Huanying Zhao

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

Abstractα-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.

Silencing of PINK1 induces mitophagy via mitochondrial permeability transition in dopaminergic MN9D cells.

Accumulation of dysfunctional Mitochondria has been implicated in the pathogenesis of Parkinsons disease (PD). Mutations in PTEN-induced kinase 1 (PINK1), which encodes a putative mitochondrial serine/threonine kinase, have been identified in early-onset forms of PD. Recent data showed that the loss of PINK1 function led to oxidative stress, mitochondrial damage and autophagic elimination of damaged mitochondria. But the precise mechanism of autophagy induced by loss of PINK1 is unclear. In this study, we found that in mouse dopaminergic MN9D cells, down-regulation of PINK1 by RNA interference resulted in induction of mitochondrial autophagy (mitophagy), abnormal mitochondrial morphology, partial loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Mitophagy in these cells was associated with the up-regulation of autophagy activator Beclin 1 and opening of mitochondrial permeability transition (MPT) pore. These findings suggest that PINK1 may regulate mitophagy through controlling MPT pore opening and general autophagy regulators.

Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model.

Parkinsons disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.

THE ROLE OF Ret RECEPTOR TYROSINE KINASE IN DOPAMINERGIC NEURON DEVELOPMENT

Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent trophic factors identified for promoting survival and function of dopaminergic (DA) neurons in the midbrain. Ret, a member of the receptor tyrosine kinase (RTK) superfamily transduces GDNF signaling. The role of Ret in the development of DA neurons is not clear however. Here we demonstrate the involvement of Ret in the DA neuron development both in vitro and in vivo. The dopamine transporter (DAT) gene was clearly induced in rat embryonic neural precursors that had been transfected with Ret. Temporary blockade of Ret expression in embryos using Ret antisense oligonucleotides (Ret-AS-ODN) in vivo led to reduced striatal DA content and a decrease of tyrosine hydroxylase (TH) positive fibers in the striatum. Additionally, some DA neurons in the substantia nigra (SN) underwent apoptotic cell death following the Ret-AS-ODN treatment. Taken together, the data suggest that normal function of Ret is required in vivo for the maturation of DA neurons, in particular for cell survival and fiber innervation. We further demonstrated Ret-induced expression of DAT in vitro.

DJ-1 upregulates tyrosine hydroxylase gene expression by activating its transcriptional factor Nurr1 via the ERK1/2 pathway

Loss-of-function DJ-1 mutations have been linked to autosomal recessive early-onset Parikinsonism. However, the putative function of DJ-1 is not completely understood. Previous studies indicate that DJ-1 overexpression results in upregulation of the tyrosine hydroxylase gene. The mechanism by which DJ-1 affects tyrosine hydroxylase expression remains elusive. In the present study, we show that DJ-1 overexpression induces ERK1/2 activation, along with increased tyrosine hydroxylase expression. The L166P DJ-1 mutant, which has been identified as being responsible for familial Parkinsonism, did not have this effect. Moreover, suppression of ERK1/2 phosphorylation by the pharmacological inhibitor U0126 partially abolished the regulating effect of DJ-1 on tyrosine hydroxylase. Nurr1, a transcriptional factor for tyrosine hydroxylase, can be phosphorylated by ERK1/2 and translocate to the nucleus, where it is activated. Thus, we measured nuclear translocation of Nurr1. Confocal microscopy and Western blotting revealed that Nurr1 translocated to the nucleus and was activated by overexpression of wild-type DJ-1, but not of its L166P mutant. Knockdown of Nurr1 gene expression abolished the effect on tyrosine hydroxylase induced by DJ-1. Taken together, these data suggest that DJ-1 upregulates tyrosine hydroxylase expression by activating its transcription factor Nurr1 via the ERK1/2 pathway.

Calcium-related signaling pathways contributed to dopamine-induced cortical neuron apoptosis

Accumulating pathological evidence showing layer-specific neuronal reduction, dendrite deficits and brain volume loss have implicated an apoptotic process in schizophrenia, but the exact mechanism remains elusive. Dopamine hyperactivity at D2 receptor sites was considered as an important mechanism in schizophrenia pathogenesis. Recently, a newly identified D1 and D2 receptor heterooligomer activated by the specific agonist SKF83959 has been shown to stimulate phospholipase C-related intracellular calcium release in the brain. In this study, we intend to test the hypothesis that overstimulation of this calcium-related signaling pathway by high concentration of dopamine and SKF83959 is capable of inducing cortical neuronal apoptosis through calcium disturbance. Our experimental results demonstrated that 10-100μM dopamine and 10-50μM SKF83959 treatments for 72h were able to induce cortical neuronal apoptosis via the D1 and D2 receptor heterooligomer mediated calcium overload and mitochondria dysfunction-dependent pathways. Meanwhile, we found that although 24h dopamine and SKF83959 treatments have not produced major apoptosis, they induced significant neuronal dendrite retraction as well as reduction of neurotrophic molecules such as phosphorylated AKT, ERK and Bcl-2 through PLC-sensitive pathways. Taken together, prolonged stimulation of dopamine and SKF83959 in cortical neurons can reduce dendrite extension at early stage and induce neuronal apoptosis later on through PLC-calcium related pathways, which might provide important apoptotic mechanisms for schizophrenia pathogenesis.

