{'fraction_non_alphanumeric': 0.035938966320253035, 'fraction_numerical': 0.022696465190643358, 'mean_word_length': 4.8983189831898315, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca2+ levels to α-synuclein toxicity in Parkinson's disease models

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca2+ fluxes, arguing for an involvement of deregulated Ca2+ homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca2+/Mn2+ ATPase PMR1 (plasma membrane-related Ca2+-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca2+ homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca2+ levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca2+ levels is pivotal for its cytotoxicity and requires PMR1.

a-Synuclein (aSyn) is a small, natively unfolded protein that is abundantly expressed in the central nervous system. It constitutes the major structural component of the intracellular protein inclusions termed Lewy bodies that define a pathological hallmark of Parkinson's disease (PD). 1 Mutations in or duplication and triplication of the gene coding for aSyn all result in familial PD. [2][3][4][5][6] An increasing body of evidence points towards a role for Ca 2 þ ions and Ca 2 þ -dependent processes in the pathology of PD in general 7,8 and aSyn-mediated neuronal death during PD in particular. [9][10][11][12] The underlying mechanisms, however, remain enigmatic. A valuable tool to explore such pending questions are humanized yeast models based on heterologous expression of human aSyn and pathogenic mutants, as they have not only recapitulated several features of PD but have also allowed to identify novel and evolutionaryconserved mediators and processes involved in the cytocidal consequences of aSyn. [13][14][15][16][17] As the regulation of Ca 2 þ homeostasis is highly conserved between yeast and mammals -with the advantage of reduced complexity and redundancy in yeast 18-20 -we heterologously expressed human aSyn in yeast to elucidate its effect on Ca 2 þ homeostasis and cell death. We could identify the Golgi-resident Ca 2 þ /Mn 2 þ ATPase PMR1 (plasma membrane-related Ca 2 þ -ATPase 1) as mediator of aSyn -driven changes in Ca 2 þ homeostasis and cytotoxicity in yeast, nematodes and flies.

Results
Heterologous expression of aSyn in yeast disrupts Ca 2 þ homeostasis. We first quantified the basal cytosolic Ca 2 þ levels in yeast cells equipped with the Ca 2 þ -dependent reporter protein aequorin at standard external [Ca 2 þ ] of B1 mM in the culture medium. In this setting, heterologous expression of aSyn provoked an elevation of the cytosolic Ca 2 þ -concentration, [Ca 2 þ ] cyt , within the first 24 h of expression that subsided after 2 days of culturing (Figure 1a). Simultaneous determination of oxidative stress based on the superoxide-driven conversion of dihydroethidium to fluorescent ethidium (DHE-Eth) demonstrated that the rise in [Ca 2 þ ] cyt triggered by aSyn ( Figure 1a) coincided with an increase in oxidative stress. However, the [Ca 2 þ ] cyt boost developed well before the massive production of reactive oxygen species (ROS), which started after 24 h of aSyn expression (Figure 1b), and subsequent cell death (Figure 1c). This points towards a sequential course of events, where aSyn-directed [Ca 2 þ ] cyt increase occurs upstream of ROS generation and death. Immunoblot analysis revealed that aSyn (driven by a galactose-promoter) was well expressed throughout the experiment, accumulating around 8 h after promoter induction (Figure 1e). The transient, aSyn-induced increase in [Ca 2 þ ] cyt was not related to a different localization of the protein itself, as -in line with ] cyt responses were observed for 70 s. Data was normalized to maximum peak amplitude of control cells. Mean±S.E.M., n ¼ 6. (e) Western blot analysis of aSyn expression in WT cells. Cells were harvested at indicated time points after induction of galactose-driven expression. Blots were probed with antibodies directed against FLAG-epitope to detect FLAG-tagged aSyn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control. (f) Fluorescence microscopic analysis of WT cells expressing GFP-tagged aSyn (aSyn GFP ) or harbouring the corresponding vector control (Ctrl. GFP ) at indicated time points. (g) Determination of basal cytosolic Ca 2 þ levels using aequorin-based luminescence measurement of yeast cells expressing human aSyn or harbouring the empty vector (Ctrl.) after growth for 12 h, 20 h or 24 h on galactose media (promoter induction) supplemented or not with 2 mM ethylene glycol tetraacetic acid (EGTA). Data has been normalized to equally treated vector control cells. Mean ± S.E.M., n ¼ 6; **Po0.01 and *Po0.05. (h) Flow cytometric quantification of oxidative stress by assessing the ROS-driven conversion of dihydroethidium to ethidium (DHE-Eth) upon expression of aSyn for indicated time and supplementation of media with 2 mM EGTA. Mean ± S.E.M., n ¼ 4-8; ***Po0.001 and **Po0.01. (i) Survival determined by clonogenicity of yeast cells expressing aSyn or harbouring the empty vector control for 48 h and supplementation of galactose medium with 2 mM EGTA. Cells were plated on YEPD agar plates. Mean ± S.E.M., n ¼ 12; ***Po0.001 previous studies 15 -green fluorescent protein (GFP)-tagged aSyn was prominently detectable at the plasma membrane within the first 2 days of expression ( Figure 1f).
In addition to the disturbance in basal [Ca 2 þ ] cyt , aSyn altered the cellular response to high extracellular Ca 2 þ pulses. Upon challenge with 150 mM Ca 2 þ , a rapid and transient increase in [Ca 2 þ ] cyt was detectable that was largely amplified upon expression of aSyn (Figure 1d).
Addition of 2 mM ethylene glycol tetraacetic acid (a Ca 2 þ chelator) to the culture medium partly inhibited the increase in basal [Ca 2 þ ] cyt and at the same time ameliorated aSyninduced accumulation of ROS and cell death (Figures 1g-i). Similar results were obtained using another Ca 2 þ chelator, BAPTA-AM (1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester), which is cell-permeable and acts as intracellular Ca 2 þ sponge (Supplementary Figures S1A and B). Thus, inhibition of the rise in basal [Ca 2 þ ] cyt attenuates subsequent ROS accumulation and death, indicating a causal link between disturbances in Ca 2 þ homeostasis and aSyn-inflicted cellular demise.
Oxidative stress and concomitant oxidation of DNA, proteins and lipids are essentially involved in neuronal cell death in various models of PD. [21][22][23] Therefore, we tested whether the thiolic antioxidant N-acetylcysteine (NAC), a precursor of glutathione that has been shown to be protective in several PD-associated neurodegenerative scenarios, [24][25][26][27] could block aSyn-induced generation of ROS and eventual cell death. Although supplementation with NAC did not prevent the first increase in ROS generation detectable within 24 h, it clearly inhibited massive ROS production and subsequent cell death occurring after 2 days of aSyn expression (Figures 2a-c). Basal [Ca 2 þ ] cyt levels were largely unaffected by the addition of NAC (Figure 2d). By contrast, overexpression of cytosolic or mitochondrial superoxide dismutase (Sod1p or Sod2p, respectively) had no effect on aSyn-induced oxidative stress (Supplementary Figures S2A  and B), arguing in favour of a rather broad pattern of cellular oxidative stress (that cannot by antagonized by overexpression of superoxid dismutases) as a cause of aSyn-induced cell death. aSyn cytotoxicity in yeast is facilitated by PMR1. To identify molecular determinants for aSyn toxicity and dysregulation of Ca 2 þ homeostasis, we monitored aSyn-induced consequences in numerous deletion mutants known to influence Ca 2 þ transport or signalling. Yeast codes for various homologues of mammalian Ca 2 þ channels, transporters, sensors and buffers, including the plasma membrane-located voltage-dependent Ca 2 þ channel Cch1p/Mid1p, the secretory pathway and Golgi-resident Ca 2 þ -ATPase Pmr1p, the sarcoendoplasmic reticulum Ca 2 þ -ATPase Cod1p, the vacuolar H þ /Ca 2 þ exchanger Vcx1p, the plasma membrane Ca 2 þ -ATPase Pmc1p, the vacuolar cation channel Yvc1p -a putative homologue of the mammalian transient receptor potential canonical channelsor the general calcium sensor calmodulin as well as calcineurin and calmodulin-dependent kinases. [18][19][20] Using deletion mutants of these calcium-related regulators and automated quantification of ROS production, we evaluated the contribution of these proteins to aSyn cytotoxicity. This approach identified PMR1 as a mediator of aSyn-triggered ROS production (Figures 3a and b). In addition, the aSyndriven development of apoptotic and necrotic markersassessed using AnnexinV/propidium iodide (PI) co-staining, which allows the discrimination between early apoptotic (AnnV þ ), late