A cell culture model for monitoring α-synuclein cell-to-cell transfer

Reyes, Juan F.; Olsson, Tomas; Lamberts, Jennifer T.; Devine, Michael J.; Kunath, Tilo; Brundin, Patrik

doi:10.1016/j.nbd.2014.07.003

Cited by 76 publications

(76 citation statements)

References 52 publications

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“…Although these observations seem to go against the general consensus of the literature, several possible explanations might justify these results: (i) the concentration of aSyn-GFP resulting from cell release is too low; (ii) aSyn molecules that find their way inside the cell might be rapidly degraded or re-released—in which case experiments would need to be carried out in a shorter time frame; (iii) molecules enter the cells in a very low number, and consequently have no visible influence on the fluorescence signal; (iv) GFP affects the uptake of or diffusion of aSyn, since it is a larger protein than aSyn itself. Control experiments in which H4 were co-cultured with H4 producing GFP seem to support the last explanation, since no uptake of GFP was observed (Figure 5B), however, other studies reported diffusion of GFP into cells in similar conditions (Reyes et al, 2015). It should be noted that in other studies using extracellular aSyn, internalization was observed after as little as 5 min for monomeric aSyn and 1 h for aSyn fibrils (Lee et al, 2008), which suggests that 10 h is an appropriate time scale for such an experiment.…”

Section: Resultssupporting

confidence: 53%

“…However, these studies rely either on the use of previously prepared conditioned medium (Lee et al, 2008, 2010), i.e., medium in which cells released aSyn, or on culturing a populations expressing tagged aSyn together with another population expressing another fluorescent molecule (Reyes et al, 2015). This platform allows us to monitor the spreading of aSyn without the need for using recombinantly produced protein.…”

Section: Resultsmentioning

confidence: 99%

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A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies

et al. 2016

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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.

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Section: Resultssupporting

confidence: 53%

Section: Resultsmentioning

confidence: 99%

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies

et al. 2016

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“…In the presence of the dynamin inhibitor dynasore, instead of the expected perinuclear accumulation, the ␣-syn immunoreactivity was excluded from the cell interior and retained at the periphery adjacent to the plasma membrane. Despite the qualitatively different intracellular localization, the overall signal for each ␣-syn isoform was not greatly reduced, indicating normal cell surface binding and sequestration, and consistent with a blockade of dynamindependent endocytosis (69,(72)(73)(74). Interestingly, the dynasore treatment accentuated the differential accumulation of the non-phosphorylated and phosphorylated WT ␣-syn, raising the possibility that the endocytosed ␣-syn may normally undergo trafficking to organelles with protein degradation machinery.…”

Section: Discussionmentioning

confidence: 74%

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization

Samuel

Flavin²,

Iqbal

et al. 2016

Journal of Biological Chemistry

144

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Although trace levels of phosphorylated ␣-synuclein (␣-syn) are detectable in normal brains, nearly all ␣-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on ␣-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of ␣-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated ␣-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated ␣-syn isoforms were detected earlier, although the fibrils remained phosphatase-and protease-sensitive. Membrane binding of ␣-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT ␣-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P ␣-syn binding was greatly increased, and A53T ␣-syn was slightly lower, implicating distal effects of the carboxyl-on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of ␣-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated ␣-syn isoforms, a potential means for misfolded extracellular or lumenal ␣-syn to access cytosolic ␣-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of ␣-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of ␣-syn misfolding.

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“…In these human neurons an increase of alphasynuclein levels was found and the pathological phenotype was reverted when the mutation causing the Gaucher pathology was corrected (Schöndorf et al, 2014). Similarly, iPSCs from PD patients carrying an alpha-synuclein triplication have been shown to transmit alpha-synuclein pathology to neighbour N2a cells in a non-mixed co-culture experiment (Reyes et al, 2014). These results, not involving cell to cell contact, are in agreement with previous data from other groups showing that alpha-synuclein aggregates are secreted by cells through exosomes into the media in a calcium-dependent manner and subsequently uptaken by naïve cells (Alvarez-Erviti et al, 2011;Emmanouilidou et al, 2010).…”

Section: Cellular Models Of Alpha-synuclein Pathology And/or Spreadingmentioning

confidence: 92%

Parkinson's disease as a member of Prion-like disorders

Herva

Spillantini

2015

Virus Research

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A cell culture model for monitoring α-synuclein cell-to-cell transfer

Cited by 76 publications

References 52 publications

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization

Parkinson's disease as a member of Prion-like disorders

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