Positive biocompatibility of several graphene derivatives with dopaminergic cells at long term culture

The emerging carbon nanomaterial graphene (G) and its oxidized derivative graphene oxide (GO) have recently gained considerable attention in biomedical applications such as cancer therapy or biosensors. It has for example been demonstrated that G has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells (Li et al., 2013). This way, G could acts as a physical support or scaffold to promote axonal sprout as a “deceleration” support for the DA cells derived from neural stem cells. Since GO in its multilayer form and with multiples carboxilate and epoxy groups seems to shows interesting biological properties (Yang et al., 2013) the aim of the present work has been to test different graphene derivatives searching for the best scaffold to be used in stem cell differentiation. For this purpose we have tested the cytotoxicity of GO and reduced GO, and specifically its biocompatibility with SN4741, a dopaminergic cells line derived from mouse substance nigra, measuring the effect in the cells at long term culture. The cells were cultured in Dulbecco’s modified Eagle’s medium 10% FCS (Gibco) to about 80% confluence. Cells were incubated applying 1.000 cells in 96-well microliter plates with graphene using three chemically different types of GO as powders and films: 1) GO, which is hydrophilic; 2) partially reduced GO (PRGO) which is hydrophobic and 3) fully reduced GO (FRGO), also hydrophobic, in five concentrations: 1 mg/ml; 0.1 mg/ml; 0.05 mg/ml; 0.02 mg/ml and 0.01 mg/ml, in each type of graphene. Cells were cultured with GO and cell viability was determined after 24 hours, 1 week and 2 weeks using the MTT assay (Roche) and cytotoxicitity was determined by the lactate dehydrogenase (LDH) (Roche) assay measured at 560nm. The results demonstrated positive biocompatibility between the G-derivatives and SN4741 cells. We conclude that the use of our G-derivative scaffolds can enhance the neural differentiation towards neurons (TH positive) providing a cell growth microenvironments and appropriate synergistic cell guidance cues. This findings demonstrated that biocompatibility of scaffolds is a pre-requisite for generation of successful clinical application of graphene. It could offer a platform for neural stem cells and a promising approach for neural regeneration in the research of neurological diseases like PD. Long-term studies on the biological effects of graphene will now be performed for the development of therapeutic treatment as the goal. (Refs: Li N., Zhang Q, Gao S. et a., 2013, Nature/Sci Rep. 3:1604. doi: 10.1038/srep01604; Yan K., Li Y., Tan X., et al., 2013, Small., 9(9-10): 1492-1503)1. Universidad de Malaga. Campus de Excelencia Internacional Andalucia Tech, España. 2. The Norwegian Research Council (grant nº 215086, Oslo, Noruega. 3. Karolinska Institute Reasearch Fund, Estocolmo, Suecia

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line- derived neurotrophic factor

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease

Les neurotrofines en el Sistema Nerviós Central: Efectes neuroprotectors i implicacions

La formació de les connexions i 1'estabilització dels circuits neuronals són crítiques en el desenvolupament del sistema nerviós, ja que són la base de les seves funcions, com són 1'aprenentatge i la memòria. Durant el desenvolupament es produeix un excès de cèl·lules nervioses que seran seleccionades en funció de la seva habilitat per establir contactes sinàptics amb les cèl·lules diana, mitjançant un procés de mort cel·lular fisiològica que serveix per seleccionar i estabilitzar els circuits neuronals. L'estudi dels mecanismes implicats en la regulació de la supervivència és important també per entendre i intentar trobar possibles tractaments per als processos de neurodegeneració selectiva que hi ha en alguns trastorns neurològics

Applying Microsatellite Multiplex PCR Analysis (MMPA) for Determining Allele Copy-Number Status and Percentage of Normal Cells within Tumors

The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs) that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques. Microsatellite multiplex PCR analysis (MMPA) should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques

Dkk1 Regulates Ventral Midbrain Dopaminergic Differentiation and Morphogenesis

Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1+/− embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1−/− embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis

Translational molecular imaging and drug development in Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder that primarily affects elderly people and constitutes a major source of disability worldwide. Notably, the neuropathological hallmarks of PD include nigrostriatal loss and the formation of intracellular inclusion bodies containing misfolded α-synuclein protein aggregates. Cardinal motor symptoms, which include tremor, rigidity and bradykinesia, can effectively be managed with dopaminergic therapy for years following symptom onset. Nonetheless, patients ultimately develop symptoms that no longer fully respond to dopaminergic treatment. Attempts to discover disease-modifying agents have increasingly been supported by translational molecular imaging concepts, targeting the most prominent pathological hallmark of PD, α-synuclein accumulation, as well as other molecular pathways that contribute to the pathophysiology of PD. Indeed, molecular imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) can be leveraged to study parkinsonism not only in animal models but also in living patients. For instance, mitochondrial dysfunction can be assessed with probes that target the mitochondrial complex I (MC-I), while nigrostriatal degeneration is typically evaluated with probes designed to non-invasively quantify dopaminergic nerve loss. In addition to dopaminergic imaging, serotonin transporter and N-methyl-D-aspartate (NMDA) receptor probes are increasingly used as research tools to better understand the complexity of neurotransmitter dysregulation in PD. Non-invasive quantification of neuroinflammatory processes is mainly conducted by targeting the translocator protein 18 kDa (TSPO) on activated microglia using established imaging agents. Despite the overwhelming involvement of the brain and brainstem, the pathophysiology of PD is not restricted to the central nervous system (CNS). In fact, PD also affects various peripheral organs such as the heart and gastrointestinal tract - primarily via autonomic dysfunction. As such, research into peripheral biomarkers has taken advantage of cardiac autonomic denervation in PD, allowing the differential diagnosis between PD and multiple system atrophy with probes that visualize sympathetic nerve terminals in the myocardium. Further, α-synuclein has recently gained attention as a potential peripheral biomarker in PD. This review discusses breakthrough discoveries that have led to the contemporary molecular concepts of PD pathophysiology and how they can be harnessed to develop effective imaging probes and therapeutic agents. Further, we will shed light on potential future trends, thereby focusing on potential novel diagnostic tracers and disease-modifying therapeutic interventions

Graphene derivative scaffolds facilitate in vitro cell survival and maturation of dopaminergic SN4741 cells

The emerging carbon nanomaterial Graphene (G), in the form of scaffold structure, has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells, providing a promising approach for neural regeneration. We propose the use of G as a scaffold to re-address the dopaminergic (DA) neurons and the residual axons from dead or apoptotic DA neurons in Parkinson´s disease (PD). G could act as a physical support to promote the axonal sprout as a “deceleration” support for the DA cells derived from neural stem cells or DA direct cell conversion, allowing the propagation of nerve impulses. We cultured a clonal substantia nigra (SN) DA neuronal progenitor cell line (SN4741) in presence of G as scaffold. This cell line derived from mouse embryos was cultured in Dulbecco’s modified Eagle’s medium/10% FCS to about 80% confluence. Cells were incubated in three chemically different G derivatives and two different presentation matrixes as powder and films: 1) G oxide (GO); 2) partially reduced GO (PRGO) which is hydrophobic; and 3) fully reduced GO (FRGO). Cell viability was determined using the MTT assay after adding the following G concentrations: 1mg/ml; 0.1mg/ml; 0.05mg/ml; 0.02mg/ml and 0.01mg/ml, in each type of GO. To study cellular morphology and assessment of cell engraftment into GO films (GO film, PRGO film, FRGO film), we analyzed the immunostaining of the anti-rabbit neuron-specific DNA-binding protein (NeuN) antibody, the anti-rat Beta-3-tubulin antibody in combination with the mitochondrial marker mouse anti-ATP synthase antibody, and the anti-rabbit DCX as immature neuronal marker. Hoechst label was used as nuclei marker. Reactive oxidative species (ROS) were measured by flow cytometry to study the influence of G on the cell redox-state. With this purpose, cells were loaded with dihydroethidium. The mitochondrial membrane potential after JC-1 incubation was studied by flow cytometry. Our results show an increase of survival and metabolism (30-40%) at low concentrations of PRGO and FRGO (0.05-0.01 mg/ml) respect to the higher concentration (1 mg/ml), while no changes were seen in the GO group. LDH concentration was measured in the supernatant using a COBAS analyzer showing a neuroprotective action at low concentrations. Furthermore, either PRGO film or FRGO film show an increase in the effective anchorage capacity to nest into the G matrix and in the maturation of the SN 4741 cells. We conclude that the use of G scaffolds in the research of neurological diseases like PD could offer a powerful platform for neural stem cells, direct cell conversion techniques and neural tissue engineering.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Norwegian Research Council (grant nº 215086

Combinatorial ECM Arrays Identify Cooperative Roles for Matricellular Proteins in Enhancing the Generation of TH+ Neurons From Human Pluripotent Cells

The development of efficient cell culture strategies for the generation of dopaminergic neurons is an important goal for transplantation-based approaches to treat Parkinson’s disease. To identify extracellular matrix molecules that enhance differentiation and might be used in these cell cultures we have used micro-contact printed arrays on glass slides presenting 190 combinations of 19 extracellular matrix molecules selected on the basis of their expression during embryonic development of the ventral midbrain. Using long-term neuroepithelial stem cells (Lt-NES), this approach identified a number of matricellular proteins that enhanced differentiation, with the combination of Sparc, Sparc-like (Sparc-l1) and Nell2 increasing the number of tyrosine hydroxylase+ neurons derived from Lt-NES cells and, critically for further translation, human pluripotent stem cells

The graphene oxide species induce a different biological response in SN4741 Parkinson cell line

Introduction: Graphene Oxide (GO)has recently emerged as a reliable material to create scaffolds for the neural tissue because of its biocompatibility, electroconductive and physicochemical properties. Graphene is a 2-dimensional material consisting of rings of carbon atoms with an excellent electrical conductivity originating in the sp2 hybridized carbons network. Nevertheless, there is not a consensus which kind of graphene oxide is most useful of benefit.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Graphene oxide and reduced derivates, as powder or film scaffolds, differentially promote dopaminergic neuron differentiation and survival.

Emerging scaffold structures made of carbon nanomaterials, such as graphene oxide (GO) have shown efficient bioconjugation with common biomolecules. Previous studies described that GO promotes the differentiation of neural stem cells and may be useful for neural regeneration. In this study, we examined the capacity of GO, full reduced (FRGO), and partially reduced (PRGO) powder and film to support survival, proliferation, differentiation, maturation, and bioenergetic function of a dopaminergic (DA) cell line derived from the mouse substantia nigra (SN4741). Our results show that the morphology of the film and the species of graphene (GO, PRGO, or FRGO) influences the behavior and function of these neurons. In general, we found better biocompatibility of the film species than that of the powder. Analysis of cell viability and cytotoxicity showed good cell survival, a lack of cell death in all GO forms and its derivatives, a decreased proliferation, and increased differentiation over time. Neuronal maturation of SN4741 in all GO forms, and its derivatives were assessed by increased protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), the glutamate inward rectifying potassium channel 2 (GIRK2), and of synaptic proteins, such as synaptobrevin and synaptophysin