Primary neuronal cells express endogenous rαS and neuronal marker MAP2.

<p>A) Immunoblot of cell lysate of primary neuronal cells after 14 days shows immuno-reactivity for MAP2 and αS. B) Cells were fixed and fluorescently labeled; red: phalloidin ^alexa647, cyan: αS ^alexa594, green: anti-MAP2 ^alexa488, blue: DAPI; confocal microscopy; scale bar 10 μm. C) Cells were treated with 100 μM labeled hαS ^alexa488 and unlabeled hαS monomers (ratio 1:3) for six hours and processed as in B; red: phalloidin ^alexa647, green: exogenous hαS ^alexa488, cyan: anti-αS ^alexa594 (endogenous rαS and exogenous hαS), blue: DAPI; confocal microscopy; scale bar 5 μm.</p

hαS interferes with intercellular communication.

<p>Networks of primary cortical rat neurons are electrically stimulated before (day 0) and after administration of 100 μM hαS. A) The first 15 milliseconds after stimulation are dominated by action potentials that were directly triggered by the stimulus pulse (early response; E). These early action potentials are then synaptically propagated through the network and neurons at other electrodes respond up to 300 milliseconds after the stimulation (late response; L). Time progress is visualized by shifting color from blue (day 0) to red (day 5), every line represents the averaged result of 12 stimulation periods (600 stimulations). B) Average development of early and late response after addition of 50 or 100 μM αS, normalized to baseline values. C) Average activity for neural networks treated with 50 μM αS. n = 9 electrodes in three different preparations for each condition.</p

hαS does not aggregate into amyloid fibrils in R12 medium.

<p>A) Fluorescence intensity of the amyloid binding dye ThT as a function of the hαS concentration after incubation in R12 medium for eight days at 37°C (blue) and hαS fibril control sample (red). B) Fluorescence spectra of 1,8-ANS incubated in R12 medium for eight days at 37°C in the presence of 100 μM hαS (red) and without hαS (blue). As a reference the fluorescence spectrum of 1,8-ANS in 10 mM Tris-HCl, pH 7.4 is shown (purple). C) Representative atomic force microscopy (AFM) height image of a 50 μM hαS sample after eight days of incubation in R12 medium indicating that the samples contained small structures. D) In AFM images of the deposited R12 medium with no added hαS similar structures were found as those observed in C). E) Native western blot showing hαS after incubation in 10mM Tris buffer or R12 medium for 0 and 5 days at 37°C.</p

Exogenous hαS induces αS deposit formation in primary cortical cells.

<p>Cultured cortical cells were exposed to 100 μM hαS monomers for one, five and seven days. Cells were fluorescently labeled with actin^{phalloidin647}: red and anti-αS ^alexa594: green; confocal microscopy; A) After hαS exposure, αS deposits were observed in the cells. Note the actin positive staining of deposit (middle arrow), confirming the intracellular location. B-C) αS deposits formed in control cells. D-F) αS deposits formed in cells exposed to exogenous hαS. G-H) Magnification of αS immunolabeled cell cultures at day seven. I) The number of αS deposits per cell; n = 3 for all groups; paired student’s t-test: * indicates P<0.05; scale bar 5 μm (A), 50 μm (B-F) and 10 μm (G-H); αS deposits are indicated by white arrows.</p

Exogenous hαS decreases neuronal activity and synchronicity of neuronal action potential firing pattern.

<p>Rat primary cortical cells were cultured for three weeks and subsequently exposed to either 100 μM hαS or BSA. Neuronal activity was recorded using microelectrode arrays. A) An example of baseline neuronal activity. On the vertical axis, all 60 electrodes are indicated and each tick in the corresponding row represents an action potential. During baseline recordings, started 30 minutes before hαS administration, on average around 50 of the 60 electrodes were active and all activity patterns included network bursts (synchronous firing at all or most electrodes, visualized as ‘vertical columns’). B) Recording of the same network four days post hαS administration. C-D) The average number of active electrodes and Array wide firing rate (AWFR; the summed activity of all electrodes in one hour time bins, divided by the bin length) were registered for eight days from eight αS treated cultures (blue) and three BSA (red) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193763#pone.0193763.ref095" target="_blank">95</a>] treated cultures. E) Neuronal synchronicity slightly increased for two days and then decreased, as indicated by the development of the burstiness index. BSA treated cultures initially showed a similar pattern but here the burstiness recovered after four days F) The action potential shapes and the activity (dots; inset) of a representative neuron at different timepoints (color shift; day 0 = blue, day 7 = red) after addition of αS. G) Examples of neurons with unchanged and changed wave shapes. Top row, overlay of action potentials from every hour color coded as in F. Middle row, correlation coefficients at every hour. Bottom row, action potential amplitudes at every hour. Note the drop in correlation coefficient and amplitude in the last hours in right versus left panel. H) Cumulative histogram showing the fraction of all neurons, with reproducible baseline action potential shape (n = 115), that became inactive in time. Blue indicates all neurons, red shows the fraction of neurons for which the action potential shape changed before becoming inactive. C-E: Error bars represent SEM; n = 8 independent experiments.</p