大鼠中脑发育早期Nurr1时空模式及与TH表达的相关性

目的:观察孤儿核受体相关因子1(Nuclear receptor-related factor1,Nurr1)在大鼠胚胎中脑发育过程中的时空表达模式及与酪氨酸羟化酶(tyrosine hydroxylase,TH)表达的相关性。方法:制备大鼠胚胎E12.5、E13.5、E14.5d中脑脑片,行Nurr1/TH的免疫荧光双标记。在荧光显微镜下分别计数每个视野下TH、Nurr1单标神经元和Nurr1/TH双标神经元的数量,用图像处理系统和Stata7.0软件对所得数据进行统计学分析。结果:Nurr1/TH双标神经元在E12.5d时较少,随着发育时间的推移逐渐增加,至E14.5d时TH阳性细胞中几乎均有Nurr1的表达。结论:本研究结果进一步从形态学的角度提示转录因子Nurr1在胚胎发育过程中对中脑多巴胺(dopamine,DA)能神经元的发育起重要作用。     

P>核受体相关因子1基因过表达诱导神经干细胞分化为多巴胺能神经元/P>

目的 探讨核受体相关因子1（Nurr1）基因对大鼠中脑神经干细胞(NSCs)体外诱导分化为多巴胺能神经元的作用。方法 通过基因重组技术构建pEGFP-N1-Nurr1真核表达载体,用电穿孔法将重组质粒转染第3代NSCs,用荧光显微镜观察转染效率,免疫印迹法检测基因表达效果;并对转染后的NSCs进行体外分化培养3周,用免疫荧光双标技术检测酪氨酸羟化酶（TH）和微管相关蛋白-2（MAP-2）的表达。结果电穿孔法转染NSCs 48h后转染效率达35%,1周后免疫印迹法检测到Nurr1蛋白高表达,约为对照组的5倍;Nurr1基因转染的NSCs分化为TH阳性神经元的比率为15.45%,明显高于对照组,而MAP-2阳性神经元数量与对照组相似。结论Nurr1基因过表达可以诱导NSCs向TH阳性的多巴胺能神经元分化。     

胶质细胞源性神经营养因子促进中脑源神经干细胞增殖和向多巴胺能神经元分化

目的:探讨胶质细胞源性神经营养因子(GDNF)对中脑源神经干细胞(mNSCs)增殖、分化能力和表达垂体同源盒家族因子3(Pitx3)、孤儿核受体相关因子1(Nurr1)和酪氨酸羟化酶(TH)蛋白的影响。方法:取E14.5d大鼠中脑组织体外培养原代mNSCs,增殖培养传三代后进行分化实验;使用免疫荧光方法检测培养分化的mNSCs表达Pitx3、Nurr1和TH蛋白。结果:在GDNF作用下,体外培养的mNSCs克隆球直径增大,数量增多;分化的神经元中Pitx3、Nurr1和TH蛋白表达增加(P0.05)。结论:在体外培养条件下,GDNF有促进mNSCs增殖和向多巴胺能神经元分化的作用。     

pitx3和nurr1基因在不同年龄大鼠不同脑区的表达变化

目的:探讨正常不同年龄SD大鼠不同脑区pitx3、nurr1基闪的表达变化.方法:使用RT-PCR、免疫印迹法和免疫荧光方法检测pitx3、nurr1基因在正常不同年龄SD大鼠不同脑区转录与翻译水平的表达.结果:RT-PCR、免疫印迹法检测显示,pitx3、nurr1基因在各年龄组火鼠中脑、大脑皮质、海马、纹状体、嗅球和下丘脑均有表达,其中在海马和中脑表达较高;在各年龄组中新生组表达最高,并随着年龄的增长表达呈下凋趋势;免疫荧光检测E16.5 d组和新生组的中脑和海马区观察到有大量Pitx3或Nurr1单标细胞和Pitx3/TH或Nurrl/TH双标细胞.结论:pitx3、nurr1基凶的表达小仅与中脑多巴胺能神经元,而且可能与儿茶酚胺类神经元的发育和生存维持有关;老年期pitx3、nurr1基因表达下降可能与脑老化和神经退行性变疾病.如帕金森病和老年性痴呆等有关.     

