The Role of Alpha-Synuclein in Melanin Synthesis in Melanoma and Dopaminergic Neuronal Cells

The relatively high co-occurrence of Parkinson’s disease (PD) and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR)-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM), the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn) that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR) and inhibit tyrosine hydroxylase (TH), both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA), led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB) light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in melanoma cells and in dopaminergic neuronal cells.     

Unique Pattern of Gene Expression in the Erythroid Precursor Cells

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU-E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt-genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt-genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU-E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt-genes may be induced after the stimulation of erythropoietin.     

Short-Term Withdrawal of Mitogens Prior to Plating Increases Neuronal Differentiation of Human Neural Precursor Cells

Background

Human neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.

Principal Findings

We demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. In this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.

Conclusions

These results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.     

Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2

The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC) characteristically express the transmembrane proteoglycan nerve-glia antigen 2 (NG2) and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding) in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice), or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA) receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal synapses.     

Paradoxical Sensitivity to an Integrated Stress Response Blocking Mutation in Vanishing White Matter Cells

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2’s alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4^A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2α on GEF by a contravening EIF2S1/eIF2α^S51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4^A391D and the related severe VWM EIF2B4^R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2α genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.     

Neuronal Differentiation and Extensive Migration of Human Neural Precursor Cells following Co-Culture with Rat Auditory Brainstem Slices

Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry.     

Gene Expression and Regulation of Higher Plants Under Soil Water Stress

Higher plants not only provide human beings renewable food, building materials and energy, but also play the most important role in keeping a stable environment on earth. Plants differ from animals in many aspects, but the important is that plants are more easily influenced by environment than animals. Plants have a series of fine mechanisms for responding to environmental changes, which has been established during their long-period evolution and artificial domestication. The machinery related to molecular biology is the most important basis. The elucidation of it will extremely and purposefully promote the sustainable utilization of plant resources and make the best use of its current potential under different scales. This molecular mechanism at least includes drought signal recognition (input), signal transduction (many cascade biochemical reactions are involved in this process), signal output, signal responses and phenotype realization, which is a multi-dimension network system and contains many levels of gene expression and regulation. We will focus on the physiological and molecular adaptive machinery of plants under soil water stress and draw a possible blueprint for it. Meanwhile, the issues and perspectives are also discussed. We conclude that biological measures is the basic solution to solving various types of issues in relation to sustainable development and the plant measures is the eventual way.Key Words: Higher plants, soil water stress, gene regulatory network, drought, anti-drought gene resources, signal, ion homeostasis, physiological mechanisms.     

(Pro)renin Receptor Mediates Both Angiotensin II-Dependent and -Independent Oxidative Stress in Neuronal Cells

The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.     

Tetanus Toxin Hc Fragment Induces the Formation of Ceramide Platforms and Protects Neuronal Cells against Oxidative Stress

Tetanus toxin (TeTx) is the protein, synthesized by the anaerobic bacteria Clostridium tetani, which causes tetanus disease. TeTx gains entry into target cells by means of its interaction with lipid rafts, which are membrane domains enriched in sphingomyelin and cholesterol. However, the exact mechanism of host membrane binding remains to be fully established. In the present study we used the recombinant carboxyl terminal fragment from TeTx (Hc-TeTx), the domain responsible for target neuron binding, showing that Hc-TeTx induces a moderate but rapid and sustained increase in the ceramide/sphingomyelin ratio in primary cultures of cerebellar granule neurons and in NGF-differentiated PC12 cells, as well as induces the formation of ceramide platforms in the plasma membrane. The mentioned increase is due to the promotion of neutral sphingomyelinase activity and not to the de novo synthesis, since GW4869, a specific neutral sphingomyelinase inhibitor, prevents neutral sphingomyelinase activity increase and formation of ceramide platforms. Moreover, neutral sphingomyelinase inhibition with GW4869 prevents Hc-TeTx-triggered signaling (Akt phosphorylation), as well as the protective effect of Hc-TeTx on PC12 cells subjected to oxidative stress, while siRNA directed against nSM2 prevents protection by Hc-TeTx of NSC-34 cells against oxidative insult. Finally, neutral sphingomyelinase activity seems not to be related with the internalization of Hc-TeTx into PC12 cells. Thus, the presented data shed light on the mechanisms triggered by TeTx after membrane binding, which could be related with the events leading to the neuroprotective action exerted by the Hc-TeTx fragment.     

Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.     

上调hMLH1基因表达对卵巢癌药物敏感性的影响

构建携带错配修复基因hMLH1编码序列全长的真核表达质粒pCAN—hMLHl,并探讨其对卵巢癌细胞顺铂耐药的逆转作用。应用基因重组技术将pET28-hMLHl中的目的基因hMLHl定向克隆到真核表达载体pCAN,经酶切及测序鉴定：分别将pCAN—hMLHl和空质粒pCAN转染进卵巢癌耐药细胞SKOV3／DDP,同时以对顺铂敏感的sKOV3细胞和未转染的SKOV3／DDP细胞作为对照：应用RT-PCR和Westemblo凇测转染前后细胞内hMLHlmRNA和蛋白的表达变4Jc;四甲基偶氮唑蓝（MTT）比色法检测转染前后sKOv3／DDP细胞对顺铂敏感性的变化;Hoechst染色检测转染前后细胞的凋亡。结果提示：pCAN—hMLHl重组质粒经酶切及测序鉴定,表明真核表达质粒构建正确;采用脂质体法转染sKOv3／DDP细胞后,RT-PCR和Westernblot检测到耐药细胞内hMLHl的表达增强：MTT结果显示转染重组质粒后sKOv3／DDP细胞对顺铂的敏感性显著增加;Hoechst染色观察到转染后耐药细胞的凋亡明显增强。该研究成功构建了pCAN．hMLHl重组质粒,在sKOV3／DDP细胞中进行表达,并能增强耐药细胞对顺铂的敏感性,促进耐药细胞的凋亡。     

Regulation of the Manganese Superoxide Dismutase and Inducible Nitric Oxide Synthase Gene in Rat Neuronal and Glial Cells

Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O₂⁻) and nitric oxide (NO^•). Neurotoxicity mediated by NO^• may result from the reaction of NO^• with O₂, leading to formation of peroxynitrite (ONOO⁻). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1β, interferon-γ (IFN-γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.     

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.     

Changes in Sensitivity of Leukemia P388 Cells to Oxidative Stress and Platidiam upon Tumor Growth

We studied the effect of oxidative stress induced by hyperoxia, hydrogen peroxide, or menadione on leukemia P388 cells isolated from mice with early (4 days) and late (7 days) stages of tumor growth. Oxidative stress was shown to inhibit cell division and to induce apoptosis. The seven-day leukemia cells feature lower proliferative potential and higher sensitivity to oxidative stress and platidiam.     

食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定

间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程。胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型。该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCs。结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞。将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性。     

Direct Reprogramming of Rat Neural Precursor Cells and Fibroblasts into Pluripotent Stem Cells

Background

Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes.

Methodology and Principal Findings

iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency.

Conclusions/Significance

Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease.