Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons

Abstract

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrP^C-overexpressing mice. In addition, α-synuclein binds strongly on PrP^C-expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.

Keywords: Amyloid spreading; Microfluidic devices; Prnp; Synuclein.

Fig. 1

p-α-Synuclein pathology in the telencephalon of Prnp ^0/0, Prnp ^+/+ and Tga20 mice injected with mouse α-synuclein fibrils in the postcommissural striatum. a-c Schemes illustrating the location of p-α-synuclein deposits (asterisks) shown in panels (d–o). d-o High power photomicrographs showing p-α-synuclein labelling in the striatum (d–f); neocortical layer V (g–i); amygdala (j–l) and S. nigra (m–o) of Prnp ^0/0 (d, g, j, m), Prnp ^+/+ (e, h, k, n) and Tga20 (f, i, l, o) mice. Note the relevant accumulation of p-α-synuclein labelling in intracellular deposits of retrograde-labelled projecting neurons. Scale bar: d = 100 μm pertains to e–o

Fig. 2

Increased α-synuclein labelling as LBL aggregates in the neocortex in Tga20 mice. a–c Examples of p-α-synuclein aggregates in the motor cortex of Prnp ^0/0, Prnp ^+/+ and Tga20 mice injected with mouse α-synuclein fibrils in the postcommissural striatum. Note the relevant accumulation of p-α-synuclein labelling in intracellular deposits of retrograde-labelled neurons in the cortical layer V of Tga20 mice. The LBL and LNL aggregates can be clearly seen over the pale background in the cortex. d Graph illustrating the quantification of the neuronal aggregates in the different genotypes. Each count represents one section. (Mice number Prnp ^+/+ (n = 5); Prnp ^0/0 (n = 4) and Tga20 (n = 6)). In addition, the mean ± SEM is also plotted. *P < 0.05 and ***P < 0.01, ANOVA Bonferroni post hoc test. Scale bar: a = 100 μm pertains to b and c

Fig. 3

Analysis of α-synuclein transport using microfluidic devices. a 2D representation of the two PDMS devices used in the present study. b, c Double immunofluorescence photomicrographs illustrating TUJ1/GFAP (b) or TUJ1/olig2 (c) staining in primary cultured neurons in the devices. d Primary cortical cultures of the Prnp ^+/+ were maintained in the devices for 7 days. Then, mouse α-synuclein fibrils were added to A reservoir (asterisk), and their transport to B reservoir was analysed with immunocytochemistry (d–h) and western blot (i). d Examples of double-labelled neurons (TUJ1/α-synuclein) in B reservoir (indicated with camera icon) showing α-synuclein labelling (arrows). e, f Examples of p-α-synuclein-labelled neurons and neurites (arrows) in A reservoir (indicated with camera icon). g, h Examples of p-α-synuclein-labelled neurons and axons (arrows) in B reservoir (indicated with camera icon). i Western blot showing the presence of α-synuclein (17 kDa band) in both cellular extracts (A and B) and the absence in the culture medium of B in contrast to A reservoir, avoiding passive fluid transport. Membranes were reblotted with an antibody against β-tubulin or TUJ1 for standardization and characterization. Scale bar: b, d, e, g and h = 40 μm

Fig. 4

Determination of α-synuclein transport in neuronal cultures from mouse embryos carrying differing Prnp dosages. Recombinant mouse α-synuclein protofibrils were added to A reservoir (asterisk), and its binding to neurons in A reservoir as well as their transport towards B were analysed by western blot in cellular extracts. a–d Examples of double-labelled Prnp ^0/0 (a, b) and Prnp ^+/+ (c, d) neurons (TUJ1/α-synuclein) in B reservoir showing discrete cytoplasmatic α-synuclein labelling (arrows). e Examples of western blot determination of one device for each Prnp genotype for α-synuclein. Anti-β-tubulin and anti-PrP^C monoclonal antibodies were used for standardization and genotype characterization. Notice that PrP^C was present in WT and Tga20 cells at the time of treatment. PrP^C is downregulated shortly after plating neurons, and their levels increased over time in culture (see also [68] for details). f–g Densitometric analysis (see the “Material and Methods” section for details) were performed, and quantification was represented as the ratio between α-synuclein/β-tubulin detected in A reservoir after protofibril treatment (b) and ratio between α-synuclein/β-tubulin detected in B vs A reservoirs (c). Results for each device are represented by a single plot in the scatter plot. In addition, mean ± SEM is also plotted

Fig. 5

Increased binding of α-synuclein in Prnp-transfected HEK293 cells. a Western blot shows increased labelling of the 17 KDa α-synuclein band in HEK293 cells transfected with mouse Prnp-encoding plasmid in contrast to mock-transfected cells. Anti-β-tubulin monoclonal antibody was used for standardization, and anti-PrP^C antibody was used to check PrP^C overexpression after transfection. Notice that HEK293 cells showed a very low endogenous PrP^C expression. The upper bands observed after protofibril treatment corresponded to non-monomeric forms of α-synuclein. b Examples of double-labelled GFP/α-synuclein HEK293 cells after transfection of PrP^C-IRES-GFP. Note the presence of the relevant labelling in the two transfected cells in comparison to the disperse α-synuclein labelling in non-transfected cells (arrows). c–e Fluorescence photomicrographs showing examples of double-labelled cells PrP^C (d) overexpressing cells and α-synuclein (c). HEK293 cells were transfected with Prnp-encoding plasmid (c, d) or mock-transfected cells (e). Arrows in c and d point to double-labelled cells, and the asterisk in d labels a PrP^C-positive/α-synuclein-negative HEK293 cell. Arrows in e point to α-synuclein labelling in mock-transfected cells. f–h High magnification photomicrograph illustrating the distribution of α-synuclein (f) in PrP^C-transfected HEK293 cells (g). Notice the relevant colocalization in several domains of the transfected cell including the plasma membrane (arrows in g and h). Scale bars: b, e = 25 μm. c, f = 25 μm belongs to d; g and h, respectively

Fig. 6

a Scheme of the ΔCC, ΔHR maps and the full-length PrP^C. b, c Fluorescence photomicrographs showing examples of double-labelled PrP^C/α-synuclein cells (arrows). d, e Fluorescence photomicrographs showing examples of double-labelled cultures using the 6H4 PrP^C and α-synuclein antibodies. Note the absence of double-labelled cells in these examples (arrows) and the background staining of α-synuclein labelling in non-transfected cells. h–k Fluorescence photomicrographs showing examples of double-labelled ΔHR/α-synuclein cells (arrows) after treatment over the labelling of non-transfected cells. Scale bar: b, d, f, h and j = 40 μm pertains to c and e, respectively