Expression of the LRRK2 gene in the midbrain dopaminergic neurons of the substantia nigra

A hallmark of Parkinson's disease (PD) is the progressive loss of the A9 midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta. Recently, multiple causative mutations have been identified in the leucine-rich repeat kinase 2 (LRRK2) gene for both familial and sporadic PD cases. Therefore, to investigate functional roles of LRRK2 in normal and/or diseased brain, it is critical to define LRRK2 expression in mDA neurons. To address whether LRRK2 mRNA and protein are expressed in mDA neurons, we purified DA neurons from the tyrosine hydroxylase (TH)-GFP transgenic mouse using FACS-sorting and analyzed the expression of LRRK2 and other mDA markers. We observed that all mDA markers tested in this study (TH, Pitx3, DAT, Nurr1 and Lmx1a) are robustly expressed only in GFP(+) cells, but not in GFP(-) cells. Notably, LRRK2 was expressed in both GFP(+) and GFP(-) cells. Consistent with this, our immunohistochemical analyses showed that LRRK2 is expressed in TH-positive mDA neurons as well as in surrounding TH-negative cells in the rat brain. Importantly, in the midbrain region, LRRK2 protein was preferentially expressed in A9 DA neurons of the substantia nigra, compared to A10 DA neurons of the ventral tegmental area. However, LRRK2 was also highly expressed in the cortical and hippocampal regions. Taken together, our results suggest that LRRK2 may have direct functional role(s) in the neurophysiology of A9 DA neurons and that dysfunction of these neurons by mutant LRRK2 may directly cause their selective degeneration.     

外源性Nurr1基因过表达对SK-N-SH细胞分化作用的研究

为研究外源性Nurr1基因过表达对转染细胞的作用,探讨Nurr1基因调控多巴胺能神经元分化的机制。本研究应用:(1)pBK- RSV -Nurr1质粒经脂质体法转染SK- N- SH细胞,G418筛选;用RT PCR和免疫细胞化学方法检测基因转染细胞Nurr1mRNA及Nurr1蛋白的表达; (2)all- trans- RA、9- cis- RA、forskolin诱导基因转染细胞,RT PCR及免疫荧光细胞化学方法检测基因转染细胞MAP2 和TH的表达。结果显示: (1)经RT -PCR检测,基因转染细胞表达Nurr1mRNA;免疫细胞化学染色结果显示基因转染细胞表达Nurr1蛋白,说明外源性Nurr1基因在转染细胞内过表达;基因转染细胞形态与正常细胞相比没有明显变化,生长速度略有降低并表达成熟神经元的特异性标识物MAP2,但不表达多巴胺能神经元的特异性标识物TH。(2)尽管RA、forskolin诱导可促进Nurr1基因转染的SK- N- SH细胞进一步向成熟神经元分化,但不能诱导基因转染细胞表达TH。结论:单纯的Nurr1过表达可以促进SK- N- SH细胞向成熟神经元方向分化,但不足以激活TH基因转录。TH基因的转录激活还需要除Nurr1以外的其它细胞(或神经元)的环境或因子的共同作用。     

核受体相关因子1基因转染神经干细胞移植治疗帕金森病

目的 探讨转染核受体相关因子1(Nurr1)基因的神经干细胞(NSCs)移植至帕金森病(PD)大鼠纹状体后向多巴胺能神经元的分化及对PD大鼠行为的影响.方法 应用脑立体定位技术构建单侧PD大鼠模型.将PD大鼠随机分为3组,每组8只.假手术组注射生理盐水,NSCs组移植未转染的NSCs,Nurr1组先将重组质粒载体pEGFP-N1-Nurr1转染NSCs,用RT-PCR方法及免疫荧光染色检测Nurr1基因的表达效果,然后用转染Nurr1 基因的NSCs进行移植.细胞用DIL标记后移植至PD大鼠右侧纹状体中,术后2周起用阿朴吗啡诱发旋转试验观测移植后大鼠行为改善情况,12周后用免疫荧光技术检测移植细胞中酪氨酸羟化酶(TH)的表达.结果 重组质粒转染后Nurr1基因能在NSCs中过表达.移植后假手术组和NSCs组PD大鼠的旋转圈数均无下降趋势,两者差异无统计学意义(P＞0.05);NSCs组移植区可见DIL阳性细胞,但TH免疫阳性细胞较少,平均每视野为(3.21±0.40)个;Nurr1组移植后PD大鼠的旋转圈数从第6周开始下降,移植区DIL/TH双标神经元每视野达(9.28±1.09)个.结论 Nurr1基因过表达能诱导NSCs在PD大鼠纹状体内分化为TH阳性的多巴胺能神经元,并在一定程度上改善大鼠的行为功能.     

