Brain-Derived Neurotrophic Factor Protects Dopamine Neurons Against 6-Hydroxydopamine and N-Methyl-4-Phenylpyridinium Ion Toxicity: Involvement of the Glutathione System

Brain-derived neurotrophic factor (BDNF) has recently been shown to enhance the survival of dopamine neurons in cultures derived from the embryonic rat mesencephalon. We now extend this study by demonstrating that, in addition to the effect of sustaining survival of dopaminergic neurons, BDNF also confers protection against the neurotoxic effects of 6-hydroxydopamine (6-OHDA) and N-methyl-4-phenylpyridinium ion (MPP+). Exposure of mesencephalic cultures to either 6-OHDA or MPP+ resulted in a loss of 70-80% of dopaminergic neurons, as determined by tyrosine hydroxylase (TH) immunocytochemistry. In BDNF-treated cultures, loss of TH-positive cells after exposure to either toxin was reduced to only 30%. To facilitate biochemical measurements, we studied SH-SY5Y dopaminergic neuroblastoma cells. BDNF was found to protect these cells from the dopaminergic neurotoxins, 6-OHDA and MPP+. Indicative of oxidative stress, treatment of SH-SY5Y cells with 10 microM 6-OHDA for 24 h caused a fivefold increase in the levels of oxidized glutathione (GSSG). Pretreatment with BDNF for 24 h completely prevented the rise in GSSG. Further examination revealed that BDNF increased the activity of the protective enzyme, glutathione reductase, by 100%. In contrast, BDNF had no effect on the activity of catalase. These results add further impetus to exploring the therapeutic potential of BDNF in animal models of Parkinson's disease.     

Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.     

Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into gamma-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.     

Neurotrophic and neuroprotective effects of the neuregulin glial growth factor-2 on dopaminergic neurons in rat primary midbrain cultures

Glial growth factor-2 (GGF2) and other neuregulin (NRG) isoforms have been shown to play important roles in survival, migration, and differentiation of certain neural and non-neural cells. Because midbrain dopamine (DA) cells express the NRG receptor, ErbB4, the present study examined the potential neurotrophic and/or neuroprotective effects of GGF2 on cultured primary dopaminergic neurons. Embryonic day 14 rat mesencephalic cell cultures were maintained in serum-free medium and treated with GGF2 or vehicle. The number of tyrosine hydroxylase-positive (TH+) neurons and high-affinity [3H]DA uptake were assessed at day in vitro (DIV) 9. Separate midbrain cultures were treated with 100 ng/mL GGF2 on DIV 0 and exposed to the catecholamine-specific neurotoxin 6-hydroxydopamine (6-OHDA) on DIV 4. GGF2 treatment significantly increased DA uptake, the number of TH+ neurons, and neurite outgrowth when compared to the controls in both the serum-free and the 6-OHDA-challenged cultures. Furthermore, three NRG receptors were detected in the midbrain cultures by western blot analysis. Immunostaining for glial fibrillary acidic protein revealed that GGF2 also weakly promoted mesencephalic glial proliferation in the midbrain cultures. These results indicate that GGF2 is neurotrophic and neuroprotective for developing dopaminergic neurons and suggest a role for NRGs in repair of the damaged nigrostriatal system that occurs in Parkinson's disease.     

Activin A Protects Midbrain Neurons in the 6-Hydroxydopamine Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) and a subsequent loss of dopamine (DA) within the striatum. Despite advances in the development of pharmacological therapies that are effective at alleviating the symptoms of PD, the search for therapeutic treatments that halt or slow the underlying nigral degeneration remains a particular challenge. Activin A, a member of the transforming growth factor β superfamily, has been shown to play a role in the neuroprotection of midbrain neurons against 6-hydroxydopamine (6-OHDA) in vitro, suggesting that activin A may offer similar neuroprotective effects in in vivo models of PD. Using robust stereological methods, we found that intrastriatal injections of 6-OHDA results in a significant loss of both TH positive and NeuN positive populations in the SNpc at 1, 2, and 3 weeks post-lesioning in drug naïve mice. Exogenous application of activin A for 7 days, beginning the day prior to 6-OHDA administration, resulted in a significant survival of both dopaminergic and total neuron numbers in the SNpc against 6-OHDA-induced toxicity. However, we found no corresponding protection of striatal DA or dopamine transporter (DAT) expression levels in animals receiving activin A compared to vehicle controls. These results provide the first evidence that activin A exerts potent neuroprotection in a mouse model of PD, however this neuroprotection may be localized to the midbrain.     

Neuroprotective effect of BDNF in young and aged 6-OHDA treated rat model of Parkinson disease

Brain derived neurotrophic factor (BDNF) has been shown to exert trophic effects on dopaminergic neurons against 6-hydroxydopamine (6-OHDA) in young rat. Since the degeneration of substantia nigra dopaminergic neurons that occurs in Parkinson's disease is more often than not confined to elderly individuals, it is of interest to determine whether the effects of BDNF against 6 hydroxydopamine (6-OHDA) in young rats can be extended to aged animals. 6-hydroxydopamine was stereotaxically injected into the striatum of young (3-months) and aged (24-months) rats, which were treated two hours earlier with BDNF. 6-OHDA results in almost complete destruction of substantia nigra pars compacta dopaminergic neurons. BDNF injection significantly changed apomorphine induced rotations from 132 +/- 15 to 181 +/- 10, staircase test from 73 +/- 2% to 61 +/- 3%, initiation time from 7 +/- 2 to 12 +/- 1 sec, and disengage time from 80 +/- 7 to 90 +/- 5 sec in young and aged animals, respectively. It is concluded that BDNF causes the limited behavior recovery of striatal DA systems from 6-OHDA toxicity in aged animals.     

Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development

Recent studies of mouse mutant aphakia have implicated the homeobox gene Pitx3 in the survival of substantia nigra dopaminergic neurons, the degeneration of which causes Parkinson's disease. To directly investigate a role for Pitx3 in midbrain DA neuron development, we have analysed a line of Pitx3-null mice that also carry an eGFP reporter under the control of the endogenous Pitx3 promoter. We show that the lack of Pitx3 resulted in a loss of nascent substantia nigra dopaminergic neurons at the beginning of their final differentiation. Pitx3 deficiency also caused a loss of tyrosine hydroxylase (TH) expression specifically in the substantia nigra neurons. Therefore, our study provides the first direct evidence that the aphakia allele of Pitx3 is a hypomorph and that Pitx3 is required for the regulation of TH expression in midbrain dopaminergic neurons as well as the generation and/or maintenance of these cells. Furthermore, using the targeted GFP reporter as a midbrain dopaminergic lineage marker, we have identified previously unrecognised ontogenetically distinct subpopulations of dopaminergic cells within the ventral midbrain based on their temporal and topographical expression of Pitx3 and TH. Such an expression pattern may provide the molecular basis for the specific dependence of substantia nigra DA neurons on Pitx3.     

Transplantation of novel human GDF5-expressing CHO cells is neuroprotective in models of Parkinson's disease

Growth/differentiation factor 5 (GDF5) is a neurotrophic factor that promotes the survival of midbrain dopaminergic neurons in vitro and in vivo and as such is potentially useful in the treatment of Parkinson's disease (PD). This study shows that a continuous supply of GDF5, produced by transplanted GDF5-overexpressing CHO cells in vivo, has neuroprotective and neurorestorative effects on midbrain dopaminergic neurons following 6-hydroxydopamine (6-OHDA)-induced lesions of the adult rat nigrostriatal pathway. It also increases the survival and improves the function of transplanted embryonic dopaminergic neurons in the 6-OHDA-lesioned rat model of PD. This study provides the first proof-of-principle that sustained delivery of GDF5 in vivo may be useful in the treatment of PD.     

Evaluation of Estrogen Neuroprotective Effect on Nigrostriatal Dopaminergic Neurons Following 6-Hydroxydopamine Injection into the Substantia Nigra Pars Compacta or the Medial Forebrain Bundle

Studies involving estrogen treatment of ovariectomized rats or mice have attributed to this hormone a neuroprotective effect on the substantia nigra pars compacta (SNpc) neurons. We investigated the effect of estradiol replacement in ovariectomized rats on the survival of dopaminergic mesencephalic cell and the integrity of their projections to the striatum after microinjections of 1 microg of 6-hydroxydopamine (6-OHDA) into the right SNpc or medial forebrain bundle (MFB). Estradiol replacement did not prevent the reduction either in the striatal concentrations of DA and metabolites or in the number of nigrostriatal dopaminergic neurons following lesion with 1 microg of 6-OHDA into the SNpc. Nevertheless, estradiol treatment reduced the decrease in striatal DA following injection of 1 microg of 6-OHDA into the MFB. Results suggest therefore that estrogen protect nigrostriatal dopaminergic neurons against a 6-OHDA injury to the MFB but not the SNpc. This may be due to the distinct degree of lesions promoted in these different rat models of Parkinson's disease.     

Neuroprotective effects of Astragaloside IV in 6-hydroxydopamine-treated primary nigral cell culture

Parkinson's disease (PD) is caused by a progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress and neural degeneration are suggested to be involved in the pathogenesis of Parkinson's disease. In the present study, Astragaloside IV (AS-IV) extracted from the dried root of Astragalus membranaceus, a well-known Chinese medicine used for the treatment of neurodegenerative diseases, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. By examining the effect of AS-IV on 6-hydroxydopamine (6-OHDA)-induced loss of dopaminergic neurons in primary nigral culture, we found that AS-IV pretreatment significantly and dose-dependently attenuated 6-OHDA-induced loss of dopaminergic neurons. Neuronal fiber length studies showed that massive neuronal cell death with degenerated neurons was observed in those cultures incubated with 6-OHDA, whereas in AS-IV co-treatments most dopaminergic neurons were seen to be intact and sprouting. In flow cytometric analysis, AS-IV resulted in a marked and dose-dependent rescue in tyrosine hydrolase (TH)-immunopositive cells from 6-OHDA-induced degeneration of dopaminergic neurons. Double immunofluorescence revealed that AS-IV treatment alone at concentrations of 100 and 200 μM increased the level of TH and NOS (nitrite oxide synthase) immunoreactivities; however, the protective effect of AS-IV on TH and NOS immunopositive cells in 6-OHDA treated nigral cell cultures was only seen at a concentration of 100 μM. These findings show that AS-IV can protect dopaminergic neurons against 6-OHDA-induced degeneration. Besides the neuroprotective effect, AS-IV alone promoted neurite outgrowth and increased TH and NOS immunoreactive of dopaminergic neurons. The neuroprotective and neurosprouting effects of AS-IV are specific for dopaminergic neurons and it has therapeutic potential in the treatment of PD.     

Effect of Intranigral Injection of GDNF and EGF on the Survival and Possible Differentiation Fate of Progenitors and Immature Neurons in 6-OHDA-Lesioned Rats

We investigated the survival and the possible differentiation fate of the progenitors and immature neurons in the pars compacta of the substantia nigra (SNc) by intranigral injection of a glial cell line-derived neurotropic factor (GDNF) or glial cell line-derived neurotropic factor plus epidermal growth factor (EGF + GDNF) in 6-hydroxydopamine (6-OHDA)-lesioned rats. First, we performed behavioral tests by postural asymmetry and forelimb akinesia on the rats injected with 6-OHDA in striatum at day 7, and selected the qualified model according to the results. Then, intranigral GDNF or EGF + GDNF treatment was administered in the qualified PD model rats. On day 21, behavioral tests were performed with these rats; and then the rats were sacrificed for analyses of β-tubulin isotype-III (Tuj1), nestin, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) by immunohistochemistry and Western blotting. The results indicated that GDNF could promote the survival of the progenitor cells and immature neurons in rat SNc following 6-OHDA lesion. Moreover, EGF is capable of enhancing the survival effect of GDNF on the progenitor cells and immature neurons in SNc. On day 21, rapid functional recovery from the lesion-induced behavioral asymmetries was observed in the GDNF or EGF + GDNF-treated rats, and the numbers of TH-positive neurons increased in SNc, suggesting that the rats might generate new dopaminergic neurons. Thus, our study provides the new insight that the progenitors and immature neurons in SNc of 6-OHDA-lesioned rats might be able to differentiate toward the dopaminergic neurons fate subsequent to treatment with GDNF or EGF + GDNF.     

Melatonin Regulates the Viability and Differentiation of Rat Midbrain Neural Stem Cells

(1) Neurogenesis driven by neural stem cells (NSCs) is regulated by physiological and pathological factors. Melatonin (MT) has profound neurotrophic and neuroprotective effects. Hence, we studied the role of MT in regulating the viability and differentiation of NSCs derived from rat ventral midbrain. (2) NSCs were isolated from the rat ventral midbrain. The viability of NSCs was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium assay. The differentiation of NSCs was examined by analyzing the expression of the neural markers, MT receptors, brain derived neurotropic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) with semi-quantitative RT-PCR, immunofluorescence cytochemistry, and Western blot. (3) Our results showed that MT could promote the viability of NSCs. In addition, MT could significantly elevate the mRNA and protein levels of tyroxine hydroxylase (TH), a marker of dopaminergic neurons, and decrease the expression of the astrocytes maker glial fibrillary acidic protein (GFAP). MT also increased the production of BDNF and GDNF in the cultured NSCs. Meanwhile, we first found that two subtypes of MT receptors, MT1 and MT2, were expressed in the ventral midbrain NSCs. (4) These results demonstrated that MT could induce NSCs to differentiate into dopaminergic neurons and decrease astrocyte production. These findings also suggest that MT could offer a beneficial tool in guiding directional differentiation of NSCs.     

6-OHDA制备的帕金森病大鼠黑质神经元的超微结构改变

目的观察6-羟多巴胺(6-OHDA)单侧注射制备的帕金森病(PD)大鼠多巴胺(DA)能神经元的超微结构改变。方法单侧微量注射6-OHDA制备PD大鼠模型,用免疫荧光组织化学方法观察正常侧与6-OHDA注射侧黑质酪氨酸羟化酶(TH)阳性神经细胞及神经纤维的变化;并利用免疫电镜技术观察大鼠正常侧与注射侧黑质致密部DA能神经元的超微结构。结果免疫荧光法显示注射侧黑质致密部TH阳性细胞数和网状部TH阳性纤维面积与正常侧的百分比平均值分别为21.83%,23.19%。免疫电镜显示:TH免疫反应阳性产物表达于PD大鼠正常侧DA能神经元的高尔基复合体质膜面及胞质内,电子密度较高,注射侧很少见或几乎未见,且注射侧线粒体嵴有不同程度的溶解,呈空泡样变或髓样变,粗面内质网脱颗粒。结论6-OHDA可引起DA能神经元发生凋亡的超微结构改变。     

Changes in the Serum Urate Level Can Predict the Development of Parkinsonism in the 6-Hydroxydopamine Animal Model

Epidemiological studies indicate that a higher plasma level of uric acid (UA) associates with the reduced risk of Parkinson’s disease (PD). To confirm the role of UA as a biomarker for PD, we evaluated changes in the serum UA level in the 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonism in rat. For this purpose, 6-OHDA was administered in the medial forebrain bundle by stereotaxic surgery. According to the apomorphine-induced rotational test, the increased intensity of behavioral symptoms as a function of time was associated with the further reduction of UA level. On the other hand, the level of UA increased in the midbrain of the injured hemisphere. The level of reduction in the serum UA level of rats with severe and moderate symptoms was significantly higher than that of rats with mild symptoms. The immunohistofluorescence and biochemical analyses showed that the serum UA level was also correlated with the death of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNc), reduced level of striatal dopamine, and severity of oxidative stress in the midbrain. The rats with mild symptoms also showed a significant decrease in TH-positive neurons and striatal dopamine level. These findings suggest a positive correlation between the level of reduction in the serum urate level and severity of 6-OHDA-induced Parkinsonism. In addition, our findings indicated that UA had no marked neuroprotective effects, at least at concentrations obtained in this study. On the other hand, UA was introduced as a biomarker for PD, as a significant decline was observed in the serum UA level of rats with mild behavioral symptoms but with significant dopaminergic cell death in the SNc.     

Xenopus XsalF: anterior neuroectodermal specification by attenuating cellular responsiveness to Wnt signaling

Here we show that XsalF, a frog homolog of the Drosophila homeotic selector spalt, plays an essential role for the forebrain/midbrain determination in Xenopus. XsalF overexpression expands the domain of forebrain/midbrain genes and suppresses midbrain/hindbrain boundary (MHB) markers and anterior hindbrain genes. Loss-of-function studies show that XsalF is essential for the expression of the forebrain/midbrain genes and for the repression of the caudal genes. Interestingly, XsalF functions by antagonizing canonical Wnt signaling, which promotes caudalization of neural tissues. XsalF is required for anterior-specific expressions of GSK3beta and Tcf3, genes encoding antagonistic effectors of Wnt signaling. Loss-of-function phenotypes of GSK3beta and Tcf3 mimic those of XsalF while injections of GSK3beta and Tcf3 rescue loss-of-function phenotypes of XsalF. These findings suggest that the forebrain/midbrain-specific gene XsalF negatively controls cellular responsiveness to posteriorizing Wnt signals by regulating region-specific GSK3beta and Tcf3 expression.     

Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons

Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson's disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 μM) significantly attenuated 6-OHDA (50 μM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.     

6-羟基多巴胺损毁大鼠内侧前脑束早期黑质-纹状体系统的铁水平与多巴胺能神经元退变的关系

应用快速周期伏安法(fast cyclic vohammetry,FCV)、原子吸收分光光度法及免疫组织化学方法,观察了6-羟基多巴胺(6-hydroxydopamine,6-OHDA)单侧损毁大鼠内侧前脑束(medial forebrain bundle,MFB)早期黑质(sub-stantia nigra,sN)铁水平与多巴胺(dopamine,DA)神经元损伤的变化,以及纹状体(striatum,Str)的DA释放。结果如下：6-OHDA单侧损毁大鼠MFB1d和3d后,SN的酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞分别下降了45％和66％;与正常鼠和未损毁侧相比,损毁侧SN的铁染色增强,铁浓度增加,而Str的DA释放量不变;6-OHDA损毁后1d与3d组相比,损毁侧的铁染色、铁浓度及DA释放量差别无显著性。上述结果表明,6-OHDA单侧损毁大鼠MFB的早期阶段,SN的DA能神经元数目中等程度减少时,铁染色及铁浓度即有增加,由于DA能神经系统有强大的代偿功能,使得Str的DA释放量仍趋于正常。     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.     

Auricular Vagus Nerve Stimulation Exerts Antiinflammatory Effects and Immune Regulatory Function in a 6-OHDA Model of Parkinson’s Disease

According to epidemiologic studies, smoking appears to downregulate the prevalence of Parkinson’s disease (PD), possibly due to antiinflammatory mechanisms via activation of α7 nicotinic acetylcholine receptors (α7 nAChRs). This receptor also appears to play a role in T-cell differentiation. Recently, it has become apparent that the innate immune system participates in PD pathogenesis. The aim of this study was to evaluate the effects of auricular vagus nerve stimulation (aVNS) on substantia nigra (SN) dopaminergic neurodegeneration and the associated neuroinflammation and immune responses in a rat PD model. Adult male Wistar rats were unilaterally administered 6-hydroxydopamine (6-OHDA) to the medial forebrain bundle, followed by aVNS treatment after surgery. Following motor behavioral tests, the expression of tyrosine hydroxylase (TH) in the SN and the levels of inflammatory cytokines in the ventral midbrain were evaluated. In addition, changes in the trends of subsets of CD4+ T lymphocytes in the SN were measured by immunofluorescence staining. Western blotting was used to evaluate the α7 nAChR protein level. Compared with 6-OHDA treats rats, aVNS treatment significantly improved motor deficits, increased TH and α7 nAChR expression, and reduced the levels of inflammatory cytokines (tumor necrosis factor-a (TNF-α) and interleukin-1β (IL-1β)) (p?<?0.05). Additionally, aVNS increased the numbers of regulatory T (Treg) cells while decreasing T helper (Th)17 cells. aVNS exerted neuroprotective effects against dopaminergic damage, possibly by suppressing the evolution of inflammation and modulating innate immune responses. Thus, aVNS may be a potential promising therapy in the future.