apoptotic/secondary necrotic (AnnV þ /PI þ ) and necrotic (PI þ ) cells -demonstrated that deletion of PMR1 completely inhibited aSyn-driven increase in apoptotic and necrotic populations (Figures 3c and d). By contrast, the absence of CCH1, MID1, PMC1, VCX1, YVC1, or COD1 did neither alter aSyn-instigated ROS-accumulation nor cell death markers as compared with wild type (WT; Figures 3a-d). Notably, deletion of these genes did not compromise aSyn expression (Figure 3e). Determination of survival using clonogenic survival plating assays showed that the absence of Pmr1p potently inhibited aSyn-induced cell death ( Figure 3f). In addition, we generated a conditional PMR1 mutant by replacement of the promoter region of PMR1 with a tetO promoter that is active in the absence of doxycycline and prevents gene expression upon addition of 10 mg/ml of the chemical. 28 Treatment of cells with doxycycline and thus depletion of Pmr1p largely reduced aSyn-induced ROS production, confirming that Pmr1p is essentially involved in aSyn cytotoxicity (Figures 3g and h).
To test whether aSyn has any effect on the localization and/or expression levels of Pmr1p, we performed fluorescence microscopy as well as immunoblot analyses of yeast cells endogenously expressing a Pmr1p-GFP fusion protein.
No obvious effect on Pmr1p protein levels or Golgi localization was apparent (Figures 4a and b). Using reverse transcription quantitative PCR, we could detect a slight upregulation of PMR1 mRNA (normalized to actin mRNA) after 14 h of aSyn expression ( Figure 4c).
We next analysed whether the rise in basal [Ca 2 þ ] cyt triggered by aSyn is connected to the upregulation of a specific Ca 2 þ influx system that has been shown to be stimulated in scenarios such as the accumulation of misfolded proteins in the endoplasmic reticulum, 29 different defects in vesicular trafficking 30,31 and depletion of Ca 2 þ from the endoplasmic reticulum, for example, upon PMR1 deficiency. 32,33 In all of these cases, a high rate of Ca 2 þ uptake via upregulation/activation of a high-affinity Ca 2 þ influx system that involves Cch1p and Mid1p is triggered. 34 Resultant elevated basal [Ca 2 þ ] cyt levels lead to the activation of Ca 2 þ signalling pathways essential for initiation of compensatory mechanisms and subsequent cell survival. 34 Using quantitative PCR, we could indeed detect that after 14 h of expression, aSyn significantly amplified CCH1 mRNA levels and slightly upregulated MID1 mRNA ( Figure 4d). As shown for PMR1 mRNA levels (Figure 4c), this effect faded after 24 h (Figure 4d). Given these results, we tested whether proteins involved in the calmodulin/calcineurin pathway and thus responsible for cellular Ca 2 þ sensing might alter aSyn-instigated cytotoxicity. Determination of ROS production demonstrated that aSyn toxicity was mostly unaffected by deletion of genes coding for the calcineurin-responsive zinc finger transcription factor Crz1p and the calmodulindependent kinases Cmk1p and Cmk2p, while the absence of Cnb1p, the regulatory subunit of calcineurin (and to some extent the absence of Cna2p, one of two isoforms of the catalytic subunit of calcineurin), did aggravate and expedite aSyn-induced oxidative stress ( Figure 4e). Thus, an intact and functional Ca 2 þ signalling pathway that essentially involves calcineurin might contribute to a compensatory mechanism that partly counteracts and/or delays the toxic consequences of aSyn expression.
Pmr1p mediates aSyn-induced dysregulation of Ca 2 þ homeostasis. Next, we analysed whether the absence of proteins mediating cellular Ca 2 þ transport modified the aSyn-induced raise in cytosolic Ca 2 þ levels. Consistent with a link between aSyn-mediated toxicity and dysregulation of Ca 2 þ -homeostasis, expression of aSyn increased basal [Ca 2 þ ] cyt in all deletion mutants except in Dpmr1 cells ( Figure 5a). In line with previous studies, 35    an important route to detoxify excess manganese, providing Mn 2 þ for protein glycosylation in the Golgi complex. 36 Thus, deletion of PMR1 results in (i) depletion of endoplasmic reticulum Ca 2 þ stores, 33 which in turn causes elevated [Ca 2 þ ] cyt via increased Ca 2 þ uptake, as well as in (ii) hypersensitivity to manganese. 36 Defects in cellular manganese homeostasis as well as exposure to manganese have been associated with PD and PD-like syndroms in humans 37,38 and several model systems. [39][40][41][42][43] Thus, we performed spotting assays on galactose plates containing high levels of manganese, demonstrating that exposure to manganese slightly aggravated aSyn-instigated cytotoxicity in concentrations that did not affect isogenic control cells (2 mM and 4 mM Mn 2 þ ions, respectively; Figure 6a). Upon PMR1 deletion, Mn 2 þ was highly toxic (Figures 6a and b). Furthermore, we generated strains that overexpress Pmr1p alone or in combination with aSyn and subjected these strains to spotting and clonogenic survival assays on galactose plates with and without manganese. Notably, high levels of Pmr1p were per se toxic to yeast cells, and combined overexpression of Pmr1p and aSyn killed 495% of all cells (Figures 6b and c). Complementation analyses in PMR1-deficient cells demonstrated that expression of Pmr1p could (i) restore aSyn cytotoxicity and (ii) supress manganese toxicity (Figures 6b  and c).
To further investigate the contribution of Ca 2 þ versus Mn 2 þ transport activity of Pmr1p to the toxic consequences of aSyn expression, we additionally transfected WT and PMR1deficient cells with two point mutants of Pmr1p that were defective for transport of either Ca 2 þ ions (Pmr1p D53A ) or Mn 2 þ ions (Pmr1p Q783A ). 36,44 Compared with native Pmr1p, the overexpression of these point mutants in the background of WT cells still was less toxic than that of native Pmr1p, and both mutated variants were slightly less effective in enforcing aSyn cytotoxicity as determined in spotting assays and survival plating on galactose plates with and without addition of Mn 2 þ (Figures 7a and b). Conducting the same experiments in PMR1-deleted cells demonstrated that native Pmr1p and Pmr1p Q783A , which displays a selective loss of Mn 2 þ transport, both efficiently reinstated aSyn toxicity, whereas the Pmr1p D53A variant deficient in Ca 2 þ transport partly lost this ability (Figures 7a and b). Native Pmr1p as well as Pmr1p D53A expectedly suppressed manganese toxicity of PMR1-deficient cells, whereas Pmr1p Q783A did not (Figures  7a and b). Expression of the Pmr1p variants and aSyn was confirmed in all strains (Figure 7c). These data indicate that the Ca 2 þ transport activity of Pmr1p -rather than that of Mn 2 þ -contributes to aSyn-induced cell killing.
aSyn neurotoxicity in nematodes and flies requires PMR1. To test and challenge our finding in vivo, we analysed the effects of PMR1 depletion on aSyn neurotoxicity in the nematode Caenorhabditis elegans and in the fruit fly Drosophila melanogaster. Nematodes expressing human aSyn directed by the dopamine transporter (dat-1) promoter were examined for survival of dopaminergic neurons. In WT nematodes, expression of aSyn resulted in the death of B40% of the dopaminergic neurons, while only B20% died  (Figure 8a). Quantification of cytosolic Ca 2 þ levels in aSyn-expressing dopaminergic neurons using the Ca 2 þ -sensitive fluorescent reporter protein GCaMP2.0 45 revealed that aSyn elevated the resting [Ca 2 þ ] cyt in WT nematodes but not in pmr-1(tm1840) mutants (Figure 8b). In flies, the pan-neuronal elav-GAL4-driven expression of human aSyn significantly enhanced organismal death of male and female animals upon treatment with manganese. This effect was largely revised by RNA interference (RNAi)-mediated depletion of the Drosophila homologue of PMR1 (SPoCk) (Figures 8c and  d). The absence of SPoCk did not affect the expression of aSyn ( Figure 8e). As aSyn is known to provoke locomotive deficits and the selective loss of tyrosine hydroxylasepositive dopaminergic neurons in Drosophila PD models, 16,46 we tested for a possible involvement of SPoCk in these neurotoxic consequences. The aSyn-induced decline in negative geotaxis (which drives flies to walk upwards after being tapped to the bottom of a vial) was prevented by depletion of SPoCk (Figure 8f). Furthermore, expression of aSyn caused a significant loss of dopaminergic neurons in defined clusters of the brain, and this effect was absent when SpoCk was depleted (Figures 8g and h). Thus, the toxic consequences of aSyn expression in yeast, nematodes and flies essentially involve the Ca 2 þ ATPase PMR1.

Discussion
Diverse Ca 2 þ signals govern a myriad of vital functions, including mechanisms of fundamental neuronal biology such as synaptic transmission, plasticity, regulated neurite outgrowth and synaptogenesis as well as pivotal generic processes like proliferation, transcription, differentiation and apoptosis. In particular, the mitochondrial cell death pathway is susceptible to elevated calcium concentrations. 47 Here, we establish that aSyn cytotoxicity is governed through sequentially occurring events, where the PMR1-dependent generation of a [Ca 2 þ ] cyt increase precedes a burst of oxidative radicals that ultimately triggers cell death. In fact, the cytocidal effects of aSyn are reduced by treatment of cells with Ca 2 þ chelators or by PMR1 deletion as well as via treatment with the generic antioxidant NAC. Thus, whether aSyn is able to trigger elevated basal cytosolic Ca 2 þ levels appears crucial for the subsequent cellular death. However, the transient [Ca 2 þ ] cyt peaks following high external Ca 2 þ or glucose pulses in aSyn-expressing cells, while remaining a good predictor of toxicity, are not fully stringent, as under specific conditions (e.g., upon deletion of COD1 or CCH1), high Ca 2 þ pulses were able to trigger massive transient [Ca 2 þ ] cyt peaks without aSyn expression ( Figure 5 and Supplementary Figure S3). The prominent role of Ca 2 þ in aSyn-triggered cell death predicts that cellular Ca 2 þ sensing/signalling mechanisms may modulate the detrimental effects of aSyn expression. In fact, disruption of calcineurin signalling results in exacerbated toxicity, suggesting a compensatory mechanism based on the recognition of abnormal Ca 2 þ levels that partly counteracts the toxic consequences of aSyn. Cellular survival depends on tightly controlled Ca 2 þ fluxes between cellular organelles as well as across the plasma membrane. Impaired Ca 2 þ homeostasis and dysfunctional Ca 2 þ signalling are implicated in a broad variety of neurodegenerative diseases besides PD, including Alzheimer's disease, Huntington's disease, Glaucoma, Amyotrophic Lateral Sclerosis, Epilepsy and even the psychiatric disorder Schizophrenia. 48 Upon Ca 2 þ overload, mitochondria readily sequester and accumulate Ca 2 þ , which leads to enhanced production of ROS, and subsequently to dissipation of mitochondrial transmembrane potential, opening of the mitochondrial permeability transition pore and cellular demise. 47 For aSyn-induced cell death in particular, where the lethal role of mitochondria has been clearly established, 14,49-51 the molecular axis of toxicity might therefore converge in this organelle. Our findings establish Pmr1p as a conserved mediator of aSyn cytotoxicity in yeast, nematodes and flies and indicate a toxic role for Ca 2 þ in the pathology of PD. Enhancement of cytosolic calcium levels upon aSyn expression seems crucial for subsequent toxicity, a deadly road that requires PMR1.
Analysis of S. cerevisiae survival, oxidative stress and apoptotic and necrotic changes. To determine survival, oxidative stress, phosphatidylserine externalization and loss of membrane integrity upon expression of aSyn, cells from overnight cultures were inoculated in SCD to OD 600 0.1, grown to midlog phase and shifted to SCG for induction of aSyn expression. Clonogenic survival plating was performed as previously described. 53,54 Briefly, a CASY cell counter (Schärfe System, Reutlingen, Germany) was used to measure the cell counts and 500 cells were either plated on full-media YEPD (1% yeast extract, 2% Bacto peptone, 4% glucose; Difco) agar plates (to repress further galactose-driven expression of aSyn and/or Pmr1p and point mutants) or on selective galactose agar plates (to induce expression of aSyn and/or Pmr1p) supplemented or not with 1 mM, 2 mM or 4 mM MnCl 2 as indicated. Colony-forming units were quantified after 2 days (YEPD plates) or 3 days (galactose plates) of growth using a Scanalyser Colony Counter (LemnaTec, Wuerselen, Germany). To measure the level of cellular oxidative stress, cultures were subjected to DHE staining at indicated time points, followed by quantification using a fluorescence reader or flow cytometry as previously described. 55 Externalization of phosphatidylserine and loss of membrane integrity was quantified after 48 h of aSyn expression using Annexin V/propidium iodide co-staining as previously described. 55 For quantifications using flow cytometry (FACSAria, BD Biosciences, Schwechat, Austria), 30 000 cells were evaluated and analysed with BD FACSDiva software. Same cells were visualized via epifluorescence microscopy on a Zeiss Axioskop microscope (Zeiss, Vienna, Austria). Notably, at least four different clones were tested after plasmid transformation to rule out clonogenic variations. For experiments with the Ca 2 þ chelators ethylene glycol tetraacetic acid and BAPTA-AM (Sigma, Vienna, Austria) and the antioxidant NAC (Sigma), cultures were either grown for 7 h after the shift on SCG for induction of aSyn expression and then supplemented with 2 mM ethylene glycol tetraacetic acid or 380 mM BAPTA-AM or treated with 20 mM and 30 mM NAC directly after the shift. For spotting assays, cells were grown in SCG for 24 h, adjusted to 5 Á 10 6 cells/ml and spotted in fivefold serial dilutions onto glucose (expression repressed) and galactose (expression induced) agar plates supplemented or not with indicated concentrations of Mn 2 þ .
S. cerevisiae cytosolic Ca 2 þ measurement. [Ca 2 þ ] cyt were measured using yeast strains carrying the vector pYX212 encoding the bioluminescent protein aequorin under the control of a TPI promoter. For analysis of resting, basal [Ca 2 þ ] cyt and of the cellular response to high doses of external Ca 2 þ , cells expressing aSyn under a galactose-inducible promoter (pESC-His) and equipped with pYX212-cytAEQ were inoculated in SCD to OD 600 0.1, grown to midlog phase and shifted to SCG for induction of aSyn expression. At indicated time points, an equivalent of 6 Á 10 6 cells was transferred into a 96 well plate and harvested by centrifugation. The pellets were resuspended in 200 ml SCG containing 4 mM coelenterazine and incubated for 1 h in the dark. To remove excess coelenterazine, the cells were washed once with fresh SCG and subsequently incubated for further 30 min. A LUMIstar Galaxy Luminometer (BMG Labtechnologies, Offenburg, Germany) was used to measure basal [Ca 2 þ ] cyt as well as the response of [Ca 2 þ ] cyt to external Ca 2 þ shocks. The basal luminescence was measured per well in 0.5-s intervals for 25 s, whereas for kinetic luminescence measurements the signal was recorded for 70 s. In order to investigate the kinetics of the cellular response to an external Ca 2 þ shock, a pump injected 40 ml of a 0.8 M CaCl 2 solution into each well. During all measurements, the plate was shaken and incubated at 28 1C. The luminescence signal was normalized to the OD 600 of each well and reported in relative luminescence units.
For analysis of glucose-induced transients after glucose starvation, yeast cells constitutively expressing aSyn (using the pGGE181 plasmid) transformed with pYX212-cytAEQ were grown in selective medium with 2% glucose. Cells taken from stationary-phase pre-cultures were used to inoculate a new culture. When cultures reached an OD 600 of ± 1.2, one OD 600 unit of cells was plated on concanavaline A-coated coverslips and incubated at 30 1C for 1 h. Cells were subsequently washed with 0.1 M 2-(N-morpholino) ethanesulphonic acid (MES)/Tris, pH 6.5, which is a nutrient-free buffer, and again incubated for 1 h at 30 1C with 0.1 M MES/ Tris pH 6.5 supplemented with 5 mM wt coelenterazine (Promega, Mannheim, Germany). Excess of coelenterazine was removed by washing the cells three times with 0.1 M MES/Tris pH 6.5, and coverslips were mounted in a thermostated perfusion chamber (30 1C). Cells were initially perfused with 0.1 M MES/Tris pH 6.5, followed by 0.1 M MES/Tris pH 6.5 supplemented with 10 mM CaCl 2 . Cells were then stimulated by addition of 80 mM glucose to induce a transient elevation of cytosolic Ca 2 þ (TECC response) after glucose starvation. 56 At the end of the experiment, cells were lysed in a Ca 2 þ -rich hypotonic medium (10 mM CaCl 2 in H 2 O) containing 0.5% Triton X-100. The recorded aequorin luminescence data were calibrated offline into cytosolic Ca 2 þ values using the following algorithm [Ca 2 þ ] cyt ¼ ((L/L max ) 1/3 þ [118(L/L max ) 1/3 À 1])/ (7 Â 10 6 À [7 Â 10 6 (L/L max ) 1/3) ]), where L is the luminescence intensity at any time point and L max is the integrated luminescence. 57 S. cerevisiae immunoblot analysis. Immunoblot analysis of whole-cell extracts was performed as described. 53 Blots were probed with monoclonal antibodies against FLAG-epitope (Sigma), GFP (Sigma), glyceraldehyd-3-phosphate dehydrogenase (Sigma) and Aequorin (Abcam, Cambridge, UK) and the respective peroxidase-conjugated affinity-purified secondary antibodies (Sigma).
Statistical analysis. A one-way ANOVA followed by a Bonferroni post-hoc test was used to calculate P-values. For survival of Drosophila, a two-way ANOVA with time and strain as independent factors followed by a Bonferroni post-hoc test was used.
C. elegans strains and genetics. We followed standard procedures for C. elegans strain maintenance. 58 Nematode-rearing temperature was kept at 20 1C, unless noted otherwise. The following strains were used in this study: N2: WT Bristol isolate (wt), pmr-1(tm1840), Ex[p dat-1 GCamP2.0], pmr-1(tm1840)X;Ex[p dat- C. elegans neurodegeneration analysis. Seven-day-old animals were used for aSyn-induced neurodegeneration quantification. The four CEP dopaminergic neurons in the worm of the head were scored as described previously. 59 Experiments were repeated four times, and statistical analyses were performed using the GraphPad Prism software package (GraphPad Software, San Diego, USA). Analysis of variance (ANOVA) was used for comparisons of multiple groups of values (in both approaches of neurodegeneration analysis), followed by Bonferroni multiple-group comparison tests.
C. elegans monitoring of cytosolic Ca 2 þ levels. For intracellular Ca 2 þ monitoring experiments, transgenic animals expressing the Ca 2 þ reporter GCaMP2.0 45 in dopaminergic neurons were examined under a Zeiss AxioImager Z2 epifluorescence microscope (Zeiss, Thessaloniki, Greece). The four CEP dopaminergic neurons in the worm of the head were imaged. Only neurons of very initial stages of degeneration (based on morphological features using DIC microscopy) were used for analysis, as the expression of GCaMP2.0 ceases during later stages of neurodegeneration. The emission intensity of GCaMP2.0 was calculated by using the ImageJ software (http://rsb.info.nih.gov/ij/).