褪黑素和6-羟褪黑素保护神经细胞抗缺血再灌注损伤比较研究

目的研究褪黑素(Mel)和6-羟褪黑素(6-OHMel)神经保护作用及作用机理。方法体外培养N2a细胞,模拟缺血再灌注(OGSD),加入Mel和6-OHMel,检测以下指标:①细胞生存能力:MTT法、乳酸脱氢酶释放;②细胞凋亡分析:DNA片断化,细胞色素C,Caspase3活性;③活性氧(ROS)和线粒体跨膜电位。结果①Mel和6-OHMel都能减轻OGSD诱导的N2a细胞损伤,Mel的作用强于6-OHMel。②Mel和6-OHMel均能抑制细胞色素C释放,但6-OHMel强于Mel。③Mel和6-OHMel都能稳定线粒体跨膜电位,但Mel作用时间比6-OHMel长。④Mel和6-OHMel能清除ROS,6-OHMel表现为直接作用,Mel表现为间接作用。⑤Mel和6-OHMel均能抑制caspase3的活性,但是作用时间不同。6-OHMel表现在OGSD后12h,Mel在OGSD后24h。结论Mel和6-OHMel的神经保护作用与其抗氧化、稳定线粒体功能相关,Mel的作用机制更复杂。     

抑制beclin-1信号通路对缺血再灌注后神经母细胞瘤N2a细胞自噬的影响

目的观察抑制beclin-1信号通路对缺血再灌注后神经母细胞瘤N2a细胞自噬的影响。方法体外培养神经母细胞瘤系N2a细胞,脂质体转染beclin-1的miRNA干扰质粒,以缺氧、缺营养方式模拟缺血再灌注,甲基噻唑基四唑法检测各组N2a细胞活性,免疫印迹法测定各组N2a细胞beclin-1、微管相关蛋白1轻链3(LC3)、天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)的表达,电镜观察该细胞自噬活性。结果在缺血90min、再灌注24h干扰组较相应假干扰组细胞活性明显升高(P<0.05);干扰组beclin-1和Caspase3表达水平明显低于相应的假干扰组(P<0.05),但两组之间LC3表达无明显差异(P>0.05);而在缺血30min再灌注24h细胞活性,beclin-1、Caspase3和LC3的表达真假干扰组间无明显差异(P>0.05)。电镜下在正常组没有观察到细胞自噬现象;在缺血30min和90min再灌注24h,真假干扰组均观察到明显的细胞自噬现象,且两组间无明显差异。结论缺血90min,再灌注24h时,干扰beclin-1的表达虽不能明显改变N2a细胞自噬水平,却减少了凋亡,有利细胞存活。     

体外模拟缺血再灌注诱导神经细胞线粒体功能改变的研究

目的观察线粒体功能失调在缺血再灌注后神经细胞凋亡中的作用.方法(1)采用体外培养神经母细胞瘤细胞株N2a,模拟缺血再灌注(先缺氧缺营养90 min,然后正常培养不同时间);(2)采用琼脂糖凝胶电泳检测细胞凋亡情况;(3)通过MTT法、细胞色素C释放和跨膜压的改变判断线粒体的功能;(4)Cas-pase 3活性测定采用水解其可见光底物.结果(1)N2a细胞缺血再灌注12 h即出现明显DNA片段化,24h更明显;(2)线粒体琥珀酸脱氢酶活性缺血再灌注24 h明显降低;跨膜电位在缺血再灌注1 h先短暂下降,3 h明显升高,6 h后降低,以后再没回升.缺血再灌注3 h细胞色素C开始释放,6 h达到高峰,并持续到24 h;(3)Caspase 3活性在缺血再灌注10 h升高,24 h达到高峰.线粒体通透性转换孔抑制剂cyclosporine A只能抑制部分caspase 3的活性和DNA片段化改变,而caspase 8抑制剂虽不能完全抑制caspase 3的活性,但能完全抑制DNA的片段化.结论缺血再灌注诱导神经细胞凋亡存在caspase 3依赖性和非依赖性两条途径,线粒体功能失调在缺血再灌注引起的N2a细胞凋亡过程中可能起到凋亡早期的启动和随后的信号放大作用.     