转录因子Pitx3和Nurr1在中脑多巴胺能神经元终末分化中的表达特点

目的:探讨转录因子垂体同源盒家族因子3(Pitx3)和孤儿核受体相关因子1(Nurr1)在多巴胺(DA)能神经元终末分化中的表达特点。方法:采用免疫荧光方法检测体外培养的中脑源性神经干细胞(mNSCs)诱导分化后和大鼠胚胎发育至出生腹侧中脑Pitx3、Nurr1和酪氨酸羟化酶(TH)的表达。结果:(1)体外培养的mNSCs诱导分化48 h的Map-2阳性细胞不表达Pitx3、Nurr1和TH;分化7 d的TH阳性细胞均表达转录因子Pitx3和Nurr1;(2)在大鼠腹侧中脑黑质(SN)、腹侧被盖区(VTA)和中缝背核(DRN)可见大量TH与Pitx3(或Nurr1)共表达的神经元;(3)Pitx3和Nurr1阳性细胞主要分布于E16.5、P0大鼠SN、VTA和DRN区,其中Pitx3阳性细胞还少量分布于腹侧中脑非DA能神经元区域,Nurr1阳性细胞在腹侧中脑分布范围较Pitx3更广泛。结论:转录因子Pitx3、Nurr1在中脑DA能神经元的终末分化和生存维持中起重要作用。     

GDNF acutely modulates excitability and A-type K(+) channels in midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) prevents lesion-induced death of midbrain dopaminergic neurons, but its function in normal brain remains uncertain. Here we show that GDNF acutely and reversibly potentiated the excitability of cultured midbrain neurons by inhibiting transient A-type K(+) channels. The effects of GDNF were limited to large, tyrosine hydroxylase (TH)-positive dopaminergic neurons, and were mediated by mitogen associated protein (MAP) kinase. Application of GDNF also elicited a MAP kinase-dependent enhancement of the excitability in dopaminergic neurons in midbrain slice. These results demonstrate an acute regulation of GDNF on ion channels and its underlying signaling mechanism, and reveal an unexpected role of GDNF in normal midbrain dopaminergic neurons.     

大鼠生后视网膜发育过程中Nurr1与TH的相关关系研究

目的:观察核受体相关因子1(nuclear receptor-related factor 1,Nurr1)与多巴胺能神经细胞特异性标记物酪氨酸羟化酶(TH)在生后大鼠视网膜发育过程中的表达变化,探明Nurr1与视网膜多巴胺能神经元的相关关系。方法:石蜡包埋组织切片,免疫组织化学双重标记。结果:Nurr1在视网膜发育过程中的表达出现了显著的动态变化,Nurr1阳性产物主要表达在内核层细胞,生后3～7 d达到高峰,之后随着细胞的成熟阳性表达又逐渐减少,成熟的视网膜组织内仅见少量Nurr1阳性细胞,在视网膜神经细胞从幼稚到成熟的分化过程中仅见个别TH阳性细胞。Nurr1与TH的免疫组化双标结果显示两蛋白可以共表达在同一细胞中,但众多的Nurr1阳性细胞不表达TH。结论:Nurr1在大鼠视网膜多巴胺能神经细胞及非多巴胺能神经细胞从幼稚到成熟的分化过程中可能具有重要的调控作用。     

Enriched NCAM-positive cells form functional dopaminergic neurons in the rat model of Parkinson's disease

We describe a method of generating an enriched population of NCAM-positive cells from a human teratocarcinoma cell line (NTera2/D1) and their differentiation into midbrain dopaminergic neurons in the absence of the caudalizing factor retinoic acid (RA). NTera2 cells were induced to form embryoid bodies and then to generate nestin-positive cells on treatment with serum-free defined medium supplemented with neurotrophic factors. We enriched the neuroprogenitor population by magnetic sorting of the nestin-positive cells using the antibody to neural cell adhesion molecule (NCAM). These cells were expanded by exposing them to the signaling molecule sonic hedgehog (SHH) in conjunction with fibroblast growth factor-8 (FGF-8). The predifferentiated cells when analyzed by RT-PCR showed expression of dopaminergic markers such as Nurr1, Engrailed-1, aromatic amino decarboxylase (AADC), VMAT2, tyrosine hydroxylase (TH), and dopamine transporter (DAT). These cells also stained positively for protein markers such as nestin, NCAM, MAP-2, and TH. We further demonstrated that when transplanted into the brain of Parkinsonian rats, these neuroprogenitor cells did not form tumors but differentiated into dopaminergic neurons, as revealed by TH immunolabeling. The origin of transplanted cells were further confirmed by positive immunolabeling with anti-human nuclei. Our results suggest that enriching the neuroprogenitor population by magnetic sorting prevents tumor formation and is a prerequisite before cell replacement therapy for Parkinson's disease.     

Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells

During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the 'right' therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and beta-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT-PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K(+). Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH(+) cells which may be used in cell therapy for PD in future.     

Transforming growth factor beta is required for differentiation of mouse mesencephalic progenitors into dopaminergic neurons in vitro and in vivo: ectopic induction in dorsal mesencephalon

Tissue engineering is a prerequisite for cell replacement as therapeutic strategy for degenerative diseases, such as Parkinson's disease. In the present study, we investigated regional identity of mesencephalic neural progenitors and characterized their development toward ventral mesencephalic dopaminergic neurons. We show that neural progenitors from ventral and dorsal mouse embryonic day 12 mesencephalon exhibit regional identity in vitro. Treatment of ventral midbrain dissociated neurospheres with transforming growth factor beta (TGF-beta) increased the number of Nurr1- and tyrosine hydroxylase (TH)-immunoreactive cells, which can be further increased when the spheres are treated with TGF-beta in combination with sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). TGF-beta differentiation signaling is TGF-beta receptor-mediated, involving the Smad pathway, as well as the p38 mitogen-activated protein kinase pathway. In vivo, TGF-beta2/TGF-beta3 double-knockout mouse embryos revealed significantly reduced numbers of TH labeled cells in ventral mesencephalon but not in locus coeruleus. TH reduction in Tgfbeta2(-/-)/Tgfbeta3(+/-) was higher than in Tgf-beta2(+/-)/Tgf-beta3(-/-). Most importantly, TGF-beta may ectopically induce TH-immunopositive cells in dorsal mesencephalon in vitro, in a Shh- and FGF8-independent manner. Together, the results clearly demonstrate that TGF-beta2 and TGF-beta3 are essential signals for differentiation of midbrain progenitors toward neuronal fate and dopaminergic phenotype.     

Alpha-chemokines regulate proliferation, neurogenesis, and dopaminergic differentiation of ventral midbrain precursors and neurospheres

Increasing evidence suggests that alpha-chemokines serve several important functions in the nervous system, including regulation of neuroimmune responses, neurotransmission, neuronal survival, and central nervous system development. In this study, we first examined the function of two alpha-chemokines, chemokine ligand (CXCL) 6 and CXCL8, and their receptors, CXCR1 and CXCR2, in the developing rat ventral midbrain (VM). We found that CXCR2 and CXCL6 are regulated during VM development and that CXCL6 promotes the differentiation of nurr77-related receptor (Nurr1)+ precursors into dopaminergic (DA) neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in Homo sapiens, enhanced progenitor cell division, neurogenesis, and tyrosine hydroxylase-positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited activities similar but not identical to those of CXCL8. TH+ cells derived from chemokine-treated VM neurospheres coexpressed Nurr1 and VMAT and were functionally active, as shown by calcium (Ca(2+)) fluxes in response to AMPA. In conclusion, our data demonstrate that CXCL1, CXCL6, and CXCL8 increase the number of DA neurons in VM precursor and neurosphere cultures by diverse mechanisms. Thus, alpha-chemokines may find an application in the preparation of cells for drug development or Parkinson's disease cell replacement therapy.     

Specification of midbrain dopamine neurons from primate pluripotent stem cells

By sequentially applying sonic hedgehog (C25II) and CHIR99021 (GSK3β inhibitor) to induce the midbrain floor plate (FP) progenitors and fibroblast growth factor 8 (FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for the generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells and induced pluripotent stem cells (PSCs). We found that CHIR99021 specifies diencephalon to hind brain fates in a concentration-dependent manner and only a narrow concentration range of CHIR99021 at a particular window is necessary to induce the midbrain FP progenitors, expressing Corin, En1, FoxA2, and Lmx1a. FGF8 enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of tyrosine hydroxylase, Lmx1a/b, FoxA2, FoxP1, Nurr1, and En1 as well as typical electrophysiological properties. More than half of these DA neurons expressed A9 DA neuron markers Girk2 and ALDH1a1. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy.