Reduced Cytotoxicity by Repetitive mRNA Transfection in Differentiated Neurons.

Background and Objectives: mRNA-based protein expression technology has been used to express functional proteins. We have previously generated dopamine neurons from rat-embryo derived neural precursor cells (NPCs) through repeated transfection of synthetic transcription factor mRNA encoding dopamine-inducible genes. However, NPCs began to die approximately 10 d post-transfection. In this study, we examined a long-term transfection protocol that did not affect cell viability. Methods and Results: Experiments were performed in eight groups sorted according to the start date of mRNA transfection. mRNA was transfected into NPCs daily for 21 d and live cell images of each group were recorded. NPCs which were differentiated for more than five days showed sustained gene expression and appreciable viability despite daily mRNA transfection for 21 d. Conclusions: Repeated mRNA transfection requires cells with a sufficient differentiation period.

Efficient transdermal delivery of functional protein cargoes by a hydrophobic peptide MTD 1067.

The skin has a protective barrier against the external environment, making the transdermal delivery of active macromolecules very difficult. Cell-penetrating peptides (CPPs) have been accepted as useful delivery tools owing to their high transduction efficiency and low cytotoxicity. In this study, we evaluated the hydrophobic peptide, macromolecule transduction domain 1067 (MTD 1067) as a CPP for the transdermal delivery of protein cargoes of various sizes, including growth hormone-releasing hexapeptide-6 (GHRP-6), a truncated form of insulin-like growth factor-I (des(1-3)IGF-I), and platelet-derived growth factor BB (PDGF-BB). The MTD 1067-conjugated GHRP-6 (MTD-GHRP-6) was chemically synthesized, whereas the MTD 1067-conjugated des(1-3)IGF-I and PDGF-BB proteins (MTD-des(1-3)IGF-I and MTD-PDGF-BB) were generated as recombinant proteins. All the MTD 1067-conjugated cargoes exhibited biological activities identical or improved when compared to those of the original cargoes. The analysis of confocal microscopy images showed that MTD-GHRP-6, MTD-des(1-3)IGF-I, and MTD-PDGF-BB were detected at 4.4-, 18.8-, and 32.9-times higher levels in the dermis, respectively, compared to the control group without MTD. Furthermore, the MTD 1067-conjugated cargoes did not show cytotoxicity. Altogether, our data demonstrate the potential of MTD 1067 conjugation in developing functional macromolecules for cosmetics and drugs with enhanced transdermal permeability.

Usefulness of Diffusion Tensor Imaging in Unexplained Ipsilateral Hemiplegia.

Ipsilateral hemiparesis is a rare and challenging sign in clinical neurological practice. Although the etiology of this manifestation is poorly understood, recent studies have attempted to probe the pathomechanism of this sign with advanced radiological techniques. Additional knowledge about the lesion and unraveling the pathomechanisms causing neurological impairments are important to predict the prognosis and clinical course and to aid in rehabilitation. Therefore, we present a case of a patient with a traumatic subdural hematoma on the left hemisphere and left spastic hemiparesis. Using diffusion tensor imaging (DTI), we concluded that the right corticospinal tract injury caused by compression of the cerebral peduncle accounted for the ipsilateral hemiparesis, also known as Kernohan's notch phenomenon. Thus, this case report highlights the usefulness of the newer radiological techniques, such as DTI, to identify the pathomechanisms of neurological presentations.

Upper Extremity Deep Vein Thrombosis After Botulinum Toxin Injection: A Case Report.

Botulinum toxin (BoNT) injection is widely used to improve spasticity. However, after the treatment, the patient may experience pain, inflammation, swelling and redness at the injection site. In this case, we addressed deep vein thrombosis (DVT) after BoNT treatment of the upper limb. A male aged 37 years had spasticity and dystonia in his left upper extremity. BoNT-A 100 U was injected into the left biceps brachii and an equal amount into the brachialis to relieve spasticity. After three days, he developed redness and painful swelling in the left upper arm and the next day, through the upper extremity computed tomography venography, DVT was identified in the left cephalic vein. The thrombus resolved after the anticoagulation therapy with rivaroxaban (Xarelto). We hypothesized the role of mainly three mechanisms in the development of DVT in this case: repetitive strenuous activity, relative stasis due to reduced muscle tone, and possible direct mechanical damage to the vessel wall.

Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain.

Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.

Fluazinam targets mitochondrial complex I to induce reactive oxygen species-dependent cytotoxicity in SH-SY5Y cells.

Although the underlying cause of Parkinson's disease (PD) is not well characterized, epidemiological studies suggest that exposure to agricultural chemicals is a risk factor for PD. Fluazinam (FZN) is a new active ingredient for the control of grey mould, belonging to the novel broad spectrum phenylpyridinamine fungicides. We used human neuroblastoma SH-SY5Y cells to investigate mechanisms of dopaminergic cell death in response to FZN. FZN treatment produced dose-dependent cytotoxicity, and decreased the tyrosine hydroxylase (TH) expression in SH-SY5Y cells. We provided evidence for the occurrence of oxidative stress and oxidative damage during FZN exposure on dopaminergic cells through the measurement of reactive oxygen species (ROS) in cells with DCFH-DA. The cytotoxic effects of FZN appear to involve an increase in ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FZN treatment, dopamine (DA) levels decreased in both cell and culture media, and oxidative effects of FZN were blocked by NAC pretreatment. We show that cell death in response to FZN was due to apoptosis since FZN exposure results in an increased in cytochrome c release into the cytosol and activated caspase-3 through p38 and JNK signaling. Furthermore, the blocking of p38 or JNK signaling inhibits FZN-induced cell death. Phosphorylation of mitogen-activated protein kinases precedes cytochrome c release and caspase-3 activation. This cellular response is characteristic of mitochondrial dysfunction. Therefore, we also investigated the effect of FZN on mitochondrial complex I activity in FZN-treated cell. Interestingly, we show that FZN inhibited the complex I activity. Thus in this study, we report a new mode of action by which the fungicide FZN could triggers apoptosis.

Fluazinam-induced apoptosis of SH-SY5Y cells is mediated by p53 and Bcl-2 family proteins.

A number of epidemiological studies have demonstrated a strong association between the incidence of neurodegenerative disease and pesticide exposure. Fluazinam (FZN) is a preventative fungicide from the pyridinamine group that was introduced in the 1990 s and that quickly established itself as a new standard for the control of blight caused by Phytophthora infestans in potatoes. We used human neuroblastoma SH-SY5Y cells to investigate mechanisms of neuronal cell death in response to FZN and showed that FZN was cytotoxic to SH-SY5Y cells in a concentration- and time-dependent manner. Additionally, we showed that FZN treatment significantly decreased the neuron numbers including dopaminergic neurons and mitochondrial complex I activity. The cytotoxic effects of FZN were associated with an increase in reactive oxygen species (ROS) generation because pretreatment with N-acetyl cysteine, an anti-oxidant, reduced cell death. We showed that neuronal cell death in response to FZN was due to apoptosis because FZN increased cytochrome C release into the cytosol and activated caspase-3 through the accumulation of p53. FZN also reduced the levels of Bcl-2 protein but increased the levels of Bax. Our results provide insight into the molecular mechanisms of FZN-induced apoptosis in neuronal cells.

Akt/GSK3ß signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells.

Fipronil (FPN) is a phenylpyrazole insecticide acted on insect gamma-aminobutyric acid (GABA) receptors. Although action of FPN is restricted on insect neuronal or muscular transmitter system, a few studies have assessed the effects of this neurotoxicant on neuronal cell death. To determine the mechanisms underlying FPN-induced neuronal cell death, we investigated whether reactive oxygen species (ROS) plays a role in FPN-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. FPN was cytotoxic to these cells and its cytotoxicity showed a concentration-dependent manner. Additionally, FPN treatment significantly decreased the tyrosine hydroxylase (TH) expression without change of glutamic acid decarboxylase 65 (GAD65) expression. FPN-induced dopaminergic cell death involved in increase of ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FPN treatment, dopamine (DA) levels decreased significantly in both cell and culture media, and oxidative effects of DA were blocked by NAC pretreatment. We showed that cell death in response to FPN was due to apoptosis since FPN increased cytochrome c release into the cytosol and activated caspase-3. It also led to nuclear accumulation of p53 and reduced the level of Bcl-2 protein in a concentration-dependent manner. Additionally, FPN altered the level of Akt/glycogen synthase kinase-3 (GSK3ß) phosphorylation. FPN reduced the Akt phosphorylation on Ser473, and in parallel with the inactivation of Akt, phosphorylation of GSK3ß on Ser9 which inactivates GSK3ß, decreased after treatment with FPN. Furthermore, inhibition of the GSK3ß signal protected the cell against FPN-induced cell death. These results suggest that regulation of GSK3ß activity may control the apoptosis induced by FPN-induced oxidative stress associated with neuronal cell death.

Complete rooming-in care of newborn infants.

PURPOSE: In Kyung Hee East-West Neo Medical Center, Seoul, Korea, efforts to raise rooming-in care success rate have been undertaken since when the hospital was established in 2006. We intended to analyze our experience over the past 3 years of period and to discuss the advantages of rooming-in. METHODS: We analyzed the rooming-in practice rate, failure rate, and the breast feeding rate. Subjects were 860 normal healthy neonates from June 2006 to June 2009. RESULTS: Among these 860 cases, 83 babies were required separation out of rooming-in in the middle of the course. Among these 83 cases, 70 cases had to stop the course due to poor condition of babies and 13 cases due to maternal condition. 70 cases of infant's causes consist of 68 cases of NICU admission and 2 cases of poor feeding support. The other 13 cases of separation include refusal by maternal condition. Therefore the success rate of rooming-in for the last 3 years was 90.3%, that is 777 cases among the total 860 cases. The percentage of exclusive breast feeding was 64%, that of mixed feeding with breast and formula feeding was 25%, and formula feeding only was 11%. CONCLUSION: We experienced successful rooming-in care for the last 3 years. Nursery facilities should educate and encourage the advantages of rooming-in, including the good formation of attachment between mother and infant, emotional stability, protection from infection, and increased breast feeding rate so that rooming-in care can be fully established.

Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival.

Effective dopamine (DA) neuron differentiation from neural precursor cells (NPCs) is prerequisite for precursor/stem cell-based therapy of Parkinson's disease (PD). Nurr1, an orphan nuclear receptor, has been reported as a transcription factor that can drive DA neuron differentiation from non-dopaminergic NPCs in vitro. However, Nurr1 alone neither induces full neuronal maturation nor expression of proteins found specifically in midbrain DA neurons. In addition, Nurr1 expression is inefficient in inducing DA phenotype expression in NPCs derived from certain species such as mouse and human. We show here that Foxa2, a forkhead transcription factor whose role in midbrain DA neuron development was recently revealed, synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain-specific gene expression, and neuronal maturation. Thus, the combinatorial expression of Nurr1 and Foxa2 in NPCs efficiently yielded fully differentiated nigral (A9)-type midbrain neurons with clearly detectable DA neuronal activities. The effects of Foxa2 in DA neuron generation were observed regardless of the brain regions or species from which NPCs were derived. Furthermore, DA neurons generated by ectopic Foxa2 expression were more resistant to toxins. Importantly, Foxa2 expression resulted in a rapid cell cycle exit and reduced cell proliferation. Consistently, transplantation of NPCs transduced with Nurr1 and Foxa2 generated grafts enriched with midbrain-type DA neurons but reduced number of proliferating cells, and significantly reversed motor deficits in a rat PD model. Our findings can be applied to ongoing attempts to develop an efficient and safe precursor/stem cell-based therapy for PD.

Proneural bHLH neurogenin 2 differentially regulates Nurr1-induced dopamine neuron differentiation in rat and mouse neural precursor cells in vitro.

Roles of Nurr1 and neurogenin 2 (Ngn2) have been shown in midbrain dopamine (DA) neuron development. We present here rat and mouse species-dependent differences of Nurr1 and Ngn2 actions in DA neuron differentiation. Nurr1 exogene expression caused an efficient generation of tyrosine hydroxylase (TH)-positive DA cells from rat neural precursor cells (NPCs). Nurr1-induced TH+ cell yields were low and highly variable depending on the origins of NPCs in mouse cultures. Coexpression of Ngn2 repressed Nurr1-induced generation of TH+ cells in rat cultures. In clear contrast, a robust enhancement in Nurr1-induced DA cell yields was observed in mouse NPCs by Ngn2. These findings imply that DA neurons may develop differently in the midbrains of these two species.

Bcl-XL/Bax proteins direct the fate of embryonic cortical precursor cells.

In the developing mouse brain, the highest Bcl-X(L) expression is seen at the peak of neurogenesis, whereas the peak of Bax expression coincides with the astrogenic period. While such observations suggest an active role of the Bcl-2 family proteins in the generation of neurons and astrocytes, no definitive demonstration has been provided to date. Using combinations of gain- and loss-of-function assays in vivo and in vitro, we provide evidence for instructive roles of these proteins in neuronal and astrocytic fate specification. Specifically, in Bax knockout mice, astrocyte formation was decreased in the developing cortices. Overexpression of Bcl-X(L) and Bax in embryonic cortical precursors induced neural and astrocytic differentiation, respectively, while inhibitory RNAs led to the opposite results. Importantly, inhibition of caspase activity, dimerization, or mitochondrial localization of Bcl-X(L)/Bax proteins indicated that the differentiation effects of Bcl-X(L)/Bax are separable from their roles in cell survival and apoptosis. Lastly, we describe activation of intracellular signaling pathways and expression of basic helix-loop-helix transcriptional factors specific for the Bcl-2 protein-mediated differentiation.

Acquisition of in vitro and in vivo functionality of Nurr1-induced dopamine neurons.

Neural precursor cells provide an expandable source of neurons and glia for basic and translational applications. However, little progress has been made in directing naive neural precursors toward specific neuronal fates such as midbrain dopamine (DA) neurons. We have recently demonstrated that transgenic expression of the nuclear orphan receptor Nurr1 is sufficient to drive dopaminergic differentiation of forebrain embryonic rat neural precursors in vitro. However, Nurr1-induced DA neurons exhibit immature neuronal morphologies and functional properties and are unable to induce behavioral recovery in rodent models of Parkinson's disease (PD). Here, we report on the identification of key genetic factors that drive morphological and functional differentiation of Nurr1-derived DA neurons. We show that coexpression of Nurr1, Bcl-XL, and Sonic hedgehog (SHH) or Nurr1 and the proneural bHLH factor Mash1 is sufficient to drive naive rat forebrain precursors into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neuron in vitro. On transplantation into the striatum of Parkinsonian rats, precursor cells engineered with Nurr1/SHH/Bcl-XL or Nurr1/Mash1 survived in vivo and differentiated into mature DA neurons that can reverse the behavioral deficits in the grafted animals.

Differential actions of the proneural genes encoding Mash1 and neurogenins in Nurr1-induced dopamine neuron differentiation.

The steroid receptor-type transcription factor Nurr1 has a crucial role in the development of the mesencephalic dopamine (DA) neurons. Although ectopic expression of Nurr1 in cultured neural precursor cells is sufficient in establishing the DA phenotype, Nurr1-induced DA cells are morphologically and functionally immature, suggesting the necessity of additional factor(s) for full neuronal differentiation. In this study, we demonstrate that neurogenic basic helix-loop-helix (bHLH) factors Mash1, neurogenins (Ngns) and NeuroD play contrasting roles in Nurr1-induced DA neuronal differentiation. Mash1, but not Ngn2, spatially and temporally colocalized with aldehyde dehydrogenase 2 (AHD2), a specific midbrain DA neuronal progenitor marker, in the early embryonic ventral mesencephalon. Forced expression of Mash1 caused immature Nurr1-induced DA cells to differentiate into mature and functional DA neurons as judged by electrophysiological characteristics, release of DA, and expression of presynaptic DA neuronal markers. By contrast, atonal-related bHLHs, represented by Ngn1, Ngn2 and NeuroD, repressed Nurr1-induced expression of DA neuronal markers. Domain-swapping experiments with Mash1 and NeuroD indicated that the helix-loop-helix domain, responsible for mediating dimerization of bHLH transcription factors, imparts the distinct effect. Finally, transient co-transfection of the atonal-related bHLHs with Nurr1 resulted in an E-box-independent repression of Nurr1-induced transcriptional activation of a reporter containing Nurr1-binding element (NL3) as well as a reporter driven by the native tyrosine hydroxylase gene promoter. Taken together, these findings suggest that Mash1 contributes to the generation of DA neurons in cooperation with Nurr1 in the developing midbrain whereas atonal-related bHLH genes inhibit the process.

In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.

Human embryonic stem (hES) cells, due to their capacity of multipotency and self-renewal, may serve as a valuable experimental tool for human developmental biology and may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hES cells to replace the selectively lost midbrain dopamine (DA) neurons in Parkinson's disease. Here, we report the development of an in vitro differentiation protocol to derive an enriched population of midbrain DA neurons from hES cells. Neural induction of hES cells co-cultured with stromal cells, followed by expansion of the resulting neural precursor cells, efficiently generated DA neurons with concomitant expression of transcriptional factors related to midbrain DA development, such as Pax2, En1 (Engrailed-1), Nurr1, and Lmx1b. Using our procedure, the majority of differentiated hES cells (> 95%) contained neuronal or neural precursor markers and a high percentage (> 40%) of TuJ1+ neurons was tyrosine hydroxylase (TH)+, while none of them expressed the undifferentiated ES cell marker, Oct 3/4. Furthermore, hES cell-derived DA neurons demonstrated functionality in vitro, releasing DA in response to KCl-induced depolarization and reuptake of DA. Finally, transplantation of hES-derived DA neurons into the striatum of hemi-parkinsonian rats failed to result in improvement of their behavioral deficits as determined by amphetamine-induced rotation and step-adjustment. Immunohistochemical analyses of grafted brains revealed that abundant hES-derived cells (human nuclei+ cells) survived in the grafts, but none of them were TH+. Therefore, unlike those from mouse ES cells, hES cell-derived DA neurons either do not survive or their DA phenotype is unstable when grafted into rodent brains.