褪黑素在缺血再灌注引起神经元凋亡的保护作用

目的探讨模拟缺血再灌注引起神经元凋亡的途径和褪黑素(m elaton in,MT)抗凋亡的作用机理。方法建立原代培养大鼠小脑颗粒细胞的体外模拟缺血(Oxygen G lucose Deprivation,OGD)再灌注模型,测定培养液LDH活性和细胞DNA琼脂糖凝胶电泳;利用Rhodam ine123和激光共聚焦显微镜观察线粒体膜电位的变化;ELISA检测细胞浆中细胞色素C水平;用同样指标观察MT对其损伤的保护作用。结果在体外模拟缺血再灌注模型中培养液LDH活性增加(P<0.05),琼脂糖凝胶电泳DNA出现梯状条带,线粒体膜电位明显降低,细胞浆中细胞色素C含量增加(P<0.05),MT对上述现象有明显的抑制作用。结论(1)缺血再灌注引起的神经元凋亡机理之一是通过线粒体凋亡途径;(2)MT可通过阻止线粒体凋亡途径而保护模拟缺血再灌注诱导小脑颗粒细胞的凋亡。     

线粒体在缺血再灌注继发的神经元细胞凋亡中的作用

目的 观察线粒体功能失调在缺血再灌后神经元凋亡中的作用。方法(1)采用体外培养神经母细胞瘤细胞株N2a细胞，体外模拟缺血再灌注(缺氧90min，然后再灌不同时间)；(2)采用琼脂糖凝胶电泳检测细胞凋亡情况；(3)MTT法、细胞色素c释放和跨膜压的改变判断线粒体的功能；(4)caspase-3活性测定采用水解其可见光底物。结果(1)N2a细胞缺血再灌12h即出现明显DNA片段化，24h更明显；(2)线粒体酶活性降低．跨膜电位在缺氧再灌1h先短暂下降再升高然后又明显降低。细胞色素c缺氧再灌3h开始下降，6h达到高峰．持续到24h；(3)caspase-3活性在缺氧再灌10h升高，24h达到高峰，线粒体通透性转换孔形成抑制剂CsA只能抑制部分caspasc-3的活性和DNA片段化改变；而caspase-8抑制剂虽不能完全抑制caspase-3的活性但能完全抑制DNA的片段化。结论N2a凋亡尚存在有caspase-3依赖性和非依赖性两条途径线粒体功能失调在缺氧再灌引起的N2a细胞凋亡过程中可能起到凋亡早期的启动和随后的信号放大作用。     

Beclin-1、LC3-Ⅱ在缺血后处理大鼠再灌注中的表达及意义

目的研究自噬相关蛋白Beclin-1、LC3-Ⅱ在缺血后处理大鼠脑缺血再灌注海马中的表达及意义。方法根据Zea-Longa线栓法制作大脑中动脉缺血再灌注损伤模型,将实验动物随机分为3组:假手术组、缺血再灌注组、缺血后处理组,缺血后处理组再按不同缺血后处理时间分为15 s、30 s、1 min 3个亚组。缺血2 h再灌注24h后用免疫组织化学法检测自噬相关蛋白Beclin-1、LC3-Ⅱ的表达情况,透射电镜观察神经细胞中自噬和溶酶体的激活以及细胞超微结构的变化。结果与缺血再灌注组相比,缺血后处理组神经行为学评分明显降低(P<0.05),脑梗死体积明显减小(P<0.01),Beclin-1、LC3-Ⅱ蛋白表达细胞数明显增加(P<0.01),自噬体形成和溶酶体激活量增加;缺血30 s亚组相比较15 s和1 min亚组上述指标(神经行为学评分除外)(P>0.05),差异亦有显著性(P<0.01)。结论脑缺血后处理的抗缺血再灌注损伤作用可能与上调自噬相关蛋白Beclin-1、LC3-Ⅱ的表达有关。     

6-羟基褪黑素保护神经细胞抗缺血再灌注损伤的作用机制

目的探讨6-羟基褪黑素的神经保护作用及作用机理。方法体外培养神经母细胞瘤N2a细胞，缺糖、缺血清和缺氧（OGSD）90min，再正常培养不同时间，同时加入6-羟基褪黑素，观察细胞生存能力（MTT法）、线粒体跨膜电位变化（荧光标记）、线粒体细胞色素C释放（western blot）、caspase3活性（荧光底物）以及细胞内活性氧（荧光标记）的产生。结果6-羟基褪黑素抑制细胞色素C释放，抑制caspase3激活，清除细胞内活性氧，稳定线粒体跨膜电位。结论6-羟基褪黑素通过直接抗氧化作用和抑制线粒体凋亡途径促进神经细胞生存。     

