α-Synuclein (αS) aggregation and deposition are associated with the onset and progression of Parkinson’s disease (PD) and other synucleinopathies. Here, we explored the role of the cellular prion protein (PrPC) in the cytotoxic cascade induced by αS prefibrillar oligomers and fibrils in human iPSC-derived dopaminergic neurons, primary rat cortical neurons and human neuroblastoma cells. Using high resolution confocal microscopy and both siRNA-mediated PrPC silencing and blockade with an anti-PrPC antibody, we showed that PrPC mediates the rapid recruitment of αS aggregates at the plasma membrane, resulting in massive Ca2+ influx and membrane permeabilisation, with only a minor contribution to the release of toxic oligomers from aS fibrils and the downstream events leading to cell death. aS aggregates neurotoxicity was largely associated with the presence of extracellular Ca2+, that directly promoted the cell membrane recruitment and penetration of αS oligomers inside neurons, allowing them to cause neuronal death after prolonged times of exposure with the cells.
Research Article
Cellular prion protein and calcium ions trigger the neurotoxicity of α-synuclein aggregates
https://doi.org/10.21203/rs.3.rs-5230539/v1
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synucleinopathies
protein aggregation
amyloid
toxic oligomers
Lewy bodies
prion-like
PD
protein misfolding
neurodegeneration
fibrils
The aberrant misfolding, aggregation and accumulation of aggregates of αS protein in intracellular assemblies called Lewy bodies is the major neuropathological hallmark of a range of neurodegenerative diseases collectively referred to as α-synucleinopathies (McCann et al., 2014; Goedert et al., 2017). Fibrillar aggregates of αS have been identified as the major constituent of such inclusions, but increasing evidence indicates that small oligomeric assemblies, formed early during the aggregation process or released by mature fibrils, are the most neurotoxic species, playing a crucial role in the pathogenesis (Winner et al., 2011; Cremades et al., 2012; Fusco et al., 2017; Luth et al., 2019; Yang et al., 2020; Cascella et al., 2021; Bigi et al., 2021; Bigi et al., 2022; Emin et al., 2022).
A well-defined type of αS prefibrillar oligomers referred to as type B* oligomers (OB*), possessing a rudimental b-sheet structure, featuring a large exposure of hydrophobic groups and a weak binding of thioflavin T, has been previously described for its ability to bind to neuronal membranes through the unstructured αS N-terminal domain, and later insert its b-sheet core in the interior of the bilayer (Fusco et al., 2017; Cascella et al., 2019; Cascella et al., 2021). This OB*-membrane interaction results into the entry of Ca2+ ions from the cell medium to the cytosol, as a consequence of its destabilization, and also in membrane permeabilization to large organic molecules such as calcein and OB* themselves (Fusco et al., 2017; Cascella et al., 2021). OB* can form transiently during aS fibril formation (Cremades et al., 2012), but also be stabilized by specific protocols (Chen et al., 2015; Camino et al., 2020). OB* can further aggregate to form short and long fibrils (SF and LF, respectively), whose neurotoxicity was recently correlated to their ability to bind to the neuronal membranes and release small oligomeric assemblies, showing the same features as OB* (Cascella et al., 2021; Cascella et al., 2022; Bigi et al., 2022). Moreover, it was recently found that SF are able to induce PD-like pathology by a spreading process when injected in the brain of healthy mice (Froula et al., 2019). Importantly, such spreading from neuron to neuron, resulting from the extracellular release and internalization of the pathogenic species, can be described as a prion-like propagation, with harmful aggregates acting as templates for the conversion of native αS into neurotoxic conformers (Angot et al., 2010; Prusiner et al., 2015; Candelise et al., 2019; Cascella et al., 2022; Bigi et al., 2022; Bigi et al., 2023a).
The cellular uptake of these pathogenic species has been described to occur through an array of mechanisms (Brás et al., 2021; Cascella et al., 2022). A growing body of evidence suggests that extracellular αS aggregates can act as ligands for specific cell surface receptors, that could in turn mediate their cytotoxicity (Kim et al., 2013; Ferreira et al., 2017; Brás et al., 2021). Among them, several studies were focused on the investigation of the putative role of PrPC, a widely described receptor for amyloid-β (Aβ) oligomers and tau oligomers and fibrils associated with Alzheimer’s disease (AD), thus representing a relevant conformation-specific sensor for disease-associated proteins (Wang et al., 2008; Lauren et al., 2009; Resenberger et al., 2011; Ganzinger et al., 2014; Scott-McKean et al., 2016; Cascella and Cecchi, 2021). PrPC has been reported to act as a receptor for αS fibrils, facilitating their uptake through an endocytic pathway and facilitating the cell-to-cell spreading of such harmful conformers (Aulić et al., 2017; Urrea et al., 2018). Other works revealed a role for PrPC in mediating the interaction and cellular uptake of αS oligomers in neuronal cells and their resulting neurotoxicity (Ferreira et al., 2017; Corbett et al., 2020). In particular, in hippocampal neurons from brain slices, αS oligomers form a complex with a specific N-terminal portion of PrPC containing residues 93-109, leading to the metabotropic glutamate receptors 5 (mGluR5)-mediated phosphorylation of the Fyn kinase, in turn responsible for the activation of the glutamatergic N-methyl-D-aspartate receptors (NMDARs) composed of the GluN2B subunit (Ferreira et al., 2017). Such activation leads to a massive Ca2+ dysregulation and synaptic dysfunction culminating in long term potentiation (LTP) impairment (Ferreira et al., 2017). Consistently, the interaction between αS oligomers and the fragment containing residues 95−111 of PrPC in micron-sized condensates was confirmed in vitro (Rösener et al., 2020).
Intriguingly, contrasting results emerged from a different work in which surface plasmon resonance reported a lack of interaction between PrPC and αS oligomers (La Vitola et al., 2019). In the same study, wild-type (Prnp+/+) and knockout (Prnp0/0) PrPC mice showed no significant differences in terms of memory impairment and neuroinflammation, suggesting a PrPC-independent mechanism of αS neurotoxicity (La Vitola et al., 2019). Thus, contradictory results emerged from the literature, and the contribution of PrPC in the pathogenesis of synucleinopathies remains to be further validated.
Here, we investigated the role of PrPC as a receptor on the neuronal membrane for highly stable and well characterized αS aggregates, and as a mediator of their downstream neurotoxic effects. We have applied confocal and stimulated emission depletion (STED) microscopy and an array of probes of cellular toxicity to reveal that αS oligomers and fibrils colocalize with PrPC, inducing a prompt and specific Ca2+ influx, followed by a later PrPC-independent Ca2+ permeabilization of the plasma membrane, leading to mitochondrial dysfunction. Consistently, we also show that PrPC modulates the release of oligomeric species from αS fibrils, responsible for later detrimental effects on neuronal cells. We finally demonstrate that the ability of αS oligomers and fibrils to interact with and permeabilize the plasma membrane, culminating in a significant impairment of neuronal viability after prolonged incubation times with the cells, were directly triggered by extracellular Ca2+ ions.
αS prefibrillar oligomers and short fibrils bind to the plasma membrane
We have recently demonstrated the different ability of OB* and SF, added to the extracellular medium, to aberrantly interact with neuronal cells and to affect their viability, with OB* being the most rapid in inducing neurotoxicity (Cascella et al. 2021; Bigi et al., 2021). Here, taking advantage of super-resolution stimulated emission depletion (STED) microscopy, we shed light into the interaction and degree of localization of OB* and SF at the surface of the neuronal membrane following 60 min of incubation with primary rat cortical neurons (Fig. 1A and Fig. S1A). The degree of colocalization between OB* (labelled with the mouse monoclonal 211 anti-aS Abs, green channel) and the plasma membrane (labelled with tetramethylrhodamine-conjugated wheat germ agglutinin, red channel), quantified as Pearson's correlation coefficient, appeared to be 0.49±0.01 whereas that of SF was significantly lower (0.21±0.02). The Manders' overlap coefficients, indicating the pixel fraction of the red channel overlapping with the green one (reported as M1) and vice versa (M2) were estimated to be 0.29±0.02 and 0.76±0.02, respectively, for OB*, and 0.28±0.01 and 0.35±0.01 for SF (Fig. 1A). This indicates that the large majority (76%) of OB* are recruited at the plasma membrane, whereas this value goes down to 35% for SF.
Substantially similar results were obtained in SH-SY5Y neuroblastoma cells (Fig. S1B), a widely used cellular model that recapitulates many features of human dopaminergic neurons, which are the most affected neurons in PD patients. In this case, the Pearson's correlation coefficients appeared to be 0.49±0.02 and 0.26±0.02 for OB* and SF, respectively, whereas the M1 and M2 of the Menders’ overlap coefficients were 0.11±0.02 and 0.97±0.01 for OB* and 0.10±0.01 and 0.77 ±0.04, respectively, for SF (Fig. S1B).
PrPC recruits αS prefibrillar oligomers and short fibrils at the membrane
We thus investigated whether the specific sites of early recruitment of αS aggregates at the neuronal membrane were represented by PrPC. This protein is present in many neuronal cell lines, including SH-SY5Y cells, that express appreciable levels of PrPC (Rezvani et al., 2020; Thom et al., 2022). The degree of co-localization between PrPC and αS conformers was evaluated by immunostaining analysis with mouse monoclonal anti-PrPC antibody(Ab, red channel) and fluorescently 488-labelled αS aggregates (0.3 μM, monomer equivalents, green channel) following a very short time of incubation (10 min) with SH-SY5Y cells (Fig. 1B). The value of the Pearson’s correlation coefficient for PrPC and OB* appeared to be significantly higher (0.86±0.01) than that observed for PrPC and SF (0.30±0.02) (Fig. 1B). M1 and M2 appeared to be 0.42±0.06 and 0.97±0.02 for OB*, respectively, and 0.04±0.01 and 0.86±0.03 for SF, respectively (Fig. 1B). These data indicate that the totality of αS aggregates (97% of OB* and 86% of SF) localize in the proximity of the 42% and 4%, respectively, of the exposed PrPC.
To collect stronger evidence that aS oligomer recruitment is mediated by PrPC at the plasma membrane, we used RNA interference. SH-SY5Y cells were transfected with a siRNA-negative control or with a siRNA against PrPC for 72 h. A significant reduction (~ 40%) of the exposed PrPC was observed in cells silenced with the PrPC-specific siRNA relative to those treated with the control siRNA (Fig. 1C). The binding of αS OB* and SF to the membrane after a 10 min incubation at 0.3 μM (monomer equivalents), estimated through the Pearson’s correlation coefficient, decreased significantly in both cases following PrPC silencing relative to the treatment with the control siRNA (Fig. 1D).
To corroborate our evidence with a different technique, we performed a dose-response analysis in which SH-SY5Y neuroblastoma cells were pre-treated with increasing concentrations of an anti-PrPC Ab raised against the extracellular N-terminal domain of PrPC with the aim to conceal possible aS binding sites. Cells were then exposed for 10 min to 0.3 μM of OB* and SF and a marked reduction of both OB* and SF bound to the cell membrane was observed in the presence of the anti-PrPC Ab, especially at 1:1 molar ratio (Fig. S2A,B). Accordingly, when membrane proteins exposed at the outer leaflet of the bilayer in SH-SY5Y cells were removed through a mild protease pre-treatment with trypsin, we observed a significant reduction of the green fluorescent signal arising from both OB* and SF through immunostaining (Fig. S3).
The binding of OB* to PrPC was further assessed in more relevant cellular models such as human iPSC-derived dopaminergic neurons and primary rat cortical neurons. Mature dopaminergic neurons expressing the dopaminergic neuron marker tyrosine hydroxylase (TH), and microtubule associated protein 2 (MAP-2), were obtained from human iPSC-derived neuron progenitors after 14-18 days of culture in neuronal basal medium (Fig. 2A). Confocal microscope images showed a remarkable signal from fluorescent OB* (green channel) on the plasma membrane (red channel) of both human dopaminergic and primary rat cortical neurons following a 10 min incubation at 0.3 µM (Fig. 2B), indicating the presence of a large amount of aS oligomers bound to the neuronal surface. This was strongly decreased by the neuron pre-treatment with anti-PrPC Ab (OB*:anti-PrPC Ab molar ratio of 1:0.5) by 47% in iPSC-derived dopaminergic neurons and by 39% in primary cortical neurons (Fig. 2B).
Taken together, these data indicate that PrPC plays a crucial role in the early recruitment of αS prefibrillar oligomers and SF to the neuronal membrane.
PrPC modulates the release of αS oligomers from short fibrils
We have recently demonstrated that SF, either in suspension in vitro or upon their interaction with neuronal membranes, are able to release toxic oligomeric species structurally similar to OB*, responsible for detrimental effects on neuronal cells and detectable inside the cells with the anti-oligomer polyclonal A11 Ab (Cascella et al., 2021; Bigi et al., 2021; Bigi et al., 2022). This Ab was previously shown to recognize toxic oligomers from various proteins, including αS, but not their monomeric or fibrillar conformations(Kayed et al., 2003; Cascella et al., 2021). Considering this evidence, we evaluated here the possible role of PrPC in the internalization of aS oligomeric species released by SF upon prolonged incubation with cultured cells, as compared to those directly added to the culture medium (OB*). iPSC-derived dopaminergic neurons exposed to 0.3 μM SF for 6 h showed a significant increase in the intracellular A11-positive signal with respect to untreated cells (Fig. 2C), indicating a penetration inside the cells of aS oligomers released over time by SF, as previously shown (Cascella et al. 2021). As a positive control, OB* were directly added to the culture medium, validating the internalization of aS oligomers (Fig. 2C). When the anti-PrPC Ab was pre-added for 30 min to the cell culture medium at an equimolar concentration with respect to SF and OB* (0.3 µM), a significant reduction (by 24±11% and 37±15%, respectively) was observed in the internalization of oligomeric conformers into the cytosol of dopaminergic neurons (Fig. 2A,B). This observation suggests that the extent to which oligomers are released by fibrillar species and taken up by neurons is correlated with the amount of aS recruited by PrPC.
PrPC mediates the early Ca2+ dyshomeostasis induced by αS oligomers and short fibrils
A growing body of evidence indicates that Ca2+ dyshomeostasis is a crucial early event in the complex neuropathological cascade evoked by aS species added to the extracellular cell medium (Danzer et al., 2007; Angelova et al., 2016; Cascella et al., 2021; Ito et al., 2023). Thus, we firstly monitored with confocal microscopy and the Fluo-4 probe the kinetic of Ca2+ entry in primary rat cortical neurons treated with OB* and SF at 0.3 µM (monomer equivalents) for different lengths of time (0, 5, 15, 60 and 180 min). aS OB* determined a ca. 7-fold increase of intracellular Ca2+ levels in neurons, indicating a dramatic disruption of neuronal Ca2+ homeostasis (Fig. 3A). aS SF also induced Ca2+ dysregulation, but the increase of intracellular Ca2+ levels was lower (ca. 5/6-fold) and delayed (Fig. 3A). The normalized Ca2+-derived fluorescence was plotted as a function of time, obtaining an exponential trend for OB*, with a half-maximal increase of Ca2+-derived fluorescence occurring after 7.2 min, whereas SF determined a sigmoidal trend with an extremely rapid lag phase and the half-maximal increase of the green fluorescent signal occurring after 10 min of treatment (Fig. 3A).
The silencing of PrPC in SH-SY5Y cells with its specific siRNA delayed the increase of intracellular Ca2+ levels induced by 0.3 µM OB* (Fig. 3B). Exponential and sigmoidal trends were evident for OB* without and with PrPC silencing, respectively, with half-maximum increases occurring at 14.5 min and 33.7 min, respectively. However, the Ca2+ influx was reduced only at the early times of Ca2+ influx (up to 30 min), without a consistent recovery for longer times of exposure (60–180 min). To confirm these observations with a different technique, we performed a dose-response analysis in which SH-SY5Y cells were pre-treated for 30 min with increasing concentrations of the anti-PrPC Ab and then exposed for 15 min to 0.3 μM OB* and SF (Fig. S5). In such conditions, we revealed a significant reduction of the aberrant Ca2+ entry evoked by both OB* and SF at 1:0.5 and 1:1 molar ratios (Fig. 3C,D). The effect of PrPC blockade by its specific Ab was also monitored over time (Fig. S4A,B). The presence of an OB*:anti-PrPC Ab molar ratio of 1:0.5 reduced only the very early phases of Ca2+ influx (up to 10 min), without inhibiting its entry for longer times of exposure (15–180 min), and delaying the half-maximum increase by 29 min, from 11 min to 40 min (Fig. S4B). When the OB*:anti-PrPC Ab molar ratio was 1:1, the Ca2+ dyshomeostasis evoked by OB* was significantly reduced both at early and prolonged incubation times, but a slight and progressive increase over time was still evident (Fig. S6B). Additionally, the removal of membrane-exposed proteins by mild trypsin treatment also delayed the cellular Ca2+ influx evoked by both OB* and SF by 27 and 9 minutes, respectively, without changing again the outcome at late time points (Fig. S5A,B).
Overall, these data suggest that PrPC plays a crucial role in the early recruitment of αS prefibrillar oligomers and, consequently, partially mediates the early phases of neuronal Ca2+ dyshomeostasis, with a minor effect in the later ones.
PrPC inhibition reduces in part the cellular uptake of aS oligomers and membrane permeabilization to calcein
A trypsin-mediated removal of the exposed membrane proteins was also found to reduce the interaction and internalization of OB* and, to a minor extent, SF with neuronal cells after the removal of the exposed membrane proteins (Fig. S6A-C). Accordingly, in such conditions we also observed a minor perturbation of membrane permeability as assessed in SH-SY5Y cells loaded with the fluorescent probe calcein acetoxymethyl (AM) ester (Papadopoulos et al., 1994). Indeed, a significant release of this fluorescent probe in the culture medium, indicating membrane disruption, was observed after 60 min of treatment with OB* and, to a lesser extent, SF (Fig. S6D,E), in good agreement with previously reported data (Cascella et al., 2021). The removal of membrane-associated proteins by trypsin before cell treatment with OB* prevented the leakage of calcein; by contrast, calcein release induced by SF was very marginally affected by such pre-treatment (Fig. S6D,E).
We finally tested whether PrPC blockade with its specific Ab could reduce the impairment of mitochondrial functionality evoked by OB* and SF in SH-SY5Y cells after prolonged times of exposure; to this aim, cells were pre-treated for 30 min with the anti-PrPC Ab at αS:Ab molar ratios of 1:0.5 and 1:1, and then treated with OB* and SF at 0.3 μM for 24 h (Fig. 3E). In the presence of OB* and SF, the ability of neuroblastoma cells to reduce the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was significantly decreased (by 25% and 14%, respectively, as compared to untreated cells, taken as 100%), in good agreement with previously reported evidence (Cascella et al., 2021). The presence of anti-PrPC Ab did not induce any significant reduction of αS-induced cytotoxicity (Fig. 3E).
Taken together, therefore, our data indicate that the removal or blockade of the exposed membrane proteins, and in particular PrPC, affects the early membrane perturbation induced by harmful αS conformers, resulting in Ca2+ influx, aS oligomer uptake and permeability to large organic molecules such as calcein, without reducing their neurotoxic capability after prolonged times of exposure.
Extracellular Ca2+ promotes the internalization of αS aggregates in neurons
Calcium ions can bind to the residues at the C-terminus of aS monomer neutralizing their negative charge and increasing aS interaction with synaptic vesicles and phospholipid membranes (Lautenschläger et al., 2018). Thus, in a physiological context, aS acts as a calcium-dependent modulator of vesicle homeostasis at the pre-synaptic terminal (Lautenschläger et al., 2018; Byrd et al., 2023). Here we investigated whether extracellular Ca2+ ions modulate the binding of aS aggregates to the neuronal membrane of primary rat cortical neurons and their subsequent internalization into the cytosol. To this purpose, we used STED microscopy and the mouse monoclonal 211 anti-aS Ab on neurons exposed for 60 min to culture medium containing OB* at 0.3 µM in the presence of physiological Ca2+ levels (2 mM), reproducing the experimental setting adopted in our previous works (Fusco et al. 2017; Cascella et al. 2021), and observing the binding of such small and globular aS aggregates (green fluorescent punctae) both on the plasma membrane and inside the cytosol (Fig. 4A), in good agreement with foregoing findings. By contrast, a lower Ca2+ concentration (0.5 mM) in the culture medium containing OB* significantly reduced the amount of oligomers bound to the neuronal membrane (by 38±17%) and those internalized by the cells (by 46±24%; Fig. 4B). Consistently, neurons treated for 60 min with SF at 0.3 µM in the presence of physiological Ca2+ levels(2 mM) showed SF bound to the membrane and a number of smaller and globular αS species into the cytosol (Fig. 4A). A lower Ca2+ concentration (0.5 mM) in the culture medium containing SF evoked a significant reduction (by 27±10%) of the amount of fibrillar conformers interacting with the neuronal membranes and of the aggregates internalized by neurons (56±31%), as depicted in Fig. 4B. A consistent trend was also observed in SH-SY5Y cells exposed to OB* under different concentrations of Ca2+ ions and in the presence of the BAPTA Ca2+ chelator (Fig. 4C,D). In particular, the 211 Ab recognized OB* interacting with the surface of the plasma membrane and internalized by the cells in the presence of physiological calcium levels (2 mM), resulting in an increase of fluorescence by 889±79% as compared to untreated cells, taken as 100±12%. The progressive reduction of the Ca2+ levels in the extracellular medium gave rise to an equally progressive reduction of OB*-derived fluorescence, that was particularly evident when Ca2+ was totally removed from the extracellular medium, also taking advantage of the Ca2+ chelator BAPTA: in such condition, a reduction of the αS-derived fluorescence by 500±190% was observed (Fig. 4C,D). A very significant reduction was also revealed when the exposed PrPC was concealed by adding its specific Ab to the extracellular medium, both with 2 mM Ca2+ (by 420±110%) and in its absence (by 550±130%; Fig. 4C,D).
To confirm this evidence, we evaluated the perturbation of the plasma membrane integrity evoked αS species occurring in a medium with low calcium levels. A significant leakage of intracellular calcein - low green fluorescence signal - was shown in primary rat cortical neurons upon a 60 min addition to the complete culture medium of 0.3 µM aS OB* and SF (Fig. 5A,B). This condition reproduced again our previous experimental setting, in which aS species were added to a medium containing physiological Ca2+ levels (Fusco et al., 2017; Cascella et al., 2019; Cascella et al., 2021). By contrast, a lower Ca2+ concentration in the culture medium (0.5 mM instead of 2 mM) significantly reduced the leakage of calcein observed after 60 min of treatment with aS OB* and SF (by 16% and 20%, respectively; Fig. 5A,B), indicating a lower propensity of aS aggregates to permeabilize neuronal membrane in such conditions. Similar results were obtained with SH-SY5Y cells, in which we progressively reduced the Ca2+ levels in the extracellular medium (Fig. 5A,B). After 60 min of incubation of aS species with the cells, the most pronounced effect was obtained when the reduction of calcium concentration was accompanied by the BAPTA Ca2+ chelator or by PrPC blockade using its specific Ab. Consistently, cell treatment with OB* at 0.3 µM in the presence of physiological Ca2+ levels evoked a significant reduction of their mitochondrial activity (by 23±4%), as revealed by the MTT reduction inhibition assay, that was not observed when the experiment was repeated in extracellular medium without Ca2+ (Fig. 5C).
Overall, these data suggest that extracellular Ca2+ ions promote the interaction of aS aggregates with neuronal cell membranes, thereby influencing their ability to induce membrane alteration and the neurotoxic cascade finally culminating in cell death.
The relationship between PrPC and misfolded neurotoxic aggregates has not yet been fully understood. Several independent studies demonstrated that PrPC acts as a cell surface receptor for a range of pathologically relevant oligomeric and fibrillar assemblies, including those of Aβ, tau, αS and TDP-43, modulating both their cell-to-cell transmission and inherent neurotoxicity (Laurén et al., 2009; Ganzinger et al., 2014; Aulić et al., 2017; Urrea et al., 2018; Corbett et al., 2020; Rezvani Boroujeni et al., 2020; Brás et al., 2021; Scialò et al., 2021; Liu et al., 2022), (Resenberger et al., 2011; Legname and Scialò, 2020; Scialò et al., 2021). Other reports, however, have proposed that misfolded protein oligomers either interact with PrPC without representing an essential component for oligomer-induced toxicity (Balducci et al., 2010; Calella et al., 2010; Kessels et al., 2010) or do not interact altogether (la Vitola et al., 2019). At present, therefore, the existence of an oligomer-PrPC interaction and its role in oligomer-induced toxicity is an unresolved issue.
In this manuscript we have investigated the role of PrPC in the neurotoxic cascade evoked by well-defined and characterized αS oligomeric and fibrillar conformers, both in the earlier and in the later phases of the neurotoxic cascade. We firstly demonstrated the ability of PrPC to recruit such aberrant species at the neuronal membrane, as revealed by the high degree of colocalization between PrPC and OB* and, to a minor extent, PrPC and SF. Consistently, we observed a significant decrease in the binding of OB* and SF to the neuronal membrane both after silencing PrPC and after masking the membrane protein using an Ab raised against the N-terminal domain of mammalian PrPC. This observation is in good agreement with previously reported evidence obtained with αS conformers produced with different aggregation protocols from those adopted here (Aulić et al., 2017; Urrea et al., 2018; Thom et al., 2022), but is not in accordance with other findings indicating the inability of PrPC to recuit αS species (La Vitola et al., 2019). In agreement with the first view, αS oligomers were described to interact with human PrPC with high affinity, with amino acid residues 95−111 driving their condensation (Rösener et al., 2020), as well as for their ability to induce neurotoxicity (Ferreira et al., 2017).
In addition to showing an important role of PrPC in recruiting aS assemblies on the cell membrane, we also demonstrate here that PrPC mediates the initial perturbations of fundamental cellular conditions, such as Ca2+ dyshomeostasis induced by αS oligomeric and fibrillar assemblies, without contributing to the later events leading to cell death. Indeed, PrPC blockade significantly reduces the early Ca2+ entry evoked by αS oligomers and partially their internalization as well as membrane disruption, but not the final levels of intracellular Ca2+ at late time points or other late neurotoxic effects, such as mitochondrial impairment. This suggests a generic and PrPC-independent destabilization of the neuronal membrane at prolonged times of exposure. Thus, the direct or indirect interaction between αS assemblies and PrPC, that is essential in the very early phases of the neurotoxic cascade of αS species with the cells, is not mandatory to mediate their late and sustained detrimental effects.
Moreover, the scenario emerging from our findings is reasonably in line with an enormous body of evidence indicating that the interaction of αS aggregates with the lipid component of biological membranes strongly promotes neuronal toxicity (Grey et al., 2011; Fusco et al., 2017; Iyer et al., 2019; Runwal et al., 2021; Ito et al., 2023; Mansueto et al., 2023). This could explain the capability of the analysed αS species to evoke a delayed neurotoxic pathway even when PrPC is blocked or removed from the plasma membrane. We also show that the blockade of PrPC with its specific Ab is able to reduce, at least in part, the internalization of A11-positive toxic oligomers directly added to the culture medium of dopaminergic neurons (OB*) and, to a minor extent, released by SF (Cascella et al., 2021; Bigi et al., 2021; Bigi et al., 2022; Bigi et al., 2023), suggesting that the release of αS oligomers from mature fibrils is partially modulated by PrPC. Collectively, our evidence indicates that the PrPC receptor-mediated mechanism can explain only in part the pathogenic cascade evoked by αS OB* and SF. This suggests that their sustained neurotoxic effects should be also associated to a generic mechanism of pore formation and membrane leakage with the loss of its permeability, in turn responsible for an aberrant influx of calcium ions from the extracellular space. Consistently, given the prominent role of calcium dyshomeostasis in the neurotoxic cascade evoked by αS aggregates (Danzer et al., 2007; Benilova et al., 2016; Cascella et al., 2021), we extended our investigation on the contribution of the extracellular Ca2+ pool in the neurotoxicity of OB* and SF, even in the presence of PrPC blockade, in order to dissect the role of both Ca2+ ions and PrPC in the pathogenic cascade. In this regard, it has been previously shown that Ca2+ ions can bind to the residues at the C terminus of αS monomer neutralizing their negative charge, thus increasing αS interaction with synaptic vesicles and phospholipid membranes (Lautenschläger et al., 2018), and a similar behaviour should be hypothesized also for αS misfolded aggregates. Consistently, taking advantage of the high-resolution power of STED microscopy we show here that the extracellular Ca2+ supports the interaction of αS aggregates with the neuronal membrane and the consequent permeabilization of the lipid bilayer, followed by their internalization into the cytosol. This in turn results in a further increase of Ca2+ dyshomeostasis contributing to downstream neuronal dysfunction, finally culminating in cell death, thus trapping neuronal cells in a vicious cycle that exacerbates αS aggregates neurotoxicity. This body of evidence shed light in more detail into the previously characterized molecular mechanisms of αS neurotoxicity (Fusco et al., 2017; Cascella et al., 2021).
Taken together, our findings support a main role for PrPC in mediating the recruitment of aS oligomers and short fibrils on the neuronal membrane and the early phases of αS-induced neurotoxicity, but not the later ones, suggesting the involvement of other membrane elements, such as lipid components, in the cytotoxic cascade evoked by αS species. We also demonstrate that the interaction of harmful aS assemblies with the neuronal membrane and the subsequent loss of its physiological permeability in the presence of functional PrPC are directly favored by extracellular Ca2+, that modulates both the aggregates interaction with neuronal membranes and their downstream effects. In conclusion, our evidence strongly suggests that the PrPC receptor-mediated mechanism contributes significantly, but is not strictly necessary, to trigger irreversible neuronal dysfunction, that should be rather attributed to a collection of mechanisms, all co-existing in the complex pathological cascade leading to synucleinopathies.
Purification of aS monomers and formation of aS OB* and SF
The recombinant human αS was over-expressed in E. coli BL21 cells (Agilent, UK) and purified as a monomeric fraction as described previously (Chenet al., 2015). OB* were prepared as described previously (Chen et al., 2015; Fusco et al., 2017). SF were prepared by incubating monomeric αS at 70 μM (1 mg/ml) in PBS buffer pH 7.4 (0.1 M ionic strength) containing 0.01% NaN3 at 37 °C, under constant agitation for 4-6 day as described previously (Cascella et al., 2021; Bigi et al., 2021). The concentration of αS aggregates was estimated by measuring the absorbance at 275 nm using e275 = 5600 M-1 cm-1 after disaggregating an aliquot by the addition of guanidinium chloride to a final concentration of 4 M.
Cell culture
Primary rat cortical neurons were purchased from Thermo Fisher Scientific, plated and maintained in neuronal basal plus medium (Gibco, Thermo Fisher Scientific) supplemented with GlutaMAX (Gibco, Thermo Fisher Scientific) at the concentration of 0.5 mM and 2% (v/v) and a serum-free complement, B-27 (Gibco, Thermo Fisher Scientific) in a 5.0% CO2 humidified atmosphere at 37 °C. The experiments were performed 12-16 days after plating, as previously reported (Bigi et al., 2024).
Authenticated human neuroblastoma SH-SY5Y cells were purchased from A.T.C.C. and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), F-12 Ham with 25 mM HEPES and NaHCO3 (1:1) supplemented with 10% fetal bovine serum (FBS), 1.0 mM glutamine and 1.0% penicillin and streptomycin solution. Cells were maintained in a 5.0% CO2 humidified atmosphere at 37° C and grown until 80% confluence for a maximum of 20 passages and routinely tested negative from mycoplasma contamination (Capitini et al., 2023).
Human iPSC-derived dopaminergic progenitors were purchased from Axol Bioscience. The company obtains human cell resources from cell repositories, who guarantee that all human cell collections are performed at certified facilities under the highest ethical standards and protocols, in compliance with the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Discrete legal consent form was obtained and the donors’ or clinics’ rights to hold research uses, for any purpose, or further commercialization use were waved. Human iPSC-derived dopaminergic neuron progenitors were plated on 12-well plates containing glass coverslips coated with poly-D-lysine plus surebond-XF solution, according to manufacturer’s instructions. Then the maturation to dopaminergic neurons was achieved starting from day 1, by growing the cells in differentiation medium at 37 °C with 5% CO2, as previously reported (Cascella et al., 2021). On day 5 post-plating, the differentiation medium was replaced with the maintenance medium, which was changed every two days. The resulting iPSC-derived dopaminergic neurons were characterized and analyzed between day 14 and day 18 of culturing, as described in the Supplementary Information.
Immunocytochemical analysis of differentiated iPSC-derived dopaminergic neurons
iPSC-derived dopaminergic neurons were characterized between day 14 and day 18 of culturing. The cells were fixed in 4% (v/v) paraformaldehyde at room temperature and blocked with fetal bovine serum in 0.1 % Triton X-100 for 30 min. Coverslips were incubated with 1:300 diluted rabbit anti-MAP-2 Abs (ab32454, Abcam) and 1:200 diluted mouse anti-tyrosine hydroxylase (TH) Abs (sc-25269, Santa Cruz Biotechnology) in blocking solution overnight at 4 °C. After washing with PBS, the cells were incubated with 1:500 diluted Alexa-Fluor-568-conjugated anti-rabbit secondary Abs and 1:500 diluted Alexa-Fluor-514-conjugated anti-mouse secondary Abs for 90 min at room temperature. Finally, coverslips were incubated in PBS containing DAPI for 15 min at room temperature. Fluorescence emission was detected after double excitation at 568 nm and 514 nm by the TCS SP8 scanning confocal microscope described in the Supplementary text.
Stimulated Emission Depletion (STED) microscopy analysis of aS binding to neuronal membranes
STED xyz images (i.e., z-stacks acquired along 3 directions: x, y, and z axes) of primary rat cortical neurons were acquired by using an SP8 STED 3X confocal microscope (Leica Microsystems), as reported previously (Bigi et al., 2020; Cascella et al., 2021; Bigi et al., 2021). In a set of experiments, primary rat cortical neurons were treated with OB* and SF at 0.3 µM (monomer equivalents) for 60 min. In another set of experiments, primary rat cortical neurons were treated for 60 min with the same αS species in culture medium containing different Ca2+ concentrations (2 mM and 0.5 mM). After treatment, cell membranes were counterstained with 0.01 mg/ml WGA, Tetramethylrhodamine Conjugate (W849, Thermo Fisher Scientific), and aS was detected with 1:125 diluted mouse monoclonal 211 anti-αS IgG1 Abs (sc-12767, Santa Cruz Biotechnology) and then with 1:500 Alexa Fluor 514-goat anti mouse IgG1 secondary Abs (A-31555, Thermo Fisher Scientific). Fluoromount-G™ (00-4958-02, Fisher Scientific) was used as mounting medium. Fluorescence emission was detected after double excitation at 550 nm and 514 nm. STED xyz images were acquired in bidirectional mode with a Leica SP8 STED 3X confocal microscope system. Tetramethylrhodamine fluorophore was excited with a 550 nm-tuned white light laser (WLL) and emission collected from 564 to 599 nm, Alexa Fluor 514 was excited with a 510 nm-tuned WLL and emission collected from 532 to 551 nm. Frame sequential acquisition was applied to avoid fluorescence overlap. 650 nm Gated pulsed-STED were applied, and a gating between 0.3 to 6 ns to avoid collection of reflection and autofluorescence. Images were acquired with Leica HC PL APO CS2 100x/1.40 oil STED White objective, and de-convolved with Huygens Professional software (Scientific Volume Imaging B.V., version 18.04), as previously reported (Bigi et al., 2021). Z- series stacks were obtained from 5 μm neuron slices. Images were collected at 0.1 μm intervals. The degree of co-localization between aS species and the plasma membrane was determined in regions of interest (ROIs) by using the ImageJ software, version 1.52t (NIH) and the JACoP (Just Another Co-localization Plugin) plugin. We determined both the Pearson’s correlation coefficient, an indicator of the correlation of the pixel intensities in the two channels (Dunn et al., 2011), and the Mandes’ overlap coefficients (M1 and M2), (Adler et al., 2012) which reflect the co-occurrence, intended as the fraction of pixels in the first channel overlapping with those of the second one.
