Dopamine-Induced Conformational Changes in Alpha-Synuclein

Background: Oligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinson's disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7]. Methodology/Principal Findings: Here, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species. Conclusion/Significance: Our results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinson's disease

mTOR: A Link from the Extracellular Milieu to Transcriptional Regulation of Oligodendrocyte Development

Oligodendrocyte development is controlled by numerous extracellular signals that regulate a series of transcription factors that promote the differentiation of oligodendrocyte progenitor cells to myelinating cells in the central nervous system. A major element of this regulatory system that has only recently been studied is the intracellular signalling from surface receptors to transcription factors to down-regulate inhibitors and up-regulate inducers of oligodendrocyte differentiation and myelination. The current review focuses on one such pathway: the mTOR (mammalian target of rapamycin) pathway, which integrates signals in many cell systems and induces cell responses including cell proliferation and cell differentiation. This review describes the known functions of mTOR as they relate to oligodendrocyte development, and its recently discovered impact on oligodendrocyte differentiation and myelination. A potential model for its role in oligodendrocyte development is proposed

aSyn adopts different conformations in primary neuronal cultures.

<p>Primary cortical neurons were transfected with Myc-aSyn-V5 and immunocytochemistry was performed as in 1. A. Intensity image. B. Representative FLIM image showing the conformation of aSyn varies throughout the processes. C. Lifetime scale. D. Analysis of the contribution of intermolecular interactions for the lifetimes registered for aSyn. Dilution of the intermolecular interactions with untagged WT aSyn shows the majority of the detected interactions result from intramolecular interactions, i.e., different conformational states of aSyn (n = 3, 30–40 cells per condition).</p

aSyn secondary structure alterations induced by DA.

<p>Conformational changes of aSyn in the absence (....) and in the presence of 10 µM (- - -); 100 µM (-- --) and 1000 µM (___) of dopamine were monitored by CD. The concentration of aSyn used was 70 µM and the spectra were taken at 37°C in a 0.1 mm path quartz cuvette at 25°C. The inset graphs show the percentage of random coil and beta-sheet as a function of DA concentration and was calculated after deconvolution of the CD spectra according to the description in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006906#s2" target="_blank">material and methods</a> section (average of 5 independent experiments +/− SD).</p

Compounds tested in the FLIM assay in primary neurons.

<p>Compounds with different activities were used and their effects on the conformation of aSyn were assessed via the FLIM assay. Only dopamine affected the conformation of aSyn (+) (*n = 3, 30–50 cells per condition, p<0.01).</p

Expression of aSyn in primary cortical neurons.

<p>A. Schematic of the Myc-aSyn-V5 construct. B. Immunocytochemistry with anti-Myc (green) and anti-V5 (red) antibodies showing expression throughout the cell (including in the nucleus).</p

Dopamine receptor agonists and antagonists do not alter aSyn conformation.

<p>A. D1 and D2 receptors are present in primary cortical neurons. DIV7 neurons were immunostained against D1 and D2 receptors and observed via fluorescence microscopy. B. FLIM study with dopamine agonists/antagonists showing that these drugs do not alter aSyn conformation (*n = 3, 25–30 cells per condition, p<0.01; unpaired, double sided t test). C. FLIM image of an example compound, SKF38393, showing aSyn conformation is not altered.</p

Dopamine enters cells to modulate aSyn conformation.

<p>A. ICC of cells treated with DA or DA + nomifensine (catecholamine uptake blocker) showing DA is able to enter cells. B. Quantification of the fluorescence intensity in A (∼200 cells) (*p<0.01, t test). C. FLIM analysis showing that DA needs to enter cells to alter aSyn conformation (n = 3, 25–30 cells per condition, *p<0.01; unpaired, double sided t test). D. Representative FLIM images of C.</p

DA induces conformational changes in aSyn in neuronal cell lines.

<p>A. FLIM study showing DA alters aSyn conformation in cell lines of neuronal origin (H4 and MES23.5) but not in HEK cells (*n = 3, 30–40 cells per condition, p<0.01; unpaired, double sided t test). B. Native PAGE showing the presence of certain oligomeric species (arrows) is altered in DA treated H4 cells (representative immunoblot shown, n = 3). C.Triton X-100 fractionation showing DA does not alter the soluble vs. insoluble aSyn fraction in H4 cells (T-total, S-supernatant, P-pellet) (representative immunoblot shown, n = 3).</p