-channel antagonists, could correspond to those underlying the dendritic release of dopamine.     

Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson's disease models

The striatum is a major forebrain nucleus that integrates cortical and thalamic afferents and forms the input nucleus of the basal ganglia. Striatal projection neurons target the substantia nigra pars reticulata (direct pathway) or the lateral globus pallidus (indirect pathway). Imbalances between neural activity in these two pathways have been proposed to underlie the profound motor deficits observed in Parkinson's disease and Huntington's disease. However, little is known about differences in cellular and synaptic properties in these circuits. Indeed, current hypotheses suggest that these cells express similar forms of synaptic plasticity. Here we show that excitatory synapses onto indirect-pathway medium spiny neurons (MSNs) exhibit higher release probability and larger N-methyl-d-aspartate receptor currents than direct-pathway synapses. Moreover, indirect-pathway MSNs selectively express endocannabinoid-mediated long-term depression (eCB-LTD), which requires dopamine D2 receptor activation. In models of Parkinson's disease, indirect-pathway eCB-LTD is absent but is rescued by a D2 receptor agonist or inhibitors of endocannabinoid degradation. Administration of these drugs together in vivo reduces parkinsonian motor deficits, suggesting that endocannabinoid-mediated depression of indirect-pathway synapses has a critical role in the control of movement. These findings have implications for understanding the normal functions of the basal ganglia, and also suggest approaches for the development of therapeutic drugs for the treatment of striatal-based brain disorders.     

Positron emission tomography after MPTP: observations relating to the cause of Parkinson's disease

The dopa analogue 6-fluorodopa (6-FD) used with positron emission tomography (PET) allows in vivo visualization of dopamine and its metabolites in nigrostriatal nerve endings. We have now found abnormal 6-FD scans in four subjects exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). None had parkinsonism. The results suggest subclinical damage to the nigrostriatal pathway. This is the first direct evidence that dopaminergic impairment can exist without clinical deficits. Here we discuss this finding in the context of the hypothesis that Parkinson's disease may stem from clinically silent damage to the substantia nigra, followed by slow attrition of neurones in this region because of its particular vulnerability to cell loss as a normal consequence of ageing.     

Activation of NMDA receptors induces dephosphorylation of DARPP-32 in rat striatal slices

In the caudate-putamen the glutamatergic cortical input and the dopaminergic nigrostriatal input have opposite effects on the firing rate of striatal neurons. Although little is known of the biochemical mechanisms underlying this antagonism, one action of dopamine is to stimulate the cyclic AMP-dependent phosphorylation of DARPP-32 (dopamine and cAMP-regulated phospho-protein, of relative molecular mass 32,000 (32K]. This phosphorylation converts DARPP-32 from an inactive molecule into a potent inhibitor of protein phosphatase-1. Here we show that activation of the NMDA (N-methyl-D-aspartate) subclass of glutamate receptors reverses the cAMP-stimulated phosphorylation of DARPP-32 in striatal slices through NMDA-induced dephosphorylation of DARPP-32. Thus, the antagonistic effects of dopamine and glutamate on the excitability of striatal neurons are reflected in antagonistic effects of these neurotransmitters on the state of phosphorylation of DARPP-32. Our results indicate that stimulation of NMDA receptors leads to the activation of a neuronal protein phosphatase, presumably the calcium-dependent phosphatase calcineurin, and show, in an intact cell preparation, that signal transduction in the nervous system can be mediated by protein dephosphorylation.