Analysis of aS species co-localizing with PrPC
SH-SY5Y cells seeded on glass coverslips were treated for 10 min with OB* and SF at 0.3 μM (monomer equivalents). In a set of experiments, the cells were pre-treated with 0.05% trypsin on ice for 10 min, washed twice with PBS and then OB* were added to the culture medium of for 10 min at 0.3 μM (monomer equivalents). After fixation in ice-cold methanol for 20 min at -20 °C, cells were incubated with 1:250 diluted rabbit anti-aS polyclonal Abs (ab52168, Abcam), together with 1:250 diluted mouse anti-PrPC monoclonal Abs (sc47730, Santa Cruz Biotechnology) for 60 min at 37°C, and then with 1:1000 diluted Alexa Fluor 488- and 633- conjugated anti-rabbit and anti-mouse secondary Abs (A-11034 and A-21052, Thermo Fisher Scientific) for 60 min at 37°C. Fluorescence emission was detected after double excitation at 488 and 633 nm by the TCS SP8 scanning confocal microscopy system described in the Supplementary text. The colocalization of aS species with PrPC was estimated in regions of interest, as reported above.
RNA Interference for PrPC silencing and immunocytochemical analysis of PrPC-silenced SH-SY5Y cells
SH-SY5Y cells seeded on glass coverslips were transfected with 25 nM Stealth RNAi, referred to as control siRNA (Thermo Fisher Scientific), or with 25 nM siRNA against PrPC (Thermo Fisher Scientific), using Lipofectamine 3000 (Life Technologies, Thermo Fisher Scientific), according to the manufacturer’s instructions, with 7 μL of Lipofectamine and 10 μL of 5 mg/L transferrin in DMEM for 3 h in a 5% CO2 humidified atmosphere at 37 °C. Then, the transfection medium was replaced with fresh complete one, in which cells were incubated for 72 h, as reported previously (Bigi et al., 2023b). 72 h after the beginning of silencing, cells were washed with PBS, fixed in 2% (v/v) paraformaldehyde for 10 min at room temperature and then incubated for 60 min at 37 °C with 1:250 diluted mouse monoclonal anti-PrPC Ab and then for 60 min at 37 °C with 1:1000 diluted Alexa Fluor 633-conjugated anti-mouse secondary Ab.
Confocal microscopy analysis of aS binding to cell membranes
In a set of experiments, SH-SY5Y cells silenced with PrPC siRNA or with control siRNA were treated with OB* and SF for 10 min at 0.3 μM (monomer equivalents). In another set of experiments, primary cortical neurons and iPSC-derived dopaminergic neurons were pre-treated with 0.15 μM mouse monoclonal anti-PrPc Ab (sc-47730, Santa Cruz Biotechnology), with a αS : anti-PrPc Ab molar ratio of 1:0.5, and then treated with OB* at 0,3 µM for 10 min. After incubation, SH-SY5Y cells and primary rat cortical neurons were washed with PBS, counterstained with 5.0 μg/ml Alexa Fluor 633-conjugated wheat germ agglutinin (Thermo Fisher Scientific), and fixed with 2% (v/v) paraformaldehyde. To detect only the oligomers bound to the cell surface, the cellular membrane was not permeabilized, thus preventing Ab internalisation, as reported previously (Cascella et al., 2019). After washing twice with PBS,the presence of αS species bound to cellular membranes was detected with 1:250 diluted rabbit polyclonal anti-aS Abs (ab52168 Abcam), and subsequently with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse secondary Abs. iPSC-derived dopaminergic neurons were then incubated with 1:400 diluted mouse anti-MAP-2 Abs (ab11267, Abcam) and 1:250 diluted rabbit polyclonal anti-aS Abs (ab52168 Abcam) in blocking solution overnight at 4 °C. After washing with PBS, cells were incubated with 1:500 diluted Alexa-Fluor-568-conjugated anti-rabbit secondary Abs (Thermo Fisher Scientific) and 1:500 diluted Alexa-Fluor-514-conjugated anti-mouse secondary Abs (Thermo Fisher Scientific) for 90 min at room temperature. Fluorescence emission was detected after double excitation at 488 nm and 633 nm, or 514 nm and 568 nm, by the Leica SP8 STED 3X confocal microscope system described in the Supplementary text. Three-four apical sections were then projected as a single composite image by superimposition, and the degree of co-localisation of αS species with the cell membranes was determined as reported above.
Confocal microscopy analysis of the penetration of aS species
In a set of experiments, OB* and SF at 0.3 μM (monomer equivalents) were added to the culture medium of iPSC-derived dopaminergic neurons for 6 h, previously pre-treated or not with 0.15 μM mouse monoclonal anti-PrPc Ab (sc-47730, Santa Cruz Biotechnology), with a αS : anti-PrPc Ab molar ratio of 1:0.5. The cells were treated with 0.01 mg/ml WGA, Tetramethylrhodamine Conjugate, fixed in 4% (v/v) paraformaldehyde at room temperature and blocked with fetal bovine serum in 0.1% Triton X-100 for 30 min. Coverslips were then incubated with 1:250 diluted rabbit anti-oligomer A11 polyclonal Abs (AHB005 Thermo Fisher Scientific) in blocking solution overnight at 4 °C. After washing with PBS, the cells were incubated with 1:500 diluted Alexa-Fluor-568-conjugated anti-rabbit secondary Abs (Thermo Fisher Scientific) and 1:500 diluted Alexa-Fluor-514-conjugated anti-mouse secondary Abs (Thermo Fisher Scientific) for 90 min at room temperature. Fluorescence emission was detected after double excitation at 514 nm and 568 nm by the TCS SP8 scanning confocal microscopy system described in the Supplementary text.
In another set of experiments, primary rat cortical neurons were treated for 60 min with OB* and SF at 0,3 µM (monomer equivalents) in culture medium containing different Ca2+ concentrations (2 mM, and 0.5 mM). After incubation, the cells were washed with PBS, counterstained with 0.01 mg/ml WGA, Tetramethylrhodamine Conjugate, fixed with 2% (v/v) paraformaldehyde and then permabilized in PBS supplemented with 0.5% bovine serum albumin (BSA) and 3% glycerol. The presence of αS species was detected with 1:250 diluted mouse monoclonal 211 Abs, and subsequently with 1:500 diluted Alexa Fluor 514-conjugated anti-mouse secondary Abs (A-11034, Thermo Fisher Scientific). Fluorescence emission was detected after double excitation at 514 nm and 568 nm by the Leica SP8 STED 3X confocal microscope system described above, and expressed in arbitrary units.
In another set of experiments, aS OB* were added to the culture medium of SH-SY5Y cells seeded on glass coverslips for 60 min at 0.3 μM (monomer equivalents) in the presence of physiological Ca2+ levels (2 mM) or lower Ca2+ levels (1, 0.5 and 0 mM), also in the presence of the Ca2+ chelator BAPTA at 10 µM, in the absence or in the presence of a pre-treatment for 30 min with the anti-PrPC Ab at OB*:Ab molar ratio of 1:1. Cellular membranes were counterstained with 633-conjugated wheat germ agglutinin and αS with 1:250 diluted mouse monoclonal 211 anti-αS IgG1 Abs (sc-12767, Santa Cruz Biotechnology) and with 1:1000 diluted Alexa-Fluor-488-conjugated anti-mouse secondary Abs. Then, fluorescence emission was detected after double excitation at 488 and 633 nm by the TCS SP8 scanning confocal microscopy system described in the Supplementary Text, and expressed as the percentage of that observed in untreated cells, taken as 100%.
Measurementof intracellular Ca2+
aS OB* and SF were added to the culture medium of primary rat cortical neurons seeded on glass coverslips for 0, 5, 15, 60, and 180 min at 0.3 μM (monomer equivalents). In a set of experiments, SH-SY5Y cells were transfected with control siRNA, or with a siRNA against PrPC as reported in the previous section, and then treated with OB* at 0.3 µM (monomer equivalents) for 0, 5, 10, 15, 30, 60, and 180 min. In another set of experiments, SH-SY5Y cells were pre-treated with different concentrations of mouse monoclonal anti-PrPC Ab (sc47730, Santa Cruz Biotechnology) (from 0.03 μM to 0.3 μM), with αS:anti-PrPc Ab molar ratios of 1:0.1, 1:0.25, 1:0.5, and 1:1, and then treated with OB* and SF at 0,3 µM for 15 min. After treatments, cells were loaded with 4.5 µM Fluo-4 AM fluorescent probe (Thermo Fisher Scientific) for 10 min, as reported previously (Bigi et al., 2023b). The florescence was then measured by the TCS SP8 scanning confocal microscopy system described in the Supplementary text.
Analysis of the kinetics of intracellular Ca2+ influx
The different intracellular fluorescence intensities associated with Ca2+ influx were plotted versus the time elapsed after aS addition to the cellular culture medium and the resulting kinetic plots were analysed with a procedure of best fitting using a single exponential or a sigmoidal function of the forms:
F(t) = F(eq) + A exp (-k t)
where F(t) is the intracellular fluorescence at time t as a percentage of that observed in untreated cells, F(eq) is the same fluorescence at the apparent equilibrium (time ¥), A is the amplitude of the exponential fluorescence change as a percentage of that observed in untreated cells, and k is the apparent rate constant in s-1.
where F(t) is the intracellular fluorescence at time t as a percentage of that observed in untreated cells, F(0) is the same fluorescence at time zero, F(eq) is the same fluorescence at the apparent equilibrium (time ¥), A is the amplitude of the fluorescence change as a percentage of that observed in untreated cells, k is the apparent rate constant in s-1 and B is the slope of the sigmoidal function at time t.
MTT reduction assay
SH-SY5Y cells were pre-treated for 30 min with mouse anti-PrPC monoclonal Abs (sc47730, Santa Cruz Biotechnology), with αS:anti-PrPC Ab molar ratios of 1:0.5, and 1:1, and then treated with OB* and SF at 0,3 μM (monomer equivalents) for 24 h. After treatment, the cell culture medium was removed, cells were washed with PBS and the MTT assay was assessed as previously reported (Cascella et al., 2021). In another set of experiments, the viability of SH-SY5Y cells treated for 24 h with OB* at 0.3 µM (monomer equivalents) in the presence of physiological Ca2+ levels (2 mM) or in the absence of Ca2+. Cell viability was expressed as the percentage of MTT reduction in treated cells as compared to those untreated (taken as 100%).
Alteration of membrane permeability
The membrane integrity disruption was assessed in primary rat cortical neurons seeded on glass coverslips as previously described (Cascella et al., 2021). Briefly, cells were loaded with 1.0 μM calcein-AM (Thermo Fisher Scientific) for 10 min at 37 °C and then treated for 60 min with aS species at 0.3 μM (monomer equivalents) in culture medium containing different Ca2+ concentrations (2 mM and 0.5 mM). In another set of experiments SH-SY5Y cells in the presence of physiological Ca2+ levels (2 mM) or lower Ca2+ levels (1, 0.5 and 0 mM), also in the presence of the BAPTA Ca2+ chelator at 10 µM, or in the presence of a pre-treatment for 30 min with the anti-PrPC Ab at OB*:Ab molar ratio of 1:1. Cells were treated for 60 min with aS OB* at 0.3 μM (monomer equivalents). The analysis was performed after excitation at 488 nm by the TCS SP8 scanning confocal microscopy system described in the Supplementary text.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). All data were expressed as means ± standard error of mean (S.E.M). Comparisons between the different groups were performed by Student t test, or by one-way analysis of variance (ANOVA) followed by Bonferroni’s post comparison test. P-values lower than 0.05, 0.01 and 0.001 were considered to be statistically significant, very significant and extremely significant, respectively.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author.
Author Contributions
Alessandra Bigi: conceptualization, methodology, investigation, validation, formal analysis, data curation, visualisation, writing—original draft, writing—review and editing, funding acquisition. Liliana Napolitano: investigation. Giuliana Fusco: investigation, writing – review & editing. Alfonso De Simone: funding acquisition, writing – review & editing, supervision. Fabrizio Chiti: funding acquisition, writing – review & editing, supervision. Roberta Cascella: conceptualization, methodology, formal analysis, writing—review and editing, supervision, funding acquisition. Cristina Cecchi: conceptualization, methodology, writing—original draft, writing—review and editing, supervision, project administration, funding acquisition.
Funding
This research was supported by #NEXTGENERATIONEU (NGEU) and funded by the Italian MUR, National Recovery and Resilience Plan (NRRP), project Investment PE8 - Project Age-It: “Ageing Well in an Ageing Society” (D.R. 1557 11.10.2022; to A.B. and F.C.) and project MNESYS (PE0000006) - A Multiscale integrated approach to the study of the nervous system in health and disease (D.R. 1553 11.10.2022; to R.C. and C.C.). Work was also supported by University of Florence (Fondi di Ateneo to A.B., R.C., C.C. and F.C.; RICTD2024-2025 project VANGUARD to R.C).
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