线粒体分裂抑制剂通过抑制自噬加重OGD/R损伤

目的探讨线粒体分裂抑制剂(Mdivi-1)处理对原代培养的皮层神经元缺血再灌注损伤的作用及其机制研究。方法在原代培养的小鼠皮层神经元上建立氧糖剥夺/复氧复糖(OGD/R)模型模拟体内缺血再灌注损伤,根据再灌注时间长短检测自噬的发生情况,并进一步选取自噬发生最明显的时间点。然后分别采用自噬抑制剂-3-MA、自噬激动剂-雷帕霉素(rapamycin)、Mdivi-1以及联合rapamycin和Mdivi-1进行干预,分别从mRNA以及蛋白水平检测自噬相关分子的改变,并同时检测神经元损伤情况。结果 OGD/R处理后,神经元自噬随再灌注时间增长而上调,在OGD 2 h/R 24 h时,Beclin 1以及微管相关轻链蛋白3 II/I(LC3 II/I)比值均达峰值(P0.001,P0.01)。细胞活力结果显示,rapamycin可减轻OGD/R损伤(P0.01),Mdivi-1则会加重OGD/R损伤(P0.05),但rapamycin可通过增加自噬来减轻Mdivi-1造成的损伤(P0.05)。蛋白检测结果显示,OGD/R处理后,Mdivi-1可明显降低神经元细胞内Beclin 1和LC3 II的表达(P0.05)。结论 Mdivi-1可能通过抑制自噬的发生,从而加重神经元OGD/R损伤。     

肢体缺血预处理对脑缺血再灌注损伤大鼠自噬的影响

目的探讨肢体缺血预处理对脑缺血再灌注损伤大鼠自噬的影响。方法将60只Wistar大鼠随机分为假手术组(Sham组)、缺血再灌注组(I/R组)、肢体缺血预处理组(LIPC组)、3-甲基嘌呤组(3-MA组),每组15只。制作脑缺血再灌注、肢体缺血预处理及3-MA干预大鼠模型,在脑缺血2 h再灌注24 h后进行神经功能缺陷评分和脑梗死体积测定,HE染色观察细胞形态学改变,Western Bloting法检测自噬相关蛋白Beclin-1、Cathepsin B的表达。结果与I/R组比较,LIPC组神经功能缺陷评分降低(P<0.05),脑梗体积明显减小(P<0.05),细胞损伤、坏死减轻(P<0.05),Beclin-1、Cathepsin B的蛋白表达明显减弱(P<0.05)。结论 LIPC对缺血再灌注损伤大脑具有保护作用,其机制可能与减弱自噬水平有关。     

褪黑素在培养的小脑颗粒细胞模拟缺血再灌注中的抗氧化作用研究

目的探讨褪黑素(Melatonm,MT)在模拟缺血再灌注中对神经元的保护机制,从而为褪黑素的临床应用提供可靠的理论依据.方法分离SD乳鼠的小脑颗粒细胞进行原代培养,待细胞发育成熟后建立体外模拟缺血再灌注模型.采用相差显微镜进行细胞的形态学观察;通过TBA荧光法和联苯三酚自氧化法来检测模拟缺血再灌注过程中细胞内丙二醛(MDA)生成量、超氧化物歧化酶(SOD)的活性变化以及褪黑素在模拟缺血再灌注过程中对二者的影响.结果模拟缺血(OGD)后再灌注12 h的细胞,胞内MDA的生成比在OGD后再灌注6 h明显增多(P＜0.05),OGD后再灌注12 h MDA生成最多随后降低;经OGD处理后细胞内SOD的活性较正常组明显降低(P＜0.05),OGD后再灌注24 h降到最低随后升高.MT能显著降低细胞内MDA的生成(P＜0.05),并存在着一定的剂量依赖关系.SOD的活性在加入MT后也明显提高(P＜0 05).结论MT可能通过其抗氧化作用在模拟缺血(OGD)再灌注中起到神经保护作用.     

Melatonin receptors are anatomically organized to modulate transmission specifically to cone pathways in the retina of Xenopus laevis

Melatonin receptors have been identified in several retinal cell types, including photoreceptors, horizontal cells, amacrine cells, and ganglion cells. Recent reports suggest that melatonin potentiates signaling from rods to inner retinal neurons. However, the organization of the melatonin receptors mediating this action in the outer plexiform layer (OPL) is not clear. To assess melatonin receptor localization in the OPL, double-label confocal immunohistochemistry for Mel1a or Mel1b melatonin receptors was performed in combination with markers for cone photoreceptors (calbindin, XAP-1) and ON bipolar cells (guanine nucleotide binding protein alpha, Goα) on the retina of Xenopus laevis. Both Mel1a and Mel1b receptors were specifically associated with processes contacting the pedicles of cones, but localized to processes from different sets of second-order neurons. Mel1a receptors localized to the large axonal processes of horizontal cells, while Mel1b receptors localized to the dendrites of OFF bipolar cells. Both receptors also localized to third-order amacrine and ganglion cells and their processes in the inner plexiform layer. This study indicates that Mel1a and Mel1b melatonin receptors are expressed specifically in the Xenopus OPL to modulate transmission from cones to horizontal cells and OFF bipolar cells, respectively; they are second-order neurons that predominantly contact ribbon synapses and display OFF responses to light. When combined with results from recent physiological studies, the current results suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons across species, although the precise mechanisms by which melatonin enhances this transmission are likely to vary in a species-dependent manner.     

Metabolism and toxicity of methyl iodide in primary dissociated neural cell cultures

The metabolism and the toxicity of methyl iodide (Mel) has been studied in primary dissociated neuronal and glial murine cell cultures to further characterize the mechanisms of monohalomethane neurotoxicity. Measurement of intracellular glutathione (GSH) concentrations in cerebellar and cerebral cultures revealed GSH levels (21.6 +/- 1.9 and 29.1 +/- 1.9 nmol/mg protein, respectively) close to brain GSH levels measured in vivo. A GSH-depleting effect of Mel was demonstrated, with an ED50 for a 5 min exposure of 0.2 and 0.5 mM for glial and mixed (neurons + glia) cultures, respectively. Mel-induced GSH depletion was correlated with its neurotoxicity as the two powerful protective agents of monohalomethane toxicity, 3-amino-1-[m-(trifluoromethyl) phenyl]-2-pyrazoline (BW 755C, 1 mM) and nordihydroguaiaretic acid (NDGA, 10 microM) provided a 20-fold protection against depletion of GSH levels following Mel exposure. When glia and neurons from cerebral cultures were exposed in suspension to increasing concentrations of Mel for 30 min at 37 degrees C, a concentration-dependent increase in the production of formaldehyde resulted. Formaldehyde appeared to be an indicator of Mel metabolism as its production was decreased by sulfasalazine, a compound which was shown to be an inhibitor of the glutathione-S-transferases in this culture system. Since BW 755C and NDGA had no effect on formaldehyde production, while sulfasalazine as well as semicarbazide, a protective agent against formaldehyde-producing toxicants, failed to protect the cells against Mel toxicity, mechanism(s) of Mel neurotoxicity appeared independent of the GSH-mediated metabolism of this compound. It is concluded that GSH-mediated metabolic biotransformation is not necessary for the neurotoxicity of the monohalomethanes, that GSH depletion may act as a starting point in the chain of events leading to neural cell death, and that glia may be more sensitive than neurons to this primary effect. Moreover, these results demonstrate the value of primary dissociated neuronal cell cultures for studies of biochemical mechanisms of neurotoxicity.     

NFAT5 protects astrocytes against oxygen–glucose–serum deprivation/restoration damage via the SIRT1/Nrf2 pathway

Nuclear factor of activated T cells (NFAT) is a multifunctional cytokine family. NFAT5 was recently reported to be involved in many neuronal functions, but its specific function remains unclear. In this study, our aim is to investigate whether NFAT5 overexpression can protect astrocytes against oxygen–glucose–serum deprivation/restoration (OGSD/R) damage. In vivo, rats were subjected to ischemia–reperfusion injury, resulting in increased water content, infarct volume, and expression of NFAT5 protein in rat spinal cord. After primary culture for spinal cord astrocytes, the in vitro OGSD/R model was established. The results of the CCK8 assay and flow cytometry showed that, in the OGSD/R group, astrocyte cell viability was downregulated, but astrocyte apoptosis increased. Caspase 3 activity increased as well. Levels of NFAT5, as detected by real-time quantitative PCR and western blot, decreased under OGSD/R, as did SIRT1. Commercial kits for activity assays were used to show that OGSD/R inhibited SIRT1 activation but accelerated SOD activation after OGSD/R. Next, pcDNA-NFAT5 or NFAT5 siRNA was transfected into astrocytes. Overexpression of NFAT5 not only promoted the survival of the astrocytes and SIRT1 activation under OGSD/R but also inhibited cell apoptosis and SOD activation. Moreover, overexpression of NFAT5 apparently diminished histone acetylation and promoted the nuclear transport of Nrf2. Our results show that NFAT5 protects spinal astrocytes in a manner that depends on activation of the SIRT1/Nrf2 pathway. These findings present a novel potential molecular mechanism for NFAT5 therapy in the context of spinal cord injury.     

Cytosolic labile zinc: a marker for apoptosis in the developing rat brain

Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.     

Neuroprotective effect of melatonin against ischemia/reperfusion-induced neuronal apoptosis in mouse cerebellum

BACKGROUND: Some experiments have demonstrated that melatonin (N-aceyl-5-methoxytryptamine, Mel) has antioxidation. However, whether it has neuroprotective effect in the ischemia/reperfusion injury of central nervous system is unclear. OBJECTIVE: To observe the protective effect of Mel on ischemia/reperfusion-induced cerebellar neuronal apoptosis of rats, and the action mechanism. DESIGN: Controlled observation experiment. SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Eight Sprague-Dawley rats aged 7–8 days and weighing 10–12 g were provided by Medical Experimental Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Anti-cytochrome C monoclonal antibody was purchased from R & D Company; 7-dichlorodihydrofluorescein diacetate(DCFH-DA), rhodamine 123 and Mel were purchased from Sigma Company (USA). Lactate dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng Bioengineering Institute. METHODS: This experiment was carried out in the laboratory for Department of Biochemistry and Molecule Biology, Tongji Medical College between October 2002 and March 2004. Cerebellar neurons of rats were cultured in vitro. After oxygen-glucose deprivation (OGD) for 90 minutes, 1×10–4,1×10–6, 1×10–9 mol/L Mel was added, respectively, namely high-, middle-, and low-concentration Mel groups. Cells, which were cultured by OGD, served as model group, and control group, in which OGD intervention was omitted, was set. ①Cytochrome C level of mitochondrial cells in each group was detected by ELISA method. ②LDH activity in the cell culture fluid was measured, and cell membrane permeability change was analyzed. The cells in the Mel group with the lowest LDH activity served as Mel treatment group, i.e. cells were cultured with OGD, and then Mel was added; Meanwhile, Mel prevention group was set, i.e. Mel was added before OGD. Intervention was not changed in the model group and control group. ③ DNA level was analyzed and cell apoptosis was observed by agarose gel electrophoresis(AGE). ④Mitochondrial transmembrane potential of cells, and apoptotic way in each group were analyzed by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: ①Mitochondrial cytochrome C level of cerebellar nerve cells. ②LDH activity of cerebellar nerve cells. ③ DNA AGE results. ④Mitochondrial transmembrane potential change. RESULTS: ①Mitochondrial cytochrome C level of cerebellar nerve cells: cytochrome C was obviously released at 6 hours of OGD-reperfusion. Mel inhibited the release of cytochrome C in dose-dependent manner. ②LDH activity of cerebellar nerve cells: LDH activity (A value) was significantly lower in the high- and middle-concentration Mel groups than in the model group (P < 0.05). LDH activity (A value) in the low-concentration Mel group was 0.415 0±0.012 9, indicating that Mel could decrease LDH activity of OGD-treated cell supernatant and promote membrane stablization in dose-dependent manner. ③AGE results of DNA: 1×10–9 mol/L was considered as the best concentration of melatonin. Cell DNA was extracted for AGE. Results presented typical ladder shape, indicating apoptosis appeared, while apoptosis was lessened in the Mel treatment group and Mel prevention group.④Mitochondrial transmembrane potential change: Experimental results showed that green fluorescein was evenly distributed in cerebellar granule cells cultured normally, and the axons of neurons were very clear. The body of neurons was condensed and the axons disappeared after cerebellar granule cells undergoing OGD injury. Mel could completely reverse the effect of OGD. CONCLUSION: Mel can enhance cerebellar neuronal membrane stabilization of rats in dose-dependent manner, and suppress OGD-induced apoptosis of cerebellar granule cells by preventing against mitochondrial apoptosis.     

Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor

Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.