Prion protein facilitates synaptic vesicle release by enhancing release probability.
Bottom Line: Here we investigated wild-type PrP(C) signalling in synaptic function as well as the effects of a disease-relevant mutation within PrP(C) (proline-to-leucine mutation at codon 101).The expression of the mutated PrP(C) leads to reduction of both parameters compared with wild-type PrP(C).Wild-type PrP(C) enhances synaptic release probability and quantal content but reduces the size of the ready-releasable vesicle pool.
Affiliation: MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK.Show MeSH
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Mentions: To further investigate synaptic vesicles at NMJ synapses we used electron microscopy (EM) to measure presynaptic vesicle sizes. As the Drosophila NMJ harbours two different kinds of boutons, namely 1s and 1b, both exhibiting different vesicular sizes, a subdivision of EM images into 1s and 1b boutons was made as shown before (41). The images in Figure 3A illustrate representative 1s and 1b boutons with release sites [long arrows: active zones (AZs)] and vesicles and higher magnification images for genotypes indicated. Bouton types were identified by size, number of synapses and AZs and size of the sub-synaptic reticulum (41) (Fig. 3A). Figure 3B shows average relative cumulative frequency histograms and histograms for vesicle diameters demonstrating an increased probability of larger diameters and hence can explain a right-shift in mEJC amplitude distributions in PrP3F4 and PrPP101L expressing larvae relative to their respective controls (PrP3F4 versus UAS Ctrl: D = 0.131, P < 0.0001, PrPP101L versus UAS Ctrl: D = 0.191, P < 0.0001, K–S test, n = 9–17 boutons). Furthermore, the difference between vesicle diameters from PrP3F4 and PrPP101L boutons shows also significant differences (PrPP101L versus PrP3F4: D = 0.137, P < 0.0001). Mean total vesicular sizes provide the best comparison to the mean mEJC amplitudes shown above (Fig. 2). Figure 3C illustrates the increase in mean total diameters in PrPP101L (n = 16–17 boutons) and PrP3F4 larvae (n = 9–17 boutons) relative to their UAS controls but also the difference between PrPP101L and PrP3F4 expressing larvae (*P < 0.05, **P < 0.01, Student's t-test). As these values are composed of 1s and 1b type boutons (equal number of each bouton type), we decided next to test whether prion protein effects could be observed at both bouton types. We subdivided the boutons according to previously published characteristics (number of synapses and AZs and size of the sub-synaptic reticulum) and vesicle diameter values for 1s and 1b boutons of ∼45 and ∼38 nm, respectively (41). Expression of either prion protein led to an increase in mean vesicle diameter in both types of boutons compared with their UAS controls (Fig. 3D, *P < 0.05, Student's t-test, n = 4–10 boutons). Interestingly, the number of AZs per bouton type did not differ between the genotypes (data not shown). The overall shift in the distribution of synaptic vesicle diameter in 1s and 1b boutons towards larger values suggests that PrP3F4/PrPP101L is directly or indirectly involved in regulating vesicle size. Based on the changes in vesicular diameter we calculated the increase in volume which increased in PrP3F4 expressing larvae by 60 and 50% in 1s and 1b boutons, respectively, whereas PrPP101L mutants showed an increase of 30 and 41% in 1s and 1b boutons, respectively. These increases are comparable with data from electrophysiological recordings although one has to consider an overestimation of measured mEJC amplitudes due to non-detectable smaller mEJCs (42). The data also imply that the mutated form of prion protein (PrPP101L) exhibits a diminished and altered function compared with PrP3F4 signalling. Together, this data suggest that PrP3F4 has an endogenous function in vesicle biogenesis and positively controls vesicle size and transmitter release at the Drosophila NMJ.Figure 3.
Affiliation: MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK.