A novel microfluidic cell co-culture platform for the study of the molecular mechanisms of Parkinson's Disease and other synucleinopathies

Copyright © 2016 Fernandes, Chutna, Chu, Conde and Outeiro. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Although, the precise molecular mechanisms underlying Parkinson's disease (PD) are still elusive, it is now known that spreading of alpha-synuclein (aSyn) pathology and neuroinflammation are important players in disease progression. Here, we developed a novel microfluidic cell-culture platform for studying the communication between two different cell populations, a process of critical importance not only in PD but also in many biological processes. The integration of micro-valves in the device enabled us to control fluid routing, cellular microenvironments, and to simulate paracrine signaling. As proof of concept, two sets of experiments were designed to show how this platform can be used to investigate specific molecular mechanisms associated with PD. In one experiment, naïve H4 neuroglioma cells were co-cultured with cells expressing aSyn tagged with GFP (aSyn-GFP), to study the release and spreading of the protein. In our experimental set up, we induced the release of the contents of aSyn-GFP producing cells to the medium and monitored the protein's diffusion. In another experiment, H4 cells were co-cultured with N9 microglial cells to assess the interplay between two cell lines in response to environmental stimuli. Here, we observed an increase in the levels of reactive oxygen species in H4 cells cultured in the presence of activated N9 cells, confirming the cross talk between different cell populations. In summary, the platform developed in this study affords novel opportunities for the study of the molecular mechanisms involved in PD and other neurodegenerative diseases.JF was supported by FCT (SFRH/BD/73908/2010). TO is supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB). The work was also supported by FCT through the Associated Laboratory IN—Institute of Nanoscience and Nanotechnology and the research project EXCL/CTM-NAN/0441/2012.info:eu-repo/semantics/publishedVersio

Extracellular alpha-synuclein: Sensors, receptors, and responses

Synucleinopathies are a group of progressive neurodegenerative diseases known for the accumulation of insoluble aggregates containing the protein alpha-synuclein (aSyn). Recently, it has been assumed that pathology spreads in the brain during disease progression, implying that, at some point in the process, aSyn may exist outside of cells. In this context, extracellular-aSyn (e-aSyn) might transduce signals to the inside of the cells it interacts with, and/or be internalized by different types of cells through the extracellular matrix. Both negatively charged lipids and membrane receptors have been hypothesized as modulators of the loss of cellular homeostasis and cytotoxicity, and of the internalization of e-aSyn. Internalized e-aSyn causes the disruption of multiple cellular processes such as the autophagy lysosomal pathway (ALP), mitochondrial function, endoplasmic reticulum (ER)-stress, UPR activation, or vesicular transport. These processes happen not only in neurons but also in glial cells, activating inflammatory or anti-inflammatory pathways that can affect both neuronal function and survival, thereby affecting disease progression. In this review, we explore possible effects e-aSyn, all the way from the extracellular matrix to the nucleus. In particular, we highlight the glial-neuronal relationship as this is particularly relevant in the context of the spreading of aSyn pathology in synucleinopathies

Elevated alpha-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson´s disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson´s disease progression, particularly in the context of bioenergetic dysfunction.Fil: Oliveira, L. M. A.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Falomir Lockhart, Lisandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Botelho, M. G.. Max-Planck-Institut für biophysikalische Chemie; Alemania. Universidade Federal do Rio de Janeiro; BrasilFil: Lin, K. H.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Wales, P.. Universität Göttingen; AlemaniaFil: Koch, J. C.. Universität Göttingen; AlemaniaFil: Gerhardt, Elizabeth. Universität Göttingen; AlemaniaFil: Taschenberger, H.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Outeiro, T. F.. Universität Göttingen; AlemaniaFil: Lingor, P.. Universität Göttingen; AlemaniaFil: Schüele, B.. The Parkinson’s Institute; Estados UnidosFil: Arndt Jovin, D. J.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Jovin, T. M.. Max-Planck-Institut für biophysikalische Chemie; Alemani

Saccharomyces cerevisiae as a toxicological model to study synthetic cannabinoids and its pyrolysis products

Poster presented at the 7th European Academy of Forensic Science Conference. Prague, 6-11 September 2015"Synthetic cannabinoids are among the major psychoactive drugs widespread as safe and legal alternatives to cannabis. They are commercially available as herbal incense products intended for smoke. This has led most of developed countries to concentrate efforts in order to ban the so called “legal highs”. Despite of their increasing use, there is still a lack of information on both synthetic and natural ingredients, pharmacokinetic properties and toxic effects. In fact some of the substances seem to have stronger toxicological effects when compared to their legal counterpart. Toxicological assays are paramount to know how harmful these new substances are, helping increase public awareness since several hospitalization cases have been reported due to consumption. To tackle the new challenges posed by novel drugs worldwide, we developed an approach using Saccharomyces cerevisiae as a model to investigate the toxicity of pyrolysis products of synthetic cannabinoids. S. cerevisiae.

Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is pathologically defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons in the brain. Predominately pre-synaptic, aSyn has been reported in various subcellular compartments in experimental models. Indeed, nuclear alpha-synuclein (aSynNuc) is evident in many models, the dysregulation of which is associated with altered DNA integrity, transcription and nuclear homeostasis. However, the presence of aSynNuc in human brain cells remains controversial, yet the determination of human brain aSynNuc and its pathological modification is essential for understanding synucleinopathies. Here, using a multi-disciplinary approach employing immunohistochemistry, immunoblot, and mass-spectrometry (MS), we confirm aSynNuc in post-mortem brain tissue obtained from DLB and control cases. Highly dependent on antigen retrieval methods, in optimal conditions, intra-nuclear pan and phospho-S129 positive aSyn puncta were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations in all cases examined. Furthermore, an increase in nuclear phospho-S129 positive aSyn immunoreactivity was apparent in DLB cases compared to controls, in both neuronal and non-neuronal cell types. Our initial histological investigations identified that aSynNuc is affected by epitope unmasking methods but present under optimal conditions, and this presence was confirmed by isolation of nuclei and a combined approach of immunoblotting and mass spectrometry, where aSynNuc was approximately tenfold less abundant in the nucleus than cytoplasm. Notably, direct comparison of DLB cases to aged controls identified increased pS129 and higher molecular weight species in the nuclei of DLB cases, suggesting putative pathogenic modifications to aSynNuc in DLB. In summary, using multiple approaches we provide several lines of evidence supporting the presence of aSynNuc in autoptic human brain tissue and, notably, that it is subject to putative pathogenic modifications in DLB that may contribute to the disease phenotype

Tribology of metal cutting: newly formed underside of chip

This investigation provides comprehensive knowledge regarding metal cutting tribology with the purpose of re-examining the role of Oxygen in the process mechanics. Special purpose apparatus and tribological tools were designed and used to conduct experimental cutting tests on pure metals under Oxygen-rich surrounding medium. It was observed that chip curl, sticking and sliding zones are intimately connected with Oxygen concentration. The main research contribution of this study however, is the assessment of friction coefficient as a function of the process parameters, rather than a constant numeric value, resulting from complex phenomena at the contact interface between the cutting tool and the underside of the newly formed chip

Formation of Toxic Oligomeric α-Synuclein Species in Living Cells

Background: Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinson's disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells. Methodology/Principal Findings: Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein. Conclusions/Significance: This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies

Cellular Prion Protein Mediates alpha-Synuclein Uptake, Localization, and Toxicity In Vitro and In Vivo

Background The cellular prion protein (PrPC) is a membrane-bound, multifunctional protein mainly expressed in neuronal tissues. Recent studies indicate that the native trafficking of PrPC can be misused to internalize misfolded amyloid beta and α-synuclein (aSyn) oligomers. Objectives We define PrPC's role in internalizing misfolded aSyn in α-synucleinopathies and identify further involved proteins. Methods We performed comprehensive behavioral studies on four transgenic mouse models (ThySyn and ThySynPrP00, TgM83 and TgMPrP00) at different ages. We developed PrPC-(over)-expressing cell models (cell line and primary cortical neurons), used confocal laser microscopy to perform colocalization studies, applied mass spectrometry to identify interactomes, and determined disassociation constants using surface plasmon resonance (SPR) spectroscopy. Results Behavioral deficits (memory, anxiety, locomotion, etc.), reduced lifespans, and higher oligomeric aSyn levels were observed in PrPC-expressing mice (ThySyn and TgM83), but not in homologous Prnp ablated mice (ThySynPrP00 and TgMPrP00). PrPC colocalized with and facilitated aSyn (oligomeric and monomeric) internalization in our cell-based models. Glimepiride treatment of PrPC-overexpressing cells reduced aSyn internalization in a dose-dependent manner. SPR analysis showed that the binding affinity of PrPC to monomeric aSyn was lower than to oligomeric aSyn. Mass spectrometry-based proteomic studies identified clathrin in the immunoprecipitates of PrPC and aSyn. SPR was used to show that clathrin binds to recombinant PrP, but not aSyn. Experimental disruption of clathrin-coated vesicles significantly decreased aSyn internalization. Conclusion PrPC's native trafficking can be misused to internalize misfolded aSyn through a clathrin-based mechanism, which may facilitate the spreading of pathological aSyn. Disruption of aSyn-PrPC binding is, therefore, an appealing therapeutic target in α-synucleinopathies. <br