Establishment of an immortalized GABAergic neuronal progenitor cell line from embryonic ventral mesencephalon in the rat

Effective cell replacement therapies for neurological disease require neuron-restricted precursors as grafted cells. The problem of obtaining sufficient grafts for transplantation can be resolved by creating an appropriate immortalized cell line. In the present study, a thermally controlled immortalized GABAergic neuronal progenitor cell line (RMNE6) was established from E13 rat ventral mesencephalon cells immortalized using the temperature-sensitive mutant of SV40 large T antigen (ts-TAg). RMNE6 cells proliferated rapidly and expressed a neuron-like phenotype at the permissive temperature (33 degrees C), but eventually stopped growing at the non-permissive temperature (39 degrees C). Expression of the neuronal markers PSA-NCAM, beta-tubulin III and MAP2 by RMNE6 cells was confirmed by RT-PCR or immunocytochemistry. Furthermore, these cells exhibited functional GABAergic neuron properties, as evidenced by the expression of glutamate decarboxylase (GAD) as well as the synthesis and release of the neurotransmitter GABA in a calcium-dependent manner. Moreover, RMNE6 cells spontaneously expressed and secreted several neurotrophic factors, such as NGF, BDNF, NT-3, NT-4/5, and GDNF. The cells survived well and kept expression of SV40 Tag, GAD65/67 and GABA in the striatum, at least 28 days after being transplanted in the rat brain. Tumorigenesis assays confirmed the safety of the immortalized cell line in vivo. Taken together, the results support the use of RMNE6 cells as an ideal cell model for transplantation research aimed at the treatment and prevention of neurodegenerative disease.

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

To silence the expression of α-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability. We identified two 19-nucleotide stretches within the coding region of the α-synuclein gene and designed three sets of oligonucleotides to generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR™/H1/TO vector and transfected into MN9D dopaminergic cells. α-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide (MTT). The mRNA and protein levels of α-synuclein gene were significantly down-regulated in pSH2/α-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/α-SYNtransfected cells showed no significant difference. Silencing α-synuclein expression does not affect cell proliferation but may decrease cell viability. Our results demonstrated pSH2/α-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse α-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of α-synuclein and examining its role in Parkinson’s disease (PD) pathogenesis. α-Synuclein may be important for the viability of MN9D cells, and loss of α-synuclein may induce cell injury directly or indirectly. 使用载体介导的RNA 干扰(RNA interference, RNAi)方法抑制MN9D 细胞中的α-synuclein 基因表达, 并检测其对细胞增殖和活力的影响. 在α-synuclein 的开放读码区选择两段19 个核苷酸的片断, 并选择一段与小鼠基因无同源性的阴性对照序列, 分别设计合成单链寡核苷酸, 退火融合成双链 寡核苷酸. 将双链 寡核苷酸克隆入pENTR/H1/TO 质粒载体, 鉴定正确后, 转染MN9D 细胞, 通过筛选获得稳定的细胞克隆. 使用RT-PCR、Real-time PCR、免疫细胞化学染色和Western blot 方法检测细胞内的α-synuclein 的表达水平, 鉴定RNAi 的效率. 利用生长曲线和MTT法分别检测比较各组细胞的增殖和活力. 转染pSH2/α-SYN的细胞与正常和转染pSH/CON的MN9D细胞相比, α-synuclein mRNA及蛋白水平均明显减少; 而转染pSH1/α-SYN的细胞α-synuclein mRNA及蛋白水平与两组对照细胞没有明显差别. 抑制gα-synuclein 表达不影响细胞增殖, 但可以降低细胞活力. 结果表明, SH2/α-SYN是有效的小干扰RNA (small interfering RNA, siRNA)序列, 通过载体介导的RNAi方法可以有效抑制MN9D细胞中的α-synuclein表达, 这为进一步研究α-synuclein蛋白的生理功能及其在帕金森病(Parkinson’s disease, PD)发病中的作用提供了良好的细胞模型; α-synuclein在维持MN9D细胞活力中起到重要作用, α-synuclein功能缺失会导致细胞直接或间接的损伤.

DJ-1/PARK7, But Not Its L166P Mutant Linked to Autosomal Recessive Parkinsonism, Modulates the Transcriptional Activity of the Orphan Nuclear Receptor Nurr1 In Vitro and In Vivo.

Although mutations of DJ-1 have been linked to autosomal recessive Parkinsonism for years, its physiological function and the pathological mechanism of its mutants are not well understood. We report for the first time that exogenous application of DJ-1, but not its L166P mutant, enhances the nuclear translocation and the transcriptional activity of Nurr1, a transcription factor essential for dopaminergic neuron development and maturation, both in vitro and in vivo. Knockdown of DJ-1 attenuates Nurr1 activity. Further investigation showed that signaling of Raf/MEK/ERK MAPKs is involved in this regulatory process and that activation induced by exogenous DJ-1 is antagonized by U0126, an ERK pathway inhibitor, indicating that DJ-1 modulates Nurr1 activity via the Raf/MEK/ERK pathway. Our findings shed light on the novel function of DJ-1 to enhance Nurr1 activity and provide the first insight into the molecular mechanism by which DJ-1 enhances Nurr1 activity.

Human umbilical cord blood-derived non-hematopoietic stem cells suppress lymphocyte proliferation and CD4, CD8 expression

One concern in the use of transplantation of non-hematopoietic stem cells from human umbilical cord blood (CB-nHSCs) is the possibility of rejection by the hosts immune system. This study shows that both CB-nHSCs and their progenies after passaging, neuronal differentiation or IFN-gamma treatment have no significant effects on proliferation of xenogenic T lymphocytes. CB-nHSCs transplanted into the striatum of SD rat are shown to induce a lower level of CD4 and CD8 expression in the brain and in the peripheral blood and to survive better in the brain than SH-SY5Y cells. The results indicate that both undifferentiated and differentiated CB-nHSCs all have weak immunogenicity.